WO2008155139A2 - Procédés et moyens pour la production de plantes à résistance au stress améliorée - Google Patents

Procédés et moyens pour la production de plantes à résistance au stress améliorée Download PDF

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WO2008155139A2
WO2008155139A2 PCT/EP2008/005213 EP2008005213W WO2008155139A2 WO 2008155139 A2 WO2008155139 A2 WO 2008155139A2 EP 2008005213 W EP2008005213 W EP 2008005213W WO 2008155139 A2 WO2008155139 A2 WO 2008155139A2
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plant
udp
glycosyltransferase
nucleic acid
seq
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PCT/EP2008/005213
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WO2008155139A3 (fr
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Olivier Van Aken
Frank Van Breusegem
Vanesa Tognetti
Inge De Clercq
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Vib Vzw
Universiteit Gent
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Publication of WO2008155139A3 publication Critical patent/WO2008155139A3/fr

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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
    • 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
    • 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
    • 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
    • C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—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 biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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 relates generally to the field of plant molecular biology and concerns a method for improving plant growth characteristics. More specifically, the present invention concerns a method for increasing stress resistance comprising increasing the level of activity of an auxin UDP-glycosyltransferase in a plant. The present invention also concerns plants having an increased level of activity of an auxin UDP-glycosyltransferase, which plants have increased yield and increased stress resistance relative to corresponding wild-type plants.
  • Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.
  • a trait of particular economic interest is yield.
  • Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production and more. Root development, nutrient uptake and stress tolerance are also important factors in determining yield. Optimizing one of the above mentioned factors may therefore contribute to increasing crop yield.
  • Abiotic stress conditions such as shortage or excess of solar energy, water and nutrients, salinity, high and low temperature, pollution (e.g., heavy metals), and UV-light such as UV-B radiation (280-320 nm)
  • UV-light such as UV-B radiation (280-320 nm)
  • the physiological response of a plant to these stresses arises out of changes in cellular gene expression.
  • Drought, heat, cold, and salinity constitute stresses which share a common important consequence for plant growth, which concerns water availability. Plants are exposed during their entire life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against lack of water. However, if the severity and duration of the drought conditions are too important, the effects on plant development, growth and yield of most crop plants are severe. Since high salt content in some soils result in less available water for cell intake, its effect is similar to those observed under drought conditions. Likewise, under freezing temperatures, plant cells loose water as a result of ice formation that starts in the apoplast and withdraws water from the symplast (McKersie and Leshem (1994) Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers). Generally speaking, the molecular mechanisms of a plant's response to each of these stress conditions are the same.
  • the ability to increase plant yield would have many applications in areas such as agriculture, including in the production of ornamental plants, arboriculture, horticulture and forestry. Increasing yield may also find use in the production of algae for use in bioreactors (for the biotechnological production of substances such as pharmaceuticals, antibodies or vaccines, or for the bioconversion of organic waste) and other similar areas.
  • Uridine diphosphate (UDP) glycosyltransferases mediate the transfer of glycosyl residues from the activated nucleotide sugars to acceptor molecules (aglycones), thus regulating properties of the acceptors such as their bioactivity, solubility and transport within the cell and throughout the organism.
  • UDP-glycosyltransferases form a multigene family of more than 100 different members in Arabidopsis thaliana with a least 14 distinct evolutionary groups. Moreover, the individual members of these groups have different substrate specificities (Ross et al. (2001) Genome Biology 2(2), reviews 3004.1-3004.6).
  • the inventors have surprisingly found that increasing the expression of an auxin UDP- glycosyltransferase in plants leads to plants with increased yield and increased stress resistance relative to corresponding wild-type plants.
  • One embodiment of the present invention concerns a method for increasing the yield of a plant, comprising increasing the level of activity of an auxin UDP-glycosyltransferase in said plant.
  • the plant yield comprises or consists of the biomass yield of said plant.
  • the increase in plant yield is correlated to an increase in stress resistance of said plant, in particular to an increase in the resistance against any one of abiotic stresses such as drought, heat, cold, and salinity or biotic stresses such as a pathogen infection or weed infestation.
  • another aspect of the invention is a method for increasing the stress resistance of a plant, comprising increasing the level of activity of an auxin UDP-glycosyltransferase.
  • Said stress can be a biotic stress like a pathogen infection or weed infestation or an abiotic stress like drought, heat, cold, and salinity.
  • said abiotic stress can be drought.
  • said biotic stress can be a fungal infection, in particular an infection with a necrotrophic fungal pathogen, such as Botrytis cinerea.
  • the increase in the level of activity of said auxin UDP-glycosyltransferase is effected by increasing the expression of a nucleic acid/gene encoding an auxin UDP-glycosyltransferase, in said plant.
  • the plant has been transformed with a chimeric nucleic acid comprising:
  • a nucleic acid/gene encoding an auxin UDP-glycosyltransferase can be any nucleic acid/gene:
  • the nucleic acid/gene encoding an auxin UDP-glycosyltransferase comprises any nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with one of the nucleotide sequences selected among SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17; provided that said nucleic acid/gene encodes a polypeptide exhibiting an auxin UDP- glycosyltransferase activity.
  • increasing the expression of the above- described nucleic acid/gene is carried out by introducing a genetic modification in the plant or plant cell.
  • said genetic modification consists in introducing, in a plant cell, an exogenous nucleic acid/gene encoding an auxin UDP-glycosyltransferase.
  • said genetic modification consists in introducing, in the plant cell, an exogenous nucleic acid which alters the expression of an endogenous nucleic acid/gene encoding an auxin UDP-glycosyltransferase.
  • said genetic modification consists in introducing, in the plant cell, an exogenous regulatory element which increases the expression of an endogenous nucleic acid/gene encoding an auxin UDP- glycosyltransferase.
  • Another embodiment of the present invention concerns the use of the methods of the invention to increase the stress resistance of a plant, in particular abiotic stress such as drought, heat, cold, and salinity and biotic stresses such as a pathogen infection or weed infestation.
  • abiotic stress such as drought, heat, cold, and salinity
  • biotic stresses such as a pathogen infection or weed infestation.
  • Another embodiment of the present invention concerns the use of the methods of the invention to increase the yield of seeds in crops, particularly in the case of plants subjected to stress conditions.
  • the use of the methods of the invention applies to crops such as cotton, canola, and rice.
  • Another embodiment of the present invention concerns the non-Arabidopsis thaliana plants obtainable by the methods of the present invention, such as plants comprising a chimeric nucleic acid as defined in the invention.
  • the plant is a dicotyledonous plant such as cotton or canola.
  • the plant is a monocotyledonous plant such as rice.
  • FIG. 1 Plant branching in Arabidopsis thaliana plants which over-express UGT74E2 (UGT74E2 OE ).
  • the position and size of the UGT74E2 protein are indicated by an arrowhead.
  • Arabidopsis thaliana were grown in controlled environments under 16h photoperiod of 100 ⁇ mol E m-2 s— 1. Day/night temperature and relative humidity were 22/18 °C and 60/50%, respectively. When plants were 4 weeks old, watering was withheld. The photograph was taken after 8 days of drought treatment.
  • CoI-O wild-type plants In Figure 4B, the left row are CoI-O wild-type plants, the right row are UGT74E2 OE lines.
  • FIG. 5 Water use efficiency of UGT74E2 OE Arabidopsis thaliana plants. Plants of wild-type (CoI-O) and two independent UGT74E2 OE lines (OE3 and OEl 3, indicated in picture as UGTx OE3.10 and UGTx OE 13.8) of Arabidopsis thaliana were grown under control conditions during 2 weeks. Then, wild-type and transgenic plants were separated into 2 groups. One group ('Control') was further grown under control conditions (2.00 g H 2 O/ g dry soil), a second group ('Mild (drought)') was subjected to mild drought stress conditions (1.50 g H 2 O / g dry soil) during 3,5 weeks. Next watering of both groups was stopped for 13 days.
  • FIG. 7 Disease responses of UGT74E2 OES (A), UGT74E2 OEU (C) and wild-type (B and D) plants 7 days after infection with Botrytis cinerea. Individual leaves on each plant were drop- inoculated with 20 ⁇ L (A and B) or lO ⁇ L (C and D) of a 10 6 spore suspension.
  • CoI-O designates the non-transgenic Arabidopsis thaliana ecotype Columbia plant
  • UHT74E2 OE or "OE” designates the corresponding transgenic plant lines.
  • the current invention is based on the finding that increasing the expression of a nucleic acid/gene encoding an UDP glycosyltransferase with a substrate specificity for auxins, in a plant, renders said plant more resistant to stress, particularly abiotic stress and biotic stress, in comparison to a corresponding wild-type plant.
  • the term “gene” means any DNA fragment comprising a DNA region (the "transcribed DNA region") that is transcribed into a RNA molecule (e.g., a mRNA) in a cell under control of suitable regulatory regions, e.g., a plant expressible promoter region.
  • a gene may thus comprise several operably linked DNA fragments such as a promoter, a 5' untranslated leader sequence, a coding region, and a 3' untranslated region comprising a polyadenylation site.
  • An endogenous plant gene is a gene which is naturally found in a plant species.
  • a chimeric gene is any gene which is not normally found in a plant species or, alternatively, any gene in which the promoter is not associated in nature with part or all of the transcribed DNA region (a "heterologous"
  • DNA region or with at least one other regulatory regions of the gene.
  • 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 comprise the gene/nucleic acid of interest.
  • corresponding wild-type or wild-type define a plant (or a plant cell) which differs from the plant (or plant cell) to which the methods of the invention has been applied only (or essentially only) by the fact that it does not exhibit an increased expression of an auxin UDP- glycosyltransferase nucleic acid/gene according to the invention.
  • the "corresponding wild- type” or wild-type” plant is generally an isogenic line of the plant line which has been transformed.
  • the "corresponding wild-type” or wild-type” plant has not been transformed with an exogenous nucleic acid/gene according to the invention (i.e. an exogenous nucleic acid encoding an auxin UDP-glycosyltransferase and/or an exogenous regulatory element).
  • a method for increasing the yield of a plant comprising increasing the level of activity of an auxin UDP glycosyltransferase in said plant is provided.
  • the terms “increased yield” and “increasing the yield” as defined herein are taken to mean an increase in any one or more of the following, each relative to wild-type plants: (i) increased biomass (weight) of one or more parts of a plant, particularly aboveground (harvestable) parts, increased root biomass or increased biomass of any other harvestable part; (ii) increased total seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant or on an individual seed basis; (iii) increased number of (filled) seeds; (iv) increased seed size, which may also influence the composition of seeds; (v) increased seed volume, which may also influence the composition of seeds (including oil, protein and carbohydrate total content and composition); (vi) increased oil content in the seed; (vii) increased amylase content; (viii) increased content of any other molecule of interest; (ix) increased individual seed area; (x) increased individual seed length; (xi) increased harvest index, which is expressed as a ratio of the yield of harvest
  • a yield increase may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, among others.
  • a yield increase may be manifested by an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in the seed filling rate, increase in thousand kernel weight, among others.
  • An increase in yield may also result in modified architecture, or may occur as a result of modified architecture.
  • a yield increase may be manifested by an increase in one or more of the following: number of plants per hectare or acre, number of bolls, number of branches, number of flowers, content of fiber per boll, among others.
  • a yield increase may be manifested by an increase in one or more of the following: number of plants per hectare or acre, number of pods per plant, number of inflorescences per plant, oil content per seed, among others.
  • a yield increase may be manifested by an increase in one or more of the following: number of plants per hectare or acre, number of pods per plant, oil content per seed among others.
  • the methods of the invention produces plants having increased biomass.
  • a method for increasing plant biomass comprises increasing the level of activity of an auxin UDP glycosyltransferase, in said plant.
  • the increased growth rate may specifically concern one or more parts of a plant (including seeds), or may concern substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle.
  • the life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, flowering time and speed of seed maturation.
  • An increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible (a similar effect may be obtained with earlier flowering time). If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period).
  • the growth rate may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of rice plants followed by, for example, the sowing and optional harvesting of soy bean, potato or any other suitable plant).
  • Harvesting additional times from the same rootstock in the case of some crop plants may also be possible.
  • Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre due to an increase in the number of times (for instance within a year) that any particular plant may be grown and harvested.
  • An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened.
  • the growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
  • an increase in plant yield, biomass yield, and/or growth rate occurs regardless of whether the transgenic plant obtained by the methods of the invention is under non-stress conditions or whether the plant is exposed to stress conditions.
  • the methods of the invention produces plants having an increased growth rate, under stress conditions such as abiotic stress conditions or biotic stress conditions.
  • stress conditions such as abiotic stress conditions or biotic stress conditions.
  • the method according to the present invention results in plants having increased yield, particularly seed yield, relative to corresponding wild-type plants when the plants are cultivated under stress conditions, in particular abiotic stress conditions or biotic stress conditions.
  • a method for increasing plant biomass of a plant subjected to stress conditions comprises increasing the level of activity of an auxin UDP-glycosyltransferase, in said plant.
  • non-stress conditions are those optimal environmental conditions encountered in the greenhouse.
  • stress conditions are those environmental conditions that are significantly different from those normally encountered by a plant for a given geographical location and season.
  • the methods of the invention produces plants having increased stress resistance relative to corresponding wild-type-plants.
  • another aspect of the invention concerns a method for increasing the stress resistance of a plant, which method comprises increasing the level of activity of an auxin UDP- glycosyltransferase in said plant.
  • stress resistance applied to plants, means that the plants are capable of growing normally under environmental conditions in which corresponding wild-type plants show reduced growth, metabolism, viability, productivity, vigour, and/or male or female sterility.
  • tolerance encompasses protection against stress ranging from a delay to substantially a complete inhibition of alteration in cellular metabolism, reduced cell growth and/or cell death caused by stress conditions.
  • the stress to which the plants obtained by the methods of the invention are resistant may be any biotic and/or abiotic (environmental) stress to which a plant is usually exposed.
  • Typical "abiotic" or environmental stresses include temperature stresses caused by atypical high or low temperatures (hot or cold); salt stress (salinity); water stress (drought or excess water), UV radiation stress.
  • Sunlight does not only contain radiation of the appropriate wavelengths for photosynthesis: radiation of shorter wavelengths, such as ultraviolet-B radiation (UV-B, 280-320 nm) is also present. UV-B is damaging to living organisms since cellular components such as proteins and nucleic acids absorb this energy-rich radiation.
  • UV-B radiation Due to reduction of the protecting ozone layer in the stratosphere, concern about the effects of UV-B radiation on plants has increased. Many studies have shown deleterious UV-B effects such as reduced photosynthesis, biomass reduction, decreased protein synthesis and impaired chloroplast function, as well as damage to DNA. In addition, the expression of many genes is changed by UV-B irradiation: e.g., transcription of defense genes is increased, whereas mRNA levels for chloroplastic genes decline. Chemicals may also cause abiotic stresses.
  • Biotic stresses are typically those stresses caused by pathogens or pests, such as bacteria, viruses, fungi, insects, nematodes, mycoplasms and mycoplasm-like organisms or by weeds.
  • Mild stress is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow completely and for which the plant retains its capacity to resume growth. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the typical stresses to which a plant may be exposed.
  • a method for increasing abiotic or biotic stress resistance of a plant comprises increasing the level of activity of an auxin UDP-glycosyltransferase in said plant.
  • the invention encompasses a method for increasing the plant resistance to different kinds of abiotic stresses such as, for instance, drought, excess of water, heat, cold, excess or lack of light, and UV radiation, in particular UV-B radiations.
  • abiotic stresses can be those related to water availability such as drought, heat, cold, salinity.
  • a method for increasing drought resistance of a plant which method comprises increasing the level of activity of an auxin UDP- glycosyltransferase in said plant.
  • the invention further encompasses a method for increasing the plant resistance to different kinds of biotic stresses such as weed infestations or pathogen infections such as, for instance, infections by fungi such as necotrophic fungi like Botrytis cinerea.
  • a method for increasing fungal disease resistance of a plant comprises increasing the level of activity of an auxin UDP- glycosyltransferase in said plant.
  • the methods of the invention should not be understood as being limited to an increase in resistance regarding a single stress but, advantageously, encompassing resistance regarding several stresses, for instance, an increase in resistance to drought and hot or cold, or the alternance of hot and cold.
  • a further example of resistance to more than one kind of stress is a resistance to drought and salt-stress (salinity). It should also be understood that the different kinds of stress (to which the plant is subjected) can occur simultaneously or alternatively.
  • Uridine diphosphate (UDP) glycosyltransferases (UDP-glycosyltransferases) (EC 2.4.1) mediate the transfer of a glycosyl group from a UDP-sugar to a small hydrophobic molecule.
  • the amino acid sequence of the UDP-glycosyltransferase has the following carboxy-terminal consensus sequence: [FW]-X(2)-[QL]-X(2)-[LIVMYA]-[LIMV]-X(4 I 6)-[L VG AC]-[L VF YAH M]- [LIVMF]-[STAGCM]-[HNQ]-[STAGC]-G-X(2)-[STAG]-X(3)-[STAGL]-[LIVMFA]-X(4,5)-[PQR]-
  • auxins are a class of plant growth substances often called as phytohormones or plant hormones. Auxins play an essential role in coordination of many growth and behavioral processes in the plant life cycle.
  • Naturally-occurring auxins include indole-3 -acetic acid (IAA), 4-chloro- indoleacetic acid, phenylacetic acid (PAA) and indole-butyric acid (IBA).
  • IAA indole-3-acetic acid
  • PAA phenylacetic acid
  • IBA indole-butyric acid
  • IAA indole-3-acetic acid
  • IAA indole-3-acetic acid
  • It generates the majority of auxin effects in intact plants, and is the most potent native auxin.
  • an UDP-glycosyltransferase with a substrate specificity for auxins and "an auxin UDP-glycosyltransferase” are meant to be synonymous.
  • An assay may be carried out to determine if a particular polypeptide shows an auxin UDP- glycosyltransferase activity.
  • Several assays are available in the art to determine auxin UDP- glycosyltransferase activity.
  • a suitable assay for determining auxin UDP- glycosyltransferase activity is the following one: The reaction mix in a volume of 0.1 ml contains 50 mM HEPES pH 7.6, 2.5 mM MgSO4, 10 mM KCl, 5 mM UDP-glucose, 14.4 mM 2-mercaptoethanol.
  • Samples are prepared with 1 mM of auxin, with or without 2 ⁇ g purified UDP-glycosyltransferase which has to be tested, and incubated for 3 hours at 30°C. Reactions are terminated by adding 10 ⁇ l trifluoroacetic acid. 10 ⁇ l of each sample is injected by means of a SpectraSystem ASlOOO autosampler (Thermo Separation Products, Riviera Beach, FL), cooled at 4°C, onto a reversed phase Luna Cl 8(2) column (2.1 x 150 mm, 3 ⁇ m; Phenomenex, Torrance, CA).
  • a gradient separation (SpectraSystem PlOOOXR HPLC pump, Thermo Separation Products) is run from 0.1% aqueous triethylammonium acetate (TEAA; solvent A, pH 5) to acetonitrile (0.1% TEAA;
  • MS scans (m/zl00 - m/z700) using an electrospray ionization source, operated in the negative mode, coupled to an LCQ Classic (Thermo Quest, San Jose, CA) mass spectrometer, are taken using the following conditions: spray voltage 4.5 kV; sheath gas 63 (arb); capillary temperature 265°C.
  • the auxin UDP-glycosyltransferase of the invention has a specificity for the plant auxins selected among indole-3 -butyric acid (IBA) and indole-3-acetic acid (IAA).
  • said auxin UDP-glycosyltransferase has a higher specificity for IBA than for IAA.
  • said auxin UDP-glycosyltransferase has a higher specificity for IAA than for IBA.
  • Auxin UDP-glycosyltransferase polypeptides or homologues thereof may be identified from the publicly available databases using routine techniques well known in the art, such as by sequence comparison and preferentially in combination with the determination of the substrate specificity wherein the substrate is a plant auxin.
  • Methods for the comparison of sequences are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA.
  • GAP uses the algorithm of Needleman and Wunsch (J. MoI Biol. (1970) 48: 443-453) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • the BLAST algorithm (Altschul et al. (1990) J. MoI. Biol. 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information.
  • 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. MoI Biol. 48: 443-453).
  • RNA sequences are the to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
  • “Homologues” of a polypeptide encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified polypeptide from which they are derived.
  • amino acids of the polypeptide may be replaced by other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break ⁇ -helical structures or ⁇ -sheet structures).
  • Conservative substitution tables are well known in the art (see for example
  • homologues are orthologous sequences and paralogous sequences, two special forms of homology which encompass evolutionary concepts used to describe ancestral relationships of genes.
  • paralogous relates to gene-duplications within the genome of a species leading to paralogous genes.
  • Paralogues of auxin UDP-glycosyltransferase may easily be identified by performing a BLAST analysis against a set of sequences from the same species as the query sequence.
  • orthologues in, for example, monocot plant species may easily be found by performing a so-called reciprocal blast search, in combination with the determination of the auxin substrate specificity of the UDP-glycosyltransferase. This may be done by a first blast involving blasting the sequence in question (for example, SEQ ID NO: 1 or SEQ ID NO:3) against any sequence database, such as the publicly available NCBI database which may be found at: http://www.ncbi.nlm.nih.gov.
  • BLASTn or TBLASTX may be used when starting from nucleotide sequence, or BLASTP or TBLASTN when starting from the protein, with standard default values.
  • the BLAST results may be filtered.
  • the full-length sequences of either the filtered results or the non-filtered results are then BLASTed back (second BLAST) against the sequences of the organism from which the sequence in question is derived.
  • the results of the first and second BLASTs are then compared.
  • An orthologue is found when the results of the second BLAST give as hits with the highest similarity the query auxin UDP-glycosyltransferase nucleic acid or auxin UDP-glycosyltransferase polypeptide.
  • auxin UDP-glycosyltransferase If for a specific query sequence the highest hit is a paralogue of auxin UDP-glycosyltransferase then such query sequence is also considered a homologue of auxin UDP-glycosyltransferase, provided that this homologue has a substrate specificity for auxins, hi the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize the clustering.
  • orthologues are depicted in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1, SEQ ID NO: 13, 1 SEQ ID NO: 5 and SEQ ID NO: 17.
  • auxin UDP-glycosyltransferase polypeptides which are suitable for the methods of the invention, include: the Arabidopsis thaliana amino acid sequences SEQ ID NO: 2 and 4; - the Oryza sativa amino acid sequence SEQ ID NO: 6; the Populus trichocarpa amino acid sequence SEQ ID NO: 8; - the Beta vulgaris amino acid sequence SEQ ID NO: 10; the Gossypium amino acid sequence SEQ ID NO: 12; the Vitis vinifera amino acid sequence SEQ ID NO: 14; the Zea mays amino acid sequence SEQ ID NO: 16; the Solanum tuberosum amino acid sequence SEQ ID NO: 18;
  • polypeptides falling under the definition of "auxin UDP- glycosyltransferase polypeptide or homologues thereof are not to be limited to those which amino acid sequence is represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 18, but that any UDP-glycosyltransferase polypeptide having as substrate an auxin such as IAA and IBA, is suitable for use in the methods of the invention.
  • a homologue of a polypeptide may be in the form of a "substitutional variant" of a polypeptide, i.e. where at least one residue in an amino acid sequence has been removed and a different residue inserted in its place.
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1 to 10 amino acid residues.
  • amino acid substitutions comprise conservative amino acid substitutions. Conservative substitution tables are readily available in the art. The table below gives examples of conserved amino acid substitutions.
  • a homologue of a polypeptide may also be in the form of an "insertional variant" of a polypeptide, i.e. where one or more amino acid residues are introduced into a predetermined site in a polypeptide. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues.
  • N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)- ⁇ -tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydro folate reductase, Tag- 100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
  • a transcriptional activator as used in the yeast two-hybrid system
  • phage coat proteins phage coat proteins
  • (histidine)- ⁇ -tag glutathione S-transferase-tag
  • protein A maltose-binding protein
  • dihydro folate reductase dihydro folate reductase
  • Tag- 100 epitope
  • Homologues of a polypeptide in the form of "deletion variants" of a polypeptide are characterized by the removal of one or more amino acids from a polypeptide, for instance from one to 10 amino acids.
  • Amino acid variants of a protein may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include Ml 3 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
  • auxin UDP-glycosyltransferase polypeptide or homologue thereof may be a derivative.
  • derivatives include peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise substitutions, deletions or additions of naturally and non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide.
  • Derivatives of a polypeptide encompass peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise amino acid residues, either naturally occurring or non-naturally occurring compared to the amino acid sequence of a naturally-occurring form of the polypeptide, which have been altered, glycosylated, acylated, or prenylated.
  • the auxin UDP-glycosyltransferase polypeptide or homologue thereof may be encoded by an alternative splice variant of an auxin UDP-glycosyltransferase nucleic acid/gene.
  • the term "alternative splice variant” as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the biological activity of the protein is retained, which may be achieved by selectively retaining functional segments of the protein.
  • Such splice variants may be found in nature or may be man-made. Methods for making such splice variants are known in the art.
  • the homologue of an auxin UDP-glycosyltransferase polypeptide may also be encoded by an allelic variant of a nucleic acid encoding an auxin UDP-glycosyltransferase polypeptide.
  • allelic variants exist in nature, and encompassed within the methods of the present invention is the use of the natural alleles of a nucleic acid encoding an auxin UDP-glycosyltransferase.
  • Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp.
  • auxin UDP-glycosyltransferase polypeptide or homologue thereof is encoded by an auxin UDP-glycosyltransferase nucleic acid/gene. Therefore the term "auxin UDP- glycosyltransferase nucleic acid/gene" as defined herein is any nucleic acid/gene encoding an auxin UDP-glycosyltransferase polypeptide or a homologue thereof as defined above.
  • nucleic acid/gene encoding an auxin UDP-glycosyltransferase according to the invention can be any nucleic acid/gene:
  • the nucleic acid/gene encoding an auxin UDP- glycosyltransferase according to the invention comprises a nucleic acid selected among:
  • nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with one of the nucleotide sequences selected among SEQ ID NO: 5,
  • nucleotide sequence hybridizing in stringent hybridization conditions with one of the nucleotide sequences selected among SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,
  • SEQ ID NO: 8 SEQ ID NO: 10 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or
  • SEQ ID NO: 18 any nucleic acid encoding a polypeptide which amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 2 or SEQ ID NO:
  • SEQ ID NO: 8 SEQ ID NO: 10 , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO:
  • nucleic acid/gene encodes a polypeptide exhibiting an auxin UDP- glycosyltransferase activity.
  • the nucleic acid/gene encoding an auxin UDP- glycosyltransferase comprises any nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with one of the nucleotide sequences selected among SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17; provided that said nucleic acid/gene encodes a polypeptide exhibiting an auxin UDP- glycosyltransferase activity.
  • the nucleic acid/gene encoding an auxin UDP-glycosyltransferase comprises any nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with one of the nucleotide sequences SEQ ID NO: 1 or SEQ ID NO: 3; provided that said nucleic acid/gene encodes a polypeptide exhibiting an auxin UDP- glycosyltransferase activity.
  • the nucleic acid/gene encoding an auxin UDP-glycosyltransferase comprises any nucleotide sequence encoding a polypeptide with an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with one of the amino acid sequences selected among SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18; provided that said polypeptide exhibits an auxin UDP glycosyltransferase activity.
  • the nucleic acid/gene encoding an auxin UDP-glycosyltransferase comprises any nucleotide sequence encoding a polypeptide with an amino acid sequence having at most 10, at most 5, or at most 4 amino acid differences compared to one of the amino acid sequences selected among SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18; provided that said polypeptide exhibits an auxin UDP glycosyltransferase activity.
  • the coding region of the nucleic acid/gene encoding an auxin UDP-glycosyltransferase may have been modified according to the preferred codon usage of the plant to which the methods of the invention are applied.
  • variants of auxin UDP- glycosyltransferase nucleic acids/genes are also encompassed within the methods of the invention.
  • variants of auxin UDP-glycosyltransferase nucleic acid/genes include portions of an auxin UDP-glycosyltransferase nucleic acid/gene and/or nucleic acids capable of hybridizing with an auxin UDP-glycosyltransferase nucleic acid/gene.
  • a variant of an auxin UDP-glycosyltransferase nucleic acid/gene can be a nucleic acid capable of hybridizing under reduced stringency conditions, or under stringent conditions, with an auxin UDP-glycosyltransferase nucleic acid/gene.
  • hybridization is a process wherein substantially homologous complementary nucleotide sequences anneal to each other.
  • the hybridization process can occur entirely in solution, i.e. both complementary nucleic acids are in solution.
  • the hybridization process can also occur with one of the complementary nucleic acids immobilized to a matrix such as magnetic beads, Sepharose beads or any other resin.
  • the hybridization process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilized by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips).
  • the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
  • the stringency of hybridization is influenced by conditions such as temperature, salt concentration, ionic strength and hybridization buffer composition. "Stringent hybridization conditions" as used herein means that hybridization will generally occur if there is at least 95%, for instance at least 97% sequence identity between the probe and the target sequence.
  • Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1 x SSC at approximately 65 °C.
  • Other hybridization and wash conditions are well known and are exemplified in Sambrook et ai, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989), particularly chapter 11.
  • the variant of an auxin UDP- glycosyltransferase nucleic acid/gene has a nucleic acid sequence capable of hybridizing, e.g. in stringent hybridization conditions, to nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17, or to a portion of any of the aforementioned nucleic acid sequences; provided that said nucleic acid/gene encodes a polypeptide exhibiting an auxin UDP- glycosyltransferase activity.
  • the variant of a nucleic acid/gene variant has a nucleic acid sequence capable of hybridizing to nucleic acid SEQ ID NO: 1 or SEQ ID NO: 3.
  • the expression of a nucleic acid/gene encoding an auxin UDP-glycosyltransferase polypeptide or a homologue or fragment thereof may be modulated by introducing a genetic modification in the plant and/or plant cell.
  • RNA which is biologically active i.e., which is either capable of interaction with another nucleic acid or which is capable of being translated into a biologically active polypeptide or protein.
  • a gene is said to encode an RNA when the end product of the expression of the gene is biologically active RNA, such as an antisense RNA or a ribozyme.
  • a gene is said to encode a protein when the end product of the expression of the gene is a functionally or biologically active protein or polypeptide.
  • the modulation of the expression of the nucleic acid/gene encoding an auxin UDP-glycosyltransferase according to the invention consists in increasing said expression by introducing a genetic modification in the plant and/or a plant cell.
  • Said genetic modification can consist in introducing, in a plant cell, an exogenous nucleic acid molecule such as a chimeric nucleic acid according to the invention.
  • exogenous as used herein in reference to a nucleic acid molecule and a transgenic plant cell, means a nucleic acid molecule originating from outside the plant cell.
  • An exogenous nucleic acid molecule can be, for example, the coding sequence of a nucleic acid molecule encoding an auxin UDP-glycosyltransferase or an exogenous regulatory element such as a constitutive regulatory element or an element which enhances a gene's expression.
  • An exogenous nucleic acid molecule can have a naturally occurring or non-naturally occurring nucleotide sequence and can be a heterologous nucleic acid molecule derived from a different organism or a different plant species than the plant cell into which the nucleic acid molecule is introduced or can be a nucleic acid molecule derived from the same plant species as the plant cell into which it is introduced.
  • said genetic modification consists in introducing, in a plant cell, an exogenous nucleic acid/gene encoding an auxin UDP-glycosyltransferase.
  • Said exogenous nucleic acid/gene may or may not be integrated in the plant cell's genome.
  • transient expression of the nucleic acid/gene occurs in the plant cell.
  • said exogenous nucleic acid/gene is integrated in the plant cell's genome either at the locus of an auxin UDP-glycosyltransferase gene or at a different locus.
  • the locus of a gene as defined herein is taken to mean a genomic region, which includes the gene of interest and 10 kb up- or down- stream of the coding region.
  • exogenous nucleic acid/gene encoding an auxin UDP- glycosyltransferase can be introduced in the plant cell.
  • a genetic modification consists in introducing, in the plant cell, an exogenous nucleic acid which alters the expression of at least one endogenous nucleic acid/gene encoding an auxin UDP-glycosyltransferase.
  • the alteration consists in an increase of the expression of an endogenous nucleic acid/gene encoding an auxin UDP-glycosyltransferase; and the exogenous nucleic acid is an exogenous regulatory element.
  • the genetic modification consists in introducing, in a plant cell, an exogenous nucleic acid molecule comprising an exogenous regulatory element which is integrated in an auxin UDP-glycosyltransferase gene locus in the plant cell's genome.
  • auxin UDP-glycosyltransferase there may be several genes encoding an auxin UDP-glycosyltransferase in a given plant's genome.
  • Arabidopsis thaliana possesses at least five genes encoding an auxin UDP-glycosyltransferase (UGT75B1, UGT75B2, UGT84B1, UGT84B2, UGT74E2). Therefore, at least one to possibly all of the loci, ranging from only one, two, three, four, five, or more than five of the auxin UDP-glycosyltransferase loci of the plant can have integrated the exogenous regulatory element according to the invention. Furthermore, it can also happen that the exogenous nucleic acid according to the invention is also, or only, integrated in a locus which is not that of an auxin UDP-glycosyltransferase gene.
  • the exogenous regulatory element is different from the one which naturally controls the expression of the auxin UDP-glycosyltransferase gene at the locus in which said exogenous regulatory element is integrated.
  • An exogenous regulatory element can be a constitutive regulatory element or an element which enhances the expression of a gene which is operably linked to said regulatory element.
  • An exogenous regulatory element useful in a transgenic cell plant of the invention can also be an inducible regulatory element, which is a regulatory element that confers conditional expression upon an operatively linked nucleic acid molecule, where expression of the operatively linked nucleic acid molecule is increased in the presence of a particular inducing agent or stimulus as compared to expression of the nucleic acid molecule in the absence of the inducing agent or stimulus.
  • Particularly useful inducible regulatory elements include stress-inducible regulatory elements, such as abiotic stress-inducible regulatory elements like the regulatory elements inducible by drought, cold, heat, and/or salinity.
  • Methods for increasing the expression of genes or gene products are well documented in the art and include, for example, expression driven by appropriate promoters such as constitutive promoters or inducible promoters, the use of transcription enhancers or translation enhancers.
  • Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to increase the expression of an auxin UDP-glycosyltransferase nucleic acid or variant thereof.
  • endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution, or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
  • polypeptide expression it is generally desirable to include a polyadenylation region at the 3 '-end of a polynucleo tide-coding region.
  • the polyadenylation region may be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or from any other eukaryotic gene.
  • An intron sequence may also be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature mRNA that accumulates in the cytosol.
  • Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) MoI. Cell. Biol. 8: 4395-4405 ; Callis et al. (1987) Genes Dev. 1 : 1183-1200).
  • Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit.
  • said genetic modification consists in introducing, in the plant cell, an exogenous nucleic acid/gene encoding an auxin UDP- glycosyltransferase as defined above, as well as introducing an exogenous nucleic acid which alters the expression of an endogenous nucleic acid/gene encoding an auxin UDP- glycosyltransferase as defined above.
  • the genetic modification may be introduced, for example, by any one (or more) of the following methods: transposon insertion mutagenesis, T-DNA insertion mutagenesis, T-DNA activation tagging, TILLING, site-directed mutagenesis, directed evolution, and homologous recombination; or by introducing and expressing in a plant a nucleic acid encoding an auxin UDP-glycosyltransferase polypeptide or a homologue thereof.
  • a step of selecting for modified expression of a nucleic acid encoding an auxin UDP-glycosyltransferase polypeptide or a homologue thereof which modification in expression gives plants having increased stress resistance and increased yield.
  • T-DNA activation tagging involves insertion of T-DNA, usually containing a promoter (there may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10 kb up- or down stream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene.
  • a promoter usually also be a translation enhancer or an intron
  • regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter.
  • the promoter is typically embedded in a T-DNA. This T-DNA is randomly inserted into the plant genome, for example, through Agrobacterium infection and leads to an increased expression of the genes near the inserted T-DNA.
  • the resulting transgenic plants show dominant phenotypes due to over- expression of genes close to the introduced promoter.
  • the promoter to be introduced may be any promoter capable of directing expression of a gene in the desired organism, in this case a plant.
  • constitutive, tissue-preferred, cell type-preferred and inducible promoters are all suitable for use in T-DNA activation tagging.
  • a genetic modification may also be introduced in the locus of an auxin UDP- glycosyltransferase gene using the technique of TILLING (Targeted Induced Local Lesions In Genomes).
  • TILLING Tueted Induced Local Lesions In Genomes. This is a mutagenesis technology useful to generate and/or identify, and to eventually isolate, mutagenised variants of an auxin UDP-glycosyltransferase nucleic acid/gene.
  • TILLING also allows selection of plants carrying such mutant variants. TILLING combines high-density mutagenesis with high-throughput screening methods.
  • T-DNA activation tagging, TILLING, site-directed mutagenesis and directed evolution are examples of technologies that enable the generation of novel alleles and auxin UDP- glycosyltransferase variants, in particular alleles and variants with an increased UDP- glycosylation of auxins.
  • Homologous recombination allows introduction in a genome of a selected nucleic acid at a defined selected position.
  • Homologous recombination is a standard technology used routinely in biological sciences for lower organisms such as yeast or the moss Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offringa et al. (1990) EMBO J. 9(10): 3077-84) but also for crop plants, for example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; Iida and Terada (2004) Curr. Opin. Biotech. 15(2): 132-8). General methods of homologous recombination are disclosed in patent application WO 2006/105946.
  • the nucleic acid to be introduced (which may be an auxin UDP-glycosyltransferase nucleic variant with increased enzymatic activity as hereinbefore defined) is targeted to the locus of an auxin UDP-glycosyltransferase gene.
  • a method for increasing stress resistance of a plant which method comprises increasing the specific activity of an auxin UDP-glycosyltransferase polypeptide in said plant.
  • Specific activity of an UDP-glycosyltransferase is defined as the amount of substrate the enzyme converts (reactions catalyzed), per mg of polypeptide in the enzyme preparation, per unit of time.
  • the invention also provides chimeric nucleic acids and vectors to facilitate introduction and/or expression of the nucleic acids/genes useful in the methods according to the invention.
  • a chimeric nucleic acid useful for the methods of the invention comprises:
  • a chimeric nucleic acid useful for the methods of the invention comprises the following operably- linked sequences:
  • - optionally, a 3' polyadenylation and transcript termination region.
  • Chimeric nucleic acids useful in the methods according to the present invention may be obtained using recombinant DNA technology well known to persons skilled in the art.
  • the chimeric nucleic acids may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells.
  • Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding an auxin UDP-glycosyltransferase).
  • the sequence of interest is operably linked to one or more control sequences (at least to a promoter).
  • control sequence at least to a promoter.
  • promoter at least to a promoter.
  • transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • additional regulatory elements i.e. upstream activating sequences, enhancers and silencers
  • a transcriptional regulatory sequence of a classical prokaryotic gene in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences.
  • regulatory element also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
  • operably linked refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
  • any type of promoter may be used to drive expression of the nucleic acid sequence.
  • the promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus.
  • an inducible promoter is a stress-inducible promoter, i.e. a promoter activated when a plant is exposed to various stress conditions, such as drought, salt, UV radiation, high light, cold, high temperature, pathogens, weed infestation...
  • stress-inducible when used in combination with promoters, refers herein to a promoter that is expressed predominantly under specific stress conditions and in at least one tissue or organ. Regulation of transgenes by stress-inducible promoters not only allows avoiding yield penalties provoked by a continuous over-expression, but could also increase stress tolerance and yield. Many ex'amples show that stress-inducible promoters might be useful for molecular breeding for stress tolerance (Raza Ahmad et al.
  • the promoter may be a tissue- or organ-preferential promoter, i.e. one that is capable of preferentially initiating transcription in certain tissues or organs, such as the leaves, roots, seed tissue, etc, or even a tissue- or organ-specific promoter, i.e. one that is capable of initiating transcription in certain tissues or organs only.
  • tissue- and organ-specific promoters that can be used to drive the expression of an auxin UDP- glycosyltransferase nucleic acid according to the invention are shown in the Table below:
  • the promoter is a plant-expressible promoter.
  • plant-expressible promoter means a DNA sequence that is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S promoter (" ⁇ 35S") (Hapster et al. (1988) MoI. Gen. Genet.
  • the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also tissue-specific or organ- specific promoters including but not limited to seed-specific promoters (e.g., WO89/03887), organ-primordia specific promoters (An et al. (1996) Plant Cell 8: 15-30), stem-specific promoters (Keller et al. (1988) EMBO J. 7(12): 3625-3633), leaf specific promoters (Hudspeth et al. (1989) Plant MoI. Biol.
  • mesophyl-specific promoters such as the light- inducible Rubisco promoters
  • root-specific promoters such as the light- inducible Rubisco promoters
  • tuber-specific promoters such as the tuber-specific promoters
  • vascular tissue specific promoters such as the vascular tissue specific promoters (Peleman et al. (1989) Gene 84: 359-369)
  • stamen-selective promoters WO 89/10396, WO 92/13956
  • dehiscence zone specific promoters WO 97/13865
  • the auxin UDP-glycosyltransferase nucleic acid or variant thereof can be operably linked to a constitutive promoter.
  • constitutive refers to a promoter that is expressed predominantly in at least one tissue or organ and predominantly at any life stage of the plant. The promoter can also be expressed predominantly throughout the plant.
  • a suitable constitutive promoter capable of preferentially expressing the nucleic acid throughout the plant has a comparable or identical expression profile to the 35S promoter.
  • Another suitable constitutive promoter capable of preferentially expressing the nucleic acid throughout the plant is the 35S promoter. Examples of other constitutive promoters that may also be used to drive expression of an auxin UDP-glycosyltransferase nucleic acid according to the invention are shown in Table 4 below.
  • terminator sequences may also be used in the chimeric nucleic acids introduced into a plant.
  • 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. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
  • the chimeric nucleic acid may optionally comprise a selectable marker gene.
  • selectable marker gene includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are tra ⁇ sfected or transformed with a nucleic acid construct of the invention. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection.
  • selectable marker genes include genes conferring resistance to antibiotics (such as nptll that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin), to herbicides (for example bar which provides resistance to glufosinate; aroA or gox providing resistance against glyphosate), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source).
  • Visual marker genes result in the formation of colour (for example ⁇ -glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof).
  • the present invention also encompasses plants obtainable by the methods according to the present invention.
  • the present invention therefore provides plants obtainable by the methods according to the present invention, which plants have an increased expression of auxin UDP- glycosyltransferase.
  • the invention also provides a method for the production of transgenic plants having increased yield and/or increased stress resistance, comprising introduction and expression in a plant of a chimeric nucleic acid leading to an increased expression of auxin UDP-glycosyltransferase.
  • the present invention provides a method for the production of transgenic plants having increased yield and increased stress resistance, which method comprises: (i) introducing and expressing in a plant or plant cell a chimeric nucleic acid comprising a nucleic acid encoding an auxin UDP-glycosyltransferase or variant thereof; and (ii) cultivating the plant cell under conditions promoting plant growth and development.
  • the chimeric nucleic acid of the invention may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a particular feature of the present invention, the nucleic acid is introduced into a plant by transformation.
  • transformation encompasses the transfer of an exogenous nucleic acid molecule into a host cell, irrespective of the method used for transfer.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a chimeric nucleic acid of the present invention and a whole plant regenerated therefrom.
  • 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 nucleic acid molecule 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 plants is now a routine technique.
  • any of several transformation methods may be used to introduce the nucleic acid/gene of interest into a suitable ancestor cell. 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 microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens et al. (1982) Nature 296: 72-74 ; Negrutiu et al. (1987) Plant. MoI. Biol. 8: 363-373); electroporation of protoplasts (Shillito et al. (1985) Bio/Technol.
  • Transgenic rice plants expressing an auxin UDP-glycosyltransferase nucleic acid/gene can be produced via Agrobacte ⁇ um-mediated transformation using any of the well known methods for rice transformation, such as described in any of the following: European patent application EP 1198985 Al ; Aldemita and Hodges (1996) Planta 199: 612-617 ; Chan et ⁇ l. (1993) Plant.
  • 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.
  • putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organization.
  • 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).
  • 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.
  • the present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof.
  • the present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibits the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
  • the invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stem cultures, rhizomes, tubers and bulbs.
  • the invention furthermore relates to products directly derived from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
  • Gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants comprising the chimeric nucleic acids of the present invention, which are produced by traditional breeding methods are also included within the scope of the present invention.
  • the plants obtained by the methods described herein may be further crossed by traditional breeding techniques with other plants to obtain stress tolerant progeny plants comprising the chimeric nucleic acids of the present invention.
  • a further embodiment of the present invention concerns a plant such as a non- Arabidopsis plant, comprising a chimeric nucleic acid comprising:
  • a further embodiment of the present invention concerns a plant such as a non- Arabidopsis plant, comprising a chimeric nucleic acid comprising the following operably- linked sequences:
  • the methods according to the present invention result in plants having increased yield and increased stress resistance, as described hereinbefore. These advantageous growth characteristics may also be combined with other economically advantageous traits, such as further yield-enhancing traits, further tolerance to various stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
  • the methods and means described herein are believed to be suitable for all plant cells and plants, both dicotyledonous and monocotyledonous plant cells and plants including but not limited to cotton, Brassica vegetables, oilseed rape, wheat, corn or maize, barley, alfalfa, peanuts, sunflowers, rice, oats, sugarcane, soybean, turf grasses, barley, rye, sorghum, sugar cane, vegetables (including chicory, lettuce, tomato, zucchini, bell pepper, eggplant, cucumber, melon, onion, leek), tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, but also plants used in horticulture, floriculture or forestry (poplar, fir, eucalyptus etc.).
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agropyron spp., Allium spp., Amaranthus spp., Ananas comosus, Annona spp., Apium graveolens, Arabidopsis thaliana, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena sativa, Averrhoa carambola, Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp., Cadaba farinosa, Camellia sinensis, Canna indica, Capsicum s
  • the plant is a monocotyledonous plant, such as sugar cane or rice.
  • the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.
  • nucleic acid or protein comprising a sequence of nucleotides or amino acids
  • a chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.
  • SEQ ID NO: 1 corresponds to the nucleotide sequence of A. thaliana UDP-glycsolytransferase
  • SEQ ID NO: 2 corresponds to the amino acid sequence of Arabidopsis thaliana UDP- glycsolytransferase UGT74E2.
  • SEQ ID NO: 3 corresponds to the nucleotide sequence of Arabidopsis thaliana UDP- glycosyltransferase UGT84B1.
  • SEQ ID NO: 4 corresponds to the amino acid sequence of Arabidopsis thaliana UDP- glycosyltransferase UGT84B 1.
  • SEQ ID NO: 5 corresponds to the nucleotide sequence of Oryza sativa UDP- glycosyltransferase TC272040.
  • SEQ ID NO: 6 corresponds to the amino acid sequence of Oryza sativa UDP- glycosyltransferase TC272040.
  • SEQ ID NO: 7 corresponds to the nucleotide sequence of Populus trichocarpa UDP- glycosyltransferase TC39681.
  • SEQ ID NO: 8 corresponds to the amino acid sequence of Populus trichocarpa UDP- glycosyltransferase TC39681.
  • SEQ ID NO: 9 corresponds to the nucleotide sequence of Beta vulgaris UDP- glycosyltransferase TC2334.
  • SEQ ID NO: 10 corresponds to the amino acid sequence of Beta vulgaris UDP- glycosyltransferase TC2334.
  • SEQ ID NO: 11 corresponds to the nucleotide sequence of Gossypium UDP-glucosyltransferase
  • SEQ ID NO: 12 corresponds to the amino acid sequence of Gossypium UDP- glycosyltransferase TC38948.
  • SEQ ID NO: 13 corresponds to the nucleotide sequence of Vitis vinifera UDP- glycosyltransferase TC38971.
  • SEQ ID NO: 14 corresponds to the amino acid sequence of Vitis vinifera UDP- glycosyltransferase TC38971.
  • SEQ ID NO: 15 corresponds to the nucleotide sequence of Zea mays UDP-glycosyltransferase
  • SEQ ID NO: 16 corresponds to the amino acid sequence of Zea mays UDP-glycosyltransferase
  • SEQ ID NO: 17 corresponds to the nucleotide sequence of Solarium tuberosum UDP- glycosyltransferase TCl 31428.
  • SEQ ID NO: 18 corresponds to the amino acid sequence of Solarium tuberosum UDP- glycosyltransferase TC131428.
  • a BX815725 full-length GSLT cDNA template provided by Genoscope (Centre National de Sequencage, Evry, France) was used as template for PCR reactions with PLATINUM Pfx DNA polymerase (Invitrogen).
  • the forward and reverse primers were as follows:
  • the PCR product was cloned into pDONR221 (Invitrogen).
  • UGT74E2 was cloned by recombination from the Gateway®-compatible pDONR221
  • the resulting construct was used to transform Arabidopsis thaliana CoI-O by
  • Arabidopsis thaliana CoI-O seeds were sterilized by overnight incubation with chlorine gas (100 ml 12% NaOCl and 3 ml 37% HClO). Plants were grown on 4.3 g/1 Murashige-Skoog (Duchefa, Haarlem, The Netherlands), 0.5 g/1 MES, 0.1 g/1 myo-inositol, 20 g/1 sucrose, 7 g/1 plant tissue culture agar plant tissue culture agar (LabM, Bury, UK), 0.5 mg/1 nicotinic acid, 0.5 mg/1 pyridoxin and 1 mg/1 thiamin at 22°C and 65 ⁇ E/m /s radiation in a 16-h-light/8-h-dark photoperiod.
  • Arabidopsis plants were transformed using Agrobacterium mediated floral dip transformation as described by Clo ⁇ gh and Bent (Plant J. (1998) 16:735-43). Transformants with Kanamycine resistance gene were selected on MS medium with 35 mg/1 Kanamycin (Sigma-Aldrich, St. Louis, MO, USA).
  • UGT74E2 was cloned by recombination from pDONR221 into pK7WG2 (Karimi et al. (2002) Trends Plant Sci. 7: 193-195) and transformed into Arabidopsis thaliana CoI-O by Agrobacterium tumefaciens mediated floral dipping (Clough and Bent (1998) Plant J. 16:735- 43).
  • the different transgenic lines displayed variably increased transgene expression levels (Figure IA).
  • UGT74E2 protein levels in wild-type Arabidopsis thaliana CoI-O and UGT74E2 OE3 and UGT74E2 OE13 plants were assessed by Western blot analysis on protein extracts (30 ⁇ g) from 4 weeks old leafs of the plants. Protein extracts were resolved by SDS-PAGE (12%), and analyzed by immunoblotting with rabbit antisera raised against UGT74E2 specific peptides (Eurogentec SA, Belgium). As a secondary antibody, anti-rabbit IgG antibody conjugated with horseradish peroxidase (GE Healthcare UK limited, UK) was used.
  • UGT74E2 OE leaves had a dark green color (Figure 1C). Chlorophyll content was measured and revealed a 15% increase in chlorophyll and carotene concentrations. After inflorescence emergence several independent UGT74E2 OE lines developed a clear shoot branching phenotype, in correlation with the highest expression levels of UGT74E2 ( Figures 1 A, C, D). hi the F2 progeny plants, the shoot branching phenotype again emerged and was strictly correlated with high transgene expression levels. Mature UGT74E2 OE plants were also shorter in stature than wild-type plants ( Figure 1C).
  • the primary inflorescence (the inflorescence that first arises from the rosette) is usually outgrown by its side branches in UGT74E2 OE plants. This is in contrast to wild-type plants, where the primary apex dominates its side branches. Loss of apical dominance is a phenotype often linked to altered auxin homeostasis (Estelle and Somerville
  • IBA-glc was detectable in all genotypes and it was significantly more abundant in both UGT74E2 OE lines ( Figure 2). Surprisingly, free IBA levels in UGT74E2 OE plants were also increased compared to wild-type plants ( Figure 2), suggesting that there is a complex interplay in the homeostasis of IBA and its conjugates.
  • UGT74E2 OE plants have improved salt-stress tolerance
  • wild type CoI-O and two independent UGT74E2 OE lines were subjected to high salinity stress.
  • a new set of wild type and UGT74E2 OE seeds were grown on Jiffy-7 in separate pots for 2 weeks under normal watering conditions. After 2 weeks, all pots were irrigated in a controlled manner by bringing the total weight of each pot (plastic container, soil and plant) to a target weight of 63 g with water, 2-3 times per week, to ensure that all plants received a similar watering regime. After 3.5 weeks, all pots were watered to the target weight a last time and then separated into a control group (further watering, 6 plants per line) and salt-treated group (500 mM NaCl, 6 plants per line).
  • Wild-type CoI-O and two independent transformant UGT74E2 plants were grown for 2 weeks under normal watering conditions. Then, wild-type and transgenic plants were separated into 2 groups. One group was further grown under control conditions (2.00 g H 2 O/ g dry soil), a second group was subjected to mild drought stress conditions (1.50 g H 2 O/ g dry soil) during 3,5 weeks. Soil water content was determined by weighing the soil before and after drying (4 days at 180°C). Changes in pot plus plant weight were corrected by adding water on a daily basis (Granier et al. (2006) New Phytol. 169: 623-635). After that, both groups were stopped being watered for 13 days. The total weight (pot and plant) was recorded at different time points. Both UGT74E2 OE lines showed a higher water use efficiency than the wild-type plants (Figure 5).
  • the UGT74E2 promoter is activated in the shoot after infection with Botrytis cinerea
  • the 1500 base pair upstream of the UGT74E2 start codon was cloned by recombination from pDONR221 into pBGWFS7 (Karimi et al., 2002, supra), generating a transcriptional GFP- GUS (green fluorescent protein - ⁇ -glucuronidase) fusion.
  • the construct was transformed into Arabidopsis CoI-O by Agrobacterium-mediated floral dipping (Clough and Bent (1998) Plant J. 16:735-43). Multiple transformants with a single insertion locus were selected by segregation analysis.
  • Two independent promoterUGT74E2 Two independent promoterUGT74E2: . GFP-GUS lines were grown in vitro on MS agar medium for 20 days.
  • the plants were sprayed with a suspension of 10 6 spores ml "1 in 25 M KH 2 PO 4 and 50 M glucose. GUS activity was determined 14 hours after infection.
  • Whole plantlets were harvested and incubated in 90% acetone at 4°C for one hour, washed in 10OmM Tris.HCl/50mM NaCl (pH 7.0), preincubated in 2 mM K 3 [Fe(CN) 6 ], and subsequently incubated in 4mM X-gluc (in 2 mM K 3 [Fe(CN) 6 ]) at 37°C for overnight (Beeckman and Engler, 1994). The seedlings were washed and cleared overnight in lactic acid.
  • the UGT74E2 promoter was generally little active in the shoot tissue, with only a low level of expression in the hydathodes and very particularly along the borders of the leaf base and the petiole. In contrast, the promoter is strongly activated in young and old leaves 14 hours after infection with Botrytis (Figure 6).
  • UGT74E2 E plants have decreased disease symptoms after infection with Botrytis cinerea
  • the UGT74 E plants were tested for their response to Botrytis cinerea, a necrotrophic fungal pathogen.
  • Wild-type and two independent UGT74E2 OE lines were grown in separate pots on Jiffy-7 soil in a controlled growth chamber at 21 0 C with 70 % relative humidity and 8h light/ 16h dark cycle. Plants were watered by subirrigation.
  • Botrytis cinerea was cultured on potato dextrose agar (PDA) and incubated at 18 °C. Conidia were collected from 3 -week-old cultures by placing agar slices containing fungal material in sterile water and shaking to release the spores. The suspension was passed through a filter to separate the fungal material from pieces of agar.
  • PDA potato dextrose agar
  • the UGT74E2 coding sequence was transferred by recombination from pDONR221 to the 6xHIS pDEST17 vector (Invitrogen, Carlsbad, CA, USA).
  • the expression vector was transformed into Escherichia coli BL21 DE3 pLys.
  • a culture of 100 ml LB + 100 ⁇ g/ml ampicillin was inoculated and grown overnight at 37°C and 220rpm. This culture was diluted to a total of 500 ml with OD600 of 0.125, grown for 2 hours at room temperature and 220 rpm and induced with 0.2 mM isopropyl-1-thio- ⁇ -D-galactopyranoside.
  • the reaction mix in a volume of 0.1 ml contained 50 mM HEPES pH 7.6, 2.5 mM MgSO4, 10 mM KCl, 5 mM UDP-glucose, 14.4 mM 2-mercaptoethanol.
  • Samples were prepared with 1 mM of auxin, with or without 2 ⁇ g purified UGT74E2 polypeptide, and incubated for 3 hours at 30°C. Reactions were terminated by adding 10 ⁇ l trifluoroacetic acid.
  • a SpectraSystem UV6000LP detector (Thermo Separation Products) measured UV/Vis absorption between 200 and 450 nm with a scan rate of 5 Hz.
  • Full MS scans (m/zl00 - m/z700) using an electrospray ionization source, operated in the negative mode, coupled to an LCQ Classic (Thermo Quest, San Jose, CA) mass spectrometer, were taken using the following conditions: spray voltage 4.5 kV; sheath gas 63 (arb); capillary temperature 265 0 C.

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Abstract

D'une manière générale, la présente invention porte sur le domaine de la biologie moléculaire végétale et sur un procédé permettant d'améliorer les caractéristiques de croissance végétale. Plus spécifiquement, la présente invention porte sur un procédé permettant d'augmenter la résistance au stress. Le procédé consiste à augmenter l'expression d'un acide nucléique/gène codant pour une auxine UDP-glycosyltransférase dans une plante. La présente invention concerne également des plantes munies d'une activité d'auxine UDP-glycosyltransférase accrue et présentant un rendement accru et/ou une résistance accrue au stress par rapport aux plantes sauvages correspondantes.
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CN104805058A (zh) * 2014-01-23 2015-07-29 上海欧易生物医学科技有限公司 一种稳定表达外源性glp-1基因的人脐带源间充质干细胞株
CN104818258A (zh) * 2015-03-04 2015-08-05 中国农业科学院棉花研究所 陆地棉糖基转移酶GhUGT85O1及其编码基因和应用
CN110747210A (zh) * 2019-12-09 2020-02-04 安徽农业大学 茶树糖基转移酶基因ugt91q2在提高植物抗寒性上的应用
CN113046373A (zh) * 2021-03-24 2021-06-29 中国热带农业科学院热带生物技术研究所 甘蔗UDP-糖基转移酶基因ShUGT2及其应用

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104805058A (zh) * 2014-01-23 2015-07-29 上海欧易生物医学科技有限公司 一种稳定表达外源性glp-1基因的人脐带源间充质干细胞株
CN104818258A (zh) * 2015-03-04 2015-08-05 中国农业科学院棉花研究所 陆地棉糖基转移酶GhUGT85O1及其编码基因和应用
CN110747210A (zh) * 2019-12-09 2020-02-04 安徽农业大学 茶树糖基转移酶基因ugt91q2在提高植物抗寒性上的应用
CN113046373A (zh) * 2021-03-24 2021-06-29 中国热带农业科学院热带生物技术研究所 甘蔗UDP-糖基转移酶基因ShUGT2及其应用

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