WO1999054471A1 - Sequences de polynucleotides et leur utilisation dans une methode de production de plantes a nombre accru de stomates - Google Patents

Sequences de polynucleotides et leur utilisation dans une methode de production de plantes a nombre accru de stomates Download PDF

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WO1999054471A1
WO1999054471A1 PCT/GB1999/001191 GB9901191W WO9954471A1 WO 1999054471 A1 WO1999054471 A1 WO 1999054471A1 GB 9901191 W GB9901191 W GB 9901191W WO 9954471 A1 WO9954471 A1 WO 9954471A1
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plants
polynucleotide
seq
plant
increased number
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PCT/GB1999/001191
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Frederique Marianne Van Der Lee
Peter Christiaan Sijmons
Alistair Maculloch Hetherington
Geoffrey Heys Holroyd
Julie Elizabeth Gray
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Zeneca Limited
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Priority to CA002324442A priority Critical patent/CA2324442A1/fr
Priority to EP99918107A priority patent/EP1080196A1/fr
Priority to JP2000544803A priority patent/JP2002512035A/ja
Priority to KR1020007011507A priority patent/KR20010042772A/ko
Priority to BR9909765-6A priority patent/BR9909765A/pt
Priority to AU36151/99A priority patent/AU3615199A/en
Publication of WO1999054471A1 publication Critical patent/WO1999054471A1/fr

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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • 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
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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8225—Leaf-specific, e.g. including petioles, stomata
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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention alleviates the aforesaid problems by providing plants which specifically respond to elevated carbon dioxide concentrations by increasing the number of stomata on their leaf surfaces. In the absence of other limiting factors this would be expected to increase carbon dioxide uptake for photosynthesis and present a greater effective surface area for water loss resulting in increased transpiration and thereby counteracting calcium deficiency. Data from carbon isotope discrimination studies in wheat, barley, rice and Phaseolus vulgaris indicate that genotypes with lower stomatal resistance are higher yielding.
  • This invention also relates to polynucleotide sequences and variants thereof which are capable of regulating gene expression, particularly in the stomatal guard cells.
  • genes in plants is controlled by a number of regulatory components, including nucleic acid and protein elements. Where the plant gene exists as double stranded DNA, the primary steps of expression involve the production of a messenger RNA by a polymerase enzyme. The initiation of this part of the expression process is controlled by a region commonly referred to as the "promoter".
  • the promoter lies upstream (5') of the protein encoding region and may be constitutive or tissue-specific, developmentally-regulated and/or inducible.
  • the core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence which defines the transcription start point for the structural gene.
  • the precise length of the core promoter region is indefinite but it is usually easily recognisable. Such a region is normally present, with some variation, in all promoters.
  • the base sequences lying between the various well-characterised “boxes” appear to be of lesser importance.
  • the presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. Furthermore, the core promoter region is insufficient to provide full promoter activity.
  • Manipulation of crop plants to alter and/or improve phenotypic characteristics may require the expression of heterologous genes in plant tissues.
  • Such genetic manipulation therefore relies on the availability of means to drive and to control gene expression as required; for example, on the availability and use of suitable promoters which are effective in plants and which regulate gene expression so as to give the desired effect(s) in the transgenic plant. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, plant or environment. Promoters (and other regulatory components) from bacteria, viruses, fungi and plants have been used to control gene expression in plant cells.
  • a range of naturally-occurring promoters are known to be operative in plants and have been used to drive the expression of heterologous (both foreign and endogenous) genes in plants: for example, the constitutive 35S cauliflower mosaic virus promoter, the ripening-enhanced tomato polygalacturonase promoter (Bird et al, 1988, Plant Molecular Biology, 11:651-662), the E8 promoter (Diekman & Fischer, 1988, EMBO, 7:3315-3320) and the fruit specific 2A1 1 promoter (Pear et al, 1989, Plant Molecular Biology, 13:639-651) and many others.
  • the constitutive 35S cauliflower mosaic virus promoter the ripening-enhanced tomato polygalacturonase promoter (Bird et al, 1988, Plant Molecular Biology, 11:651-662), the E8 promoter (Diekman & Fischer, 1988, EMBO, 7:3315-3320) and the fruit specific 2A1 1 promote
  • a method of producing plants with an increased number of stomata relative to control like plants comprising the steps of: (i) inhibiting in plant material the production of fatty acids which stimulate the synthesis of the 14-3-3 class of transcription factors, or otherwise preventing the fatty acids from stimulating the synthesis of the said factors; (ii) selecting the thus inhibited material; and (iii) regenerating the thus selected material into plants and selecting from the population of regenerants those plants having an increased number of stomata relative to control like plants.
  • the invention further provides a method of producing plants with an increased number of stomata relative to control like plants comprising the steps of: (i) inhibiting the function, or otherwise disrupting the activity, of an endogenous gene comprising a polynucleotide sequence depicted as SEQ LD No 1 or SEQ LD No 2 or SEQ ID No 8. (ii) selecting the thus inhibited material; (iii) regenerating the thus selected material into plants and selecting from the population of regenerants those plants having an increased number of stomata relative to control like plants.
  • the endogenous gene may comprise a polynucleotide sequence which is complementary to one which when incubated at a temperature of between 60 and 65 °C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ LD No. 1 or SEQ LD No 2 or SEQ ID No 8.
  • Also provided is a method of producing plants with an increased number of stomata relative to control like plants comprising the steps of: (i) transforming plant material with a polynucleotide comprising the sequence depicted as SEQ LD No 1 or 2 or SEQ LD No 8. (ii) selecting the thus transformed material;
  • the polynucleotide used in the above mentioned method may comprise one which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0 3 strength citrate buffered saline containing 0 1% SDS still hybridises with the sequence depicted in SEQ ID No 1 or SEQ ID No 2 or SEQ ID No 8 It is particularly preferred that the polynucleotide used in the method is in antisense orientation Plants with an increased number of stomata may be selected on the basis of a difference between non transformed control plants and the thus transformed plants when both are subjected post germination to at least one of the following: (l) elevated carbon dioxide concentration; (n) elevated calcium; (in) extremes of temperature or pressure; (IV) reduced water availability, (v) elevated environmental pollutant gases, such as ozone, oxides of nitrogen or sulphur, and (vi) elevated light
  • the present invention also provides morphologically normal fertile whole plants and the seed and progeny thereof regenerated from the material desc ⁇ bed above and having an increased number of stomata relative to a control like plants.
  • Plants transformed according to the methods of the present invention may include; soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, ⁇ ce, pine, poplar, apple, grape, vines, cucumbers, peppers, citrus and nut plants.
  • the present invention additionally provides the use of a polynucleotide comp ⁇ smg the sequence depicted as SEQ LD No 1 or SEQ ID No 2 or SEQ LD No 8 in a method of producing plants with an increased number of stomata relative to control like plants
  • Other polynucleotides which can be used in this method may compose a sequence which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by ⁇ nsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybndises with the sequence depicted in SEQ LD No.
  • the polynucleotides for use in this method may be under expression control of a plant operable promoter and may further comprise a transc ⁇ ption termination region which is downstream of the protein encoding region of the said polynucleotide.
  • the following promoters may be used: CaMV35S; FMV35S; NOS, OCS and E9. More preferably the promoter may be a stomatal guard cell specific promoter. Even more preferably the promoter may be comprised by the polynucleotide sequence depicted as SEQ ID No 2 or SEQ ID No 8. Also provided is an isolated polynucleotide comprising the sequence depicted as SEQ ID No 2 or SEQ ID No 8..
  • the present invention still further provides an expression regulatory sequence comprising the sequence depicted as SEQ ID No 2 or SEQ ID No 8. Surprisingly, it has been found that this sequence is capable of providing for expression of heterologous genes in the stomatal guard cell.
  • the regulatory sequences provided in the present invention can be used in combination with the polynucleotides and methods described above. The person skilled in the art will however, recognise that these regulatory sequences can be used in combination with any other polynucleotide in any method where transcription is particularly required in the stomatal guard cell.
  • a polynucleotide which comprises a sequence which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID No. 1, but does not so hybridise when the said temperature is between 65 and 70°C.
  • the polynucleotide may be used to produce plants which increase their stomatal index (as described below) rather than decrease it when subjected to conditions of elevated carbon dioxide concentration.
  • the polynucleotide may comprise the sequence depicted in SEQ LD No. 2 or SEQ LD No 8.
  • the protein encoding region comprised by the polynucleotide may be bounded by a plant operable promoter and terminator.
  • promoters and terminators which are per se not germane to the invention, are well known to the skilled man and include, for example, the CaMV35S, FMV35S, NOS, OCS and E9 (derived from the small subunit of RUBISCO) promoters and terminators. It is particularly preferred, however, that the protein encoding region of the polynucleotide according to the invention is under expression control of a stomatal guard cell specific promoter.
  • the skilled man understands that the term “specific” does not necessarily mean “solely restricted to” so that expression of the said sequence cannot be found anywhere else within the plant regenerated from material transformed so as to comprise such a region.
  • the invention also includes a plant transformation vector comprising the present inventive polynucleotide.
  • the protein encoding region (or a substantial part of it) may be in an anti-sense orientation when compared with that depicted in SEQ ID Nos. 1, 2 and 8, so that the RNA product of the region is capable of causing - in plant material comprising it - suppression of endogenous genes with which the protein encoding region exhibits substantial identity.
  • substantially is meant at least 70% identical when related to sequence.
  • the invention further provides the translational product encoded by the polynucleotide of the invention, particularly in the case that it has the activity of a fatty acid elongase.
  • the invention still further provides plant material which has been transformed with the polynucleotide or vector of the invention, or a polynucleotide comprising a sequence which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ LD No. 1.
  • the plant material may have been, or may subsequently be further transformed with a polynucleotide comprising a region encoding a protein capable of providing the plant with resistance or tolerance to herbicides, insects, desiccation and/or fungal, bacterial or viral infections.
  • the protein capable of providing for herbicide resistance may be selected from the group consisting of glyphosate oxido-reductase (GOX), 5-enol-pyravyl-3-phosphoshikimate synthetase (EPSPS), phosphinothricin acetyl transferase (PAT), hydroxyphenyl pyruvate dioxygenase (HPPD), glutathione S transferase (GST), cytochrome P450, Acetyl-COA carboxylase (ACCase), Acetolactate synthase (ALS), protoporphyrinogen oxidase (PROTOX), dihydropteroate synthase, polyamine transport proteins, superoxide dismutase (SOD), bromoxynil nitrilase, phytoene desaturase (PDS), the product of the tfdA gene obtainable from Alcaligenes eutrophus, and known mutagenised or otherwise modified variants of the said proteins.
  • the polynucleotide with which the plant material may be transformed may comprise 5' of the protein encoding regions which encode: (i) a peptide which is capable of targeting the translation products of the regions to plastids such as chloroplasts, mitochondria, other organelles or plant cell walls; and/or (ii) non-translated translational enhancing sequences.
  • the polynucleotide may be codon-optimised, or otherwise altered to enhance at least transcription once it is incorporated into plant material.
  • the polynucleotide used to transform the material may be modified in that mRNA instability encoding motifs and/or fortuitous splice regions may be removed, or plant preferred codons may be used so that expression of the thus modified polynucleotide in a plant yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the unmodified polynucleotide in the organism in which the protein encoding regions of the unmodified polynucleotide are endogenous, with the proviso that if - in respect of the herbicide resistance conferring regions - the thus modified polynucleotide composes plant preferred codons, the degree of identity between the protein encoding regions within the modified polynucleotide and like protein encoding regions endogenously contained within the said plant and encoding substantially the same protein is less than about 70%
  • Transformation techniques are well known and include particle mediated biohstic transformation, Agrobacterium-mediated transformation, protoplast transformation
  • the invention still further provides a morphologically normal fertile whole plant regenerated from the mateoal mentioned in the paragraph immediately preceding the last and the progeny of such plants, the seed of such plants and progeny, and parts of such plants and progeny
  • the transformed inventive plants include small grain cereals, oil seed crops, fibre plants, fruit, vegetables, plantation crops and trees.
  • Such plants include soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, oce, pine, poplar, apple, grape, cucumbers, peppers, citrus and nut plants
  • Particularly preferred parts include cut flowers.
  • the invention still further provides a method of producing plants with an increased number of stomata relative to control plants composing the steps of:
  • the production of the fatty acids, which stimulate or otherwise enhance the synthesis of the transcription factors is inhibited by either sense cosuppression of an endogenous plant gene encoding a protein involved in the biosynthetic pathway of the fatty acids, or else by anti-sense inhibition of the expression of the same gene.
  • Anti-sense inhibition techniques are well know, developed and used routinely by persons skilled in the art. Usually, the inhibition technique is effected in the plant through the production of an antisense mRNA which is complementary to and capable of hybridising with the sense mRNA produced by the endogenous gene.
  • the method of the present invention may comprise the steps of:
  • the inventive plants typically contain at least 10% more stomata than do prior art like plants. It is prefeoed that they contain at least 15% more stomata, more preferred that they contain at least 20% more stomata, still more prefeoed that they contain at least 25% more stomata, and yet still more preferred that they contain at least 30% more stomata.
  • mismatch which becomes represented in the endogenous gene via the action of DNA repair and replication enzymes.
  • the mismatch typically occurs within a region of the gene encoding the active site of an enzyme, the activity of which is consequentially abolished or at least severely curtailed.
  • any gene suppression technique can be applied.
  • the person skilled in the art is also free to use techniques available within the art to enhance the efficacy of suppression of the desired gene. On such method involves the use of an inverted repeat sequence and is described in the International Application, PCT publication number WO98/53083 which is incorporated herein by reference.
  • the skilled man may, however, prefer to transform plant material with a polynucleotide comprising a sequence which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID No. 1.
  • the plant material may be transformed with a polynucleotide which comprises a sequence which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1 % SDS still hybridises with the sequence depicted in SEQ ID No. 1, but does not so hybridise when the said temperature is between 65 and 70°C.
  • a particularly prefeoed polynucleotide for use in this method comprises the sequence depicted in SEQ ID No. 2 or in SEQ LD No 8.
  • the plants with an increased number of stomata may be selected on the basis of a difference between non transformed control plants and the thus transformed plants when both are subjected post germination to at least one of the following: (i) elevated carbon dioxide concentrations, (ii) elevated calcium; (iii) extremes of temperature or pressure; (iv) reduced water availability; (v) elevated environmental pollutant gases, such as ozone, oxides of nitrogen or sulphur, for example; (vi) elevated light conditions.
  • the said difference may be selected from the group consisting of: (i) delayed flowering; (ii) altered growth characteristics; and (iii) an elevated stomatal index.
  • the plants may be selected on the basis of resistance to an antibiotic, which resistance is produced by an antibiotic resistance conferring gene which has been co-introduced into the plant material together with genes capable of increasing the number of stomata.
  • an antibiotic which resistance is produced by an antibiotic resistance conferring gene which has been co-introduced into the plant material together with genes capable of increasing the number of stomata.
  • Stomatal index which in essence is the number of stomata in a particular area of a leave (for example) when expressed as a percentage of the total number of cells contained within that area. Stomatal index may be defined as follows:
  • SI stomatal frequency/ epidermal cell frequency + stomatal frequency X 100.
  • the invention also includes plants - and their progeny - which eventually result from the crossing of plants of the invention with control non transformed sexually compatible plants.
  • the plants or progeny may be homozygous for the transgene.
  • the invention still further includes plants which result from the method disclosed above, the progeny of such plants, the seed of such plants and progeny, and parts of such plants and progeny. Particularly prefeoed parts are fruits, flowers and seeds.
  • the invention still further includes the use of the polynucleotide or vector of the invention in a method for the production of plants which have an increased number of stomata relative to non transformed control plants.
  • the invention will be further apparent from the following description, taken together with the associated Figures and Sequence Listings.
  • SEQ LD No. 1 shows the sequence of a fatty acid elongase gene designated FAE-1 which is expressed in seeds.
  • SEQ ID No. 1 can be found on page 316 of The Plant Cell, Vol 7, 1995.
  • SEQ ID No. 2 shows the sequence of a gene which may be related to - but which is not obviously derivable from - the sequence depicted in SEQ LD No. 1.
  • SEQ LD Nos. 3 and 4 depict PCR primers which were used in the provision of the vectors pFL30 and pFL44.
  • SEQ LD Nos 5, 6 depict GUS gene primers.
  • SEQ ID No 7 depicts a primer for use with pfu polymerase.
  • SEQ LD No 8 shows the sequence of a gene which may be related to - but which is not obviously derivable from - the sequence depicted in SEQ LD No. 2.
  • Figure 1 depicts schematically the proof reading PCR DNA clone of Arabidopsis C24, line 590, which is cloned into the pGEMT vector (Promega).
  • Figure 2 depicts the vector pMOG553.
  • Figure 3 depicts schematicall the relationship between the clones pFL44 and pFL30.
  • Figure 4 depicts the pFL13 scematic drawing.
  • Figure 5 depicts the cloning scheme of pMOG1017.
  • Figure 6 depicts Arabidopsis leaves transformed with the construct pMOG1017 following histochemical staining for GUS activity in the guard cells. In this figure, the "black spots" represent the staining which is naturally blue in colour.
  • Figure 7 is a magnification of the leaf shown in figure 6 more clearly showing that the stianing is present in the stomata, more specifically in the guard cells
  • This Example illustrates the provision of a gene involved in the control of stomatal number
  • the gene was initially isolated through a promoter trap screen in Arabidopsis ecotype C24.
  • the trapping construct, pMOG553 consisted of the transcobed sequence of the ⁇ Glucuromdase enzyme synthesis gene (GUS) linked to the Hygromycin B phosphotransferase gene (hyg) both from E-coh Expression of this construct through an endogenous 'trapped' promoter allowed localisation of gene expression through staining for GUS activity and an antibiotic selection marker for isolation of mutants.
  • GUS reporter gene a primary mutant (Ml) plant (it and the progeny thereof hereinafter being designated Tag 590) exhibiting guard cell specific expression was identified, mdicaUng that a guard cell specific gene had been disrupted.
  • pMOG553 promoterless GUS on T-DNA.
  • pMOG553 is depicted schematically m Figure 2.
  • Genomic DNA was isolated from Tag 590 plant material (rosette leaves). The copy No. of the T-DNA insert was determined by restriction enzyme analysis of the said genomic DNA using EcoR V, Msc I with the 5' part of the GUS gene being used as a probe. This restriction regime yielded a single band on suitable electrophoresis. An inverse PCR was performed on the gDNA of Tag590 using the following GUS gene primers (according to the method described by Barthels, see p. 2131, Fig. 7 of that document).
  • primer 7 5'-GTA.ATG.CTC.TAC.ACC.ACG.CCG-3' (SEQ LD No. 5).
  • primerS 5'-CTT.TCC.CAC.CAA.CGC.TGA.TC-3' (SEQ LD No. 6).
  • the following primers were used to in the production of a PCR fragment which was generated on genomic DNA from original TAG590 using pfu polymerase (Proofreading capacity). This fragment was then cloned into the pGEMT making the construct labelled as pFL44.
  • GUS 9 S'-CAG.AAA.CTT.ACG.TAC.ACT.TTT.C-S' (SEQ ID 7)
  • pFL44 does not contain any recognizable promoter sequences, it has been shown that it gives specific expression when transformed to plants indicating that it comprises some tissue specific regulatory elements.
  • the PCR fragment (pFL44) has been completely sequenced on both strands. It appears that the GUS tag has inserted within a gene with extensive similarity but clearly non- identity to an Arabidopsis gene that has been previously identified by transposon tagging experiments and is designated as FAE I.
  • the sequence depicted in SEQ LD No. 2 contains at least two exons and does have putative translation initiation and termination points but appears to encode a peptide significantly shorter than FAE I (110 and 12 amino acids missing at the N- and C- terminal ends respectively). Therefore it is possible that there are additional coding regions that are not within this cloned fragment.
  • the GUS tag has inserted 3' to the second exon (180bp 3' to the putative translation stop codon) this is probably close to the end of the transcribed sequence or alternatively maybe within an intron. It is unlikely that another gene could be within this distance from the Fae-like coding sequences.
  • the cooesponding region of the genomic clone (pFL30) has been sequenced using the same Internal primers. This longer clone is sequenced with a view (i) to identifying further exons, if they exist; and (ii) to characterise the potential gene promoter regions which may lie upstream of the identified coding sequence.
  • FAE I is thought to encode a fatty acid elongase necessary for the production of very long chain fatty acids.
  • a transposon tagged FAE I mutant fails to accumulate fatty acids longer than C18 (i.e. 20:0, 20: 1 and 22: 1) in its seed.
  • FAE I is thought to encode a seed specific ketoacyl synthase which catalyses the condensation reaction with malonyl CoA.
  • a region of protein having approximately 50 amino acid in FAE I has been identified which shares some sequence similarity to regions within other plant malonyl CoA condensing enzymes (e.g. CHS and STS).
  • CHS and STS e.g. CHS and STS.
  • At the DNA level there is some similarity to 4 Arabidopsis ESTS, T6700, T44939, T44368 and ATTS 1282. These homologies lie entirely within the proposed coding regions of the SEQ ID No. 2 sequence.
  • Tag 590 gene encodes a putative fatty acid elongase which is expressed specifically in developing guard cells. This enzyme plays a role in determining stomatal density in response to altered carbon dioxide concentration (described below).
  • the plasmid pFL44 was digested with SnaBI(Located in 5' end of GUS gene) providing a 1.8 kb fragment. This fragment was cloned in the SnaBI/Smal site of pFL7 (which is a multicopy construct harbouring a promoterless GUS gene and 35S terminator) replacing 5 'end of GUS producing a construct labelled the "new multicopy construct". From this "new multicopy construct" a EcoRI/BamHI fragment harbouring the following elements was cloned in pMOG800; a Tag590 "promoter"; GUS gene and 35S terminator.
  • pMOG1017 was used for the Arabidopsis C24 transformation following which 40 transgenic lines were generated and their leaves were histochemically tested for GUS expression. 14 Lines show clearly specific expression of the GUS gene in the stomata only see photo's 1 or 2. The remaining 26 lines did not show clear expression and were thought to be low or non expressors.
  • Controlled environment chambers were used to grow plants under ambient (350- 450ppm) and elevated (ambient +650ppm) carbon dioxide. Under these conditions flower and leaf appearance, plant development and flowering time were all recorded. Wild type C24 were used as untransformed controls and 35S constitutive GUS expressors were used as GUS positive controls. Plants were transfeoed to the chambers at 16 or 35 days after exposure to lighting (dal). In the case of the 16 day age group, plants were grown on to 35 days whilst the older plants were grown to seed set before termination of the experiments. Initiation of flowering was recorded for individual plants up to 67 days. On harvest all plants were photographed.
  • Xantopren dental impression material (Dental Links Products) was used to take impressions of the abaxial epidermis (Weyers and Johansen 1985) from 2 to 3 leaves per plant, choice of leaf being based on comparative size rather than leaf number.
  • Optically clear acetone based varnish was used to make positives from the Xantopren impressions.
  • Stomatal and epidermal cell counts per unit area (9.2 ). were taken from three different parts of each positive under light microscopy at x200 magnification.
  • Guard cells are known to exhibit two distinct responses to elevated carbon dioxide (CO?).
  • CO? elevated carbon dioxide
  • the other is a developmental response and is manifested in certain species by a reduction in the number of stomata in plants grown under elevated CO?.
  • the results described below come from experiments in which the phenotype of the Tag 590 plants and C24 controls were compared under elevated and ambient CO?.
  • the objective of this experiment was to use more carefully controlled growth conditions to investigate the TAG 590 phenotype more accurately.
  • an ambient level of CO? was maintained for the duration of the experiment.
  • the CO? level was kept at 650 ppm above ambient. This was a higher concentration that used in Experiment 1 but was chosen as it more accurately reflects the CO 2 regimes employed by commercial growers.
  • the seeds were germinated in a growth room, potted on and at 45 days old were transfeoed to growth cabinets. They were then grown for a further 22 days and at the end of the experiment flowering was recorded as was stomatal number. It is important to note that stomatal number was only recorded in leaves which had grown during the experimental treatment.
  • the delayed flowering of the tagged plants in elevated CO? was seen again in this experiment. All the tagged lines flowered later than the controls in both elevated and ambient treatments (delayed by approximately 7 days).
  • the results of the stomatal number determination are summarised in Table 2 where it is apparent that stomatal number was reduced in control plants grown in elevated CO? but that in both tagged lines it was increased thus confirming the results of the previous experiment which indicated that stomatal numbers increased in response to elevated CO 2 .
  • the delay in flowering observed in the tagged mutant lines may result from a secondary effect of the gene disruption. That is, the altered stomatal density in these plants causes changes in the rate of carbon assimilation which in turn affects the initiation of the floral meristem.

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Abstract

L'invention porte sur une méthode de production de plantes à nombre accru de stomata par rapport à des plantes de référence consistant: (i) à inhiber dans le matériel végétal la production des acides gras qui stimulent la synthèse de la classe 14-3-3 de facteurs de transcription, ou sinon empêcher les acides gras de stimuler la synthèse desdits facteurs; (ii) à sélectionner le matériel ainsi inhibé; et (iii) à régénérer le matériel ainsi sélectionné dans des plantes. Ladite inhibition peut s'obtenir par co-suppression sens ou par inhibition antisens d'un gène endogène comprenant une séquence complémentaire d'une séquence incubée à une température comprise entre 60 et 65 °C dans une solution saline à 0,3 et à 0,1 % de SDS renforcée par du citrate, puis rincée par la même solution à la même température, et qui s'hybride néanmoins à la séquence représentée par SEQ ID NO 1. Les séquences préférées utilisables dans ce procédé sont représentées par SEQ ID N° 1 et 2.
PCT/GB1999/001191 1998-04-20 1999-04-19 Sequences de polynucleotides et leur utilisation dans une methode de production de plantes a nombre accru de stomates WO1999054471A1 (fr)

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CA002324442A CA2324442A1 (fr) 1998-04-20 1999-04-19 Sequences de polynucleotides et leur utilisation dans une methode de production de plantes a nombre accru de stomates
EP99918107A EP1080196A1 (fr) 1998-04-20 1999-04-19 Sequences de polynucleotides et leur utilisation dans une methode de production de plantes a nombre accru de stomates
JP2000544803A JP2002512035A (ja) 1998-04-20 1999-04-19 増加した数の気孔を有する植物を産生する方法におけるポリヌクレオチド配列およびその使用
KR1020007011507A KR20010042772A (ko) 1998-04-20 1999-04-19 폴리누클레오티드 서열 및 이들을 기공의 수가 증가된식물을 생산하는데 사용하는 방법
BR9909765-6A BR9909765A (pt) 1998-04-20 1999-04-19 Sequências de polinucleotìdios e seu uso em um processo de produção de plantas com um número aumentado de stomata
AU36151/99A AU3615199A (en) 1998-04-20 1999-04-19 Polynucleotide sequences and their use in a method of producing plants with an increased number of stomata

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GBGB9808304.1A GB9808304D0 (en) 1998-04-20 1998-04-20 Improvements in or relating to organic compounds

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WO2001011061A2 (fr) * 1999-08-04 2001-02-15 The University Of British Columbia Regulation de la transcription embryonnaire dans des plantes
WO2001090364A2 (fr) * 2000-05-24 2001-11-29 The University Of British Columbia Acide nucleique codant un enzyme biosynthetique d'acide gras possedant une chaine tres longue et appartenant a une plante
WO2001090386A2 (fr) * 2000-05-24 2001-11-29 The University Of British Columbia Region regulatrice de genes promouvant une transcription precoce specifique de graines
WO2001090387A2 (fr) * 2000-05-24 2001-11-29 The University Of British Columbia Region regulatrice de gene qui favorise la transcription specifique de la racine et utilisation de cette region
US6784342B1 (en) 1999-08-04 2004-08-31 The University Of British Columbia Regulation of embryonic transcription in plants
WO2005033319A3 (fr) * 2003-10-02 2005-08-25 Monsanto Technology Llc Amelioration de l'andainage des cultures dans les plantes transgeniques
EP2511292A1 (fr) * 2009-12-07 2012-10-17 Kyoto University Agent d'augmentation des stomates, polypeptide, méthode d'augmentation du nombre et/ou de la densité des stomates d'un végétal et méthode d'augmentation du rendement d'un végétal
US8536095B2 (en) 2008-07-03 2013-09-17 Monsanto Technology Llc Combinations of derivatized saccharide surfactants and etheramine oxide surfactants as herbicide adjuvants

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WO2007086402A1 (fr) * 2006-01-25 2007-08-02 Osaka University Facteur de régulation stomatique végétal
BR112016028069A2 (pt) * 2014-06-06 2017-10-24 Univ Cornell composições e processos para impedimento de alimentação por psilídeos
CN115820683B (zh) * 2022-09-28 2024-04-05 西南大学 家蚕Cyp9a20基因及其应用

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001011061A2 (fr) * 1999-08-04 2001-02-15 The University Of British Columbia Regulation de la transcription embryonnaire dans des plantes
WO2001011061A3 (fr) * 1999-08-04 2001-06-07 Univ British Columbia Regulation de la transcription embryonnaire dans des plantes
US6784342B1 (en) 1999-08-04 2004-08-31 The University Of British Columbia Regulation of embryonic transcription in plants
WO2001090387A2 (fr) * 2000-05-24 2001-11-29 The University Of British Columbia Region regulatrice de gene qui favorise la transcription specifique de la racine et utilisation de cette region
WO2001090386A2 (fr) * 2000-05-24 2001-11-29 The University Of British Columbia Region regulatrice de genes promouvant une transcription precoce specifique de graines
WO2001090364A3 (fr) * 2000-05-24 2002-06-13 Univ British Columbia Acide nucleique codant un enzyme biosynthetique d'acide gras possedant une chaine tres longue et appartenant a une plante
WO2001090386A3 (fr) * 2000-05-24 2002-06-20 Univ British Columbia Region regulatrice de genes promouvant une transcription precoce specifique de graines
WO2001090387A3 (fr) * 2000-05-24 2002-06-27 Univ British Columbia Region regulatrice de gene qui favorise la transcription specifique de la racine et utilisation de cette region
WO2001090364A2 (fr) * 2000-05-24 2001-11-29 The University Of British Columbia Acide nucleique codant un enzyme biosynthetique d'acide gras possedant une chaine tres longue et appartenant a une plante
WO2005033319A3 (fr) * 2003-10-02 2005-08-25 Monsanto Technology Llc Amelioration de l'andainage des cultures dans les plantes transgeniques
EP2272967A1 (fr) * 2003-10-02 2011-01-12 Monsanto Technology LLC Accumulation de traits d'amélioration de plantes agricoles dans des plantes transgéniques
US8536095B2 (en) 2008-07-03 2013-09-17 Monsanto Technology Llc Combinations of derivatized saccharide surfactants and etheramine oxide surfactants as herbicide adjuvants
US9351486B2 (en) 2008-07-03 2016-05-31 Monsanto Technology Llc Combinations of derivatized saccharide surfactants and etheramine oxide surfactants as herbicide adjuvants
EP2511292A1 (fr) * 2009-12-07 2012-10-17 Kyoto University Agent d'augmentation des stomates, polypeptide, méthode d'augmentation du nombre et/ou de la densité des stomates d'un végétal et méthode d'augmentation du rendement d'un végétal
EP2511292A4 (fr) * 2009-12-07 2013-05-01 Univ Kyoto Agent d'augmentation des stomates, polypeptide, méthode d'augmentation du nombre et/ou de la densité des stomates d'un végétal et méthode d'augmentation du rendement d'un végétal
US9115367B2 (en) 2009-12-07 2015-08-25 Kyoto University Stomata-increasing agent, polypeptide, method for increasing number and/or density of stomata in plant, and method for increasing yield of plant

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