AU782180B2 - Methods, compositions and genetic sequences for modulating flowering in plants, and plants genetically modified to flower early and tardily - Google Patents

Description

WO 01/02589 PCT/CAOO/00801 METHODS, COMPOSITIONS AND GENETIC SEQUENCES FOR MODULATING FLOWERING IN PLANTS, AND PLANTS GENETICALLY MODIFIED TO FLOWER EARLY AND TARDILY BACKGROUND OF THE INVENTION a) Field of the invention The invention relates to methods, compositions and genetic sequences to modulate flowering in plants and to plants genetically modified to flower early and plants genetically modified to flower tardily.

b) Brief description of the prior art In plants, the transition from vegetative to reproductive growth involves complex interactions between several endogenous biochemical pathways. These pathways are continuously evaluating the environmental conditions and the state of growth of the plant. When adequate conditions are met, cross-talk between pathways will ultimately result in the formation of a floral meristem.

For a long time, plant scientists have tried to control floral induction. The results of grafting experiments performed about 70 years ago led to the proposal of a hypothesis which states that a flower inducer, i.e. "a florigen", is synthesized in the leaves and translocated to the shoot apex to induce the development of the flower meristem. However, despite considerable research efforts, the search for the hypothetical "florigen" hormone was unsuccessful. Molecules such as cytokinins, gibberellins and carbon assimilates have also been proposed to act as flowering promoters in some species For instance; U.S. patents Nns .,523.28?R1 6,020,288 and 6,057,157 disclose various methods and compositions for inducing, accelerating and prolonging flowering in plants or enhancing their growth.

However, some of the molecules or compositions described in these patents were found to be inactive or even inhibitory to flower formation in other plant species.

Some others are also known to affect the biomass or the plant morphology.

12-hydroxyjasmonic acid (see Fig. 1A) is a natural metabolite and was first isolated from the leaves of Solanum tuberosum (potato) (Yoshihara et al. (1989), WO 01/02589 PCT/CA00/00801 2 Agric. Biol. Chem. 53: 2835). The biosynthesis of 12-hydroxyjasmonic acid has not been studied at the biochemical level but recent studies suggest that jasmonic acid is converted to 12-hydroxyjasmonic acid by a single oxidation step catalyzed by the jasmonic acid 12-hydroxylase (Yoshihara et al. (1996), Plant Cell Physiol.

37: 586). 11-hydroxyjasmonic acid (see Fig. 1B) is also a natural metabolite for which the mechanism of biosynthesis have not been described either. However, based on the results obtained for the in vivo synthesis of 12-hydroxyjasmonic acid, one can predict that a jasmonic acid 11-hydroxylase converts jasmonic acid or methyljasmonic acid into the 11-hydroxylated compounds.

Although many functions have been associated with jasmonates metabolites such as 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid, these metabolites have never been associated with flower formation. For instance, U.S. patent No 5,935,809, suggests the use of jasmonate for inducing plant defense mechanisms. U.S. patent No 5,814,581 describes a plant growth promoter composition comprising jasmonate and brassinolide as active ingredients and Japanese patent application No 00292220 published April 3, 1990, Yoshihara et al. (1989), Agric. Biol. Chem. 53: 2835-2837, Matsuki et al. (1992), Biosci. Biotech. Biochem. 56: 1329.; and Koda and Okazawa (1988), Plant Cell Physiol. 29: 969), suggest the use of 12-hydroxyjasmonic acid for inducing tuber formation in potatoes. None of these documents disclose nor suggest that compounds of the jasmonates family are involved in flower formation pathways.

Accordingly, there is a need for effective methods and compositions to modulate flowering, particularly for plants which are used in the food-processing industry and plants with a horticultural value. There is also a need for plants genetically modified to flower early and for plants genetically modified to flower tardiiy as weii as for methods for produciiny suui y geiilaiy IIluuifi;e planiis.

SUMMARY OF THE INVENTION The present invention relates to the modulation of flowering in plants. More particularly, the present invention pertains to methods, compositions and genetic sequences for modulating flowering in plants and to plants genetically modified to flower early and to plants genetically modified to flower tardily.

004646740v2.doc 2a In one aspect, there is provided a method for modulating flowering in a plant, comprising modifying in said plant the endogenous level of at least one compound selected from the group consisting of 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, sulfate ester of 12-hydroxyjasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, sulfate ester of 12-hydroxymethyljasmonic acid, 11-hydroxyjasmonic acid, glucoside of 11-hydroxyjasmonic acid, sulfate ester of 11-hydroxyjasmonic acid, 11-hydroxymethyljasmonic acid, glucoside of 11-hydroxymethyljasmonic acid, sulfate ester of 11-hydroxymethyljasmonic acid, and mixtures thereof.

In one embodiment of this aspect, there is provided a method for modulating flowering in a plant wherein flowering of said plant is induced by increasing in said plant the endogenous level of at least one flowering inducing compound selected from the group consisting of 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, methyljasmonic acid, 12hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, 11hydroxyjasmonic acid, glucoside of 1-hydroxyjasmonic acid, 11hydroxymethyljasmonic acid, and glucoside of 11-hydroxymethyljasmonic acid, said flowering induction and said endogenous level increase being compared to a S*2 corresponding plant wherein the endogenous level of said at least one compound 20 has not been modified.

In another embodiment of this aspect of the invention, there is provided a method for modulating flowering in a plant wherein flowering of said plant is delayed by lowering in said plant the endogenous level of at least one compound selected from the group consisting of 12-hydroxyjasmonic acid, glucoside of 12hydroxyjasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12hydroxymethyljasmonic acid, 11-hydroxyjasmonic acid, glucoside of 11hydroxyjasmonic acid, 11-hydroxymethyljasmonic acid, and glucoside of 12hydroxymethyljasmonic acid, said flowering delay and said lower endogenous level being compared to a corresponding plant wherein the endogenous level of said at least one compound has not been modified.

004646740v2.doc 2k In a further embodiment of this aspect of the invention there is provided a composition when used for inducing flowering in a plant comprising a flowering inducing effective amount of a compound selected from the group consisting of 12hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, 12hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, 11hydroxyjasmonic acid, glucoside of 11-hydroxyjasmonic acid, 11hydroxymethyljasmonic acid, glucoside of 11-hydroxymethyljasmonic acid, salts thereof, and mixtures thereof, in combination with a diluent or a carrier such that an induction in flowering of said plant occurs when compared to a corresponding plant in the absence of said composition.

In yet a further embodiment of this aspect of the invention there is provided a composition when used for delaying flowering in a plant comprising a flowering delaying effective amount of an inhibitor or of an inactivator of a compound selected from the group consisting of 12-hydroxyjasmonic acid, glucoside of 12- 15 hydroxyjasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12hydroxymethyljasmonic acid, 1-hydroxyjasmoic acid, glucoside of 11hydroxymethyljasmonic acid, 11-hydroxymethyjasmonic acid, and glucoside of 11hydroxyjasmonic acid, 11-hydroxymethyljasmonic acid, and glucoside of 11hydroxymethyljasmonic acid, in combination with a diluent or a carrier such that a delay in flowering of said plant occurs when compared to a corresponding plant in 20 the absence of said composition.

*o o*oo WO 01/02589 PCT/CA00/00801 3 According to an aspect of the invention, there is provided a method for modulating flowering in a plant. The method comprises the step of modifying in said plant the endogenous level of at least one compound of the jasmonate family, and more particularly compounds selected from the group consisting of jasmonic acid, jasmonic acid-tyrosine conjugate, jasmonic acid-tryptophan conjugate, jasmonic acid-phenylalanine conjugate, jasmonic acid-isoleucine conjugate, jasmonic acid-leucine conjugate, jasmonic acid-valine conjugate, 12hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, sulfate ester of 12hydroxyjasmonic acid, methyljasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, sulfate ester of 12hydroxymethyljasmonic acid, 11-hydroxyjasmonic acid, glucoside of 11hydroxyjasmonic acid, sulfate ester of 11-hydroxyjasmonic acid, 11hydroxymethyljasmonic acid, glucoside. of 11-hydroxymethyljasmonic acid, sulfate ester of 11-hydroxymethyljasmonic acid, and mixtures thereof.

According to another aspect of the invention, flowering of a plant is induced by increasing in the plant the endogenous level of at one flowering inducing compound selected from the previously mentioned jasmonate family compounds excluding sulfate ester of 12-hydroxyjasmonic acid, sulfate ester of 12hydroxymethyljasmonic acid, sulfate ester of 11-hydroxyjasmonic acid, and sulfate ester of 11-hydroxymethyljasmonic acid. In a preferred embodiment, this can be achieved by: a) applying to the plant at least one flowering inducing compound and/or salts thereof; b) applying to the plant at least one inhibitor of a sulfotransferase suffonating 12hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid; appnninn to the nlanf at I~=at nn ctimlllator nf an hydrnoy!ase hydroy!ating r- jasmonic acid and/or methyljasmonic acid; d) increasing in the plant the endogenous level of an hydroxylase hydroxylating jasmonic acid and/or methyljasmonic acid; and/or e) lowering in the plant the endogenous level of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid.

WO 01/02589 PCT/CA00/00801 4 Alternatively, flowering of a plant can be delayed by lowering the endogenous level in the plant of at least one of the above mentioned flowering inducing compounds. According to an embodiment of the invention this can be achieved by: a) applying to the plant an inhibitor and/or an inactivator of at least one of the flowering inducing compounds; b) applying to the plant at least one stimulator of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic; c) applying to the plant at least one inhibitor of an hydroxylase hydroxylating jasmonic acid and/or methyljasmonic acid; d) lowering in the plant the endogenous level of an hydroxylase hydroxylating jasmonic acid and/or methyljasmonic acid; and/or e) increasing in the plant the endogenous level of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid.

According to another aspect of the invention, compositions for modulating flowering are provided. In an embodiment of the invention, a composition for inducing flowering in a plant is provided, comprising a flowering inducing effective amount of at least one of the previously mentioned flowering inducing compounds or salts thereof, in combination with a diluent or a carrier such that an induction in flowering of the plant occurs when compared to a corresponding plant in the absence of the flowering inducing composition. Similarly, in another embodiment, is provided a flowering delaying composition for delaying flowering in a plant, the composition comprising a flowering delaying effective amount of an inhibitor or of an inactivator of the previously mentioned jasmonate family compounds excluding sulfate ester of 12-hydroxyjasmonic acid, sulfate ester of 12ilydiuxyrreiyijasmonic acid, suifate esier of 1 i-iydruxyjdsiliuiiiL auid, and suifaie ester of 11-hydroxymethyljasmonic acid, in combination with a diluent or a carrier such that a delay in flowering of said plant occurs when compared to a corresponding plant in the absence of the flowering delaying composition.

According to a further aspect of the invention, there are provided genetically modified plants. In an embodiment, a plant is genetically modified to flower early when compared to a corresponding plant not genetically modified. The genetically WO 01/02589 PCT/CA00/00801 modified plant exhibits an increased endogenous level of at least one compound selected from the previously mentioned jasmonate family compounds, excluding sulfate ester of 12-hydroxyjasmonic acid, sulfate ester of 12hydroxymethyljasmonic acid, sulfate ester of 11-hydroxyjasmonic acid, and sulfate ester of 11-hydroxymethyljasmonic acid, when compared to a corresponding nongenetically modified plant. In another embodiment a plant is genetically modified to flower tardily when compared to a corresponding plant not genetically modified, the genetically modified plant exhibiting a lowered level of at least one compound selected from the previously mentioned jasmonate family compounds excluding sulfate ester of 12-hydroxyjasmonic acid, sulfate ester of 12hydroxymethyljasmonic acid, sulfate ester of 11-hydroxyjasmonic acid, and sulfate ester of 11-hydroxymethyljasmonic acid.

In another aspect, the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a plant hydroxyjasmonic acid sulfotransferase, and more particularly a plant 11or 12-hydroxyjasmonic acid sulfotransferase. Preferably, the nucleotide sequence is selected from the group consisting of SEQ ID NO:1, nucleotide sequences having at least 50% similarity with SEQ ID NO:1, SEQ ID NO:2 nucleotide sequences having at least 50% similarity with SEQ ID NO:2, and sequences hybridizing under low stringency conditions to one or more of these sequences Advantageously, these sequences are incorporated into a vector.

According to a related aspect, the invention provides transgenic plants incorporating at least one of these nucleotide sequences so that the transgenic plants are capable of flowering early or tardily. The invention also provides methods for producing such transgenic plants.

i ;r l ll^> ryfrhe lli flrA orinn a u vad lla ye i c- p, i .I in plants without decreasing yield or modifying plant morphology. According to the invention it is possible to inhibit flowering in crop plants such as sugarcane, sugar beets or lettuce, just to mention a few, and thereby increase the taste, sweetness, and tenderness of these agricultural products. On the other hand, it is also possible according to the present invention, to induce flowering which is an 004646740v2.doc 6 advantage of great economic importance for horticultural plants and some crop plants such as cauliflower and broccoli.

Other objects and advantages of the present invention will be apparent upon reading the following non-restrictive description of several preferred embodiments, made with reference to the accompanying drawings and to the enclosed examples.

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1B show the chemical structures of 12-hydroxyjasmonic acid (Fig. 1A) and 11-hydroxyjasmonic acid (Fig. 1B).

Figures 2A and 2B are pictures showing the effect on flowering time of a LO TSGI A I4I I VVIL I I I.-IIUIVy U I UAyJoI III I I.au FIy. d III 1dUIUV Ic ldll dIcl, 20 when compared to a treatment with water (Fig. 2A).

Figure 3 is a picture showing the phenotype of transgenic Arabidopsis plants expressing AtST2a gene under the control of a constitutive promoter when compared to wild type non-transgenic plant S5, S6, S9 and S16 indicate independent transgenic lines.

Figure 4 is a Western blot of protein extracts from the plants shown in Fig.

3 probed with anti-AtST2a antibodies. MW: Molecular weight markers; WT: wild type plants; S5, S6, S9 and S16: independent transgenic lines.

004646740v2.doc 6a Figure 5 is a picture showing the phenotype of transgenic Arabidopsis plants expressing the AtST2a gene in the antisense orientation under the control of a constitutive promoter (TL 7-2-5) when compared to non transgenic plants (WT).

Figure 6 is a picture showing the effect of methyljasmonic acid treatment on the flowering time of wild type Arabidopsis thaliana plants (WT C24) and on transgenic Arabidopsis thaliana plants expressing the AtST2a gene in the antisense orientation under the control of a constitutive promoter (TL 7-2-5).

Figure 7: Shows nucleotide sequence of AtST2a gene (SEQ ID NO 1) taken from Arabidopsis thaliana database at Stanford University (clone number MOJ9, gene MOJ9.16 and the EST 119G6T7) and the GenBank

TM

database (accession number AB010697, nucleotides 53936 to 55015).

S* e e *o ~'2-O92OO oIle Richard ROBIC (Be)514 845 b51U; UHI'ellul CA0000801 7 Figure 8: Shows the deduced amino acid sequence (SEQ ID NO 3) of the Protein encoded by the AtST2a gene shown in Fig. 7.

Figure 9: -Shows the nucleotide sequence of AtST2b gene (SEQ ID NO 2) taken from Arabidopsis thaliana database at Stanford University (clone number MOJ9, gene MO.19.15) and the GenBankT database (accession number AB0 10697, nucleotldes 50627 to 51670).

Figure 10: Shows the deduced amino acid sequence (SEQ ID NO 4) of the protein encoded by the AtS T2b gene shown in Fig. 9.

Figure I11 is a Northern blot of plants mRNA extracts showing the effect of various I 2-hydroxyjasmonate concentrations on the expression of the AtST2a gene.

Figure 12 is a Northern blot of plants mRNA extracts showing the effect of the photoperiod on the expression of the AtST*2a gene.

DETAILED DESCRIPTION OF THE INVENTION A) Definlitions In order to provide an even clearer and more consistent understanding of the specification and the claims, including the scope given herein to such terms, the following definitions are provided: 1 1-hydroxyjas monic actid; 3-Oxo-2-(4-hydroxy-2-pentenyl)-cyclopentane- 1-acetic acid. Its chemical structure is shown In Fig. 16.

I1-hyd roxyjas manic acid glucoside: 3-Oxo-2-(4-D-giucopyranosyloxy- 2-pentenyi)-cyclopentane-1 -acetic acid 11 -hydroxyjasmonic acid sulfate: 3-Oxo-2-(4-hydroxysulfonyoxy-2pentenyl)-cyclopentane- 1-acetic acid I 2-hydroxyjasmon ic acid; 3-Oxo-2-(5-hydroxy-2-pentenyI)-cyclopentane- 1-acetic acid. Its chemical structure is shown In Fig. lA.

I 2-hydroxyjasmonic acid glucoslde: 3-Oxo-2-(5-j3-D-glucopyranosyloxy- 2-.pentenyl)-cyclopentane-1 -acetic acid.

1 2-hyd roxyjasmonic acid sulfate: 3-Oxo -2-(5-hyd roxysulfonyloxy-2pentenyl)-cyclopentane-1 -acetic acid.

AMENDED SHEET AMENDED SHEET WO 01/02589 PCT/CA00/00801 8 Antisense: Refers to nucleic acids molecules capable of regulating the expression of a corresponding gene in a plant. An antisense molecule as used herein may also encompass a gene construct comprising a structural genomic gene, a cDNA gene or part thereof in reverse orientation relative to its or another promoter. Typically antisense nucleic acid sequences are not templates for protein synthesis but yet interact with complementary sequences in other molecules (such as a gene or RNA) thereby causing the function of those molecules to be affected.

Delay or retard or tardily: When used in conjunction with the term flowering, it refers to the increase of the time of vegetative growth before flowering of a plant. A flowering delay may be observed when compared with a corresponding plant where flowering has not been delayed.

Effective amount: Refers to the amount or concentration of a suitable compound that is administered to a plant such that the compound induces or delays flowering of a plant.

Exogenous nucleic acid: A nucleic acid sequence (such as cDNA, cDNA fragments, genomic DNA fragments, antisense RNA, oligonucleotide) which is not normally part of a plant genome. The "exogenous nucleic acid" may be from any organism or purely synthetic. Typically, the "exogenous nucleic acid sequence" encodes a plant gene such as a AtST2a, AtST2b or functional homologues of these genes.

Expression: The process whereby an exogenous nucleic acid, such as a nucleic acid sequence encoding a gene, is transcribed into a mRNA and afterwards translated into a peptide or a protein, in order to carry out its function, if any.

Flowering: Refers to the appearance of a flower bud in a plant. As it is knnwn the flower bud will eventually mati Furp tn flnw.r Functional homologue: Refers to a molecule having at least 50%, more preferably at least 55%, even more preferably at least 60%, still more preferably at least 65-70%, and yet even more preferably greater than 85% similarity at the level of nucleotide or amino acid sequence to at least one or more regions of a given nucleotide or amino acid sequence. According to preferred embodiments of the present invention, the terms "functional homologue" refer to proteins or nucleic WO 01/02589 PCT/CA00/00801 9 acid sequences encoding an enzyme having a substantially similar biological activity as 11- or 12-hydroxyjasmonate sulfotransferase and isoenzyme(s) thereof.

Such a functional homologue may exist naturally or may be obtained following a single or multiple amino acid substitutions, deletions and/or additions relative to the naturally occurring enzyme(s) using methods and principles well known in the art. A functional homologue of a protein may or may not contain post-translational modifications such as covalently linked carbohydrate, if such modification is not necessary for the performance of a specific function. It should be noted, however, that nucleotide or amino acid sequences may have similarities below the above given percentages and still encode a 11- or 12-hydroxyjasmonate sulfotransferase-like molecule, and such molecules may still be considered within the scope of the present invention where they have regions of sequence conservation.

Genetic/nucleotide sequence: These terms are used herein in their most general sense and encompass any contiguous series of nucleotide bases encoding directly, or via a complementary series of bases, a sequence of amino acids comprising a hydroxyjasmonic acid sulfotransferase molecule, and more particularly a 11- or 12-hydroxyjasmonic acid sulfotransferase. Such a sequence of amino acids may constitute a full-length 11- or 12-hydroxyjasmonic acid sulfotransferase such as is set forth in SEQ ID No:1 and SEQ ID No:2 or an active truncated form thereof or a functional mutant, derivative, part, fragment, homologue or analogue thereof, or may correspond to a particular region such as an N-terminal, C-terminal or internal portion of the enzyme.

Genetic modification or genetic engineering: Refers to the introduction of an exogenous nucleic acid into one or more plant cells to create a genetically modified olant. Methods for genetically modifying a plant are well known in the art.

In some cases, in may be preferable that the genetic modification is permanent such that the genetically modified plant may regenerate into whole, sexually competent, viable genetically modified plants. A plant genetically modified in a permanent manner would preferably be capable of self-pollination or crosspollination with other plants of the same species, so that the exogenous nucleic WO 01/02589 PCT/CA0/00801 acid, carried in the germ line, may be inserted into or bred into agriculturally useful plant varieties.

Endogenous level(s): Refers to the concentration of a given substance which is normally found in a plant (intrinsic) at a given time and stage of growth.

Reference herein is made to the altering of the endogenous level of a compound or of an enzyme activity relating to an elevation or reduction in the compound's level or enzyme activity of up to 30% or more preferably of 30-50%, or even more preferably 50-75% or still more preferably 75% or greater above or below the normal endogenous or existing levels. The levels of a compound or the levels of activity of an enzyme can be assayed using known method and techniques.

Isolated nucleic acid molecule: Means a genetic sequence in a nonnaturally-occurring condition. Generally, this means isolated away from its natural state or formed by procedures not necessarily encountered in its natural environment. More specifically, it includes nucleic acid molecules formed or maintained in vitro, including genomic DNA fragments, recombinant or synthetic molecules and nucleic acids in combination with heterologous nucleic acids such as heterologous nucleic acids fused or operably-linked to the genetic sequences of the present invention. The term "isolated nucleic acid molecule" also extends to the genomic DNA or cDNA or part thereof, encoding a hydroxyjasmonic acid sulfotransferase, preferably a 11- or 12-hydroxyjasmonic acid sulfotransferase, or a functional mutant, derivative, part, fragment, homologue or analogue of 11- or 12-hydroxyjasmonic acid sulfotransferase in reverse orientation relative to its or another promoter. It further extends to naturally-occurring sequences following at least a partial purification relative to other nucleic acid sequences. The term isolated nucleic acid molecule as used herein is understood to have the same meanina a nutr.leic acid isolate Induce or increase: When used in conjunction with the term flowering, it refers to the reduction of the time of vegetative growth before flowering of a plant.

A flowering induction may be observed when compared with a corresponding plant wherein flowering has not been induced.

Modulation: Refers to the process by which a given variable is regulated to a certain proportion. According to preferred embodiments of the present invention, WO 01/02589 PCT/CA00/00801 11 the term "modulate" refers in some cases to induction and in other cases delay, of flowering of a plant.

Plant: refers to a whole plant or a part of a plant comprising, for example, a cell of a plant, a tissue of a plant, an explant, or seeds of a plant. This term further contemplates a plant in the form of a suspension culture or a tissue culture including, but not limited to, a culture of calli, protoplasts, embryos, organs, organelles, etc.

Similarity/Complementarity: In the context of nucleic acid sequences, these terms mean a hybridizable similarity under low, alternatively and preferably medium and alternatively and most preferably high stringency conditions, as defined below. Such a nucleic acid is useful, for example, in screening hydroxyjasmonic acid sulfotransferase genetic sequences, preferably a 11- or 12hydroxyjasmonic acid sulfotransferase genetic sequences from various sources or for monitoring an introduced genetic sequence in a transgenic plant. The preferred oligonucleotide is directed to a conserved hydroxyjasmonic acid sulfotransferase, preferably a 11- or 12-hydroxyjasmonic acid sulfotransferase genetic sequence or a sequence conserved within a plant genus, plant species and/or plant cultivar or variety.

Stringency: For the purpose of defining the level of stringency, reference can conveniently be made to Maniatis et al. (1982) at pages 387-389, and especially paragraph 11. A low stringency is defined herein as being in 4-6X SSC/1% SDS at 37-45 °C for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridization, altemative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 1-4X SSC/0.5-1% SDS at greater than or equal to r h 1 r kink e+rinnont nnritinncna nrl o r e rl harain tM ha n 1-1X SSC/0.1-1.0% SDS at greater than or equal to 60" C. for 1-3 hours.

Transformed plant: Refers to introduction of an exogenous nucleic acid, typically a gene, into a whole plant or a part thereof, and expression of the exogenous nucleic acid in the plant.

PCT/CA00/00801 WO 01/02589 12 Transgenic plant: Refers to a whole plant or a part thereof stably transformed with an exogenous nucleic acid introduced into the genome of an individual plant cell using genetic engineering methods.

Vector: A self-replicating RNA or DNA molecule which can be used to transfer an RNA or DNA segment from one organism to another. Vectors are particularly useful for manipulating genetic constructs and different vectors may have properties particularly appropriate to express protein(s) in a recipient during cloning procedures and may comprise different selectable markers. Bacterial plasmids are commonly used vectors. Preferably, the vectors of the invention are capable of facilitating transfer of a nucleic acid into a plant cell and/or facilitating integration into a plant genome.

B) General overview of the invention The present inventors have now discovered that compounds of the jasmonate family are involved in the flowering of plants. They have also characterized the biological function of two highly homologous genes from A.

thaliana (AtST2a and AtST2b) which encode enzymes that inactivate by sulfonation the biological activity of 11-hydroxyjasmonic acid and 12hydroxyjasmonic acid. The inventors have also determine that expression of the AtST2a gene in under the control of photoperiod. These properties suggest that flowering could be induced or delayed, and yield to the elaboration of the following model which is given for purposes of clarification and not to limit the scope of the present invention.

WO 01/02589 PCT/CA00/00801 13 Proposed model for the control of flower induction in plants:

INACTIVE

12-hydroxyjasmonate sulfate 11-hydroxyjasmonate sulfate Short days expression of AtST2a and AtST2b 12-hydroxyJasmonate Flowering 11-hydroxyjasmonate Long days no expression ACTIVE of AtST2a and AtST2b Jasmonate 12-hydroxylase Jasmonic acid Jasmonate 11-hydroxylase Using methods, compositions and genetically modified plants the present application demonstrates that flowering can actually be modulated in plants.

According to an aspect of the invention, flowering of a plant is modulated by modifying in the plant the endogenous level of at least one compound of the jasmonate family selected from the group consisting of jasmonic acid, jasmonic acid-tyrosine conjugate, jasmonic acid-tryptophan conjugate, jasmonic acidphenylalanine conjugate, jasmonic acid-isoleucine conjugate, jasmonic acidleucine conjugate, jasmonic acid-valine conjugate, 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, sulfate ester of 12-hydroxyjasmonic acid, methyljasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12hydroxymethyljasmonic acid, sulfate ester of 12-hydroxymethyljasmonic acid, 11hydroxyjasmonic acid, glucoside of 11-hydroxyjasmonic acid, sulfate ester of 11hydroxyjasmonic acid, 11-hydroxymethyljasmonic acid, glucoside of 11hydroxymethyijasmonic acid, and suifate esier oi i-iydluxyiielyiiiyioiic acii In practice, flowering modulation is achieved in two different ways: it is induced or it is delayed. Although many approaches may be used to achieve these effects, the approaches described hereinafter are preferably used according to the invention.

WO 01/02589 PCT/CA00/00801 14 1) Chemical approach i) Flowering induction According to the invention, flowering is induced by increasing in a plant the endogenous level of at least one given flowering inducing compound of the jasmonate family selected preferably from the group consisting of jasmonic acid, jasmonic acid-tyrosine conjugate, jasmonic acid-tryptophan conjugate, jasmonic acid-phenylalanine conjugate, jasmonic acid-isoleucine conjugate, jasmonic acidleucine conjugate, jasmonic acid-valine conjugate, 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, methyljasmonic acid, 12hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, 11hydroxyjasmonic acid, glucoside of 11-hydroxyjasmonic acid, 11hydroxymethyljasmonic acid, and glucoside of 11-hydroxymethyljasmonic acid. All these compounds have been tested for their biological activity and all of them have been shown to induce flowering at various levels (data not shown). More preferably, levels of 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid are increased. The flowering induction and the endogenous level increased is detectable when compared to a corresponding plant in which the endogenous level of said compound has not been modified. However, it could be preferable in some cases to genetically modify a plant to induce its flowering prior to apply thereto a product or composition further inducing its flowering. The increase of the endogenous level would then have to be compared with the endogenous level of the genetically modified plant in which flowering has previously been induced.

According to a preferred embodiment of the invention, the endogenous level of a selected jasmonate compound is increased by: a) applying to the plant at least one selected jasmonate compound and/or salts thereof: b) applying to the plant at least one inhibitor of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic; and/or c) applying to the plant at least one stimulator of an hydroxylase hydroxylating jasmonic acid and/or methyljasmonic acid.

More preferably, the selected jasmonate compound which is applied is 12hydroxyjasmonic acid or 11-hydroxyjasmonic acid. However, amino acid WO 01/02589 PCT/CA00/00801 conjugates, glucosides, sulfate esters, salts, derivatives or any others natural or chemically synthesized compounds having a similar biological activity on flowering induction, is suitable according to the invention. For instance, inactive compounds such sulfate ester of 12-hydroxyjasmonic acid, sulfate ester of 12hydroxymethyljasmonic acid, sulfate ester of 11-hydroxyjasmonic acid, and sulfate ester of 11-hydroxymethyljasmonic acid could be applied to a plant and it is possible that the bacterial or fungal flora of the plant or of its soil would hydrolyze these compounds in active jasmonate compounds. Any of the above mentioned compounds can be applied in a pure form or as a mixture of a plurality of compounds.

Inhibitors of hydroxyjasmonic acid sulfotransferase(s) should prevent in vivo inactivation of the flower-inducing molecule by sulfonation. To the contrary, stimulators of jasmonic acid hydroxylase(s) should help in the production of jasmonate family compound(s).

The above mentioned jasmonate compounds, stimulators and/or inhibitors can be part of a composition for inducing flowering in a plant. Such a composition would comprise a flowering inducing effective amount of at least one selected jasmonate compound, in combination with a diluent or a carrier. The compound(s) and their amount would be selected such that an early flowering of the plant would occur following application of the flower inducing composition when compared to a corresponding plant in the absence of said compound(s).

The carrier or diluent can be a solvent such as water, oil or alcohol. The composition may also comprise others active agents such as fertilizers and growth regulators. The inducing composition may also be formulated with emulsifying agents in the presence or absence of fungicides or insecticides, if required. The orecise amount of comoound emDloyed in the oractice of the present invention will depend upon the type of response desired, the formulation used and the type of plant treated. In the following examples, the plant culture medium was supplemented with about 10 pM of 12-hydroxyjasmonate or with 50 pM methyljasmonic acid for flowering induction.

WO 01/02589 PCT/CA00/00801 16 i) Flowering retardation According to the invention, flowering is delayed by lowering the endogenous level in a plant of at least one given compound of the jasmonate family selected preferably from the group consisting of jasmonic acid, jasmonic acid-tyrosine conjugate, jasmonic acid-tryptophan conjugate, jasmonic acid-phenylalanine conjugate, jasmonic acid-isoleucine conjugate, jasmonic acid-leucine conjugate, jasmonic acid-valine conjugate, 12-hydroxyjasmonic acid, glucoside of 12hydroxyjasmonic acid, methyljasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, 11-hydroxyjasmonic acid, glucoside of 11-hydroxyjasmonic acid, 11-hydroxymethyljasmonic acid, and glucoside of 11hydroxymethyljasmonic acid. More preferably, the levels of 12hydroxymethyljasmonic acid and of 11-hydroxymethyljasmonic acid are reduced.

The flowering delay and the endogenous level lowering is detectable when compared to a corresponding plant in which the endogenous level of the compound has not been modified. However, it could be preferable in some cases to genetically modify a plant to delay its flowering prior to apply thereto a product or composition further delaying its flowering. The lowering of the endogenous level would then have to be compared with the endogenous level of the genetically modified plant in which flowering has previously been delayed.

According to a preferred embodiment of the invention, the endogenous level of a selected jasmonate compound is lowered by: a) applying to the plant at least one inhibitor and/or an inactivator of a selected jasmonate compound; b) applying to the plant at least one stimulator of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid; and/or annlvinn tn thp. nl;nt at peast nne inhihitor nf nn hyrrnvyyA.c hywrnwdiatinm jasmonic acid and/or methyljasmonic acid.

Inhibitor(s) and/or an inactivator(s) of jasmonate compounds should block or inhibit the biological activity of jasmonate compound(s).

Stimulator(s) of hydroxyjasmonic acid sulfotransferase(s) should stimulate in vivo inactivation of the flower-inducing molecule by sulfonation. To the contrary, 2 1 0 9 2 0 0 1 ,bic Richard ROBIC (6e)514 845 031d;u JLL..IC~ CA0000801 17 inhibitors of jasmonic acid hydroxyla-se(s) should prevent the production of hydroxylated jasmonate cornpound(s).

As for the flowering compounds, the above stimulators and/or inhibitors can be applied in a pure form, as a mixture of a plurality of compounds or be part of .a flowering delaying composition.

2) Molecular approach In accordance with the present invention, genetic sequences encoding a plant hydroxyjsmonic acid suffotransferase have been identified, cloned and used to generate transgenic plants.

SEQ 1D NO 1 (Fig. 7: GenBank~m: accession number AB010697, nuofeotides 53936 to 55015; and Stanford University Arabidopsis thaliana database: clone number MOJ9, gene MOJ9. 16 and EST 11 9G6T7) corresponds to the gene AtST2a in Arabidopsis thaliana. SEQ ID NO 3 (Fig. 8) is an amino acid sequence deduced from SEQ ID NO 1. This amino acid sequence is of public domain and comes from the Kazusa Arabidopsis Opening Site (KADS) of the Kazusa DNA Research Institute (KDRI) (http://www.kazusa.or.ln/kaoS/: clone number MOJ9, gene M0.19.16). The present inventors have found that the AtST2a gene from Arabidopsis tlialiana encodes a sulfotransferase that sulfonates 12hydroxyjasmonic acid and, I 1-hydroxyjasmonic acid with high specificity. Although not shown, results obtained demonstrated that this hydroxyjasmonic acid sulfotransferase exhibits high affinity for its substrate with a Kmn value of 11 pM for 12-hydroxyjasmonic acid and 60 pM for 11 -hydroxyjasmonic acid. The enzyme did not accept structurally related compounds. such as cucurbic acid, arachidanyl alcohol or prostaglandins. Maximum enzyme activity was observed at pH 7.5 In Tris/HOI buffer and did not require divalent cations for activity. The purified recombinant protein expressed in E. coli migrated in SDS-PAGE at a position corresponding to approximately. 35,000 daltons (see Fig. 4).

SEQ ID NO 2 (Fig. 9; GenBankm: accession number A1301 0697, nucleotides '50627 to 51670; and Stanford University Arabidopsis thallana database: clone number MOJ9 gene MOJ9.15), corresponds to the gene AtST2b in Arabidopsis thaliana. SEQ ID NO 4. (Fig. 10) is an amino acid sequence deduced from SEQ ID AMENDED SHEET AMENDED SHEET Cmnf-.nrcoait ')1.qon. ?n:11 WO 01/02589 PCT/CA00/00801 18 NO 1. This amino acid sequence is of public domain and comes from the Kazusa Arabidopsis Opening Site (KAOS) of the Kazusa DNA Research Institute (KDRI) (http://www.kazusa.or.jp/kaos/; clone number MOJ9, gene MOJ9.15). Amino acid sequence alignment between SEQ ID NOS 3 and 4 indicates that they share amino acid sequence identity and 92% similarity, suggesting that AtST2a and AtST2b are functional homologues encoding isoenzymes.

Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a plant hydroxyjasmonic acid sulfotransferase enzyme. More particularly, the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence preferably selected from the group consisting of SEQ ID NO:1, nucleotide sequences having at least 50% similarity with SEQ ID NO:1, SEQ ID NO:2, nucleotide sequences having at least 50% similarity with SEQ ID NO:2, and sequences hybridizing under low stringency conditions to one or more of theses sequences.

The nucleic acid molecules contemplated herein may exist in either orientation alone or in combination with a vector and preferably an expressionvector capable of facilitating transfer and expression of the nucleic acid into the plant cell and/or facilitating integration into the plant genome. Such a vector may, for example, be adapted for use in electroporation, microprojectile bombardment, Agrobacterium-mediated transfer or insertion via DNA or RNA viruses. The vector and/or the nucleic acid molecule contained therein may or may not need to be stably integrated into the plant genome. The vector may also replicate and/or express in prokaryotic cells. Preferably, the vector molecules or parts thereof are capable of integration into the plant genome. The nucleic acid molecule and/or the vector mav additionally contain a Dromoter seauence capable of directina expression of the nucleic acid molecule in a plant cell. The nucleic acid molecule and/or the vector may also be introduced into the cell by any number of means such as those described above. The vector may also comprise a genetic sequence encoding a ribozyme capable of cleaving a hydroxyjasmonic acid sulfotransferase mRNA transcript.

WO 01/02589 PCT/CA00/00801 19 The present invention is exemplified using nucleic acid sequences derived from Arabidopsis thaliana since this plant is commonly studied in and it represents a convenient and easily accessible source of material. However, one skilled in the art will immediately appreciate that similar sequences can be isolated from any number of sources such as other plants or certain microorganisms fungi or bacteria). All such nucleic acid sequences encoding directly or indirectly a hydroxyjasmonic acid sulfotransferase are encompassed by the present invention regardless of their source. Examples of other suitable sources of genes encoding hydroxyjasmonic acid sulfotransferase include, but are not limited to Brassica napus, Brassica oleracea and Brassica juncea.

i) Flowering induction An aspect of the invention contemplates a plant genetically modified to flower early when compared to a corresponding plant not genetically modified, wherein the genetically modified plant has an increased endogenous level of at least one given compound of the jasmonate family selected preferably from the group consisting of jasmonic acid, jasmonic acid-tyrosine conjugate, jasmonic acid-tryptophan conjugate, jasmonic acid-phenylalanine conjugate, jasmonic acidisoleucine conjugate, jasmonic acid-leucine conjugate, jasmonic acid-valine conjugate, 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, methyljasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12hydroxymethyljasmonic acid, 11-hydroxyjasmonic acid, glucoside of 11hydroxyjasmonic acid, 11-hydroxymethyljasmonic acid, and glucoside of 11hydroxymethyljasmonic acid, when compared to the corresponding non-genetically modified plant.

in a preferred emboudiiwi, ihe u ogenuo:3 U of I. '1 compound is increased by: a) increasing in the plant the endogenous level of an hydroxylase hydroxylating jasmonic acid and/or methyljasmonic acid; and/or b) lowering in the plant the endogenous level of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid.

WO 01/02589 PCT/CA00/00801 According to a preferred embodiment of the invention this is achieved by genetically modifying the plant so as to lower the expression of the sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid, and functional homologues of this sulfotransferase. More preferably, the plant is modified for inhibiting or blocking the expression of at least one gene selected from the group consisting of AtST2a, AtST2b and functional homologues of AtST2a or of AtST2b.

Many methods for inhibiting expression of genes in plants are well known in the art, such as techniques using ribozymes, targeted mutagenesis,

T-DNA

insertion mutagenesis, and antisense techniques to name a few, and these methods could be used to reduce the present invention in practice. According to a preferred embodiment of the invention, the expression of one of the above mentioned gene is inhibited by providing a transgenic plant expressing an exogenous nucleic acid sequence antisense to this gene. More preferably, the endogenous level of the sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid is lowered by expressing into a genetically modified plant an exogenous nucleic acid sequence, the exogenous nucleic acid sequence encoding i) for a nucleic acid sequence antisense to a gene encoding at least one of said sulfotransferases or ii) for a nucleic acid sequence antisense to a fragment of this gene.

Accordingly, another aspect of the invention contemplates a method for producing a transgenic plant with reduced endogenous or existing hydroxyjasmonic acid sulfotransferase activity, such transgenic plant thereby being capable of flowering early. Preferably, the altered level would be less than the endogenous or existing level of activity in a comparable non-transgenic plant.

mo-iman-t the methnd r. mnrises the steps of: a) introducing into a cell of a suitable plant an exogenous nucleic acid molecule comprising a sequence of nucleotides antisense to a sequence encoding a plant hydroxyjasmonic acid sulfotransferase, preferably a 11- or 12hydroxyjasmonic acid sulfotransferase; b) regenerating a transgenic plant from the cell; and where necessary WO 01/02589 PCT/CAOO/00801 21 c) growing the transgenic plant for a time and under conditions sufficient to permit expression of the antisense sequence and thereby inhibiting expression of the hydroxyjasmonic acid sulfotransferase.

In a related embodiment, the method for producing a transgenic plant with reduced endogenous or existing hydroxyjasmonic acid sulfotransferase activity comprises the step of altering the hydroxyjasmonic acid sulfotransferase gene(s), preferably the 11- or 12-hydroxyjasmonic acid sulfotransferase gene, through modification of the endogenous sequences via homologous recombination from an appropriately altered hydroxyjasmonic acid sulfotransferase gene or derivative or part thereof introduced into the plant cell, and regenerating a transgenic plant from the cell.

ii) Flowering retardation An aspect of the invention contemplates a plant, genetically modified to flower tardily when compared to a corresponding plant not genetically modified, wherein the genetically modified plant has a lowered endogenous level of at least one given compound of the jasmonate family selected preferably from the group consisting jasmonic acid, jasmonic acid-tyrosine conjugate, jasmonic acidtryptophan conjugate, jasmonic acid-phenylalanine conjugate, jasmonic acidisoleucine conjugate, jasmonic acid-leucine conjugate, jasmonic acid-valine conjugate, 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, methyljasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12hydroxymethyljasmonic acid, 11-hydroxyjasmonic acid, glucoside of 11hydroxyjasmonic acid, 11-hydroxymethyljasmonic acid, and glucoside of 11hydroxymethyljasmonic acid, when compared to the corresponding non-genetically In a preferred embodiment, the endogenous level of the selected jasmonate compound is lowered by: a) lowering in the plant the endogenous level of an hydroxylase hydroxylating jasmonic acid and/or methyljasmonic acid; and/or b) increasing in the plant the endogenous level of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid.

WO 01/02589 PCT/CA00/00801 22 According to a preferred embodiment of the invention this is achieved by genetically modifying the plant so as to increase the expression of the sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid, or functional homologues of this sulfotransferase. More preferably, the plant is modified to increase the expression of at least one gene selected from the group consisting of AtST2a, AtST2b and functional homologues of AtST2a or of AtST2b.

Methods for increasing expression of genes in plants are well known in the art, such as activation tagging, transgenesis under the control of a strong promoter, and these methods could be used to reduce the present invention in practice. According to a preferred embodiment of the invention, the expression of one of the above-mentioned genes is increased by expressing into the plant a gene expressing the sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic under the control of a constitutive or an inducible promoter.

Accordingly, another aspect of the invention contemplates a method for producing a transgenic plant with increased endogenous or existing hydroxyjasmonic acid sulfotransferase activity, such transgenic plant thereby being capable of flowering tardily. Preferably, the altered level would be higher than the endogenous or existing level of activity in a comparably non-transgenic plant.

According to a preferred embodiment, the method comprises the step of: a) introducing into a cell of a suitable plant an exogenous nucleic acid molecule comprising a sequence of nucleotides encoding a plant hydroxyjasmonic acid sulfotransferase, preferably a 11- or 12-hydroxyjasmonic acid sulfotransferase; b) regenerating a transgenic plant from the cell; and where necessary c) growing the transgenic plant for a time and under conditions sufficient to Sh ,e nnon, r- intrn nlant hvdrnwviAsmnnir acid sulfotransferase, preferably a 11- or 12-hydroxyjasmonic acid sulfotransferase.

The details of the construction of transgenic plants are known to those skilled in the art of plant genetic engineering and do not differ in kind from those practices which have previously been demonstrated to be effective in tobacco, PCT/CA00/00801 WO 01/02589 23 petunia and other model plant species electroporation, microprojectile bombardment, Agrobacterium-mediated transfer or insertion via DNA or RNA viruses). One skilled in the art will immediately recognize the variations applicable to the methods of the present invention, such as increasing or decreasing the expression of the sulfotransferase naturally present in a target plant leading to modulation of flowering to this plant. The present invention, therefore, extends to all transgenic plants containing all or part of the nucleic acid sequence of the present invention, or antisense forms thereof and/or any homologues or related forms thereof and in particular those transgenic plants which exhibit altered flowering properties.

The transgenic plants may contain an introduced nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding a hydroxyjasmonic acid sulfotransferase. Generally, the nucleic acid would be stably introduced into the plant genome, although the present invention also extends to the introduction of a hydroxyjasmonic acid sulfotransferase nucleotide sequence within an autonomously-replicating nucleic acid sequence such as a DNA or RNA virus capable of replicating within the plant cell. The invention also extends to cut flowers and seeds from such transgenic plants.

A further aspect of the present invention is directed to an isolated plant hydroxyjasmonic acid sulfotransferase, and more particularly to an isolated hydroxyjasmonic acid sulfotransferase selected from the group of: a) an enzyme whose amino acid sequence is represented by SEQ ID NO 3 or SEQ ID NO 4; and b) functional homologues of enzyme isolated from a plant or derived from ,s a y, ,hstcitrtinn drlptinn or addition of one or several amino acids in L the amino acid sequences defined in and having similar biological activity or function(s).

These enzymes may be purified from plants or produced with routine recombinant techniques using SEQ ID NO 1, SEQ ID NO 2, or portion(s) thereof.

Various methods of purification and molecular biology techniques for producing recombinant proteins are described in the art such that a skilled technician could WO 01/02589 PCT/CA00/00801 24 obtain these enzymes without large amounts of trial and error, or complicated experimentation. Isolated hydroxyjasmonic acid sulfotransferase will provide a source of material for research to develop, for example, more active enzymes and to produce antibodies binding with affinity thereto.

Accordingly, a related aspect of the invention is directed to antibodies binding with affinity to one or more of the above mentioned hydroxyjasmonic acid sulfotransferases. Persons skilled in the art are aware that antibodies can be made against virtually any protein and should be capable of producing such antibodies using conventional techniques. Antibodies binding to hydroxyjasmonic acid sulfotransferases could be particularly useful in flowering retardation compositions and also for studying the biological activity of this type of enzymes.

EXAMPLES

The following examples are illustrative of the wide range of applicability of the present invention. The invention is not restricted to the control of flowering in Arabidopsis thaliana but can be applied to various plant species. It should readily occur that the recognition of activation or retardation of flowering using the compositions, and methods according to the present invention in connection with other plants not specifically illustrated herein, is readily within the capabilities of one skilled in the art. The following examples are intended only to illustrate the invention and are not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention.

The following experimental procedures and materials were used for the examples set fort below.

A) Materiais And Miethods Growing of plants A. thaliana plants of ecotype Columbia (ColO) and C24 were used for this study. The plants were grown in soil in a growth chamber during a 16-hour photoperiod, at a day-time temperature of 24 °C and a night-time temperature of For some experiments, the plants were grown in magenta boxes under sterile conditions according to the following protocol. Seeds of Arabidopsis WO 01/02589 PCT/CA00/00801 thaliana were sterilized for 5 minutes in a solution containing 1.5% sodium hypochlorite and 0.02% SDS, and washed five times in sterile water. Seeds were vernalized for four days at 4 Seeds were then spread on agar-solidified medium containing Murashige and Skoog salts, 1% sucrose and vitamins.

Studies using a vector: For transgenic studies a EcoR1-Hindlll cassette, from the plasmid pBI-525 comprising two CaMV 35S promoters in tandem followed by an AMV translational enhancer and a NOS terminator, was ligated to the plasmid pBl-101 which was previously digested with the same restriction endonucleases. The resulting vector called pBI-101-525 contained two CaMV 35S minimal promoters in tandem followed by an AMV translational enhancer, a NOS terminator and a kanamycin resistance gene. AtST2a cDNA (SEQ ID NO 1; Fig. 7) was cloned both in the sense and the antisense orientation at the BaMHI site in a polylinker lying downstream of the AMV enhancer. Various other promoters may be used to drive the expression of an exogenous gene in a plant. For example the ubiquitin promoter may be used for constitutive expression. Alternatively, inducible promoters may also be used such as the ethanol-inducible promoter or the glucocorticoid-inducible promoter.

Agrobacterium transformation: A. tumefaciens strain GV3101 pMP90 was transformed with the AtST2apB1-101-525 sense and antisense constructs by the method described in Gynheung et al. (1988) Biology Manual, A3:1-19.

Arabidopsis i.ar isui nI ioi: A. thaliana plants of ecotype Columbia (ColO) were transformed with Agrobacterium containing the AtST2a gene in the sense orientation by the vacuum infiltration method as described previously in Benchtold et al. (1993), CR Acad.

Sci. Paris, Life Sciences, 316: 1194. A. thaliana plants of ecotype C24 were transformed with the pBl-101-525 vector containing the AtST2a gene in the antisense orientation by the root explant method as described in Valvekans et al.

WO 01/02589 PCT/CAOO/00801 26 (1988) Proc Natl Acad Sci USA, 85: 5536. Seeds were collected from the To plants, their surface was sterilized and transformants were selected on MS salt medium containing vitamins and supplemented with 50 pg/ml of kanamycin. For phenotypic analysis of the transgenic plants, the T 2 or T 3 seeds were vernalized for four days at 4 OC. Seeds were then spread on agar-solidified medium containing Murashige and Skoog salts, 1% sucrose and vitamins. Alternatively, the vernalized seeds were planted in soil and grown in a growth chamber under a 16hour photoperiod, at a day temperature of 24 °C and a night temperature of 20 oC.

Western blot of protein extracts Protein extracts from wildtype and transgenic plants were subjected to 12% SDS-PAGE. Following electrophoresis, the proteins were transferred to nitrocellulose membranes using a Bio-Rad T semidry transblot apparatus according to the manufacturer instructions. Blots were incubated with rabbit anti- ATST2a primary antibodies. Immunodetection was carried on with alkaline phosphatase-conjugated anti rabbits antibodies and the immunodetection kit from Bio-Rad T M Quantification of endogenous levels of 12-hydroxviasmonate and 12 hvdroxviasmonate sulfate from Arabidopsis plants i) 12-hydroxyjasmonate Fresh plant material (1g) was homogenized with 10 ml methanol and 100 ng 12-( 2

H

3 )OAc-jasmonate as internal standard, the filtrate was evaporated and acetylated with Py/Ac20 at 20 *C overnight. The evaporated mixture was loaded on a 3 ml DEAE-Sephadex T M A25 columns (Acetate-form, methanol) and *h ,-,shed ,A ith I mi of methannl After washina with 3 ml of 0.1 M acetic acid in methanol, fractions eluted with 5 ml of 1 M acetic acid in methanol were collected, evaporated and separated on preparative HPLC and analyzed by GC-

MS.

HPLC: Eurospher T M 100-C 1 8 (5 pm, 250 x 4 mm), elution with a mixture methanol 0.2 acetic acid in H 2 0 (1 1) at a flow rate of 1 ml min- 1 UV detector 210 nm, fractions between Rt 5-6.5 min were evaporated.

WO 01/02589 PCT/CA00/00801 27 Derivatization: Samples were dissolved in 200 pl CHCl3/N,Ndiisopropylethylamine (1 1) and derivatized with 10 pl pentafluorobenzylbromide at 20 °C overnight. The evaporated samples were dissolved in 5 ml n-hexane and passed through a SiOH-column (500mg; Machery-NagelT). The pentafluorobenzyl esters were eluted with 7 ml n-hexane diethylether (2 1), evaporated, dissolved in 100 pl MeCN and analysed by GC-MS GC-MS: (GCQ Finnigan), 70 eV, NCI, ionization gas NH 3 source temperature 1400, column Rtx-5 (30 m x 0.25 mm, 0.25 4m film thickness), injection temperature 250 0 C, interface temperature 2750; Helium 40 cm s 1 splitless injection; column temperature program: 1 min 60°C, 250min- 1 to 1800 C, min-' to 2700 C ,1 min 270 10" min-' to 300°, 25 min 300 ii) 12-hydroxyjasmonic acid sulfate The negative ion electrospray (ES) mass spectra were obtained from a Finnigan T M MAT TSQ 7000 instrument (electrospray voltage 4 kV; heated capillary temperature 220 OC; sheath gas: nitrogen) coupled with a Micro-Tech Ultra-Plus MicroLC T M system equipped with a RP18-column (4 im, 1x100 mm, Ultrasep

M

For the HPLC a gradient system was used starting from H 2 0:CH 3 CN 90:10 (each of them containing 0.2% HOAc) to 10:90 within 15 min followed by a 10 min isocratic period at a flow rate of 70 gl min-'. The collision-induced dissociation (CID) mass spectra during the HPLC run were performed with a collision energy of eV; collision gas: argon, collision pressure: 1.8 x 10- 3 Torr. All mass spectra are averaged and background subtracted.

B) RESULTS Example 1: Flowering induction by treating A. inailana piants wiii 12hydroxyjasmonic acid Arabidopsis plants of ecotype Colombia (ColO) were grown in magenta boxes containing phytoagar and vitamins in a growth chamber under a sixteen hour photoperiod at a day-time temperature of 24 degrees and a night-time temperature of 20 degrees for a period of 18 days. The plants were then treated WO 01/02589 PCT/CA00/00801 with 10 pM of 12-hydroxyjasmonic acid (Fig. 2B) or with water as a negative control (Fig 2A) for a period of 6 days.

As seen in Figure 2B, plants treated with 12-hydroxyjasmonate flowered earlier (2 days) than the plants treated with water alone. Despite the fact that a treatment with 12-hydroxyjasmonic induces the hydroxyjasmonic acid sulfotransferase, early flowering is observed in the treated plants. The early flowering phenotype might be amplified if the treatment is coupled with an inhibitor of the hydroxyjasmonic acid sulfotransferase.

These results are of great economic importance since they show that it is possible to induce flower formation by the exogenous application of 12hydroxyjasmonate and/or others compounds of the jasmonate family to crop plants. Therefore it shows that one may induce early flowering when required by a simple application of a selected flower inducer to plants, particularly 12hydroxyjasmonate.

Example 2: Transgenic plants flowering tardily In this example, A. thaliana plants genetically modified were created by inserting therein a nucleic acid molecule encoding the AtST2a gene in the sense orientation under the control of a constitutive promoter. The results demonstrate that a higher endogenous expression of the hydroxyjasmonic acid sulfotransferase encoded by this gene is effective to delay flowering.

Figure 3 shows the phenotype of wild type non transgenic ColO Arabidopsis plants (WT) as compared to transgenic plants expressing the AtST2a gene under the control of the CaMV35S promoter 27 days after germination (S5, S6, S9, and S16). As shown in Figure 3, expression of AtST2a gene in transgenic Arabidopsis i -:i-oc Qv ihiorS rolir 1lii i i id d li. Lu -IIn ln •i ~i flowering as compared with non-transformed plants.

Fig. 4 shows a Western blot of protein extracts of these plants probed with anti-AtST2a antibodies. This figure clearly shows that the length of the delay is correlated with the level of expression of the transgene. This suggests that it is possible to vary the length of the delay by selecting transgenic lines expressing AtST2a at different levels. Delaying flowering time results in increased vegetative WO 01/02589 PCT/CA00/00801 growth and biomass which is a major advantage for crop such as lettuce, carrot, cabbage, sugar cane, sugar beet, to mention a few.

Table 1 hereinbelow also shows that a higher endogenous expression of the hydroxyjasmonic acid sulfotransferase results in higher endogenous level of 12-hydroxyjasmonate sulfate in the transgenic line S9.

TABLE 1: Wildtype (WT) Transgenic (S9) 12-hydroxyjasmonate sulfate 211 peak area/g 2234 peak arealg Example 3: Transgenic plants flowering early in non-inductive flowering conditions In this example, A. thaliana plants genetically modified were created by inserting therein a nucleic acid molecule encoding the AtST2a gene in the antisense orientation under the control of a constitutive promoter. The results demonstrate that a lower endogenous expression of the hydroxyjasmonic acid sulfotransferase is effective to induce flowering.

Figure 5 shows the phenotype of wild type Arabidopsis plants of ecotype C24 (WT) as compared to transgenic plants expressing the AtST2a gene in the antisense orientation under the control of the CAMV35S promoter (TL In this experiment, the plants were grown under short days which is non-inductive for flowering in Arabidopsis thaliana. Under these conditions the wildtype plants will flower after approximately 95 days of vegetative growth. The photograph was taken 65 days after germination and shows clearly an early flowering phenotype for the transgenic plants. As shown in this figure, inhibition of regular expression of the AtST2a gene in transgenic Arabidopsis thaliana affects flowering time since all the transgenic lines exhibited early flowering as cornped vwil. non-trsLf plants.

Table 2 hereinbelow also shows that a lower endogenous expression of the hydroxyjasmonic acid sulfotransferase results in a higher endogenous level of 12hydroxyjasmonate and in a lower endogenous level of 12-hydroxyjasmonate sulfate in plants.

WO 01/02589 PCT/CA00/00801 TABLE 2: Wildtype (WT) Transgenic (TL 7-2-5) 12-hydroxyjasmonate 7.7 ng/g 54.1 ng/g 12-hydroxyjasmonate 990 peak area/g 448 peak arealg sulfate Interestingly, apart from early flowering, the growth behavior and the size of the transgenic plants could not be distinguished from the non-transformed control plants.

Example 4: Transgenic plants flowering early under favorable flowering conditions Treatment with methyljasmonic acid of wild type Arabidopsis plants grown under favorable flowering day time conditions leads to elevated endogenous levels of both jasmonic acid and 12-hydroxyjasmonic acid (data not shown), conditions which should favor flowering. However, flowering was induced in an extent lower than what was anticipated (data not shown). As it will be explained hereinafter, a highly probable explanation for these results is that AtST2a gene expression is strongly induced under these favorable flowering day time conditions when treated with methyljasmonate thereby blocking the positive effects of the increase level of 12-hydroxyjasmonic acid.

To confirm this hypothesis, 15 days old A. thaliana plants and transgenic plants expressing the AtST2a gene in the antisense orientation under the control of a constitutive promoter were treated with 50 pM methyljasmonic acid for a period of nine days, and the plants were grown under favorable flowering day time conditions.

Figure 6 shows the effect of methyljasmonic acid treatment on the phenotype of wild type non transgenic C24 Arabidopsis plants (WT C24) as compared to transgenic plants expressing the AtST2a in the antisense orientation under the control of the CAMV35S promoter (TL 24 days after germination.

As shown in this figure, expression of AtST2a in the antisense orientation results WO 01/02589 PCT/CAOO/00801 31 in lowered levels of the AtST2 protein and allows the transgenic plants to flower early in presence of methyljasmonic acid.

This confirms that it is preferable, under certain conditions, to genetically modify a plant to induce its flowering prior to apply thereto a product further inducing its flowering.

Example 5: AtST2a gene expression is regulated by 12-hydroxyjasmonate Fifteen days-old Arabidopsis plants (ColO) were grown in magenta boxes in presence or in absence of 12-hydroxyjasmonate for a period of 24 hours. At the end of the incubation period, the plants were frozen in liquid nitrogen, ground to a fine powder and total mRNAs were extracted using the kit from the company Qiagen T M The mRNA extracts were resolved by agarose gel electrophoresis, and transferred by capillarity to a nylon membrane. The blot was probed with the sequence encoding AtST2a.

The results presented in Figure 11 show that 12-hydroxyjasmonic acid induces the expression of the AtST2a gene and that the level of expression is proportional to the amount of inducer. Furthermore, the results show that the level of expression is very low in untreated plants. The induction of AtST2a expression by its substrate suggests that the level of 12-hydroxyjasmonic acid present in the plant is tightly controlled. This result is not surprising considering the important role of 12-hydroxyjasmonic acid in the induction of flowering.

Example 6: Expression of AtST2a is under the control of photoperiod.

Fifteen days old Arabidopsis plants grown under long day conditions were transferred in the dark. At different time intervals, plants were collected, frozen in ft...h ,Ar and tntln mRNAs were extracted usinq a liqUlg IlLIUI Iel yiu iu u r. a kit from the company Qiagen

TM

The mRNA extracts were resolved by agarose gel electrophoresis, and transferred by capillarity to a nylon membrane. The blot was probed with the sequence encoding AtST2a.

The results presented in Figure 12 show that expression of AtST2a increases with time when the plants are kept in the dark reaching significant levels after 8 hours of dark treatment. This result suggests that plants monitor WO 01/02589 PCT/CAOO/00801 32 photoperiod by modulating the level of 11- and 12-hydroxyjasmonic acids. When the plants are grown under short day conditions, the increased level of expression of AtST2a leads to the sulfonation of 11- and 12-hydroxyjasmonic acids resulting in delayed flowering. When the plants are grown under long day conditions, AtST2a is not expressed and the levels of 11- and 12-hydroxyjasmonic acids increase resulting in an early flowering time.

C) CONCLUSION As shown in the above examples, AtST2a and AtST2b gene expression is induced after the application of 12-hydroxyjasmonate with a maximum of six hours after the beginning of the treatment. This pattern of induction demonstrates that the level of 12-hydroxyjasmonic acid is tightly regulated in vivo suggesting that 12hydroxyjasmonic acid plays an important role in the plant. AtST2a and AtST2b gene expression is also induced when the plants are grown in the dark. The kinetic of accumulation of AtST2a and AtST2b mRNA is slow with a maximum observed after 12 hours in the dark. Furthermore, there is a fast decrease in AtST2a and AtST2b mRNA levels when the plants are transferred back to light. Taken together, these results suggest that the biological function of AtST2a and AtST2b is to modulate the activity of 12-hydroxyjasmonic acid and 11-hydroxyjasmonic acid in relation to the photoperiod. The model presented in the section "General overview of the invention" integrates the different results obtained and tries to explain the role of the hydroxylated jasmonates and of the AtST2a and AtST2b genes in the control of flowering time. According to this model, 11- and 12hydroxyjasmonic acids are synthesized slowly into the leaves from jasmonic acid or from early fatty acids precursors. The accumulation of these metabolites up to a ^Ar r<-i nt r lin r Qho rt ilqv threshold value induces fiowering. vvWiin he Li tio l. time conditions, AtST2a and/or AtST2b will be expressed during the night and will inactivate 11- and 12-hydroxyjasmonic acids by sulfonation. This mechanism will retard flowering time until the photoperiod is favorable. When the plants are growing under long day time conditions, the level of expression of AtST2a and/or AtST2b is low and 11- and 12-hydroxyjasmonic acids will accumulate to levels sufficient to induce flowering.

WO 01/02589 PCT/CAOO/00801 33 While several embodiments of the invention have been described, it will be understood that the present invention is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention, following in general the principles of the invention and including such departures from the present disclosure as to come within knowledge or customary practice in the art to which the invention pertains, and as may be applied to the essential features hereinbefore set forth and falling within the scope of the invention or the limits of the appended claims.

obic Richard ROBIC (6e)514 U43l 031td .21-09-2001 U~I~ JulCA0000801 34 SEQUENCE LISTING <110> VARIN, LUC GIDDA, SATINDER <120> M.ETHODS, COMPOSITIONS AND GENETIC SEQUENCES FOR MODULATING FLOWERING IN PLANTS, AND PLANTS GENETICALLY MODIFIED TO FLOWER EARLY AND TARDILY (130> 29963-0002 <140> PCT/CA00/C0801 <141> 2000-07-06 ':150> CA 2,274,873 '151> 1999-07-06 ':160> <170> <210) <211> ':212> <213> 4 Patentin version 1 1077

DNA

Arabidopsis thaliana <400> 1 a tggctacct aagctagagc gggctaagct actcgttacc atgtcttcc 4aatCCggt* gatccggttg cctttctccg agtccaagaa cccggt gtga cattacacca gatctgtatt tggagagaga gacgacat cq g aagagga ac aagaagttgg t ttcggaaag caaqcatgaa tcct taaaga gcgagttcca t ttacctatt aaaaacattt caacccggct cctcgagtac agtacaaget cgttcgcaac aggtcgtgta acaacatcaa gccggggagt gcttgaagag agaccaactt qjaaagggagt aggtgaacaa gagaagtgag gagcattcca atggcgatcc caagrttctc catgtgtcac aggcaaaact agagatgttg ccaagggttt CCoatCctc aaaagCttta caaccaccct ttacgCcaac ccacttacCg cttgtgccgg at ccgagt ca gatcgggttt accagagaaa gaagaggctt cgtgaaggct gtcaaacaag tgattgggtt cgcgacgtcc gattctcttc t ggtgc caag gaaaacgacg actttcacca cttttcactt ggagatgttc t tcggttcc aacccgtttg gtgagcccag ggcccgtt tt gtcttctttt qcaactttct atcgccgagc rcgatcaaga aactatttgt cgaaagccga ctaaggagag ccaaagagat tCgtCtctcgc tccttaaccg ccaaccctca ccgatctctc taaaggaaac acacattcat tcttgctaga gggaacacat taaggtacga tagagcttcc tgtgtagctt actttgagaa caccttcaca agaagatgaa aggatggaga tcaagdcatc caccat acct tcaccggttt tgaccetgta gggtctagcc gatcgag aaa ctcttcgtgg ccaagctttt gtt gggataC ggatctcaaa t ttcaccgaa cgagaatctg tcgattcttg agtggaaaga 120 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 AMENDED SHEET Fmofanxq7pit 71. SAD. 711:31 61 21"09-2001 obic Richard ROBIC (6e)514 845 651B; obicRichrd &ROBI (6e514 45 618;CA0000801ttgtcagcct tagtggatqe caagttaggt ggatctggtc tcactttcag gttgagc 1077 <210> 2 <211> 1041 <212> DNA <213> Arabidopsis thaliana <400> 2 atggcgatcc gaaggccaag aaggagagag aaggagattc gtcctcgcca CttacccgtC tccaaccctc cccgatctct ctt a agga tt gacacattca gtcttgctag ttttgggaac Ettttaaagt trcttaggac gatctgtgta cagaactatg ggtctcactt caagtttctc aaqaagaagg gacggagaaa aagcta-:cac ccatacctaa atcggtttga acgacctcgt cqgtctagc cggt cgagaa tctccatgtg acgaagcttt acatgt tggg acgaggatct t tcctttcac gcttrgagaa agaaccggtt tcagatzgag catgtgtcac gctaagctac tcgttacctt gtctttccaa atctqgcaca tccggtttcc acctttcttc cagtccaaga tcccagtgtg 9catta cat c tgatctatat atactggaga caaagaagac cgaagaagag tctgaagaag cttgtttagg aagccegagc qagttccaag tacttatmcc aaacattttc acctggttaa tcatcaagtt gagtacaagc acattcgcaa aaggttgtgt aacaacatca tgccggggat gagagct tga atcgagacca gaacaaaagg ttggaggtga aaaggagaag tccttaagga agatgttgga aagggtttcg agtcccttc aagctttaac ccgaccaccc tttacgccaa cccacgtacc acctgtgccg cttccgag~c tactgatcgg agaggccaga acttgaagaa gagttgtgaa acaagtcaag tgagtgat tt aggcaaaagc ctctcttcct qtgcCaagct agacgacgtt tttcaccatc tcttctcaca cggaaatgt gftt cggtgcc gaacccgttt agt gagcgca atttggcecg gaaagtctta gctagcaagt agctatcgct caaattgatc ggttaactat 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1041 <210> 3 <211> 359 <212> PRT <213> Arabidopsis thaliana (400> 3 Met Ala Thx Ser Ser Met Lys Ser Ile Pro Met Ala Ile Pro Sezr Phe 1 5 10 Ser Her- Cys His Lys Leu Glu Leu Lou Lys G1lu Gly Lys Thr Arg Asp 25 AMENDED SHEET Cmnfinfofv~ 1 eon. ?n:1l ."21"'09-2001 "bi Richard ROBIC (6e)514 1845 6518; u9I:;e.Iui 14,0 JrJEA- 1 3COO O Val Pzo Lys Ala Glu Glu Asp Glu Gly Leu Ser Met Tyr Met Ala Thr His Tyr 145 Se r Thr Phe Glu Ar g 225 Trp Phe Lys Val1 305 Ph a Leu Lev Ser 1'hr Ile Pro 130 Lys Pro Ile Asp Ser 210 Gly Arg L.eu Al a 29C Asn Arg Asp Phe Phe Ile Lau 115 Lau Leu Arg Glii thr 195 Val1 ValI Glu GI u 275 Ile Lys Lys Ser Gln Gln Pro 100 As n Phe

T

yr Thr Lys 180 Phe Ser Ile Ser Lys- 260 Leu Ala Ser Gly Lev Gi y Lys Lys Arg Thr Ala Phe 165 Pro Ile Pro Gly Leu 245 Pro~ Glu As n Glu 325 Pro Phe 70 His Ser His Ser As n 150 Ala Gc-*y So r Val.

Phe 23-;0 Lys'

A*

Phe Lau Lys 313 Val1 Lys Trp Phe Gly Arg As n 135 Gi y Thr Val1 Ser Leu 215 Gi y Azg Thr Cys 295 S er Ser Glu cys Gin Thr Phe 120 Pro Asp His Lya Trp 200 Lau Pro Pro Glu 280 Ser Ile Asp Ar g Gin Ser Thr 105 As p His Val1 Lau Val1 His Asp Phe Glu 265 Gi u Phe Lys Trp Gly Ala Leu 90 Trp, Pro Asp Pro Pro 1,70 Val1 Tryr Gln Trp Lys 250 Giu Giu Asn Val 330 Tro Lys 75 Giu Lou Val1 Lou Asp Phe Tyr Thr Al.a Ci u 235 Val Len, Glu Asn Phqe 31.5 As n Cys Arg Glu Asn Lys Al a Val1 140 Leu Gly Lau Asn Phe 220 His Ph.

Arg Lau 330 Glu Tyr Gl u Thr Ile Asp Ala Se r 125 Pro Ser Ser Cys Asn 205 Asp Met Phe Lys 285 Lys Asn Lou Phe Arg Gin Val Lev.

Ser Phe Gi y Lau Arg 190 le Lou Lou Lau 2.70 Gl y Lys Arg Ser Gln Giu Tyr Leu Ala Ile s0 Val Leu Thr Phe Thr Asn Phe Glu Levu Ala 160 Lys Giu 175 Asn Pro Lys Ser Tyr Cys Gly Tyr 240 Arg Tyr 255 Alm Val. Val Leu Glu Phe Lev 320 Pro Ser 335 Gin Val. Giu Arg Lou Ser Ala Lau Val Asp Asp Lys Levu Gly Gly Ser 340 345 350 AMENDED SHEET rm~fnfqg7oif 91 Rao Vi 711 6.21"'09-2001 obic Richard ROBIC (8e)514 845 6518; ubtleiu IU ts 14:dc; ei~ A 000801 Gly Leu Thr Phe Arg Leu Ser 355 <210> 4 <211> 34? <222> PRT <213> Arabidopsis thaliana <400> 4 Met GlU Gin Tyr Ala Val Thr Ser Phe dly 145 Le u Arg Ile Leu Met 225 Phe Ala Gly Leu Ile Pbhe Ser Phe 1.30 Le u Lys As n Thr Tyr 210 Leu Leu Ile Lys Met Tyr Th r Ala Thr Asp Glu Ala Asp Pro Ser 19 5 Cys Gly Lys Pro Ser Ser Glu Lou Asp Leu Ph.

Ser Phe Thr Ile Ile Leu 100 His Pro Tyr Lys Ser Pro Ser Val 1 C-5 Phe Asp Glu Ser Arg Gly Tyr Trp Tyr Glu 245 Phe Ser Gly Gin Ser Leu Gin Gly 55 Gin Lys 70 Pro Lys Thr A--g Leu Lou Leu Tyr 135 Arg Thr 150 Glu Asn Thr Phe Val Ser Leu Leu 215 Arg Giu 230 Asp Leu Met Cys Glu Glu 25 Pro Lys 40 Phe Arg His Phe Ser Gly His Arg 105 Thr 5cr 120 Ala Asn Phe Ala Pro Ser Ile Ser 185 Ala Val 200O Ile Gly Ser Leu Lys Glu HijS 10 Glu Glu Cys Gln Th r .90 Ph.

Asn Gly Thr Val 11 Met Leu Phe Lys Asp 250 :,Ys Gly Arg Gin Ser Thr Asp Pro As n His 155 Lys Trp Leu Gly Arg 235 Ile Pro 1 1u Gly Al a Lei) Trp Pro His Val 140 ValI Val1 His Asp Pro 220 Pro Glu Gi u Ser Arg Lys Pro Leu Val Amp 125 Pro Pro Val1 Tyr Glu 205 Ph.

Glu Thr Leu Tyr Arg Glu Usp Lys 5cr 110 Lou Asp Phe Tyr Ile 190 Ala Trp Lys Azn Leu Lys Glu Ph.

Asn Azg Ile Gin Asp Val Ala Leu Ser Ser Val Pro Lou Ser cay Ala 1.60 Leu Cys 1'7 Asn Asn Phe Asp Glu His Val Lou 240 Leu Lys; 255 Lys Lou Ala Ser Ph. Leu Gly Leu Pro Phe Thr G:.u Glu Glu Glu Gin AMENDED SHEET Pmnf 2 nZ 7 0i t 71 'PD. 711:31 W2-09-2001 ",cnari is num.Iu ve UWICgSI.MJA......f CA0000801 Lys Gly Val 275 Lys Lys Leu 290 Asri Arg Phe 305 Leu Ser Pro Lou Ala Gly 260 ValI Glu Leu Ser Ser 340 Lys Ala Ile Ala 280 Val ;Asn Lys Ser 295 Phe Arg Lys Gly 310 Gln Val Glu.Arg 325 Gly Leu Thr Phe 265 Asp Ser Giu Leu Arg Leu Lys Val2 Ser 330 Leu 270 Cys Ser ?he Giu Asn Leu 285 Leu Ile Gin Asn Tyr Glu 300 Ser Asp Leu Val Asn Tyr 315 320 Ala Lou Val Asp Asp Lys 335 Ser AMENDED SHEET Empfangszeii zi-bep. Lu~di

Claims (25)

1. A method for modulating flowering in a plant, comprising modifying in said plant the endogenous level of at least one compound selected from the group consisting of 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, sulfate ester of 12-hydroxyjasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, sulfate ester of 12- hydroxymethyljasmonic acid, 11-hydroxyjasmonic acid, glucoside of 11- hydroxyjasmonic acid, sulfate ester of 11-hydroxyjasmonic acid, 11- hydroxymethyljasmonic acid, glucoside of 11-hydroxymethyljasmonic acid, sulfate ester of 11 -hydroxymethyljasmonic acid, and mixtures thereof.

2. A method of claim 1, wherein flowering of said plant is induced by increasing in said plant the endogenous level of at least one flowering inducing compound selected from the group consisting of 12-hydroxyjasmonic acid, ,i 15 glucoside of 12-hydroxyjasmonic acid, methyljasmonic acid, 12- hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, 11- hydroxyjasmonic acid, glucoside of 11-hydroxyjasmonic acid, 11- hydroxymethyljasmonic acid, and glucoside of 11-hydroxymethyljasmonic acid, said flowering induction and said endogenous level increase being compared to a 20 corresponding plant wherein the endogenous level of said at least one compound has not been modified.

3. A method of claim 2, wherein the endogenous level of said at least one flowering inducing compound is increased by: a) increasing in said plant the endogenous level of an hydroxylase hydroxylating jasmonic acid and/or methyljasmonic acid; and/or b) lowering in said plant the endogenous level of a sulfotransferase sulfonating

12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic. 004646740v2.doc 4. A method of claim 3, wherein the endogenous level of the su!fotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic is lowered by a genetic modification of said plant. 5. A method of claim 4, wherein said genetic modification comprises the step of inhibiting the expression of at least one gene selected from the group consisting of AtST2a, AtST2b and functional homologues of AtST2a or of AtST2b. 6. A method of claim 5, wherein said gene expression is inhibited by expressing into said plant an exogenous sequence coding for a nucleic acid sequence antisense to said gene, said method comprising the steps of: a) introducing into a cell of said plant an exogenous nucleic acid molecule comprising a sequence of nucleotides antisense to a sequence encoding a plant hydroxyjasmonic acid sulfotransferase; 15 b) regenerating a transgenic plant from the cell; and c) growing said transgenic plant for a time and under conditions sufficient to inhibit expression of the hydroxyjasmonic acid sulfotransferase. 7. A method of claim 6, wherein the exogenous nucleic acid molecule 20 comprises a nucleotide sequence antisense to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, nucleotide sequences having at least 50% similarity with SEQ ID NO:1, SEQ ID NO:2 and nucleotide sequences having under the control of a constitutive or an inducible promoter. 9A method of any one of claims 4 to 8, wherein the endogenous level of said at least one flowering inducing compound is further increased by a method selected from the group consisting of: 004646740v2.doc 41 a) applying to said plant at least one of said flowering inducing compounds and/or salts thereof; b) applying to said plant at least one inhibitor of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic; and c) applying to said plant at least one stimulator of an hydroxylase hydroxylating jasmonic acid and/or methyljasmonic acid. A method of any one of claims 2 to 9, wherein said plant is selected from crop plants. 11. A plant genetically modified to flower early when compared to a corresponding plant not genetically modified, wherein said genetically modified plant is obtained by the method of any one of claims 4 to 8. b) 12. A cut flower from the genetically modified plant of claim 11.

13. A composition when used for inducing flowering in a plant comprising a 20 flowering inducing effective amount of a compound selected from the group consisting of 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, 12- Shydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, 11- rrmv Anczrnn "t tn'-;e rollI hydroxymethyljasmonic acid, glucoside of 11-hydroxymethyljasmonic acid, salts thereof, and mixtures thereof, in combination with a diluent or a carrier such that an induction in flowering of said plant occurs when compared to a corresponding plant in the absence of said composition.

14. A composition of claim 13, further comprising a compound selected from the group consisting of fertilizers, growth regulators, fungicides, insecticides, emulsifying agents and mixtures thereof. 004646740v2.doc 42 A method of claim 1, wherein flowering of said plant is delayed by lowering in said plant the endogenous level of at least one compound selected from the group consisting of 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, 11-hydroxyjasmonic acid, glucoside of 11-hydroxyjasmonic acid, 11- hydroxymethyljasmonic acid, and glucoside of 12-hydroxymethyljasmonic acid, said flowering delay and said lower endogenous level being compared to a corresponding plant wherein the endogenous level of said at least one compound has not been modified.

16. A method of claim 15, wherein the endogenous level of said at least one compound is lowered by: a) lowering in said plant the endogenous level of an hydroxylase hydroxylating jasmonic acid and/or methyljasmonic acid; and/or 15 b) increasing in said plant the endogenous level of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic acid.

17. A method of claim 16, wherein the endogenous level of said sulfotransferase is increased by a genetic modification in the genome of said plant. 2 18. A method of claim 17, wherein said genetic modification comprises the steps of increasing the expression of at least one gene selected from the group r nncictinn rnf A fT9 AfQT')h cnr( ftn ir nnl hrnmrlnnin i nf A fT9-3 r\ r\f AfT9h said method comprising the steps of: a) introducing into a cell of said plant an exogenous nucleic acid molecule encoding a plant hydroxyjasmonic acid sulfotransferase; b) regenerating a transgenic plant from the cell; and c) growing said transgenic plant for a time and under conditions sufficient to permit expression of the nucleic acid sequence into an hydroxyjasmonic acid sulfotransferase. 004646740v2.doc 43

19. A method of claim 18, wherein the exogenous nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO:1, nucleotide sequences having at least 50% similarity with SEQ ID NO:1, SEQ ID NO:2 and nucleotide sequences having at least 50% similarity with SEQ ID NO:2. A method of claim 18 or 19, wherein said gene expression is increased by placing said gene under the control of a constitutive or of an inducible promoter.

21. A method of any one of claims 17 to 20, wherein the endogenous level of 0* said at least one compound is further lowered by: S 15 a) applying to said plant an inhibitor and/or an inactivator of at least one of *l* said compounds; b) applying to said plant at least one stimulator of a sulfotransferase sulfonating 12-hydroxyjasmonic acid and/or 11-hydroxyjasmonic; and/or c) applying to said plant at least one inhibitor of an hydroxylase hydroxylating 20 jasmonic acid and/or methyljasmonic acid.

22. A plant genetically modified to flower tardily when compared to a corresponding plant not genetically modified, said genetically modified plant being 0 I L- A J X -X 4 -7 4 o0* ULUCIlII Uy Lilt; I II LI IUU 1 a lly Ui IV i ,i 1lIII I I I LJ e. I. 000 0

23. A composition when used for delaying flowering in a plant comprising a flowering delaying effective amount of an inhibitor or of an inactivator of a compound selected from the group consisting of 12-hydroxyjasmonic acid, glucoside of 12-hydroxyjasmonic acid, 12-hydroxymethyljasmonic acid, glucoside of 12-hydroxymethyljasmonic acid, 11-hydroxyjasmonic acid, glucoside of 11- hydroxyjasmonic acid, 11-hydroxymethyljasmonic acid, and glucoside of 11- hydroxymethyljasmonic acid, in combination with a diluent or a carrier such that a 004646740v2.doc 44 delay in flowering of said plant occurs when compared to a corresponding plant in the absence of said composition.

24. A composition of claim 23, further comprising a compound selected from the group consisting of fertilizers, growth regulators, fungicides, insecticides, emulsifying agents and mixtures thereof. Use of an isolated or purified nucleic acid molecule encoding a plant 11- hydroxyjasmonic acid or 12-hydroxyjasmonic acid sulfotransferase for modulating flowering in a plant.

26. Use according to claim 25, wherein said isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, nucleotide sequences having at least 50% similarity with SEQ ID NO:1, 15 SEQ ID NO:2, nucleotide sequences having at least 50% similarity with SEQ ID NO:2 and nucleotide sequences complementary thereto.

27. Use according to claim 25, wherein said isolated nucleic acid molecule comprises a nucleotide sequence which hybridizes under low stringency i:i. 20 conditions to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, a complementary strand of SEQ ID NO:1, SEQ ID NO:2 and a S complementary strand of SEQ ID NO:2.

28. Use according to any one of claims 25 to 27, wherein the hydroxyjasmonic acid sulfotransferase is of Arabidopsis thaliana origin.

29. Use according to any one of claims 25 to 28, wherein the nucleic acid molecule is inserted in a vector.

30. Use according to claim 29, wherein the vector is capable of replication and expression in a plant cell. 004646740v3.doc

31. A method of any one of claims 4 to 10 or 16 to 21, wherein the hydroxyjasmonic acid sulfotransferase is a 11- or a 12- hydroxyjasmonic acid sulfotransferase.

32. An isolated or purified polypeptide having the biological activity of a plant 11-hydroxyjasmonic acid or 12-hydroxyjasmonic acid sulfotransferase.

33. A polypeptide of claim 32, encoding a sulfotransferase enzyme selected from the group consisting of: a) an enzyme whose amino acid sequence is represented by SEQ ID NO: 3 or SEQ ID NO: 4; and b) functional homologues of enzyme a) isolated from a plant, or derived from enzyme by substitution, deletion or addition of one or several amino acids in the amino acid sequences defined in a) and having similar biological activity 15 or function(s).

34. An antibody binding with affinity to a polypeptide as defined in claim 32 or 00 0 0 0 0@ 00 0 0** 9 0 0 @0* 00* 0 @0 0 0 0 0 0 *0 0 00 0 000 9 0000 0* 0 @000 4 0* 0 4 0 0000 0 000 0 0 20 35. An antibody of claim 34 when used for delaying flowering in a plant.

36. A method for modulating flowering in a plant according to claim 1,

37. A composition according to claim 13, substantially as hereinbefore described with reference to the examples.

38. A composition according to claim 23, substantially as hereinbefore described with reference to the examples. Dated 17 May 2005 Freehills Patent Trade Mark Attorneys Patent Trade Mark Attorneys for the Applicants: Luc Varin and Satinder Gidda