WO2024105148A1 - Plants with low seed glucosinolate content - Google Patents
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- WO2024105148A1 WO2024105148A1 PCT/EP2023/082018 EP2023082018W WO2024105148A1 WO 2024105148 A1 WO2024105148 A1 WO 2024105148A1 EP 2023082018 W EP2023082018 W EP 2023082018W WO 2024105148 A1 WO2024105148 A1 WO 2024105148A1
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-
- C—CHEMISTRY; METALLURGY
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Definitions
- the present invention relates to the field of agricultural products, especially crop plants and parts thereof having low IIMAMIT glucosinolate transporter activity.
- the invention relates to Brassicales plant carrying a mutation in at least one gene encoding an IIMAMIT transporter and having seeds with a low concentration of glucosinolate. Seeds of such plants are advantageous for production of seed products, e.g. seed or seed cakes, with naturally low levels of glucosinolate.
- the invention further relates to methods for production of said Brassicales plants, as well as to products prepared from plants of the invention.
- the Brassicales order of plants includes many cultivars that have provided civilization with a source of condiments, vegetables, forage crops, and the economically important crops rapeseed (Brassica napus and Brassica campestris or rapa) and mustard (Brassica juncea).
- Rapeseeds or seeds from other Brassica plants can be used as a source of seed oil. After pressing the oil out of the seeds, a protein rich cake remains. This cake is ideal for animal feed. However, the potential for this source depends on the level of glucosinolate in the remaining seed cake.
- a striking and characteristic chemical property of Brassicales plants is their high content of glucosinolates; amino acid-derived natural plant products containing a thioglucose and a sulfonated oxime. These sulphur-containing secondary metabolites are important because of the multiplicity of physiologically active products, such as nitriles, epithionitriles, oxazolidine-2-thiones, thiocyanates and isothiocyanates, derived from them upon cleavage by the hydrolytic enzyme myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1) upon plant damage.
- Glucosinolates can be found in all parts of the Brassicales plants and are toxic to mammals, thus playing a prominent function in plant defense against herbivores.
- plant organs with the highest fitness value accumulate the highest level of defense compounds to protect against herbivores and pathogens.
- the highest glucosinolate concentrations are found in reproductive organs, including seeds, siliques, flowers and developing inflorescences, followed by young leaves, the root system and fully expanded leaves.
- glucosinolates are known to be transported in the plant from maternal tissue into the seeds/embryos, where they accumulate to high levels, generating a high level of defense in the seeds.
- a further problem with “00” varieties is that the seeds, although low in glucosinolates, are not free of them.
- Pressed seed cake obtained from “00” varieties after the oil has been extracted will typically contain less than 18-24 micromoles of total glucosinolates (GSL) per gram of dry weight (as compared to traditional rapeseed meal that contains 120-150 ⁇ mol of total GSL per gram).
- GSL total glucosinolates
- palatability to ruminants sets the level of total GSL permitted at no more than 10-15 micromoles per gram of dry weight, meaning an animal feed could in theory be compounded almost entirely of “00” pressed seed cake if the seed cake is at the lower level of GSL content.
- glucosinolate importer genes which result in low total glucosinolate levels in the plant, including the seeds.
- WO 2012/004013 discloses modification of the glucosinolate importer gene GTR and discloses a reduction in glucosinolate levels of 60-70% in Brassica rapa and Brassica juncea plants.
- the obtained seed glucosinolate levels are still too high for use of the seed cakes or meals in e.g. poultry feed.
- a systemic decrease in glucosinolate content of the plant is undesirable, as this may impact its natural defense.
- UMAMIT Usually Multiple Amino acids Move In and out Transporter
- the exporter mutants do not alter the overall distribution of glucosinolates in the plant, thus, the plant retains a normal plant phenotype ensuring adequate natural defense against herbivores.
- the inventors further found that orthologous genes in Brassica napus share a similar glucosinolate transport capacity as those in Arabidopsis.
- the present invention is directed to tissue-specific glucosinolate exporters in plants, as only the exporters relevant for exporting glucosinolate from the mother plant to the seeds are modified.
- tissue-specific glucosinolate exporters By identifying and modifying those specific glucosinolate exporters, the glucosinolate level in the seeds can be specifically reduced without reducing the glucosinolate level in the remaining plant. Thus, a high level of defense remains in the mother plant.
- the inventors have thus solved the problem of providing Brassicales plants that maintain appropriate glucosinolate levels in other tissues, but which have seeds with significantly decreased levels of glucosinolates.
- the invention provides a Brassicales plant, or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 70%,
- the invention provides a plant product comprising a Brassicales plant or part thereof, or prepared from seeds of said Brassicales plant or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 9,
- the invention provides a seed cake prepared from a seed from a Brassicales plant, wherein the Brassicales plant is as described elsewhere herein.
- the invention provides a method for producing a plant product comprising a Brassicales plant or part thereof with low glucosinolate content, said method comprising the steps of a. providing a Brassicales plant or part thereof as described elsewhere herein; and b. processing said Brassicales plant or part thereof into a plant product, such as a seed oil or a seed cake.
- the invention provides a method for modifying glucosinolate content in a Brassicales plant or part thereof, said method comprising a step of modifying the functional activity or expression of at least one IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homo
- FIG. 1 Identification of source tissue and transporters critical for accumulation of glucosinolates in seeds of Arabidopsis.
- a Analysis of glucosinolate content in seeds of grafted Arabidopsis. 5-week-old wild type Col-0 (WT), biosynthetic null mutant myb28 myb29 cyp79b2 cyp79b3 (null), and transporter mutant gtr1 gtr2 (gtr) were reciprocally stem-grafted at a junction 1 cm above ground.
- Said four boxplots represent, from left to right, Col-0, ut29-1, ut29-2 and ut29-1C.
- c Expression of pSUR1 ::SUR1-mTQ2 (top) and pUT29::UT29-mVenus (bottom) in developing siliques collected on different days after pollination (DAP). Inserts show funiculus and seed at lower magnification. Scale bars: 50 ⁇ m (main panels) and 100 ⁇ m (inserts), a, Data are shown as mean ⁇ s.d.
- FIG. 1 Biochemical and biophysical characterization of Arabidopsis UMAMIT29, UMAMIT30 and UMAMIT31.
- b Membrane currents associated with 2Propenyl glucosinolate (2Prop) uptake for a representative UMAMIT29-expressing oocyte (UT29) voltage-clamped at -60 mV and superfused with 10 mM 2Prop in Kulori buffer for 2 min.
- c Charge coupling stoichiometry estimated by correlation of current-time integrals and 2Prop uptake during the clamping period indicated in b in 13 oocytes expressing UT29 (grey) and in 10 oocytes injected with H2O (black),
- d Effect of pH on UT29-mediated glucosinolate import.
- Intracellular 2Prop was quantified from UT29-expressing or H2O- injected (CO-inj) oocytes after incubation with 1 mM 2Prop at pH 7.4 and pH 5.5 with or without 0.1 mM CCCP for 60 min. e, Effect of extracellular cations on UT29- mediated 2Prop import. 91 mM choline + CI- (Choline + ) or 91 mM N-methyl-d-glutamine + CI- (NMDG + ) were used to substitute the cations in Kulori buffer (90 mM Na + CI- and 1 mM K + CI-).
- Intracellular 2Prop was quantified in UT29-expressing or CO-injected oocytes after incubation with 5 mM 2Prop at pH 7.4 for 60 min. f, Injection-based export assay of 2Prop by UT29.
- UT29-expressing (bright grey line) or H2O-injected (dark grey line) oocytes were injected with 2Prop (initial internal concentration ⁇ 2 mM).
- Glucosinolates were grouped into aliphatic and indolic according to their side chain, n, biological replicates per experiments, 3 oocytes were pooled in one replicate. Treatments were compared by one-way ANOVA analysis followed by Tukey’s post-hoc HSD test. Bars labelled with different letters are significantly different, p ⁇ 0.05).
- 4MTB 4- methylthiobutyl glucosinolate
- 2Prop 2Propenyl glucosinolate
- COOP protonophore carbonyl cyanide m-chlorophenylhydrazone
- I3M indol-3-ylmethyl glucosinolate
- GLS glucosinolates
- UT UMAMIT.
- FIG. 3 Seed trait and glucosinolate distribution in mutants of UMAMIT29, -30 and -31.
- Glucosinolate content in developing seeds and siliques without seeds i.e. silique valves, septa and funiculi
- FIG. 4 Time course of glucosinolate accumulation in developing siliques and cellular localization of UMAMIT29.
- c Cross section of siliques expressing pCYP83A1 ::CYP83A1-mVenus (top) and pCYP83B1 ::CYP83B1-mVenus (bottom) at mature green stage,
- d Funiculus-expressed transporters from transcriptomics data.
- Transporter genes were selected that showed an increase in expression in funiculi from the global stage (gFUN), heart stage (hFUN) to mature green stage (mgFUN), resulting in a list of 10 glucosinolate candidate exporters, e, Total methionine- and tryptophan-derived glucosinolates from a pool of 10 siliques without seeds (i.e.
- Four boxplots are shown for each day. Said four boxplots represent, from left to right, Col-0, ut29-1, ut29-2 and ut29-1C.
- Figure 5 Analysis of transcriptomic data of tissues from developing siliques.
- FIG. 7 Genomic loci of UMAMIT29-31 and genotypes of umamit29, -30 and -31 mutants by T-DNA insertion and CRISPR-based genome editing, a, The sgRNA sequence used to target UMAMIT31 and UMAMIT30 in a schematic representation of tandemly-linked UMAMIT29 UMAMIT30 and UMAMIT31 genomic loci. Different alleles of umamit30 (ut3O) mutants and umamit31 (ut31) mutants were used in the study.
- Wild type gene structures or sequences of UMAMIT30 (UT30) and UMAMIT31 (UT31) (top) are shown above the mutant alleles: sgRNA target site in UMAMIT31 in wild type Arabidopsis thaliana (SEQ ID NO: 23) and in UMAMIT30 in wild type Arabidopsis thaliana (SEQ ID NO: 27), and sgRNA site in mutants ut31-3 (SEQ ID NO: 24), ut31-4 (SEQ ID NO: 25), ut31-5 (SEQ ID NO: 26), ut30-2 (SEQ ID NO: 28), ut30-3 (SEQ ID NO: 29) and ut30-4 (SEQ ID NO: 30).
- FIG. 8 Distribution of glucosinolates in stem and cauline leaves of umamit29, - 30 and -31 mutants.
- Figure 9 Phylogeny of part of UMAMIT family in Malvidae. Selected part of a Maximum-likelihood inferred tree (s.d. ⁇ 0.01, optimal log-likelihood value (-35897.839)) of UMAMIT homologs from 14 species: Gossypium hirsutum, Theobroma cacao, Carica papaya, Arabidopsis thaliana, Brassica rapa, Glycine max, Manihot esculenta, Solanum lycopersicum, Zea mays, Vitis vinifera, Oryza sativa japonica, Eutrema salsugineum, Capsella rubella, and Citrus Clementina. RAxML generated bootstrap values are shown for each branch. Names of glucosinolate-producing taxa are shown in bold.
- FIG. 10 Maximum-likelihood inferred tree of UMAMIT genes in Arabidopsis and Brassica napus ZS11 orthologs. Bootstrap (1000 times) values in percent are shown for each branch.
- FIG. 11 Expression profiles of the B. napusZS11 UMAMITs genes in silique wall and seeds. Transcriptome data have been extracted from the Brassica Expression Database (Brassica EDB). One BnGTR2 required in seed glucosinolate loading by GWAS was included for validation the expression pattern.
- Figure 14 Import of 4MTB, I3M and BGLS by selected UMAMIT29 mutant variants mutated in conserved residues within the predicted substrate transporting cavity. Different letters indicate significant differences of the mean (one-way ANOVA followed by TUKEY HSD test, p ⁇ 0.05). Abbreviations: UT, UMAMIT; 4MTB, 4-methylthiobutyl glucosinolate; I3M, indole 3-ylmethyl glucosinolate; BGLS, benzyl glucosinolate.
- FIG. 15 Sequence logos of amino acid residues in the predicted substrate transporting cavities of the two major clusters of transporters of UMAMIT clade I. Differentially conserved residues (stars) were estimated using DIVERGE software. Two of the estimated residues from the DIVERGE estimation were identical among UMAMIT32 and respectively, UMAMIT30 and UMAMIT31 and therefore filtered out (grey stars). The resulting 11 residues estimated to be differentially conserved were tested experimentally for their role in glucosinolate transport activity (black stars). The sequence logos were made in JDet. The numbers constitute the residue position in UMAMIT29 and the respective residues in UMAMIT29, -30, -31 and -32 can be seen in supplementary table 1.
- Brainssicales plants refers to plants in the Brassicales order.
- Brassicales plant refers to plants of the Brassicales order.
- Non-limiting examples of Brassicales plants include plants of the Brassicaceae family, such as B. Juncea, B. napus (rape), B. carinata, B. oleracea and B. rapa.
- GSLs Glucosinolates
- GSLs amino acid-derived thioglucosidic organic anions comprising a sulfonated aldoxime moiety.
- a variable side chain depending on the parent amino acid and further side chain modifications gives the distinct chemical and biological properties for GSLs.
- GSLs Approximately 120 different GSLs have been described in the literature and they are all derived from only 8 different amino acids. The parent amino acids are conveniently used as a classification criteria.
- GSLs derived from Ala, Leu, lie, Vai and Met are called “aliphatic GSLs”, those derived from Tyr and Phe are called “aromatic GSLs” and those derived from Trp are called “indole GSLs”.
- the great variety in GSL types is caused by a number of modifications on the side chain of the parent amino acid. Especially, methionine undergoes a wide range of transformations.
- the predominant aliphatic GSLs in the Brassicaceae possess side chains derived from chain elongated forms of Met, such as aliphatic thio-GSLs 3-methylthiopropyl (3-MTP)-, 4-methylthiobutyl (4- MTB)-, 5-methylthiopentyl (5-MTP)-, 6-methylthiohexyl (6-MTH)-, 7-methylthioheptyl (7- MTH)- and 8-methylthiooctyl (8-MTO)-GSL; aliphatic sulfinyl-GSLs 3- methylsulfinylpropyl (3-MSP)-, 4-methylsulfinylbutyl (4-MSB)-, 5-methylsulfinylpentyl (5- MSP)-, 6-methylsulfinylhexyl (6-MSH)-, 7-methylsulfinylheptyl (7-MSH)- and 8- methylsulfiny
- the predominant aromatic GSLs in the Brassicaceae possess side chains derived from Phe, such as aromatic GSL 2-phenylethyl (2-PE)- GSL.
- GSLs co-occur in plants with the GSL-specific thioglucosidase myrosinase. This enzyme is physically separated from GSLs in plants, but is brought into contact with its substrate upon tissue disruption.
- the resulting hydrolysis product consists of one free glucose and one aglycone molecule per GSL molecule.
- the aglycones are unstable and readily rearrange into isothiocyanates, nitriles, thiocyanates and other more or less toxic compounds. Depending on the side chain of the parent amino acid these hydrolysis products contribute the actual biological activity of GSLs, while intact GSLs are believed to be an inactive storage form.
- glucose content of a plant or plant part refers to the total of GSLs, including aliphatic, aromatic and indole GSLs, without regard to the type of GSLs.
- total GSL content or “GSL content” of a plant or plant part means the content of total GSLs of that plant or plant part and is expressed on a molecular (nmol/g or ⁇ mol/g) basis (rather than on a weight (mg/kg) basis) as GSLs have significantly different molecular weights depending on the size of their side chain. GSL accumulation varies between tissues and develo ⁇ mental stages.
- Young leaves and reproductive tissues such as siliques and seeds contain the highest concentrations while senescing leaves contain the lowest concentrations of GSLs. Intermediate concentrations are found throughout the “large" organs such as the roots, leaves and stem. In addition, the composition of the GSL profile varies markedly between organs. In vegetative tissues, the GSL content is composed of indole and aliphatic GSLs while the aromatic are absent. In siliques and seeds, small amounts of aromatic and indole GSLs are found while the rest of the GSL content is entirely composed of aliphatic GSLs.
- glucosinolate transporter activity is intended the ability of a protein to transport glucosinolates across the cell membrane.
- Glucosinolate transporter activity can for example be measured using the Xenopus oocyte assay as described in Example 1.
- Glucosinolate transporter activity can relate to one or more different glucosinolates or to groups of glucosinolates, for example methionine-derived and tryptophan-derived glucosinolates as representatives of aliphatic and aromatic glucosinolates respectively.
- GSL transporter activity or “loss of function” with respect to GSL transportation is intended a reduction compared to the wildtype unmutated glucosinolate transporter, for example using the Xenopus oocyte assay of example 1 or by studying the GSL content in seeds in mutant plants.
- a “low” activity is a GLS transporter activity of 50% or less compared to wildtype, preferably 40% or less, more preferably 30% or less, more preferably 20% or less, more preferably 10% or less.
- no GLS transporter activity or “loss of function” is intended a GSL transporter activity reduced to 5% or less such as 3% or less, for example 1 % or less of the activity of the wild type transporter.
- a “decrease in total GSL content” or “increase in total GSL content” of a plant or plant part by the methods of the present invention is measured relative to the total GSL content of a reference plant or plant part with similar genetic background.
- Total GSL content can be measured by any appropriate method.
- Methods to quantify total GSL content and to determine GSL composition of plant material are well known in the art and include but are not limited to: HPLC-UV desulfo-method involving HPLC analysis of methanol extracts desulfated and eluted from sephadex anion exchange columns as described by, e.g., Hansen et al. (2007, Plant J.
- plant or “plants” according to the invention
- plant parts progeny of the plants which retain the distinguishing characteristics of the parents (especially the glucosinolate content in particular plant parts), such as seed obtained by selfing or crossing, e.g. hybrid seed (obtained by crossing two inbred parental lines), hybrid plants and plant parts derived thereof are encompassed herein, unless otherwise indicated.
- Plant parts refers to any part of the plant, including plant cells, plant tissues, plant organs, siliques or seed pods, seeds, severed parts such as roots, leaves, flowers, pollen, etc.).
- Wild type refers to a typical form of a plant or a gene as it most commonly occurs in nature.
- a wild type plant refers to a plant with the most common phenotype of such plant in the natural population.
- a wild type allele refers to an allele of a gene required to produce the wild- type phenotype.
- a “mutant plant” refers to a plant with a different rare phenotype of such plant in the natural population or produced by human intervention, e.g. by mutagenesis, and a “mutant allele” refers to an allele of a gene required to produce the mutant phenotype.
- a homologue or functional homologue may be any polypeptide that exhibits at least some sequence identity with a reference polypeptide and has retained at least one aspect of the original functionality.
- a functional homologue of a IIMAMIT transporter is a polypeptide sharing at least some sequence identity with said IIMAMIT transporter or a fragment thereof which has the capability to function as a glucosinolate transporter similarly to said IIMAMIT transporter.
- sequence identity as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e. a candidate sequence (e.g. a mutant sequence) and a reference sequence (such as a wild type sequence) based on their pairwise alignment.
- the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/).
- the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix.
- the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
- the Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence (e.g. a natural variant or halotype of SEQ ID NO: 1) corresponds to a given position of SEQ ID NO: 1 (reference sequence). For example, if the natural variant has two additional amino acids in the N- terminal, position 70 in the natural variant will correspond to position 68 of SEQ ID NO: 1.
- the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
- the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix.
- the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
- corresponding sequence refers to a region or residue on a second amino acid or nucleotide sequence which occupies the same (i.e. , equivalent) position as a region or residue on a first amino acid or nucleotide sequence, when the first and second sequences are optimally aligned for comparison purposes.
- a residue at a first position in a first peptide sequence does not necessarily correspond to a residue in said same first position in a second peptide sequence, but may instead correspond to a residue at a second position in the second peptide sequence that optimally aligns with the residue in said first position of said first peptide sequence, when the first and second peptide sequences are optimally aligned.
- Said alignment may be performed by any method known in the art, such as by using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.
- the parameters used may be gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix.
- missense mutation refers to a mutation/mutations in a nucleotide sequence resulting in a change from one amino acid to another in the polypeptide encoded by said nucleotide sequence.
- “Mutations” include deletions, insertions, substitutions, transversions, and point mutations in the coding and/or noncoding regions of a gene. Deletions may be of an entire gene, or of only a portion of a gene. Point mutations may concern changes of one base pair, and may result in premature stop codons, frameshift mutations, mutation of a splice site or amino acid substitutions.
- a gene comprising a mutation when compared to a wild type gene may be referred to as a “mutant gene”.
- a mutant gene generally encodes a polypeptide with a sequence different to the wild type gene, said polypeptide may be referred to as a “mutant polypeptide”.
- a mutant polypeptide may comprise an amino acid substitution, such a substitution can for example be described as “amino acid XXX at position n has been substituted to amino acid YYY” where XXX describes the amino acid at the specific position (n) of the wild type polypeptide and YYY describes the amino acid present in the mutant polypeptide at the same position when the two genes are aligned.
- amino acid XXX at position n has been substituted to amino acid YYY”
- XXX describes the amino acid at the specific position (n) of the wild type polypeptide
- YYY describes the amino acid present in the mutant polypeptide at the same position when the two genes are aligned.
- IIMAMIT transporters and “IIMAMIT exporters” are used interchangeably herein.
- Brassicales plants comprising a mutation in a gene encoding an UMAMIT transporter
- the present invention relates to Brassicales plants, or parts thereof, as well as products of said Brassicales plants and methods of producing these, wherein the Brassicales plant carries a mutation in a gene encoding an UMAMIT transporter, e.g. any of the mutations in genes encoding UMAMIT transporters described herein.
- a Brassicales plant or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an UMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as
- the glucosinolate transporter activity of an UMAMIT transporter may be measured by any method known in the art. In preferred embodiments, the glucosinolate transporter activity of an UMAMIT transporter is measured using the methods described herein in Examples 1 and 2.
- the Brassicales plant carries a mutation in at least one gene encoding an UMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 7
- the Brassicales plant carries a mutation in the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto
- the Brassicales plant carries a mutation in the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto
- the Brassicales plant carries a mutation in the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto
- the Brassicales plant carries a mutation in the IIMAMIT transporter AtUMAMIT31 as set forth in SEQ ID NO: 4 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto
- the Brassicales plant carries a mutation in the IIMAMIT transporter BnaA09G0714200ZS as set forth in SEQ ID NO: 5 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least
- the Brassicales plant carries a mutation in the IIMAMIT transporter BnaC05G0010000ZS as set forth in SEQ ID NO: 6 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least at least
- the Brassicales plant carries a mutation in the IIMAMIT transporter BnaA01G0222900ZS as set forth in SEQ ID NO: 7 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least
- the Brassicales plant carries a mutation in the IIMAMIT transporter BnaC01G0283800ZS as set forth in SEQ ID NO: 8 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least
- the Brassicales plant carries a mutation in the IIMAMIT transporter BnaC03G0332100ZS as set forth in SEQ ID NO: 9 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least
- the Brassicales plant carries a mutation in the IIMAMIT transporter BnaA09G0692700ZS as set forth in SEQ ID NO: 10 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least
- the Brassicales plant of the invention may comprise mutations in more than one gene encoding an IIMAMIT transporter.
- the Brassicales plant or part thereof carries a mutation in at least two genes encoding IIMAMIT transporters selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter Atll MAM IT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such
- the Brassicales plant or part thereof carries a mutation in at least three genes encoding IIMAMIT transporters selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 71%, such as at least 72%, such as at
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 9
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such
- the Brassicales plant or part thereof carries a mutation in at least four genes encoding IIMAMIT transporters selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at
- the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 9
- AtUMAMIT28 SEQ ID NO: 1
- AtUMAMIT29 SEQ ID NO: 2
- AtUMAMIT30 SEQ ID NO: 3
- AtUMAMIT31 SEQ ID NO: 4
- Tables 1-4 herein below The orthologs listed below are accessible in the Brassica napus pan genome database available at http://cbi.hzau.edu.cn/cgi-bin/bnapus/search (Song et al., 2021), using the identifiers listed in the below tables. The same identifier can be used to access both the genomic sequence as well as the encoded protein.
- BnaA09G0623500NG BnaA09G0623600NG, BnaC05G0007600NG, Bnascaffold2891G0004400NG, BnaA09G0624000NG, BnaC08G0554500QU,
- BnaA09G0689900SL Bnascaffold3068G0013800SL, BnaA09G0577200TA,
- BnaA09G0628700TA BnaC05G0007500TA, BnaA09G0577100TA, BnaA10G0008300TA, BnaA09G0628800TA, BnaA09G0658500WE, BnaA09G0658600WE, BnaC05G0009100WE, BnaA10G0009300WE, BnaA09G0659000WE, BnaA09G0692800ZS, BnaA09G0692700ZS, BnaA09G0714200ZS, BnaA09G0714300ZS, BnaA10G0008500ZS, BnaC05G0010000ZS, BnaA09G0634900ZY, BnaA09G0666500ZY, Bnascaffold4696G0013500ZY, BnaC05G0007300ZY, BnaA09G0635000ZY, BnaA09G0666400ZY,
- said mutant IIMAMIT transporter is a mutant glucosinolate transporter with reduced glucosinolate transporter activity compared to the wildtype protein.
- the mutated gene encodes a mutant glucosinolate transporter with reduced glucosinolate transporter activity compared to the wildtype protein.
- said reduction is a reduction of at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% of the glucosinolate transporter activity of said mutant glucosinolate transporter compared to the wildtype protein
- the glucosinolate transporter activity of an IIMAMIT transporter may be measured by any method known in the art. In preferred embodiments, the glucosinolate transporter activity of an IIMAMIT transporter is measured using the methods described herein in Examples 1 and 2.
- said mutated gene has reduced expression of the encoded glucosinolate transporter compared to the wildtype gene.
- said reduced expression is a reduction of at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% in expression of said mutated gene compared to the wildtype gene.
- Said reduced expression may be measured using methods commonly known in the art
- Said reduced gene expression and/or reduced transporter activity of the encoded IIMAMIT transporter protein results in a reduced glucosinolate content in specific parts of the Brassicales plant, such as in the seeds.
- the seeds of said Brassicales plant or part thereof have a reduced glucosinolate content compared to a Brassicales plant or part thereof of otherwise identical genotype except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
- said reduction in glucosinolate content is a reduction of at least 50%, such as at least 51%, such as at least 52%, such as at least 53%, such as at least 54%, such as at least 55%, such as at least 56%, such as at least 57%, such as at least 58%, such as at least 59%, such as at least 60%, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such
- the reduced gene expression and/or reduced transporter activity of the encoded IIMAMIT transporter protein does not materially affect the size of the seeds.
- the seeds of the Brassicales plant or part thereof having a reduced glucosinolate content have approximately the same size as seeds from a Brassicales plant or part thereof with an otherwise identical genotype except not comprising any of the mutations in the IIMAMIT exporters as defined herein, when cultivated and prepared under the same conditions,
- the reduced gene expression and/or reduced transporter activity of the encoded IIMAMIT transporter protein does not materially affect the dry weight of the seeds.
- the dry weight of the seeds of the Brassicales plant or part thereof having a reduced glucosinolate content have a slightly lower dry weight as compared to seeds from a Brassicales plant or part thereof with an otherwise identical genotype except not comprising any of the mutations in the IIMAMIT exporters as defined herein, when cultivated and prepared under the same conditions.
- the dry weight of the seeds of the Brassicales plant or part thereof having a reduced glucosinolate content have a weight that is not more than 7% lower, such as not more than 6 % lower, such as not more than 5% lower, such as not more than 4% lower, such as not more than 3% lower, such as not more than 2% lower, such as not more than 1% lower than the seeds from a Brassicales plant or part thereof with an otherwise identical genotype except not comprising any of the mutations in the IIMAMIT exporters as defined herein, when cultivated and prepared under the same conditions.
- the dry weight of the seeds of the Brassicales plant or part thereof having a reduced glucosinolate content have a weight that is between 1% and 7% lower, such as between 2% and 6% lower, such as between 3% and 5% lower, such as between 4% and 6% lower than the seeds from a Brassicales plant or part thereof with an otherwise identical genotype except not comprising any of the mutations in the IIMAMIT exporters as defined herein, when cultivated and prepared under the same conditions.
- the Brassicales plants according to the invention may have reduced glucosinolate levels in specific tissues such as in seeds, while having wildtype or near-wildtype levels in other tissues.
- the vegetative tissue of said Brassicales plant or part thereof have about the same glucosinolate content compared to a Brassicales plant or part thereof of otherwise identical genotype except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
- the Brassicales plant according to the invention has reduced glucosinolate levels in the seeds, and wildtype or near-wildtype levels in other tissues of said plant.
- the seeds have a concentration of glucosinolates of less than 18 micromoles per gram of dry weight of said seeds, such as less than 17 micromoles per gram of dry weight, such less than 16 micromoles per gram of dry weight, such as less than 15 micromoles per gram of dry weight, such less than most 14 micromoles per gram of dry weight, such as less than 13 micromoles per gram of dry weight, such as less than 12 micromoles per gram of dry weight, such as less than 11 micromoles per gram of dry weight, such as less than 10 micromoles per gram of dry weight, such as less than 9 micromoles per gram of dry weight, such as less than 8 micromoles per gram of dry weight, such as less than 7 micromoles per gram of
- the seeds have a concentration of glucosinolates of less than 100 nanomoles per gram of dry weight of said seeds, such as less than 50 nanomoles per gram of dry weight, such less than 25 nanomoles per gram of dry weight, such as less than 10 nanomoles per gram of dry weight, such less than 1 nanomoles per gram of dry weight, such less than 0.5 nanomoles per gram of dry weight, such less than 0.05 nanomoles per gram of dry weight, such less than 0.005 nanomoles per gram of dry weight of said seeds.
- the seeds do not have a measurable level of glucosinolates.
- the Brassicales plant is an oilseed crop. In some embodiments the Brassicales plant is a protein crop. In some embodiments, the Brassicales plant is an oilseed and protein crop. In some embodiments, the Brassicales plant is a brassicaceous oilseed crop. In some embodiments the Brassicales plant is a brassicaceous protein crop. In some embodiments, the Brassicales plant is a brassicaceous oilseed and protein crop.
- Oilseed and/or protein crops such as brassicaceous oilseed and/or protein crops, include, but are not limited to, the genera Brassica, Camelina, Crambe, Eruca, Raphanus, Lepidium and Thlaspi.
- the plant is of the family Brassicaceae. In some embodiments, the plant is of the genus Brassica. In some embodiments, the plant is selected from the group consisting of B. juncea, B. napus (rape), B. carinata, B. oleracea, B. rapa. In other embodiments, the plant is of the genus Lepidium, such as for example an L. campestre plant. In some embodiments, the plant is of the genus Camelina. In some embodiments, the plant is of the genus Crambe. In some embodiments, the plant is of the genus Eruca. In some embodiments, the plant is of the genus Raphanus. In some embodiments, the plant is of the genus Thlaspi.
- the Brassicales plant of the invention has not been exclusively obtained by means of an essentially biological process.
- Progeny of a Brassicales plant obtained by a technical process is herein considered as not being exclusively obtained by means of an essentially biological process, because the parent plant is obtained by a technical process.
- the Brassicales plant carries one or more mutations as disclosed elsewhere herein, wherein said one or more mutations have been induced by chemical and/or physical agents.
- the plant has been prepared by a method involving a step of induced mutagenesis or said plant is progeny of a plant prepared by a method involving a step of induced mutagenesis.
- the Brassicales plant may be a plant prepared by a method comprising the following steps or progeny of a plant prepared by a method comprising the following steps:
- the mutations according to the present invention may result in a change of the amino acid sequence of the gene encoding the IIMAMIT transporter.
- the mutation is a non-synonymous mutation.
- the mutation is a missense mutation. In some embodiments, the mutation is an insertion. In some embodiments, the mutation is a deletion. In some embodiments, the mutation is a frameshift mutation.
- the mutation lies within a promoter region of said gene(s). In some embodiments, the mutation lies within a coding region of said gene(s). In some embodiments, the mutation lies within an exonic region of said gene(s). In some embodiments, the mutation lies within a non-coding region of said gene(s). In some embodiments, the mutation lies within an intronic region of said gene(s). In some embodiments, the mutation lies within a termination sequence of said gene(s).
- the mutation is a loss-of-function mutation.
- the mutation is an insertion of a transfer DNA (T-DNA) sequence.
- the inserted sequence is pROK2 as set forth in SEQ ID NO: 21.
- the Brassicales plant comprises one or more mutations in one or more genes encoding an IIMAMIT transporter. In some embodiments, the Brassicales plant comprises at least two mutations in one or more genes encoding an IIMAMIT transporter, wherein the mutations are independently selected from the mutations described herein.
- the Brassicales plant may carry one or more amino acid mutations in IIMAMIT transporters, which abrogate glucosinolate transport activity of these transporters but still retain any other regulatory or enzymatic functions of these.
- the one or more amino acid mutations are in the AtUMAMIT29 polypeptide as set forth in SEQ ID NO: 2 or in a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, or such as at least 95% sequence identity thereto, and said Brassicales expresses said mutant AtUMAMIT29 polypeptide or a functional homolog thereof with corresponding mutations.
- one or more useful mutations for reducing or abrogating glucosinolate transport activity of AtUMAMIT29 are i. a substitution of amino acid 27 of SEQ ID NO: 2 (valine (V)) to a phenylalanine (F); ii. a substitution of amino acid 86 of SEQ ID NO:2 (methionine (M)) to a valine (V); iii. a substitution of amino acid 109 of SEQ ID NO:2 (leucine (L)) to a valine (V); iv. a substitution of amino acid 263 of SEQ ID NO:2 (glutamine (Q)) to a serine (S); v. a substitution of amino acid 267 of SEQ ID NO:2 (threonine (T)) to a tyrosine
- the Brassicales plant expresses a functional homolog of AtUMAMIT29 as set forth in SEQ ID NO: 2 with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, or such as at least 95% sequence identity thereto.
- the sequence of the functional homolog is first optimally aligned to the sequence of AtUMAMIT29 as set forth in SEQ ID NO: 2, e.g. using the Needleman-Wunsch algorithm, to identify corresponding amino acids, and one or more of the following mutations (corresponding mutations) are then performed to reduce or abrogate glucosinolate transport activity of the functional homolog of AtUMAMIT29: a.
- substitution of an arginine corresponding to the arginine in position 44 of SEQ ID NO: 2 with alanine R44A
- substitution of a tryptophan corresponding to the tryptophan in position 200 of SEQ ID NO: 2 with alanine W200A
- substitution of a glutamine corresponding to the glutamine in position 204 of SEQ ID NO: 2 substituted with alanine Q204A
- the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said
- Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 20%, such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
- the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said
- Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 20%, such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
- the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said
- Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 20%, such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
- the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said
- Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 30%, such as at the most 25%, such as at the most 20%, such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
- the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said
- the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said
- Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 20%, such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
- the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said
- the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said
- the Brassicales plant or part thereof thus carries a mutation in the AtUMAMIT29 gene as set forth in SEQ ID NO: 12 or in a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, wherein said mutated gene encodes a mutant Atll MAM IT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO: 2 or a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, except that said mutant polypeptide comprises a substitution or a corresponding substitution selected from the group consisting of V27F, M86V, L109V, Q263S T267Y, R44A, W200A and Q204A in relation to AtUMAMIT29 as set forth in SEQ ID NO:
- Said Brassicales plant or part thereof may also express a mutant AtUMAMIT29 polypeptide or a homolog thereof that carries more than one of said substitutions.
- the Brassicales plant or part thereof thus carries a mutation in the AtUMAMIT29 gene as set forth in SEQ ID NO: 12 or in a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, wherein said mutated gene encodes a mutant AtUMAMIT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, except that said mutant polypeptide comprises one or more substitutions or one or more corresponding substitutions selected from the group consisting of V
- Brassicales plants of the invention may comprise one or more additional mutations in one or more additional genes.
- the plant comprises a T-DNA insertion of SEQ ID NO: 22 (Vector_ pAC106) in the first intron of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least at least 9
- the Arabidopsis seeds having this T-DNA insertion have a Met-derived glucosinolates (GLS) content of about 34.74 ⁇ 9.04 nmol mg -1 and a Trp-derived GLS content of about 1.21 ⁇ 0.75 nmol mg -1 .
- GLS Met-derived glucosinolates
- the plant comprises a T-DNA insertion of SEQ ID NO: 22 (Vector_ pAC106) in the fourth exon of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding exon of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least
- the plant comprises a T-DNA insertion of SEQ ID NO: 22 (Vector_ pAC106) in the first exon of AtUMAMIT30 (SEQ ID NO: 13) or in a corresponding exon of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least
- the plant comprises a T-DNA insertion of SEQ ID NO: 22 (Vector_ pAC106) in the first exon of AtUMAMIT31 (SEQ ID NO: 13) or in a corresponding exon of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such
- the plant comprises a T-DNA insertion of SEQ ID NO: 21 (pROK2) in the first intron on both alleles of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 9
- the Arabidopsis seeds having this T-DNA insertion and this deletion have a Met-derived GLS content of about 12.97 ⁇ 1.25 nmol mg -1 and a Trp-derived GLS content of about 2.18 ⁇ 0.07 nmol mg -1 .
- the plant comprises a T-DNA insertion of SEQ ID NO: 21 (pROK2) in the first intron on both alleles of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 9
- the plant comprises a T-DNA insertion of SEQ ID NO: 21 (pROK2) in the first intron on both alleles of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron on all alleles of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%,
- pROK2 SEQ ID NO: 21
- the Arabidopsis seeds having this T-DNA insertion, this deletion and this insertion have a Met-derived GLS content of about 10.94 ⁇ 0.62 nmol mg -1 and a Trp-derived GLS content of about 0.13 ⁇ 0.01 nmol mg -1 .
- the plant comprises a T-DNA insertion of SEQ ID NO: 21 (pROK2) the first intron on both alleles of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%
- the Arabidopsis seeds having this T-DNA insertion, this insertion and this deletion have a Met-derived GLS content of about 17.56 ⁇ 0.89 nmol mg -1 and a Trp- derived GLS content of about 0.14 ⁇ 0.01 nmol mg -1 . Plant products and methods of producing the same
- the present prevention also provides plant products prepared from a Brassicales plant carrying a mutation in a gene encoding an IIMAMIT transporter according to the invention, e.g. any of the Brassicales plants, or parts thereof, described herein.
- the plant part is a seed.
- a plant product comprising a Brassicales plant or part thereof, or prepared from seeds of said Brassicales plant or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 9, B
- the Brassicales plant or part thereof is as defined elsewhere herein.
- Examples of useful plant products according to the invention include products prepared from the seeds of the plant, such as seed oils, seed cakes and seed meals.
- the plant product is prepared from a seed.
- the plant product is an oil, such as a seed oil.
- the plant product is a seed cake.
- the plant product is a seed meal.
- a seed cake prepared from a seed from a Brassicales plant, wherein the Brassicales plant is as described elsewhere herein.
- a seed cake prepared from a seed from a Brassicales plant wherein the Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with
- the seed has a concentration of glucosinolates of less than 18 micromoles per gram of dry weight of said seed, such as less than 17 micromoles per gram of dry weight, such less than 16 micromoles per gram of dry weight, such as less than 15 micromoles per gram of dry weight, such less than most 14 micromoles per gram of dry weight, such as less than 13 micromoles per gram of dry weight, such as less than 12 micromoles per gram of dry weight, such as less than 11 micromoles per gram of dry weight, such as less than 10 micromoles per gram of dry weight, such as less than 9 micromoles per gram of dry weight, such as less than 8 micromoles per gram of dry weight, such as less than 7 micromoles per gram of dry weight, such as less than 6 micromoles per gram of dry weight, such as less than 5 micromoles per gram of dry weight, such as less than 4 micromoles per
- the seed comprises a concentration of glucosinolate of at the most 15%, such as at the most 14%, such as at the most 13%, such as at the most 12%, such as at the most 11%, such as at the most 10%, such as at the most 9%, such as at the most 8%, such as at the most 7%, such as at the most 6%, such as at the most 5%, such as at the most 5%, such as at the most 3%, such as at the most 2%, or such as at the most 1% of the glucosinolate concentration in a seed prepared from a seed of a plant of otherwise identical genotype to said Brassicales plant except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
- the seed cake prepared from said Brassicales plant comprises a concentration of glucosinolate of at the most 15%, such as at the most 14%, such as at the most 13%, such as at the most 12%, such as at the most 11%, such as at the most 10%, such as at the most 9%, such as at the most 8%, such as at the most 7%, such as at the most 6%, such as at the most 5%, such as at the most 5%, such as at the most 3%, such as at the most 2%, or such as at the most 1% of the glucosinolate concentration in a seed cake prepared from a seed of a plant of otherwise identical genotype to said Brassicales plant except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
- the plant seed product comprises glucosinolates at a concentration of at the most 30 micromoles per gram of dry weight of said plant seed product, such as at the most 25 micromoles per gram of dry weight, such as at the most 20 micromoles per gram of dry weight, such as at the most 15 micromoles per gram of dry weight, such as at the most 14 micromoles per gram of dry weight, such as at the most 13 micromoles per gram of dry weight, such as at the most 12 micromoles per gram of dry weight, such as at the most 11 micromoles per gram of dry weight, such as at the most 10 micromoles per gram of dry weight, such as at the most 9 micromoles per gram of dry weight, such as at the most 8 micromoles per gram of dry weight, such as at the most 7 micromoles per gram of dry weight, such as at the most 6 micromoles per gram of dry weight, such as at the most 5 micromol
- the plant seed product comprises glucosinolates at a concentration of at the most 1.5 mmol per kg of plant product, such as at the most 1.25 mmol per kg of plant product, such as at the most 1 mmol per kg of plant product, 0.75 mmol per kg of plant product, such as at the most 0.5 mmol per kg of plant product, such as at the most 0.25 mmol per kg of plant product, such as at the most 0.1 mmol glucosinolates per kg of said plant seed product.
- the plant seed product does not comprise a measurable concentration of glucosinolates.
- Methods to measure glucosinolate content are well known in the art, and are described herein in the section “Definitions”.
- a method for producing a plant product comprising a Brassicales plant or part thereof with low glucosinolate content, said method comprising the steps of a. providing a Brassicales plant or part thereof as described elsewhere herein; and b. processing said Brassicales plant or part thereof into a plant product, such as a seed oil or a seed cake.
- a method for producing a plant product comprising a Brassicales plant or part thereof with low glucosinolate content, said method comprising the steps of a. providing a Brassicales plant or part thereof, wherein the Brassicales plant or part thereof carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth
- the plant seed products described herein can be particularly useful as animal feed and fodder.
- Glucosinolates are undesirable substances in animal feed and fodder, as they may cause growth retardation, reduction in performance (reduced milk and egg production), impaired reproductive activity, and impairment of liver and kidney functions.
- palatability to ruminants sets the level of total GSL permitted at no more than 10-15 micromoles per gram of dry weight.
- poultry and pigs are more sensitive to levels of GSLs than ruminants, and more than 2- 4 micromoles GSL per gram of dry weight in the feed can severely affect reproductive efficiencies in these animals.
- an animal feed comprising a plant product prepared from a Brassicales plant seed or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional
- a plant seed product prepared from a Brassicales plant or part thereof as described herein for use in animal feed.
- Said plant product may be as defined herein the section “Plant products and methods for producing same”.
- the plant product is prepared from seeds.
- the plant product is a seed cake, a seed oil or a seed meal.
- Methods for modifying the glucosinolate content of Brassicales plants, or parts thereof Brassicales plants, or parts thereof, carrying a mutation in a gene encoding an IIMAMIT transporter and having reducing glucosinolate content according to the invention may be prepared in any useful manner.
- a method for modifying glucosinolate content in a Brassicales plant or part thereof comprising a step of modifying the functional activity or expression of at least one IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homolog
- Said step of modifying the functional activity of at least one IIMAMIT transporter may be done using any useful method known in the art.
- said step of modifying said functional activity is performed by nuclease-based gene editing. In some embodiments, said step of modifying said functional activity is performed by CRISPR/Cas9 gene editing. In some embodiments, said step of modifying said functional activity is performed by gene targeting. In some embodiments, said step of modifying said functional activity is performed by transposition mutagenesis. In some embodiments, said step of modifying said functional activity is performed by transfer-DNA induced insertion. In some embodiments, said step of modifying said functional activity is performed by gene knockdown. In some embodiments, said step of modifying said functional activity is performed by RNA interference.
- said step of modifying said functional activity is performed by random mutagenesis. In some embodiments, said step of modifying said functional activity is performed using non-GMO methods. In some embodiments, the method further comprises one or more crosses of plants. Brassicales plants, or parts thereof, carrying a mutation in a gene encoding an IIMAMIT transporter and having reducing glucosinolate content according to the invention may be prepared in any useful manner.
- Brassicales plants according to the invention can be prepared by a method comprising the steps of: a. providing seeds of a Brassicales plant; b. randomly mutagenizing said Brassicales plant seeds, c. selecting Brassicales plant seeds or parts thereof carrying a mutated gene encoding a mutant IIMAMIT transporter polypeptide carrying a mutation that results in said mutant IIMAMIT transporter having low or no glucosinolate transporter activity, being expressed at low levels, or not being expressed.
- the step of modifying said functional activity further includes one or more steps of reproducing said Brassicales plants or parts thereof in order to obtain multiple Brassicales plants or parts thereof each carrying said mutation.
- Brassicales plants carrying a particular mutation in a gene encoding an IIMAMIT transporter may be prepared and identified essentially as described in international patent application WO 2018/001884 using primers and probes designed to identify a mutation in said gene.
- Brassicales plants carrying a mutation in a gene encoding an IIMAMIT transporter may also be prepared using various site directed mutagenesis methods, which for example can be based on the sequence of the coding sequence of the gene encoding an IIMAMIT transporter, such as a gene comprising the sequence according to any one of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.
- the Brassicales plant is prepared using any one of CRISPR, a TALEN, a zinc finger, meganuclease, and a DNA-cutting antibiotic as described in WO 2017/138986.
- the Brassicales plant is prepared using CRISPR/cas9 technique, e.g.
- RNA-guided Cas9 nuclease This may be done as described in Lawrenson et al., Genome Biology (2015) 16:258; DOI 10.1186/s13059-015-0826-7 except that the single guide RNA sequence is designed based on the gene sequence of a gene encoding an IIMAMIT transporter.
- Brassicales plant is prepared using a combination of both TALEN and CRISPR/cas9 techniques, e.g. using RNA- guided Cas9 nuclease. This may be done as described in Holme et al., 2017 except that the TALEN and single guide RNA sequence are designed based on the gene sequences provided herein.
- the Brassicales plant is prepared using homology directed repair, a combination of a DNA cutting nuclease and a donor DNA fragment. This may be done as described in Sun et al., 2016 except that the DNA cutting nuclease is designed based on the genes sequences provided herein.
- the objective is to provide agronomical useful Brassicales plants carrying a mutation in a gene encoding an IIMAMIT transporter.
- the mutation in the a gene encoding an IIMAMIT transporter there are additional factors which also may be considered in the art of generating a commercial Brassicales plant variety useful for preparing products from its seeds, for example seed yield and size, and other parameters that relate to seed quality and composition. Since many - if not all - relevant traits have been shown to be under genetic control, the present invention also provides modern, homozygous, high-yielding cultivars, which may be prepared from crosses with the Brassicales plants that are disclosed in the present publication.
- the skilled Brassicales breeder will be able to select and develop Brassicales plants, which - following crossings with other Brassicales plants - will result in superior cultivars.
- the breeder may utilize plants of the present invention for further mutagenesis to generate new cultivars carrying additional mutations in addition to the mutation of the gene encoding an IIMAMIT transporter.
- the invention also comprise Brassicales plants carrying a mutation in gene encoding an IIMAMIT transporter prepared from plant breeding method, including methods of selfing, backcrossing, crossing to populations, and the like. Backcrossing methods can be used with the present invention to introduce into another cultivar the mutation of the mutated gene encoding an IIMAMIT transporter.
- a way to accelerate the process of plant breeding comprises the initial multiplication of generated mutants by application of tissue culture and regeneration techniques.
- another aspect of the present invention is to provide cells, which upon growth and differentiation produce Brassicales plants carrying one or more mutations in a gene encoding an IIMAMIT transporter.
- breeding may involve traditional crossings, preparing fertile anther-derived plants or using microspore culture.
- Example 1 - Glucosinolate export by UMAMITs is essential for seed accumulation According to the optimal defence theory, plant organs with the highest fitness value such as seeds and tubers accumulate the highest level of defence compounds to protect against herbivores and pathogens. However, for humans, the often-toxic defence compounds drastically reduce the nutritional value of these edible tissues. Reduction of anti-nutritional factors by blocking the biosynthetic pathways is, however, accompanied by adverse effects on plant fitness. Since defence compounds are often translocated to edible tissues, elimination of transporters along the route from source to sink provides a strategy to reduce toxic compounds in edible parts while maintaining the defence in other tissues. Loss-of-function mutations of plasma membrane-localized importers and exporters may have fundamentally different effects on the distribution pattern within the whole plant.
- T-DNA insertion mutants, umamit29-7 (Salk_133129C), umamit29-2 (GK-007H08), umamit30-1 (SALK_146977C) and umamit31-1 (GK- 266E08) were ordered from the European Arabidopsis stock center NASC (Nottingham).
- ut29 ut31 and ut29 ut30 double mutants were generated by introducing single guide RNA (sgRNA)/Cas9 targeting UMAMIT31 and UMAMIT30 into umamit29-1 mutants, respectively.
- umamit29 umamit30 umamit31 triple mutants were generated by co-introducing a sgRNA targeting UMAMIT30 and a sgRNA targeting UMAMIT31 into umamit29-1.
- gtr1 gtr2 gtr3 umamit29 umamit30 quintuple mutants were generated by introducing sgRNA targeting UMAMIT30 into gtr1 gtr2 gtr3 umamit29-1.
- sgRNA targeting UMAMIT30 For genotyping of the transgenic lines, rosettes were collected from each plant and genomic DNA was isolated using the CTAB method. PCR amplification was performed using primers flanking the sgRNA target sites and Sanger sequencing was used to identify mutations in target genes. PCR was performed using Phusion DNA Polymerases. All primers were synthesized at the TAG Copenhagen A/S.
- pCYP83A1(2 kb 5' regulatory sequences) CYP83A 1(coding sequence)- mVen us 47
- pCYP83B1(2 kb 5' regulatory sequences) CYP83B 1 (coding sequence)- mVen us 47
- pSUR1(2 b 5' regulatory sequences) SUR1 (coding sequence)- mTurquoise2
- pUMAMIT29 5kb 5'regulatory sequences
- the genomic fragment of UMAMIT29 was amplified from genomic DNA of Arabidopsis Col-0 and cloned into the USERTM cassette that was inserted into the opened pFastRedU- mVenus plant expression vector.
- SgRNAs were designed using the CRISPR-P 2.0 (http://crispr.hzau.edu.cn/CRISPR2/).
- the sgRNAs were amplified with expression cassettes using primers containing Bsal recognition sites.
- the PCR fragments were cloned into pKIR1.1R and digested with Aarl by Golden Gate cloning for multiplex gene editing.
- the final binary vectors were transformed into Arabidopsis by Agrobacterium- mediated transformation.
- a v-shape wedge was made in the stock using a 0.5 cm long median incision along the length of the stem. Quickly, the scions were cut into a long v-shape wedge under water and were inserted inside the incision of the root stem.
- the graft junction was secured by a silicone tube and sealed by wrapping with parafilm (1 cm above and below the silicone tubing) to maintain hydraulic turgor and prevent desiccation.
- the grafted plants were covered with a clear plastic film for 7 days at 70% humidity. After 7 days, the plastic film was removed gradually 20% each day and the plants were watered regularly in order to acclimate to normal growing condition within one week. Seeds for glucosinolate extraction were harvested from senescent siliques from 8-week-old grafted plants.
- the flowers with visible white petals were defined as the onset of pollination and were labelled using sewing strings.
- the siliques were collected since day 5 after pollination. After collection, the siliques were snap frozen with liquid nitrogen and freeze dried before dissection into seeds and siliques without seeds for glucosinolate analysis.
- the developing seeds were exposed by removing one silique valve and then mounting the seeds in perfluorodecalin (Sigma) for live tissue imaging.
- siliques at mature green stage were generated by vibratome (Leica, Germany) for live tissue imaging.
- UMAMIT29- mVenus plants siliques were fixed and cleared using the ClearSee protocol (Kurihara et al. 2015). Briefly, siliques were fixed in 4% (w/v) para-formaldehyde (Sigma) in PBS (pH 6.7) with 0,1% (v/v) SR2200 cell wall stain (Renaissance Chemicals) under vacuum for 60 min.
- DNasel AMPDK1-1KT
- qRT-PCR was performed with DyNAmo Flash SYBR Green qPCR Kit (F- 415L, Thermo Scientific) and gene-specific primers. All values were normalized against transcript levels of the ACTIN2 gene (AT3G18780).
- Linear DNA templates for in vitro transcription were generated from pNB1u plasmid by PCR using Phusion High-Fidelity DNA Polymerase (NEB). PCR products were purified using the QIAquick PCR Purification Kits (Qiagen). Capped cRNA was in vitro synthesized using the mMessage mMachine T7 Kit (Ambion). Concentration of the synthesized cRNA of each transporter gene was normalized to 500 ng ⁇ L -1 and aliquoted before storage in -20 °C. Maximum one thaw cycle of the cRNAs was used for expression in Xenopus laevis oocytes.
- Xenopus oocytes were purchased from Ecocyte Bioscience (Germany). Oocytes were injected with 50.6 nL cRNA (500 ng ⁇ L' 1 ) using a Drummond NANOJECT II (Drummond scientific company, Broomall 116 Pennsylvania). Injected oocytes were incubated for 3 days at 16 °C in HEPES-based Kulori buffer (90 mM NaCI, 1 mM KOI, 1 mM MgCh, 1 mM CaCh, 5 mM HEPES pH 7.4) supplemented with gentamycin (100 pg mL -1 ). For the H2O-injected control, 50.6 nL nuclease-free water (Ambion) was injected instead of cRNA.
- HEPES-based Kulori buffer 90 mM NaCI, 1 mM KOI, 1 mM MgCh, 1 mM CaCh, 5 mM HEPES pH 7.4
- gentamycin 100
- Uptake assays in Xenopus oocytes were performed as described previously (Jorgensen et al., 2017) with some modifications.
- oocytes were pre-incubated for 5 min in 5 mL Kulori buffer (90 mM NaCI, 1 mM KCI, 1 mM MgCh, 1 mM CaCh, 5 mM MES pH 5.0), then incubated with substrate-containing Kulori buffer for a given time.
- Oocytes were washed 5 times in Milli-Q Water (20 mL each time) and homogenized with 80% methanol (containing internal standard).
- oocyte extracts were spun down at 12000 x g for 10 min at 4°C. The supernatant was diluted with water and filtered through a 0.22 ⁇ m filter plate (MSGVN2250, Merck Millipore) and analysed by LC-MS/MS as described below.
- oocytes were injected with 23 nL of 2Prop (100 mM) or a glucosinolate mixture (concentrations of individual glucosinolates in the mixture are 2mM) to achieve the initial intracellular concentration at approximately 2.2 mM or 50 pM, respectively.
- concentration of individual glucosinolates in the mixture are 2mM
- Oocytes and the external medium were separately harvested at different time points.
- the harvested oocytes were washed 5 times in Milli-Q Water (20 mL each time) and homogenized with 80% methanol. 10 ⁇ L of the external medium was sampled for quantification of glucosinolates in the media.
- the extraction, filtration and analysis by LC-MS/MS were as described below.
- the concentration of glucosinolates and amino acids inside the oocytes was calculated based on an estimated cytosolic oocyte volume of 1 ⁇ L.
- Electrodes were backfilled with a mixture of 3 M KCI and 1.5 M Acetate. Electrodes had a resistance of 280 - 1000 k ⁇ .
- UMAMIT29-expressing oocytes were clamped at -60 mV membrane potential and oocytes were continuously perfused with MES-based eKulori buffer (2 mM LaCl3, 90 mM NaCI, 1 mM KCI, 1 mM MgCl2, 1 mM CaCl2, and 10 mM MES pH 5.5). Currents were recorded under continuous perfusion in the absence and presence of 10 mM 2Prop.
- oocytes were clamped at -60 mV and currents were recorded under continuous perfusion of 10 mM 2Prop containing eKulori buffer (pH 5.5) for 2 min. Subsequently, oocytes were washed three times and homogenized with 50% methanol. Oocyte samples were prepared as described above. The amount of 2Prop in single oocyte was quantified by LC-MS/MS.
- glucosinolates alone (i.e. glucosinolates derived from plant material) was carried out in the form of desulfo-glucosinolates.
- glucosinolate and/or amino acids in Xenopus oocytes were quantified using external standard curves.
- glucosinolates For analysis of glucosinolates as desulfo-glucosinolates, chromatography was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Separation was achieved on a Kinetex 1.7u XB-C18 column (100 x 2.1 mm, 1.7 ⁇ m, 100 A, Phenomenex, Torrance, CA, USA). Formic acid (0.05%) in water and acetonitrile (supplied with 0.05% formic acid) were employed as mobile phases A and B respectively.
- the elution profile was: 0-0.5 min, 2% B; 0.5-1.2 min, 2-30% B; 1.2-2.0 min 30-100% B, 2.0-2.5 min 100% B, 2.5-2.6 min, 100-2% B and 2.6-4 min 2% B.
- the mobile phase flow rate was 400 pl min -1 .
- the column temperature was maintained at 40°C.
- the liquid chromatography was coupled to an EVOQ Elite TripleQuad mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in positive ionization mode. The instrument parameters were optimized by infusion experiments with pure standards.
- the ion spray voltage was maintained at +3500 V.
- Cone temperature was set to 300°C and cone gas to 20 psi.
- Heated probe temperature was set to 400°C and probe gas flow to 40 psi.
- Nebulizing gas was set to 60 psi and collision gas to 1.5 mTorr. Nitrogen was used as probe and nebulizing gas and argon as collision gas. Active exhaust was constantly on.
- Multiple reaction monitoring (MRM) was used to monitor analyte parent ion — > product ion transitions. Detailed values for mass transitions can be found (Jensen et al., 2015; Crocoll et al., 2016a). Both Q1 and Q3 quadrupoles were maintained at unit resolution.
- Bruker MS Workstation software (Version 8.2.1 , Bruker, Bremen, Germany) was used for data acquisition and processing. Linearity in ionization efficiencies was verified by analysing dilution series, p-hydroxybenzyl or 2Prop glucosinolate were used as internal standard.
- Diluted samples were filtered (Durapore® 0.22 ⁇ m PVDF filters (Merck Millipore, Tullagreen, Ireland) and used directly for LC-MS analysis.
- the analysis was performed as previously described (Mirza et al., 2016) with changes as detailed below. Briefly, chromatography was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Separation was achieved on a Zorbax Eclipse XDB-C18 column (100 x 3.0 mm, 1.8 ⁇ m, Agilent Technologies, Germany). Formic acid (0.05%) in water and acetonitrile (supplied with 0.05% formic acid) were employed as mobile phases A and B, respectively.
- the elution profile was: 0-1.2 min 3% B; 1.2-4.3 min 3-65% B; 4.3-4.4 min 65-100% B; 4.4-4.9 min 100% B, 4.9-5.0 min 100-3% B and 5.0-6.0 min 3% B.
- Mobile phase flow rate was 500 ⁇ L*min -1 and column temperature was maintained at 40°C.
- the liquid chromatography was coupled to an EVOQ Elite TripleQuad mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ionization source (ESI). Instrument parameters were optimized by infusion experiments with pure standards.
- the ion spray voltage was maintained at 3000 V in positive ion mode.
- Cone temperature was set to 300°C and cone gas flow to 20 psi.
- Heated probe temperature was set to 400°C and probe gas flow set to 50 psi.
- Nebulizing gas was set to 60 psi and collision gas to 1.6 mTorr.
- Nitrogen was used as both cone gas and nebulizing gas and argon as collision gas.
- Multiple reaction monitoring (MRM) was used to monitor analyte molecular ion — > fragment ion transitions: MRMs for amino acids were chosen as described for Arg and Lys. Both Q1 and Q3 quadrupoles were maintained at unit resolution.
- Bruker MS Workstation software (Version 8.2.1 , Bruker, Bremen, Germany) was used for data acquisition and processing.
- Protein sequences were retrieved from NCBI Blast using Arabidopsis UMAMIT29 as bait in 14 plant taxa (253 amino acids sequences): Gossypium hirsutum, Theobroma cacao, Carica papaya, Arabidopsis thaliana, Brassica rapa, Glycine max, Manihot esculenta, Solanum lycopersicum, Zea mays, Vitis vinifera, Oryza sativa japonica, Eutrema salsugineum, Capsella rubella, and Citrus Clementina. Sequence alignments were generated using MUSCLE 58 with default parameters and were curated with BMGE 59 .
- CYP83A1 and CYP83B1 represent biosynthesis markers for cells producing the aliphatic methionine-derived and the indolic tryptophan-derived glucosinolates, respectively.
- a time course experiment on developing Arabidopsis wild type Col-0 seeds showed that glucosinolate accumulation in seeds starts at day 8 after pollination when the embryo enters mature green stage and that seeds continue to accumulate glucosinolates thereafter and throughout seed develo ⁇ ment (Fig. 4a).
- both CYP83A1 and CYP83B1 biosynthetic markers are present in the funiculus at mature green stage (when glucosinolate accumulation begins). This is corroborated by the high expression of the glucosinolate biosynthetic and support genes in the funiculus. Moreover, our analyses show that CYP83A1 accumulates to markedly higher levels than CYP83B1 in the funiculus (Fig. 4c), which corresponds well with aliphatic glucosinolates constituting the majority (>90%) of total seed glucosinolates in Arabidopsis.
- the funiculus is the only vascular connection between the silique septum and the seed, it is regarded as part of the long-distance transport highway.
- Our findings strongly indicate that the funiculus also represents a major hub for production and export of seed-bound glucosinolates.
- the level of glucosinolates in the silique valves with funiculi and septa (hereafter silique valves) stayed constant from mature green stage until the valves began to senesce (Fig. 4b). This suggests that the silique valves and funiculi continuously produce and export glucosinolates that are destined for the seeds.
- UMAMIT29 is key for seed loading
- UMAMIT29- mVenus is present at the plasma membrane in cortex cells and the cells adjacent to the xylem vessel in funiculus as well as in the outer integument and chalazal seed coat at the mature green stage (Fig. 1c, Fig. 4f-h).
- the co-localization of UMAMIT29 with the common glucosinolate biosynthesis marker SUR1 together with its localization to the plasma membrane in cells bordering the vasculature suggests that UMAMIT29 could function as a glucosinolate exporter in source tissues such as the funiculi.
- the name of the IIMAMIT family reflects that members previously were characterized as amino acid facilitators.
- UMAMIT29 transports amino acids by exposing UMAMIT29-expressing oocytes to 13 C, 15 N-isotope-labelled glutamine and glutamate - well-established IIMAMIT substrates 27 - over a concentration range of 0.4- 10 mM and 2-20 mM for glutamine and glutamate, respectively, thereby generating inward gradients relative to the endogenous amino acid concentration. None of the amino acid gradients resulted in accumulation of detectable levels of isotope-labelled amino acids in oocytes.
- UMAMIT29 belongs to the IIMAMIT family clade I which consists of seven members: UMAMIT26 to -32. As UMAMIT30 and -31 are the other UMAMIT genes expressed along with UMAMIT29 in funiculus at the mature green stage (Fig. 5), they were screened for import activity in oocytes using 100 pM of an equimolar mixture of
- UMAMIT29- and UMAMIT30- expressing oocytes accumulated both classes of glucosinolates, whereas UMAMIT31- expressing oocytes preferred indolic glucosinolates over aliphatic glucosinolates (Fig. 2i, j).
- UMAMIT30 and UMAMIT31 to export glucosinolates in Xenopus oocytes suggests that they may function together with UMAMIT29 to export glucosinolates to seeds.
- Analysis of available data from the funiculus transcriptome atlas shows that UMAMIT31 is highly and specifically expressed in the funiculus of the developing silique with an embryo at the mature green stage, whereas UMAMIT30 is generally lowly expressed (Fig. 5).
- seeds of single mutants of umamit30 have wild type glucosinolate profile and level whereas umamit31 mutants have a -50% reduction of indolic glucosinolates specifically (Fig. 3a, b).
- Glucosinolate analysis of seeds of double mutants of the tandemly-linked UMAMIT29 and UMAMIT31 showed that the aliphatic glucosinolate level of umamit29 umamit31 is the same as for umamit29 single mutants (-80% reduction of wild type level), while the level of indolic glucosinolates in seeds of umamit29 umamit31 is reduced to -5% of wild type level (Fig. 3b).
- the remaining aliphatic glucosinolates in umamit29 umamit31 mutants suggests that a different transporter accounts for glucosinolate transport in the absence of these two IIMAMIT transporters.
- UMAMIT30 transcripts are upregulated > 4-fold in the siliques (Fig. 6), suggesting that expression of UMAMIT30 is induced in this mutant in order to transport glucosinolates to seeds in the absence of UMAMIT29 and UMAMIT31.
- the levels of aliphatic glucosinolates in seeds of umamit29 umamit30 double mutants were reduced an additional -50% compared to umamit29 single mutants (Fig. 3a, b, Fig. 7).
- the total glucosinolate level was - 5% of wild type (Fig. 3a, b, Fig. 7).
- glucosinolate level in the developing silique valves of umamit29 umamit30 mutants was comparable to wild type (Fig. 3d).
- the levels of total free amino acids in seeds of umamit29 and umamit31 single mutants as well as umamit29 umamit31 double mutants were not significantly different from wild type, whereas seeds of the umamit30 single mutants, umamit29 umamit30 double mutants, and umamit29 umamit30 umamit31 triple mutants displayed an increase in the levels of total free amino acids (Fig. 3e).
- the seed size and the total seed weight of umamit29 umamit30 double mutants and umamit29 umamit30 umamit31 triple mutants were both reduced, compared to wild type levels (Fig.
- Roots of single, double or triple mutants of UMAMIT29, -30 and -31 had levels of total glucosinolates comparable to wild type, contrasting the strong reduction of glucosinolates in roots of gtr1 gtr2 gtr3 mutants (Fig. 3 h-k).
- rosette and cauline leaves as well as stems of single and multiple umamit mutants showed levels of total glucosinolates comparable to wild type, whereas strong over-accumulation of glucosinolates was observed in rosette and cauline leaves of gtr1 gtr2 gtr3 mutants (Fig. 3 h-k, Fig. 8).
- the altered glucosinolate distribution pattern in gtr1 gtr2 gtr3 mutants was fully restored to wild type levels in umamit29 umamit30 gtr1 gtr2 gtr3 quintuple mutants (Fig. 3 h-k, Fig. 8).
- UMAMIT29, - 30 and -31 function as exporters of glucosinolates and that they are potential molecular targets for seed-specific elimination of glucosinolates through transport engineering.
- UMAMIT29, UMAMIT30 and UMAMIT31 as glucosinolate exporters with a key role in accumulation of glucosinolates in the seeds as evidenced by less than 6% of wild type level glucosinolates in seeds of umamit29 umamit30 umamit31 exporter mutants.
- GTR1, GTR2 and GTR3 active high-affinity IT-coupled glucosinolate importers
- UMAMIT29-31 The biochemical and biophysical characterization of UMAMIT29-31 in oocytes show that glucosinolates are exported passively along an electrochemical gradient.
- Arabidopsis where the plasma membrane potential in physiological condition is within the range of -100 to -150 mV, UMAMIT29-31 is expected to facilitate efficient export of the monovalent anionic glucosinolates out of biosynthetic cells to the acidic apoplast.
- a recent theory-based study hypothesized that the mechanism for nutrient homeostasis aimed at reaching a desired cytosolic concentration of a given nutrient, requires that at least two differently energized transporter types are involved. The presence of passive IIMAMIT exporters and IT-coupled GTR importers fulfils this requirement.
- the H + -coupled high-affinity GTR importers empty the apoplast for glucosinolates generating a strong outward chemical gradient (from cytosol to apoplast) which, together with the strong negative membrane potential, promotes efficient export of glucosinolates via the passive uniport mechanism of UMAMIT29-31.
- the glucosinolate exporters as well as the exporters of sugars (Sugars Will Eventually be Exported Transporters - SWEETs) and amino acids (UMAMITs) are all uniporters, which suggests the presence of a universal passive uniport mechanism for exporting compounds from symplasm in source tissues.
- UMAMIT29-31 belong to UMAMIT clade I proteins (UMAMIT26-32) of which UMAMIT26-31 are brassicaceous-specific (Fig. 9). This highlights the potential of transporter families involved in primary metabolism to evolve substrate specificity towards specialized metabolites as recently shown for the NPF family. Analogously, the identification of UMAMITs as exporters of glucosinolates suggests that the UMAMIT family may represent a new family of exporters of specialized metabolites.
- MYB28 and -29 are master regulators of aliphatic glucosinolates), as well as gtr 1 gtr2 mutants41 , merits future studies to investigate the relation between glucosinolates and free amino acids in seeds.
- IIMAMIT glucosinolate exporters have high biotechnological potential as molecular breeding targets for eliminating anti-nutritional glucosinolates in seed cake of brassicaceous oilseed crops through transport engineering without impacting glucosinolate defence in the rest of the plant.
- Example 2 Glucosinolate transport activity of Brassica napus UMAMIT transporters Seeds of the model plant A. thaliana and of Brassica crops accumulate glucosinolates that are produced and exported from maternal tissues. In Arabidopsis, AtUMAMIT28, AtUMAMIT29, AtUMAMIT30 and AtUMAMIT31 from the Clade I UMAMIT family are characterized bidirectional glucosinolate facilitators. Recent data showed that AtUMAMIT29, AtUMAMIT30 and AtUMAMIT31 are essential for the accumulation of glucosinolates in seeds, with a main role for AtUMAMIT29.
- AtUMAMIT28, AtUMAMIT29, AtUMAMIT30 and AtUMAMIT31 glucosinolate facilitators as a primary target for translating the umamit loss-of-function phenotype from Arabidopsis to Brassica crops.
- B. napus cultivars including the UMAMITs orthologs.
- the BnPIR Brassica napus pan-genome information resource (http:// http://cbi.hzau.edu.cn/bnapus/) was queried using the AtUMAMIT26-32 amino acids as queries to identify UMAMIT orthologs in B. napus.
- Brassica UMAMIT orthologs were named according to sequence identity to the respective Arabidopsis orthologs and to their subgenomic association as follows: Bn indicate the plant species B. napus; UMAMIT26-32 indicate whether the gene is an ortholog of which Arabidopsis UMAMIT gene. A and C indicate the subgenomic location according to the U triangle (A indicates location in the AA genome originating from B. rapa and C indicates location in the CC genome originating from B. oleracea). 1 , 2, 3 and 4 indicate amino acid sequence homology to the Arabidopsis ortholog. Genes were numbered according to decreasing amino acid sequence homology, with 1 indicating the highest level of identity.
- Full-length cDNA sequences of the 11 BnUMAMIT genes were amplified by PCR from the cDNA; due to the high sequence similarity, the primers were placed in the more-varying UTR regions. The PCR fragments were cloned into pNB1u, and verified by sequencing.
- Linear DNA templates for in vitro transcription were generated from pNB1u plasmid by PCR using Phusion High-Fidelity DNA Polymerase (NEB), according to the manufacturer’s instructions. PCR products were purified using the QIAquick PCR Purification Kits (Qiagen). Capped cRNA was in vitro synthesized using the mMessage mMachine T7 Kit (Ambion). Concentration of the synthesized cRNA of each transporter genes was normalized to 500 ng/pl. and were aliquotd before storage in -20C. Maxium one thaw cycle of the cRNAs was used for expression in Xenopus oocytes.
- Xenopus oocytes were purchased from Ecocyte Bioscience (Germany). Oocytes were injected with 50.6 nL cRNA using a Drummond NANOJECT II (Drummond scientific company, Broomall 116 Pennsylvania). cRNA (500 ng/pl) were injected into oocytes. Injected oocytes were incubated for three days at 16 °C in HEPES-based kulori buffer (90 mM NaCI, 1mM KCI, 1 mM MgCI 2 , 1 mM CaCI 2 , 5 mM HEPES pH 7.4) supplemented with gentamycin (100 pg/mL). For mock-injected control, 50.6 nL nuclease-free water (Ambion) was injected instead of cRNA.
- HEPES-based kulori buffer 90 mM NaCI, 1mM KCI, 1 mM MgCI 2 , 1 mM CaCI 2 , 5 mM HEPES pH 7.4
- Uptake assay in Xenopus oocytes was performed essentially as described previously with some modifications. Three days after cRNA injection, oocytes were pre-incubated for 5 min in 5 ml kulori buffer (90 mM NaCI, 1 mM KCI, 1 mM MgCI 2 , 1 mM CaCI 2 , 5 mM MES pH 5.0), then incubated kulori buffer containing a substrates (100pM glucosinolates) for a given time. Oocytes were washed 5 times in Milli-Q Water (20 ml each time) and homogenized with 80 % methanol (containing internal standard).
- oocyte extracts were spun down at 12000g for 10 min at 4 °C.
- the supernatant was diluted with water and filtered through a 0.22 ⁇ m filter plate 131 (MSGVN2250, Merck Millipore) and analyzed by LC-MS/MS as described below.
- the elution profile was: 0-0.5 min, 2% B; 0.5-1.2 min, 2-30% B; 1.2-2.0 min 30-100% B, 2.0-2.5 min 100% B, 2.5-2.6 min, 100-2% B and 2.6-4 min 2% B.
- the mobile phase flow rate was 400 ⁇ l min -1 .
- the column temperature was maintained at 40°C.
- the liquid chromatography was coupled to an EVOQ Elite TripleQuad mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in positive ionization mode. The instrument parameters were optimized by infusion experiments with pure standards.
- the ion spray voltage was maintained at +3500 V. Cone temperature was set to 300°C and cone gas to 20.
- Heated probe temperature was set to 400°C and probe gas flow to 40 psi.
- Nebulizing gas was set to 60 psi and collision gas to 1.5 mTorr. Nitrogen was used as probe and nebulizing gas and argon as collision gas. Active exhaust was constantly on.
- Multiple reaction monitoring (MRM) was used to monitor analyte parent ion — > product ion transitions. Detailed values for mass transitions can be found in (Jensen et al., 2015; Crocoll et al., 2016a). Both Q1 and Q3 quadrupoles were maintained at unit resolution.
- Bruker MS Workstation software (Version 8.2.1 , Bruker, Bremen, Germany) was used for data acquisition and processing. Linearity in ionization efficiencies were verified by analyzing dilution series, p-hydroxybenzyl or 2-propenyl glucosinolate were used as internal standard.
- BnUMAMIT28C1N, BnUMAMIT30A2N, BnllMAMIT30C2N, BnllmamiT30C1N and BnllmamiT28A2N are paralogs of ZS11 genes that shows highest expression in reproductive organs (silique wall and seeds) (Fig. 11).
- BnllmamiT28A2N, BnllmamiT28A3N, and BnllmamiT30A2N prefer to import 4MTB and I3M rather than progoitrin 2OH-3-but in an equal molar mixture.
- BnllmamiT28C1N import all glucosinolate (progoitrin 4MTB, I3M) into oocytes without particular preference.
- BnllmamiT glucosinolate transporters expressed in reproductive tissues were the targets for mutagenesis with BnllmamiT28C1 N as the key target, since 2OH-3-but progoitrin is the main antinutritional glucosinolates in Niklas seeds.
- Step 1 Screening for high-efficient gRNAs targeting UMAMIT genes with RNP complexes in protoplasts
- gRNA Multiple candidate gRNAs will be designed to target regions conserved across all six UMAMIT orthologs for the introduction of indel mutations, and tested together with Cas9 for their efficiency via PEG-mediated transformation of RNP complexes into protoplasts.
- gRNA will be prepared from in vitro transcription and Cas9 proteins will be purified from cell extract of Cas9-expressing E. coli strains (March et al., 1989). We will prepare protoplasts from 14-day-old cotyledons that have high regeneration ability.
- gRNA and Cas9 protein 200 ⁇ L containing 2 x 10 5 /mL protoplasts will be harvested and transfected with gRNA and Cas9 protein as well as LipofectamineTM 3000 and Plus reagentTM transfection reagents together with polyethylene glycol (PEG) 4000.
- PEG polyethylene glycol
- UMAMIT26-32 The amino acid sequences of UMAMIT clade I (UMAMIT26-32) were obtained from TAIR (https://www.arabidopsis.org) for Arabidopsis thaliana, BnPIR (http://cbi.hzau.edu.cn/bnapus/index.php) (Song et al., 2020) for Brassica napus ZS11 and Genoscope ((Belser et al., 2018), https://www.genoscope.cns.fr/externe/plants/index.html) for Brassica rapa Z1 and Brassica oleracea HDEM, phytozome 13 (https://phytozome-next.jgi.doe.gov) for Manihot esculenta, NCBI for Physcomitrella patens.
- Sequences from 27 plant species were aligned in MEGA X (https://www.megasoftware.net/)(Kumar et al., 2018) using MUSCLE with default settings (Edgar, 2004). Sequences with indels within any helices were removed, with the exceptions of AsSol/42, FaMed/43 and the root. The final alignment contained 97 sequences. Sequence logos were made using JDet (http://csbg.cnb.csic.es/JDet/) (Muth et al., 2012). Phylogenetic trees were generated in MEGAX using the neighbour- joining method (1000 bootstraps) and annotated in iTOL (Letunic and Bork, 2021).
- DIVERGE 3.0 beta 1 was used to determine differentially conserved amino acids (https://github.com/xungulab/diverge) (Gu et al., 2013).
- the multiple sequence alignment with 97 sequences was loaded into the program as well as its corresponding neighbour-joining phylogenetic tree (see above).
- the estimation of cluster-specific functional divergence was calculated based on the algorithm in Gu et al., (2013) and the final scores were listed in Table 5, below.
- Residues in bold indicate each of the 11 differentially conserved amino acids identified in the in silico analyses.
- NA Not available. * residues with a score 0 that has identical residues among UT30/31 and UT32.
- Transport assays using Xenopus oocytes were described in Xu et al., (2016). Briefly, the defolliculated Xenopus laevis oocytes (stage V or VI) were ordered from Ecocyte Bioscience and Department of Drug Design and Pharmacology, University of Copenhagen. Oocytes were injected with 50 nL RNA ( ⁇ 600 ng/ ⁇ L) using a Nanoject II (Drummond Scientific Company). For the mock (negative control) oocytes were injected with 50 nL sterilized Milli-Q® H2O.
- the injected oocytes were used for assaying after three days of incubation at 16 °C in Kulori buffer pH 7.4 (5 mM MES, 90 mM NaCI, 1 mM KCI, 1 mM CaCI 2 , 1 mM MgCl2) supplemented with gentamicin (100 pg/mL).
- the assays were performed as follows: Oocytes were first pre-incubated in Kulori buffer pH 5 (5 mM MES, 90 mM NaCI, 1 mM KCI, 1 mM CaCI 2 , 1 mM MgCh) without substrates for 5 min.
- oocytes were incubated for one hour in Kulori buffer pH 5 (5 mM MES, 90 mM NaCI, 1 mM KCI, 1 mM CaCI 2 , 1 mM MgCI 2 ) with added 4- methylthiobutyl glucosinolate (4MTB), indol-3-ylmethyl glucosinolate (I3M) and benzyl glucosinolate (BGLS) (200 pM of each).
- 4MTB 4- methylthiobutyl glucosinolate
- I3M indol-3-ylmethyl glucosinolate
- BGLS benzyl glucosinolate
- oocytes were washed in five petri dishes containing Milli-Q® H 2 O. From the final petri dish, oocytes were divided into Eppendorf tubes® with three oocytes in each. Residual H 2 O was removed from each of the tubes and the oocyte
- Glucosinolates were extracted and quantified as desulfo-glucosinolates as previously described (Crocoll et al., 2016b). Briefly, a 96 well filter plate (0.45 pM) was filled with 45 ⁇ L DEAE-Sephadex A-25 using a MultiScreen Column Loader (Merck Millipore). 300 ⁇ L H 2 O was added to each well and the plate incubated 3-4 hours at room temperature or overnight in fridge. Excess H 2 O was removed by applying 2-4 s of vacuum using a vacuum manifold.
- the mutant variants were expressed in Xenopus laevis oocytes and tested for import of a mixture of three glucosinolates, the aliphatic 4-methylthiobutyl glucosinolate (4MTB), indol-3-ylmethyl glucosinolate (I3M) and benzyl glucosinolate (BGLS). Eight of the 11 mutant variants showed reduced transport activity to all three glucosinolates (see Table 6, below).
- 4MTB 4-methylthiobutyl glucosinolate
- I3M indol-3-ylmethyl glucosinolate
- BGLS benzyl glucosinolate
- UT UMAMIT
- 4MTB 4-methylthiobutyl glucosinolate
- I3M indole 3-ylmethyl glucosinolate
- BGLS benzyl glucosinolate
- UMAMIT29#V27F, -M86V, -L109V, Q263S and - T267Y showed the most reduced activity with ⁇ 75-97% less total glucosinolate import than the wildtype UMAMIT29 when expressed in the oocytes (Table 6).
- UMAMIT29#M86V, -L109V, -Q263S and -T267Y locate in near proximity in the centre of the cavity
- UMAMIT29#V27F locates further distally at the end of helix I in the computed protein model.
- UMAMIT29#L197W showed reduction import of 4MTB and BGLS, compared to the wildtype, and the level of imported I3M was not affected.
- UMAMIT29#S289I showed reduction of I3M and BGLS import and the level of imported 4MTB is similar to wildtype (Table 6).
- UMAMIT29#M201F showed - as the only mutant variant - no reduction of any glucosinolates, but an increase of 4MTB (Table 6)
- mutant IIMAMIT transporter is a mutant glucosinolate transporter with reduced glucosinolate transporter activity compared to the wildtype protein
- said reduction is a reduction of at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as 100% of the glucosinolate transporter activity of said mutant glucosinolate transporter compared to the wildtype protein.
- Brassicales plant or part thereof according to any one of the preceding items, wherein the vegetative tissue of said Brassicales plant or part thereof have about the same glucosinolate content compared to a Brassicales plant or part thereof of otherwise identical genotype except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
- a plant product comprising a Brassicales plant or part thereof, or prepared from seeds of said Brassicales plant or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homo
- the plant product according to item 10 wherein the plant product is prepared from a seed, such as wherein the plant product is an oil, a seed cake or a seed meal.
- a method for modifying glucosinolate content in a Brassicales plant or part thereof comprising a step of modifying the functional activity or expression of at least one IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%
- step of modifying said functional activity of said at least one of IIMAMIT transporter is performed by nuclease-based gene editing, such as by CRISPR/Cas9 gene editing, by random mutagenesis, by gene targeting, by transposition mutagenesis, by transfer-DNA induced insertion, or by gene knockdown, such as by RNA interference.
- a method for producing a plant product comprising a Brassicales plant or part thereof with low glucosinolate content said method comprising the steps of a. providing a Brassicales plant or part thereof according to any one of items 1 to 9; and b. processing said Brassicales plant or part thereof into a plant product, such as a seed oil or a seed cake.
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Abstract
The present invention provides Brassicales plants, or parts thereof, having a low UMAMIT glucosinolate transporter activity. In particular, Brassicales plants carrying a mutation in a gene encoding an UMAMIT transporter and having a low seed glucosinolate content are provided. Furthermore, plant products prepared from said Brassicales plants, or parts thereof, are described, as well as methods of producing said plants.
Description
Plants with low seed glucosinolate content
Technical field
The present invention relates to the field of agricultural products, especially crop plants and parts thereof having low IIMAMIT glucosinolate transporter activity. In particular, the invention relates to Brassicales plant carrying a mutation in at least one gene encoding an IIMAMIT transporter and having seeds with a low concentration of glucosinolate. Seeds of such plants are advantageous for production of seed products, e.g. seed or seed cakes, with naturally low levels of glucosinolate. The invention further relates to methods for production of said Brassicales plants, as well as to products prepared from plants of the invention.
Background
The Brassicales order of plants, including the Brassicaceae or Cruciferae family, includes many cultivars that have provided mankind with a source of condiments, vegetables, forage crops, and the economically important crops rapeseed (Brassica napus and Brassica campestris or rapa) and mustard (Brassica juncea).
Rapeseeds or seeds from other Brassica plants can be used as a source of seed oil. After pressing the oil out of the seeds, a protein rich cake remains. This cake is ideal for animal feed. However, the potential for this source depends on the level of glucosinolate in the remaining seed cake.
A striking and characteristic chemical property of Brassicales plants is their high content of glucosinolates; amino acid-derived natural plant products containing a thioglucose and a sulfonated oxime. These sulphur-containing secondary metabolites are important because of the multiplicity of physiologically active products, such as nitriles, epithionitriles, oxazolidine-2-thiones, thiocyanates and isothiocyanates, derived from them upon cleavage by the hydrolytic enzyme myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1) upon plant damage. Glucosinolates can be found in all parts of the Brassicales plants and are toxic to mammals, thus playing a prominent function in plant defense against herbivores.
According to the optimal defense theory, plant organs with the highest fitness value, accumulate the highest level of defense compounds to protect against herbivores and
pathogens. Accordingly, the highest glucosinolate concentrations are found in reproductive organs, including seeds, siliques, flowers and developing inflorescences, followed by young leaves, the root system and fully expanded leaves. In particular, glucosinolates are known to be transported in the plant from maternal tissue into the seeds/embryos, where they accumulate to high levels, generating a high level of defense in the seeds.
In the 1970s, traditional breeding generated a multiple-loci-dependent B. napus cultivar with reduced glucosinolate content in all parts of the plant, including the seeds. This “00” (“double low”) variety and its descendants have subsequently become the most widely grown rapeseed cultivars across the northern hemisphere. The prolonged selection bottleneck caused by this single source has, however, created a limited genetic diversity for future B. napus breeding programs and limits interspecific hybridization. This poses a serious problem for B. napus breeders striving to improve yield and disease resistance as well as to introduce novel traits such as drought tolerance through interspecific hybridizations.
A further problem with “00” varieties is that the seeds, although low in glucosinolates, are not free of them. Pressed seed cake obtained from “00” varieties after the oil has been extracted will typically contain less than 18-24 micromoles of total glucosinolates (GSL) per gram of dry weight (as compared to traditional rapeseed meal that contains 120-150 μmol of total GSL per gram). For use in compound feed, palatability to ruminants sets the level of total GSL permitted at no more than 10-15 micromoles per gram of dry weight, meaning an animal feed could in theory be compounded almost entirely of “00” pressed seed cake if the seed cake is at the lower level of GSL content. However, it has recently been found that poultry and pigs are both much more sensitive to levels of GSLs than ruminants, and more than 2-4 micromoles GSL per gram of dry weight in the feed can severely affect reproductive efficiencies in these animals. A truly “zero GSL” variety would be of significant commercial advantage to animal feed compounders, producers of pressed seed cake and the growers by increasing quantities of seed cake that can be included in compound feeds and removing the need for continual monitoring of GSL levels in their products.
For humans, the presence of toxic compounds in the edible parts of the plants reduce their nutritional value. Reduction of anti-nutritional factors by blocking the biosynthetic pathways, however, is often accompanied with adverse effects on plant fitness due to e.g. increased susceptibility to biotic or abiotic stresses.
The general concept of reducing glucosinolate in seeds by modification is known. This has been done by modification of glucosinolate importer genes, which result in low total glucosinolate levels in the plant, including the seeds. For example, WO 2012/004013 discloses modification of the glucosinolate importer gene GTR and discloses a reduction in glucosinolate levels of 60-70% in Brassica rapa and Brassica juncea plants. However, the obtained seed glucosinolate levels are still too high for use of the seed cakes or meals in e.g. poultry feed. Additionally, a systemic decrease in glucosinolate content of the plant is undesirable, as this may impact its natural defense.
There is thus a significant commercial need for Brassicales plants with tissue-specific decreases in glucosinolate content, e.g. restricted to the seeds that maintain appropriate glucosinolate levels in other tissues.
Summary
The inventors have identified members of a brassicaceous-specific clade of Usually Multiple Amino acids Move In and out Transporter (UMAMIT) which are essential for accumulation of glucosinolates in seeds of in Arabidopsis thaliana. Surprisingly, the main function of UMAMIT29, UMAMIT30 and UMAMIT31 was discovered by the inventors to be export of glucosinolates to the seeds. The inventors found that single, double and triple exporter mutants had significantly decreased levels (up to 95% reduction) of glucosinolates in seeds, which is much lower than what has been obtained previously. The exporter mutants do not alter the overall distribution of glucosinolates in the plant, thus, the plant retains a normal plant phenotype ensuring adequate natural defense against herbivores. The inventors further found that orthologous genes in Brassica napus share a similar glucosinolate transport capacity as those in Arabidopsis.
The present invention is directed to tissue-specific glucosinolate exporters in plants, as only the exporters relevant for exporting glucosinolate from the mother plant to the seeds are modified. By identifying and modifying those specific glucosinolate exporters,
the glucosinolate level in the seeds can be specifically reduced without reducing the glucosinolate level in the remaining plant. Thus, a high level of defense remains in the mother plant.
The inventors have thus solved the problem of providing Brassicales plants that maintain appropriate glucosinolate levels in other tissues, but which have seeds with significantly decreased levels of glucosinolates.
In one aspect, the invention provides a Brassicales plant, or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby said Brassicales plant, or part thereof, expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
In another aspect, the invention provides a plant product comprising a Brassicales plant or part thereof, or prepared from seeds of said Brassicales plant or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such
as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
In a further aspect, the invention provides a seed cake prepared from a seed from a Brassicales plant, wherein the Brassicales plant is as described elsewhere herein.
In some aspects, the invention provides a method for producing a plant product comprising a Brassicales plant or part thereof with low glucosinolate content, said method comprising the steps of a. providing a Brassicales plant or part thereof as described elsewhere herein; and b. processing said Brassicales plant or part thereof into a plant product, such as a seed oil or a seed cake.
In another aspect, the invention provides a method for modifying glucosinolate content in a Brassicales plant or part thereof, said method comprising a step of modifying the functional activity or expression of at least one IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto.
Description of Drawings
Figure 1. Identification of source tissue and transporters critical for accumulation of glucosinolates in seeds of Arabidopsis. a, Analysis of glucosinolate content in seeds of grafted Arabidopsis. 5-week-old wild type Col-0 (WT), biosynthetic null mutant myb28 myb29 cyp79b2 cyp79b3 (null), and transporter mutant
gtr1 gtr2 (gtr) were reciprocally stem-grafted at a junction 1 cm above ground. Seeds were harvested from mature siliques of the scion and total glucosinolates (GLS) were quantified from a pool of 10 seeds (n>3 plants per group, except 2 plants in WT/null grafts), b, Total GLS from a pool of 10 seeds from Col-0, umamit29-1(ut29-1), umamit29-2 (ut29-2) and plants complemented with pUT29(6kb)-UT29 (genomic fragment)-mVenus (ut29-1C) (data are representative of three independent lines) at different days after pollination (DAP) (n=6 plants per group). Four boxplots are shown for each day. Said four boxplots represent, from left to right, Col-0, ut29-1, ut29-2 and ut29-1C. c, Expression of pSUR1 ::SUR1-mTQ2 (top) and pUT29::UT29-mVenus (bottom) in developing siliques collected on different days after pollination (DAP). Inserts show funiculus and seed at lower magnification. Scale bars: 50 μm (main panels) and 100 μm (inserts), a, Data are shown as mean ± s.d. Letters indicate significant differences by a non-parametric Kruskal-Wallis test followed by Dunn’s test (p < 0.05) b, Time course data were fitted to linear models and pairwise comparisons between genotypes performed on estimated marginal means (EMMs). Seed glucosinolate accumulation in Col-0 and ut29-1C differ significantly from ut29-1 and ut29-2 (Tukey adjusted p< 0.0001). Abbreviations: DAP = days after pollination; GLS = glucosinolates; SUR1=SUPERROOT1 ; UT = UMAMIT; WT = wild type.
Figure 2. Biochemical and biophysical characterization of Arabidopsis UMAMIT29, UMAMIT30 and UMAMIT31. a, Glucosinolate accumulation in Xenopus laevis oocytes expressing UMAMIT29 (UT29) or injected with water (H20-inj). Oocytes were incubated with 4MTB (64 pM) and I3M (34 pM) in Kulori buffer at pH 5.5 (mean ± s.d., n = 15). b, Membrane currents associated with 2Propenyl glucosinolate (2Prop) uptake for a representative UMAMIT29-expressing oocyte (UT29) voltage-clamped at -60 mV and superfused with 10 mM 2Prop in Kulori buffer for 2 min. c, Charge coupling stoichiometry estimated by correlation of current-time integrals and 2Prop uptake during the clamping period indicated in b in 13 oocytes expressing UT29 (grey) and in 10 oocytes injected with H2O (black), d, Effect of pH on UT29-mediated glucosinolate import. Intracellular 2Prop was quantified from UT29-expressing or H2O- injected (CO-inj) oocytes after incubation with 1 mM 2Prop at pH 7.4 and pH 5.5 with or without 0.1 mM CCCP for 60 min. e, Effect of extracellular cations on UT29- mediated 2Prop import. 91 mM choline+ CI- (Choline+) or 91 mM N-methyl-d-glutamine+ CI- (NMDG+) were used to substitute the cations in Kulori buffer (90 mM Na+CI- and 1 mM K+CI-). Intracellular 2Prop was quantified in UT29-expressing or CO-injected
oocytes after incubation with 5 mM 2Prop at pH 7.4 for 60 min. f, Injection-based export assay of 2Prop by UT29. UT29-expressing (bright grey line) or H2O-injected (dark grey line) oocytes were injected with 2Prop (initial internal concentration ~2 mM). Export activity was measured by quantifying intracellular and extracellular 2Prop content over time in oocytes incubated in Kulori buffer (pH 7.4) (mean ± s.d., n = 5). g, Uptake by UT29-expressing oocytes of 0.5 mM 4MTB in the presence or absence of excess glutamine at pH 5. h, Uptake of 0.5 mM 4MTB by UT29-expressing oocytes in the presence or absence of excess 2Prop (10mM). i, j, Oocytes expressing UT29, -30 and -31 transporters were assayed for import activity using an equimolar mixture (0.1 mM for each glucosinolate) of glucosinolates (GLS) consisting of 11 aliphatic glucosinolates and 1 indolic glucosinolate as substrates, k, I, exported glucosinolates from L/T-expressing oocytes injected with the glucosinolate mixture used in i and j (initial intracellular concentration of ~50 pM for each glucosinolate) and incubated in Kulori buffer (pH 5.5) for 5 hours (mean ± s.d., n = 4). Glucosinolates were grouped into aliphatic and indolic according to their side chain, n, biological replicates per experiments, 3 oocytes were pooled in one replicate. Treatments were compared by one-way ANOVA analysis followed by Tukey’s post-hoc HSD test. Bars labelled with different letters are significantly different, p<0.05). Abbreviations: 4MTB = 4- methylthiobutyl glucosinolate; 2Prop = 2Propenyl glucosinolate; COOP = protonophore carbonyl cyanide m-chlorophenylhydrazone; I3M = indol-3-ylmethyl glucosinolate; GLS = glucosinolates; UT = UMAMIT.
Figure 3. Seed trait and glucosinolate distribution in mutants of UMAMIT29, -30 and -31. a, b, Total content of methionine- (a) and tryptophan-derived (b) glucosinolates (GLS) in seeds from A rabidopsis wild type Col-0 (Col-0), umamit29-1 (ut29-1), umamit30-1 (ut30-1) and umamit31-1 (ut31-1) single mutants, as well as ut29- 1 ut30-2, ut29-1 ut30-3, ut29-1 ut31-2 double mutants and ut29-1 ut30-3 ut31-5, ut29-1 ut30-2 ut31-3 triple mutants (mean ± s.d., n=6). c, d, Glucosinolate content in developing seeds and siliques without seeds (i.e. silique valves, septa and funiculi) of umamit mutants. Glucosinolate content in seeds (c) and siliques without seeds (d) at different silique positions counted from the first silique on the stem, e, Free amino acids in seeds (mean ± s.d., n>6 per genotype), f, Seed area of individual seeds of different genotypes (n=250-300 per genotype) as estimated from quantifying the area outline of individual seed, g, Total weight of 500 seeds from each genotype (mean ± s.d., n=3). h-k, Glucosinolate content in roots (h, i) and rosette leaves (j, k) of umamit single,
double and triple mutants as well as of ut29-1 ut30-5 gtr1 gtr2 gtr3 mutants (mean ± s.d., n>6 per genotype), a-k, Treatments were compared by one-way ANOVA analysis followed by Tukey’s post-hoc HSD test. Bars labelled with different letters are significantly different (P < 0.05). Abbreviations: GLS = glucosinolates; UT = IIMAMIT; FW = fresh weight.
Figure 4. Time course of glucosinolate accumulation in developing siliques and cellular localization of UMAMIT29. a, b, Total glucosinolates (GLS) from a pool of 10 developing seeds (a) and in the corresponding developing silique without seeds (including silique valves, repla and funiculi) (b) from wild type Arabidopsis Col-0 at different days after pollination (n=6 plants per group), c, Cross section of siliques expressing pCYP83A1 ::CYP83A1-mVenus (top) and pCYP83B1 ::CYP83B1-mVenus (bottom) at mature green stage, d, Funiculus-expressed transporters from transcriptomics data. Transporter genes were selected that showed an increase in expression in funiculi from the global stage (gFUN), heart stage (hFUN) to mature green stage (mgFUN), resulting in a list of 10 glucosinolate candidate exporters, e, Total methionine- and tryptophan-derived glucosinolates from a pool of 10 siliques without seeds (i.e. silique valves including septa and funiculi) from wild type Col-0 (Col- 0), umamit29- 1 (ut29-1), umamit29-2 (ut29-2) and plants complemented with pUT29(6kb)-UT29 (genomic fragment)-mVenus (ut29-1C) (Data are representative of three independent lines) at different days after pollination (n=6 plants per group). Four boxplots are shown for each day. Said four boxplots represent, from left to right, Col-0, ut29-1, ut29-2 and ut29-1C. f-h, Cellular localization of UMAMIT29-mVenus at day 8 after pollination in living funiculi, f, UMAMIT29-mVenus accumulation in the funiculus. Note that the seed is detached in this view, thus exposing the funiculus optimally for live imaging at high resolution, g, Maximum intensity projection of a Z-stack through the funiculus shown in panel f at larger magnification, h, Single plane of the Z-stack showing that Umami-T29-mVenus is localized at the plasma membrane. Insert: Magnification of UMAMIT29-mVenus signal surrounding the chloroplasts in pUT29::UT29-mVenus plants. Green: UT29-mVenus, magenta: chlorophyll autofluorescence. Scale bars: A: 250 μm, B and C: 50 μm, C insert: 10μm.
Figure 5. Analysis of transcriptomic data of tissues from developing siliques.
Tissue-specific transcript enrichment of UMAMIT Clade I genes in developing seeds and funiculi. Heatmap with hierarchal clustering analysis showing relative mRNA levels
of IIMAMIT family Clade I across subregions of the seeds and the funiculus when the embryo enter at the globular (g), heart (h) and mature green (mg) stages. Hierarchal cluster analysis was performed using Origin with default settings. Abbreviation: EP = embryo proper; SUS = suspensor; PEN = peripheral endosperm; MCE = micropylar endosperm; CZE = chalazal endosperm; CZSC = chalazal seed coat; SC = distal seed coat; FUN = funiculus.
Figure 6. Relative expression levels of UMAMIT clade I genes in wild type Col-0 versus ut29-1 ut31-2 mutants (n=3). Expression levels were normalized against the reference gene actin (At3g18780). Data are means ± s.d. Data point outside the lines (in bright grey) are significantly differentially expressed in two genotypes. Student T- test, two tale, (p<0,05).
Figure 7. Genomic loci of UMAMIT29-31 and genotypes of umamit29, -30 and -31 mutants by T-DNA insertion and CRISPR-based genome editing, a, The sgRNA sequence used to target UMAMIT31 and UMAMIT30 in a schematic representation of tandemly-linked UMAMIT29 UMAMIT30 and UMAMIT31 genomic loci. Different alleles of umamit30 (ut3O) mutants and umamit31 (ut31) mutants were used in the study. Wild type gene structures or sequences of UMAMIT30 (UT30) and UMAMIT31 (UT31) (top) are shown above the mutant alleles: sgRNA target site in UMAMIT31 in wild type Arabidopsis thaliana (SEQ ID NO: 23) and in UMAMIT30 in wild type Arabidopsis thaliana (SEQ ID NO: 27), and sgRNA site in mutants ut31-3 (SEQ ID NO: 24), ut31-4 (SEQ ID NO: 25), ut31-5 (SEQ ID NO: 26), ut30-2 (SEQ ID NO: 28), ut30-3 (SEQ ID NO: 29) and ut30-4 (SEQ ID NO: 30). Sanger sequencing of the PCR products denoting the detection of the DNA fragment flanking the loci targeted by sgRNAs in each mutant allele are shown, b, Transcript levels of UMAMIT29, UMAMIT30 and UMAMIT31 in Col-0 and T-DNA insertion lines ut29-1, ut29-2, ut30-1, ut31-1 as determined by quantitative real-time PCR with reverse transcription. Values are mean ± s.d. (n = 4, representing 2 independent experiments with 2 biological repeats each). Quantitative real-time RT-PCR data are relative to ACTIN2(ACT2) gene (AT3G18780).
Figure 8. Distribution of glucosinolates in stem and cauline leaves of umamit29, - 30 and -31 mutants. Content of methionine-derived (Met-derived) (a, c) and tryptophane-derived (Trp-derived) (b, d) glucosinolates (GLS) in the first internode (from base of the stem to the first node) (a, b) and the cauline leaves (c, d) of umamit single, double and triple mutants as well as of ut29-1 ut30-5 gtr1 gtr2 gtr3 mutants
(mean ± s.d., n>6 per genotype), a, b, c indicate significant differences determined by two-way ANOVA followed by post-hoc Tukey's HSD test for all pairwise comparisons (p<0.05).
Figure 9. Phylogeny of part of UMAMIT family in Malvidae. Selected part of a Maximum-likelihood inferred tree (s.d. <0.01, optimal log-likelihood value (-35897.839)) of UMAMIT homologs from 14 species: Gossypium hirsutum, Theobroma cacao, Carica papaya, Arabidopsis thaliana, Brassica rapa, Glycine max, Manihot esculenta, Solanum lycopersicum, Zea mays, Vitis vinifera, Oryza sativa japonica, Eutrema salsugineum, Capsella rubella, and Citrus Clementina. RAxML generated bootstrap values are shown for each branch. Names of glucosinolate-producing taxa are shown in bold.
Figure 10. Maximum-likelihood inferred tree of UMAMIT genes in Arabidopsis and Brassica napus ZS11 orthologs. Bootstrap (1000 times) values in percent are shown for each branch.
Figure 11. Expression profiles of the B. napusZS11 UMAMITs genes in silique wall and seeds. Transcriptome data have been extracted from the Brassica Expression Database (Brassica EDB). One BnGTR2 required in seed glucosinolate loading by GWAS was included for validation the expression pattern.
Figure 12. Clone (A) and characterization (B) of UMAMIT orthologous genes in B. napus Niklas. A) Relative expression of 11 UMAMIT orthologs mRNAs in Niklas from different tissues (leaf, stem, flower, silique at three develoμmental stages: «young» silique, less than 20 DAP, «old» silique, between 30 and 40 DAP, senescent mature silique and dormant seeds.) Relative to the expression of the Actin reference gene (n = 3 plants). Results are representative of two independent experiments. (B) 4MTB (4- methylthiobutylglucosinolate), 2OH-3But (2(R)-2-Hydroxy-3-butenyl glucosinolate) and I3M (lndol-3-ylmethyl glucosinolate) uptake at pH 5 by oocytes expressing UMAMIT (n = 5 batches of three oocytes). Results are representative of three independent experiments.
Figure 13. Sequence logo of the 51 residues predicted to constitute substrate transporting cavity based on amino acid sequences of 96 transporters of the UMAMIT
clade I. The % conservation of each residue related to all 96 sequences is shown. Four of the amino acid residues are 100% conserved and marked with black dots. The sequence logo was made in JDet.
Figure 14. Import of 4MTB, I3M and BGLS by selected UMAMIT29 mutant variants mutated in conserved residues within the predicted substrate transporting cavity. Different letters indicate significant differences of the mean (one-way ANOVA followed by TUKEY HSD test, p < 0.05). Abbreviations: UT, UMAMIT; 4MTB, 4-methylthiobutyl glucosinolate; I3M, indole 3-ylmethyl glucosinolate; BGLS, benzyl glucosinolate.
Figure 15. Sequence logos of amino acid residues in the predicted substrate transporting cavities of the two major clusters of transporters of UMAMIT clade I. Differentially conserved residues (stars) were estimated using DIVERGE software. Two of the estimated residues from the DIVERGE estimation were identical among UMAMIT32 and respectively, UMAMIT30 and UMAMIT31 and therefore filtered out (grey stars). The resulting 11 residues estimated to be differentially conserved were tested experimentally for their role in glucosinolate transport activity (black stars). The sequence logos were made in JDet. The numbers constitute the residue position in UMAMIT29 and the respective residues in UMAMIT29, -30, -31 and -32 can be seen in supplementary table 1.
Detailed description
Definitions
The term “Brassicales plants” as used herein refers to plants in the Brassicales order.
The terms "approximately" and “about” when used herein in relation to numerical values preferably means ±10%, more preferably ±5%, yet more preferably ±1%.
The term “Brassicales plant” as used herein refers to plants of the Brassicales order. Non-limiting examples of Brassicales plants include plants of the Brassicaceae family, such as B. Juncea, B. napus (rape), B. carinata, B. oleracea and B. rapa.
“Glucosinolates” (abbreviated herein as “GSLs” or “GLSs”), as used herein, refers to amino acid-derived thioglucosidic organic anions comprising a sulfonated aldoxime
moiety. A variable side chain depending on the parent amino acid and further side chain modifications gives the distinct chemical and biological properties for GSLs. Approximately 120 different GSLs have been described in the literature and they are all derived from only 8 different amino acids. The parent amino acids are conveniently used as a classification criteria. GSLs derived from Ala, Leu, lie, Vai and Met are called “aliphatic GSLs”, those derived from Tyr and Phe are called “aromatic GSLs” and those derived from Trp are called “indole GSLs”. The great variety in GSL types is caused by a number of modifications on the side chain of the parent amino acid. Especially, methionine undergoes a wide range of transformations. The predominant aliphatic GSLs in the Brassicaceae possess side chains derived from chain elongated forms of Met, such as aliphatic thio-GSLs 3-methylthiopropyl (3-MTP)-, 4-methylthiobutyl (4- MTB)-, 5-methylthiopentyl (5-MTP)-, 6-methylthiohexyl (6-MTH)-, 7-methylthioheptyl (7- MTH)- and 8-methylthiooctyl (8-MTO)-GSL; aliphatic sulfinyl-GSLs 3- methylsulfinylpropyl (3-MSP)-, 4-methylsulfinylbutyl (4-MSB)-, 5-methylsulfinylpentyl (5- MSP)-, 6-methylsulfinylhexyl (6-MSH)-, 7-methylsulfinylheptyl (7-MSH)- and 8- methylsulfinyloctyl (8-MSO)-GSL; aliphatic hydroxy-GSLs 3-hydroxypropyl (3-OHP)- and 4-hydroxybutyl (4-OHB)-GSL; aliphatic benzoyloxy-GSLs 3-benzoyloxypropyl (3- BZOP)- and 4-benzoyloxybutyl (4-BZOB)-GSL, and aliphatic alkenyl-GSLs 2-propenyl (2-P)- and 3-butenyl (3-B)-GSL. The predominant aromatic GSLs in the Brassicaceae possess side chains derived from Phe, such as aromatic GSL 2-phenylethyl (2-PE)- GSL. Lower amounts of GSLs with indolylic side chains derived from Trp, such as indol-GSL indol-3-ylmethyl (i3M)-GSL, also occur. GSLs co-occur in plants with the GSL-specific thioglucosidase myrosinase. This enzyme is physically separated from GSLs in plants, but is brought into contact with its substrate upon tissue disruption. The resulting hydrolysis product consists of one free glucose and one aglycone molecule per GSL molecule. The aglycones are unstable and readily rearrange into isothiocyanates, nitriles, thiocyanates and other more or less toxic compounds. Depending on the side chain of the parent amino acid these hydrolysis products contribute the actual biological activity of GSLs, while intact GSLs are believed to be an inactive storage form.
As used herein, “glucosinolate content” of a plant or plant part refers to the total of GSLs, including aliphatic, aromatic and indole GSLs, without regard to the type of GSLs. Thus the “total GSL content” or “GSL content” of a plant or plant part means the content of total GSLs of that plant or plant part and is expressed on a molecular
(nmol/g or μmol/g) basis (rather than on a weight (mg/kg) basis) as GSLs have significantly different molecular weights depending on the size of their side chain. GSL accumulation varies between tissues and develoμmental stages. Young leaves and reproductive tissues such as siliques and seeds contain the highest concentrations while senescing leaves contain the lowest concentrations of GSLs. Intermediate concentrations are found throughout the “large" organs such as the roots, leaves and stem. In addition, the composition of the GSL profile varies markedly between organs. In vegetative tissues, the GSL content is composed of indole and aliphatic GSLs while the aromatic are absent. In siliques and seeds, small amounts of aromatic and indole GSLs are found while the rest of the GSL content is entirely composed of aliphatic GSLs.
By “glucosinolate transporter activity” is intended the ability of a protein to transport glucosinolates across the cell membrane. Glucosinolate transporter activity can for example be measured using the Xenopus oocyte assay as described in Example 1. Glucosinolate transporter activity can relate to one or more different glucosinolates or to groups of glucosinolates, for example methionine-derived and tryptophan-derived glucosinolates as representatives of aliphatic and aromatic glucosinolates respectively.
By “low or no” GSL transporter activity or “loss of function” with respect to GSL transportation is intended a reduction compared to the wildtype unmutated glucosinolate transporter, for example using the Xenopus oocyte assay of example 1 or by studying the GSL content in seeds in mutant plants. A “low” activity is a GLS transporter activity of 50% or less compared to wildtype, preferably 40% or less, more preferably 30% or less, more preferably 20% or less, more preferably 10% or less. By “no” GLS transporter activity or “loss of function” is intended a GSL transporter activity reduced to 5% or less such as 3% or less, for example 1 % or less of the activity of the wild type transporter.
A “decrease in total GSL content” or “increase in total GSL content” of a plant or plant part by the methods of the present invention is measured relative to the total GSL content of a reference plant or plant part with similar genetic background. Total GSL content can be measured by any appropriate method. Methods to quantify total GSL content and to determine GSL composition of plant material are well known in the art and include but are not limited to: HPLC-UV desulfo-method involving HPLC analysis
of methanol extracts desulfated and eluted from sephadex anion exchange columns as described by, e.g., Hansen et al. (2007, Plant J. 50 (5): 902-910); analysis of intact GSLs by MALDI-TOF mass spectrometry as described by, e.g., Botting et al. (2002, J. Agric. Food Chem. 50 (5): 983-988); near-infrared reflectance spectroscopy as described by, e.g., Font et al. (2005, J. Agric. Sci. 143: 65-73); methods yielding spectrophotometrically active degradation products as summarized by, e.g., Clarke (2010, Anal. Methods 2: 310-325); HPLC mass spectrometry analysis of intact glucosinolates as described by, e.g., Rochfort et al. (2008, Phytochemistry 69: 1671).
Whenever reference to a “plant” or “plants” according to the invention is made, it is understood that also plant parts, progeny of the plants which retain the distinguishing characteristics of the parents (especially the glucosinolate content in particular plant parts), such as seed obtained by selfing or crossing, e.g. hybrid seed (obtained by crossing two inbred parental lines), hybrid plants and plant parts derived thereof are encompassed herein, unless otherwise indicated.
“Plant parts”, as used herein, refers to any part of the plant, including plant cells, plant tissues, plant organs, siliques or seed pods, seeds, severed parts such as roots, leaves, flowers, pollen, etc.).
“Wild type” (also written “wildtype” or “wild-type”), as used herein, refers to a typical form of a plant or a gene as it most commonly occurs in nature. A “wild type plant” refers to a plant with the most common phenotype of such plant in the natural population. A “wild type allele” refers to an allele of a gene required to produce the wild- type phenotype. By contrast, a “mutant plant” refers to a plant with a different rare phenotype of such plant in the natural population or produced by human intervention, e.g. by mutagenesis, and a “mutant allele” refers to an allele of a gene required to produce the mutant phenotype.
A homologue or functional homologue may be any polypeptide that exhibits at least some sequence identity with a reference polypeptide and has retained at least one aspect of the original functionality. Herein, a functional homologue of a IIMAMIT transporter is a polypeptide sharing at least some sequence identity with said IIMAMIT transporter or a fragment thereof which has the capability to function as a glucosinolate transporter similarly to said IIMAMIT transporter.
The term “sequence identity” as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e. a candidate sequence (e.g. a mutant sequence) and a reference sequence (such as a wild type sequence) based on their pairwise alignment. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence (e.g. a natural variant or halotype of SEQ ID NO: 1) corresponds to a given position of SEQ ID NO: 1 (reference sequence). For example, if the natural variant has two additional amino acids in the N- terminal, position 70 in the natural variant will correspond to position 68 of SEQ ID NO: 1.
For purposes of the present invention, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment).
The terms “corresponding sequence”, “corresponding region” or "corresponding residue", as is generally understood in the art, refers to a region or residue on a second amino acid or nucleotide sequence which occupies the same (i.e. , equivalent) position as a region or residue on a first amino acid or nucleotide sequence, when the first and second sequences are optimally aligned for comparison purposes. Thus, a residue at a first position in a first peptide sequence does not necessarily correspond to a residue in said same first position in a second peptide sequence, but may instead correspond to a residue at a second position in the second peptide sequence that optimally aligns with the residue in said first position of said first peptide sequence, when the first and second peptide sequences are optimally aligned. Said alignment may be performed by any method known in the art, such as by using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The parameters used may be gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix.
The term “missense mutation” as used herein refers to a mutation/mutations in a nucleotide sequence resulting in a change from one amino acid to another in the polypeptide encoded by said nucleotide sequence.
"Mutations" include deletions, insertions, substitutions, transversions, and point mutations in the coding and/or noncoding regions of a gene. Deletions may be of an entire gene, or of only a portion of a gene. Point mutations may concern changes of one base pair, and may result in premature stop codons, frameshift mutations, mutation of a splice site or amino acid substitutions. A gene comprising a mutation when compared to a wild type gene may be referred to as a “mutant gene”. In the present invention a mutant gene generally encodes a polypeptide with a sequence different to the wild type gene, said polypeptide may be referred to as a “mutant polypeptide”. A mutant polypeptide may comprise an amino acid substitution, such a substitution can for example be described as “amino acid XXX at position n has been substituted to amino acid YYY” where XXX describes the amino acid at the specific position (n) of the wild type polypeptide and YYY describes the amino acid present in the mutant polypeptide at the same position when the two genes are aligned.
The term “IIMAMIT transporters” and “IIMAMIT exporters” are used interchangeably herein.
Brassicales plants comprising a mutation in a gene encoding an UMAMIT transporter
The present invention relates to Brassicales plants, or parts thereof, as well as products of said Brassicales plants and methods of producing these, wherein the Brassicales plant carries a mutation in a gene encoding an UMAMIT transporter, e.g. any of the mutations in genes encoding UMAMIT transporters described herein.
Thus, in one aspect of the invention is provided a Brassicales plant, or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an UMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant UMAMIT transporter, wherein said mutant UMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
The glucosinolate transporter activity of an UMAMIT transporter may be measured by any method known in the art. In preferred embodiments, the glucosinolate transporter activity of an UMAMIT transporter is measured using the methods described herein in Examples 1 and 2.
In some embodiments, the Brassicales plant carries a mutation in at least one gene encoding an UMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4,
BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as
at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter AtUMAMIT31 as set forth in SEQ ID NO: 4 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter BnaA09G0714200ZS as set forth in SEQ ID NO: 5 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as
at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter BnaC05G0010000ZS as set forth in SEQ ID NO: 6 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter BnaA01G0222900ZS as set forth in SEQ ID NO: 7 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter BnaC01G0283800ZS as set forth in SEQ ID NO: 8 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at
least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter BnaC03G0332100ZS as set forth in SEQ ID NO: 9 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant carries a mutation in the IIMAMIT transporter BnaA09G0692700ZS as set forth in SEQ ID NO: 10 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
The Brassicales plant of the invention may comprise mutations in more than one gene encoding an IIMAMIT transporter.
Thus, in some embodiments the Brassicales plant or part thereof carries a mutation in at least two genes encoding IIMAMIT transporters selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
Accordingly, in some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter Atll MAM IT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at
least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such
as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the IIMAMIT transporter AtUMAMIT31 as set forth in SEQ ID NO: 4 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the IIMAMIT transporter AtUMAMIT31 as set forth in SEQ ID NO: 4 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the IIMAMIT transporter AtUMAMIT31 as set forth in SEQ ID NO: 4 or a respective
functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in at least three genes encoding IIMAMIT transporters selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
Accordingly, in some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least
86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least
98%, such as at least 99% sequence identity thereto, the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the II MAM IT transporter AtUMAMIT31 as set forth in SEQ ID NO: 4 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, the gene encoding the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as
at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the II MAM IT transporter AtUMAMIT31 as set forth in SEQ ID NO: 4 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, the gene encoding the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least
84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the II MAM IT transporter AtUMAMIT31 as set forth in SEQ ID NO: 4 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the Brassicales plant or part thereof carries a mutation in at least four genes encoding IIMAMIT transporters selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto.
Accordingly, in some embodiments, the Brassicales plant or part thereof carries a mutation in the gene encoding the IIMAMIT transporter AtUMAMIT28 as set forth in SEQ ID NO: 1 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least
86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, the gene encoding the IIMAMIT transporter AtUMAMIT29 as set forth in SEQ ID NO: 2 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, the gene encoding the IIMAMIT transporter AtUMAMIT30 as set forth in SEQ ID NO: 3 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and the gene encoding the IIMAMIT transporter AtUMAMIT31 as set forth in SEQ ID NO: 4 or a respective functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
Several orthologs of AtUMAMIT28 (SEQ ID NO: 1), AtUMAMIT29 (SEQ ID NO: 2), AtUMAMIT30 (SEQ ID NO: 3), AtUMAMIT31 (SEQ ID NO: 4), which may be useful for the purposes of the present invention are listed in Tables 1-4 herein below. The orthologs listed below are accessible in the Brassica napus pan genome database available at http://cbi.hzau.edu.cn/cgi-bin/bnapus/search (Song et al., 2021), using the identifiers listed in the below tables. The same identifier can be used to access both the genomic sequence as well as the encoded protein. Table 1 - Useful Brassicales orthologs of AtUmamiT28
Table 2 - Useful Brassicales orthologs of AtUmamiT29
Table 3 - Useful Brassicales orthologs of AtUmamiT30
Table 4 - Useful Brassicales orthologs of AtUmamiT31
In some embodiments, is thus provided a Brassicales plant or part thereof carrying a mutation in a gene encoding an IIMAMIT transporter selected from the group consisting of BnaA09G0679100GG, BnaA09G0679000GG, BnaC05G0008000GG, BnaA09G0647600GG, BnaA09G0647700GG, BnaA10G0008000GG,
BnaA09G0623500NG, BnaA09G0623600NG, BnaC05G0007600NG, Bnascaffold2891G0004400NG, BnaA09G0624000NG, BnaC08G0554500QU,
BnaA09G0664600QU, BnaC08G0554400QU, BnaA10G0008300QU,
Bnascaffold2503G0022000QU, BnaA09G0664700QU, BnaA09G0724700SL, BnaA09G0689800SL, BnaA09G0724800SL, BnaC05G0007600SL,
BnaA09G0689900SL, Bnascaffold3068G0013800SL, BnaA09G0577200TA,
BnaA09G0628700TA, BnaC05G0007500TA, BnaA09G0577100TA,
BnaA10G0008300TA, BnaA09G0628800TA, BnaA09G0658500WE, BnaA09G0658600WE, BnaC05G0009100WE, BnaA10G0009300WE, BnaA09G0659000WE, BnaA09G0692800ZS, BnaA09G0692700ZS, BnaA09G0714200ZS, BnaA09G0714300ZS, BnaA10G0008500ZS, BnaC05G0010000ZS, BnaA09G0634900ZY, BnaA09G0666500ZY, Bnascaffold4696G0013500ZY, BnaC05G0007300ZY, BnaA09G0635000ZY, BnaA09G0666400ZY, BolC5g28871H, BolC8g52858H, BraA10g42362Z, BraA09g42228Z and BraA09g42227Z.
In some embodiments, is provided a Brassicales plant or part thereof carrying a mutation in a gene encoding an II MAM IT transporter selected from the group consisting of BnaC02G0303900GG, BnaA02G0239900GG, BnaC02G0303800GG, BnaC02G0289800NG, BnaA02G0248400NG, BnaA02G0248300NG, BnaC02G0286300QU, BnaA02G0247800QU, BnaA02G0247700QU, BnaC02G0286400QU, BnaC02G0230500SL, BnaA02G0281700SL, BnaC02G0230600SL, BnaC02G0254900TA, BnaA02G0214900TA, BnaC02G0255000TA, BnaC02G0326100WE, BnaA02G0260700WE, BnaA02G0260600WE, BnaC02G0326200WE, BnaC02G0337200ZS, BnaA02G0251300ZS, BnaC02G0337100ZS, BnaA02G0251200ZS, BnaA02G0278400ZY, BnaA02G0278500ZY, BnaC02G0162700ZY, BnaC02G0162600ZY, BolC2g09864H and BraA02g07429Z.
In some embodiments, is provided a Brassicales plant or part thereof carrying a mutation in a gene encoding an II MAM IT transporter selected from the group consisting of BnaC03G0326100GG, BnaA03G0226900GG, BnaA01G0215700GG, BnaC01G0262700GG, BnaA03G0226800GG, BnaC01G0263000GG, BnaC03G0325800GG, BnaC03G0326000GG, BnaC02G0304200GG, BnaA02G0240100GG, BnaC03G0251900NG, BnaA03G0272800NG, BnaA01G0189100NG, BnaA02G0248600NG, BnaC03G0251700NG, BnaC02G0290200NG, BnaA03G0272700NG, BnaC05G0297600NG, BnaC05G0297300NG, BnaC03G0252000NG, BnaC03G0317800QU, BnaC03G0317900QU, BnaC03G0317600QU, BnaA03G0263200QU, BnaC01G0188500QU, BnaA03G0262800QU, BnaA01G0168400QU, BnaC01G0188900QU, BnaC02G0286800QU, BnaA03G0263100QU, BnaA02G0248000QU, BnaC03G0209800SL, BnaA01G0193300SL,
BnaA03G0197300SL, BnaC02G0231000SL, BnaA02G0281500SL, BnaC01G0253100SL, BnaC03G0210100SL, BnaA03G0269600TA, BnaA03G0269800TA, BnaA02G0215100TA, BnaA03G0269900TA, BnaC02G0255300TA, BnaC01G0212600TA, BnaC01G0213100TA, BnaC03G0292000TA, BnaC03G0291900TA, BnaA01G0144700TA, BnaC03G0291700TA, BnaC03G0265500WE, BnaC01G0224300WE, BnaA03G0285900WE, BnaA02G0260400WE, BnaC02G0326500WE, BnaC03G0265600WE, BnaA01G0142400WE, BnaC03G0265300WE, BnaA03G0286100WE, BnaC01G0224600WE, BnaA03G0286000WE, BnaA01G0222900ZS, BnaC03G0331600ZS, BnaC03G0332100ZS, BnaA02G0251500ZS, BnaC03G0332000ZS, BnaC02G0337600ZS, BnaA03G0275500ZS, BnaA03G0275200ZS, BnaC01G0283800ZS, BnaC01G0283500ZS, BnaA03G0275300ZS, BnaC02G0163100ZY, BnaA03G0295200ZY, BnaC03G0268300ZY, BnaC03G0268600ZY, BnaC03G0268500ZY, BnaC01G0208900ZY, BnaA03G0295000ZY, BnaA03G0295100ZY, BnaC01G0209300ZY, BnaA01G0220000ZY, BnaA02G0278700ZY, BolC3g16132H, BolC2g09869H, BolC3g16128H, BolC3g16133H, BolC1g03106H, BolC1g03101H, BraA03g12335Z, BraA03g12337Z, BraA02g07432Z and BraA01g02441Z.
In some embodiments, is provided a Brassicales plant or part thereof carrying a mutation in a gene encoding an II MAM IT transporter selected from the group consisting of BnaA02G0240000GG, BnaC02G0304100GG, Bnascaffold1465G0001500GG, BnaA09G0014000GG, BnaC02G0290100NG, BnaA09G0016000NG, BnaA02G0248500NG, BnaC09G0009500QU, BnaA02G0247900QU, BnaA09G0011000QU, BnaC02G0286700QU, BnaA09G0009300SL, Bnascaffold966G0010300SL, BnaA02G0281600SL, BnaC02G0230900SL, BnaA02G0215000TA, BnaC09G0008700TA, BnaA09G0005700TA, BnaC02G0255200TA, BnaC02G0326400WE, BnaC09G0008200WE, BnaA02G0260500WE, BnaA09G0011300WE, BnaC09G0002100ZS, BnaC02G0337500ZS, Bnascaffold0025G0022100ZS, BnaA02G0251400ZS, BnaA09G0019300ZS, Bnascaffold0025G0022000ZS, BnaC09G0001300ZY, BnaC02G0163000ZY, BnaA02G0278600ZY, BnaA09G0018200ZY, BolC9g53044H, BolC2g09868H, BraA02g07431Z and BraA09g35773Z.
The mutations of the present invention preferably change the activity and/or expression of the encoded II MAM IT transporter.
In some embodiments, said mutant IIMAMIT transporter is a mutant glucosinolate transporter with reduced glucosinolate transporter activity compared to the wildtype protein. In some embodiments, the mutated gene encodes a mutant glucosinolate transporter with reduced glucosinolate transporter activity compared to the wildtype protein.
Accordingly, in some embodiments, said reduction is a reduction of at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% of the glucosinolate transporter activity of said mutant glucosinolate transporter compared to the wildtype protein. The glucosinolate transporter activity of an IIMAMIT transporter may be measured by any method known in the art. In preferred embodiments, the glucosinolate transporter activity of an IIMAMIT transporter is measured using the methods described herein in Examples 1 and 2.
In some embodiments, said mutated gene has reduced expression of the encoded glucosinolate transporter compared to the wildtype gene.
Accordingly, in some embodiments, said reduced expression is a reduction of at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at
least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% in expression of said mutated gene compared to the wildtype gene. Said reduced expression may be measured using methods commonly known in the art, such as by quantitative reverse transcription PCR (RT-qPCR). In preferred embodiments, said reduced expression is measured using the methods described herein in Examples 1 and 2.
Said reduced gene expression and/or reduced transporter activity of the encoded IIMAMIT transporter protein results in a reduced glucosinolate content in specific parts of the Brassicales plant, such as in the seeds.
Thus, in some embodiments, the seeds of said Brassicales plant or part thereof have a reduced glucosinolate content compared to a Brassicales plant or part thereof of otherwise identical genotype except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions. In some embodiments, said reduction in glucosinolate content is a reduction of at least 50%, such as at least 51%, such as at least 52%, such as at least 53%, such as at least 54%, such as at least 55%, such as at least 56%, such as at least 57%, such as at least 58%, such as at least 59%, such as at least 60%, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100%.
In some embodiments, the reduced gene expression and/or reduced transporter activity of the encoded IIMAMIT transporter protein does not materially affect the size of the seeds.
Thus, in some embodiments, the seeds of the Brassicales plant or part thereof having a reduced glucosinolate content have approximately the same size as seeds from a Brassicales plant or part thereof with an otherwise identical genotype except not comprising any of the mutations in the IIMAMIT exporters as defined herein, when cultivated and prepared under the same conditions,
In some embodiments, the reduced gene expression and/or reduced transporter activity of the encoded IIMAMIT transporter protein does not materially affect the dry weight of the seeds. However, in other embodiments, the dry weight of the seeds of the Brassicales plant or part thereof having a reduced glucosinolate content have a slightly lower dry weight as compared to seeds from a Brassicales plant or part thereof with an otherwise identical genotype except not comprising any of the mutations in the IIMAMIT exporters as defined herein, when cultivated and prepared under the same conditions. In some embodiments, the dry weight of the seeds of the Brassicales plant or part thereof having a reduced glucosinolate content have a weight that is not more than 7% lower, such as not more than 6 % lower, such as not more than 5% lower, such as not more than 4% lower, such as not more than 3% lower, such as not more than 2% lower, such as not more than 1% lower than the seeds from a Brassicales plant or part thereof with an otherwise identical genotype except not comprising any of the mutations in the IIMAMIT exporters as defined herein, when cultivated and prepared under the same conditions.
In some embodiments, the dry weight of the seeds of the Brassicales plant or part thereof having a reduced glucosinolate content have a weight that is between 1% and 7% lower, such as between 2% and 6% lower, such as between 3% and 5% lower, such as between 4% and 6% lower than the seeds from a Brassicales plant or part thereof with an otherwise identical genotype except not comprising any of the mutations in the IIMAMIT exporters as defined herein, when cultivated and prepared under the same conditions.
The Brassicales plants according to the invention may have reduced glucosinolate levels in specific tissues such as in seeds, while having wildtype or near-wildtype levels in other tissues.
Thus, in some embodiments, the vegetative tissue of said Brassicales plant or part thereof have about the same glucosinolate content compared to a Brassicales plant or part thereof of otherwise identical genotype except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
In some embodiments, the Brassicales plant according to the invention has reduced glucosinolate levels in the seeds, and wildtype or near-wildtype levels in other tissues of said plant. In some embodiments, the seeds have a concentration of glucosinolates of less than 18 micromoles per gram of dry weight of said seeds, such as less than 17 micromoles per gram of dry weight, such less than 16 micromoles per gram of dry weight, such as less than 15 micromoles per gram of dry weight, such less than most 14 micromoles per gram of dry weight, such as less than 13 micromoles per gram of dry weight, such as less than 12 micromoles per gram of dry weight, such as less than 11 micromoles per gram of dry weight, such as less than 10 micromoles per gram of dry weight, such as less than 9 micromoles per gram of dry weight, such as less than 8 micromoles per gram of dry weight, such as less than 7 micromoles per gram of dry weight, such as less than 6 micromoles per gram of dry weight, such as less than 5 micromoles per gram of dry weight, such as less than 4 micromoles per gram of dry weight, such as less than 3 micromoles per gram of dry weight, such as less than 2 micromoles per gram of dry weight, such as less than 1 micromoles per gram of dry weight of said seeds.
In some embodiments, the seeds have a concentration of glucosinolates of less than 100 nanomoles per gram of dry weight of said seeds, such as less than 50 nanomoles per gram of dry weight, such less than 25 nanomoles per gram of dry weight, such as less than 10 nanomoles per gram of dry weight, such less than 1 nanomoles per gram of dry weight, such less than 0.5 nanomoles per gram of dry weight, such less than 0.05 nanomoles per gram of dry weight, such less than 0.005 nanomoles per gram of dry weight of said seeds.
In some embodiments, the seeds do not have a measurable level of glucosinolates.
In some embodiments, the Brassicales plant is an oilseed crop. In some embodiments the Brassicales plant is a protein crop. In some embodiments, the Brassicales plant is an oilseed and protein crop.
In some embodiments, the Brassicales plant is a brassicaceous oilseed crop. In some embodiments the Brassicales plant is a brassicaceous protein crop. In some embodiments, the Brassicales plant is a brassicaceous oilseed and protein crop.
Oilseed and/or protein crops, such as brassicaceous oilseed and/or protein crops, include, but are not limited to, the genera Brassica, Camelina, Crambe, Eruca, Raphanus, Lepidium and Thlaspi.
In some embodiments, the plant is of the family Brassicaceae. In some embodiments, the plant is of the genus Brassica. In some embodiments, the plant is selected from the group consisting of B. juncea, B. napus (rape), B. carinata, B. oleracea, B. rapa. In other embodiments, the plant is of the genus Lepidium, such as for example an L. campestre plant. In some embodiments, the plant is of the genus Camelina. In some embodiments, the plant is of the genus Crambe. In some embodiments, the plant is of the genus Eruca. In some embodiments, the plant is of the genus Raphanus. In some embodiments, the plant is of the genus Thlaspi.
In some embodiments, the Brassicales plant of the invention has not been exclusively obtained by means of an essentially biological process. Progeny of a Brassicales plant obtained by a technical process is herein considered as not being exclusively obtained by means of an essentially biological process, because the parent plant is obtained by a technical process.
In one embodiment the Brassicales plant carries one or more mutations as disclosed elsewhere herein, wherein said one or more mutations have been induced by chemical and/or physical agents.
In one embodiment the plant has been prepared by a method involving a step of induced mutagenesis or said plant is progeny of a plant prepared by a method involving a step of induced mutagenesis.
Thus, the Brassicales plant may be a plant prepared by a method comprising the following steps or progeny of a plant prepared by a method comprising the following steps:
• Mutagenizing Brassicales plants or parts thereof, for example with a chemical mutagenizing agent such as NaN3
• Selecting Brassicales plants carrying any of the mutations as disclosed elsewhere herein.
Useful mutations
The mutations according to the present invention may result in a change of the amino acid sequence of the gene encoding the IIMAMIT transporter. Thus, in preferred embodiments, the mutation is a non-synonymous mutation.
In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is an insertion. In some embodiments, the mutation is a deletion. In some embodiments, the mutation is a frameshift mutation.
In some embodiments, the mutation lies within a promoter region of said gene(s). In some embodiments, the mutation lies within a coding region of said gene(s). In some embodiments, the mutation lies within an exonic region of said gene(s). In some embodiments, the mutation lies within a non-coding region of said gene(s). In some embodiments, the mutation lies within an intronic region of said gene(s). In some embodiments, the mutation lies within a termination sequence of said gene(s).
In some embodiments, the mutation is a loss-of-function mutation.
In some embodiments, the mutation is an insertion of a transfer DNA (T-DNA) sequence. In some embodiments, the inserted sequence is pROK2 as set forth in SEQ ID NO: 21.
In some embodiments, the Brassicales plant comprises one or more mutations in one or more genes encoding an IIMAMIT transporter. In some embodiments, the Brassicales plant comprises at least two mutations in one or more genes encoding an IIMAMIT transporter, wherein the mutations are independently selected from the mutations described herein.
The Brassicales plant may carry one or more amino acid mutations in IIMAMIT transporters, which abrogate glucosinolate transport activity of these transporters but still retain any other regulatory or enzymatic functions of these. In some embodiments, the one or more amino acid mutations are in the AtUMAMIT29 polypeptide as set forth in SEQ ID NO: 2 or in a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, or such as at least 95% sequence identity thereto, and said Brassicales expresses said mutant AtUMAMIT29 polypeptide or a functional homolog thereof with corresponding mutations.
In particular, one or more useful mutations for reducing or abrogating glucosinolate transport activity of AtUMAMIT29 are i. a substitution of amino acid 27 of SEQ ID NO: 2 (valine (V)) to a phenylalanine (F); ii. a substitution of amino acid 86 of SEQ ID NO:2 (methionine (M)) to a valine (V); iii. a substitution of amino acid 109 of SEQ ID NO:2 (leucine (L)) to a valine (V); iv. a substitution of amino acid 263 of SEQ ID NO:2 (glutamine (Q)) to a serine (S); v. a substitution of amino acid 267 of SEQ ID NO:2 (threonine (T)) to a tyrosine
(Y); vi. a substitution of amino acid 44 of SEQ ID NO: 2 (arginine (R)) to an alanine (A); vii. a substitution of amino acid 200 of SEQ ID NO: 2 (tryptophan (W)) to an alanine
(A); viii. a substitution of amino acid 204 of SEQ ID NO: 2 (glutamine (Q)) to an alanine (A).
In some embodiments, the Brassicales plant expresses a functional homolog of AtUMAMIT29 as set forth in SEQ ID NO: 2 with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, or such as at least 95% sequence identity thereto. In such embodiments, the sequence of the functional homolog is first optimally aligned to the sequence of AtUMAMIT29 as set forth in SEQ ID NO: 2, e.g. using the Needleman-Wunsch algorithm, to identify corresponding amino acids, and one or more of the following mutations (corresponding mutations) are then performed to reduce or abrogate glucosinolate transport activity of the functional homolog of AtUMAMIT29:
a. substitution of a valine corresponding to the valine in position 27 of SEQ ID NO: 2 with phenylalanine (V27F); b. substitution of a methionine corresponding to the methionine in position 86 of SEQ ID NO: 2 with valine (M86V); c. substitution of a leucine corresponding to the leucine in position 109 of SEQ ID NO: 2 with valine (L109V), d. substitution of a glutamine corresponding to the glutamine in position 263 of SEQ ID NO: 2 with serine (Q263S); e. substitution of a threonine corresponding to the threonine in position 267 of SEQ ID NO: 2 with tyrosine (T267Y); f. substitution of an arginine corresponding to the arginine in position 44 of SEQ ID NO: 2 with alanine (R44A); g. substitution of a tryptophan corresponding to the tryptophan in position 200 of SEQ ID NO: 2 with alanine (W200A); h. substitution of a glutamine corresponding to the glutamine in position 204 of SEQ ID NO: 2 substituted with alanine (Q204A).
In some embodiments, the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said mutated gene encodes a mutant Atll MAM IT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as
at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, except that said mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 27 of SEQ ID NO:2 (valine (V)) to a phenylalanine (F)) or said mutant functional homolog of AtUMAMIT29 comprises a substitution of a valine (V) corresponding to the valine of amino acid 27 of SEQ ID NO:2 to a phenylalanine (F). Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 20%, such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
In some embodiments, the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said mutated gene encodes a mutant Atll MAM IT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, except that said mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 86 of SEQ ID NO:2 (methionine (M)) to a valine (V) or said mutant functional homolog of AtUMAMIT29 comprises a substitution of a methionine (M) corresponding to the methionine of amino acid 86 of SEQ ID NO:2 to a valine (V). Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 20%,
such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
In some embodiments, the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said mutated gene encodes a mutant Atll MAM IT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, except that said mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 109 of SEQ ID NO:2 (leucine (L)) to a valine (V) or said mutant functional homolog of AtUMAMIT29 comprises a substitution of a leucine (L) corresponding to the leucine of amino acid 109 of SEQ ID NO: 2 to a valine (V). Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 20%, such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
In some embodiments, the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such
as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said mutated gene encodes a mutant Atll MAM IT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, except that said mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 263 of SEQ ID NO:2 (glutamine (Q)) to a serine (S) or said mutant functional homolog of AtUMAMIT29 comprises a substitution of a glutamine (Q) corresponding to the glutamine of amino acid 263 of SEQ ID NO: 2 to a serine (S). Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 30%, such as at the most 25%, such as at the most 20%, such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
In some embodiments, the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said mutated
gene encodes a mutant AtUMAMIT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, except that said mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 267 of SEQ ID NO: 2 (threonine (T)) to a tyrosine (Y) or said mutant functional homolog of AtUMAMIT29 comprises a substitution of a threonine (T) corresponding to the threonine of amino acid 267 of SEQ ID NO: 2 to a tyrosine (Y). Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
In some embodiments, the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said mutated gene encodes a mutant Atll MAM IT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at
least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, except that said mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 44 of SEQ ID NO: 2 (arginine (R)) to an alanine (A) or said mutant functional homolog of AtUMAMIT29 comprises a substitution of an arginine (R) corresponding to the arginine of amino acid 44 of SEQ ID NO: 2 to an alanine (A). Said mutant polypeptide may have a mean total glucosinolate import activity of at the most 20%, such as at the most 15%, such as at the most 10%, such as at the most 5% compared to a corresponding wild type UMAMIT29 polypeptide.
In some embodiments, the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said mutated gene encodes a mutant Atll MAM IT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, except that said mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 200 of SEQ ID NO: 2 (tryptophan (W)) to an alanine (A) or said mutant functional homolog of AtUMAMIT29 comprises a substitution of a tryptophan (W) corresponding to the tryptophan of amino acid 200 of SEQ ID NO: 2 to an alanine (A).
In some embodiments, the Brassicales plant carries a mutation in the AtUMAMIT29 gene (SEQ ID NO: 12) or in a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, wherein said mutated gene encodes a mutant Atll MAM IT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, except that said mutant AtUMAMIT29 polypeptide comprises a substitution of amino acid 204 of SEQ ID NO: 2 (glutamine (Q)) to an alanine (A) or said mutant functional homolog of AtUMAMIT29 comprises a substitution of a glutamine (Q) corresponding to the glutamine of amino acid 204 of SEQ ID NO: 2 to an alanine (A).
In some embodiments, the Brassicales plant or part thereof thus carries a mutation in the AtUMAMIT29 gene as set forth in SEQ ID NO: 12 or in a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, wherein said mutated gene encodes a mutant Atll MAM IT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO: 2 or a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, except that said mutant polypeptide comprises a substitution or a corresponding substitution
selected from the group consisting of V27F, M86V, L109V, Q263S T267Y, R44A, W200A and Q204A in relation to AtUMAMIT29 as set forth in SEQ ID NO: 2.
Said Brassicales plant or part thereof may also express a mutant AtUMAMIT29 polypeptide or a homolog thereof that carries more than one of said substitutions. In some embodiments, the Brassicales plant or part thereof thus carries a mutation in the AtUMAMIT29 gene as set forth in SEQ ID NO: 12 or in a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, wherein said mutated gene encodes a mutant AtUMAMIT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO:2 or a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, except that said mutant polypeptide comprises one or more substitutions or one or more corresponding substitutions selected from the group consisting of V27F, M86V, L109V, Q263S T267Y, R44A, W200A and Q204A in relation to AtUMAMIT29 as set forth in SEQ ID NO: 2.
In addition to the herein described one or more mutations in one or more genes encoding an IIMAMIT transporter, further Brassicales plants of the invention may comprise one or more additional mutations in one or more additional genes.
Specific examples of Brassicales plants comprising a mutation in a gene encoding an UMAMIT transporter
In some embodiments, the plant comprises a T-DNA insertion of SEQ ID NO: 22 (Vector_ pAC106) in the first intron of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as
at least 98%, such as at least 99% sequence identity thereto. The Arabidopsis seeds having this T-DNA insertion have a Met-derived glucosinolates (GLS) content of about 34.74± 9.04 nmol mg-1 and a Trp-derived GLS content of about 1.21 ± 0.75 nmol mg-1.
In some embodiments, the plant comprises a T-DNA insertion of SEQ ID NO: 22 (Vector_ pAC106) in the fourth exon of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding exon of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
In some embodiments, the plant comprises a T-DNA insertion of SEQ ID NO: 22 (Vector_ pAC106) in the first exon of AtUMAMIT30 (SEQ ID NO: 13) or in a corresponding exon of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto. The Arabidopsis seeds having this T-DNA insertion have a Met-derived GLS content of about 128.76± 15.80 nmol mg-1 and a Trp-derived GLS content of about 2.13± 0.42 nmol mg-1.
In some embodiments, the plant comprises a T-DNA insertion of SEQ ID NO: 22 (Vector_ pAC106) in the first exon of AtUMAMIT31 (SEQ ID NO: 13) or in a corresponding exon of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as
at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto. The Arabidopsis seeds having this T-DNA insertion have a Met-derived GLS content of about 154.83± 7.01 nmol mg-1 and Trp-derived GLS content of about 0.64± 0.43 nmol mg-1.
In some embodiments, the plant comprises a T-DNA insertion of SEQ ID NO: 21 (pROK2) in the first intron on both alleles of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, and a deletion of 11 nucleotides at positions 1048-1059 on both alleles of AtUMAMIT30 (SEQ ID NO: 13), or in corresponding positions on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto. The Arabidopsis seeds having this T-DNA insertion and this deletion have a Met-derived GLS content of about 12.97± 1.25 nmol mg-1 and a Trp-derived GLS content of about 2.18± 0.07 nmol mg-1.
In some embodiments, the plant comprises a T-DNA insertion of SEQ ID NO: 21 (pROK2) in the first intron on both alleles of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, a deletion of 11 nucleotides at positions 1048-1059 on both alleles of AtUMAMIT30 (SEQ ID NO: 13) or in corresponding positions on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, a deletion of 3 nucleotides at positions 57-59 on both alleles of AtUMAMIT31 (SEQ ID NO: 14) or in corresponding positions on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto. The Arabidopsis seeds having this T-DNA insertion and these deletions have a Met-derived GLS content of about 10.94± 0.62 nmol mg-1 and Trp-derived GLS content of about 0.13± 0.01 nmol mg-1.
In some embodiments, the plant comprises a T-DNA insertion of SEQ ID NO: 21 (pROK2) in the first intron on both alleles of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron on all alleles of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, a deletion of 9 nucleotides at positions 1044-1052 on both alleles of AtUMAMIT30 (SEQ ID NO: 13) or in corresponding positions on all alleles of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, an insertion of 4 nucleotides at positions 57-59 on both alleles of AtUMAMIT31 (SEQ ID NO: 14) or in corresponding positions on all alleles of a functional homolog thereof with at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto. The Arabidopsis seeds having this T-DNA insertion, this deletion and this insertion have a Met-derived GLS content of about 10.94± 0.62 nmol mg-1 and a Trp-derived GLS content of about 0.13± 0.01 nmol mg-1.
In some embodiments, the plant comprises a T-DNA insertion of SEQ ID NO: 21 (pROK2) the first intron on both alleles of AtUMAMIT29 (SEQ ID NO: 12) or in a corresponding intron on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, an insertion of 1 nucleotide at positions 1051 on both alleles of AtUMAMIT30 (SEQ ID NO: 13) or in corresponding positions on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, a deletion of 12 nucleotides at positions 60-71 on both alleles of AtUMAMIT31 (SEQ ID NO: 14) or in corresponding positions on all alleles of a functional homolog thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto. The Arabidopsis seeds having this T-DNA insertion, this insertion and this deletion have a Met-derived GLS content of about 17.56± 0.89 nmol mg-1 and a Trp- derived GLS content of about 0.14± 0.01 nmol mg-1.
Plant products and methods of producing the same
The present prevention also provides plant products prepared from a Brassicales plant carrying a mutation in a gene encoding an IIMAMIT transporter according to the invention, e.g. any of the Brassicales plants, or parts thereof, described herein.
In some embodiments, the plant part is a seed.
In one aspect is thus provided a plant product comprising a Brassicales plant or part thereof, or prepared from seeds of said Brassicales plant or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
In some embodiments, the Brassicales plant or part thereof is as defined elsewhere herein.
Examples of useful plant products according to the invention include products prepared from the seeds of the plant, such as seed oils, seed cakes and seed meals.
Thus, in some embodiments, the plant product is prepared from a seed. In some embodiments, the plant product is an oil, such as a seed oil. In some embodiments, the plant product is a seed cake. In some embodiments, the plant product is a seed meal.
In one aspect is provided a seed cake prepared from a seed from a Brassicales plant, wherein the Brassicales plant is as described elsewhere herein.
In some embodiments, is thus provided a seed cake prepared from a seed from a Brassicales plant, wherein the Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
In some embodiments, the seed has a concentration of glucosinolates of less than 18 micromoles per gram of dry weight of said seed, such as less than 17 micromoles per gram of dry weight, such less than 16 micromoles per gram of dry weight, such as less than 15 micromoles per gram of dry weight, such less than most 14 micromoles per gram of dry weight, such as less than 13 micromoles per gram of dry weight, such as
less than 12 micromoles per gram of dry weight, such as less than 11 micromoles per gram of dry weight, such as less than 10 micromoles per gram of dry weight, such as less than 9 micromoles per gram of dry weight, such as less than 8 micromoles per gram of dry weight, such as less than 7 micromoles per gram of dry weight, such as less than 6 micromoles per gram of dry weight, such as less than 5 micromoles per gram of dry weight, such as less than 4 micromoles per gram of dry weight, such as less than 3 micromoles per gram of dry weight, such as less than 2 micromoles per gram of dry weight, such as less than 1 micromoles per gram of dry weight of said seed.
In some embodiments, the seed comprises a concentration of glucosinolate of at the most 15%, such as at the most 14%, such as at the most 13%, such as at the most 12%, such as at the most 11%, such as at the most 10%, such as at the most 9%, such as at the most 8%, such as at the most 7%, such as at the most 6%, such as at the most 5%, such as at the most 5%, such as at the most 3%, such as at the most 2%, or such as at the most 1% of the glucosinolate concentration in a seed prepared from a seed of a plant of otherwise identical genotype to said Brassicales plant except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
In some embodiments, the seed cake prepared from said Brassicales plant comprises a concentration of glucosinolate of at the most 15%, such as at the most 14%, such as at the most 13%, such as at the most 12%, such as at the most 11%, such as at the most 10%, such as at the most 9%, such as at the most 8%, such as at the most 7%, such as at the most 6%, such as at the most 5%, such as at the most 5%, such as at the most 3%, such as at the most 2%, or such as at the most 1% of the glucosinolate concentration in a seed cake prepared from a seed of a plant of otherwise identical genotype to said Brassicales plant except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
In some embodiments, the plant seed product comprises glucosinolates at a concentration of at the most 30 micromoles per gram of dry weight of said plant seed product, such as at the most 25 micromoles per gram of dry weight, such as at the most 20 micromoles per gram of dry weight, such as at the most 15 micromoles per gram of dry weight, such as at the most 14 micromoles per gram of dry weight, such as
at the most 13 micromoles per gram of dry weight, such as at the most 12 micromoles per gram of dry weight, such as at the most 11 micromoles per gram of dry weight, such as at the most 10 micromoles per gram of dry weight, such as at the most 9 micromoles per gram of dry weight, such as at the most 8 micromoles per gram of dry weight, such as at the most 7 micromoles per gram of dry weight, such as at the most 6 micromoles per gram of dry weight, such as at the most 5 micromoles per gram of dry weight, such as at the most 5 micromoles per gram of dry weight, such as at the most 3 micromoles per gram of dry weight, such as at the most 2 micromoles per gram of dry weight, or such as at the most 1 micromoles glucosinolates per gram of dry weight of said plant seed product.
In some embodiments, the plant seed product comprises glucosinolates at a concentration of at the most 1.5 mmol per kg of plant product, such as at the most 1.25 mmol per kg of plant product, such as at the most 1 mmol per kg of plant product, 0.75 mmol per kg of plant product, such as at the most 0.5 mmol per kg of plant product, such as at the most 0.25 mmol per kg of plant product, such as at the most 0.1 mmol glucosinolates per kg of said plant seed product.
In some embodiments, the plant seed product does not comprise a measurable concentration of glucosinolates. Methods to measure glucosinolate content are well known in the art, and are described herein in the section “Definitions”.
In some aspects is also provided a method for producing a plant product comprising a Brassicales plant or part thereof with low glucosinolate content, said method comprising the steps of a. providing a Brassicales plant or part thereof as described elsewhere herein; and b. processing said Brassicales plant or part thereof into a plant product, such as a seed oil or a seed cake.
In some embodiments is thus provided a method for producing a plant product comprising a Brassicales plant or part thereof with low glucosinolate content, said method comprising the steps of a. providing a Brassicales plant or part thereof, wherein the Brassicales plant or part thereof carries a mutation in at least one gene encoding an IIMAMIT
transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed; and b. processing said Brassicales plant or part thereof into a plant product, such as a seed oil or a seed cake.
The plant seed products described herein can be particularly useful as animal feed and fodder. Glucosinolates are undesirable substances in animal feed and fodder, as they may cause growth retardation, reduction in performance (reduced milk and egg production), impaired reproductive activity, and impairment of liver and kidney functions. For use in compound feed, palatability to ruminants sets the level of total GSL permitted at no more than 10-15 micromoles per gram of dry weight. However poultry and pigs are more sensitive to levels of GSLs than ruminants, and more than 2- 4 micromoles GSL per gram of dry weight in the feed can severely affect reproductive efficiencies in these animals. Further, for rapeseed meal or press cakes, there is a recommendation to restrict the total glucosinolate content to 1-1.5 mmol per kg feed for
monogastric animals, and to even lower concentrations in feed for young animals (Alexander et al., 2008).
Thus, in some embodiments, it is provided an animal feed comprising a plant product prepared from a Brassicales plant seed or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
In some embodiments it is provided a plant seed product prepared from a Brassicales plant or part thereof as described herein for use in animal feed.
Said plant product may be as defined herein the section “Plant products and methods for producing same”. In preferred embodiments, the plant product is prepared from seeds. In even more preferred embodiments, the plant product is a seed cake, a seed oil or a seed meal.
Methods for modifying the glucosinolate content of Brassicales plants, or parts thereof Brassicales plants, or parts thereof, carrying a mutation in a gene encoding an IIMAMIT transporter and having reducing glucosinolate content according to the invention may be prepared in any useful manner.
In one aspect is thus provided a method for modifying glucosinolate content in a Brassicales plant or part thereof, said method comprising a step of modifying the functional activity or expression of at least one IIMAMIT transporter selected from the
group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
Said step of modifying the functional activity of at least one IIMAMIT transporter may be done using any useful method known in the art.
In some embodiments, said step of modifying said functional activity is performed by nuclease-based gene editing. In some embodiments, said step of modifying said functional activity is performed by CRISPR/Cas9 gene editing. In some embodiments, said step of modifying said functional activity is performed by gene targeting. In some embodiments, said step of modifying said functional activity is performed by transposition mutagenesis. In some embodiments, said step of modifying said functional activity is performed by transfer-DNA induced insertion. In some embodiments, said step of modifying said functional activity is performed by gene knockdown. In some embodiments, said step of modifying said functional activity is performed by RNA interference.
In some embodiments, said step of modifying said functional activity is performed by random mutagenesis. In some embodiments, said step of modifying said functional activity is performed using non-GMO methods. In some embodiments, the method further comprises one or more crosses of plants.
Brassicales plants, or parts thereof, carrying a mutation in a gene encoding an IIMAMIT transporter and having reducing glucosinolate content according to the invention may be prepared in any useful manner.
For example, Brassicales plants according to the invention can be prepared by a method comprising the steps of: a. providing seeds of a Brassicales plant; b. randomly mutagenizing said Brassicales plant seeds, c. selecting Brassicales plant seeds or parts thereof carrying a mutated gene encoding a mutant IIMAMIT transporter polypeptide carrying a mutation that results in said mutant IIMAMIT transporter having low or no glucosinolate transporter activity, being expressed at low levels, or not being expressed.
In some embodiments, the step of modifying said functional activity further includes one or more steps of reproducing said Brassicales plants or parts thereof in order to obtain multiple Brassicales plants or parts thereof each carrying said mutation.
In particular, Brassicales plants carrying a particular mutation in a gene encoding an IIMAMIT transporter may be prepared and identified essentially as described in international patent application WO 2018/001884 using primers and probes designed to identify a mutation in said gene.
Brassicales plants carrying a mutation in a gene encoding an IIMAMIT transporter may also be prepared using various site directed mutagenesis methods, which for example can be based on the sequence of the coding sequence of the gene encoding an IIMAMIT transporter, such as a gene comprising the sequence according to any one of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. In one embodiment, the Brassicales plant is prepared using any one of CRISPR, a TALEN, a zinc finger, meganuclease, and a DNA-cutting antibiotic as described in WO 2017/138986. In one embodiment, the Brassicales plant is prepared using CRISPR/cas9 technique, e.g. using RNA-guided Cas9 nuclease. This may be done as described in Lawrenson et al., Genome Biology (2015) 16:258; DOI 10.1186/s13059-015-0826-7 except that the single guide RNA sequence is designed based on the gene sequence of a gene encoding an IIMAMIT transporter. In one embodiment, Brassicales plant is prepared using a combination of both TALEN and CRISPR/cas9 techniques, e.g. using RNA-
guided Cas9 nuclease. This may be done as described in Holme et al., 2017 except that the TALEN and single guide RNA sequence are designed based on the gene sequences provided herein.
In one embodiment, the Brassicales plant is prepared using homology directed repair, a combination of a DNA cutting nuclease and a donor DNA fragment. This may be done as described in Sun et al., 2016 except that the DNA cutting nuclease is designed based on the genes sequences provided herein.
In one embodiment of the invention, the objective is to provide agronomical useful Brassicales plants carrying a mutation in a gene encoding an IIMAMIT transporter. In addition to the mutation in the a gene encoding an IIMAMIT transporter, there are additional factors which also may be considered in the art of generating a commercial Brassicales plant variety useful for preparing products from its seeds, for example seed yield and size, and other parameters that relate to seed quality and composition. Since many - if not all - relevant traits have been shown to be under genetic control, the present invention also provides modern, homozygous, high-yielding cultivars, which may be prepared from crosses with the Brassicales plants that are disclosed in the present publication. The skilled Brassicales breeder will be able to select and develop Brassicales plants, which - following crossings with other Brassicales plants - will result in superior cultivars. Alternatively, the breeder may utilize plants of the present invention for further mutagenesis to generate new cultivars carrying additional mutations in addition to the mutation of the gene encoding an IIMAMIT transporter.
The invention also comprise Brassicales plants carrying a mutation in gene encoding an IIMAMIT transporter prepared from plant breeding method, including methods of selfing, backcrossing, crossing to populations, and the like. Backcrossing methods can be used with the present invention to introduce into another cultivar the mutation of the mutated gene encoding an IIMAMIT transporter.
A way to accelerate the process of plant breeding comprises the initial multiplication of generated mutants by application of tissue culture and regeneration techniques. Thus, another aspect of the present invention is to provide cells, which upon growth and differentiation produce Brassicales plants carrying one or more mutations in a gene
encoding an IIMAMIT transporter. For example, breeding may involve traditional crossings, preparing fertile anther-derived plants or using microspore culture.
Examples
Example 1 - Glucosinolate export by UMAMITs is essential for seed accumulation According to the optimal defence theory, plant organs with the highest fitness value such as seeds and tubers accumulate the highest level of defence compounds to protect against herbivores and pathogens. However, for humans, the often-toxic defence compounds drastically reduce the nutritional value of these edible tissues. Reduction of anti-nutritional factors by blocking the biosynthetic pathways is, however, accompanied by adverse effects on plant fitness. Since defence compounds are often translocated to edible tissues, elimination of transporters along the route from source to sink provides a strategy to reduce toxic compounds in edible parts while maintaining the defence in other tissues. Loss-of-function mutations of plasma membrane-localized importers and exporters may have fundamentally different effects on the distribution pattern within the whole plant. For instance, in Arabidopsis, mutation of the H+-coupled GLUCOSINOLATE TRANSPORTERS (GTRs) - functioning as importers of glucosinolate defence compounds from the apoplast - resulted not only in elimination of glucosinolate accumulation in seeds (that have no de novo synthesis) but also led to major alterations in the glucosinolate distribution pattern across the whole plant. The latter is due to the flow of apoplast-accumulated glucosinolates to distant organs along the transpiration stream. In contrast, blocking the mechanism that export defence compounds to the apoplast prior to their import into symplasm may provide a strategy for eliminating accumulation of anti-nutritional compounds in sink tissues, without affecting defence distribution. However, the potential of exporter engineering remains unexplored due to a general lack of knowledge on exporters of specialized metabolites. Here, we identify and characterize members of the Usually Multiple Amino Acids Move In and out Transporter (UMAMIT) family, UMAMIT29, -30 and -31 as glucosinolate exporters and show that mutants of these exporters specifically block accumulation of glucosinolates in Arabidopsis seeds without altering the defence distribution in the rest of the plant.
Material and methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) (N7000) gtr1 gtr27 and gtr1 gtr2 gtr39 gtr1, line SAIL_801_G03; gtr2, line SAIL_20_B07; gtr3, line GK-099B01) have been described previously. T-DNA insertion mutants, umamit29-7 (Salk_133129C), umamit29-2 (GK-007H08), umamit30-1 (SALK_146977C) and umamit31-1 (GK- 266E08) were ordered from the European Arabidopsis stock center NASC (Nottingham). Loss of expression of targeted genes was confirmed by qRT-PCR (Extended Data Figure 8b). Other mutants were generated by CRISPR-Cas9-mediated genome editing. ut29 ut31 and ut29 ut30 double mutants were generated by introducing single guide RNA (sgRNA)/Cas9 targeting UMAMIT31 and UMAMIT30 into umamit29-1 mutants, respectively. umamit29 umamit30 umamit31 triple mutants were generated by co-introducing a sgRNA targeting UMAMIT30 and a sgRNA targeting UMAMIT31 into umamit29-1. gtr1 gtr2 gtr3 umamit29 umamit30 quintuple mutants were generated by introducing sgRNA targeting UMAMIT30 into gtr1 gtr2 gtr3 umamit29-1. For genotyping of the transgenic lines, rosettes were collected from each plant and genomic DNA was isolated using the CTAB method. PCR amplification was performed using primers flanking the sgRNA target sites and Sanger sequencing was used to identify mutations in target genes. PCR was performed using Phusion DNA Polymerases. All primers were synthesized at the TAG Copenhagen A/S.
For microscopy, pCYP83A1(2 kb 5' regulatory sequences): CYP83A 1(coding sequence)- mVen us47 , pCYP83B1(2 kb 5' regulatory sequences): CYP83B 1 (coding sequence)- mVen us47 , pSUR1(2 b 5' regulatory sequences): SUR1 (coding sequence)- mTurquoise2 and pUMAMIT29 (5kb 5'regulatory sequences): UMAMIT29 (genomic sequence)-mVenus expressing plants were grown under long-day conditions. Different genotypes were grown side by side for metabolite extraction and bio-imaging sample collection at light condition: 16 h, 21°C; darkness: 8 h, 21°C; 55% humidity; light intensity 100-140 μmol m-2 s-1.
Plasmid constructions
The genomic fragment of UMAMIT29 (from 4220bp upstream of the start codon and until stop codon TGA) was amplified from genomic DNA of Arabidopsis Col-0 and cloned into the USER™ cassette that was inserted into the opened pFastRedU- mVenus plant expression vector. SgRNAs were designed using the CRISPR-P 2.0
(http://crispr.hzau.edu.cn/CRISPR2/). The sgRNAs were amplified with expression cassettes using primers containing Bsal recognition sites. The PCR fragments were cloned into pKIR1.1R and digested with Aarl by Golden Gate cloning for multiplex gene editing. The final binary vectors were transformed into Arabidopsis by Agrobacterium- mediated transformation.
Grafting of Arabidopsis inflorescence stem
Grafting of Arabidopsis inflorescence stem was performed as previously described (Nisar et al., 2012) with minor modifications. When the primary inflorescence meristem reached a height of 5 cm above the rosette, plants with floral stems with a similar thickness were selected from more than 30 plants per genotype. Stem with inflorescences (scions) was removed from the rootstock (1-2 cm of the stem left on the stock) by making a horizontal cut and a small droplet of water was immediately placed on the stock stem to prevent any desiccation. The scion was cut horizontally and placed in a petri dish containing water to prevent air from entering the vascular stream. When both scions and stocks were ready, a v-shape wedge was made in the stock using a 0.5 cm long median incision along the length of the stem. Quickly, the scions were cut into a long v-shape wedge under water and were inserted inside the incision of the root stem. The graft junction was secured by a silicone tube and sealed by wrapping with parafilm (1 cm above and below the silicone tubing) to maintain hydraulic turgor and prevent desiccation. The grafted plants were covered with a clear plastic film for 7 days at 70% humidity. After 7 days, the plastic film was removed gradually 20% each day and the plants were watered regularly in order to acclimate to normal growing condition within one week. Seeds for glucosinolate extraction were harvested from senescent siliques from 8-week-old grafted plants.
Labelling flowers for analysis of protein localization and glucosinolates in developing siliques
The flowers with visible white petals were defined as the onset of pollination and were labelled using sewing strings. To quantify glucosinolate content and profile in developing siliques, the siliques were collected since day 5 after pollination. After collection, the siliques were snap frozen with liquid nitrogen and freeze dried before dissection into seeds and siliques without seeds for glucosinolate analysis. For protein localization in developing siliques of SUR1-mTurquoise2 (Goedhart et al., 2012) plants, the developing seeds were exposed by removing one silique valve and then mounting
the seeds in perfluorodecalin (Sigma) for live tissue imaging. For CYP83A1-mVenus and CYP83B1-mVenus plants, 30 μm sections of siliques at mature green stage were generated by vibratome (Leica, Germany) for live tissue imaging. For UMAMIT29- mVenus plants, siliques were fixed and cleared using the ClearSee protocol (Kurihara et al. 2015). Briefly, siliques were fixed in 4% (w/v) para-formaldehyde (Sigma) in PBS (pH 6.7) with 0,1% (v/v) SR2200 cell wall stain (Renaissance Chemicals) under vacuum for 60 min. For young siliques (<16 days after pollination), a slit was cut in both silique valves before fixation to facilitate fixative entry. On older siliques, the valves already showed dehiscence and thus allowed fixative entry. The fixed siliques were washed twice in PBS and transferred to ClearSee solution (10% (w/v) xylitol, 15% (w/v) sodium deoxycholate, 25% (w/v) urea). The siliques were cleared for a minimum of 1 month and the ClearSee solution was exchanged 3 times. On the day of imaging, one silique valve was gently removed and the cleared specimen mounted in ClearSee for imaging. All images were acquired on a Leica SP5-X confocal point scanning laser microscope (Leica microsystems). mTurquoise2 was excited at 458 nm and emission recorded at 468-500 nm, while mVenus was excited at 514 nm and emission recorded at 525-560 nm.
Plant RNA Extraction and Quantitative PCR
Using a spectrum Plant Total RNA Kit (Sigma), total RNA was extracted from pools of 3 siliques at positions 5, 6 and 7 counting from the first silique on the stem of each plant. The extracted RNA was DNase treated using DNasel (AMPDK1-1KT, Sigma-Aldrich). One microgram of RNA was reverse transcribed into cDNA with the iScript cDNA synthesis kit (#1708891, Bio-Rad). To quantify glucosinolate biosynthetic gene expression, qRT-PCR was performed with DyNAmo Flash SYBR Green qPCR Kit (F- 415L, Thermo Scientific) and gene-specific primers. All values were normalized against transcript levels of the ACTIN2 gene (AT3G18780).
Biochemical characterization of UMAMITs in Xenopus laevis oocytes
Linear DNA templates for in vitro transcription were generated from pNB1u plasmid by PCR using Phusion High-Fidelity DNA Polymerase (NEB). PCR products were purified using the QIAquick PCR Purification Kits (Qiagen). Capped cRNA was in vitro synthesized using the mMessage mMachine T7 Kit (Ambion). Concentration of the synthesized cRNA of each transporter gene was normalized to 500 ng μL-1 and
aliquoted before storage in -20 °C. Maximum one thaw cycle of the cRNAs was used for expression in Xenopus laevis oocytes.
Xenopus oocytes were purchased from Ecocyte Bioscience (Germany). Oocytes were injected with 50.6 nL cRNA (500 ng μL'1) using a Drummond NANOJECT II (Drummond scientific company, Broomall 116 Pennsylvania). Injected oocytes were incubated for 3 days at 16 °C in HEPES-based Kulori buffer (90 mM NaCI, 1 mM KOI, 1 mM MgCh, 1 mM CaCh, 5 mM HEPES pH 7.4) supplemented with gentamycin (100 pg mL-1). For the H2O-injected control, 50.6 nL nuclease-free water (Ambion) was injected instead of cRNA.
Uptake assays in Xenopus oocytes were performed as described previously (Jorgensen et al., 2017) with some modifications. Three days after cRNA injection, oocytes were pre-incubated for 5 min in 5 mL Kulori buffer (90 mM NaCI, 1 mM KCI, 1 mM MgCh, 1 mM CaCh, 5 mM MES pH 5.0), then incubated with substrate-containing Kulori buffer for a given time. Oocytes were washed 5 times in Milli-Q Water (20 mL each time) and homogenized with 80% methanol (containing internal standard). Subsequently, oocyte extracts were spun down at 12000 x g for 10 min at 4°C. The supernatant was diluted with water and filtered through a 0.22 μm filter plate (MSGVN2250, Merck Millipore) and analysed by LC-MS/MS as described below.
For export assays, 48 hours after cRNA injection, oocytes were injected with 23 nL of 2Prop (100 mM) or a glucosinolate mixture (concentrations of individual glucosinolates in the mixture are 2mM) to achieve the initial intracellular concentration at approximately 2.2 mM or 50 pM, respectively. After washing once in 5 mL Kulori buffer (pH 7.4) some oocytes were harvested for time 0 samples and the remaining oocytes were incubated in Kulori buffer (pH 7.4) in a 96-well U-bottom microtiter plate (Greiner Bio-One) (3 oocytes per well containing 100 μL buffer). Oocytes and the external medium were separately harvested at different time points. The harvested oocytes were washed 5 times in Milli-Q Water (20 mL each time) and homogenized with 80% methanol. 10 μL of the external medium was sampled for quantification of glucosinolates in the media. The extraction, filtration and analysis by LC-MS/MS were as described below. For both import and export assays, the concentration of glucosinolates and amino acids inside the oocytes was calculated based on an estimated cytosolic oocyte volume of 1 μL.
Two-electrode voltage-clamp electrophysiology
2Prop-induced currents in UMAMIT29-expressing oocytes were recorded using the two-electrode voltage-clamp technique (TEVC) on automated Roboocyte2 (Multichannel Systems, Reutlingen, Germany). Electrodes were backfilled with a mixture of 3 M KCI and 1.5 M Acetate. Electrodes had a resistance of 280 - 1000 kΩ. To test electrogenecity of UMAMIT29-mediated 2Prop import, UMAMIT29-expressing oocytes were clamped at -60 mV membrane potential and oocytes were continuously perfused with MES-based eKulori buffer (2 mM LaCl3, 90 mM NaCI, 1 mM KCI, 1 mM MgCl2, 1 mM CaCl2, and 10 mM MES pH 5.5). Currents were recorded under continuous perfusion in the absence and presence of 10 mM 2Prop.
For current-voltage (l-V) relationship, currents were recorded while clamping the membrane potential of oocytes stepwise from -80 mV to 0 mV in 20 mV increments for 100 msec before and after addition of 10 mM 2Prop.
To verify that 2Prop is imported into UMAMIT29-expressing oocytes during electrophysiology measurements, oocytes were clamped at -60 mV and currents were recorded under continuous perfusion of 10 mM 2Prop containing eKulori buffer (pH 5.5) for 2 min. Subsequently, oocytes were washed three times and homogenized with 50% methanol. Oocyte samples were prepared as described above. The amount of 2Prop in single oocyte was quantified by LC-MS/MS.
For measuring changes in membrane potential in response to changes in a monovalent anion (CI-) and cation (Na+) or in response to addition of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) in Kulori buffer, non-injected oocytes were incubated in the various uptake buffers for 5 min before membrane potential was recorded using the TEVC technique on Roboocyte2 (Multichannel Systems, Reutlingen, Germany).
Analysis of glucosinolates by LC-MS/MS
In general, to achieve the lowest matrix effect and to increase sensitivity for targeted quantification by LC-MS/MS, quantification of glucosinolates alone (i.e. glucosinolates derived from plant material) was carried out in the form of desulfo-glucosinolates. To analyse glucosinolate and/or amino acids in Xenopus oocytes in the transport assays,
intact glucosinolates and glucosinolate-amino acid mixtures were quantified using external standard curves.
For analysis of glucosinolates as desulfo-glucosinolates, chromatography was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Separation was achieved on a Kinetex 1.7u XB-C18 column (100 x 2.1 mm, 1.7 μm, 100 A, Phenomenex, Torrance, CA, USA). Formic acid (0.05%) in water and acetonitrile (supplied with 0.05% formic acid) were employed as mobile phases A and B respectively. The elution profile was: 0-0.5 min, 2% B; 0.5-1.2 min, 2-30% B; 1.2-2.0 min 30-100% B, 2.0-2.5 min 100% B, 2.5-2.6 min, 100-2% B and 2.6-4 min 2% B. The mobile phase flow rate was 400 pl min-1. The column temperature was maintained at 40°C. The liquid chromatography was coupled to an EVOQ Elite TripleQuad mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in positive ionization mode. The instrument parameters were optimized by infusion experiments with pure standards. The ion spray voltage was maintained at +3500 V. Cone temperature was set to 300°C and cone gas to 20 psi. Heated probe temperature was set to 400°C and probe gas flow to 40 psi. Nebulizing gas was set to 60 psi and collision gas to 1.5 mTorr. Nitrogen was used as probe and nebulizing gas and argon as collision gas. Active exhaust was constantly on. Multiple reaction monitoring (MRM) was used to monitor analyte parent ion — > product ion transitions. Detailed values for mass transitions can be found (Jensen et al., 2015; Crocoll et al., 2016a). Both Q1 and Q3 quadrupoles were maintained at unit resolution. Bruker MS Workstation software (Version 8.2.1 , Bruker, Bremen, Germany) was used for data acquisition and processing. Linearity in ionization efficiencies was verified by analysing dilution series, p-hydroxybenzyl or 2Prop glucosinolate were used as internal standard.
For analysis free amino acids in plant material and analysis of intact glucosinolates with or without profiling amino acids, chromatography was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Plant and oocyte extracts were prepared in 85% methanol. The supernatant was collected after centrifugation 12,000 x g for 10 min. An aliquot of the supernatant was mixed 1 :10 with 13C, 15N labelled amino acids (Algal amino acids 13C, 15N, Isotec, Miamisburg, US) at a concentration of 1 μg mL-1. Diluted samples were filtered (Durapore® 0.22μm PVDF filters (Merck Millipore, Tullagreen, Ireland) and used directly for LC-MS analysis. The analysis was performed as previously described (Mirza et al., 2016) with changes as detailed below. Briefly,
chromatography was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Separation was achieved on a Zorbax Eclipse XDB-C18 column (100 x 3.0 mm, 1.8 μm, Agilent Technologies, Germany). Formic acid (0.05%) in water and acetonitrile (supplied with 0.05% formic acid) were employed as mobile phases A and B, respectively. The elution profile was: 0-1.2 min 3% B; 1.2-4.3 min 3-65% B; 4.3-4.4 min 65-100% B; 4.4-4.9 min 100% B, 4.9-5.0 min 100-3% B and 5.0-6.0 min 3% B. Mobile phase flow rate was 500 μL*min-1 and column temperature was maintained at 40°C. The liquid chromatography was coupled to an EVOQ Elite TripleQuad mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ionization source (ESI). Instrument parameters were optimized by infusion experiments with pure standards. The ion spray voltage was maintained at 3000 V in positive ion mode. Cone temperature was set to 300°C and cone gas flow to 20 psi. Heated probe temperature was set to 400°C and probe gas flow set to 50 psi. Nebulizing gas was set to 60 psi and collision gas to 1.6 mTorr. Nitrogen was used as both cone gas and nebulizing gas and argon as collision gas. Multiple reaction monitoring (MRM) was used to monitor analyte molecular ion — > fragment ion transitions: MRMs for amino acids were chosen as described for Arg and Lys. Both Q1 and Q3 quadrupoles were maintained at unit resolution. Bruker MS Workstation software (Version 8.2.1 , Bruker, Bremen, Germany) was used for data acquisition and processing. Individual amino acids in the sample were quantified by the respective 13C, 15N-labelled amino acid internal standard except for tryptophan (which was quantified by 13C, 15N-Phe). Tryptophan was quantified using 13C, 15N-Phe applying a response factor of 0.42, asparagine and glutamine were quantified using 13C, 15N-Asp and 13C, 15N-Glu applying a response factor of 1.0 and 0.36, respectively. Glucosinolate quantification was performed by external dilution series and linearity in ionization efficiencies was verified by analysing dilution series.
Molecular Phylogenetic analysis by Maximum Likelihood method
Protein sequences were retrieved from NCBI Blast using Arabidopsis UMAMIT29 as bait in 14 plant taxa (253 amino acids sequences): Gossypium hirsutum, Theobroma cacao, Carica papaya, Arabidopsis thaliana, Brassica rapa, Glycine max, Manihot esculenta, Solanum lycopersicum, Zea mays, Vitis vinifera, Oryza sativa japonica, Eutrema salsugineum, Capsella rubella, and Citrus Clementina. Sequence alignments were generated using MUSCLE58 with default parameters and were curated with BMGE59. Maximum likelihood analysis was conducted with the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/). The tree with Optimal log-likelihood (-35897.839) is
shown (Figure 9). Bootstrap values are located at each node and were calculated from 1000 replicates. The resulting tree was visualized using iTOL.
Statistical analysis
No statistical methods were used to predetermine sample size. The investigators were not blinded to allocation in experiment and assessment. Data were analysed and plotted using Excel (Microsoft), Origin2021 software (OriginLab), or RStudio. Comparison between two groups were carried out by two-tailed Student’s t test or Mann-Whitney II test depending on the variance homogeneity and the normality of residuals. One-way analysis of variance was used to compare three or more groups, and then a post hoc multiple comparisons test was performed. The P value was calculated by comparing models, and factor level reductions revealed substantial differences across groups. Data are plotted onto the graphs whenever possible, and the number of samples is indicated in the corresponding figure legends.
Results
Source tissue for seed glucosinolates
Lack of knowledge on the site of synthesis of seed-bound glucosinolates has so far hampered the mining for exporter gene candidates using transcriptomes from source tissue. Thus, we first set out to identify the source tissues producing seed-bound glucosinolates. In Arabidopsis, glucosinolates have been detected in phloem sap and externally-applied p-hydroxybenzyl glucosinolate was shown to translocate from rosette leaves or roots to seeds. This suggests that rosette leaves and roots may contribute to seed glucosinolate content. To test this hypothesis, we grafted floral tissue (i.e. upper stem with flowers) of a biosynthetic null mutant myb28 myb29 cyp79b2 cyp79b3 as scion onto wild type vegetative tissue (i.e. lower stem with rosette and roots) as stock. Less than 6% of the glucosinolate levels in seeds from wild type homografts were detected in seeds of the biosynthetic null scion grafted on wild type stock, whereas reciprocal grafts with wild type scion on biosynthetic null stock accumulated as many glucosinolates in the seeds as wild type homografts (Fig. 1a). Additionally, we performed reciprocal grafting with gtr1 gtr2 mutants that previously have shown very low levels of glucosinolates in seeds. Glucosinolates were not detected in seeds from gtr1 gtr2 scion grafted onto wild type stock, whereas reciprocal grafting of wild type floral tissue on gtr1 gtr2 mutant stock accumulated the same glucosinolate level in the seeds as wild type homografts (Fig. 1a). Collectively, this data shows that vegetative
tissues are not the major source for seed glucosinolates in Arabidopsis and that de novo synthesis and transport within reproductive tissues are sufficient to supply the seeds with glucosinolates.
To identify the major site producing seed-bound glucosinolates within the reproductive tissues, we investigated the coordination between the accumulation of seed glucosinolates with the presence of CYP83A1 and CYP83B1 , which represent biosynthesis markers for cells producing the aliphatic methionine-derived and the indolic tryptophan-derived glucosinolates, respectively. A time course experiment on developing Arabidopsis wild type Col-0 seeds showed that glucosinolate accumulation in seeds starts at day 8 after pollination when the embryo enters mature green stage and that seeds continue to accumulate glucosinolates thereafter and throughout seed develoμment (Fig. 4a). Notably, both CYP83A1 and CYP83B1 biosynthetic markers are present in the funiculus at mature green stage (when glucosinolate accumulation begins). This is corroborated by the high expression of the glucosinolate biosynthetic and support genes in the funiculus. Moreover, our analyses show that CYP83A1 accumulates to markedly higher levels than CYP83B1 in the funiculus (Fig. 4c), which corresponds well with aliphatic glucosinolates constituting the majority (>90%) of total seed glucosinolates in Arabidopsis. As the funiculus is the only vascular connection between the silique septum and the seed, it is regarded as part of the long-distance transport highway. Our findings strongly indicate that the funiculus also represents a major hub for production and export of seed-bound glucosinolates. The level of glucosinolates in the silique valves with funiculi and septa (hereafter silique valves) stayed constant from mature green stage until the valves began to senesce (Fig. 4b). This suggests that the silique valves and funiculi continuously produce and export glucosinolates that are destined for the seeds.
UMAMIT29 is key for seed loading
Based on the proposed role of the funiculus in production of seed glucosinolates, we selected genes encoding transmembrane proteins in funiculus with the highest relative expression at mature green stage (Fig. 4d) and measured glucosinolate levels in seeds from the corresponding knockout mutants. Two independent mutant alleles of UMAMIT29 showed an overall 80% reduction in total glucosinolate level in the seeds (Fig. 1b) and no strong reduction in glucosinolate levels in mature silique valves compared to the wild type (Fig. 4e). UMAMIT29 tagged with yellow fluorescent protein
mVenus complemented the mutant phenotype, thus demonstrating that UMAMIT29 is critical for accumulation of glucosinolates in seeds (Fig. 1b).
We analysed the cellular localization in siliques during seed develoμment of mVenus- tagged fusion protein of UMAMIT29 in relation to an mTurquoise2 (mTQ2) fusion protein of the biosynthetic marker enzyme SUPERROOT 1 (SUR1), which is common for both aliphatic and indolic glucosinolates. Confocal Z-stack images show that in developing siliques with the embryo at the mature green stage and onward, SUR1- mTQ2 accumulates to high levels in all cells in the funiculus (Fig. 1c). UMAMIT29- mVenus is present at the plasma membrane in cortex cells and the cells adjacent to the xylem vessel in funiculus as well as in the outer integument and chalazal seed coat at the mature green stage (Fig. 1c, Fig. 4f-h). The co-localization of UMAMIT29 with the common glucosinolate biosynthesis marker SUR1 together with its localization to the plasma membrane in cells bordering the vasculature suggests that UMAMIT29 could function as a glucosinolate exporter in source tissues such as the funiculi.
Glucosinolate-transporting UMAMITs
Next, we biochemically characterized UMAMIT29 in oocytes of Xenopus laevis. At pH 5.5 (that mimics the acidic apoplast), UMAMIT29-expressing oocytes imported 4- methylthiobutyl glucosinolate (4MTB) and indol-3-ylmethyl glucosinolate (I3M), i.e. the major methionine-derived and tryptophan-derived glucosinolates in Arabidopsis seeds, whereas H2O-injected control oocytes did not (Fig. 2a). Two Electrode Voltage Clamp (TEVC) electrophysiology showed that the application of 10 mM of the standard 2Propenyl glucosinolate (2Prop) induced positive currents in UMAMIT29-expressing oocytes clamped at -60 mV and not in H2O-injected oocytes (Fig. 2b). Thus, UMAMIT29-mediated glucosinolate import results in net inward movement of negative charge. As organic anions, glucosinolates carry one negative charge per molecule. Accordingly, we observed that the magnitude of the positive current measured by TEVC is proportional to the amount of 2Prop imported. By quantifying the total amount of 2Prop-induced positive charges and the imported 2Prop molecules in individual UMAMIT29-expressing oocytes over the course of 2 minutes, we estimated the stoichiometry between net charges moved across the membrane and imported 2Prop molecules to be 0.90±0.23 (Fig. 2c), indicating that UMAMIT29-mediated 2Prop import follows a uniport mechanism. Notably, the intracellular concentration of 2Prop imported by UMAMIT29 reached a maximum plateau at approximately 30% of the extracellular
concentration within 60 minutes of incubation (Fig. 2a). This finding supports a uniport transport mechanism in which the import of glucosinolates along its concentration gradient is opposed by the strength of the negative membrane potential of the oocytes.
To further investigate the transport mechanism, we conducted a series of experiments in which the external glucosinolate concentration was kept constant and glucosinolate import was monitored under varying membrane potentials. Firstly, a current-voltage (I- V) curve (-80 to 0 mV) conducted on UMAMIT29-expressing oocytes perfused with 10 mM 2Prop showed an increase in the positive 2Prop-induced currents upon depolarization of the oocyte membrane potential, i.e. a weakening of the opposing negative membrane potential led to an increased influx of glucosinolates. Additionally, we measured increased 2Prop uptake by UMAMIT29 in oocytes, in which the membrane potential was depolarized by exchanging 95% of CI- in the external buffer with gluconate or by the presence of the protonophore carbonyl cyanide m- chlorophenylhydrazone (CCCP) in the uptake medium at pH 5.5 (Fig. 2d). Conversely, we showed reduction in 2Prop uptake in oocytes, in which the membrane potential was hyperpolarized by replacing the cations, i.e. K+ and Na+, in the uptake buffer with either a quaternary amine choline+ or a secondary amine N-methyl-d-glucamine+ (NMDG+) (Fig. 2e). Combined, these results indicate that UMAMIT29-mediated import of monovalent glucosinolate anions follows a uniport mechanism controlled by the electrochemical gradient across the membrane.
To test whether UMAMIT29 can facilitate glucosinolate export, we injected 2Prop directly into UMAMIT29-expressing and control oocytes (intracellular 2Prop concentration ~2 mM). In UMAMIT29-expressing oocytes, the level of intracellular 2Prop decreased over time, which was accompanied by a concomitant increase in 2Prop in the extracellular medium (Fig. 2f), whereas control oocytes showed no export activity. Notably, the export (and import) of 2Prop by UMAMIT29 is enhanced when the external media pH is 5.5 (mimicking the acidic apoplast) compared to 7.4 (Fig. 2d), suggesting a regulatory role of protons on UMAMIT29 activity.
The name of the IIMAMIT family reflects that members previously were characterized as amino acid facilitators. We investigated if UMAMIT29 transports amino acids by exposing UMAMIT29-expressing oocytes to 13C,15N-isotope-labelled glutamine and glutamate - well-established IIMAMIT substrates27 - over a concentration range of 0.4-
10 mM and 2-20 mM for glutamine and glutamate, respectively, thereby generating inward gradients relative to the endogenous amino acid concentration. None of the amino acid gradients resulted in accumulation of detectable levels of isotope-labelled amino acids in oocytes. In export assays, where oocytes were injected with 13C, 15N- isotope-labelled glutamine or glutamate, no export to the media by UMAMIT29- expressing oocytes was detected. Instead, we observed the formation of intracellular isotope-labelled aspartate in both UMAMIT29-expressing and control oocytes accompanied by a strong reduction of the isotope-labelled glutamine and glutamate in the oocytes. This suggests that part of the injected isotope-labelled amino acids are metabolically converted inside the oocytes and therefore unavailable to UMAMIT29. Since we can measure the transport of glucosinolates and not amino acids by UMAMIT29, we investigated if the external amino acids could affect glucosinolate import. We found that up to 1000x excess glutamine (100 mM) did not inhibit 4MTB uptake. In comparison, uptake of 4MTB is completely outcompeted by 10x excess of 2Prop (Fig. 2g, h). The data indicate that UMAMIT29-mediated import and export of glucosinolates are not affected by the presence of glutamine and glutamate at concentrations within the physiological range.
Arabidopsis UMAMIT29 belongs to the IIMAMIT family clade I which consists of seven members: UMAMIT26 to -32. As UMAMIT30 and -31 are the other UMAMIT genes expressed along with UMAMIT29 in funiculus at the mature green stage (Fig. 5), they were screened for import activity in oocytes using 100 pM of an equimolar mixture of
11 aliphatic glucosinolates and one indolic glucosinolate. UMAMIT29- and UMAMIT30- expressing oocytes accumulated both classes of glucosinolates, whereas UMAMIT31- expressing oocytes preferred indolic glucosinolates over aliphatic glucosinolates (Fig. 2i, j). Export activity of UMAMIT29-31 was demonstrated by monitoring glucosinolate release to media five hours after injection of the equimolar mixture of aliphatic and indolic glucosinolates into oocytes (intracellular concentration ~50 μM for each glucosinolate) (Fig.2 k, i). Similar to the substrate preferences observed in the import assay, UMAMIT31 exported indolic glucosinolate more favourably than aliphatic glucosinolates while UMAMIT29 and UMAMIT30 showed broad substrate specificity (Fig. 2k, i). Our finding suggests that not only UMAMIT29 but also UMAMIT30 and UMAMIT31 are glucosinolate uniporters with a capacity to passively facilitate efflux along the electrochemical gradient from cytosol (e.g. of the source cell) to apoplast.
UMAMITs’ role in seed loading
The ability of UMAMIT30 and UMAMIT31 to export glucosinolates in Xenopus oocytes suggests that they may function together with UMAMIT29 to export glucosinolates to seeds. Analysis of available data from the funiculus transcriptome atlas shows that UMAMIT31 is highly and specifically expressed in the funiculus of the developing silique with an embryo at the mature green stage, whereas UMAMIT30 is generally lowly expressed (Fig. 5). In agreement with the pattern of expression and specificity, seeds of single mutants of umamit30 have wild type glucosinolate profile and level whereas umamit31 mutants have a -50% reduction of indolic glucosinolates specifically (Fig. 3a, b). Glucosinolate analysis of seeds of double mutants of the tandemly-linked UMAMIT29 and UMAMIT31, showed that the aliphatic glucosinolate level of umamit29 umamit31 is the same as for umamit29 single mutants (-80% reduction of wild type level), while the level of indolic glucosinolates in seeds of umamit29 umamit31 is reduced to -5% of wild type level (Fig. 3b). The remaining aliphatic glucosinolates in umamit29 umamit31 mutants suggests that a different transporter accounts for glucosinolate transport in the absence of these two IIMAMIT transporters. Notably, in umamit29 umamit31 mutants, UMAMIT30 transcripts are upregulated > 4-fold in the siliques (Fig. 6), suggesting that expression of UMAMIT30 is induced in this mutant in order to transport glucosinolates to seeds in the absence of UMAMIT29 and UMAMIT31. Indeed, the levels of aliphatic glucosinolates in seeds of umamit29 umamit30 double mutants were reduced an additional -50% compared to umamit29 single mutants (Fig. 3a, b, Fig. 7). In seeds of umamit29 umamit30 umamit31 triple mutants, the total glucosinolate level was - 5% of wild type (Fig. 3a, b, Fig. 7). In contrast, the glucosinolate level in the developing silique valves of umamit29 umamit30 mutants was comparable to wild type (Fig. 3d). The levels of total free amino acids in seeds of umamit29 and umamit31 single mutants as well as umamit29 umamit31 double mutants were not significantly different from wild type, whereas seeds of the umamit30 single mutants, umamit29 umamit30 double mutants, and umamit29 umamit30 umamit31 triple mutants displayed an increase in the levels of total free amino acids (Fig. 3e). The seed size and the total seed weight of umamit29 umamit30 double mutants and umamit29 umamit30 umamit31 triple mutants were both reduced, compared to wild type levels (Fig. 3f and g). In conclusion, the substrate preferences of UMAMIT29, -30 and -31, as determined in oocytes, are consistent with the distinct reduction of specific glucosinolates in seeds of the different umamit
mutants, supporting a critical role of these UMAMITs in transport of glucosinolates to seeds.
Next, to evaluate the transport engineering potential of IIMAMIT exporters, we examined the impact of eliminating the three IIMAMIT exporters on accumulation of glucosinolates in other organs i.e. root, rosette leaves, stem and cauline leaves. Roots of single, double or triple mutants of UMAMIT29, -30 and -31 had levels of total glucosinolates comparable to wild type, contrasting the strong reduction of glucosinolates in roots of gtr1 gtr2 gtr3 mutants (Fig. 3 h-k). Similarly, rosette and cauline leaves as well as stems of single and multiple umamit mutants showed levels of total glucosinolates comparable to wild type, whereas strong over-accumulation of glucosinolates was observed in rosette and cauline leaves of gtr1 gtr2 gtr3 mutants (Fig. 3 h-k, Fig. 8). Interestingly, the altered glucosinolate distribution pattern in gtr1 gtr2 gtr3 mutants was fully restored to wild type levels in umamit29 umamit30 gtr1 gtr2 gtr3 quintuple mutants (Fig. 3 h-k, Fig. 8). The in planta data support that UMAMIT29, - 30 and -31 function as exporters of glucosinolates and that they are potential molecular targets for seed-specific elimination of glucosinolates through transport engineering.
Discussion
In this study, we have identified UMAMIT29, UMAMIT30 and UMAMIT31 as glucosinolate exporters with a key role in accumulation of glucosinolates in the seeds as evidenced by less than 6% of wild type level glucosinolates in seeds of umamit29 umamit30 umamit31 exporter mutants. We previously identified the active high-affinity IT-coupled glucosinolate importers (GTR1, GTR2 and GTR3) and showed that elimination of seed glucosinolates in the gtr1 gtr2 gtr3 triple mutants was accompanied by altered accumulation patterns of glucosinolates in the rest of the plant. The suppression of this phenotype in the umamit29 umamit30 gtr1 gtr2 gtr3 quintuple mutant provides genetic evidence that the UMAMITs function as the exporters that deliver glucosinolates to the apoplast.
The biochemical and biophysical characterization of UMAMIT29-31 in oocytes show that glucosinolates are exported passively along an electrochemical gradient. In Arabidopsis, where the plasma membrane potential in physiological condition is within the range of -100 to -150 mV, UMAMIT29-31 is expected to facilitate efficient export of the monovalent anionic glucosinolates out of biosynthetic cells to the acidic apoplast. A recent theory-based study hypothesized that the mechanism for nutrient homeostasis
aimed at reaching a desired cytosolic concentration of a given nutrient, requires that at least two differently energized transporter types are involved. The presence of passive IIMAMIT exporters and IT-coupled GTR importers fulfils this requirement. Accordingly, the H+-coupled high-affinity GTR importers empty the apoplast for glucosinolates generating a strong outward chemical gradient (from cytosol to apoplast) which, together with the strong negative membrane potential, promotes efficient export of glucosinolates via the passive uniport mechanism of UMAMIT29-31. Interestingly, the glucosinolate exporters as well as the exporters of sugars (Sugars Will Eventually be Exported Transporters - SWEETs) and amino acids (UMAMITs) are all uniporters, which suggests the presence of a universal passive uniport mechanism for exporting compounds from symplasm in source tissues.
UMAMIT29-31 belong to UMAMIT clade I proteins (UMAMIT26-32) of which UMAMIT26-31 are brassicaceous-specific (Fig. 9). This highlights the potential of transporter families involved in primary metabolism to evolve substrate specificity towards specialized metabolites as recently shown for the NPF family. Analogously, the identification of UMAMITs as exporters of glucosinolates suggests that the UMAMIT family may represent a new family of exporters of specialized metabolites. Interestingly, the substrate specificity of the three UMAMITs exporters, with UMAMIT29 and -30 having broad substrate specificity and UMAMIT31 being specific for indolic glucosinolates, is mirrored in the three GTR importers with GTR1 and GTR2 having broad substrate specificity and GTR3 favouring indolic glucosinolates, indicating co- evolution of substrate preferences across these distinct transporter families. Previous studies on amino acid export activity of UMAMITs using Xenopus oocytes and Saccharomyces cerevisiae cells have reported different outcomes, which may reflect difficulties in measuring transport activity of primary metabolites that are concomitantly being metabolized by the heterologous host expressing the transporter. The minor alteration of free amino acid levels in seeds of various umamit mutants and myb28 myb29 (MYB28 and -29 are master regulators of aliphatic glucosinolates), as well as gtr 1 gtr2 mutants41 , merits future studies to investigate the relation between glucosinolates and free amino acids in seeds.
Based on the overlapping presence of UMAMIT29, -30 and -31 with glucosinolate biosynthetic marker enzymes in the funiculus at the mature green stage, we propose that the funiculus acts as a major glucosinolate source tissue from where UMAMIT29, - 30 and -31 export de novo synthesized seed-bound glucosinolates into the apoplast, and that the secondarily activated high-affinity IT-coupled GTRs subsequently import
them into the phloem via which the glucosinolates are translocated to the seeds. Generally, understanding the seed loading process has been hampered due to lack of tools as transporter mutants of primary metabolites such as sucrose and amino acids - for which importers and exporters are already known - often have pleiotropic effects. The latter is typically not the case for specialized metabolite transporters. The glucosinolate IIMAMIT exporters, along with the GTR importers, are essential for seed accumulation and provide powerful molecular tools to dissect the process of seed loading to the embryo.
Transport engineering that uses altered expression of transporter genes to change metabolite distribution patterns, has resulted in the develoμment of improved agronomic traits (Moore et al., 2015; Krattinger et al., 2016; Kim et al., 2021). Here, we show that umamit29 umamit30 umamit31 exporter mutants do not alter the overall distribution of glucosinolates in the plant, which is in contrast to either reduction (roots, stems) or strong over-accumulation (rosette, cauline leaves, silique valves) in the gtr1 gtr2 gtr3 glucosinolate importer mutants. Hence, from an agricultural perspective, IIMAMIT glucosinolate exporters have high biotechnological potential as molecular breeding targets for eliminating anti-nutritional glucosinolates in seed cake of brassicaceous oilseed crops through transport engineering without impacting glucosinolate defence in the rest of the plant.
Example 2 - Glucosinolate transport activity of Brassica napus UMAMIT transporters Seeds of the model plant A. thaliana and of Brassica crops accumulate glucosinolates that are produced and exported from maternal tissues. In Arabidopsis, AtUMAMIT28, AtUMAMIT29, AtUMAMIT30 and AtUMAMIT31 from the Clade I UMAMIT family are characterized bidirectional glucosinolate facilitators. Recent data showed that AtUMAMIT29, AtUMAMIT30 and AtUMAMIT31 are essential for the accumulation of glucosinolates in seeds, with a main role for AtUMAMIT29. We selected AtUMAMIT28, AtUMAMIT29, AtUMAMIT30 and AtUMAMIT31 glucosinolate facilitators as a primary target for translating the umamit loss-of-function phenotype from Arabidopsis to Brassica crops.
The initial work focused on reducing glucosinolate content in seeds of Brassica napus (B. napus, AACC, 2n=38). B. napus is an allotetraploid species that is the result of the natural hybridization between two diploid progenitors B. rapa (AA, 2n=20) and B.
oleracea. (CC, 2n=18) -7500 years ago. During evolution, domestication and breeding practices, genome duplication, chromosomal rearrangement and deletion events resulted in big variation of copy number and gene structure among B. napus cultivars, including the UMAMITs orthologs.
Material and methods
Identification of Arabidopsis UMAMIT Clade I orthologs.
The BnPIR: Brassica napus pan-genome information resource (http:// http://cbi.hzau.edu.cn/bnapus/) was queried using the AtUMAMIT26-32 amino acids as queries to identify UMAMIT orthologs in B. napus.
Brassica UMAMIT nomenclature
Brassica UMAMIT orthologs were named according to sequence identity to the respective Arabidopsis orthologs and to their subgenomic association as follows: Bn indicate the plant species B. napus; UMAMIT26-32 indicate whether the gene is an ortholog of which Arabidopsis UMAMIT gene. A and C indicate the subgenomic location according to the U triangle (A indicates location in the AA genome originating from B. rapa and C indicates location in the CC genome originating from B. oleracea). 1 , 2, 3 and 4 indicate amino acid sequence homology to the Arabidopsis ortholog. Genes were numbered according to decreasing amino acid sequence homology, with 1 indicating the highest level of identity.
Phylogenetic tree
Sequences were aligned using CLASWX with a gap open penalty of -2.9, gap extend of 0 and hydrophobicity multiplier of 1.2. Poorly aligned regions were trimmed manually. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model. The tree with the highest log likelihood (- 9060,10) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1,5532)). The rate variation model allowed for some sites to be evolutionarily invariable ([+l], 0,84%
sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA X.
Cloning
Total RNA was extracted from different tissues of B. napus at different develoμmental stages and used for cDNA synthesis using the Superscript III First-Strand Synthesis kit (Invitrogen) using oligo(dT) primers. Full-length cDNA sequences of the 11 BnUMAMIT genes were amplified by PCR from the cDNA; due to the high sequence similarity, the primers were placed in the more-varying UTR regions. The PCR fragments were cloned into pNB1u, and verified by sequencing.
Expression in Xenopus laevis oocytes
Linear DNA templates for in vitro transcription were generated from pNB1u plasmid by PCR using Phusion High-Fidelity DNA Polymerase (NEB), according to the manufacturer’s instructions. PCR products were purified using the QIAquick PCR Purification Kits (Qiagen). Capped cRNA was in vitro synthesized using the mMessage mMachine T7 Kit (Ambion). Concentration of the synthesized cRNA of each transporter genes was normalized to 500 ng/pl. and were aliquotd before storage in -20C. Maxium one thaw cycle of the cRNAs was used for expression in Xenopus oocytes.
Xenopus oocytes were purchased from Ecocyte Bioscience (Germany). Oocytes were injected with 50.6 nL cRNA using a Drummond NANOJECT II (Drummond scientific company, Broomall 116 Pennsylvania). cRNA (500 ng/pl) were injected into oocytes. Injected oocytes were incubated for three days at 16 °C in HEPES-based kulori buffer (90 mM NaCI, 1mM KCI, 1 mM MgCI2, 1 mM CaCI2, 5 mM HEPES pH 7.4) supplemented with gentamycin (100 pg/mL). For mock-injected control, 50.6 nL nuclease-free water (Ambion) was injected instead of cRNA.
Uptake assay in Xenopus oocytes was performed essentially as described previously with some modifications. Three days after cRNA injection, oocytes were pre-incubated for 5 min in 5 ml kulori buffer (90 mM NaCI, 1 mM KCI, 1 mM MgCI2, 1 mM CaCI2, 5 mM MES pH 5.0), then incubated kulori buffer containing a substrates (100pM glucosinolates) for a given time. Oocytes were washed 5 times in Milli-Q Water (20 ml each time) and homogenized with 80 % methanol (containing internal standard).
Subsequently, oocyte extracts were spun down at 12000g for 10 min at 4 °C. The
supernatant was diluted with water and filtered through a 0.22 μm filter plate 131 (MSGVN2250, Merck Millipore) and analyzed by LC-MS/MS as described below.
Desulfo-glucosinolate analysis by LC-MS/TripleQuad
Samples were 10-fold diluted with deionized water and subjected to analysis by liquid chromatography coupled to mass spectrometry. Chromatography was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Separation was achieved on a Kinetex 1.7u XB-C18 column (100 x 2.1 mm, 1.7 μm, 100 A, Phenomenex, Torrance, CA, USA). Formic acid (0.05%) in water and acetonitrile (supplied with 0.05% formic acid) were employed as mobile phases A and B respectively. The elution profile was: 0-0.5 min, 2% B; 0.5-1.2 min, 2-30% B; 1.2-2.0 min 30-100% B, 2.0-2.5 min 100% B, 2.5-2.6 min, 100-2% B and 2.6-4 min 2% B. The mobile phase flow rate was 400 μl min-1. The column temperature was maintained at 40°C. The liquid chromatography was coupled to an EVOQ Elite TripleQuad mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in positive ionization mode. The instrument parameters were optimized by infusion experiments with pure standards. The ion spray voltage was maintained at +3500 V. Cone temperature was set to 300°C and cone gas to 20. Heated probe temperature was set to 400°C and probe gas flow to 40 psi. Nebulizing gas was set to 60 psi and collision gas to 1.5 mTorr. Nitrogen was used as probe and nebulizing gas and argon as collision gas. Active exhaust was constantly on. Multiple reaction monitoring (MRM) was used to monitor analyte parent ion — > product ion transitions. Detailed values for mass transitions can be found in (Jensen et al., 2015; Crocoll et al., 2016a). Both Q1 and Q3 quadrupoles were maintained at unit resolution. Bruker MS Workstation software (Version 8.2.1 , Bruker, Bremen, Germany) was used for data acquisition and processing. Linearity in ionization efficiencies were verified by analyzing dilution series, p-hydroxybenzyl or 2-propenyl glucosinolate were used as internal standard.
Results
Phylogenic analysis of the sequences from the well-assembled ZS11 reference genome (Song et al., 2020) indicate it encodes four UMAMIT28, four UMAMIT29, eleven AtUMAMIT30 and six AtUMAMIT31 orthologs (Fig. 10). We could not assign Arabidopsis orthologs for BnaA09G0692800ZS, BnaA09G0714300ZS, BnaC02G0337200ZS and BnaA02G0251300ZS, which were grouped at the outer layer within UMAMIT26-29 subclade and were distinguished from the UMAMIT30 and the
UMAMIT31 subclade. We did not identify homologs of AtUMAMIT26 and AtUMAMIT27 in Brassica napus.
Among the 27 IIMAMIT in ZS11 , four anotated genes (BnaC02G0337200ZS, BnaA02G0251300ZS, Bnacaffold0025G0022000ZS and Bnascaffold0025G0022100ZS) were excluded in the further analysis since they have mutations that cause the presence of early stop codons resulting in very short proteins.
To focus on the BnallMAMIT-expressing genes most likely to function in seed loading of glucosinolates, we analyzed the expression of the 23 IIMAMIT orthologs in seeds and siliques ZS11 cultivar by mining transcriptomic data in Brassica EDB (https://brassica.biodb.org/) (Fig. 11).
Transcript analyses for the expression of each UMAMIT-expressing gene in different tissues and develoμmental stages showed that BnllMAMIT28A1, BnllMAMIT28C1, BnllMAMIT30A2, BnllMAMIT30C2 were among the highest expression in silique walls at both podding and maturation stage, while BnllmamiT30C1, BnllmamiT28C1 and BnllmamiT28A2 were highly expressed in seeds (Fig. 11). In contrast, almost no transcripts corresponding to (BnllmamiT29C1 , BnllmamiT29A1 , BnllmamiT30C5, BnllmamiT30A3, BnllmamiT30C6, BnllmamiT30C4, BnllmamiT30A1, BnllmamiT30A4, BnllmamiT30C3, BnllmamiT30A5, BnllmamiT31C2, BnllmamiT31C1 , BnllmamiT31A1 , BnllmamiT31A2, BnllmamiT28A3, BnllmamiT32A2, BnllmamiT32C3, BnllmamiT32C1, BnllmamiT32A1, BnllmamiT32C2, BnllmamiT32A3) and were detected in seeds and silique walls.
Based on the above in silico analysis of IIMAMIT expression in the ZS11 cultivar, we embarked to clone the silique and/or seed enriched UMAMITs and test their glucosinolate transport activities. In a high glucosinolates 0 variety Niklas, we isolated cDNA from leave, stem, young silique, old silique and mature silique as well as dry seeds. Eleven IIMAMIT orthologs coding sequence were cloned by gene specific primers (Fig. 12A). Five of them (BnUMAMIT28C1N, BnUMAMIT30A2N, BnllMAMIT30C2N, BnllmamiT30C1N and BnllmamiT28A2N) are paralogs of ZS11 genes that shows highest expression in reproductive organs (silique wall and seeds) (Fig. 11). We did not detect the transcript of paralogs of the siliques-expressing
BnllMAMIT28A1ZS in Niklas, instead, the BnllMAMIT28A3N were identified which transcripts are not shown in ZS11 database.
Next, we tested glucosinolate transport activity of the Niklas orthologs by heterologous expression in Xenopus oocytes. Interestingly, out of eleven cloned IIMAMIT genes, the six UMAMITs (BnUMAMIT28A3N, BnUMAMIT28C1 N, BnUMAMIT30A2N, BnllMAMIT30C2N, BnllmamiT30C1N and BnllmamiT28A2N) that are highly expressed in silique and/or seed in both Niklas and ZS11 import glucosinolates into oocytes with different activity and substrate specificity (Fig. 12B), the other five orthologs did not show import of glucosinolates. Among the six functional transporters, BnllmamiT28A2N, BnllmamiT28A3N, and BnllmamiT30A2N prefer to import 4MTB and I3M rather than progoitrin 2OH-3-but in an equal molar mixture. In contrast, BnllmamiT28C1N import all glucosinolate (progoitrin 4MTB, I3M) into oocytes without particular preference. The six BnllmamiT glucosinolate transporters expressed in reproductive tissues were the targets for mutagenesis with BnllmamiT28C1 N as the key target, since 2OH-3-but progoitrin is the main antinutritional glucosinolates in Niklas seeds.
Example 3 - Reduction of glucosinolate levels in Brassica napus seeds by mutation of UMAMIT glucosinolate exporters
We will generate multiple umamit mutants in 0 variety Brassica napus Niklas through DNA-free genome-editing technology that targeting all the six UMAMIT orthologs (i.e. BnUMAMIT28A3N, BnUMAMIT28C1 N, BnUMAMIT30A2N, BnUMAMIT30C2N, BnUmamiT30C1 N and BnUmamiT28A2N). Alternatively, we will use EMS mediated mutagenesis to create a TILLING population in Niklas and screen for single loss-of- function mutants of the individual orthologs. Multiple mutants will be generated through crossing and stacking of mutations through generations.
Step 1 : Screening for high-efficient gRNAs targeting UMAMIT genes with RNP complexes in protoplasts
Multiple candidate gRNAs will be designed to target regions conserved across all six UMAMIT orthologs for the introduction of indel mutations, and tested together with Cas9 for their efficiency via PEG-mediated transformation of RNP complexes into protoplasts. gRNA will be prepared from in vitro transcription and Cas9 proteins will be purified from cell extract of Cas9-expressing E. coli strains (March et al., 1989). We will
prepare protoplasts from 14-day-old cotyledons that have high regeneration ability. 200 μL containing 2 x 105/mL protoplasts will be harvested and transfected with gRNA and Cas9 protein as well as Lipofectamine™ 3000 and Plus reagent™ transfection reagents together with polyethylene glycol (PEG) 4000. We will evaluate the efficiency of gRNA using the Indel Detection by Amplicon Analysis (IDAA) method that is based on analysis of fluorescence-labelled PCR products using capillary electrophoresis.
Step 2: Regeneration of UMAMIT mutant lines from protoplasts
The efficiency of tissue regeneration from genome-edited protoplasts is cultivar- dependent and relatively low. Thus, we will compare and test multiple existing protocols for regeneration of whole plants from transfected protoplasts of rapeseed, to develop a method for the Niklas cultivar. The most efficient gRNAs targeting UMAMIT orthologs (from Step 2) will together with Cas9 in RNP complexes be transformed into rapeseed protoplasts. Calluses will be regenerated from individual protoplasts through hormone induction containing 2,4-D, BAP and NAA, followed by NAA, GA3 and 2iP treatment to induce shoots and then roots. Massive screening of seedlings will be carried out to identify successfully genome-edited UMAMIT orthologs using the IDAA method (see above) using the gene-specific primers generated in Step 1 . The genome- editing will be confirmed via Sanger sequencing. The genome-edited UMAMIT mutant seedlings will be transferred to soil and grow in climate chambers with 16 hours light/8 hours dark at 22°C until seeds are mature. We will analyze the level and profile of glucosinolates in the obtained homozygous UMAMIT mutant lines to evaluate the result of the genome-editing.
Example 4 - Identification of key amino acid residues in AtUMAMIT29 fortransport of glucosinolates
Materials and Methods
Mining amino acid sequences of UMAMIT homologs
The amino acid sequences of UMAMIT clade I (UMAMIT26-32) were obtained from TAIR (https://www.arabidopsis.org) for Arabidopsis thaliana, BnPIR (http://cbi.hzau.edu.cn/bnapus/index.php) (Song et al., 2020) for Brassica napus ZS11 and Genoscope ((Belser et al., 2018), https://www.genoscope.cns.fr/externe/plants/index.html) for Brassica rapa Z1 and Brassica oleracea HDEM, phytozome 13 (https://phytozome-next.jgi.doe.gov) for
Manihot esculenta, NCBI for Physcomitrella patens. Sequences of the other plant species were adopted from (Zhao et al., 2021), except for MaCap/01 (NCBI ID: XP_023632698.1), MaCar/15 (NCBI ID: XP_021905388.1) and MaCar/16 (NCBI ID: XP_021887563.1), which were retrieved from NCBI.
Multiple sequence alignment and phylogenetic analysis
Sequences from 27 plant species were aligned in MEGA X (https://www.megasoftware.net/)(Kumar et al., 2018) using MUSCLE with default settings (Edgar, 2004). Sequences with indels within any helices were removed, with the exceptions of AsSol/42, FaMed/43 and the root. The final alignment contained 97 sequences. Sequence logos were made using JDet (http://csbg.cnb.csic.es/JDet/) (Muth et al., 2012). Phylogenetic trees were generated in MEGAX using the neighbour- joining method (1000 bootstraps) and annotated in iTOL (Letunic and Bork, 2021).
Estimation of differentially conserved amino acids using Diverge 3.0 beta 1 DIVERGE 3.0 beta 1 (from now on referred to as DIVERGE) was used to determine differentially conserved amino acids (https://github.com/xungulab/diverge) (Gu et al., 2013). The multiple sequence alignment with 97 sequences was loaded into the program as well as its corresponding neighbour-joining phylogenetic tree (see above). The estimation of cluster-specific functional divergence (corresponding to the amount of differential conservation), was calculated based on the algorithm in Gu et al., (2013) and the final scores were listed in Table 5, below.
Table 5 - The amino acid residues predicted to constitute the substrate transporting cavity of Arabidopsis UMAMIT29 and UMAMIT32.
0000 0 0 9 s 992
Residues in bold indicate each of the 11 differentially conserved amino acids identified in the in silico analyses. NA: Not available. * residues with a score 0 that has identical residues among UT30/31 and UT32.
Protein modelling and analysis
Structures of the Arabidopsis UMAMIT29 (Uniprot ID: Q9M131) and AtUMAMIT32 (Uniprot ID: Q9LI65) were modelled using RaptorX from (http://raptorx.uchicago.edu/ContactMap/)(Ma et al., 2015; Wang et al., 2017; Wang et al., 2016) and AlphaFold (https://alphafold.ebi. ac.uk/)(Senior et al., 2020; Jumper et al., 2021). Protein models were depicted by The PyMOL Molecular Graphics System, Version 2.4 Schrodinger, LLC (https ://pymol.org/2/). The residues constituting the active site of UMAMIT29 and UMAMIT32, respectively, were determined using the CAVER webtool v1.0 (https://loschmidt.chemi.muni.cz/caverweb/) (Stourac et al., 2019) followed by manual inspection and minor revisions in PyMOL 2.4.
Generation of UMAMIT29 mutant variants
DNA fragments of UT29_UT32-11 i.e., UMAMIT29 with the 11 differentially conserved residues from UMAMIT32, and UT29_clade1-3cons i.e., the UMAMIT29 with the 3 UMAMIT-conserved residues mutated into alanine were ordered from Twist
Bioscience. UMAMIT29 single residue mutant variants were generated through USER cloning. Linear DNA template for in vitro transcription was obtained from PCR amplification of the pNB1u plasmids using Phusion High-Fidelity DNA polymerase (NEB) and PCR product purified using the E.Z.N.A® Cycle Pure Kit (Omega Bio-tek). Template DNA was in vitro transcribed using the mMessage mMachine™ T7 transcription kit (InVitrogen). The RNA transcripts (~600 ng/μL) were aliquoted into 10 μL per tube and kept at -18 °C until use.
Measurement of transport activity in Xenopus oocytes
Transport assays using Xenopus oocytes were described in Xu et al., (2016). Briefly, the defolliculated Xenopus laevis oocytes (stage V or VI) were ordered from Ecocyte Bioscience and Department of Drug Design and Pharmacology, University of Copenhagen. Oocytes were injected with 50 nL RNA (~600 ng/μL) using a Nanoject II (Drummond Scientific Company). For the mock (negative control) oocytes were injected with 50 nL sterilized Milli-Q® H2O. The injected oocytes were used for assaying after three days of incubation at 16 °C in Kulori buffer pH 7.4 (5 mM MES, 90 mM NaCI, 1 mM KCI, 1 mM CaCI2, 1 mM MgCl2) supplemented with gentamicin (100 pg/mL).
The assays were performed as follows: Oocytes were first pre-incubated in Kulori buffer pH 5 (5 mM MES, 90 mM NaCI, 1 mM KCI, 1 mM CaCI2, 1 mM MgCh) without substrates for 5 min. Then oocytes were incubated for one hour in Kulori buffer pH 5 (5 mM MES, 90 mM NaCI, 1 mM KCI, 1 mM CaCI2, 1 mM MgCI2) with added 4- methylthiobutyl glucosinolate (4MTB), indol-3-ylmethyl glucosinolate (I3M) and benzyl glucosinolate (BGLS) (200 pM of each). After 1 hour, oocytes were washed in five petri dishes containing Milli-Q® H2O. From the final petri dish, oocytes were divided into Eppendorf tubes® with three oocytes in each. Residual H2O was removed from each of the tubes and the oocytes homogenized in 50 μL 50% containing an internal standard of 1.25 pM sinigrin (62.5 μmol per sample). The tubes were placed at -18 °C overnight.
Sample preparation for LC/MS analysis
Glucosinolates were extracted and quantified as desulfo-glucosinolates as previously described (Crocoll et al., 2016b). Briefly, a 96 well filter plate (0.45 pM) was filled with 45 μL DEAE-Sephadex A-25 using a MultiScreen Column Loader (Merck Millipore). 300 μL H2O was added to each well and the plate incubated 3-4 hours at room temperature or overnight in fridge. Excess H2O was removed by applying 2-4 s of vacuum using a vacuum manifold. After centrifugation for >15 min at >20.000x g at 4 °C and placed on ice, all supernatant was added to the DEAE-sephadex and vacuum was applied for 2-4 s. The wells were then washed two times with 100 μL 70% methanol and two times with 100 μL H2O using 2-4 s of vacuum. In the final washing step, the plate was spun shortly at 5900 RPM. 20 μL sulphatase were added to each well and
the plate incubated overnight at room temperature. Desulfo-glucosinolates were eluted with 90 μL of H2O at 5900 RPM. The plate was kept at -18 °C until LC/MS analysis.
Data analysis and statistics
Plots and statistics were made with R studio (version 2021.09.0+351). Statistics were done by first perform a one-way ANOVA test of the compound to the RNA followed by a Tukey Honest Significant Differences (Tukey HSD) test for a multiple pairwise comparison of the mean for each of the RNAs (P<0.05).
Results
Substrate transporting cavity of UMAMIT29
As there is yet no experimentally determined structure available for any member of the IIMAMIT family, we generated a model of UMAMIT29 in an apparent occluded conformation by the ab initio protein modelling tool RaptorX (Ma et al., 2015; Wang et al., 2016; Wang et al., 2017). Based on our model, we proposed a substrate transporting cavity of UMAMIT29 defined by helix 1-4 and 6-9 and selected 51 residues as putative substrate binding sites based on solvent accessibility. We hypothesized that the transport cavity contains highly conserved residues that are key for the transport activity. To test this, we aligned 97 protein sequences consisting of homologs of IIMAMIT Clade I from 27 plant species and created sequence logos containing the 51 residues identified in the structural analysis. Four residues within the binding cavity - R44, G82, W200, Q204 - are 100% conserved among all the sequences in the multiple sequence alignment (Figure 13). This suggests that these residues are crucial for functional or structural features of the transporter proteins. When mutating, respectively, R44, W200 and Q204 into alanine in UMAMIT29 (UMAMIT29#3CON), import activity of aliphatic 4-methylthiobutyl glucosinolate (4MTB) and benzyl glucosinolate (BGLS) was reduced by over 80% and indol-3-ylmethyl glucosinolate (I3M) was reduced by approx. 50% (Figure 14). Interestingly, altering only R44 (UMAMIT29#R44A) reduced all tested glucosinolate transport activity by 88-98% (Figure 14). Identification of essential residues for glucosinolate transport activity within the predicted substrate transporting cavity supports our model.
Key residues for glucosinolate transport activity of UMAMIT29
Phylogenetic analysis of the above 97 sequences shows that the homologs of brassicaceous-specific, glucosinolate-transporting UMAMITs and non-brassicaceous-
specific, non-glucosinolate-transporting UMAMITs fall into two different clusters. We hypothesized that differentially conserved residues located in the binding cavity of the proteins within the two clusters may reveal specificity-determining positions.
Analysis of the sequence logos of the 51 predicted substrate transporting cavity residues between the two clusters identify 13 differentially conserved residues (Figure 15; Table 5). Two amino acid residues amongst the 13 positions were identical between the non-glucosinolate-transporting UMAMIT32 and the glucosinolate- transporting UMAMIT30 and -31 and were therefore not considered further.
Subsequently, we set out to examine the contributions of the 11 differentially conserved residues to glucosinolate transport activity by generating 11 UMAMIT29 mutant variants in which each of the 11 residues was individually changed into the corresponding residue from UMAMIT32. The glucosinolate substrate specificity of the mutant variants were compared to UMAMIT29 that shows broad substrate specificity towards methionine-derived aliphatic glucosinolates and tryptophan-derived indolic glucosinolates. The mutant variants were expressed in Xenopus laevis oocytes and tested for import of a mixture of three glucosinolates, the aliphatic 4-methylthiobutyl glucosinolate (4MTB), indol-3-ylmethyl glucosinolate (I3M) and benzyl glucosinolate (BGLS). Eight of the 11 mutant variants showed reduced transport activity to all three glucosinolates (see Table 6, below).
Table 6 - Glucosinolate import activity of mutant variants of UMAMIT29.
UT: UMAMIT; 4MTB: 4-methylthiobutyl glucosinolate; I3M: indole 3-ylmethyl glucosinolate; BGLS: benzyl glucosinolate
Among the eight mutant variants, UMAMIT29#V27F, -M86V, -L109V, Q263S and - T267Y showed the most reduced activity with ~75-97% less total glucosinolate import than the wildtype UMAMIT29 when expressed in the oocytes (Table 6).
Although all these mutant variants have big reductions in total glucosinolate import activity, UMAMIT29#M86V, -L109V, -Q263S and -T267Y locate in near proximity in the centre of the cavity, whereas UMAMIT29#V27F locates further distally at the end of helix I in the computed protein model.
UMAMIT29#L197W showed reduction import of 4MTB and BGLS, compared to the wildtype, and the level of imported I3M was not affected. UMAMIT29#S289I showed reduction of I3M and BGLS import and the level of imported 4MTB is similar to wildtype (Table 6). UMAMIT29#M201F showed - as the only mutant variant - no reduction of any glucosinolates, but an increase of 4MTB (Table 6)
As we only exchange a single amino acid residue in UMAMIT29 with the corresponding amino acid residue in the homolog UMAMIT32, we anticipate that the UMAMIT29 mutant variants are being functionally expressed. This is supported by the observation that variant UMAMIT29#S289I shows specific increase of 4MTB import and over 40% reduction of import of the I3M and BGLS, and that variant UMAMIT29#L197W imports less 4MTB but similar level of I3M and BGLS, but more I3M compared to wild type. The alteration of glucosinolate substrate specificity of these UMAMIT29 mutant variants suggests that these residues are critical for glucosinolate binding.
Items
1. A Brassicales plant, or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
2. The Brassicales plant or part thereof according to item 1 , wherein said mutant IIMAMIT transporter is a mutant glucosinolate transporter with reduced glucosinolate transporter activity compared to the wildtype protein, and wherein said reduction is a reduction of at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as 100% of the glucosinolate transporter activity of said mutant glucosinolate transporter compared to the wildtype protein.
3. The Brassicales plant or part thereof according to any one of the preceding items, wherein said mutated gene has reduced expression of the encoded glucosinolate transporter compared to the wildtype gene, and wherein said reduced expression is a reduction of at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% in expression of said mutated gene compared to the wildtype gene.
4. The Brassicales plant or part thereof according to any one of the preceding items, wherein the seeds of said Brassicales plant or part thereof have a reduced
glucosinolate content compared to a Brassicales plant or part thereof of otherwise identical genotype except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions, and wherein said reduction in glucosinolate content is a reduction of at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% or such as at least 95%.
5. The Brassicales plant or part thereof according to any one of the preceding items, wherein the vegetative tissue of said Brassicales plant or part thereof have about the same glucosinolate content compared to a Brassicales plant or part thereof of otherwise identical genotype except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
6. The Brassicales plant or part thereof according to any one of the preceding items, wherein the mutation is selected from the group consisting of a missense mutation, a frameshift mutation, an insertion and a deletion.
7. The Brassicales plant or part thereof according to any one of the preceding items, wherein said plant is of the genus Brassica or the genus Lepidium, such as wherein said plant is a B. Juncea, B. napus (rape), B. carinata, B. oleracea, B. rapa, or L. campestre plant.
8. The Brassicales plant or part thereof according to any one of the preceding items, wherein said part is a seed.
9. The Brassicales plant or part thereof according to item 8, wherein the seed has a concentration of glucosinolates of less than 18 micromoles per gram of dry weight of said seed, such as less than 17 micromoles per gram of dry weight, such less than 16 micromoles per gram of dry weight, such as less than 15 micromoles per gram of dry weight, such less than most 14 micromoles per gram of dry weight, such as less than 13 micromoles per gram of dry weight, such as less than 12 micromoles per gram of dry weight, such as less than 11 micromoles per gram of dry weight, such as less than 10 micromoles per gram of dry weight, such as less than 9 micromoles per gram of dry weight, such as less than 8 micromoles per
gram of dry weight, such as less than 7 micromoles per gram of dry weight, such as less than 6 micromoles per gram of dry weight, such as less than 5 micromoles per gram of dry weight, such as less than 4 micromoles per gram of dry weight, such as less than 3 micromoles per gram of dry weight, such as less than 2 micromoles per gram of dry weight, such as less than 1 micromoles per gram of dry weight of said seed.
10. A plant product comprising a Brassicales plant or part thereof, or prepared from seeds of said Brassicales plant or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
11. The plant product according to item 10, wherein the plant product is prepared from a seed, such as wherein the plant product is an oil, a seed cake or a seed meal.
12. A seed cake prepared from a seed from a Brassicales plant, wherein the Brassicales plant is according to any one of items 1 to 9.
13. A method for modifying glucosinolate content in a Brassicales plant or part thereof, said method comprising a step of modifying the functional activity or expression of at least one IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in
SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto. The method according to item 13, wherein said step of modifying said functional activity of said at least one of IIMAMIT transporter is performed by nuclease-based gene editing, such as by CRISPR/Cas9 gene editing, by random mutagenesis, by gene targeting, by transposition mutagenesis, by transfer-DNA induced insertion, or by gene knockdown, such as by RNA interference. A method for producing a plant product comprising a Brassicales plant or part thereof with low glucosinolate content, said method comprising the steps of a. providing a Brassicales plant or part thereof according to any one of items 1 to 9; and b. processing said Brassicales plant or part thereof into a plant product, such as a seed oil or a seed cake.
Sequence overview
07
09
11
13
15
16
18
0
A
C
T
C
T
T
T
G
T
C
G
A
A
T
A
G
T
A
A
T
T
A
T
A
T
G
A
G
T
A
T
T
G
T
A
1
G G A A T T A A T A A A C G A A T C A C T C T A C T G A A T T A T A T
2
T T A T T T G C A T T A T G A T T A A T T C T T C A A A T T T T G G T
3
C T T A A A A T G C C T A G T A A G T A A A T T A A G A T C A C T T A
4
T C T A T A A T A T T T T T A A T T G G T A G A C G G A C A T T T T T
6
A T A C G C T C G T T G A T T A C A A G T A A T T C A A C A T A T C G
7
A
T
A
C
T
A
G
G
T
T
A A A A T A A A A T T A A T C C T A A T C A G A T
9
C A T T A A G A T T G C A T C C A T T G
31
32
33
35
36
37
38
39
0
C A T T C C A C C A G A G C T A A G G T C T G A T T T G A A T G A C G
1
T G T G C C T A C G G T T G T A T A A T T T G C A C C C T G A G T T T
2
A G C T G A A A G T T T A A G A C T A T C T A T C C G C T G T T G T T
3
G T T G T A A C C C G C T C C T T G G C C T C C G G G C A A G T C T A
4
A C G A T C G C C G A A T G A A A A G C C A C T T C T C C G T C A G G
5
T G G T G C A G G T G A A T A A G A G G T A T G G C C C G G G C T T T
7
C T G T C A C A C C A A T T C T T G G A T G T G G G T C A A C A C A C
8
G A T A G T A G C T C T C C A T C G G C T C T G A A T C T G G G T C A
9
G G G G C A C T C C G T A A T A C G A G C G A A C T C C T T G T A C C
50
51
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Claims
Claims
1. A Brassicales plant, or part thereof, wherein said Brassicales plant is an oilseed and/or a protein crop, and wherein said plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant II MAM IT transporter, wherein said mutant IIMAMIT transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed.
2. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said Brassicales plant carries a mutation in at least two genes encoding IIMAMIT transporters selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto.
3. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said Brassicales plant carries a mutation in at least three genes encoding IIMAMIT transporters selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30
as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto. he Brassicales plant or part thereof according to any one of the preceding claims, wherein said Brassicales plant carries a mutation in at least four genes encoding IIMAMIT transporters selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto. he Brassicales plant or part thereof according to any one of the preceding claims, wherein said IIMAMIT transporter is selected from the group consisting of BnaA09G0679100GG, BnaA09G0679000GG, BnaC05G0008000GG, BnaA09G0647600GG, BnaA09G0647700GG, BnaA10G0008000GG, BnaA09G0623500NO, BnaA09G0623600NO, BnaC05G0007600NQ, Bnascaffold2891G0004400NQ, BnaA09G0624000NQ, BnaC08G0554500QU, BnaA09G0664600QU, BnaC08G0554400QU, BnaA10G0008300QU, Bnascaffold2503G0022000QU, BnaA09G0664700QU, BnaA09G0724700SL, BnaA09G0689800SL, BnaA09G0724800SL, BnaC05G0007600SL, BnaA09G0689900SL, Bnascaffold3068G0013800SL, BnaA09G0577200TA, BnaA09G0628700TA, BnaC05G0007500TA, BnaA09G0577100TA, BnaA10G0008300TA, BnaA09G0628800TA, BnaA09G0658500WE, BnaA09G0658600WE, BnaC05G0009100WE, BnaA10G0009300WE,
BnaA09G0659000WE, BnaA09G0692800ZS, BnaA09G0692700ZS, BnaA09G0714200ZS, BnaA09G0714300ZS, BnaA10G0008500ZS, BnaC05G0010000ZS, BnaA09G0634900ZY, BnaA09G0666500ZY, Bnascaffold4696G0013500ZY, BnaC05G0007300ZY, BnaA09G0635000ZY, BnaA09G0666400ZY, BolC5g28871 H, BolC8g52858H, BraA10g42362Z, BraA09g42228Z and BraA09g42227Z. he Brassicales plant or part thereof according to any one of the preceding claims, wherein said IIMAMIT transporter is selected from the group consisting of BnaC02G0303900GG, BnaA02G0239900GG, BnaC02G0303800GG, BnaC02G0289800NG, BnaA02G0248400NG, BnaA02G0248300NG, BnaC02G0286300QU, BnaA02G0247800QU, BnaA02G0247700QU, BnaC02G0286400QU, BnaC02G0230500SL, BnaA02G0281700SL, BnaC02G0230600SL, BnaC02G0254900TA, BnaA02G0214900TA, BnaC02G0255000TA, BnaC02G0326100WE, BnaA02G0260700WE, BnaA02G0260600WE, BnaC02G0326200WE, BnaC02G0337200ZS, BnaA02G0251300ZS, BnaC02G0337100ZS, BnaA02G0251200ZS, BnaA02G0278400ZY, BnaA02G0278500ZY, BnaC02G0162700ZY, BnaC02G0162600ZY, BolC2g09864H and BraA02g07429Z. he Brassicales plant or part thereof according to any one of the preceding claims, wherein said IIMAMIT transporter is selected from the group consisting of BnaC03G0326100GG, BnaA03G0226900GG, BnaA01G0215700GG, BnaC01G0262700GG, BnaA03G0226800GG, BnaC01G0263000GG, BnaC03G0325800GG, BnaC03G0326000GG, BnaC02G0304200GG, BnaA02G0240100GG, BnaC03G0251900NG, BnaA03G0272800NG, BnaA01G0189100NG, BnaA02G0248600NG, BnaC03G0251700NG, BnaC02G0290200NG, BnaA03G0272700NG, BnaC05G0297600NG, BnaC05G0297300NG, BnaC03G0252000NG, BnaC03G0317800QU, BnaC03G0317900QU, BnaC03G0317600QU, BnaA03G0263200QU, BnaC01G0188500QU, BnaA03G0262800QU, BnaA01G0168400QU, BnaC01G0188900QU, BnaC02G0286800QU, BnaA03G0263100QU, BnaA02G0248000QU, BnaC03G0209800SL, BnaA01G0193300SL, BnaA03G0197300SL, BnaC02G0231000SL, BnaA02G0281500SL, BnaC01G0253100SL, BnaC03G0210100SL, BnaA03G0269600TA,
BnaA03G0269800TA, BnaA02G0215100TA, BnaA03G0269900TA, BnaC02G0255300TA, BnaC01G0212600TA, BnaC01G0213100TA, BnaC03G0292000TA, BnaC03G0291900TA, BnaA01G0144700TA, BnaC03G0291700TA, BnaC03G0265500WE, BnaC01G0224300WE, BnaA03G0285900WE, BnaA02G0260400WE, BnaC02G0326500WE, BnaC03G0265600WE, BnaA01G0142400WE, BnaC03G0265300WE, BnaA03G0286100WE, BnaC01G0224600WE, BnaA03G0286000WE, BnaA01G0222900ZS, BnaC03G0331600ZS, BnaC03G0332100ZS, BnaA02G0251500ZS, BnaC03G0332000ZS, BnaC02G0337600ZS, BnaA03G0275500ZS, BnaA03G0275200ZS, BnaC01G0283800ZS, BnaC01G0283500ZS, BnaA03G0275300ZS, BnaC02G0163100ZY, BnaA03G0295200ZY, BnaC03G0268300ZY, BnaC03G0268600ZY, BnaC03G0268500ZY, BnaC01G0208900ZY, BnaA03G0295000ZY, BnaA03G0295100ZY, BnaC01G0209300ZY, BnaA01G0220000ZY, BnaA02G0278700ZY, BolC3g16132H, BolC2g09869H, BolC3g16128H, BolC3g16133H, BolC1g03106H, BolC1g03101 H, BraA03g12335Z, BraA03g12337Z, BraA02g07432Z and BraA01g02441Z. he Brassicales plant or part thereof according to any one of the preceding claims, wherein said IIMAMIT transporter is selected from the group consisting of BnaA02G0240000GG, BnaC02G0304100GG, Bnascaffold1465G0001500GG, BnaA09G0014000GG, BnaC02G0290100NG, BnaA09G0016000NG, BnaA02G0248500NG, BnaC09G0009500QU, BnaA02G0247900QU, BnaA09G0011000QU, BnaC02G0286700QU, BnaA09G0009300SL, Bnascaffold966G0010300SL, BnaA02G0281600SL, BnaC02G0230900SL, BnaA02G0215000TA, BnaC09G0008700TA, BnaA09G0005700TA, BnaC02G0255200TA, BnaC02G0326400WE, BnaC09G0008200WE, BnaA02G0260500WE, BnaA09G0011300WE, BnaC09G0002100ZS, BnaC02G0337500ZS, Bnascaffold0025G0022100ZS, BnaA02G0251400ZS, BnaA09G0019300ZS, Bnascaffold0025G0022000ZS, BnaC09G0001300ZY, BnaC02G0163000ZY, BnaA02G0278600ZY, BnaA09G0018200ZY, BolC9g53044H, BolC2g09868H, BraA02g07431Z and BraA09g35773Z. he Brassicales plant or part thereof according to any one of the preceding claims, wherein said mutant IIMAMIT transporter is a mutant glucosinolate transporter
with reduced glucosinolate transporter activity compared to the wildtype protein. The Brassicales plant or part thereof according to claim 9, wherein said reduction is a reduction of at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as 100% of the glucosinolate transporter activity of said mutant glucosinolate transporter compared to the wildtype protein. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said mutated gene has reduced expression of the encoded glucosinolate transporter compared to the wildtype gene. The Brassicales plant or part thereof according to claim 11, wherein said reduced expression is a reduction of at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% in expression of said mutated gene compared to the wildtype gene. The Brassicales plant or part thereof according to any one of the preceding claims, wherein the seeds of said Brassicales plant or part thereof have a reduced glucosinolate content compared to a Brassicales plant or part thereof of otherwise identical genotype except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions. The Brassicales plant or part thereof according to claim 13, wherein said reduction in glucosinolate content is a reduction of at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% or such as at least 95%. The Brassicales plant or part thereof according to any one of the preceding claims, wherein the vegetative tissue of said Brassicales plant or part thereof have about the same glucosinolate content compared to a Brassicales plant or part thereof of otherwise identical genotype except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
16. The Brassicales plant or part thereof according to any one of the preceding claims, wherein the mutation is selected from the group consisting of a missense mutation, an insertion or a deletion.
17. The Brassicales plant or part thereof according to any one of the preceding claims, wherein the Brassicales plant or part thereof carries a mutation in the AtUMAMIT29 gene as set forth in SEQ ID NO: 12 or in a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, wherein said mutated gene encodes a mutant AtUMAMIT29 polypeptide, wherein said mutant AtUMAMIT29 is AtUMAMIT29 as set forth in SEQ ID NO: 2 or a functional homolog thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity thereto, except that said mutant polypeptide comprises one or more substitutions selected from the group consisting of a. a valine corresponding to the valine in position 27 of SEQ ID NO: 2 substituted with phenylalanine (V27F); b. a methionine corresponding to the methionine in position 86 of SEQ ID NO: 2 substituted with valine (M86V); c. a leucine corresponding to the leucine in position 109 of SEQ ID NO: 2 substituted with valine (L109V), d. a glutamine corresponding to the glutamine in position 263 of SEQ ID NO: 2 substituted with serine (Q263S); e. a threonine corresponding to the threonine in position 267 of SEQ ID NO: 2 substituted with tyrosine (T267Y); f. an arginine corresponding to the arginine in position 44 of SEQ ID NO: 2 substituted with alanine (R44A); g. a tryptophan corresponding to the tryptophan in position 200 of SEQ ID NO: 2 substituted with alanine (W200A); and h. a glutamine corresponding to the glutamine in position 204 of SEQ ID NO: 2 substituted with alanine (Q204A).
18. The Brassicales plant or part thereof according to any one claims 1 to 16, wherein the mutation is a frameshift mutation.
19. The Brassicales plant or part thereof according to any one of the preceding claims, wherein the mutation lies within a promoter region, a coding region, such as an exonic region, a non-coding region, such as an intronic region, and/or a termination sequence of said gene(s).
20. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said mutation is a loss-of-function mutation.
21. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said mutation is an insertion of a T-DNA sequence, such as pROK2 as set forth in SEQ ID NO: 21 or Vector_ pAC106 as set forth in SEQ ID NO: 22.
22. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said plant is of the family Brassicaceae.
23. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said plant is of a genus selected from the group consisting of Brassica, Camelina, Crambe, Eruca, Lepidium, and Thlaspi.
24. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said plant is a B. Juncea, B. napus (rape), B. carinata, B. oleracea, B. rapa, or L. campestre plant.
25. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said part is a seed.
26. The Brassicales plant or part thereof according to claim 25, wherein the seeds have approximately the same size as seeds from a Brassicales plant or part thereof with an otherwise identical genotype except not comprising a mutation in any of the transporters defined in claims 1-8, when said plants are cultivated and prepared under the same conditions.
27. The Brassicales plant or part thereof according to any one of claims 25 to 26, wherein the seeds have a concentration of glucosinolates of less than 18 micromoles per gram of dry weight of said seeds, such as less than 17 micromoles
per gram of dry weight, such less than 16 micromoles per gram of dry weight, such as less than 15 micromoles per gram of dry weight, such less than most 14 micromoles per gram of dry weight, such as less than 13 micromoles per gram of dry weight, such as less than 12 micromoles per gram of dry weight, such as less than 11 micromoles per gram of dry weight, such as less than 10 micromoles per gram of dry weight, such as less than 9 micromoles per gram of dry weight, such as less than 8 micromoles per gram of dry weight, such as less than 7 micromoles per gram of dry weight, such as less than 6 micromoles per gram of dry weight, such as less than 5 micromoles per gram of dry weight, such as less than 4 micromoles per gram of dry weight, such as less than 3 micromoles per gram of dry weight, such as less than 2 micromoles per gram of dry weight, such as less than 1 micromoles per gram of dry weight of said seeds. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said plant has not been exclusively obtained by means of an essentially biological process. The Brassicales plant or part thereof according to any one of the preceding claims, wherein said plant is a brassicaceous oilseed crop and/or a brassicaceous protein crop. A plant product comprising a Brassicales plant or part thereof, or prepared from seeds of said Brassicales plant or part thereof, wherein said Brassicales plant carries a mutation in at least one gene encoding an IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1, AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto, whereby said Brassicales plant, or part thereof expresses at least one mutant IIMAMIT transporter, wherein said mutant IIMAMIT
transporter has low or no glucosinolate transporter activity, is expressed at low levels, or is not expressed. The plant product according to claim 30, wherein said Brassicales plant or part thereof is as defined according to any one claims 1 to 29. The plant product according to any one of claims 30 to 31 , wherein the plant product is an oil, such as a seed oil. The plant product according to any one of claims 30 to 31 , wherein the plant product is a seed cake or a seed meal. The plant product according to any one of claims 30 to 33, wherein the plant product is prepared from a seed, optionally wherein said seed has a concentration of glucosinolates of less than 18 micromoles per gram of dry weight of said seeds, such as less than 17 micromoles per gram of dry weight, such less than 16 micromoles per gram of dry weight, such as less than 15 micromoles per gram of dry weight, such less than most 14 micromoles per gram of dry weight, such as less than 13 micromoles per gram of dry weight, such as less than 12 micromoles per gram of dry weight, such as less than 11 micromoles per gram of dry weight, such as less than 10 micromoles per gram of dry weight, such as less than 9 micromoles per gram of dry weight, such as less than 8 micromoles per gram of dry weight, such as less than 7 micromoles per gram of dry weight, such as less than 6 micromoles per gram of dry weight, such as less than 5 micromoles per gram of dry weight, such as less than 4 micromoles per gram of dry weight, such as less than 3 micromoles per gram of dry weight, such as less than 2 micromoles per gram of dry weight, such as less than 1 micromoles per gram of dry weight of said seed. The plant product according to claim 34, wherein said plant product comprises glucosinolates at a concentration of at the most 30 micromoles per gram of dry weight of said plant seed product, such as at the most 25 micromoles per gram of dry weight, such as at the most 20 micromoles per gram of dry weight, such as at the most 15 micromoles per gram of dry weight, such as at the most 14 micromoles per gram of dry weight, such as at the most 13 micromoles per gram
of dry weight, such as at the most 12 micromoles per gram of dry weight, such as at the most 11 micromoles per gram of dry weight, such as at the most 10 micromoles per gram of dry weight, such as at the most 9 micromoles per gram of dry weight, such as at the most 8 micromoles per gram of dry weight, such as at the most 7 micromoles per gram of dry weight, such as at the most 6 micromoles per gram of dry weight, such as at the most 5 micromoles per gram of dry weight, such as at the most 5 micromoles per gram of dry weight, such as at the most 3 micromoles per gram of dry weight, such as at the most 2 micromoles per gram of dry weight, or such as at the most 1 micromoles glucosinolates per gram of dry weight of said plant seed product.
36. The plant product according to claim 34 for use as animal feed.
37. A seed cake prepared from a seed from a Brassicales plant, wherein the Brassicales plant is according to any one of claims 1 to 29.
38. An animal feed comprising the plant product according to claim 34 or the seed cake according to claim 37.
39. The seed cake according to claim 37, wherein the seed cake prepared from said Brassicales plant comprises a concentration of glucosinolate of at the most 15%, such as at the most 10%, or such as at the most 5% of the glucosinolate concentration in a seed cake prepared from a seed of a plant of otherwise identical genotype to said Brassicales plant except not comprising a mutation in any of said genes, when cultivated and prepared under the same conditions.
40. A method for modifying glucosinolate content in a Brassicales plant or part thereof, said method comprising a step of modifying the functional activity or expression of at least one IIMAMIT transporter selected from the group consisting of AtUMAMIT28 as set forth in SEQ ID NO: 1 , AtUMAMIT29 as set forth in SEQ ID NO: 2, AtUMAMIT30 as set forth in SEQ ID NO: 3, AtUMAMIT31 as set forth in SEQ ID NO: 4, BnaA09G0714200ZS as set forth in SEQ ID NO: 5, BnaC05G0010000ZS as set forth in SEQ ID NO: 6, BnaA01G0222900ZS as set forth in SEQ ID NO: 7, BnaC01G0283800ZS as set forth in SEQ ID NO: 8, BnaC03G0332100ZS as set forth in SEQ ID NO: 9, BnaA09G0692700ZS as set
forth in SEQ ID NO: 10, and respective functional homologs thereof with at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity thereto. 41. The method according to claim 40, wherein said step of modifying said functional activity of said at least one of IIMAMIT transporter is performed by nuclease-based gene editing, such as by CRISPR/Cas9 gene editing, by random mutagenesis, by gene targeting, by transposition mutagenesis, by transfer-DNA induced insertion, or by gene knockdown, such as by RNA interference.
42. A method for producing a plant product comprising a Brassicales plant or part thereof with low glucosinolate content, said method comprising the steps of a. providing a Brassicales plant or part thereof according to any one of claims 1 to 29; and b. processing said Brassicales plant or part thereof into a plant product, such as a seed oil or a seed cake.
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| Application Number | Priority Date | Filing Date | Title |
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| EP22207870 | 2022-11-16 | ||
| EP22207870.1 | 2022-11-16 | ||
| EP23178512 | 2023-06-09 | ||
| EP23178512.2 | 2023-06-09 |
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| WO2024105148A1 true WO2024105148A1 (en) | 2024-05-23 |
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| ANONYMOUS: "WAT1-related protein - Arabidopsis thaliana (Mouse-ear cress) | UniProtKB | UniProt", 24 February 2020 (2020-02-24), XP093039747, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A5S9S0B5/entry#function> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Arabidopsis thaliana x Arabidopsis arenosa | UniProtKB | UniProt", 12 October 2022 (2022-10-12), XP093039756, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A8T1Z5L7/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Brassica campestris (Field mustard) | UniProtKB | UniProt", 5 December 2018 (2018-12-05), XP093039820, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A398APN0/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Brassica campestris (Field mustard) | UniProtKB | UniProt", 5 December 2018 (2018-12-05), XP093039826, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A398APN0/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Brassica campestris (Field mustard) | UniProtKB | UniProt", 5 December 2018 (2018-12-05), XP093039828, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A397ZZ72/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Brassica cretica (Mustard) | UniProtKB | UniProt", 12 October 2022 (2022-10-12), XP093039812, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A8S9S6W1/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Brassica cretica (Mustard) | UniProtKB | UniProt", 12 October 2022 (2022-10-12), XP093039833, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A8S9K391/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Brassica napus (Rape) | UniProtKB | UniProt", 29 September 2021 (2021-09-29), XP093039825, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A816RR16/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Brassica oleracea (Wild cabbage) | UniProtKB | UniProt", 13 February 2019 (2019-02-13), XP093039796, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A3P6H1B2/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Brassica oleracea (Wild cabbage) | UniProtKB | UniProt", 13 February 2019 (2019-02-13), XP093039832, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A3P6H1B2/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein - Brassica oleracea (Wild cabbage) | UniProtKB | UniProt", 13 February 2019 (2019-02-13), XP093039836, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A3P6F239/entry> [retrieved on 20230418] * |
| ANONYMOUS: "WAT1-related protein At4g01450-like isoform X1 [Brassica napus] - Protein - NCBI", 23 June 2022 (2022-06-23), XP093039821, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/protein/XP_048601679> [retrieved on 20230418] * |
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