WO2020030783A2 - Means and methods for drought tolerance in crops - Google Patents

Means and methods for drought tolerance in crops Download PDF

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WO2020030783A2
WO2020030783A2 PCT/EP2019/071426 EP2019071426W WO2020030783A2 WO 2020030783 A2 WO2020030783 A2 WO 2020030783A2 EP 2019071426 W EP2019071426 W EP 2019071426W WO 2020030783 A2 WO2020030783 A2 WO 2020030783A2
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plant
protein
plants
sugarcane
drought
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PCT/EP2019/071426
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French (fr)
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WO2020030783A3 (en
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Dirk Gustaaf INZÉ
Marcelo MENOSSI
Vanessa Regina GONÇALVES
Giovanna Vieira GUIDELLI
Hilde Nelissen
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Vib Vzw
Universiteit Gent
Universidade Estadual De Campinas - Unicamp
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Priority to BR112021002264-3A priority Critical patent/BR112021002264A2/en
Publication of WO2020030783A2 publication Critical patent/WO2020030783A2/en
Publication of WO2020030783A3 publication Critical patent/WO2020030783A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates to the field of agriculture, more particularly to the field of plant molecular biology, even more particularly to the field of improving or maintaining the productivity of plants under environmental stress conditions.
  • the present invention provides chimeric genes and constructs which can be used to enhance the drought tolerance in plants and crops.
  • Biotechnology and molecular breeding techniques are useful tools to enhance crop productivity under drought stress.
  • engineering crops for drought tolerance remains a major challenge (Wang et al. (2003) Planta 218, 1-14; Wang et al. (2016) Front. Plant Sci. 7:67; Hu and Xiong (2014) Annu. Rev. Plant Biol. 65, 715-741).
  • This is not only due to the complexity of the plant responses to water deficit (Hu and Xiong (2014) Annu. Rev. Plant Biol. 65, 715-741; Wang et al. (2003) Planta 218, 1-14; Wang et al. (2016) Front. Plant Sci.
  • Drought tolerance is a highly desired trait for breeders and therefore Applicant provides herein chimeric gene constructs comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a sugarcane HIPP, NRX, HP, RTNL, TPX2, SEC61 , RNS3 or ZnF protein and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • said promoter is a constitutive promoter.
  • said sugarcane HIPP protein has at least a 90% homology to SEQ ID No. 2
  • said sugarcane HP protein has at least a 90% homology to SEQ ID No.
  • said sugarcane NRX protein at least 90% homology to SEQ ID No. 5
  • said sugarcane RTNL protein at least 90% homology to SEQ ID No. 6
  • said sugarcane TPX2 protein at least 90% homology to SEQ ID No. 7
  • said sugarcane SEC61 protein at least 90% homology to SEQ ID No. 8
  • said sugarcane RNS3 protein at least 90% homology to SEQ ID No. 9
  • said sugarcane ZnF protein at least 90% homology to SEQ ID No. 10.
  • Another object of current application is to provide the use of any of the chimeric genes disclosed in current application or of a recombinant vector comprising any of said chimeric genes to increase the drought tolerance of plants.
  • Said plant can be crops, more particularly C4 plants, cereals or grasses.
  • the methods disclosed herein are methods of producing a plant with increased drought tolerance as compared to a corresponding wild type plant, the method comprises introducing any of the chimeric genes disclosed herein or the recombinant vector comprising any of said chimeric genes in a plant and selecting a plant with a stable expression of said chimeric gene.
  • said method further comprises a step of quantifying the drought tolerance of the transformed plant lines and/or a step of isolating a plant from the population of transformed plant lines with increased drought tolerance compared to a plant without said chimeric gene construct.
  • FIG. 1 Response of transgenic Arabidopsis lines to severe drought stress.
  • Two weeks-old Arabidopsis thaliana plants over-expressing (A) ScSec61 , (B) ScTpx2, (C) ScRNS3, (D) ScZnF and empty vectors plants were exposed to water deprivation for 2 weeks and then irrigated. The photographs were taken just before and after (1 day) watering; the survival rate in each sample was quantified and shown in the graphs.
  • Three different over-expressing lines for each gene OE1, OE2, OE3
  • empty vector events were randomized in the same tray.
  • FIG. 1 Survival assay under severe stress, (a) Arabidopsis plants before and after rewatering (b) Graphic showing the survival rate. Arabidopsis plants constitutively overexpressing the genes ScRTNL, ScNRX or ScHIPP presented a higher survival rate compared to the controls under dehydration. Survival rates in percentages (numbers on the bars) and standard error (bars) were calculated from results of two independent experiments. Controls: WT and 3 events from empty vector (EV1, 2 and 3).
  • FIG. 7 Physiological analysis of sugarcane plants overexpressing the ScHIPP gene.
  • Leaf relative water content (RWC) (a, b) and chlorophyll content (SPAD index) (c, d) in sugarcane plants under drought (a, c) and rehydration conditions (b, d).
  • Three transgenic events were evaluated: ScHIPP-OEl, 2 and 3.
  • WT plants were used as control.
  • Rehydrated treatment represents drought-stressed plants after rewatering.
  • Percentages represent reductions/increases in the parameters under drought/rewatering compared to well-watered condition for each event.
  • FIG. 8 Physiological analysis of sugarcane plants overexpressing the ScNRX gene.
  • Leaf relative water content (RWC) (a) and chlorophyll content (SPAD index) (b) in sugarcane plants under drought conditions.
  • Three transgenic events were evaluated: ScNRX-OEl, 2 and 3. WT plants were used as control. Percentages represent reductions/increases in the parameters under drought compared to well- watered condition for each event.
  • FIG. 9 Physiological analysis of sugarcane plants overexpressing the ScHP gene.
  • Leaf relative water content (RWC) (a, b) in sugarcane plants under drought (a) and rehydration conditions (b).
  • Three transgenic events were evaluated: ScHP-OEl, 2 and 3.
  • WT plants were used as control.
  • Rehydrated treatment represents drought-stressed plants after rewatering.
  • Percentages represent reductions/increases in the parameters under drought/rewatering compared to well-watered condition for each event.
  • FIG. 10 Growth rate of biometric traits in sugarcane transgenic plants overexpressing the ScHP gene.
  • Figure 11 Final length (in mm) of leaf 4 of corn plants overexpressing ScTpx2 compared to wild-type corn plants in well-watered (left) or in drought conditions.
  • FIG. 12 Biomass (in g) of corn plants overexpressing ScTpx2 compared to wild-type corn plants in well-watered (left) or in drought conditions. Biomass was collected when leaf 4 was fully mature.
  • FIG. 13 Leaf Elongation Rate (LER) in mm/h for WT corn plants and corn plants overexpressing ScTpx2 in well-watered or in drought conditions.
  • Applicant discloses eight sugarcane genes of which the expression is upregulated under drought stress. Data is provided that overexpression of these genes confers increased tolerance towards periods of drought in dicots and/or in monocots.
  • a chimeric gene comprising the following operably linked DNA elements: a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein selected from the list consisting of a HI PP protein, a H P protein, a NRX protein, a RTNL protein, a SEC61 protein, a RNS3 protein, a ZnF protein and a TPX2 protein; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • a chimeric gene comprising the following operably linked DNA elements: a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein selected from the list consisting of SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9 and SEQ ID No: 10; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • H IPP proteins are metallochaperones that contain a heavy-metal-associated domain (FIMA) and a C-terminal isoprenylation motif.
  • the heavy-metal-associated domain (FIMA, pfam00403.6) is a conserved domain of approximately 30 amino acid residues comprising two cysteine residues that are important in binding and transfer of metal ions, such as copper, cadmium, cobalt and zinc.
  • the FIMA domain of the ScH IPP protein disclosed herein is depicted in SEQ ID No: 1: MDCEGCERRVKSAVKSMRGVTSVAVNPKQSKCTVTG.
  • Isoprenylation also known as farnesylation
  • farnesylation is a post-translational protein modification that involves addition of a C-terminal hydrophobic anchor that is important for interaction of the protein with membranes or other proteins. This occurs via covalent thioether binding of a 15-carbon farnesyl or 20- carbon geranylgeranyl group to the cysteine residue of a C-terminal CaaX motif (also known as the isoprenylation motif), where 'C' is cysteine, 'a' is an aliphatic amino acid, and 'X' is any amino acid (de
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a H I PP protein comprising an isoprenylation motif and a H MA domain with a sequence identity of at least 70%, at least 75%, at least 80%, at least 83%, at least 86%, at least 88%, at least 91%, at least 94%, at least 97%, at least 98% or 100% to SEQ I D No: 1; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • H I PP protein of sugarcane in disclosed, from here on referred to as ScH IPP or SEQ I D No: 2:
  • sequence identity of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (xlOO) divided by the number of positions compared.
  • a gap i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues.
  • the alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch (1970) J Mol Biol. 48: 443-453).
  • sequence alignment can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wisconsin, USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3. Sequences are indicated as "essentially similar" when such sequence have a sequence identity of at least about 75%, particularly at least about 80 %, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially are identical.
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScHI PP or a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 2; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • Non-limiting examples of functional homologues of ScH IPP are H IPP20 from Sorghum bicolor (Sequence
  • Homologs of a protein encompass peptides, oligopeptides and polypeptides having amino acid substitutions, deletions and/or insertions, preferably by a conservative change, relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived; or in other words, without significant loss of function or activity.
  • Orthologs and paralogs which are well-known terms by the skilled person, define subcategories of homologs and encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogs are genes within the same species that have originated through duplication of an ancestral gene; orthologs are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologues and paralogues include phylogenetic methods, sequence similarity and hybridization methods.
  • the HP protein of sugarcane from here on referred to as ScHP is characterized by SEQ ID No: 3:
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScHP or a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 3; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • Non-limiting examples of functional homologues of ScHP are hypothetical protein ZEAMM B73_Zm00001d031877 from Zea mays (Sequence I D: ON M03873.1), hypothetical protein Zm00014a_032104 from Zea mays (Sequence I D: PWZ57059.1), protein TsetseEP from Zea mays (Sequence I D: XP_008666540.1), hypothetical protein SORBI_3007G164500 from Sorghum bicolor (Sequence I D: EES14054.1).
  • N RX (nucleoredoxin) proteins belong to the Thioredoxin superfamily of proteins. Nrxs have been shown to play an interesting role as a protective mechanism of antioxidant systems controlling the status of ROS-scavenging enzymes such as catalase. However, to the best of Applicant's knowledge its overexpression has not yet been linked to increased drought tolerance in plants.
  • N RX proteins are characterized by the presence of a short C-terminal domain rich in cysteines and histidines. This domain is referred to as the Cl domain (pfam03107).
  • the Cl domain of ScN RX as depicted herein is SEQ I D No: 4: H RH ELSIVSDKSGGGPYICCECEEQGLGWAYQCIACGYEIH LRC.
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a N RX protein comprising a Cl domain with a sequence identity of at least 70%, at least 75%, at least 80%, at least 83%, at least 86%, at least 88%, at least 91%, at least 94%, at least 97% or 100% to SEQ I D No: 4; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • NRX protein of sugarcane is disclosed, from here on referred to as ScN RX or SEQ ID No: 5.
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScNRX or a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 5; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • Non-limiting examples of functional homologues of ScNRX are disulfide isomerase from Saccharum hybrid cultivar R570 (Sequence ID: AGT16827.1), nucleoredoxin 2 from Sorghum bicolor (Sequence I D: XP_002456473.1), protein disulfide isomerase from Zea mays (Sequence I D: N P_001131397.1), uncharacterized protein LOC100382831 from Zea mays (Sequence I D: N P_001169000.1), protein disulfide isomerase from Zea mays (Sequence I D: ACG38694.1), protein disulfide isomerase isoform XI from Zea mays (Sequence ID: XP_008654313.1), protein disulfide isomerase isoform X3 from Zea mays (Sequence I D: XP_008654315.1), hypothetical protein SETIT_001322mg from Setaria italica (Se
  • RTN L protein stands for reticulon-like protein. Proteins of the reticulon family are present in all eukaryotic organisms examined and range in size from 200 to 1,200 amino acids. The vertebrate proteins of this family are called reticulons (RTNs). Reticulon homologs from non-chordate taxa have been classified into six reticulon-like protein subfamilies (RTN L), including the plant subfamily of RTNLs named RTN LB (Oertle and Schwab 2003 Trends Cell Bio 13: 187-194).
  • RTN L reticulon-like protein subfamilies
  • RHD reticulon homology domain
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 6; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScRTNL and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • Non-limiting examples of functional homologues of ScRTNL are reticulon-like protein B2 from Sorghum bicolor (Sequence ID: XP_002438444.1), reticulon from Zea mays (Sequence ID: ACG36034.1), uncharacterized protein LOC100191184 from Zea mays (Sequence ID: NP_001130091.1), Reticulon-like protein B2 from Zea mays (Sequence ID: PWZ16372.1), Reticulon from Zea mays (Sequence ID: AQK84022.1), hypothetical protein GQ55_4G143000 from Panicum hallii var.
  • TPX2 stands for Targeting Protein for Xklp2.
  • Xklp2 is a kinesin-like protein localized on centrosomes throughout the cell cycle and on spindle pole microtubules during metaphase.
  • TPX2 is a microtubule- associated protein that mediates the binding of the C-terminal domain of Xklp2 to microtubules. It is phosphorylated during mitosis in a microtubule-dependent way.
  • the TPX2 family represents a conserved region (pfam06886) approximately 60 residues long within the eukaryotic targeting protein for Xklp2 (TPX2). Very little is known about TPX2 family members in plants. In this application it is disclosed that overexpression of a sugarcane TPX2 (from here on referred to as ScTPX2 or SEQ ID No: 7) confers increased tolerance towards drought in both monocots and dicots.
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID No: 7; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • chimeric genes comprising a functional homologue of ScTPX2 will also lead to increased drought tolerance when expressed in plants.
  • the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScTPX2 and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • Non-limiting examples of functional homologues of ScTPX2 are protein TPX2 from Sorghum bicolor (Sequence I D: XP_002460013.1), protein TPX2 from Setaria italic (Sequence I D: XP_004956528.1), hypothetical protein PAHAL_2G221100 from Panicum hallii (Sequence I D: PAN 11841.1), uncharacterized protein LOC100277148 from Zea mays (Sequence I D: N P_001144271.1), hypothetical protein GQ55_2G214000 from Panicum hallii var. hallii (Sequence I D: PUZ70269.1), TPX2 from Zea mays (Sequence I D: ONM21467.1).
  • SEC61 stands for SU PPRESSORS OF SECRETION-DEFECTIVE 61 BETA.
  • the sugarcane SEC61 (from here on referred to as ScSEC61 or SEQ ID No: 8) confers increased tolerance towards drought in plants.
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 8; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • chimeric genes comprising a functional homologue of ScSEC61 will also lead to increased drought tolerance when expressed in plants.
  • the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScSEC61 and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • Non-limiting examples of functional homologues of ScSEC61 are protein SEC61 from Zea mays (Sequence I D: N P_001151680), protein SEC61 from Sorghum bicolor (Sequence I D: XP_002455808.1), protein SEC61 from Panicum hallii (Sequence I D: XP_025812251.1), protein SEC61 from Glycine max (Sequence I D: XP_003517012.1), protein SEC61 from Glycine soja (Sequence ID: RZC29809.1).
  • RNS3 stands for ribonuclease 3.
  • the sugarcane RNS3 (from here on referred to as ScRNS3 or SEQ I D No: 9) confers increased tolerance towards drought in plants.
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 9; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • chimeric genes comprising a functional homologue of ScRNS3 will also lead to increased drought tolerance when expressed in plants.
  • the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScRNS3 and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • Non-limiting examples of functional homologues of ScRNS3 are extracellular ribonuclease LE from Sorghum bicolor (Sequence I D: XP_002462742.1), knotted l induced 1 precursor from Zea mays (Sequence I D: N P_001106070.2), ribonuclease 1 from Glycine max (Sequence I D: XP_003517989.1), hypothetical protein GLYMA_01G048200 from Glycine max (Sequence ID: KRH74869.1).
  • ScZnF or SEQ I D No: 10 confers increased tolerance towards drought in plants.
  • a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 10; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • chimeric genes comprising a functional homologue of ScZnF will also lead to increased drought tolerance when expressed in plants.
  • the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScZnF and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
  • Non-limiting examples of functional homologues of ScZnF are uncharacterized protein LOC8060005 from Sorghum bicolor (Sequence ID: XP_002466564.1), uncharacterized protein LOC100282293 from Zea mays (Sequence ID: NP_001278519.1), uncharacterized protein LOC112897867 from Panicum hallii (Sequence ID: XP_025822040.1).
  • a “chimeric gene” or “chimeric construct” is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably linked to, or associated with, a nucleic acid sequence that codes for an mRNA, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid coding sequence.
  • the regulatory nucleic acid sequence of the chimeric gene is not normally operably linked to the associated nucleic acid sequence as found in nature.
  • operably linked refers to a linkage in which the promoter or regulatory sequence is contiguous with the gene of interest to control the gene of interest (i.e. initiate the transcription of the gene of interest), as well as a promoter that act in trans or at a distance to control the gene of interest.
  • a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter and allows transcription elongation to proceed through the DNA sequence.
  • Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or adapters or linkers inserted instead of using restriction endonucleases known to one of skill in the art.
  • nucleic acid molecules in an organism must be linked operably to or comprise a suitable promoter which expresses said nucleic acid molecule at the right point in time and with the required spatial expression pattern.
  • a promoter that enables the initiation of gene transcription in a host cell is referred to as being "active".
  • the promoter can be operably linked to a reporter gene after which the expression level and pattern of the reporter gene can be assayed.
  • Suitable well-known reporter genes include for example beta- glucuronidase, beta-galactosidase or any fluorescent or luminescent protein.
  • promoter activity refers to the extent of transcription of a polynucleotide sequence, homologue, variant or fragment thereof that is operably linked to the promoter whose promoter activity is being measured.
  • the promoter activity is assayed by measuring the enzymatic activity of the beta-glucuronidase or beta- galactosidase.
  • promoter strength may also be assayed by quantifying mRNA levels or by comparing mRNA levels of the nucleic acid, with mRNA levels of housekeeping genes such as 18S rRNA, using methods known in the art, such as Northern blotting with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).
  • the promoter is an exogenous promoter, i.e. a regulatory nucleic acid sequence which differs from the sequence to which said nucleic acid molecule is associated in nature. This is equivalent as saying that said promoter or regulatory nucleic acid sequence to which the nucleic acid molecule is operably linked differs from the promoter or regulatory nucleic acid sequence operably linked or associated with said nucleic acid molecule in the natural environment.
  • a non-limiting example of an exogenous promoter for expression of a gene of interest in plants is the 35S promoter.
  • the "35S promoter” or the “cauliflower mosaic virus (CaMV) 35S promoter” is a constitutive or constant active promoter that directs high-level expression in a wide range of cells under a wide range of conditions and in most plant tissues including monocots.
  • Examples of other constitutive plant promoters useful for expressing heterologous, modified or non-modified polypeptides in plant cells include, but are not limited to, the plant ubiquitin (Ubi) promoter, the ethylene response factor (ERF) promoter, the nopaline synthase promoter and the octopine synthase promoter.
  • the promoter being part of the chimeric genes described above is a constitutive promoter.
  • said promoter is not a 35S promoter.
  • said promoter is a root specific promoter or a shoot specific promoter or a meristem specific promoter or a leaf specific promoter or a promoter driving expression in the growth zone of the leaves.
  • said promoter is selected from the list consisting of Gmubil (Glymal0g39780), Gmubi2 (Glymal3gl7830.1), Gmubi3 (Glyma20g27950.1), Gmubi4 (Glymal3g24470.1), Gmubi5
  • Gmubi8 (Glymal3g24500.1), Gmubi6 (Glyma07g32020.1), Gmubi7 (Glymal7g04690.1), Gmubi8
  • Glymal0g05830.1 Gmubi9 (Glymal3g20200), GmubilO (Glymal5gl3650.1), GmERFl
  • GmERF4 (Glyma20gl6920.1), GmERF2 (Glyma20gl6910.1), GmERF3 (Glymallg03900.1), GmERF4
  • GmERF5 (Glyma01g41530.1), GmERF5 (Glyma05g05180.1), GmERF6 (Glyma05g05130.1), GmERF7
  • Glymal9g43820.1 GmERF8 (Glyma20g34570.1), GmERF9 (Glymal0g33060.1) and GmERFlO (Glymal7gl5460.1) as referred to by Fgruandez-Garcia et al 2010 (BMC Plant Biology 10:237) which is hereby inserted by reference.
  • said promoter is selected from the list consisting of GmCons4, GmCons6, GmConslO, GmRootl, GmRoot2, GmRoot3, GmRoot5, GmRoot6, GmRoot7, GmRoot8, GmSeed2, GmSeed3, GmSeed5, GmSeed6, GmSeed7, GmSeed8, GmSeedlO, GmSeedll, GmFABl, GmFAB2, GmFAB3, GmFAB5, GmFAB8, GmFAB9, GmFABlO, GmFABll, GmFAB17, GmWRKY13, GmWRKY17, GmWRKY21, GmWRKY27, GmWRKY43, GmWRKY54, GmWRKY67, GmWRKY79, GmWRKY80, GmWRKY82, GmWRKY85 and GmWRKY162 as referred by Gun
  • terminal encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription.
  • the terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
  • said chimeric gene constructs are transformed or introduced and expressed in said plants.
  • expression means the transcription of a specific gene or specific genes or specific genetic construct.
  • expression in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
  • introduction of genes in plants or transformation encompass the transfer of an exogenous polynucleotide or foreign genes into a host cell, irrespective of the method used for transfer.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • the polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome.
  • the resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
  • Transformation of plant species is now a fairly routine technique.
  • any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microinjection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al.
  • Transformed or transgenic plants are preferably produced via Agrobacterium- mediated transformation.
  • An advantageous transformation method is the transformation in planta.
  • Agrobacteria it is possible, for example, to allow the Agrobacteria to act on plant seeds or to inoculate the plant meristem with Agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed Agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743).
  • Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP1198985, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.
  • nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al (1984) Nucl. Acids Res. 12- 8711).
  • Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an Agrobacteria solution and then culturing them in suitable media.
  • plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
  • the transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 (Nature Biotechnology 22 (2), 225-229). Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21 , 20-28. Further biotechnological progress has been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225- 229).
  • the genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
  • plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
  • the plant material obtained in the transformation is, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
  • a further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
  • the transformed plants are screened for the presence of a selectable marker such as the ones described above.
  • putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation.
  • expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or Tl) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
  • transgenic plant for the purposes of the invention is understood as meaning that the nucleic acids or chimeric gene constructs used in the method of the invention are not present in, or originating from, the genome of said plant, or are present in the genome of said plant but not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously.
  • transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified.
  • Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, heterologous expression of the nucleic acids takes place.
  • Preferred transgenic plants are mentioned herein.
  • a vector, a recombinant vector or an expression cassette comprising on of the chimeric genes from the application is herein provided.
  • the term "vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • the vector may be of any suitable type including, but not limited to, a phage, virus, plasmid, phagemid, cosmid, bacmid or even an artificial chromosome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell).
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome.
  • the markers may a) code for protection against a biocide, such as antibiotics, toxins, heavy metals, certain sugars or the like; b) provide complementation, by imparting prototrophy to an auxotrophic host: or c) provide a visible phenotype through the production of a novel compound in the plant.
  • NPTII neomycin phosphotransferase
  • HPT hygromycin phosphotransferase
  • CAT chloramphenicol acetyltransferase
  • NPTII neomycin phosphotransferase
  • HPT hygromycin phosphotransferase
  • CAT chloramphenicol acetyltransferase
  • gentamicin resistance gene gentamicin resistance gene.
  • suitable markers are b-glucuronidase, providing indigo production, luciferase, providing visible light production, Green Fluorescent Protein and variants thereof, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance.
  • certain preferred vectors are capable of directing the expression of certain genes of interest.
  • Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, and the like as desired and according to a particular host organism (e.g. plant cell).
  • a recombinant vector according to the present invention comprises at least one "chimeric gene" or "expression cassette".
  • Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof of the present invention operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as plant cells, to be transformed.
  • the promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell.
  • expression cassette refers to any recombinant expression system for the purpose of expressing a chimeric gene described above in vitro or in vivo, constitutively or inducibly, in any cell, including, in addition to plant cells, prokaryotic, yeast, fungal, insect or mammalian cells.
  • the term includes linear and circular expression systems.
  • the term includes all vectors.
  • the cassettes can remain episomal or integrate into the host cell genome.
  • the expression cassettes can have the ability to self- replicate or not (i.e., drive only transient expression in a cell).
  • the term includes recombinant expression cassettes that contain only the minimum elements needed for transcription of the recombinant nucleic acid.
  • plant as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
  • fodder or forage legumes include fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g.
  • Avena sativa Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp.
  • Flelianthus annuus Flemerocallis fulva
  • Hibiscus spp. Flordeum spp. (e.g. Flordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g.
  • Applicant discloses that overexpression of a sugarcane gene encoding a HIPP protein, a HP protein, a NRX protein, a RTNL protein, a SEC61 protein, a RNS3 protein, a ZnF protein or a TPX2 protein (which are defined in detail by the description above) confers increased tolerance towards periods of drought both in dicots and/or in monocots.
  • the Examples section clearly demonstrates this by using a chimeric gene construct to overexpress one of said sugarcane genes. Consequently said chimeric genes, uses thereof and methods comprising the step of expressing one of said chimeric genes are part of current disclosure.
  • a plant, seed, plant part and/or plant tissue in which the expression or the expression level of an endogenous gene encoding a HIPP protein or a functional homologue thereof, a HP protein or a functional homologue thereof, a NRX protein or a functional homologue thereof, a RTNL protein or a functional homologue thereof or a TPX2 protein or a functional homologue thereof, a SEC61 protein or a functional homologue thereof, a RNS3 protein or a functional homologue thereof or a ZnF protein or a functional homologue thereof is increased or enhanced.
  • Said HIPP protein, HP protein, NRX protein, RTNL protein, TPX2 protein, SEC61 protein, RNS3 protein and ZnF protein are those described earlier in this application and more particularly are defined by SEQ ID No: 2, 3, 5, 6, 7, 8, 9 and 10 respectively.
  • said plant, seed, plant part and/or plant tissue is a sugarcane plant, sugarcane seed, sugarcane plant part and/or sugarcane plant tissue and the endogenous genes referred to above are endogenous sugarcane genes.
  • said increased or enhanced expression or expression level means an expression or expression level that is at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold or 10-fold higher than that of a control plant as defined herein.
  • the person skilled in the art is familiar with techniques to establish a higher expression level of an endogenous gene in a plant cell, including a sugarcane plant cell.
  • a non-limiting example is the use of CRISPR-Cas mediated genome engineering.
  • CRISPR-Cas technology is primarily known as a molecular mutagenesis tool
  • variants of the technology are able to modify the expression of target genes.
  • This method of regulating and more particularly increasing the expression of endogenous genes in a plant or plant cell by using a nuclease-inactive Cas protein directly or indirectly fused to a transcription activator is well-established and the skilled person is directed to a non-exhaustive lists of documents including WO2014197568A2, WO2014197748A2, W02015006294A2 or W02014089290A1 for all details.
  • a plant, seed, plant part and/or plant tissue in which the expression or the expression level of an endogenous gene encoding a HIPP protein or a functional homologue thereof, a HP protein or a functional homologue thereof, a NRX protein or a functional homologue thereof, a RTNL protein or a functional homologue thereof, a TPX2 protein or a functional homologue thereof, a SEC61 protein or a functional homologue thereof, a RNS3 protein or a functional homologue thereof or a ZnF protein or a functional homologue thereof is increased or enhanced.
  • said plant, seed, plant part, plant tissue is genetically engineered and the expression or expression level of one of said genes encoding SEQ ID No: 2, 3, 5, 6, 7, 8, 9 or 10 or a functional homologues thereof is increased or enhanced through CRISPR-Cas technology.
  • said plant, seed, plant part, plant tissue is a sugarcane plant, sugarcane seed, sugarcane plant part or sugarcane plant tissue.
  • said plant, seed, plant part, plant tissue is a soybean plant, soybean seed, soybean plant part or soybean plant tissue.
  • the use of one of the chimeric genes or recombinant vectors comprising said chimeric genes disclosed in the application is provided to increase drought tolerance in plants.
  • said plants are crops.
  • said plants are dicotyledonous plants, even more particularly leguminous plants such as soy or soybean.
  • said plants are C4 plants, cereals or grasses.
  • C4 plants refer to plants that use the C4 carbon fixation pathway to increase their photosynthetic efficiency by reducing or suppressing photorespiration, which mainly occurs under low atmospheric CO2 concentration, high light, high temperature, drought, and salinity.
  • C4 carbon fixation or the Hatch-Slack pathway is a photosynthetic process in C4 plants. It is the first step in extracting carbon from carbon dioxide to be able to use it in sugar and other biomolecules. It is one of three known processes for carbon fixation.
  • C4 refers to the 4-carbon molecule that is the first product of this type of carbon fixation.
  • C4 fixation is an elaboration of the more common C3 carbon fixation and is believed to have evolved more recently.
  • C4 overcomes the tendency of the enzyme RuBisCO to wastefully fix oxygen rather than carbon dioxide in the process of photorespiration. This is achieved by ensuring that RuBisCO works in an environment where there is a lot of carbon dioxide and very little oxygen.
  • CO2 is shuttled via malate or aspartate from mesophyll cells to bundle-sheath cells. In these bundle-sheath cells CO2 is released by decarboxylation of the malate.
  • C4 plants use PEP carboxylase to capture more CO2 in the mesophyll cells.
  • PEP Carboxylase (3 carbons) binds to CO2 to make oxaloacetic acid (OAA). The OAA then makes malate (4 carbons). Malate enters bundle sheath cells and releases the CO2.
  • OAA oxaloacetic acid
  • C4 plants are able to more efficiently fix carbon in drought, high temperatures, and limitations of nitrogen or CO2. Since the more common C3 pathway does not require this extra energy, it is more efficient in the other conditions.
  • Non-limiting example of such C4 plants are important crops such as maize, sorghum and sugarcane.
  • “Drought tolerance” is the degree to which a plant is adapted to or can cope with drought conditions.
  • the term “increased drought tolerance” or “enhanced drought tolerance” as used herein refers to an enhanced ability and detectable change of the genetically modified plants described in current application (compared to wild type or control transformants) to tolerate a period of drought or low- water conditions (water deprivation/depletion leading to for example (without the purpose of limiting) visible leaf wilting symptoms in control plants, loss of turgor, or reduction of photosynthesis rate) and to recover subsequently. In most cases this will lead to a reduced overall yield loss, as more plants per m 2 survive and/or the yield of the surviving plants is not significantly or less reduced compared to control plants.
  • drought tolerance can be assessed in controlled environments (green house or growth chambers) by placing at least about 10 transformants per transformation event and at least 10 control plants for various time periods (ranging from 1-4 weeks or more) into the environment without watering them, until leaf wilting or loss of turgor is caused on control plants, and subsequently watering the plants again for 1-2 weeks, while their recovery phenotype is analyzed.
  • Transformants with drought tolerance survive at least 2, 3, 4, 5, 6, 7 days, preferably at least 2-5 days longer without water than control transformants (e.g. transformed with an empty vector) or wild type plants do under the same conditions, and which show irreversible tissue damage.
  • the recovery of transformants is at least about 2-5 times higher than that of the control plants (e.g. with 20% control recovery, 40-100% survival in transformants).
  • Drought tolerance is often linked to salt tolerance, since both are associated with regulation of osmotic potential and turgor.
  • the described uses and methods to increase drought tolerance in plants are uses and methods to increase salt tolerance in plants.
  • drought tolerance is mostly a synonym for maintaining yield under periods of drought or reducing the reduction in yield associated with drought. Therefore, in one aspect, increased drought tolerance means a biomass production that is between 3 and 10%, between 5 and 20%, between 8 and 40%, between 12 and 45% or between 15 and 50% greater than the biomass production of non-tolerant drought stressed plants.
  • increased drought tolerance means a biomass production that is at least 20%, at least 50%, at least 75% or at least 100% higher than the biomass production of non-tolerant drought stressed plants.
  • a method for producing a plant with increased drought tolerance as compared to a corresponding wild type or control plant comprises introducing one of the chimeric genes or one of the recombinant vectors comprising one of said chimeric genes in a plant or transforming a plant with one of the chimeric genes or one of the recombinant vectors comprising one of said chimeric genes, and selecting a plant with a stable expression of said chimeric gene.
  • Also provided herein is a method for producing a sugarcane or soybean plant with increased drought tolerance as compared to a corresponding control sugarcane plant, said method comprises increasing the expression of an endogenous gene encoding any of SEQ ID No: 2, 3, 5, 6, 7, 8, 9 or 10 or a functional homologue thereof.
  • said sugarcane or soybean plant with increased drought tolerance is a genetically engineered sugarcane or soybean plant.
  • said increased expression is established using CRISPR-Cas technology.
  • Also provided herein is a method for producing a sugarcane plant or plant cell with increased drought tolerance as compared to a corresponding control sugarcane plant, said method comprises:
  • RNAs, the nuclease-null Cas protein, and the transcriptional regulator protein or domain are expressed and co-localize to said target DNA sequence and wherein the transcriptional regulator protein or domain increases the expression or expression level of a sugarcane gene encoding SEQ ID No: 2, 3, 5, 6, 7, 8, 9 or 10 or a functional homologue thereof.
  • said increased or enhanced expression or expression level means an expression or expression level that is at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold or 10-fold higher than that of a control plant as defined herein.
  • target nucleic acid refers to a nucleic acid sequence or site within a nucleic acid molecule that is recognized and to which a guide RNA sequence is designed to target, e.g. have complementarity, where hybridization between a target nucleic acid and a guide sequence promotes the formation of a CRISPR complex.
  • Cas protein refers to a protein comprising a nucleic acid (e.g., RNA) binding domain and an effector domain (e.g., Cas9, such as Streptococcus pyogenes Cas9).
  • the nucleic acid binding domains interact with a first nucleic acid molecules either having a region capable of hybridizing to a desired target nucleic acid (e.g., a guide RNA) or allows for the association with a second nucleic acid having a region capable of hybridizing to the desired target nucleic acid (e.g., a crRNA).
  • CRISPR proteins can also comprise nuclease domains (i.e., DNase or RNase domains), additional DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, as well as other domains.
  • Cas protein also refers to proteins that form a complex that binds the first nucleic acid molecule referred to above.
  • nuclease-null or “nuclease inactive" Cas protein refers to a Cas protein that can still bind to its specific nucleic acid binding site but does not have functionality or activity anymore and thus is not able to nick or cleave the nucleic acid molecule on which it binds.
  • exogenous or "heterologous” as used herein refers to any material originated outside of an organism, tissue, or cell.
  • endogenous refers to substances (e.g. genes) originating from within an organism, tissue, or cell.
  • the methods of current application further comprise a step of quantifying the drought tolerance of the transformed or genetically engineered plant lines and/or a step of isolation of a plant from the population of transformed or genetically engineered plant lines with increased drought tolerance compared to a plant without said chimeric gene construct or a suitable control plant.
  • said drought tolerance is determined by measuring the relative water content, the photosynthesis rate, the stomatal conductance, the transpiration rate, the chlorophyll content and/or the biomass of said transformed and control plant lines.
  • control plants are routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest.
  • the control plant is typically of the same plant species or even of the same variety as the plant to be assessed.
  • the control plant may also be a nullizygote of the plant to be assessed. Nullizygotes are individuals missing the transgene by segregation.
  • a "control plant” as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
  • Example 1 Selection of sugarcane genes putatively involved in drought stress tolerance
  • Leaf relative water status and chlorophyll levels are physiological parameters related with drought tolerance (Silva et al., 2007). These traits are evaluated in above described transgenic sugarcane plants under water deficit conditions. After 21 days of drought, ScHIPP-OE2 and 3 plants showed relative water content (RWC) of 53 and 69%, respectively, significantly higher than WT ( ⁇ 17%). Additionally, WT plants reduced on average 79% of RWC under drought in relation to well-watered treatment, while for ScHIPP- OE2 and 3 events, the reduction was less than half of this value (35 and 11%, respectively) (Figure 7).
  • Drought tolerant sugarcane plants tend to present higher RWC under water limitation. This parameter indicates cell hydration level, which is essential to maintain plant metabolism and growth under water deficit (Silva et al., 2007; Silva et al., 2011).
  • sugarcane ScNRX-OE plants have enhanced capacity to retain water under drought, which may contributes for better photosynthesis performance under drought and after rehydration observed in these plants.
  • the overexpression of ScHP gene showed to be related with improved water retention under rewatering conditions, which is accompanied by higher CO2 assimilation rate in the same environment.
  • Sugarcane ScHIPP-OE plants showed to be able to maintain higher RWC levels under stress, suffering less during stress, thus enabling better photosynthetic recuperation capacity after rewatering.
  • the sugarcane genes described in Example 1 are also overexpressed and evaluated in maize plants. Constructs comprising the sugarcane gene fused to a His-tag under control of the UBI promoter were made and transformed according to Coussens et al (2012 J Exp Bot 63:4263-4273). The primary transformants were backcrossed to B104 plants and the T1 plants (segregating 1:1) were sown for phenotypic analysis. Half of the plants were watered as normal, while no water was administered to the other half. When the effects of leaf rolling were visible (approximately six days after the appearance of leaf 4) the stressed plants were rewatered with the same volumes as the well-watered plants. The length of leaf four was measured daily, allowing for leaf growth rate and duration calculations and when leaf four was fully grown, the seedling biomass was determined.
  • Arabidopsis thaliana ecotype Col-0 was used in this study. Seeds were incubated at 4°C in the dark during 3 or 4 days for stratification before germination. Plants were grown at 24 °C and 16 h light (cool white fluorescent; ⁇ 120 mE m-2 s-1). Transformations of Arabidopsis were performed by the floral dip method (Clough and Bent, 1998). The first primary inflorescence was clipped to favour the growth of multiple secondary inflorescences. Plants were selected by spraying a Finale solution (0.1% Finale with 0.01% Silwet L-77) every 4 days. After two weeks, transgenic plants were transferred separately in pots with soil. Leaves were collected for DNA extraction.
  • T1 selected plants were confirmed by PCR with primers for the bar gene, and with primers into the 35S promoter (5'CTATCCTTCGCAAGACCCTTCCT3') and into the NOS terminator pGWB608 (5'AACGATCGGGGAAATTCGAGCTC3').
  • at least one amplicon for each gene constructed was verified by sequencing. Plants confirmed for T-DNA integration were subsequently cultivated in Murashige-Skoog medium (Sigma-Aldrich, USA) containing 1% agar and 50mM of glufosinate-ammonium to identify single copy events, presenting a 3:1 (tolerant:sensitive) segregation ratio. The single copy events were further selected to identify homozygous transgenic lines.
  • Seeds were surface-sterilized by the vapor-phase method. Briefly, seeds were placed in microcentrifuge tubes inside a desiccator jar containing a Beaker with 200 ml of bleach. Every hour 1 ml of hydrochloric acid was added to the bleach and the chlorine gas was maintained for five hours. For seeds sown directly in the soil the sterilization process was not used.
  • Trizol (Life technologies, USA) protocol was used as described by the manufacturer.
  • the possible DNA contamination was digested with RNase-free DNase I (Qiagen, USA) and the RNA was additionally purified using RNeasy Mini Kit (Qiagen, USA).
  • Complementary DNA was synthesized using Superscript III enzyme (Life Technologies, USA) starting with lpg of RNA.
  • the RT-PCR was performed with ImI of cDNA using Taq polymerase (Life technologies, USA) under the following conditions: lx95°C 2 min; 35x95°C 30 sec, 60°C 30 sec, 72°C 60s/kb; lx72°C 10 min; lxl2°C final step.
  • the primer forward used was specific for sugarcane genes and primer reverse inside the NOS terminator region for all genes (5 ' CCGGCAACAGGATTCAATCT 3 ' ).
  • Seedlings were sown in separated pots (55mm) filled with jiffy-7 (Jiffypot, Netherlands). Three different T3 homozygous events for each gene and two empty vector events were randomized in the same tray (35 pots), grown under normal conditions (16h light, at 22°C). After 10 days of well-watered conditions, the weight of all pots was normalized until the maximum water capacity and the watering was withheld for approximately two weeks. The positions of the pots were randomly changed every day to avoid differential water loss among the pots. When the majority of the plants showed clear symptoms of wilting, the plants were re-watered and one day after survivors was counted.
  • the pGVG vector was used for sugarcane transformation. Briefly, this vector presents a Gateway cassette under control of ZmUbil promoter and CaMV 35S terminator for gene overexpression or silencing. Additionally, a FLAG-tag sequence was inserted upstream the CaMV 35S terminator for C-terminal fusion with the target protein. This vector was validated using GUS staining and qRT-PCR assays and showed to be able to efficient and fast overexpression or silencing of genes in sugarcane plants.
  • Sugarcane transformation was validated using GUS staining and qRT-PCR assays and showed to be able to efficient and fast overexpression or silencing of genes in sugarcane plants.
  • the meristematic region from shoot apex of six-months-old sugarcane plants was used to produce embryogenic calli.
  • This material was cultivated in MS maintenance medium [4.33 g/L MS salts (Murashige and Skoog, 1962), 1 mL/L MS vitamins, 0.15 g/L citric acid, 0.5 g/L casein hydrolysate, 25 g/L sucrose, 12 g/L mannitol, 100 mg/L proline, 3 mg/L 2-4 dichlorophenoxyacetic acid (2,4-D) and 2.8 g/L phytagel] at 26 °C in the dark, until the generation of embryogenic calli.
  • MS maintenance medium [4.33 g/L MS salts (Murashige and Skoog, 1962), 1 mL/L MS vitamins, 0.15 g/L citric acid, 0.5 g/L casein hydrolysate, 25 g/L sucrose, 12 g/L mannitol,
  • the selected genes already inserted into pENTR/D-TOPO vector were transferred to pGVG destination vector, using Gateway recombination.
  • the constructs were inserted into EHA105 A. tumefaciens strain by heat shock. Bacterial cultures were incubated with sugarcane calli under vacuum pressure for five minutes and transferred to co-cultivation medium (4.33 g/L MS salts, 1 mL/L MS vitamins, 3 mg/L 2,4-D, 0.15 g/L citric acid, 25 g/L sucrose and 3.5 g/L phytagel) at 22 °C, in the dark for 3 days.
  • co-cultivation medium (4.33 g/L MS salts, 1 mL/L MS vitamins, 3 mg/L 2,4-D, 0.15 g/L citric acid, 25 g/L sucrose and 3.5 g/L phytagel
  • the calli were kept in resting medium (4.33 g/L MS salts, 1 mL/L MS vitamins, 3 mg/L 2,4-D, 0.5 g/L casein hydrolysate, 0.15 g/L citric acid, 25 g/L sucrose, 100 mg/L proline, 2.8 g/L phytagel and 200 mg/mL timentin) at 26 °C, in the dark for 6 days.
  • resting medium 4.33 g/L MS salts, 1 mL/L MS vitamins, 3 mg/L 2,4-D, 0.5 g/L casein hydrolysate, 0.15 g/L citric acid, 25 g/L sucrose, 100 mg/L proline, 2.8 g/L phytagel and 200 mg/mL timentin
  • the transformed calli were transferred to a selective regeneration medium [4.33 g/L MS salts, 1 mL/L MS vitamins, 25 g/L sucrose, 5 mg/mL CuS04, 1 mg/mL benzylaminopurine (BAP), 7 g/L agar, 200 mg/mL timentin and 40 mg/L geneticin] at 26 °C, during 14 days with 16 h photoperiod.
  • the transgenic events were kept in medium without fitohormones (4.33 g/L MS salts, 1 mL/L MS vitamins, 25 g/L sucrose, 7 g/L agar, 200 mg/mL timentin and 40 mg/L geneticin) to induce growth and rooting. Plants transformed with pGVG empty vector and wild-type plants were used as negative controls.
  • NPTII neomycin phosphotransferase
  • the selected transgenic events were transferred to 415 mL plastic pots containing substrate and vermiculite (1:1), kept in culture room at 25 °C and photoperiod 12h for two weeks and moved to the greenhouse for two more weeks, to acclimatization. After that, plants were transferred to 18 L individual pots equalized with a mixture of soil, substrate and vermiculite (65, 30 and 5%, respectively) and kept under normal irrigation for 3 months. Drought stress assay were performed with selected genes and WT as control, using a randomized complete block design. The experiment was divided in independent blocks, each one corresponding to one gene. Each block contained 40 plants: five plants to each event (three events per gene and WT) and treatment (irrigated and drought).
  • the irrigated treatment refers to plants kept at pot capacity (PC - water content) 80% and the drought treatment, at PC 30%. Soil water percentage was calculated collecting 10 soil samples. This material was dried in drying oven at 70 °C for five days and then soaked with water until weight stabilization (PC 100%). The average weights were used to determined soil water percentage under PC 100%. On day one of the assay, sugarcane plants were soaked with water to achieve PC 100%. The pots weights average was measured and the weights for irrigated and drought treatments were inferred.
  • Total chlorophyll content estimation was performed at the end of drought and rehydration in the leaf +2 and +1, respectively, using a chlorophyll meter SPAD-502Plus (Konica Minolta, Japan). Three measurements in the middle of the leaf (without midrib) were taken for each plant and the average was used in the analyses.
  • Biometric agronomic traits considered yield components were taken at the beginning of the assay, end of drought and end of rehydration. Stalk circumference was measured in the base of the plant and the height was considered from base until leaf +1 insertion. The biometric data were plotted as growth rates during the stress assay. By the end of rehydration period, roots were carefully removed from the soil, washed and photographed for further root development analysis. The shoot and root of all plants were harvested, weighted (fresh weight) and dried at 60 °C for 15 days to obtain biomass. Samples of fresh root and leaf tissues (leaf +1) were collected for further biochemical and molecular analyses. Leaf +2 was collected at the end of drought stress.
  • Drought stress test was performed with five biological replicates. However, two outliers were identified with GraphPad Outlier calculator (Grubb's test) and excluded from statistical analysis. The data were evaluated using ANOVA (p-value ⁇ 0.05), followed by a least significant difference test (LSD, p-value ⁇ 0.05) to compare means. The analyses were conducted using the package Agricolae in R software.

Abstract

The present invention relates to the field of agriculture, more particularly to the field of plant molecular biology, even more particularly to the field of improving or maintaining the productivity of plants under environmental stress conditions. The present invention provides chimeric genes and constructs which can be used to enhance the drought tolerance in plants and crops.

Description

MEANS AND METHODS FOR DROUGHT TOLERANCE IN CROPS
Field of the invention
The present invention relates to the field of agriculture, more particularly to the field of plant molecular biology, even more particularly to the field of improving or maintaining the productivity of plants under environmental stress conditions. The present invention provides chimeric genes and constructs which can be used to enhance the drought tolerance in plants and crops.
Introduction
Environmental stresses limit plant growth and crop productivity (Mahajan and Tuteja (2005) Arch. Biochem. Biophys. 444, 139-158; Lobell et al. (2011) Science 333, 616-620). Drought is considered to be the most deleterious abiotic stress, affecting crop productivity worldwide (Wang et al. (2003) Planta 218, 1-14; Rampino et al. (2006) Plant Cell Environ. 29, 2143-2152). Plants have evolved various drought tolerance strategies, such as changes in life cycle, modulation of growth and development to match with water supply, regulation of whole plant functions to balance resource allocation for growth and stress adaptation, and evolution of stress signal perception for rapid and long-term expression of stress tolerance (Hirayama and Shinozaki (2010) Plant J. 61, 1041-1052; Hu and Xiong (2014) Annu. Rev. Plant Biol. 65, 715-741; You and Chan (2015) Front. Plant Sci. 6:1092). The expanding knowledge helps to identify key genes associated with drought tolerance and maintenance of growth under water deficit condition in various crops including sugarcane (Wang et al. (2003) Planta 218, 1-14; 2016; Yamaguchi and Blumwald (2005) Trends Plant Sci. 10, 615-620; Hu and Xiong (2014) Annu. Rev. Plant Biol. 65, 715- 741; Augustine et al. (2015) Plant Cell Rep. 34, 247-263; Rampino et al. (2006) Plant Cell Environ. 29, 2143-2152). Biotechnology and molecular breeding techniques are useful tools to enhance crop productivity under drought stress. Despite the availability of molecular tools and strategies and advancements in our understanding of stress responses, engineering crops for drought tolerance remains a major challenge (Wang et al. (2003) Planta 218, 1-14; Wang et al. (2016) Front. Plant Sci. 7:67; Hu and Xiong (2014) Annu. Rev. Plant Biol. 65, 715-741). This is not only due to the complexity of the plant responses to water deficit (Hu and Xiong (2014) Annu. Rev. Plant Biol. 65, 715-741; Wang et al. (2003) Planta 218, 1-14; Wang et al. (2016) Front. Plant Sci. 7:67), but also due to the difficulty of identifying drought stress related genes and alleles and the subsequent development of drought tolerant varieties suitable for commercial crop production conditions (Tardieu (2012) J. Exp. Bot. 63, 25-31; Cominelli et al. (2013) N. Biotechnol. 30, 355-361). In light of the global warming, dry periods have become longer, more intense and frequent. It thus advantageous to find novel means and methods to safeguard food production under changing environmental conditions. Summary
Current application provides means and methods of producing an improved drought tolerance phenotype in a plant. Applicant has mined for genes in the C4 crop sugarcane of which the expression is highly induced under drought conditions. Chimeric gene constructs were produced and introduced in plant progenitor cells. In some cases plant lines were obtained with a significant enhanced ability to cope with periods of drought. Drought tolerance is a highly desired trait for breeders and therefore Applicant provides herein chimeric gene constructs comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a sugarcane HIPP, NRX, HP, RTNL, TPX2, SEC61 , RNS3 or ZnF protein and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant. In particular aspects, said promoter is a constitutive promoter. In other aspects, said sugarcane HIPP protein has at least a 90% homology to SEQ ID No. 2, said sugarcane HP protein has at least a 90% homology to SEQ ID No. 3, said sugarcane NRX protein at least 90% homology to SEQ ID No. 5, said sugarcane RTNL protein at least 90% homology to SEQ ID No. 6, said sugarcane TPX2 protein at least 90% homology to SEQ ID No. 7, said sugarcane SEC61 protein at least 90% homology to SEQ ID No. 8, said sugarcane RNS3 protein at least 90% homology to SEQ ID No. 9, said sugarcane ZnF protein at least 90% homology to SEQ ID No. 10. Given that said chimeric genes are first inserted in a plant transformation compatible vector and subsequently introduced in plants, recombinant vectors are provided comprising the chimeric genes disclosed in current application as well as a plant, plant cell or plant seed comprising the chimeric genes disclosed in current application.
Another object of current application is to provide the use of any of the chimeric genes disclosed in current application or of a recombinant vector comprising any of said chimeric genes to increase the drought tolerance of plants. Said plant can be crops, more particularly C4 plants, cereals or grasses.
The methods disclosed herein are methods of producing a plant with increased drought tolerance as compared to a corresponding wild type plant, the method comprises introducing any of the chimeric genes disclosed herein or the recombinant vector comprising any of said chimeric genes in a plant and selecting a plant with a stable expression of said chimeric gene. In particular aspects, said method further comprises a step of quantifying the drought tolerance of the transformed plant lines and/or a step of isolating a plant from the population of transformed plant lines with increased drought tolerance compared to a plant without said chimeric gene construct.
Figures
Figure 1. Response of transgenic Arabidopsis lines to severe drought stress. Two weeks-old Arabidopsis thaliana plants over-expressing (A) ScSec61 , (B) ScTpx2, (C) ScRNS3, (D) ScZnF and empty vectors plants were exposed to water deprivation for 2 weeks and then irrigated. The photographs were taken just before and after (1 day) watering; the survival rate in each sample was quantified and shown in the graphs. Three different over-expressing lines for each gene (OE1, OE2, OE3) and empty vector events (EV) were randomized in the same tray.
Figure 2. Survival assay under severe stress, (a) Arabidopsis plants before and after rewatering (b) Graphic showing the survival rate. Arabidopsis plants constitutively overexpressing the genes ScRTNL, ScNRX or ScHIPP presented a higher survival rate compared to the controls under dehydration. Survival rates in percentages (numbers on the bars) and standard error (bars) were calculated from results of two independent experiments. Controls: WT and 3 events from empty vector (EV1, 2 and 3).
Figure 3. Mannitol effects on root growth of transgenic plants overexpressing ScRTNL and a rtnl mutant. Primary root length, root depth, root width and root convex hull parameters were measured for each event: (a) ScRTNL-OEl and for (b) rtnl mutant. Percentages represent reductions in the parameters under drought (100 mM mannitol) compared with normal condition (control). Asterisks indicate statistical differences comparing transgenic and control group in the same treatment (Student's t-test, p < 0.05). Bars indicate the standard error (n=12). The RTNL genes from sugarcane and Arabidopsis showed to be involved with alterations in the root system architecture that can enhance drought tolerance.
Figure 4. Gas exchange analysis in sugarcane plants overexpressing the ScHIPP gene submitted to normal irrigation, drought stress and rehydration, (a) Photosynthesis rate - A. (b) Stomatal conductance
- gs. (c) Transpiration rate - E. The measures were taken under drought (D, filled symbols) and irrigated (I, open symbols) treatment. PC 100% represents water capacity of 100%. WT plants were used as control. Asterisks indicate statistical differences [ANOVA, followed by LSD test (p-value < 0.05)] between transgenic and control group in drought-stressed and rehydrated plants. Statistical differences between transgenic and control group under normal irrigation were not considered. Data are presented as mean and error bars represent the standard error (n=3).
Figure 5. Gas exchange analysis in sugarcane plants overexpressing the ScNRX gene submitted to normal irrigation, drought stress and rehydration, (a) Photosynthesis rate - A. (b) Stomatal conductance
- gs. (c) Transpiration rate - E. The measures were taken under drought (D, filled symbols) and irrigated (I, open symbols) treatment. PC 100% represents water capacity of 100%. WT plants were used as control. Asterisks indicate statistical differences [ANOVA, followed by LSD test (p-value < 0.05)] between transgenic and control group in drought-stressed and rehydrated plants. Statistical differences between transgenic and control group under normal irrigation were not considered. Data are presented as mean and error bars represent the standard error (n=3). Figure 6. Gas exchange analysis in sugarcane plants overexpressing the ScHP gene submitted to normal irrigation, drought stress and rehydration, (a) Photosynthesis rate - A. (b) Stomatal conductance - gs. (c) Transpiration rate - E. The measures were taken under drought (D, filled symbols) and irrigated (I, open symbols) treatment. PC 100% represents water capacity of 100%. WT plants were used as control. Asterisks indicate statistical differences [ANOVA, followed by LSD test (p-value < 0.05)] between transgenic and control group in drought-stressed and rehydrated plants. Statistical differences between transgenic and control group under normal irrigation were not considered. Data are presented as mean and error bars represent the standard error (n=3).
Figure 7. Physiological analysis of sugarcane plants overexpressing the ScHIPP gene. Leaf relative water content (RWC) (a, b) and chlorophyll content (SPAD index) (c, d) in sugarcane plants under drought (a, c) and rehydration conditions (b, d). Three transgenic events were evaluated: ScHIPP-OEl, 2 and 3. WT plants were used as control. Rehydrated treatment represents drought-stressed plants after rewatering. Percentages represent reductions/increases in the parameters under drought/rewatering compared to well-watered condition for each event. Asterisks indicate statistical differences [ANOVA, followed by LSD test (p-value < 0.05)] between transgenic and control group in the same treatment. Data are presented as mean and error bars represent the standard error (n=3).
Figure 8. Physiological analysis of sugarcane plants overexpressing the ScNRX gene. Leaf relative water content (RWC) (a) and chlorophyll content (SPAD index) (b) in sugarcane plants under drought conditions. Three transgenic events were evaluated: ScNRX-OEl, 2 and 3. WT plants were used as control. Percentages represent reductions/increases in the parameters under drought compared to well- watered condition for each event. Asterisks indicate statistical differences [ANOVA, followed by LSD test (p-value < 0.05)] between transgenic and control group in the same treatment. Data are presented as mean and error bars represent the standard error (n=3).
Figure 9. Physiological analysis of sugarcane plants overexpressing the ScHP gene. Leaf relative water content (RWC) (a, b) in sugarcane plants under drought (a) and rehydration conditions (b). Three transgenic events were evaluated: ScHP-OEl, 2 and 3. WT plants were used as control. Rehydrated treatment represents drought-stressed plants after rewatering. Percentages represent reductions/increases in the parameters under drought/rewatering compared to well-watered condition for each event. Asterisks indicate statistical differences [ANOVA, followed by LSD test (p-value < 0.05)] between transgenic and control group in the same treatment. Data are presented as mean and error bars represent the standard error (n=3).
Figure 10. Growth rate of biometric traits in sugarcane transgenic plants overexpressing the ScHP gene.
Three independent events were subjected to drought stress (a, b) and rehydration conditions (c, d). Growth rate of main stalk height in percentage: a, c; Growth rate of main stalk circumference in percentage: b, d. WT plants were used as control. Asterisks indicate statistical differences (ANOVA, followed by LSD test, p < 0.05) between transgenic and control group in the same treatment. Data are presented as mean and error bars represent the standard error (n=3). GR: growth rate.
Figure 11. Final length (in mm) of leaf 4 of corn plants overexpressing ScTpx2 compared to wild-type corn plants in well-watered (left) or in drought conditions.
Figure 12. Biomass (in g) of corn plants overexpressing ScTpx2 compared to wild-type corn plants in well-watered (left) or in drought conditions. Biomass was collected when leaf 4 was fully mature.
Figure 13. Leaf Elongation Rate (LER) in mm/h for WT corn plants and corn plants overexpressing ScTpx2 in well-watered or in drought conditions.
Detailed description of the invention
The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein.
The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.
Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.
Sugarcane, an important source of sugar and ethanol, is a relatively high water-demanding crop and its growth is highly sensitive to water deficit (Lakshmanan and Robinson (2014) "Stress physiology: Abiotic stresses," in Sugarcane: Physiology, Biochemistry, and Functional Biology, ed P. H. Moore and F. C. Botha (Chichester: JohnWiley & Sons, Inc.), 411-434). It is estimated that sugarcane produces 8-12 ton cane per megaliter (ML) of irrigation water (Kingston (1994) "Benchmarking yield of sugarcane from estimates of crop water use," in Proceedings of the Australian Society for Sugar Cane Technologists (Bundaberg), 201-209), and water deficit can lead to productivity losses up to 60% (Robertson et al. (1999) Field Crop Res. 64, 211-227; Ramesh (2000) J. Agron. Crop Sci. 185, 83-89; Basnayake et al. (2012) J. Exp. Bot. 63, 6023-6033; Gentile et al. (2015) Front. Plant Sci. 6:58). For this reason, production areas are concentrated in regions with favorable rain regime to sugarcane growth and development (Moreira et al. (2007) "Bioenergy-successes and barriers," in Proceedings of Ises Solar World Congress 2007: Solar Energy and Human Settlement, Vols l-V (Berlin; Heidelberg), 38-45), while in other areas crop production requires supplemental or full irrigation (Walter et al. (2013) WENE 3, 70-92). The increasing incidence, duration and intensity of severe water deficit, has prompted many large sugarcane crop improvement programs to invest in water use-efficient and water stress tolerant varieties and water use-efficient crop productions systems. The increasing knowledge of stress biology coming from genetic, agronomic and molecular biology studies in various crops, including sugarcane is providing a major impetus to develop biotechnological strategies for producing water stress tolerant and commercially useful sugarcane varieties (Lakshmanan and Robinson (2014) "Stress physiology: Abiotic stresses," in Sugarcane: Physiology, Biochemistry, and Functional Biology, ed P. H. Moore and F. C. Botha (Chichester: JohnWiley & Sons, Inc.), 411-434; Augustine et al. (2015) Plant Cell Rep. 34, 247-263; Ramiro et al. (2016) Plant Biotechnol. J. 14, 1826-1837).
In this application, Applicant discloses eight sugarcane genes of which the expression is upregulated under drought stress. Data is provided that overexpression of these genes confers increased tolerance towards periods of drought in dicots and/or in monocots.
Therefore, in a first aspect, a chimeric gene is provided comprising the following operably linked DNA elements: a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein selected from the list consisting of a HI PP protein, a H P protein, a NRX protein, a RTNL protein, a SEC61 protein, a RNS3 protein, a ZnF protein and a TPX2 protein; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant. In a particular embodiment, a chimeric gene is provided comprising the following operably linked DNA elements: a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein selected from the list consisting of SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9 and SEQ ID No: 10; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
H IPP proteins (heavy metal-associated isoprenylated plant proteins) are metallochaperones that contain a heavy-metal-associated domain (FIMA) and a C-terminal isoprenylation motif. The heavy-metal- associated domain (FIMA, pfam00403.6) is a conserved domain of approximately 30 amino acid residues comprising two cysteine residues that are important in binding and transfer of metal ions, such as copper, cadmium, cobalt and zinc. The FIMA domain of the ScH IPP protein disclosed herein is depicted in SEQ ID No: 1: MDCEGCERRVKSAVKSMRGVTSVAVNPKQSKCTVTG.
Isoprenylation, also known as farnesylation, is a post-translational protein modification that involves addition of a C-terminal hydrophobic anchor that is important for interaction of the protein with membranes or other proteins. This occurs via covalent thioether binding of a 15-carbon farnesyl or 20- carbon geranylgeranyl group to the cysteine residue of a C-terminal CaaX motif (also known as the isoprenylation motif), where 'C' is cysteine, 'a' is an aliphatic amino acid, and 'X' is any amino acid (de
Abreu-Neto et al 2013 FEBS J 280: 1604-1616). The proteins of the H IPP family are found only in vascular plants and are suggested to be involved in biotic and abiotic stress conditions. They may be separated into five distinct clusters (de Abreu-Neto et al 2013 FEBS J 280: 1604-1616). In a particular aspect current application provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a H I PP protein comprising an isoprenylation motif and a H MA domain with a sequence identity of at least 70%, at least 75%, at least 80%, at least 83%, at least 86%, at least 88%, at least 91%, at least 94%, at least 97%, at least 98% or 100% to SEQ I D No: 1; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
In current application a H I PP protein of sugarcane in disclosed, from here on referred to as ScH IPP or SEQ I D No: 2:
MGI LDH LSH LCSITETKEALKLRKKRPLQTVN IKVKMDCEGCERRVKSAVKSMRGVTSVAVN PKQSKCTVTGYVEPAK
VLQRVKATGKNAEMWPYVPYALATYPYVGGAYDKKAPAGFVRSAPQAMADPSAPELKYMNM FNDDNVNACTVM
For the purpose of this invention "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (xlOO) divided by the number of positions compared. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch (1970) J Mol Biol. 48: 443-453). The computer-assisted sequence alignment above, can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wisconsin, USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3. Sequences are indicated as "essentially similar" when such sequence have a sequence identity of at least about 75%, particularly at least about 80 %, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially are identical.
Given the high degree of conservation within the plant kingdom and functional redundancy within plant protein families, it is obvious that chimeric genes comprising a functional homologue of ScH I PP will also lead to increased drought tolerance when expressed in plants. Therefore, in a particular aspect a chimeric gene is provided comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScHI PP or a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 2; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
Non-limiting examples of functional homologues of ScH IPP are H IPP20 from Sorghum bicolor (Sequence
I D: XP_002444859.1), heavy metal-associated isoprenylated plant protein 26-like from Zea mays
(Sequence I D: N P_001288682.1), farnesylated protein 2 from Zea mays (Sequence I D: ACG27273.1), uncharacterized protein LOC100281644 from Zea mays (Sequence ID: NP_001148035.1), Heavy metal- associated isoprenylated plant protein 20 from Dichanthelium oligosanthes (Sequence ID: OEL15368.1), hypothetical protein PAHAL_5G339300 from Panicum hallii (Sequence ID: PAN30182.1), hypothetical protein GQ55_5G309100 from Panicum hallii var. hallii (Sequence ID: PUZ56475.1), heavy metal- associated isoprenylated plant protein 20 from Setaria italica (Sequence ID: XP_004968816.1), Heavy metal-associated isoprenylated plant protein 22 from Dichanthelium oligosanthes (Sequence ID: OEL36532.1), heavy metal-associated isoprenylated plant protein 20 from Brachypodium distachyon (Sequence ID: XP_003574995.1), heavy metal-associated isoprenylated protein 3 from Triticum aestivum (Sequence ID: AIE40061.1), farnesylated protein 3 from Hordeum vulgare subsp. vulgare (Sequence ID: CAD70173.1), heavy metal-associated isoprenylated plant protein 26-like from Oryza brachyantha (Sequence ID: XP_006644215.1).
Homologs of a protein encompass peptides, oligopeptides and polypeptides having amino acid substitutions, deletions and/or insertions, preferably by a conservative change, relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived; or in other words, without significant loss of function or activity. Orthologs and paralogs, which are well-known terms by the skilled person, define subcategories of homologs and encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogs are genes within the same species that have originated through duplication of an ancestral gene; orthologs are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologues and paralogues include phylogenetic methods, sequence similarity and hybridization methods.
The HP protein of sugarcane, from here on referred to as ScHP is characterized by SEQ ID No: 3:
MSMRCALVVWLSLVVMAAATAAGDDKPEPRPVITPKPKPQPDQPKPTPQPYQPKPTPQPYQPKPQPEPGHPKPTP
QPNPTPEPTKPKPMPQPEPKPKPQPQPKPEPQPGPGKPKPKPPAYSPGTPGP.
Given the high degree of conservation within the plant kingdom and functional redundancy within plant protein families, it is obvious that chimeric genes comprising a functional homologue of ScHIPP will also lead to increased drought tolerance when expressed in plants. Therefore, in a particular aspect a chimeric gene is provided comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScHP or a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 3; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
Non-limiting examples of functional homologues of ScHP are hypothetical protein ZEAMM B73_Zm00001d031877 from Zea mays (Sequence I D: ON M03873.1), hypothetical protein Zm00014a_032104 from Zea mays (Sequence I D: PWZ57059.1), protein TsetseEP from Zea mays (Sequence I D: XP_008666540.1), hypothetical protein SORBI_3007G164500 from Sorghum bicolor (Sequence I D: EES14054.1).
N RX (nucleoredoxin) proteins belong to the Thioredoxin superfamily of proteins. Nrxs have been shown to play an interesting role as a protective mechanism of antioxidant systems controlling the status of ROS-scavenging enzymes such as catalase. However, to the best of Applicant's knowledge its overexpression has not yet been linked to increased drought tolerance in plants. N RX proteins are characterized by the presence of a short C-terminal domain rich in cysteines and histidines. This domain is referred to as the Cl domain (pfam03107). The Cl domain of ScN RX as depicted herein is SEQ I D No: 4: H RH ELSIVSDKSGGGPYICCECEEQGLGWAYQCIACGYEIH LRC.
In a particular aspect current application provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a N RX protein comprising a Cl domain with a sequence identity of at least 70%, at least 75%, at least 80%, at least 83%, at least 86%, at least 88%, at least 91%, at least 94%, at least 97% or 100% to SEQ I D No: 4; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
In current application a NRX protein of sugarcane is disclosed, from here on referred to as ScN RX or SEQ ID No: 5.
MAGDPGDAPEVGEGGIRSVLTMASLVDPSGN EVQFPEI DGKIIGLYFAANWYPKCEAFTPVLAAAYEQLKERGAGFE
VVLVSCDEDRPSFERFH RTMPWPAVPFGDLRCKKRLSERFQVEGIPRLVVLAPDGAVLH PDAADLVH RYGERAFPFT
AARVAELEADDQRKYASQTLEKLFSI NGKEYVNGGN EQVPISSLVGKTVGLYFSAN HCAPCIKFTAKLAAIYSI LKGKAE
DFEIVYVPMDKEEDGYLRSCSDMPWLALPYDGAPSRALARYFDVREIPTLVVVGPDGKTVTRDGRN LVNLYFDMAFP
FTDAQIRLLQEAEDEAAKEYPQSLRHRGH RHELSIVSDKSGGGPYICCECEEQGLGWAYQCIACGYEI HLRCGQNAEG
GSAGTA (SEQ ID No: 5).
Given the high degree of conservation within the plant kingdom and functional redundancy within plant protein families, it is obvious that chimeric genes comprising a functional homologue of ScN RX will also lead to increased drought tolerance when expressed in plants. Therefore, in a particular aspect a chimeric gene is provided comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScNRX or a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 5; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
Non-limiting examples of functional homologues of ScNRX are disulfide isomerase from Saccharum hybrid cultivar R570 (Sequence ID: AGT16827.1), nucleoredoxin 2 from Sorghum bicolor (Sequence I D: XP_002456473.1), protein disulfide isomerase from Zea mays (Sequence I D: N P_001131397.1), uncharacterized protein LOC100382831 from Zea mays (Sequence I D: N P_001169000.1), protein disulfide isomerase from Zea mays (Sequence I D: ACG38694.1), protein disulfide isomerase isoform XI from Zea mays (Sequence ID: XP_008654313.1), protein disulfide isomerase isoform X3 from Zea mays (Sequence I D: XP_008654315.1), hypothetical protein SETIT_001322mg from Setaria italica (Sequence I D: KQL07310.1), protein disulfide isomerase isoform X2 from Zea mays (Sequence I D: XP_008654314.1), nucleoredoxin 2 from Setaria italica (Sequence I D: XP_004970236.1), nucleoredoxin 2 from Zea mays (Sequence I D: PWZ09753.1), nucleoredoxin 1 from Zea mays (Sequence I D: AQK99915.1), nucleoredoxin 2 from Oryza brachyantha (Sequence ID: XP_006646403.1).
RTN L protein stands for reticulon-like protein. Proteins of the reticulon family are present in all eukaryotic organisms examined and range in size from 200 to 1,200 amino acids. The vertebrate proteins of this family are called reticulons (RTNs). Reticulon homologs from non-chordate taxa have been classified into six reticulon-like protein subfamilies (RTN L), including the plant subfamily of RTNLs named RTN LB (Oertle and Schwab 2003 Trends Cell Bio 13: 187-194). All family members contain the reticulon homology domain (RHD), a conserved region at the carboxy-terminal end of the molecule consisting of two hydrophobic regions flanking a hydrophilic loop (Yang and Strittmatter 2007 Genome Biol 8:234.1- 234.10). Very little is known about the subcellular localization and functions of RTN LBs. In this application it is disclosed that overexpression of a sugarcane RTN L (from here on referred to as ScRTN L or SEQ I D No: 6) confers increased tolerance towards drought in both monocots and dicots.
MADPAEENVASPPPTPAAPAEGASDPPLQPAADGASTEKVSAPAPEVRSRGFRLLGEDTSVHKALGGGKTADVLLW KDKKTSAVVIGGATVIWVLFEVLDYHLLTLISHVLIGVLAVLFLWSKATTFI KKSPPDIPVVQIPEDLVVNVSRALCNDI N R ALHLFREIAMGH DLKKFLFVIVGLWVNSVFGSSCDLLTLIYIAVLLLHTVPILYDKYQDKVDH FAGRAHTEALKQYEVLD AKVLSKIPRGPVKSKKQN (SEQ I D No: 6).
Therefore, in one aspect of this application a chimeric gene is provided comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 6; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant. Given the high degree of conservation within the plant kingdom and functional redundancy within plant protein families, it is obvious that chimeric genes comprising a functional homologue of ScRTNL will also lead to increased drought tolerance when expressed in plants. Hence, the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScRTNL and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant. Non-limiting examples of functional homologues of ScRTNL are reticulon-like protein B2 from Sorghum bicolor (Sequence ID: XP_002438444.1), reticulon from Zea mays (Sequence ID: ACG36034.1), uncharacterized protein LOC100191184 from Zea mays (Sequence ID: NP_001130091.1), Reticulon-like protein B2 from Zea mays (Sequence ID: PWZ16372.1), Reticulon from Zea mays (Sequence ID: AQK84022.1), hypothetical protein GQ55_4G143000 from Panicum hallii var. hallii (Sequence ID: PUZ60558.1), predicted protein from Hordeum vulgare subsp. vulgare (Sequence ID: BAJ94514.1), reticulon-like protein B2 from Oryza sativa (Sequence ID: XP_015643782.1), reticulon-like protein Bl from Brachypodium distachyon (Sequence ID: XP_003563831.1), reticulon from Hordeum vulgare (Sequence ID: ABB51093.1), reticulon-like protein B2-like from Glycine max (Sequence ID: NP_001239711.1).
TPX2 stands for Targeting Protein for Xklp2. Xklp2 is a kinesin-like protein localized on centrosomes throughout the cell cycle and on spindle pole microtubules during metaphase. TPX2 is a microtubule- associated protein that mediates the binding of the C-terminal domain of Xklp2 to microtubules. It is phosphorylated during mitosis in a microtubule-dependent way. The TPX2 family represents a conserved region (pfam06886) approximately 60 residues long within the eukaryotic targeting protein for Xklp2 (TPX2). Very little is known about TPX2 family members in plants. In this application it is disclosed that overexpression of a sugarcane TPX2 (from here on referred to as ScTPX2 or SEQ ID No: 7) confers increased tolerance towards drought in both monocots and dicots.
MAREMEMARKATSSPNKSSTIHTGPKSPVRNGGGCPPHKKNTTEPRGRKNEQQNVRKGGNQDMFSHDEGKRRSS TSQTSPKSQRSPRHEQPLSYNRLHTEERAIRRAGYNYQVASKINTQEIIRRFEEKLAQLMEEREIKLMRKEMVPKAQLM PAFDKPFHPQRSTRPLTVPKEPSFLRLKCCIGGEFHRHFCYNGANYKAIK (SEQ ID No: 7)
Therefore, in one aspect of this application a chimeric gene is provided comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID No: 7; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
Given the high degree of conservation within the plant kingdom and functional redundancy within plant protein families, it is obvious that chimeric genes comprising a functional homologue of ScTPX2 will also lead to increased drought tolerance when expressed in plants. Hence, the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScTPX2 and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant. Non-limiting examples of functional homologues of ScTPX2 are protein TPX2 from Sorghum bicolor (Sequence I D: XP_002460013.1), protein TPX2 from Setaria italic (Sequence I D: XP_004956528.1), hypothetical protein PAHAL_2G221100 from Panicum hallii (Sequence I D: PAN 11841.1), uncharacterized protein LOC100277148 from Zea mays (Sequence I D: N P_001144271.1), hypothetical protein GQ55_2G214000 from Panicum hallii var. hallii (Sequence I D: PUZ70269.1), TPX2 from Zea mays (Sequence I D: ONM21467.1).
SEC61 stands for SU PPRESSORS OF SECRETION-DEFECTIVE 61 BETA. The sugarcane SEC61 (from here on referred to as ScSEC61 or SEQ ID No: 8) confers increased tolerance towards drought in plants.
MVANGDAPARGSAAAAASLRRRRTTSGAAAAGGGASSMLQFYTDEAAGRKMSPNTVLI MSIGFIAVVAMLHVFGK LYRTSN (SEQ I D No: 8)
Therefore, in one aspect of this application a chimeric gene is provided comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 8; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
Given the high degree of conservation within the plant kingdom and functional redundancy within plant protein families, it is obvious that chimeric genes comprising a functional homologue of ScSEC61 will also lead to increased drought tolerance when expressed in plants. Hence, the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScSEC61 and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant. Non-limiting examples of functional homologues of ScSEC61 are protein SEC61 from Zea mays (Sequence I D: N P_001151680), protein SEC61 from Sorghum bicolor (Sequence I D: XP_002455808.1), protein SEC61 from Panicum hallii (Sequence I D: XP_025812251.1), protein SEC61 from Glycine max (Sequence I D: XP_003517012.1), protein SEC61 from Glycine soja (Sequence ID: RZC29809.1).
RNS3 stands for ribonuclease 3. The sugarcane RNS3 (from here on referred to as ScRNS3 or SEQ I D No: 9) confers increased tolerance towards drought in plants.
MASRIPLLCLLGLLLVASPAIADDSGIYYQLALLWPGAYCEQTSAGCCKPTTGVSPARDFYITGLTVYNATTDTAVTECS
NQAPYN PN LITGIGLEQYWSN IKCPSN NGQSSWKNAWKKAGACSGLDEKAYFEKALSFRSRI NPLVRLKKNGIQDDF ELYGLKAI KKVFKSGI NAEPVIQCSKGPFDKYMVYQLI FCANGNGTFM DCRRRRSTRAPRPSSSTPTRSGCSSRSSTTSP PSSTKPPTLSSCLAWPWTN DHCIYMPIVVDLERSHPI HI IH IPRLS (SEQ I D No: 9)
Therefore, in one aspect of this application a chimeric gene is provided comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 9; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
Given the high degree of conservation within the plant kingdom and functional redundancy within plant protein families, it is obvious that chimeric genes comprising a functional homologue of ScRNS3 will also lead to increased drought tolerance when expressed in plants. Flence, the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScRNS3 and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant. Non-limiting examples of functional homologues of ScRNS3 are extracellular ribonuclease LE from Sorghum bicolor (Sequence I D: XP_002462742.1), knotted l induced 1 precursor from Zea mays (Sequence I D: N P_001106070.2), ribonuclease 1 from Glycine max (Sequence I D: XP_003517989.1), hypothetical protein GLYMA_01G048200 from Glycine max (Sequence ID: KRH74869.1).
In current application it is demonstrated that ScZnF or SEQ I D No: 10 confers increased tolerance towards drought in plants.
MVRNQELEEVVPNDSDPLLGREN RESESSVELSPPQPASVSPSEI EDEETDGSSAACCRICLEAESEIGDELISPCMCKG TQQFVH RSCLDHWRSVKEGFAFSHCTTCKAQFHLRVETWEDNSWRKM KFRI FVARDVLLVFLAVQLTIAI IGAIAYFL DRDGSFRNSFSDGWDRFLSKH PIPFYYCIGVVVFFVLLGFFGLIVHCSSFN DNQDPCLAGCRNCCYGWGI LDCLPASLE ACFALVLVFIVVFAILGIAY (SEQ ID No: 10)
Therefore, in one aspect of this application a chimeric gene is provided comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a protein with a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ I D No: 10; and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
Given the high degree of conservation within the plant kingdom and functional redundancy within plant protein families, it is obvious that chimeric genes comprising a functional homologue of ScZnF will also lead to increased drought tolerance when expressed in plants. Flence, the application also provides a chimeric gene comprising a promoter controlling the expression of a gene in a plant; a DNA region encoding a functional homologue of ScZnF and a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant. Non-limiting examples of functional homologues of ScZnF are uncharacterized protein LOC8060005 from Sorghum bicolor (Sequence ID: XP_002466564.1), uncharacterized protein LOC100282293 from Zea mays (Sequence ID: NP_001278519.1), uncharacterized protein LOC112897867 from Panicum hallii (Sequence ID: XP_025822040.1).
For the purpose of the invention, a "chimeric gene" or "chimeric construct" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably linked to, or associated with, a nucleic acid sequence that codes for an mRNA, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of the chimeric gene is not normally operably linked to the associated nucleic acid sequence as found in nature.
The term "operably linked" as used herein refers to a linkage in which the promoter or regulatory sequence is contiguous with the gene of interest to control the gene of interest (i.e. initiate the transcription of the gene of interest), as well as a promoter that act in trans or at a distance to control the gene of interest. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter and allows transcription elongation to proceed through the DNA sequence. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or adapters or linkers inserted instead of using restriction endonucleases known to one of skill in the art.
To express nucleic acid molecules in an organism, said nucleic acid molecule must be linked operably to or comprise a suitable promoter which expresses said nucleic acid molecule at the right point in time and with the required spatial expression pattern. A promoter that enables the initiation of gene transcription in a host cell is referred to as being "active". To identify a promoter which is active in a host cell, the promoter can be operably linked to a reporter gene after which the expression level and pattern of the reporter gene can be assayed. Suitable well-known reporter genes include for example beta- glucuronidase, beta-galactosidase or any fluorescent or luminescent protein. The term "promoter activity" refers to the extent of transcription of a polynucleotide sequence, homologue, variant or fragment thereof that is operably linked to the promoter whose promoter activity is being measured. The promoter activity is assayed by measuring the enzymatic activity of the beta-glucuronidase or beta- galactosidase. Alternatively, promoter strength may also be assayed by quantifying mRNA levels or by comparing mRNA levels of the nucleic acid, with mRNA levels of housekeeping genes such as 18S rRNA, using methods known in the art, such as Northern blotting with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994). For the purpose of the chimeric genes disclosed herein, the promoter is an exogenous promoter, i.e. a regulatory nucleic acid sequence which differs from the sequence to which said nucleic acid molecule is associated in nature. This is equivalent as saying that said promoter or regulatory nucleic acid sequence to which the nucleic acid molecule is operably linked differs from the promoter or regulatory nucleic acid sequence operably linked or associated with said nucleic acid molecule in the natural environment.
A non-limiting example of an exogenous promoter for expression of a gene of interest in plants is the 35S promoter. The "35S promoter" or the "cauliflower mosaic virus (CaMV) 35S promoter" is a constitutive or constant active promoter that directs high-level expression in a wide range of cells under a wide range of conditions and in most plant tissues including monocots. Examples of other constitutive plant promoters useful for expressing heterologous, modified or non-modified polypeptides in plant cells include, but are not limited to, the plant ubiquitin (Ubi) promoter, the ethylene response factor (ERF) promoter, the nopaline synthase promoter and the octopine synthase promoter. In one aspect of the invention, the promoter being part of the chimeric genes described above is a constitutive promoter. In one particular embodiment, said promoter is not a 35S promoter. In other aspects, said promoter is a root specific promoter or a shoot specific promoter or a meristem specific promoter or a leaf specific promoter or a promoter driving expression in the growth zone of the leaves. In particular embodiments, said promoter is selected from the list consisting of Gmubil (Glymal0g39780), Gmubi2 (Glymal3gl7830.1), Gmubi3 (Glyma20g27950.1), Gmubi4 (Glymal3g24470.1), Gmubi5
(Glymal3g24500.1), Gmubi6 (Glyma07g32020.1), Gmubi7 (Glymal7g04690.1), Gmubi8
(Glymal0g05830.1), Gmubi9 (Glymal3g20200), GmubilO (Glymal5gl3650.1), GmERFl
(Glyma20gl6920.1), GmERF2 (Glyma20gl6910.1), GmERF3 (Glymallg03900.1), GmERF4
(Glyma01g41530.1), GmERF5 (Glyma05g05180.1), GmERF6 (Glyma05g05130.1), GmERF7
(Glymal9g43820.1), GmERF8 (Glyma20g34570.1), GmERF9 (Glymal0g33060.1) and GmERFlO (Glymal7gl5460.1) as referred to by Flernandez-Garcia et al 2010 (BMC Plant Biology 10:237) which is hereby inserted by reference. In other particular embodiments, said promoter is selected from the list consisting of GmCons4, GmCons6, GmConslO, GmRootl, GmRoot2, GmRoot3, GmRoot5, GmRoot6, GmRoot7, GmRoot8, GmSeed2, GmSeed3, GmSeed5, GmSeed6, GmSeed7, GmSeed8, GmSeedlO, GmSeedll, GmFABl, GmFAB2, GmFAB3, GmFAB5, GmFAB8, GmFAB9, GmFABlO, GmFABll, GmFAB17, GmWRKY13, GmWRKY17, GmWRKY21, GmWRKY27, GmWRKY43, GmWRKY54, GmWRKY67, GmWRKY79, GmWRKY80, GmWRKY82, GmWRKY85 and GmWRKY162 as referred by Gunadi et al 2016 (Plant Cell Tiss Organ Cult 127:145-160) which is hereby inserted by reference.
The term "terminator" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
In order to obtain plants with increased tolerance to periods of drought, said chimeric gene constructs are transformed or introduced and expressed in said plants.
The term "expression" or "gene expression" means the transcription of a specific gene or specific genes or specific genetic construct. The term "expression" or "gene expression" in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
The term "introduction" of genes in plants or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide or foreign genes into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microinjection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363- 373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1 102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein TM et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transformed or transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium- mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the Agrobacteria to act on plant seeds or to inoculate the plant meristem with Agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed Agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP1198985, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491 -506, 1993), Hiei et al. (Plant J 6 (2): 271 -282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al (1984) Nucl. Acids Res. 12- 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an Agrobacteria solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with Agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic (Feldman, KA and Marks MD (1987). Mol Gen Genet 208:1 -9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289). Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed Agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551 -558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an Agrobacteria suspension (Bechthold, N (1993). CR Acad Sci Paris Life Sci, 316: 1 194-1 199), while in the case of the "floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension (Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743). A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally in most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 (Nature Biotechnology 22 (2), 225-229). Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21 , 20-28. Further biotechnological progress has been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225- 229).
The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or Tl) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
A transgenic plant for the purposes of the invention is understood as meaning that the nucleic acids or chimeric gene constructs used in the method of the invention are not present in, or originating from, the genome of said plant, or are present in the genome of said plant but not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. Flowever, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein.
In order to express the chimeric gene of the application in plants, a vector or expression cassette is needed. A vector, a recombinant vector or an expression cassette comprising on of the chimeric genes from the application is herein provided. The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. The vector may be of any suitable type including, but not limited to, a phage, virus, plasmid, phagemid, cosmid, bacmid or even an artificial chromosome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. In addition to the replication system, there will frequently be (but not necessarily) at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts. The markers may a) code for protection against a biocide, such as antibiotics, toxins, heavy metals, certain sugars or the like; b) provide complementation, by imparting prototrophy to an auxotrophic host: or c) provide a visible phenotype through the production of a novel compound in the plant. Exemplary genes which may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrilase, and the gentamicin resistance gene. For plant host selection, non limiting examples of suitable markers are b-glucuronidase, providing indigo production, luciferase, providing visible light production, Green Fluorescent Protein and variants thereof, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance.
Additionally, certain preferred vectors are capable of directing the expression of certain genes of interest. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, and the like as desired and according to a particular host organism (e.g. plant cell). Typically, a recombinant vector according to the present invention comprises at least one "chimeric gene" or "expression cassette". Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof of the present invention operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as plant cells, to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell.
The term "expression cassette" refers to any recombinant expression system for the purpose of expressing a chimeric gene described above in vitro or in vivo, constitutively or inducibly, in any cell, including, in addition to plant cells, prokaryotic, yeast, fungal, insect or mammalian cells. The term includes linear and circular expression systems. The term includes all vectors. The cassettes can remain episomal or integrate into the host cell genome. The expression cassettes can have the ability to self- replicate or not (i.e., drive only transient expression in a cell). The term includes recombinant expression cassettes that contain only the minimum elements needed for transcription of the recombinant nucleic acid. Current application describes the expression of the chimeric genes disclosed herein in plants, more specifically in Arabidopsis, sugarcane or maize. Surprisingly because of said recombinant expression said plants demonstrated an increased tolerance towards periods of drought. This application therefore provides a plant, plant cell or plant seed comprising one of the chimeric genes described in the application. Given that the chimeric genes will be expressed in plant by making use of recombinant DNA technologies, said plant, plant cell or plant seed is a transgenic plant, transgenic plant cell or transgenic plant seed. The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
Applicants have demonstrated the technical effect of the chimeric genes disclosed herein in both monocotyledonous and dicotyledonous plants. Besides Arabidopsis, sugarcane and maize for which Applicant provides evidence in current application other plants are additionally useful in the methods of the application. These include fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Flelianthus spp. (e.g. Flelianthus annuus), Flemerocallis fulva, Hibiscus spp., Flordeum spp. (e.g. Flordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.
In current application, Applicant discloses that overexpression of a sugarcane gene encoding a HIPP protein, a HP protein, a NRX protein, a RTNL protein, a SEC61 protein, a RNS3 protein, a ZnF protein or a TPX2 protein (which are defined in detail by the description above) confers increased tolerance towards periods of drought both in dicots and/or in monocots. The Examples section clearly demonstrates this by using a chimeric gene construct to overexpress one of said sugarcane genes. Consequently said chimeric genes, uses thereof and methods comprising the step of expressing one of said chimeric genes are part of current disclosure. However, also envisaged in this application is a plant, seed, plant part and/or plant tissue in which the expression or the expression level of an endogenous gene encoding a HIPP protein or a functional homologue thereof, a HP protein or a functional homologue thereof, a NRX protein or a functional homologue thereof, a RTNL protein or a functional homologue thereof or a TPX2 protein or a functional homologue thereof, a SEC61 protein or a functional homologue thereof, a RNS3 protein or a functional homologue thereof or a ZnF protein or a functional homologue thereof is increased or enhanced. Said HIPP protein, HP protein, NRX protein, RTNL protein, TPX2 protein, SEC61 protein, RNS3 protein and ZnF protein are those described earlier in this application and more particularly are defined by SEQ ID No: 2, 3, 5, 6, 7, 8, 9 and 10 respectively. In one embodiment said plant, seed, plant part and/or plant tissue is a sugarcane plant, sugarcane seed, sugarcane plant part and/or sugarcane plant tissue and the endogenous genes referred to above are endogenous sugarcane genes. In one embodiment said increased or enhanced expression or expression level means an expression or expression level that is at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold or 10-fold higher than that of a control plant as defined herein. The person skilled in the art is familiar with techniques to establish a higher expression level of an endogenous gene in a plant cell, including a sugarcane plant cell. A non-limiting example is the use of CRISPR-Cas mediated genome engineering. Indeed, although the CRISPR-Cas technology is primarily known as a molecular mutagenesis tool, variants of the technology are able to modify the expression of target genes. This method of regulating and more particularly increasing the expression of endogenous genes in a plant or plant cell by using a nuclease-inactive Cas protein directly or indirectly fused to a transcription activator is well-established and the skilled person is directed to a non-exhaustive lists of documents including WO2014197568A2, WO2014197748A2, W02015006294A2 or W02014089290A1 for all details. Therefore, in a particular embodiment, a plant, seed, plant part and/or plant tissue is provided in which the expression or the expression level of an endogenous gene encoding a HIPP protein or a functional homologue thereof, a HP protein or a functional homologue thereof, a NRX protein or a functional homologue thereof, a RTNL protein or a functional homologue thereof, a TPX2 protein or a functional homologue thereof, a SEC61 protein or a functional homologue thereof, a RNS3 protein or a functional homologue thereof or a ZnF protein or a functional homologue thereof is increased or enhanced. More particularly, said plant, seed, plant part, plant tissue is genetically engineered and the expression or expression level of one of said genes encoding SEQ ID No: 2, 3, 5, 6, 7, 8, 9 or 10 or a functional homologues thereof is increased or enhanced through CRISPR-Cas technology. In a more particular embodiment, said plant, seed, plant part, plant tissue is a sugarcane plant, sugarcane seed, sugarcane plant part or sugarcane plant tissue. In another particular embodiment, said plant, seed, plant part, plant tissue is a soybean plant, soybean seed, soybean plant part or soybean plant tissue.
In another aspect, the use of one of the chimeric genes or recombinant vectors comprising said chimeric genes disclosed in the application is provided to increase drought tolerance in plants. In particular aspects, said plants are crops. In yet other particular aspects, said plants are dicotyledonous plants, even more particularly leguminous plants such as soy or soybean. In other particular aspects, said plants are C4 plants, cereals or grasses.
"C4 plants" as used herein refer to plants that use the C4 carbon fixation pathway to increase their photosynthetic efficiency by reducing or suppressing photorespiration, which mainly occurs under low atmospheric CO2 concentration, high light, high temperature, drought, and salinity. C4 carbon fixation or the Hatch-Slack pathway is a photosynthetic process in C4 plants. It is the first step in extracting carbon from carbon dioxide to be able to use it in sugar and other biomolecules. It is one of three known processes for carbon fixation. C4 refers to the 4-carbon molecule that is the first product of this type of carbon fixation. C4 fixation is an elaboration of the more common C3 carbon fixation and is believed to have evolved more recently. C4 overcomes the tendency of the enzyme RuBisCO to wastefully fix oxygen rather than carbon dioxide in the process of photorespiration. This is achieved by ensuring that RuBisCO works in an environment where there is a lot of carbon dioxide and very little oxygen. CO2 is shuttled via malate or aspartate from mesophyll cells to bundle-sheath cells. In these bundle-sheath cells CO2 is released by decarboxylation of the malate. C4 plants use PEP carboxylase to capture more CO2 in the mesophyll cells. PEP Carboxylase (3 carbons) binds to CO2 to make oxaloacetic acid (OAA). The OAA then makes malate (4 carbons). Malate enters bundle sheath cells and releases the CO2. These additional steps, however, require more energy in the form of ATP. Using this extra energy, C4 plants are able to more efficiently fix carbon in drought, high temperatures, and limitations of nitrogen or CO2. Since the more common C3 pathway does not require this extra energy, it is more efficient in the other conditions. Non-limiting example of such C4 plants are important crops such as maize, sorghum and sugarcane.
"Drought tolerance" is the degree to which a plant is adapted to or can cope with drought conditions. The term "increased drought tolerance" or "enhanced drought tolerance" as used herein refers to an enhanced ability and detectable change of the genetically modified plants described in current application (compared to wild type or control transformants) to tolerate a period of drought or low- water conditions (water deprivation/depletion leading to for example (without the purpose of limiting) visible leaf wilting symptoms in control plants, loss of turgor, or reduction of photosynthesis rate) and to recover subsequently. In most cases this will lead to a reduced overall yield loss, as more plants per m2 survive and/or the yield of the surviving plants is not significantly or less reduced compared to control plants. Methods to assess drought tolerance are described in detail in current application or are familiar to the person skilled in the art. For example drought tolerance can be assessed in controlled environments (green house or growth chambers) by placing at least about 10 transformants per transformation event and at least 10 control plants for various time periods (ranging from 1-4 weeks or more) into the environment without watering them, until leaf wilting or loss of turgor is caused on control plants, and subsequently watering the plants again for 1-2 weeks, while their recovery phenotype is analyzed. Transformants with drought tolerance survive at least 2, 3, 4, 5, 6, 7 days, preferably at least 2-5 days longer without water than control transformants (e.g. transformed with an empty vector) or wild type plants do under the same conditions, and which show irreversible tissue damage. In another method of estimating tolerance the recovery of transformants is at least about 2-5 times higher than that of the control plants (e.g. with 20% control recovery, 40-100% survival in transformants).
Drought tolerance is often linked to salt tolerance, since both are associated with regulation of osmotic potential and turgor. In particular aspects of current application, the described uses and methods to increase drought tolerance in plants are uses and methods to increase salt tolerance in plants. As drought conditions generally lower the yield of crops, drought tolerance is mostly a synonym for maintaining yield under periods of drought or reducing the reduction in yield associated with drought. Therefore, in one aspect, increased drought tolerance means a biomass production that is between 3 and 10%, between 5 and 20%, between 8 and 40%, between 12 and 45% or between 15 and 50% greater than the biomass production of non-tolerant drought stressed plants. In another aspect, increased drought tolerance means a biomass production that is at least 20%, at least 50%, at least 75% or at least 100% higher than the biomass production of non-tolerant drought stressed plants.
The terms "increase", "improve" or "enhance" are interchangeable and mean in the sense of the application at least a 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more yield and/or growth in comparison to control plants as defined herein.
Applicants disclose herein the potential of the chimeric genes described above to increase the tolerance of plants to periods of drought. The methods to obtain such plants with increased drought tolerance are thus also provided herein. More specifically, a method for producing a plant with increased drought tolerance as compared to a corresponding wild type or control plant is provided herein, said method comprises introducing one of the chimeric genes or one of the recombinant vectors comprising one of said chimeric genes in a plant or transforming a plant with one of the chimeric genes or one of the recombinant vectors comprising one of said chimeric genes, and selecting a plant with a stable expression of said chimeric gene.
Also provided herein is a method for producing a sugarcane or soybean plant with increased drought tolerance as compared to a corresponding control sugarcane plant, said method comprises increasing the expression of an endogenous gene encoding any of SEQ ID No: 2, 3, 5, 6, 7, 8, 9 or 10 or a functional homologue thereof. In a more particular embodiment, said sugarcane or soybean plant with increased drought tolerance is a genetically engineered sugarcane or soybean plant. In an even more particular embodiment, said increased expression is established using CRISPR-Cas technology.
Also provided herein is a method for producing a sugarcane plant or plant cell with increased drought tolerance as compared to a corresponding control sugarcane plant, said method comprises:
- introducing into said sugarcane plant or plant cell a first exogenous nucleic acid encoding one or more RNAs complementary to at least a part of a target DNA sequence, wherein said target DNA sequence encodes SEQ ID No: 2, 3, 5, 6, 7, 8, 9 or 10 or a functional homologue thereof,
- introducing into said sugarcane plant or plant cell a second exogenous nucleic acid encoding a nuclease-null or nuclease inactive Cas protein that binds to said target DNA sequence and is guided by the one or more RNAs, - introducing into said sugarcane plant or plant cell a third nucleic acid encoding a transcriptional regulator protein or domain,
wherein the one or more RNAs, the nuclease-null Cas protein, and the transcriptional regulator protein or domain are expressed and co-localize to said target DNA sequence and wherein the transcriptional regulator protein or domain increases the expression or expression level of a sugarcane gene encoding SEQ ID No: 2, 3, 5, 6, 7, 8, 9 or 10 or a functional homologue thereof.
In one embodiment said increased or enhanced expression or expression level means an expression or expression level that is at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold or 10-fold higher than that of a control plant as defined herein.
As used herein, "target nucleic acid" refers to a nucleic acid sequence or site within a nucleic acid molecule that is recognized and to which a guide RNA sequence is designed to target, e.g. have complementarity, where hybridization between a target nucleic acid and a guide sequence promotes the formation of a CRISPR complex.
The term "Cas protein" refers to a protein comprising a nucleic acid (e.g., RNA) binding domain and an effector domain (e.g., Cas9, such as Streptococcus pyogenes Cas9). The nucleic acid binding domains interact with a first nucleic acid molecules either having a region capable of hybridizing to a desired target nucleic acid (e.g., a guide RNA) or allows for the association with a second nucleic acid having a region capable of hybridizing to the desired target nucleic acid (e.g., a crRNA). CRISPR proteins can also comprise nuclease domains (i.e., DNase or RNase domains), additional DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, as well as other domains. Cas protein also refers to proteins that form a complex that binds the first nucleic acid molecule referred to above. As used herein "nuclease-null" or "nuclease inactive" Cas protein refers to a Cas protein that can still bind to its specific nucleic acid binding site but does not have functionality or activity anymore and thus is not able to nick or cleave the nucleic acid molecule on which it binds.
The term "exogenous" or "heterologous" as used herein refers to any material originated outside of an organism, tissue, or cell. Analogously, "endogenous", refers to substances (e.g. genes) originating from within an organism, tissue, or cell.
In another aspect, the methods of current application further comprise a step of quantifying the drought tolerance of the transformed or genetically engineered plant lines and/or a step of isolation of a plant from the population of transformed or genetically engineered plant lines with increased drought tolerance compared to a plant without said chimeric gene construct or a suitable control plant. In particular aspects, said drought tolerance is determined by measuring the relative water content, the photosynthesis rate, the stomatal conductance, the transpiration rate, the chlorophyll content and/or the biomass of said transformed and control plant lines.
The choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or even of the same variety as the plant to be assessed. The control plant may also be a nullizygote of the plant to be assessed. Nullizygotes are individuals missing the transgene by segregation. A "control plant" as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
The following examples are intended to promote a further understanding of the present invention. While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.
Examples
Example 1: Selection of sugarcane genes putatively involved in drought stress tolerance
To identify candidate genes potentially involved in drought tolerance we searched for differentially expressed genes after drought treatments in a sugarcane transcriptome dataset. First, we used a leaf microarray dataset containing the expression profiles of the leaf+1 (the highest expanded leaf with a visible dewlap) from 7-months old plants, grown under field conditions with irrigation (control) or rainfed (experiencing drought stress measured by physiological parameters). We selected 4 genes that were up- regulated by drought: ScSEC61 , ScRNS3, ScZnF and ScTpx2. Even though the sugarcane genome is not completely sequenced, we were able to obtain the full-length coding sequences and analysed their alignment with homologous sequences from different species of monocots.
Second, we mined for genes that are induced by water deficit in sugarcane roots. Four genes were selected, i.e. ScRTNL, ScNRX, ScHIPP and ScHP.
Example 2: Functional characterization using transgenic Arabidopsis plants
To get further insights on the role of the above described genes of which the expression is induced by drought, we over-expressed the sugarcane genes in Arabidopsis. To this end, the coding sequences of the sugarcane genes were cloned under control of the 35S cauliflower mosaic virus promoter and the expression cassettes were transferred to Arabidopsis. Three single copy and independent homozygous lines were generated for each selected gene and the expression of the transgene was confirmed by RT- PCR (data not shown). The transgenic lines overexpressing ScSEC61 , ScRNS3, ScZnF or ScTpx2 all showed higher survival rates compared to the lines transformed with the empty vector (EV) (Figure 1). Also the ScHIPP, ScRTNL and ScNRX genes presented a higher survival rate after 14 days of water suspension when overexpressed using the 35S promoter in Arabidopsis thaliana compared with controls (empty vector and WT) (Figure 2).
For the transgenic plants overexpressing ScRTNL and an Arabidopsis homozygous mutant (SALK_037546C) from the AT4G11220.1 gene, homologous to the RTNL gene from sugarcane the effects of mannitol on root growth were evaluated (Figure 2). Overexpression of ScRTNL in Arabidopsis did not affect root growth under non-stressed conditions, but enhanced root growth under severe stress (mannitol 100 mM), developing longer primary roots, a deeper and wider root system, and a larger root convex hull which would improve the uptake of water and nutrients, positively contributing to drought tolerance. Additionally, the silencing of a RTNL gene from Arabidopsis corroborates the importance of this gene class in the root development under stress conditions, showing significant reduction in root growth under mannitol stress represented by a decrease in all root parameters evaluated, but no alterations were noticed in non-stressed conditions.
Based on the positive results we have obtained from overexpressing above described genes in the dicot Arabidopsis we decided to overexpress the sugarcane genes in monocots as well, more precisely the C4 plants sugarcane and maize (see below).
Example 3. Overexpression of drought tolerance genes in sugarcane
Using the ZmUbil promoter, the above described sugarcane genes are overexpressed and evaluated in sugarcane plants. qRT-PCR experiments confirmed the overexpression of said genes in three single copy, independent transgenic sugarcane lines (data not shown). Next, a number of parameters and plant characteristics that are associated with drought tolerance are tested.
Below the results for the transgenic sugarcane plants overexpressing ScH IPP, ScHP and ScNRX are described.
3.1 Gas exchange parameters
In general, the results described in Figures 4-6 show a similar behavior of sugarcane plants regarding gas exchanges under drought (Figures 4, 5 and 6). At the beginning of the assay, gas exchange parameters are very close between the irrigated and drought groups, with some differences caused by genotype. Increasing time and intensity of water deficit there was a decline in photosynthesis rate (A), stomatal conductance (gs) and transpiration (£). Under water deficit, plants trigger stomatal closure in order to avoid water loss by transpiration, which results in decreased CO2 availability for photosynthesis (Cornic et al., 1992; Machado et al., 2009), causing the simultaneous gas exchange reductions observed in our results.
As drought becomes more severe, non-stomatal limitations take place, inhibiting CO2 assimilation by affecting photosynthetic machinery (Keck and Boyer, 1974; Farquhar and Sharkey, 1982; Irigoyen et al., 1992). During drought and rehydration, plant responses will depend on stress intensity and duration, as well as species and genotype (Campos et al., 2004; Xu et al., 2010; Lopes et al., 2011; Reis et al., 2014). In cases where drought is very intense, irreversible damages may occur, making full recovery after rehydration impossible as noticed for WT stressed plants in Figure 4. However concerning the transgenic ScHIPP-OE plants, photosynthesis rate (A), stomatal conductance (gs) and transpiration rate (E) returned to control (irrigated) levels, indicating complete recovery (Figure 4). Indeed, ScHIPP-OE plants presented significant higher foliar photosynthetic rate compared to WT plants from day 8 (7 days after drought treatment, DAT) until day 22 (when all plants were irrigated) and during all rehydration time. These periods were also accompanied by significant increase in stomatal conductance and transpiration during almost all the time. Figure 4 shows representative results for all three independent ScHIPP sugarcane lines. Sugarcane plants kept at pot capacity (PC) 80% (irrigated group) maintained their gas exchange parameters relatively constant within a variation range. Since we are focusing on drought response, the differences between the transgenic and control groups under irrigated conditions were not considered.
For sugarcane plants overexpressing the ScNRX gene (Figure 5), WT plants showed higher CO2 assimilation or photosynthesis rate (A) and stomatal conductance (gs) at the beginning of drought stress compared to the transgenic lines. However, ScNRX-OE plants presented significant higher photosynthetic performances as drought stress continued. Figure 5 shows representative results for all three independent ScNRX sugarcane lines.
For the HP gene (Figure 6), ScHP-OE plants demonstrated less clear but still statistically significant higher levels of A, gs and E in relation to WT during most of the time under drought stress. Figure 6shows representative results for all three independent ScHP sugarcane lines.
Taken together, these results provide evidence that drought-stressed transgenic sugarcane plants overexpressing HIPP, NRX and HP genes recover better and faster than WT plants during rehydration, after a long period of severe stress.
3.2 Relative water content, chlorophyll content and biometry
Leaf relative water status and chlorophyll levels are physiological parameters related with drought tolerance (Silva et al., 2007). These traits are evaluated in above described transgenic sugarcane plants under water deficit conditions. After 21 days of drought, ScHIPP-OE2 and 3 plants showed relative water content (RWC) of 53 and 69%, respectively, significantly higher than WT (~17%). Additionally, WT plants reduced on average 79% of RWC under drought in relation to well-watered treatment, while for ScHIPP- OE2 and 3 events, the reduction was less than half of this value (35 and 11%, respectively) (Figure 7). After nine days of rewatering, both the drought-stressed ScHIPP-OEl and 3 line kept higher RWC compared to WT, while ScHIPP-OE2 event presented values lower than WT. For the irrigated treatment, no statistical differences were observed between the transgenic and control groups (Figure 7).
Leaf water status of ScNRX-OEl (~89%) and 3 (~68%) plants was maintained at levels significantly elevated compared to control (~15%) after 17 days of drought stress, with lower RWC reduction under drought (0.5 and 29%, respectively) than WT (83%) (Figure 8).
Both sugarcane ScHP-OEl and OE2 plants showed a higher RWC than WT after 26 days of water deficit and six days of rewatering. Both ScHP-OE events showed also a better recuperation after rehydration overcoming WT water content (Figure 9).
Drought tolerant sugarcane plants tend to present higher RWC under water limitation. This parameter indicates cell hydration level, which is essential to maintain plant metabolism and growth under water deficit (Silva et al., 2007; Silva et al., 2011). Our results demonstrated that sugarcane ScNRX-OE plants have enhanced capacity to retain water under drought, which may contributes for better photosynthesis performance under drought and after rehydration observed in these plants. The overexpression of ScHP gene showed to be related with improved water retention under rewatering conditions, which is accompanied by higher CO2 assimilation rate in the same environment. Sugarcane ScHIPP-OE plants showed to be able to maintain higher RWC levels under stress, suffering less during stress, thus enabling better photosynthetic recuperation capacity after rewatering.
The determination of chlorophyll content using SPAD values is an efficient parameter to infer drought tolerance (Silva et al., 2013). Drought stress results in chlorophyll degradation which affects plant photosynthetic capacity (Arjenaki et al., 2012). Indeed, very low levels of chlorophyll were detected in stressed WT plants (e.g. Figure 7c, d) together with a gas exchange decline (Figure 4). These parameters show the profound and irreversible effects of drought confronting plants with the inability to restore their stomatal conductance, photosynthetic activity, transpiration and chlorophyll content after rehydration.
In contrast, all sugarcane ScHIPP-OE plants showed significant increase in chlorophyll content compared to WT under drought stress and rehydration (Figure 7c, d). Under stress, SPAD values of transgenic events were above 25 units, dropping 12 to 15% from irrigated to drought treatment, while WT plants presented SPAD index below 10 units and reduction of 78% between treatments. Besides high RWC, ScNRX-OEl (~23 units) and 3 (~23 units) plants also presented significantly higher estimated leaf chlorophyll content than WT (~10 units) under water deficit and relative reductions of 19% (event 1) and 39% (event 3), lower compared with WT (73%) (Figure 8b). This was accompanied by elevate photosynthesis rates during stress (Figure 5).
Transgenic sugarcane plants overexpressing ScHIPP and ScNRX genes presented better chlorophyll retention under drought and ScHIPP-OE plants also increased chlorophyll content after rehydration, which helps to keep chloroplast functionality, reducing stress damage and assisting photosynthesis recovery after drought (Augustine et al., 2015). These high SPAD indexes may contribute for improved photosynthesis observed in ScNRX-OE plants under prolonged water deficit and in ScNRX-OE and ScHIPP- OE plants after rewatering.
3.3 Growth rate measurements
Finally, important sugarcane growth characteristics more precisely stalk circumference and stalk height are evaluated during periods of drought and rewatering.
While no effect could be observed on main stalk height under drought stress, the ScFIP-OEl and 2 sugarcane events showed an increased growth rate in stalk circumference under drought compared with WT plants, which did not growth at all. The ScHP-OEl event also kept this high growth level during rehydration time (Figure 10b, d).
Example 4. Overexpression of drought tolerance genes in maize
The sugarcane genes described in Example 1 are also overexpressed and evaluated in maize plants. Constructs comprising the sugarcane gene fused to a His-tag under control of the UBI promoter were made and transformed according to Coussens et al (2012 J Exp Bot 63:4263-4273). The primary transformants were backcrossed to B104 plants and the T1 plants (segregating 1:1) were sown for phenotypic analysis. Half of the plants were watered as normal, while no water was administered to the other half. When the effects of leaf rolling were visible (approximately six days after the appearance of leaf 4) the stressed plants were rewatered with the same volumes as the well-watered plants. The length of leaf four was measured daily, allowing for leaf growth rate and duration calculations and when leaf four was fully grown, the seedling biomass was determined.
Below the results for the transgenic maize plants overexpressing ScTPX2 are described.
The final length of leaf 4 of ScTPX2 overexpressing maize plants was 9% shorter compared to WT maize plants under well-watered conditions. Flowever, this reduction disappeared when the plant were first deprived from water and subsequently rewatered again (Figure 11). This indicates that ScTPX2 overexpression corn plant are more tolerant to periods of drought. The same could be observed when biomass (seedling fresh weight) was measured. Although the ScTPX2 plants grew less under well- watered conditions, the significant reduction in biomass of WT plants due to water stress could not be observed in ScTPX2 overexpressing maize plants (Figure 12). These observed differences can be linked to the differences in leaf elongation rate between the plant lines (Figure 13). Under well-watered conditions a reduction in growth rate can be observed in the transgenic plants (vertical double arrow) compared to the control. However, upon rewatering the transgenic plants maintain growth for a longer time compared to wild-type plants (horizontal double arrow) (Figure 13).
Materials and methods
Transformation and Plant Growth Conditions
Arabidopsis thaliana ecotype Col-0 was used in this study. Seeds were incubated at 4°C in the dark during 3 or 4 days for stratification before germination. Plants were grown at 24 °C and 16 h light (cool white fluorescent; ~120 mE m-2 s-1). Transformations of Arabidopsis were performed by the floral dip method (Clough and Bent, 1998). The first primary inflorescence was clipped to favour the growth of multiple secondary inflorescences. Plants were selected by spraying a Finale solution (0.1% Finale with 0.01% Silwet L-77) every 4 days. After two weeks, transgenic plants were transferred separately in pots with soil. Leaves were collected for DNA extraction. All T1 selected plants were confirmed by PCR with primers for the bar gene, and with primers into the 35S promoter (5'CTATCCTTCGCAAGACCCTTCCT3') and into the NOS terminator pGWB608 (5'AACGATCGGGGAAATTCGAGCTC3'). In addition, at least one amplicon for each gene constructed was verified by sequencing. Plants confirmed for T-DNA integration were subsequently cultivated in Murashige-Skoog medium (Sigma-Aldrich, USA) containing 1% agar and 50mM of glufosinate-ammonium to identify single copy events, presenting a 3:1 (tolerant:sensitive) segregation ratio. The single copy events were further selected to identify homozygous transgenic lines. Seeds were surface-sterilized by the vapor-phase method. Briefly, seeds were placed in microcentrifuge tubes inside a desiccator jar containing a Beaker with 200 ml of bleach. Every hour 1 ml of hydrochloric acid was added to the bleach and the chlorine gas was maintained for five hours. For seeds sown directly in the soil the sterilization process was not used.
RT-PCR
To extract Arabidopsis RNA, T3 homozygous leaves about 3 weeks after germination were liquid nitrogen frozen and ground with two 2mm metal balls in a Retsch machine (Retsch, Germany). Trizol (Life technologies, USA) protocol was used as described by the manufacturer. The possible DNA contamination was digested with RNase-free DNase I (Qiagen, USA) and the RNA was additionally purified using RNeasy Mini Kit (Qiagen, USA). Complementary DNA was synthesized using Superscript III enzyme (Life Technologies, USA) starting with lpg of RNA. The RT-PCR was performed with ImI of cDNA using Taq polymerase (Life technologies, USA) under the following conditions: lx95°C 2 min; 35x95°C 30 sec, 60°C 30 sec, 72°C 60s/kb; lx72°C 10 min; lxl2°C final step. The primer forward used was specific for sugarcane genes and primer reverse inside the NOS terminator region for all genes (5'CCGGCAACAGGATTCAATCT 3').
Drought Stress Tolerance Tests:
Severe Stress in Soil
Seedlings were sown in separated pots (55mm) filled with jiffy-7 (Jiffypot, Netherlands). Three different T3 homozygous events for each gene and two empty vector events were randomized in the same tray (35 pots), grown under normal conditions (16h light, at 22°C). After 10 days of well-watered conditions, the weight of all pots was normalized until the maximum water capacity and the watering was withheld for approximately two weeks. The positions of the pots were randomly changed every day to avoid differential water loss among the pots. When the majority of the plants showed clear symptoms of wilting, the plants were re-watered and one day after survivors was counted.
Moderate Stress in Soil
We used the automated phenotyping platform WIWAM (Skirycz et al., 2011) to apply moderate drought stress treatment. Seeds from homozygous transgenic plants were sown directly in the pots. Initially, 32 pots per line were treated under control (well-watered) conditions of soil water content (2.2 g H20/g dry soil). After 10 days, 16 pots for each line received the moderate stress treatment (1.2 g H20/g dry soil) and 16 were maintained under well-watered conditions until day 21. After the treatments, the rosette and leaf area was measured using ImageJ (website imagej.nih.gov/ij/).
Sugarcane transformation vector
For sugarcane transformation the pGVG vector was used. Briefly, this vector presents a Gateway cassette under control of ZmUbil promoter and CaMV 35S terminator for gene overexpression or silencing. Additionally, a FLAG-tag sequence was inserted upstream the CaMV 35S terminator for C-terminal fusion with the target protein. This vector was validated using GUS staining and qRT-PCR assays and showed to be able to efficient and fast overexpression or silencing of genes in sugarcane plants. Sugarcane transformation
The meristematic region from shoot apex of six-months-old sugarcane plants (SP80-3280) was used to produce embryogenic calli. This material was cultivated in MS maintenance medium [4.33 g/L MS salts (Murashige and Skoog, 1962), 1 mL/L MS vitamins, 0.15 g/L citric acid, 0.5 g/L casein hydrolysate, 25 g/L sucrose, 12 g/L mannitol, 100 mg/L proline, 3 mg/L 2-4 dichlorophenoxyacetic acid (2,4-D) and 2.8 g/L phytagel] at 26 °C in the dark, until the generation of embryogenic calli. The selected genes already inserted into pENTR/D-TOPO vector were transferred to pGVG destination vector, using Gateway recombination. The constructs were inserted into EHA105 A. tumefaciens strain by heat shock. Bacterial cultures were incubated with sugarcane calli under vacuum pressure for five minutes and transferred to co-cultivation medium (4.33 g/L MS salts, 1 mL/L MS vitamins, 3 mg/L 2,4-D, 0.15 g/L citric acid, 25 g/L sucrose and 3.5 g/L phytagel) at 22 °C, in the dark for 3 days. After that, the calli were kept in resting medium (4.33 g/L MS salts, 1 mL/L MS vitamins, 3 mg/L 2,4-D, 0.5 g/L casein hydrolysate, 0.15 g/L citric acid, 25 g/L sucrose, 100 mg/L proline, 2.8 g/L phytagel and 200 mg/mL timentin) at 26 °C, in the dark for 6 days. Following the resting phase, the transformed calli were transferred to a selective regeneration medium [4.33 g/L MS salts, 1 mL/L MS vitamins, 25 g/L sucrose, 5 mg/mL CuS04, 1 mg/mL benzylaminopurine (BAP), 7 g/L agar, 200 mg/mL timentin and 40 mg/L geneticin] at 26 °C, during 14 days with 16 h photoperiod. The transgenic events were kept in medium without fitohormones (4.33 g/L MS salts, 1 mL/L MS vitamins, 25 g/L sucrose, 7 g/L agar, 200 mg/mL timentin and 40 mg/L geneticin) to induce growth and rooting. Plants transformed with pGVG empty vector and wild-type plants were used as negative controls.
Selection of transgenic events
Putative transgenic lines that survived to neomycin phosphotransferase (NPTII) selection were analyzed for transgene overexpression using qRT-PCR. Approximately twenty independent events from each gene were selected for initial screening, using a pool of leaves from three plants. Wild type and empty pGVG transformed plants (5 events) were used as control. The qRT-PCR analysis followed the methods previously described, except here it was used the enzyme iScript (Biorad), cDNA (diluition 1:20) and guanidine reagent for RNA extraction (Logemann, 1987). Gene relative quantification was performed using 2-AACT method and the endogenous polyubiquitin gene (PUB) (SCCCST2001G02.g) was used as internal control. The same primers used for RNA-Seq confirmation were used here. After screening of putative transgenic lines, three events with higher gene expression were selected for further analysis. For these events, qRT-PCR was repeated using leaves from three biological replicates (three individual plantlets) in technical triplicate. Drought tolerance assay in transgenic sugarcane
The selected transgenic events were transferred to 415 mL plastic pots containing substrate and vermiculite (1:1), kept in culture room at 25 °C and photoperiod 12h for two weeks and moved to the greenhouse for two more weeks, to acclimatization. After that, plants were transferred to 18 L individual pots equalized with a mixture of soil, substrate and vermiculite (65, 30 and 5%, respectively) and kept under normal irrigation for 3 months. Drought stress assay were performed with selected genes and WT as control, using a randomized complete block design. The experiment was divided in independent blocks, each one corresponding to one gene. Each block contained 40 plants: five plants to each event (three events per gene and WT) and treatment (irrigated and drought). The irrigated treatment refers to plants kept at pot capacity (PC - water content) 80% and the drought treatment, at PC 30%. Soil water percentage was calculated collecting 10 soil samples. This material was dried in drying oven at 70 °C for five days and then soaked with water until weight stabilization (PC 100%). The average weights were used to determined soil water percentage under PC 100%. On day one of the assay, sugarcane plants were soaked with water to achieve PC 100%. The pots weights average was measured and the weights for irrigated and drought treatments were inferred. From this point, the watering was controlled in the irrigated group at PC 80% and irrigation was suspended in the drought group until achieve PC 30%, being maintained in this level, until plants show signals of severe stress (photosynthesis close to zero and significant statistical differences between irrigated and drought group). After that, drought plants were rehydrated for 5-7 days. The effects of transgene overexpression on drought tolerance were evaluated by measuring several parameters: gas exchange, water relative content (RWC), chlorophyll content (SPAD value), biometric factors, shoot and root biomass and root development, comparing the control and transgenic groups under normal and stressed conditions, as described below.
Gas exchange parameters
Measurements of gas exchange, i.e., photosynthetic rate (A), stomatal conductance (gs) and transpiration rate (£), were taken during the experiment, using a portable photosynthesis system (LCi, ADC BioScientific Ltd.). The data were taken in the middle portion of leaf +1, from 9:00 am to 01:00 pm. Relative water content and chlorophyll content
The leaf relative water content (RWC) was determined in the last day of drought and rehydration using samples of leaf +2 and +1, respectively. Five 70 mm diameter leaf discs were collected from the middle portion of the leaf without midrib, immediately weighted (fresh weight - FW) and kept in distilled water overnight. Water excess of leaf discs were removed with filter paper and the turgid weight (TW) was recorded. Then, samples were dried at 60 °C for 48 h and the dry weight (DW) was measured. Leaf RWC percentage was calculated using the equation: RWC = [(FWDW)/(TW-DW)] x 100 (Barrs and Weatherley, 1962). Total chlorophyll content estimation was performed at the end of drought and rehydration in the leaf +2 and +1, respectively, using a chlorophyll meter SPAD-502Plus (Konica Minolta, Japan). Three measurements in the middle of the leaf (without midrib) were taken for each plant and the average was used in the analyses.
Biometry and biomass
Biometric agronomic traits considered yield components (stalk number, height and circumference) were taken at the beginning of the assay, end of drought and end of rehydration. Stalk circumference was measured in the base of the plant and the height was considered from base until leaf +1 insertion. The biometric data were plotted as growth rates during the stress assay. By the end of rehydration period, roots were carefully removed from the soil, washed and photographed for further root development analysis. The shoot and root of all plants were harvested, weighted (fresh weight) and dried at 60 °C for 15 days to obtain biomass. Samples of fresh root and leaf tissues (leaf +1) were collected for further biochemical and molecular analyses. Leaf +2 was collected at the end of drought stress.
Statistical analysis
Drought stress test was performed with five biological replicates. However, two outliers were identified with GraphPad Outlier calculator (Grubb's test) and excluded from statistical analysis. The data were evaluated using ANOVA (p-value < 0.05), followed by a least significant difference test (LSD, p-value < 0.05) to compare means. The analyses were conducted using the package Agricolae in R software.

Claims

1. A chimeric gene construct comprising the following operably linked DNA elements:
a. a promoter controlling the expression of a gene in a plant;
b. a DNA region encoding a sugarcane HP protein, NRX protein, HIPP protein, RTNL protein, TPX2 protein, SEC61 protein, RNS3 protein, ZnF protein or a functional homologue of one of said proteins;
c. a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of a plant.
2. The chimeric gene construct of claim 1 wherein said promoter is a constitutive promoter.
3. The chimeric gene construct of claim 1 or 2, wherein said sugarcane HP protein has at least a 90% homology to SEQ ID No. 3, said sugarcane NRX protein at least 90% homology to SEQ ID No. 5, said sugarcane HIPP protein at least 90% homology to SEQ ID No. 2, said sugarcane RTNL protein at least 90% homology to SEQ ID No. 6, said sugarcane TPX2 protein at least 90% homology to SEQ ID No. 7, said sugarcane SEC61 protein at least 90% homology to SEQ ID No. 8, said sugarcane RNS3 protein at least 90% homology to SEQ ID No. 9, said sugarcane ZnF protein at least 90% homology to SEQ ID No. 10.
4. A recombinant vector comprising the chimeric gene of any of claims 1-3.
5. A plant, plant cell or plant seed comprising the chimeric gene of any of claims 1-3 or the recombinant vector according to claim 4.
6. Use of the chimeric gene according to any of claims 1-3 or the recombinant vector according to claim 4 to increase the drought tolerance of plants.
7. The use according to claim 6, wherein the plants are crops.
8. The use according to claim 7, wherein the plants are C4 plants, cereals or grasses.
9. A method of producing a plant with increased drought tolerance as compared to a corresponding wild type plant, the method comprises introducing the chimeric gene according to any of claims 1- 3 or the recombinant vector according to claim 4 in a plant and selecting a plant with a stable expression of said chimeric gene.
10. The method of claim 9 further comprising a step of quantifying the drought tolerance of the transformed plant lines and/or a step of isolating a plant from the population of transformed plant lines with increased drought tolerance compared to a plant without said chimeric gene construct.
11. A plant, seed or plant part wherein the expression level of an endogenous gene encoding a HP protein or a functional homologue thereof is increased compared to a control plant.
2. The plant, seed or plant part of claim 11, wherein said plant, seed or plant part is genetically engineered and wherein said increased expression level is established through CRISPR-Cas technology.
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Cited By (2)

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CN110951752A (en) * 2019-12-31 2020-04-03 东北农业大学 Application of soybean biological regulation gene
CN110951752B (en) * 2019-12-31 2022-07-22 东北农业大学 Application of soybean biological regulation gene

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