CA2415062A1 - Methods and genetic sequences for modulating tuber formation in tuber-producing plants, and plants genetically modified to alter tuber size and number - Google Patents
Methods and genetic sequences for modulating tuber formation in tuber-producing plants, and plants genetically modified to alter tuber size and number Download PDFInfo
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Abstract
The invention relates to methods and genetic sequences to modulate the number and the size of tubers in tuber-producing plants. More particularly the present invention provides among others a genetic sequence encoding for a hydroxyjasmonic acid sulfotransferase and methods for producing transgenic plants using such a sequence.
Description
METHODS AND GENETIC SEQUENCES FOR MODULATING TUBER
FORMATION IN TUBER-PRODUCING PLANTS, AND PLANTS GENETICALLY
MODIFIED TO ALTER TUBER SIZE AND NUMBER
BACKGROUND OF THE INVENTION
a) Field of the invention The invention relates to methods and genetic sequences to modulate the number and the size of tubers in tuber-producing plants.
b) Brief description of the prior art In tuber-bearing species'such as Solanum tuberosum, grafting experiments have demonstrated that tuber formation is induced by a tuberization stimulus formed in the leaves under short day conditions and transmitted to the underground parts of the plant~~(Gregory 1956 Am .J. Bot. 43:281 and Chapman 1958 Physiol. Plant. 11:215). The tuber-inducing activity is present in the leaves, increases under short day conditions and remains constant under long-days (Koda et al, 1988 Plant Cell Physiol. 29: 1047):
Two acidic compounds having tuber-inducing activity were isolated from leaves of Solanum tuberosum kept under short day conditions. The structure of one of the active compound was determined to be 3-oxo-2- (5'-/3-D
glucopyranosyloxy-2'-Z-penfienyi)-cyclopentane-1-acetic acid (Yoshihara et al., 1989 Agric. Biol. Chem. 53: 2835). The aglycone of this glycoside is 12 hydroxyjasmonic acid (12-OH-JA), which is also referred to as tuberonic acid (Figure 1, structure of 12-~H-JAltuberonic acid).
~--i 'oH
C OOH COOW
11-hydroxyjasmonic acid 12-hydroxyjasmonic acid Figure 1 Chemical structure of 11- and 12-hydroxyjasmonic acid a
FORMATION IN TUBER-PRODUCING PLANTS, AND PLANTS GENETICALLY
MODIFIED TO ALTER TUBER SIZE AND NUMBER
BACKGROUND OF THE INVENTION
a) Field of the invention The invention relates to methods and genetic sequences to modulate the number and the size of tubers in tuber-producing plants.
b) Brief description of the prior art In tuber-bearing species'such as Solanum tuberosum, grafting experiments have demonstrated that tuber formation is induced by a tuberization stimulus formed in the leaves under short day conditions and transmitted to the underground parts of the plant~~(Gregory 1956 Am .J. Bot. 43:281 and Chapman 1958 Physiol. Plant. 11:215). The tuber-inducing activity is present in the leaves, increases under short day conditions and remains constant under long-days (Koda et al, 1988 Plant Cell Physiol. 29: 1047):
Two acidic compounds having tuber-inducing activity were isolated from leaves of Solanum tuberosum kept under short day conditions. The structure of one of the active compound was determined to be 3-oxo-2- (5'-/3-D
glucopyranosyloxy-2'-Z-penfienyi)-cyclopentane-1-acetic acid (Yoshihara et al., 1989 Agric. Biol. Chem. 53: 2835). The aglycone of this glycoside is 12 hydroxyjasmonic acid (12-OH-JA), which is also referred to as tuberonic acid (Figure 1, structure of 12-~H-JAltuberonic acid).
~--i 'oH
C OOH COOW
11-hydroxyjasmonic acid 12-hydroxyjasmonic acid Figure 1 Chemical structure of 11- and 12-hydroxyjasmonic acid a
2 Further indication that 12-OH-JA is a tuberization stimulus was obtained by Helder et al. (1993 Physiol. Plant. 88: 647) who demonstrated that 12-OH-JA and 11-OH-JA (Figure 1 ) were found in the leaves of wild type Solarium demissum that had formed tubers, but not in leaves of long-day plants where tuberization did not occur. Labeling experiments with 2-[14C] jasmonic acid (JA) provide evidence that 12-OH-JA is synthesized by the direct hydroxylation of JA (Yoshihara et al., 1996 Plant Cell Physiol. 37:
586) 11-hydroxyjasmonic acid (see Fig. 1) is also a natural metabolite for which the mechanism of biosynthesis have not been described. 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.
Many functions have been associated with jasmonates metabolites such as 12-hydroxyjasmonic acid andlor 11-hydroxyjasmonic acid. 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 Tazaki (Japanese kokai 292220 (A) published April 3 1990, and patent application no 63-242432, filed September 29, 1988); Yoshihara et al. (1989), Agric. Biol.
Chem. 53: 2835-2837, Matsuki ef al. (1992), Biosci. Biotech. Biochem. 56:
1329.;
and Koda and Okazawa (1988), Planf 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 it is possible to modulate tuber number and size by in-viva sulfonation of jasmonates:
Accordingly, there is a need for effective methods to modulate tuber size and numbers in plant producing tubers such as Solarium tuberosum. There is also a need for plants genetically modified to produce tubera of larger or smaller sizes.
586) 11-hydroxyjasmonic acid (see Fig. 1) is also a natural metabolite for which the mechanism of biosynthesis have not been described. 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.
Many functions have been associated with jasmonates metabolites such as 12-hydroxyjasmonic acid andlor 11-hydroxyjasmonic acid. 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 Tazaki (Japanese kokai 292220 (A) published April 3 1990, and patent application no 63-242432, filed September 29, 1988); Yoshihara et al. (1989), Agric. Biol.
Chem. 53: 2835-2837, Matsuki ef al. (1992), Biosci. Biotech. Biochem. 56:
1329.;
and Koda and Okazawa (1988), Planf 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 it is possible to modulate tuber number and size by in-viva sulfonation of jasmonates:
Accordingly, there is a need for effective methods to modulate tuber size and numbers in plant producing tubers such as Solarium tuberosum. There is also a need for plants genetically modified to produce tubera of larger or smaller sizes.
3 BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 shows the chemical structures of 11-hydroxyjasmonic acid and 12-hydroxyjasmonic acid.
Figure 2 is a picture showing the phenotype of tubers from transgenic Solanum chacoense plants expressing AfST2a gene under the control of a constitutive promoter when compared fo wild type non-transgenic plant (INT).
11 and 30 indicate independent transgenic lines. Ctrl1 and 2 are two independent control (non-transgenic lines).
Figure 3 shows a graphical representation of the tuber sizes of the independent transgenic lines.
Figure 4: Shows nucleotide sequence of AtST2'a gene (SEQ ID NO 1) taken from the GeneBank database (accession number NM 120783) Figure 5: Shows the deduced amino acid sequence (SEQ ID NO 3) of the protein encoded by the AfST2a gene shown in Fig. 4.
Figure 6: Shows the nucleotide sequence of AfST2b gene {SEQ ID NO 2) taken from the GeneBank database (accession number NM_120782) Figure 7: Shows the deduced amino acid sequence (SEQ ID NO 4) of the protein encoded by the AtST2b gene shown in Fig. 6.
DETAILED DESCRIPTION OF THE INVENTION
A~Definitions In order to provide an even clearer and more consistent understanding of the specification, including the scope given herein to such terms, the following definitions are provided:
11-hydroxyjasmonic acid: 3-Oxo-2-(4-hydroxy-2-pentenyl)-cyclopentane-1-acetic acid. Its chemical structure is shown in Fig. 1.
11-hydroxyjasmonic acid glucoside: 3-Oxo-2-(4-~3-D-glucopyranosyloxy-2-pentenyl)-cyclopentane-1-acetic acid 11-hydroxyjasmonic acid sulfate: 3-Oxo-2-(4-hydroxysulfonyioxy-2-pentenyl)-cyclopentane-1-acetic acid 12-hydroxyjasmonic acid: 3-Oxo-2-(5-hydroxy-2-pentenyl)-cyclopentane-1-acetic acid. Its chemical structure is shown in Fig. 1.
Figures 1 shows the chemical structures of 11-hydroxyjasmonic acid and 12-hydroxyjasmonic acid.
Figure 2 is a picture showing the phenotype of tubers from transgenic Solanum chacoense plants expressing AfST2a gene under the control of a constitutive promoter when compared fo wild type non-transgenic plant (INT).
11 and 30 indicate independent transgenic lines. Ctrl1 and 2 are two independent control (non-transgenic lines).
Figure 3 shows a graphical representation of the tuber sizes of the independent transgenic lines.
Figure 4: Shows nucleotide sequence of AtST2'a gene (SEQ ID NO 1) taken from the GeneBank database (accession number NM 120783) Figure 5: Shows the deduced amino acid sequence (SEQ ID NO 3) of the protein encoded by the AfST2a gene shown in Fig. 4.
Figure 6: Shows the nucleotide sequence of AfST2b gene {SEQ ID NO 2) taken from the GeneBank database (accession number NM_120782) Figure 7: Shows the deduced amino acid sequence (SEQ ID NO 4) of the protein encoded by the AtST2b gene shown in Fig. 6.
DETAILED DESCRIPTION OF THE INVENTION
A~Definitions In order to provide an even clearer and more consistent understanding of the specification, including the scope given herein to such terms, the following definitions are provided:
11-hydroxyjasmonic acid: 3-Oxo-2-(4-hydroxy-2-pentenyl)-cyclopentane-1-acetic acid. Its chemical structure is shown in Fig. 1.
11-hydroxyjasmonic acid glucoside: 3-Oxo-2-(4-~3-D-glucopyranosyloxy-2-pentenyl)-cyclopentane-1-acetic acid 11-hydroxyjasmonic acid sulfate: 3-Oxo-2-(4-hydroxysulfonyioxy-2-pentenyl)-cyclopentane-1-acetic acid 12-hydroxyjasmonic acid: 3-Oxo-2-(5-hydroxy-2-pentenyl)-cyclopentane-1-acetic acid. Its chemical structure is shown in Fig. 1.
4 12-hydroxyjasmonic acid glucoside: 3-Oxo-2-(5-a-D-glucopyranosyloxy-2-pentenyl)-cyclopentane-1-acetic acid.
12-hydroxyjasmonic acid suifate: 3-Oxo-2-(5-hydroxysulfonyloxy-2-pentenyl)-cyclopentane-1-acetic acid.
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 th~se molecules to be affected.
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 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.
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 acid sequences encoding an enzyme having a substantially similar biological activity as 11- or 12-hydroxyjasmonate sulfotransferase and isoenzyme 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 using methods and principles well known in the art.
A functional homologue of a protein may or may not contain post-translational
12-hydroxyjasmonic acid suifate: 3-Oxo-2-(5-hydroxysulfonyloxy-2-pentenyl)-cyclopentane-1-acetic acid.
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 th~se molecules to be affected.
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 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.
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 acid sequences encoding an enzyme having a substantially similar biological activity as 11- or 12-hydroxyjasmonate sulfotransferase and isoenzyme 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 using methods and principles well known in the art.
A functional homologue of a protein may or may not contain post-translational
5 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 canservation.
Geneticlnucleotide 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 plant. 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 cross-pollination with other plants of the same species, sv that the exogenous nucleic acid, carried in the germ line, may be inserted into or bred into agriculturally useful plant varieties.
Geneticlnucleotide 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 plant. 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 cross-pollination with other plants of the same species, sv that the exogenous nucleic acid, carried in the germ line, may be inserted into or bred into agriculturally useful plant varieties.
6 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 non-naturally-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 functions) 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 meaning as nucleic 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, the term "modulate" refers in some cases to increased or decreased tuber size
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 non-naturally-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 functions) 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 meaning as nucleic 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, the term "modulate" refers in some cases to increased or decreased tuber size
7 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.
SimilaritylComplementarity: 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 hydroxyjasmonic 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 andlor 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 % (wlv) SDS at 37-45 °C for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridization, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 1-4.X SSCI0.5-1 % (w/v) SDS at greater than or equal to 45°C for 2-3 hours or high stringent conditions considered herein to be 0.1-1X
SSCI0.1-1.0% 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.
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 proteins) in a recipient during cloning procedures and may comprise different selectable markers. Bacterial plasmids are commonly used vectors. Preferably, thie vectors of the invention are capable of facilitating transfer of a nucleic acid into a plant cell andlor facilitating integration into a plant genome.
B) General overview of the invention The present inventors have now discovered that it is possible to modulate tuber size in tuber-producing plants by increasing or decreasing the activity of a hydroxy-jasmonate sulfotransferase.
In practice, modulation of tuber size is achieved in two different ways: it is either increased or decreased. Although many approaches may be used to achieve these effects, the approaches described hereinafter are preferably used according to the invention.
1 ~ Chemical approach i) Increase tuber size Inhibitors of hydroxyjasmonic acid sulfotransferase(s) prevent in vivo inactivation of the tuber-inducing molecule by sulfonation. To the contrary, stimulators of jasmonic acid hydroxylase(s) help in the production of hydroxy-jasmonate compound(s).
The above mentioned compounds, stimulators and/or inhibitors are preferably part of a composition for modulating tuber production in plants.
The carrier or diluent is preferably a solvent such as water, oil or alcohol.
Optionally, the composition comprises others active agents such as fertilizers and growth regulators. The inducing composition is preferably formulated with emulsifying agents in the presence or absence of fungicides or insecticides, if required.
The precise amount of compound employed in the .practice of the present invention depends upon the type of response desired, the formulation used and the type of plant treated.
i) Reduce tuber size According to the invention, Inhibitors of jasmonic acid 11- and 12-hydroxylase(s) prevent the formation of the tuber-inducing molecule when compared to a corresponding plant in which the endogenous level of the compound has not been modified. The above mentioned inhibitors are preferably part of a composition for modulating tuber production in plants. The carrier or diluent is preferably a solvent such as water, oil or alcohol. Optionally, the composition comprises others active agents such as fertilizers and growth regulators. In accordance with a preferred embodiment, the inducing composition is formulated with emulsifying agents in the presence or absence of fungicides or insecticides, if required. The precise amount of compound employed in the practice of the present invention depends upon the type of response desired, the formulation used and the type of plant treated.
2) Molecular approach In accordance with the present invention, genetic sequences encoding a plant hydroxyjasmonic acid sulfotransferase have been used to generate transgenic plants.
SEQ ID NO 1 (Fig. 4 ; GeneBank: accession number NM_120783) corresponds to the gene AtST2a in Arabidopsis thaliana. SEQ ID NO 3 (Fig. 5) is an amino acid sequence deduced from SEQ ID NO 1. This amino acid sequence is of public domain and can be retrieved from Genebank, accession number NM 120783. The AtST2a gene from Arabidopsis thaliana encodes a sulfotransferase that sulfonates 12-hydroxyjasmonic acid and 11-hydroxyjasmonic acid with high specificity.
This hydroxyjasmonic acid sulfotransferase exhibits high affinity for its substrate with a Km value of 11 NM for 12-hydroxyjasmonic acid and 60 pM for 11-hydroxyjasmonic acid. The enzyme did not accept structurally related compounds such as cucurbic acid, arachidonyl alcohol or prostaglandins. Maximum enzyme activity was observed at pH 7.5 in TrislHCI buffer and did not require divalent rations for activity.
a r' SEQ ID NO 2 (Fig. 6; GeneBank: accession number NM_120782) corresponds to the gene AfST2b in Arabidopsis fhaiiana. SEQ ID NO 4 (Fig. 7) is an amino acid sequence deduced from SEQ ID NO 1. This amino acid sequence is of public domain and can be retrieved from Genebank, accession number 5 NM_120782. Amino acid sequence alignment between SEQ 1D NOS 3 and 4 indicates that they share 85% amino acid sequence identity and 92% similarity, suggesting that AtST2a and AfST2b encode isoenzymes.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or 10 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
N0:1, SEQ ID N0:2 nucleotide sequences having at least 50% similarity with SEQ
ID N0:2, and sequences hybridizing under low stringency conditions to ane 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 expression-vector 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 eiectroporation, microprojectile bombardment, Agrobacterium-mediated transferor insertion via DNA or RNA viruses. The vector andlor the nucleic acid molecule contained therein may or may not need to be stably integrated into the plant genome. The vector may also replicate andlor express in prokaryotic cells. Preferably, the vector molecules or parts thereof are capable of integration into the plant genome. The nucleic acid molecule andlor the vector may additionally contain a promoter sequence capable of directing 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. In accordance with a preferred aspect, the vector comprises a genetic sequence encoding a ribozyme capable of cleaving a hydroxyjasmonic acid sulfotransferase mRNA transcript.
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 (e.g. 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. Lxamples of other suitable sources of genes encoding hydroxyjasmonic acid sulfotransferase include, but are not limited to Brassica napes, Solanum tuberosum, Solanum demissum, Helianthus tuberoses and Astragalus complanatus i) reduce tuber size An aspect of the invention contemplates a plant genetically modified to produce smaller tubers in a larger number when compared to a corresponding plant not genetically modified, wherein the genetically modified plant has a decreased endogenous level of at least one given compound of the jasmonate family selected preferably 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, when compared to the corresponding non-genetically modified plant.
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 AfST2a or of AfST2b.
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 andlor 11-hydroxyjasmonic under the control of a constitutive or an inducible promoter.
According to a preferred embodiment, the method comprises the step of:
a) introducing into a cell of a suitable plant an exagenous nucleic acid molecule comprising a sequence of nucleotides encoding a plant hydroxyjasmonic acid sulfotransferase, preferably a 11- or 12-hydroxyj~smonic 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 permit expression of the nucleic acid sequence into a plant hydroxyjasmonic acid sulfotransferase, preferably a 11- or 12-hydroxyjasmonic acid sulfotransferase.
ii) increase tuber size 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 producing tubers of larger size Preferably, the altered level would be less than the endogenous or existing level of activity in a comparable non-transgenic plant.
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 the gene encoding 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 the gene.
According to one embodiment, the method comprises 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 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 permit expression of the antisense sequence and thereby inf~ibiting 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, 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.
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, petunia and other model plant species (e.g. electroporation, microprojectile bombardment, Agrobacterium-mediated transfer or insertion via DNA or RNA
viruses). One skilled in the art wilt 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.
w;;
The transgenic plants rnay 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.
EXAMPLE
The following examples are illustrative of the wide range of applicability of the present invention. The invention is not restricted to the control of tuber formation in Solanum chacoense but can be applied to various tuber-producing plant species. It should readily occur that the recognition of modulating tuber size according to methods of 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 example is intended only to illustrate the invention and is 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) Materials and Methods Studies using a vector:
For transgenic studies a EcoR1-Hindlll cassette, from the plasmid pBl-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 pBl-101-525 contained two CaMV 35S minimal promoters in tandem followed by an AMV translational enhancer, a NOS fierminator and a kanamycin resistance gene. AfST2a cDNA (SEQ ID NO 1; Fig. Z) 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.
Agirobacterium transformation:
A. tumefaciens strain GV3101 pMP90 was transformed with the AtST2a-pBl-101-525 sense and antisense constructs by the method described in Gynheung ef al. (1988) Biology Manual, A3:1-19.
Solanum chacoense transformation:
Solanum chacoense plants were transformed by the leaf disc method described in Horsch ef al. (1985) Science 227, 1229-1231.
Western blot ofprotein extracts Protein extracts from wildtype and transgenic plants were subjected to 12%
SDS-PAGE. Following electrophoresis, the proi;eins were transferred to nitrocellulose membranes using a Bio-Rad 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.
B) RESULTS
The inventors demonstrated that it was possible to alter the size and the number of tubers per potato plant by altering the level of the enzyme 12-OHJA
sulfotransferase. The AtST2a gene was introduced in Sotanum chacoense plants by Agrbacterium-mediated transformation. Plants were regenerated and transformed plants were selected by resistance to kanamycine. Transformation was confirmed by PCR and western blot. Transformed plants had a normal phenotype, comparable to that of the wild type plants, with the exception of the number and size of tubers that were dramatically different (Figure 2). Table 1 indicates the number and total weight of tubers harvested per individual plants of independent transgenic lines. Table 2 and Figure 3 summarize the results ~f the experiment.
Figure 3. Graphical representation of the results presented in Table 2 These results clearly indicate that it is possible to alter the size and number of tubers in a potato plant by altering the level of i:he enzyme that inhibits the tuberization molecule 12-OHJA. It is also predicted that inhibition of the expression of the endogenous level of the 12-OHJA sulfotransferase will lead to the reverse effect. By expressing the potato.12-OHJA sulfotransferase (or any enzyme with sufficient sequence similarity to this enzyme) in' the antisense orientation in transgenic potato, it should be possible to prevent, or retard the inactivation of 12-OHJA by sulfonation, and thus increase the effective concentration of active OHJA. This in turn is expected to lead to tubers with increased size.
SimilaritylComplementarity: 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 hydroxyjasmonic 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 andlor 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 % (wlv) SDS at 37-45 °C for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridization, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 1-4.X SSCI0.5-1 % (w/v) SDS at greater than or equal to 45°C for 2-3 hours or high stringent conditions considered herein to be 0.1-1X
SSCI0.1-1.0% 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.
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 proteins) in a recipient during cloning procedures and may comprise different selectable markers. Bacterial plasmids are commonly used vectors. Preferably, thie vectors of the invention are capable of facilitating transfer of a nucleic acid into a plant cell andlor facilitating integration into a plant genome.
B) General overview of the invention The present inventors have now discovered that it is possible to modulate tuber size in tuber-producing plants by increasing or decreasing the activity of a hydroxy-jasmonate sulfotransferase.
In practice, modulation of tuber size is achieved in two different ways: it is either increased or decreased. Although many approaches may be used to achieve these effects, the approaches described hereinafter are preferably used according to the invention.
1 ~ Chemical approach i) Increase tuber size Inhibitors of hydroxyjasmonic acid sulfotransferase(s) prevent in vivo inactivation of the tuber-inducing molecule by sulfonation. To the contrary, stimulators of jasmonic acid hydroxylase(s) help in the production of hydroxy-jasmonate compound(s).
The above mentioned compounds, stimulators and/or inhibitors are preferably part of a composition for modulating tuber production in plants.
The carrier or diluent is preferably a solvent such as water, oil or alcohol.
Optionally, the composition comprises others active agents such as fertilizers and growth regulators. The inducing composition is preferably formulated with emulsifying agents in the presence or absence of fungicides or insecticides, if required.
The precise amount of compound employed in the .practice of the present invention depends upon the type of response desired, the formulation used and the type of plant treated.
i) Reduce tuber size According to the invention, Inhibitors of jasmonic acid 11- and 12-hydroxylase(s) prevent the formation of the tuber-inducing molecule when compared to a corresponding plant in which the endogenous level of the compound has not been modified. The above mentioned inhibitors are preferably part of a composition for modulating tuber production in plants. The carrier or diluent is preferably a solvent such as water, oil or alcohol. Optionally, the composition comprises others active agents such as fertilizers and growth regulators. In accordance with a preferred embodiment, the inducing composition is formulated with emulsifying agents in the presence or absence of fungicides or insecticides, if required. The precise amount of compound employed in the practice of the present invention depends upon the type of response desired, the formulation used and the type of plant treated.
2) Molecular approach In accordance with the present invention, genetic sequences encoding a plant hydroxyjasmonic acid sulfotransferase have been used to generate transgenic plants.
SEQ ID NO 1 (Fig. 4 ; GeneBank: accession number NM_120783) corresponds to the gene AtST2a in Arabidopsis thaliana. SEQ ID NO 3 (Fig. 5) is an amino acid sequence deduced from SEQ ID NO 1. This amino acid sequence is of public domain and can be retrieved from Genebank, accession number NM 120783. The AtST2a gene from Arabidopsis thaliana encodes a sulfotransferase that sulfonates 12-hydroxyjasmonic acid and 11-hydroxyjasmonic acid with high specificity.
This hydroxyjasmonic acid sulfotransferase exhibits high affinity for its substrate with a Km value of 11 NM for 12-hydroxyjasmonic acid and 60 pM for 11-hydroxyjasmonic acid. The enzyme did not accept structurally related compounds such as cucurbic acid, arachidonyl alcohol or prostaglandins. Maximum enzyme activity was observed at pH 7.5 in TrislHCI buffer and did not require divalent rations for activity.
a r' SEQ ID NO 2 (Fig. 6; GeneBank: accession number NM_120782) corresponds to the gene AfST2b in Arabidopsis fhaiiana. SEQ ID NO 4 (Fig. 7) is an amino acid sequence deduced from SEQ ID NO 1. This amino acid sequence is of public domain and can be retrieved from Genebank, accession number 5 NM_120782. Amino acid sequence alignment between SEQ 1D NOS 3 and 4 indicates that they share 85% amino acid sequence identity and 92% similarity, suggesting that AtST2a and AfST2b encode isoenzymes.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or 10 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
N0:1, SEQ ID N0:2 nucleotide sequences having at least 50% similarity with SEQ
ID N0:2, and sequences hybridizing under low stringency conditions to ane 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 expression-vector 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 eiectroporation, microprojectile bombardment, Agrobacterium-mediated transferor insertion via DNA or RNA viruses. The vector andlor the nucleic acid molecule contained therein may or may not need to be stably integrated into the plant genome. The vector may also replicate andlor express in prokaryotic cells. Preferably, the vector molecules or parts thereof are capable of integration into the plant genome. The nucleic acid molecule andlor the vector may additionally contain a promoter sequence capable of directing 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. In accordance with a preferred aspect, the vector comprises a genetic sequence encoding a ribozyme capable of cleaving a hydroxyjasmonic acid sulfotransferase mRNA transcript.
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 (e.g. 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. Lxamples of other suitable sources of genes encoding hydroxyjasmonic acid sulfotransferase include, but are not limited to Brassica napes, Solanum tuberosum, Solanum demissum, Helianthus tuberoses and Astragalus complanatus i) reduce tuber size An aspect of the invention contemplates a plant genetically modified to produce smaller tubers in a larger number when compared to a corresponding plant not genetically modified, wherein the genetically modified plant has a decreased endogenous level of at least one given compound of the jasmonate family selected preferably 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, when compared to the corresponding non-genetically modified plant.
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 AfST2a or of AfST2b.
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 andlor 11-hydroxyjasmonic under the control of a constitutive or an inducible promoter.
According to a preferred embodiment, the method comprises the step of:
a) introducing into a cell of a suitable plant an exagenous nucleic acid molecule comprising a sequence of nucleotides encoding a plant hydroxyjasmonic acid sulfotransferase, preferably a 11- or 12-hydroxyj~smonic 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 permit expression of the nucleic acid sequence into a plant hydroxyjasmonic acid sulfotransferase, preferably a 11- or 12-hydroxyjasmonic acid sulfotransferase.
ii) increase tuber size 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 producing tubers of larger size Preferably, the altered level would be less than the endogenous or existing level of activity in a comparable non-transgenic plant.
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 the gene encoding 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 the gene.
According to one embodiment, the method comprises 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 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 permit expression of the antisense sequence and thereby inf~ibiting 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, 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.
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, petunia and other model plant species (e.g. electroporation, microprojectile bombardment, Agrobacterium-mediated transfer or insertion via DNA or RNA
viruses). One skilled in the art wilt 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.
w;;
The transgenic plants rnay 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.
EXAMPLE
The following examples are illustrative of the wide range of applicability of the present invention. The invention is not restricted to the control of tuber formation in Solanum chacoense but can be applied to various tuber-producing plant species. It should readily occur that the recognition of modulating tuber size according to methods of 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 example is intended only to illustrate the invention and is 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) Materials and Methods Studies using a vector:
For transgenic studies a EcoR1-Hindlll cassette, from the plasmid pBl-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 pBl-101-525 contained two CaMV 35S minimal promoters in tandem followed by an AMV translational enhancer, a NOS fierminator and a kanamycin resistance gene. AfST2a cDNA (SEQ ID NO 1; Fig. Z) 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.
Agirobacterium transformation:
A. tumefaciens strain GV3101 pMP90 was transformed with the AtST2a-pBl-101-525 sense and antisense constructs by the method described in Gynheung ef al. (1988) Biology Manual, A3:1-19.
Solanum chacoense transformation:
Solanum chacoense plants were transformed by the leaf disc method described in Horsch ef al. (1985) Science 227, 1229-1231.
Western blot ofprotein extracts Protein extracts from wildtype and transgenic plants were subjected to 12%
SDS-PAGE. Following electrophoresis, the proi;eins were transferred to nitrocellulose membranes using a Bio-Rad 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.
B) RESULTS
The inventors demonstrated that it was possible to alter the size and the number of tubers per potato plant by altering the level of the enzyme 12-OHJA
sulfotransferase. The AtST2a gene was introduced in Sotanum chacoense plants by Agrbacterium-mediated transformation. Plants were regenerated and transformed plants were selected by resistance to kanamycine. Transformation was confirmed by PCR and western blot. Transformed plants had a normal phenotype, comparable to that of the wild type plants, with the exception of the number and size of tubers that were dramatically different (Figure 2). Table 1 indicates the number and total weight of tubers harvested per individual plants of independent transgenic lines. Table 2 and Figure 3 summarize the results ~f the experiment.
Figure 3. Graphical representation of the results presented in Table 2 These results clearly indicate that it is possible to alter the size and number of tubers in a potato plant by altering the level of i:he enzyme that inhibits the tuberization molecule 12-OHJA. It is also predicted that inhibition of the expression of the endogenous level of the 12-OHJA sulfotransferase will lead to the reverse effect. By expressing the potato.12-OHJA sulfotransferase (or any enzyme with sufficient sequence similarity to this enzyme) in' the antisense orientation in transgenic potato, it should be possible to prevent, or retard the inactivation of 12-OHJA by sulfonation, and thus increase the effective concentration of active OHJA. This in turn is expected to lead to tubers with increased size.
Claims
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002415062A CA2415062A1 (en) | 2002-12-23 | 2002-12-23 | Methods and genetic sequences for modulating tuber formation in tuber-producing plants, and plants genetically modified to alter tuber size and number |
| PCT/CA2003/002009 WO2004057000A2 (en) | 2002-12-23 | 2003-12-23 | Methods and compositions for modulating tuber formation in tuber-producing plants |
| AU2003294542A AU2003294542A1 (en) | 2002-12-23 | 2003-12-23 | Methods and compositions for modulating tuber formation in tuber-producing plants |
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|---|---|---|---|
| CA002415062A CA2415062A1 (en) | 2002-12-23 | 2002-12-23 | Methods and genetic sequences for modulating tuber formation in tuber-producing plants, and plants genetically modified to alter tuber size and number |
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| AU (1) | AU2003294542A1 (en) |
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| CN116621344A (en) * | 2023-05-26 | 2023-08-22 | 海南珊海海洋科技有限公司 | Regulator for regulating coral culture water body and preparation method thereof |
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| WO2013005152A1 (en) * | 2011-07-05 | 2013-01-10 | Basf Plant Science Company Gmbh | Regulatory nucleic acid molecules for enhancing constitutive gene expression in plants |
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| JP2598811B2 (en) * | 1988-09-29 | 1997-04-09 | 日本たばこ産業株式会社 | Potato tuber formation inducer and method for inducing the same |
| CA2274873A1 (en) * | 1999-07-06 | 2001-01-06 | Satinder Gidda | Methods and compositions for modulating flowering |
-
2002
- 2002-12-23 CA CA002415062A patent/CA2415062A1/en not_active Abandoned
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2003
- 2003-12-23 WO PCT/CA2003/002009 patent/WO2004057000A2/en not_active Application Discontinuation
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| CN116621344A (en) * | 2023-05-26 | 2023-08-22 | 海南珊海海洋科技有限公司 | Regulator for regulating coral culture water body and preparation method thereof |
| CN116621344B (en) * | 2023-05-26 | 2024-04-12 | 海南珊海海洋科技有限公司 | Regulator for regulating coral culture water body and preparation method thereof |
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