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WO2003010318A2 - Desulfoglucosinolate sulfotransferases, sequences codant pour celles-ci et utilisations associees pour moduler la biosynthese de glucosinolate dans les plantes - Google Patents

Desulfoglucosinolate sulfotransferases, sequences codant pour celles-ci et utilisations associees pour moduler la biosynthese de glucosinolate dans les plantes Download PDF

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WO2003010318A2
WO2003010318A2 PCT/CA2002/001144 CA0201144W WO03010318A2 WO 2003010318 A2 WO2003010318 A2 WO 2003010318A2 CA 0201144 W CA0201144 W CA 0201144W WO 03010318 A2 WO03010318 A2 WO 03010318A2
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seq
plant
desulfoglucosinolate
glucosinolate
atstδb
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PCT/CA2002/001144
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WO2003010318A3 (fr
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Luc Varin
Diego Spertini
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Universite Concordia
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Publication of WO2003010318A3 publication Critical patent/WO2003010318A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance

Definitions

  • the present invention relates to desulfoglucosinolate sulfotransferases (DSG-STs), sequences coding the same and uses thereof for modulating glucosinolate biosynthesis in plants.
  • DSG-STs desulfoglucosinolate sulfotransferases
  • Glucosinolates are sulfated, non-volatile thioglucosides derived from aliphatic, indolyl or aromatic amino acids (Figure 1). They are found throughout the order Capparales, particularly in the Mustard family (Cruciferae), which includes important crop plants such as Brassica napus and the model species Arabidopsis thaliana.
  • glucosinolates Upon mechanical damage, infection or pest attack, cellular breakdown exposes the stored glucosinolates to catabolic enzymes (e.g. myrosinases), yielding a variety of reactive products such as isothiocyanates, organic nitriles, thiocyanates and oxazolidine-2-thiones. These reactive products contribute to the distinctive flavor and aroma of cruciferous plants and are believed to play an important role in plant protection against herbivore and pathogen attack.
  • the glucosinolate degradation products have toxic effects on animals and humans. High glucosinolate content severely diminishes the value of the rapeseed meal used as animal feed supplement due to the pungency and goitrogenic effect of their breakdown products.
  • isothiocyanates induce anti-carcinogenic enzymes, suggesting that consumption of glucosinolate- containing plants may reduce the risk of cancer development.
  • Glucosinolate biosynthesis studies have shown that N-hydroxyamino acids, aldoximes, thiohydroximates and desulfoglucosinolat.es (DSG) are precursors of glucosinolates. Their biosynthesis has been proposed to occur via three independent stages. First, the synthesis of chain-elongated amino acid occurs. Incorporation studies with radiolabeled amino acids indicated chain- elongation for methionine and phenylalanine during the synthesis of aliphatic- and 2-phenylethyl glucosinolates, respectively. In the second stage, the amino acids or their elongated analogs are first converted to their corresponding oximes and then to the thiohydroximates.
  • Glucosinolate biosynthesis is completed by S-glucosylation of the thiohydroximate to produce the desulfoglucosinolate (DSG), which is then further sulfonated.
  • the UDPG:thiohydroximate glucosyltransferase (GT) was purified from Tropaeolum majus leaves (Matsu, M. & Underhill, E. W. (1971) Phytochemistry 10, 2279-2288), Brassica napus seedlings (Reed, D. W., Davin, L., Jain, J. C, DeLuca, V., Nelson, L. & Underhill, E. W. (1993) Arch. Biochem. Biophys.
  • DSG-ST desulfoglucosinolate sulfotransferase
  • the present invention relates to the modulation of levels of glucosinolate in plants. More particularly, the present invention pertains to methods, compositions and genetic sequences for modulating biosynthesis of glucosinolate in plants and to plants genetically modified for having increased or reduced glucosinolate levels.
  • a plant genetically modified for having a modulated endogenous level of glucosinolate when compared to a corresponding non-genetically modified plant In the genetically modified plant of the invention, expression or biological activity of a desulfoglucosinolate sulfotransferase or of a functional homologue thereof is modulated when compared to a corresponding non-genetically modified plant, the desulfoglucosinolate sulfotransferase or functional homologue being selected from the group consisting of AtST5a, AtST5b, AtST5c, and functional homologues of AtST5a, AtST5b, or of AtST5c.
  • Preferred genetically modified plants consist of transgenic plants.
  • the invention also encompasses cut flowers, leafs, fruits, seeds and roots of such genetically modified plants.
  • a plant genetically modified for having an increased glucosinolate production when compared to a corresponding plant not genetically modified According to another specific aspect of the invention, there is provided a plant genetically modified for having a reduced glucosinolate production when compared to a corresponding plant not genetically modified.
  • the desulfoglucosinolate sulfotransferase or homologue thereof is encoded by a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence having at least 50% nucleotide sequence identity (more preferably at least 75% and even more preferably at least 85%) with any of SEQ ID NO: 1 , SEQ ID NO: 3, or SEQ ID NO: 5; and b) sequences as set forth in SEQ ID NOs: 1, 3 and 5.
  • the desulfoglucosinolate sulfotransferase or homologue thereof comprises an amino acid sequence selected from the group consisting of: a) sequences encoded by a nucleic acid having a sequence at least 50% identical (more preferably at least 75% and even more preferably at least 85%) to any of SEQ ID NO: 1 , SEQ ID NO: 3, or SEQ ID NO: 5; b) sequences having at least 50 % identity (more preferably at least 75% and even more preferably at least 85%) to any of SEQ ID NO: 2, 4 or 6; c) sequences having at least 50% similarity (more preferably at least 75% and even more preferably at least 85%) to any of SEQ ID NO: 2, 4 or 6; and d) sequences as set forth in SEQ ID NO: 2, 4 or 6.
  • a method for modulating a plant glucosinolate production comprises modulating in the plant expression or biological activity of a desulfoglucosinolate sulfotransferase or of a functional homologue thereof.
  • the endogenous level of glucosinolate may be modulated subsequently to a genetic modification modulating expression or biological activity of at least one of the desulfoglucosinolate sulfotransferases or functional homologue. It may also be modulated by applying to the plant a compound capable of modulating expression or biological activity of at least one of the desulfoglucosinolate sulfotransferases described herein or one of its functional homologue.
  • a related aspect of the method of the invention concerns a method for increasing production of glucosinolate by a plant, the method comprising increasing in the plant expression or biological activity of a desulfoglucosinolate sulfotransferase or functional homologue thereof selected from the group consisting of AtST5a, AtST5b, AtST5c, and functional homologues of AtST5a, AtST5b, or of AtST5c.
  • Expression or biological activity of the desulfoglucosinolate sulfotransferase or functional homologue may be increased by carrying a known method such as: i) introducing into the plant an expressible exogenous nucleic acid sequence encoding at least one of the sulfotransferases or homologue; ii) introducing into the plant an inducible or constitutive promoter in a regulatory region of a gene encoding at least one of the sulfotransferases or homologue; and iii) increasing transcriptional activity of a promoter region of a gene encoding at least one of the sulfotransferases or homologue.
  • Another related aspect of the method of the invention concerns a method for reducing production of glucosinolate by a plant, comprising reducing in the plant expression or biological activity of a desulfoglucosinolate sulfotransferase or functional homologue thereof selected from the group consisting of AtST5a, AtST5b, AtST5c, and functional homologues of AtST ⁇ a, AtST5b, or of AtST5c.
  • Expression or biological activity of the desulfoglucosinolate sulfotransferase or functional homologue may be reduced for instance by: i) expressing in the plant desulfoglucosinolate sulfotransferase antisense molecules; ii) expressing proteins inducing co-suppression of at least one of the desulfoglucosinolate sulfotransferases; iii) knocking-out or chemically mutating a gene encoding at least one the desulfoglucosinolate sulfotransferases; iv) expressing a ribozyme cleaving a desulfoglucosinolate sulfotransferase mRNA.
  • the present invention provides a transgenic plant of the Mustard family expressing a desulfoglucosinolate sulfotransferase antisense molecule such that the antisense molecule inhibits expression of the plant nucleotide sequence.
  • the antisense molecule is complementary to at least part of a nucleic acid molecule of the plant, the plant nucleic acid molecule comprising nucleotide sequence: (a) having at least 50 % nucleotide sequence identity (more preferably at least 75% and even more preferably at least 85%) with any of SEQ ID NO: 1 , SEQ ID NO: 3, or SEQ ID NO: 5; and/or (b) a sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 3, or SEQ ID NO: 5.
  • Another aspect of the present invention concerns an isolated or purified polypeptide having the biological activity of a plant desulfoglucosinolate sulfotransferase. Modulation of a plant glucosinolate production may be achieved by modulating in the plant expression or biological activity of such polypeptide(s).
  • the invention also concerns antibodies binding with affinity to a such polypeptide(s).
  • the present invention relates to a method for producing a transgenic plant with a reduced production of glucosinolate when compared to a corresponding plant not genetically modified.
  • an exogenous nucleic acid molecule comprising a sequence antisense to a plant desulfoglucosinolate sulfotransferase coding sequence is introduced into a cell of a plant.
  • another related aspect of the invention concerns antisense nucleic acid molecules specifically hybridizing to a nucleotide sequence having at least 50% nucleotide sequence identity (more preferably at least 75% and even more preferably at least 85%) with any of SEQ ID NO: 1 ; SEQ ID NO: 3; or SEQ ID NO: 5; and/or hybridizing to sequences as set forth in SEQ ID NO: 1 , 3, or 5.
  • the present invention relates to a method for producing a transgenic plant with a reduced production of glucosinolate.
  • an exogenous nucleic acid molecule comprising a sequence antisense to a plant desulfoglucosinolate sulfotransferase coding sequence is introduced into a cell of a plant under conditions sufficient to inhibit and/or block expression of the desulfoglucosinolate sulfotransferase. While the production of desulfoglucosinolate sulfotransferase(s) does not appear to be the rate limiting step in the production of glucosinolate, it is a necessary step in this biosynthesis.
  • aspects of the present invention relates to methods of modulating resistance or tolerance to a pathogen in a plant, to the manufacture of a food supplement rich in glucosinolate, to a screening assay for identifying compounds that modulate a plant endogenous level and/or biological activity of a desulfoglucosinolate sulfotransferase, and to compositions for modulating such expression or biological activity.
  • One of the greatest advantages of the present invention is that it provides nucleic acid molecules, proteins, polypeptides, antibodies, probes, and plant cells that can be used for characterizing desulfoglucosinolate sulfotransferases and modulate the cellular levels of these enzymes.
  • Figure 1 shows the general structure of glucosinolates.
  • the structure of the side-chain (R group) is illustrated for glucosinolates used in this work (prior art);
  • Figure 2 shows steps of the biosynthesis of a benzylglucosinolate from L-phenylalanine (prior art).
  • Figure 3 shows the alignment of DSG-STs from Arabidopsis.
  • Deduced amino acid sequences of AtST5a, AtST ⁇ b and AtST5c were aligned with CLUSTAL W 1.8 program (Multiple sequence alignments, Baylor College of Medicine Search Launcher, Houston, TX). Amino acid residues identical are boxed in black and conservative changes of amino acid residues are boxed in gray.
  • Black arrows indicate conserved amino acid residues involved in sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding and in catalysis.
  • PAPS 3'-phosphoadenosine 5'-phosphosulfate
  • Figure 4 shows the expression of DSG-ST genes in Arabidopsis.
  • RT-PCR reactions were performed on RNA samples using sets of primers specific to AtST5a (A), AtST5b (B) and ACT1 (C).
  • RNA sample were prepared from complete plants aged of 5 days (1), 15 days (2), 30 days (3), from flowers (4) and siliques (5).
  • 15 days-old plants (6) were exposed to MeJA vapor (7) or were treated with SA solution (8) for 24h, before RNA extraction.
  • the products of amplification were loaded on ethidium bromide-stained agarose gel. Control reactions were performed with genomic DNA using the same sets of primers (9).
  • the word “kilobase” is generally abbreviated as “kb”, the words “deoxyribonucleic acid” as “DNA”, the words “ribonucleic acid” as “RNA”, the words “complementary DNA” as “cDNA”, the words “polymerase chain reaction” as “PCR”, and the words “reverse transcription” as “RT”. Nucleotide sequences are written in the 5' to 3' orientation unless stated otherwise.
  • Antisense refers to nucleic acids molecules capable of regulating the expression of a corresponding gene in a plant by forming a stable duplex or triplex with a portion of its targeted nucleic acid sequence (DNA or RNA).
  • 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.
  • antisense nucleic acid sequences are not template 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.
  • AtST ⁇ a, AtST5b, or AtST5c desulfoglucosinolate sulfotransferase Means a polypeptide, a fragment thereof, or a functional homologue thereof encoded by a AtST5a, AtST5b, or AtST ⁇ c nucleic acid as described hereinafter.
  • AtST ⁇ a, AtST5b, or AtST ⁇ c nucleic acid means any nucleic acid encoding a polypeptide having a desulfoglucosinolate sulfotransferase biological activity or amino acid sequence substantially identical (see hereinafter) to the biological activity or amino acid sequence of any of the polypeptides which amino acid sequence is set forth in SEQ ID NO: 2, 4 or 6.
  • the nucleic acids having at least 60%, preferably at least 75%, more preferably at least 85% and most preferably at least 95% identity with SEQ ID NO: 1 , 3 or 5 are more particularly concerned.
  • a protein/polypeptide is said to be a "chemical derivative" of another protein/polypeptide when it contains additional chemical moieties not normally part of the protein/peptide, said moieties being added by using techniques well known in the art. Such moieties may improve the protein/polypeptide solubility, absorption, bioavailability, biological half life, and the like. Any undesirable toxicity and side-effects of the protein/peptide may be attenuated and even eliminated by using such moieties.
  • proteins/polypeptides can be covalently coupled to biocompatible polymers (polyvinyl-alcohol, polyethylene-glycol, etc) in order to improve stability or to decrease/increase their antigenicity.
  • Defense gene A gene that is induced and/or involved in a plant response to a pathogen challenge.
  • 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.
  • the "exogenous nucleic acid sequence” encodes a plant gene such as a AtST5a, AtST5b, or AtST5c, a functional homologue of any of these genes or an antisense molecule hybridizing thereto.
  • Fragment refers to a section of a molecule, such as protein/polypeptide or nucleic acid, and is meant to refer to any portion of the amino acid or nucleotide sequence.
  • Functional homologue refers to non-native a polypeptide or a nucleic acid molecule that possesses a functional biological activity that is substantially similar to the biological activity of a native polypeptide or a nucleic acid molecule.
  • Preferred functional homologue are polypeptides or nucleic acid molecules having a sequence "substantially identical" (see hereinafter) to the native polypeptide or a nucleic acid molecule.
  • the 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 proteinic molecule having a desired activity, and such proteinic molecules may still be considered within the scope of the present invention where they have regions of sequence conservation.
  • the term "functional homologue” is intended to the "fragments", “segments”, “variants”, “analogs” or “chemical derivatives" of a polypeptide or a nucleic acid molecule.
  • vegetal host When used with the term " vegetal host” or “plant” it refers to the introduction of an exogenous nucleic acid into one or more vegetal host (plant) cells to create a genetically modified vegetal host or plant.
  • Methods for genetically modifying vegetal host such as plants are well known in the art.
  • 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, so that the exogenous nucleic acid, carried in the germ line, may be inserted into or bred into agriculturally useful plant varieties.
  • Endogenous or Endogenous level(s) refers to a given substance or to the concentration of a given substance which is normally found in a plant (intrinsic) at a given time and stage of growth.
  • the term also includes functional homologues of a given substance or protein which may results from a mutation.
  • 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, 35, 40, 45 or 50%, or even more preferably 55, 60, 65, 70 or 75% or still more preferably 80, 85, 90, 95% 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.
  • Expression refers to the process by which gene encoded information is converted into the structures present and operating in the cell.
  • the transcribed nucleic acid is subsequently translated into a peptide or a protein in order to carry out its function if any.
  • overexpression refers to an upward deviation respectively in assayed levels of expression as compared to a baseline expression level which is the level of expression that is found under normal conditions and normal level of functioning.
  • underexpression refers to an downward deviation.
  • positioned for expression is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a NAIP polypeptide, a recombinant protein or a RNA molecule).
  • Isolated or Purified or Substantially pure Means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a protein/peptide naturally present in a living organism is not “isolated", the same polynucleotide separated from the coexisting materials of its natural state, obtained by cloning, amplification and/or chemical synthesis is “isolated” as the term is employed herein.
  • a polynucleotide or a protein/peptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism.
  • Modulation refers to the process by which a given variable is regulated to a certain proportion.
  • modulate refers in some cases to an increase and in other cases a reduction, of glucosinolate biosynthesis.
  • Nucleic acid Any DNA, RNA sequence or molecule having one nucleotide or more, including nucleotide sequences encoding a complete gene. The term is intended to encompass all nucleic acids whether occurring naturally or non-naturally in a particular cell, tissue or organism. This includes DNA and fragments thereof, RNA and fragments thereof, cDNAs and fragments thereof, expressed sequence tags, artificial sequences including randomized artificial sequences.
  • Plant or Plant entity 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, a cut flower, a leave, a fruit, a root, a pollen grain 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.
  • Polypeptide means any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.
  • Resistant or Tolerant By resistant is meant a cell or organism (such as a plant) which exhibits substantially no phenotypic changes as a consequence of an aggression by a pathogen (e.g. virus, fungus, insect, etc).
  • a pathogen e.g. virus, fungus, insect, etc.
  • tolerant By “tolerant” is meant a cell or organism which, although it may exhibit some phenotypic changes as a consequence of aggression by a pathogen, does not have a substantially decreased reproductive capacity or substantially altered metabolism.
  • 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.
  • binds means an antibody that recognizes and binds a protein but that does not substantially recognize and bind other molecules in a sample (e.g. a biological sample), that naturally includes proteins.
  • Substantially identical means a polypeptide or nucleic acid exhibiting at least 50, 55, 60, 65, 70, 75%, preferably 80 or 85%, more preferably 90, 95%, and most preferably 97 or 99% identity or similarity to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences will generally be at least 20 amino acids, preferably at least 25, 30 or 40 amino acids, more preferably at least 50 or 75 amino acids, and most preferably at least 100 amino acids.
  • the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides.
  • Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Owl 53705). This software program matches similar sequences by assigning degrees of similarity to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • 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% (w/v) SDS at 37-45 °C for 2-3 hours.
  • alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 1-4X SSC/0.5-1% (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 SSC/0.1-1.0% SDS at greater than or equal to 60° C. for 1-3 hours.
  • Transformed or Transfected or Transgenic cell refers to introduction of an exogenous nucleic acid, typically a gene or gene regulatory sequence, into a whole plant or a part thereof.
  • transformation is meant any method for introducing foreign molecules into a cell. Agrobacterium transformation, PEG treatment, lipofection, calcium phosphate precipitation, electroporation, and ballistic transformation are just a few of the teachings which may be used.
  • Transgenic plant any plant having a cell which includes a DNA sequence which has been inserted by artifice into the cell and becomes part of the genome of the plants which develops from that cell.
  • Preferred transgenic plants are those 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.
  • 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.
  • “Expression vector” defines a vector as described above designed to enable the expression of an inserted sequence following transformation into a host.
  • Vegetal Host refers to a cell, tissue, organ or organism comprising chloroplasts and capable to perform photosynthesis. This term is intended to also include hosts which have been modified in order to accomplish these functions. Algae and plants are examples of a vegetal host.
  • the present invention thus concerns the cloning and characterization of AtST5a, AtST5b and AtST5c.
  • the former two genes have been shown functionally to encode desulfoglucosinolate sulfotransferases (DSG-STs). Whereas these enzymes exhibit broad substrate specificity, they display distinct substrate preferences.
  • RT-PCR analysis reveals that the two genes are expressed constitutively in the rosette leaves, flowers and siliques.
  • AtST5c which possesses a high homology with AtST5a and AtSTSb was also shown to possess a DSG-ST activity.
  • AtST5a, AtST5b and AtST5c of the instant invention are demonstrated with Arabidopsis, the person of ordinary skill in the art will be able to produce probes to identify the corresponding genes in other plants.
  • the present invention also relates to a method of isolating DSG-STs from other plant species comprising a use of a conserved DSG-ST sequence of the present invention as a probe and a screening of a library from such other plants. i) Cloning and molecular characterization ofAtSTSa, AtST5b and AtST ⁇ c
  • AtST5a As it will be described hereinafter in the exemplification section of the invention, the inventors have cloned and characterized AtST5a, AtST5b and AtST5c, three genes from Arabidopsis thaliana encoding desulfoglucosinolate sulfotransferases.
  • the sequence of the AtST5a cDNA (SEQ ID NO: 1) and predicted amino acid sequence (SEQ ID NO: 2) is shown in the "Sequence Listing" section.
  • AtST5b cDNA SEQ ID NO: 3
  • amino acid sequence SEQ ID NO: 4
  • amino acid sequence SEQ ID NO: 5
  • amino acid sequence SEQ ID NO: 6
  • the nucleotide sequences of AtST5a, AtST5b and AtST ⁇ c can also be retrieved from the BAC clone F2P9 and F25I16 under the GenBankTM accession numbers NM_106070 (AtST5a), NM_106069 (AtST5b) and NM 01717 (AtST5c).
  • AtST5a encodes a protein of 338 amino acids long.
  • AtST5a has the following features: it has a molecular weight of about 39 219 g/mol, an isoelectric point of about 5.42; an instability index of about 31.64 (i.e. stable); an aliphatic index of about 70.68; and a grand average of hydropathicity (GRAVY) of about -0.579.
  • GRAVY grand average of hydropathicity
  • AtST5b encodes a protein of 350 amino acids long.
  • AtST5b has the following features: it has a molecular weight of about 40 465 g/mol, an isoelectric point of about 5.52; an instability index of about 37.16 (i.e. stable); an aliphatic index of about 68.54; and a grand average of hydropathicity (GRAVY) of about -0.553.
  • GRAVY grand average of hydropathicity
  • AtST5c encodes a protein of 346 amino acids long.
  • AtST5c has the following features: it has a molecular weight of about 39 912 g/mol, an isoelectric point of about 6.04; an instability index of about 38.02 (i.e. stable); an aliphatic index of about 70.69; and a grand average of hydropathicity (GRAVY) of about -0.51.
  • GRAVY grand average of hydropathicity
  • AtST5a, AtST ⁇ b and AtST5c possess conserved amino acid motifs, these motifs being probably essential for desulfoglucosinolate sulfotransferase activity.
  • conserved motifs consist of: a) DFXiVCSYPKTGTTWLKALT (SEQ ID NO:7) wherein X ⁇ is non-polar and preferably an aliphatic or aromatic amino acid, and more preferably L or F; b) LFSTHIP (SEQ ID NO:8); c) SGCKX 2 VYIWRX 3 PKDTFX4SMWTFX 5 HKE (SEQ ID NO:9) wherein X 2 is a non-polar amino acid and preferably M or I, wherein X 3 is charged and preferably acidic and most preferably E or D, wherein X is aliphatic and preferably I or V and wherein X 5 is non-polar and preferably L or A; and d) DRPX 6 VYANSAYFRKGK
  • AtST ⁇ a AtST5b
  • AtST ⁇ c AtST ⁇ c
  • other existing sequences Although not shown, none of the AtST ⁇ a, AtST ⁇ b or AtST ⁇ c nucleic or amino acid sequences were found to be highly homologous with any published sequence. The highest levels of identity ranged from 35 to 45%.
  • Standard techniques such as the polymerase chain reaction (PCR) and DNA hybridization, may be used to clone additional AtST ⁇ a, AtST ⁇ b or AtST ⁇ c homologues in other species, particularly other plant species.
  • PCR polymerase chain reaction
  • DNA hybridization DNA hybridization
  • FIG. 4 shows that AtST5a and AtST ⁇ b are constitutively expressed in all parts of the plant that have been analyzed. AtST ⁇ a does not seem to be expressed in early stage of development, contrarily to AtST ⁇ b. The requirement of only one enzyme at this stage might be in relation with the content of glucosinolates in young plants. The expression of AtST ⁇ a and AtST ⁇ b does not seem to be affected by treatment with signal molecules such as salicylic acid and jasmonic acid. Although not shown, similar results were obtained with AtST ⁇ c.
  • results of the present invention are in accordance with a proposed model suggesting that the genes encoding the enzymes of the first step in the biosynthetic pathway of glucosinolates are prone to specific regulation, whereas those coding for enzymes downstream of the oxime-step such as DSG-STs, are constitutively expressed.
  • the present inventors have been the first ones to isolate and purify desulfoglucosinolate sulfotransferases (DSG-STs). AtST ⁇ a, AtST ⁇ b and AtST ⁇ c enzymes were purified to an apparent homogeneity (at least 99% pure).
  • an aspect of the present invention concerns an isolated or purified polypeptide having the biological activity of a plant desulfoglucosinolate sulfotransferase.
  • the desulfoglucosinolate sulfotransferase may be purified from any suitable plant tissue such as seedling, flowers, fruits leaves, stem, roots, anthers and pollen grains.
  • the isolated or purified polypeptide of the invention may have a substrate specificity for glucosinolates such as desulfated derivatives of: 4-methylthiobutyl glucosinolate, 3-methylsulfinylpropyl glucosinolate, 3-methylsulfonylpropyl glucosinolate, 2-hydroxy 3-butenyl glucosinolate, allyl glucosinolate, 3-indolylmethyl glucosinolate, 4-benzoyloxybutyl glucosinolate, 2-phenylethyl glucosinolate, and benzyl glucosinolate.
  • the isolated or purified polypeptide exhibits a substrate preference for indolyl glucosinolates
  • the polypeptide comprises an amino acid sequence selected from the group consisting of: a) sequences encoded by a nucleic acid having a sequence at least 50% identical to any of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:6; b) sequences having at least 50 % identity (more preferably at least 75%) to any of SEQ ID NO:2, 4 or 6; c) sequences having at least 50% similarity (more preferably at least 75%) to any of SEQ ID NO:2, 4 or 6; and d) sequences as set forth in SEQ ID NO:2, 4 or 6.
  • the polypeptide comprises an amino acid sequence selected from the group consisting of: a) amino acid sequences as set forth in SEQ ID NO:2, 4 or 6; and b) amino acid sequences derived from (a) by substitution, deletion or addition of one or several amino acids and encoding a functional homologue of said polypeptide.
  • the polypeptide comprises at least one conserved motif as defined previously.
  • AtST ⁇ b and AtST ⁇ c have broad substrate specificity and accept all of the DSG that were tested (Table I).
  • AtST ⁇ c exhibits preference for 3-methylthiobutyl-, 3-methylsulfonylpropyl- and 2-phenylethyl DSGs while AtST ⁇ b prefers 3-methylsulfinylpropyl DSG.
  • AtST ⁇ a is not as flexible as it accepts only 3-methylthiobutyl DSG at a significant level.
  • AtST ⁇ a and AtST ⁇ b were found to be considerably less active with 2-hydroxy 3-butenyl- and DS 4-benzoyloxybutyl DSG as compared with their precursor, 4-methylthiobutyl DSG. This result suggests that sulfonation of DSGs probably takes place prior to modification of the side-chain. Although we did not test the sulfonate acceptability of the 3-methylthiopropyl DSG, its descendants 3-methylsulfinylpropyl- and allyl DSGs, were both weak substrates as compared with 4-methylthiobutyl DSG (Table I).
  • benzyl DSG was the best substrate for both AtST ⁇ a and AtST ⁇ b although its sulfated product is not a natural product of Arabidopsis.
  • This preference for benzyl DSG as compared to allyl DSG demonstrates the flexibility of the active sites of both enzymes for different side-chain structures.
  • AtST ⁇ a and AtST ⁇ b exhibit preference for their respective naturally occurring aromatic substrates, 3- indolylmethyl- and 2-phenylethyl DSGs suggests that each enzyme may have a specialized function in-vivo.
  • AtST ⁇ b and AtST ⁇ c exhibit broad substrate specificities and accept substrates which do not occur naturally in Arabidopsis support the hypothesis that these enzymes, which are involved in the two last steps of glucosinolate biosynthesis, exhibit no specificity for the structure of the side- chain.
  • the invention is also concerned with vegetal hosts, particularly plants, genetically modified for reducing (or increasing) their endogenous level of glucosinolate when compared to a corresponding non-genetically modified plant.
  • the genetic modification may consists of a gene insertion, a gene inactivation (removal, mutation, deletion, etc) and/or a modification in the promoter region of a particular gene.
  • AtST ⁇ a desulfoglucosinolate sulfotransferase
  • AtST ⁇ b desulfoglucosinolate sulfotransferase
  • AtST ⁇ c desulfoglucosinolate sulfotransferase
  • the invention is also concerned with cells and organisms genetically modified for having a higher or lower glucosinolate production.
  • cells and more particularly plant cells, that have been genetically modified to increase the levels or biological activity of a desulfoglucosinolate sulfotransferase such as AtST ⁇ a, AtST ⁇ b, AtST ⁇ c (or of a functional homologue thereof) thereby increasing the plant endogenous level of glucosinolate.
  • a desulfoglucosinolate sulfotransferase such as AtST ⁇ a, AtST ⁇ b, AtST ⁇ c (or of a functional homologue thereof) thereby increasing the plant endogenous level of glucosinolate.
  • 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, PEG transformation, Agrobactehum-vned ' ialed transfer or insertion via DNA or RNA viruses).
  • electroporation e.g. electroporation, microprojectile bombardment, PEG transformation, Agrobactehum-vned ' ialed transfer or insertion via DNA or RNA viruses.
  • PEG transformation e.g. electroporation, microprojectile bombardment, PEG transformation, Agrobactehum-vned ' ialed transfer or insertion via DNA or RNA viruses.
  • variations applicable to the methods of the present invention such as increasing or decreasing the expression of the desulfoglucosinolate sulfotransferase(s) naturally present in a target plant leading to modulation of gluco
  • the present invention 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 glucosinolate biosynthesis.
  • the nucleic acid would be stably introduced into the plant genome, although the present invention also extends to the introduction of nucleotide sequences 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.
  • plants such as cotton, rice, wheat, corn, barley, oat, lettuce, potato, tomato, tobacco, canola, soybean, pea, and banana having an increased glucosinolate production when compared to a corresponding plant not genetically modified.
  • increasing endogenous levels of glucosinolates would be advantageous in these plants (and others) because glucosinolates have potential anticancerous properties and because glucosinolates could enhance the flavor of the plant.
  • this is achieved by increasing in the plant expression or biological activity of a desulfoglucosinolate sulfotransferase or functional homologue thereof selected from the group consisting of AtST ⁇ a, AtST ⁇ b, AtST ⁇ c, and functional homologues of AtST ⁇ a, AtST ⁇ b, or of AtST ⁇ c.
  • Expression or biological activity of the desulfoglucosinolate sulfotransferase or functional homologue may be increased by any method known in the art, including but not limited to: i) introduction into the plant (or selected cells thereof) of an expressible exogenous nucleic acid sequence encoding at least one of the sulfotransferases or homologue; ii) introduction into the plant of an inducible or constitutive promoter in a regulatory region of a gene encoding at least one of the sulfotransferases or homologue; and iii) increment of the transcriptional activity of a promoter region of a gene encoding at least one of the sulfotransferases or homologue.
  • glucosinolate biosynthesis pathway e.g. cotton, rice, wheat, corn, barley, oat, lettuce, potato, tomato, tobacco, soybean, pea, and banana
  • a method for producing a transgenic plant with an increased production of glucosinolate when compared to a corresponding plant not genetically modified comprises the steps of: a) introducing an exogenous nucleic acid molecule encoding a plant desulfoglucosinolate sulfotransferase into a plant cell; b) regenerating a transgenic plant from the cell; and c) growing said transgenic plant for a time and under conditions sufficient to allow expression of the desulfoglucosinolate sulfotransferase.
  • the exogenous nucleic acid molecule comprises a sequence encoding an amino acid sequence selected from the group consisting of: a) sequences encoded by a nucleic acid having a sequence at least 50% identical (even more preferably at least 75%) to any of SEQ ID NO: 1 , SEQ ID NO: 3, or SEQ ID NO: 5; b) sequences having at least 50 % identity (even more preferably at least 75%) to any of SEQ ID NO:2, 4 or 6; c) sequences having at least 50% similarity (even more preferably at least 75%) to any of SEQ ID NO:2, 4 or 6; and d) sequences as set forth in SEQ ID NO:2, 4 or 6.
  • glucosinolate production in other plants such as Brassica napus (or other plants of the Mustard family), cabbage, turnip, broccoli, watercress, radish, horseradish, alyssum, and other plants of the cruciferous family, it may be very advantageous to reduce their glucosinolate production. Indeed, reduced endogenous levels of glucosinolates would be preferable in these plants (and others) because glucosinolates may some times be toxic to human and animals at certain levels. For instance, use of colza as fodder is presently limited by the fact that it contains high levels of glucosinolates and that it may causes diseases to ruminant if ingested in too large quantities. Also, it would be advantageous to have canola oil (used for human consumption) devoid of glucosinolates.
  • glucosinolate production is reduced by reducing in the plant expression or biological activity of a desulfoglucosinolate sulfotransferase or functional homologue thereof selected from the group consisting of AtST ⁇ a, AtST ⁇ b, AtST ⁇ c, and functional homologues of AtST ⁇ a, AtST ⁇ b, or of AtST ⁇ c.
  • a desulfoglucosinolate sulfotransferase or functional homologue thereof selected from the group consisting of AtST ⁇ a, AtST ⁇ b, AtST ⁇ c, and functional homologues of AtST ⁇ a, AtST ⁇ b, or of AtST ⁇ c.
  • Expression or biological activity of the desulfoglucosinolate sulfotransferase or functional homologue may be reduced by any method known in the art, including but not limited to: i) expression in the plant of desulfoglucosinolate sulfotransferase antisense molecules; ii) expression of proteins inducing co-suppression of at least one desulfoglucosinolate sulfotransferase; iii) knocking-out or chemically mutating a gene encoding at least one desulfoglucosinolate sulfotransferase; iv) expressing a ribozyme cleaving a desulfoglucosinolate sulfotransferase mRNA.
  • a method for producing a transgenic plant with a reduced production of glucosinolate when compared to a corresponding plant not genetically modified comprises the steps of: a) introducing into a cell of a suitable plant an exogenous nucleic acid molecule comprising a sequence antisense to a plant desulfoglucosinolate sulfotransferase coding sequence; b) regenerating a transgenic plant from the cell; and c) growing the transgenic plant for a time and under conditions sufficient to inhibit expression of the desulfoglucosinolate sulfotransferase.
  • the exogenous nucleic acid molecule comprises a nucleotide sequence antisense to a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence having at least 50% nucleotide sequence identity (even more preferably at least 75%) with any of SEQ ID NO: 1 ; SEQ ID NO: 3; or SEQ ID NO: 5; and b) sequences as set forth in SEQ ID NO: 1 , 3, or 5.
  • a related aspect of the present invention concerns antisense nucleic acid molecules which can be used for example to decrease or abrogate the expression of AtST ⁇ a, AtST ⁇ b, AtST ⁇ c (or functional homologues thereof) nucleic acid sequences or proteins.
  • Antisense nucleic acid molecules according to the present invention can be derived from AtST ⁇ a, AtST ⁇ b, AtST ⁇ c nucleic acid sequences and modified in accordance to well known methods.
  • some antisense molecules can be designed to be more resistant to degradation to increase their affinity to their targeted sequence, to affect their transport to chosen cell types or cell compartments, and/or to enhance their lipid solubility by using nucleotide analogs and/or substituting chosen chemical fragments thereof, as commonly known in the art.
  • AtST ⁇ a, AtST ⁇ b, AtST ⁇ c sequences enable the design of specific DSG-ST antisense sequences, such as of antisense sequences that will hybridize to all three DSG- STs and to functional homologue(s) thereof.
  • DSG-ST antisense sequences such as of antisense sequences that will hybridize to all three DSG- STs and to functional homologue(s) thereof.
  • glucosinolates have potential anticancerous properties. Therefore, the plants and methods of the invention could be useful for manufacturing a food supplement rich in glucosinolates.
  • manufacturing of such food supplement would comprises production of glucosinolate(s) in vitro, or in vivo, with AtST ⁇ a, AtST ⁇ b, AtST ⁇ c proteins (or functional homologues thereof) of the present invention.
  • Plant extracts enriched in glucosinolates could also be prepared and sold.
  • AtST ⁇ a, AtST ⁇ b, AtST ⁇ c (or functional homologues thereof) regular protein expression or biological activity could reduce the plant resistance to pathogens or lower induction of its defense mechanisms.
  • an increased in the plant regular protein expression or biological activity could increased the plant resistance to pathogens or results in a faster induction of its defense mechanisms.
  • polypeptides and polynucleotides of the invention may also be used for producing polyclonal or monoclonal antibodies capable of recognizing and binding the same.
  • the present invention therefore encompass such antibodies and methods for using the same. Methods for producing antibodies are well known in the art. Accordingly, the invention also features a purified antibody (monoclonal or polyclonal) that specifically binds to any or all of AtST ⁇ a, AtST ⁇ b and AtST ⁇ c proteins and/or a functional homologue thereof, such as homologous proteins in other plants species.
  • the present inventors prepared antibodies recognizing all 18 STs from Arabidopsis.
  • the antibodies of the invention may be prepared by a variety of methods using the AtST ⁇ a, AtST ⁇ b and/or AtST ⁇ c proteins or polypeptides described above.
  • the AtST ⁇ a, AtST ⁇ b and/or AtST ⁇ c polypeptide, or fragments thereof may be administered to an animal in order to induce the production of polyclonal antibodies.
  • antibodies used as described herein may be monoclonal antibodies, which are prepared using hybridoma technology (see, e.g., Hammerling et al., In Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, 1981).
  • AtST ⁇ a, AtST ⁇ b and/or AtST ⁇ c antibodies may be used for preventing the action of DGS-ST in the plant.
  • a transgenic plant could be made which expresses an AtST ⁇ a, AtST ⁇ b and/or AtST ⁇ c antibody (ex a single-chain antibody). Binding of this antibody to DGS-ST would inhibit its activity.
  • preferred antibodies are "neutralizing" antibodies.
  • neutralizing antibodies is meant antibodies that interfere with any of the biological activities of the DGS-ST.
  • the neutralizing antibody may reduce the ability of DGS-ST by preferably 50, 55, 60 or 65%, more preferably by 70, 75, 80 or 85%, and most preferably by 90, 95, 99% or more. Any standard assay, including those described herein, may be used to assess potentially neutralizing antibodies.
  • monoclonal and polyclonal antibodies are preferably tested for specific recognition by Western blot, immunoprecipitation analysis or any other suitable method.
  • the invention also relates to compositions for modulating expression or biological activity of at least one desulfoglucosinolate sulfotransferase or functional homologue selected from the group consisting of AtST ⁇ a, AtST ⁇ b, AtST ⁇ c, and functional homologues of AtST ⁇ a, AtST ⁇ b, or of AtST ⁇ c.
  • Such a composition would comprise an effective amount of at least one compound that is capable of, directly or indirectly, achieving the above mentioned desired effect, in combination with a diluent or a carrier.
  • Herbicides are example of small molecules that affect plant physiology and development by binding and inhibiting the function of specific plant proteins.
  • the compound(s) and its amount would be selected such that, following application of the composition of the invention, the desired modulation occurs into at least some of the cells of the plant when compared to a corresponding plant in the absence of the composition.
  • Subsection (ix) hereinafter provides examples of methods useful of selecting compound(s) that could be used in the composition of the invention.
  • a more specific but non restrictive examples of an inhibitor of expression or biological activity of desulfoglucosinolate sulfotransferases or functional homologues thereof include nucleic acid molecules antisense to a gene encoding at least one of the sulfotransferases or homologues; and nucleic acid molecules antisense to a portion of such gene(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, growth regulators, fungicides, insecticides, emulsifying agents and mixtures thereof.
  • the composition may also be 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 will depend upon the type of response desired, the formulation used and the type of plant treated.
  • the composition of the invention may further comprises
  • AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c may be used to facilitate the identification of molecules that increase or decrease their expression.
  • candidate molecules are added, in varying concentration, to the culture medium of cells expressing AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c mRNA.
  • AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c expression (or a functional homologues thereof) is then measured, for example, by Northern blot analysis using a AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c cDNA, or cDNA or RNA fragment, as a hybridization probe.
  • the level of expression in the presence of the candidate molecule is compared to the level of expression in the absence of the candidate molecule, all other factors (e.g. cell type and culture conditions) being equal.
  • AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c may be synthesized, purified, or substantially purified.
  • AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c expression is tested against progressively smaller subsets of the compound pool (e.g., produced by standard purification techniques such as HPLC or FPLC) until a single compound or minimal number of effective compounds is demonstrated to modulate AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c expression.
  • the effect of candidate molecules on AtST ⁇ a, AtST ⁇ b, and/or /A ST5c-biological activity may, instead, be measured at the level of translation by using the general approach described above with standard protein detection techniques, such as Western blotting or immunoprecipitation with a AtST ⁇ a, AtST ⁇ b, and/or >AfSr5c-specific antibody.
  • Another method for detecting compounds that modulate the activity of AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c is to screen for compounds that interact physically with a given AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c polypeptide.
  • the binding interaction may be measured using methods such as enzyme-linked immunosorbent assays (ELISA), filter binding assays, FRET assays, scintillation proximity assays, microscopic visualization, immunostaining of the cells, in situ hybridization, PCR, etc.
  • the method comprises an incubation of a recombinant host cell comprising a nucleic acid molecule according to the present invention with a compound and measuring the level and/or activity of the DSG-ST, in the presence versus in the absence of the compound.
  • a compound is selected when the level and/or activity in the cell is measurably different in the presence versus in the absence of the tested compound.
  • a molecule that promotes an increase in AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c (or a functional homologues thereof) expression or biological activity is considered particularly useful to the invention; such a molecule may be used, for example, to increase glucosinolate(s) production in plants which already have an endogenous production of glucosinolate(s), with the benefits associated therewith [see subsection (vi)].
  • a molecule that decreases AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c (or a functional homologues thereof) expression or biological activity may be used to reduce glucosinolate(s) production in plants, with the benefits associated therewith [see subsection (vi)].
  • the polypeptides and nucleic acid molecules of the present invention may be prepared by any suitable process. They may for instance be obtained by chemical synthesis when appropriate. They may also be prepared using biological processes involving cloning or expression vectors. Such vectors would comprise a polynucleotide sequence incorporating the nucleic acid molecule of interest such as and/or comprise a polynucleotide sequence encoding for the peptide of interest. Therefore, the present invention encompass such cloning or expression vectors and more particularly those encoding SEQ ID NO:2, 4 and/or 6, and those comprising nucleotides SEQ ID NO:1 , 3 or 5. In addition, standard techniques, such as the polymerase chain reaction (PCR) and DNA hybridization, may be used to clone additional AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c homologues in other plant species.
  • PCR polymerase chain reaction
  • DNA hybridization may be used to clone additional AtST ⁇ a, AtST ⁇ b, and/or At
  • the invention is directed to a method for producing, in vitro, a AtST ⁇ a, AtST ⁇ b, and/or AtST ⁇ c polypeptide.
  • This method comprises the step of: 1) culturing in vitro, in a suitable culture medium, a cell incorporating an expression vector as described previously; and optionally 2) collecting in the culture medium polypeptides produced by these cells.
  • Methods for producing such genetically modified cells and methods for using these cells in the production of proteins/peptides are well known in the art and will no be described in detail herein.
  • the present example concerns the cloning and characterization of AtST ⁇ a, AtST ⁇ b and AtST ⁇ c, three genes encoding desulfoglucosinolate sulfotransferases (DSG-STs).
  • AtST ⁇ a; and AtST ⁇ b and AtST ⁇ c can be retrieved from the BAC clones F2P9 and F25I16 as respectively F2P9.3 (AtST ⁇ a), F2P9.4 (AtST ⁇ b) and F25I16.7 (AtST ⁇ c) under the GenBankTM accession numbers NM_106070 (AtST ⁇ a), NM_106069 (AtST ⁇ b) and NM_101717 (AtST ⁇ c).
  • the PCR products were digested with the appropriate restriction enzymes and cloned into the corresponding sites of the bacterial expression vector pQE30TM (Qiagen).
  • the expression of AtST ⁇ a, AtST ⁇ b and AtST ⁇ c in E. coli cultures and their purification were performed as described previously in a publication describing the expression of the steroids sulfotransferases in Brassica napus (21).
  • the nickel agarose-purified proteins were desalted on Sephadex PD- 10TM columns (Pharmacia) using 25 mM Tris-HCI, pH 8.0 for AtST ⁇ a and pH 9.0 for AtST ⁇ b and AtST ⁇ c.
  • the protein concentration was evaluated using the Bradford method (Bio-Rad). The solubility and the purity of the recombinant proteins were verified by SDS-PAGE.
  • DSGs were purified by HPLC on a Waters Nova- Pak C18TM column (3.9 x 150 mm, 4 ⁇ m particle size) according to the procedure described by Hogge et al. (supra).
  • the final identification of the purified compounds was carried out by mass spectrometry using a Quattro IITM triple quadrupole (Micromass, Manchester, UK) equipped with an atmospheric source and a nanoflow probe.
  • the MS/MS fragmentation pattern of glucosinolates was characterized by a fragment at m/z 145 and the loss of a neutral of 162 Da (glucose moiety).
  • Allyl glucosinolate was purchased from Sigma. 3-Methylsulfonylpropyl, 3-methylsufinylpropyl, 4-methylthiobutyl, 2-hydroxy 3- butenyl, benzyl and 2-phenylethyl desulfoglucosinolates were kindly provided by Dr. D. W. Reed, Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada. The DSGs were prepared from their glucosinolates as described above. The concentration of individual compounds was estimated from absorbency at 227 nm using sinigrin as a reference compound.
  • Enzyme assays were performed in a 50 ⁇ L-reaction mixture containing 25 mM Tris-HCI buffer, 1.5 ⁇ M [ 35 S]PAPS (NEN Life Science Products) and 1.0 ⁇ g of recombinant protein for AtST5a at pH 8.0 and 1.0 ⁇ M [ 35 S]PAPS and 0.1 ⁇ g of protein at pH 9.0 for AtST ⁇ b and AtST5c.
  • the substrate specificity studies were conducted with substrate concentrations of 1 ⁇ M and 5 ⁇ M. Reactions mixtures were incubated for 10 min at room temperature and were stopped by freezing at -80°C.
  • Seeds of Arabidopsis thaliana (L.) Heyne ecotype Columbia (Col-0) were surface sterilized, plated on a germination medium (Marlon & Browse (1991) Plant Cell Reports 10, 235-239) and grown under long day conditions (16 hr light, 22°C : 8 hr dark 20°C). 15-days old seedlings were treated with SA at a final concentration of 100 ⁇ M, or exposed to MeJa vapors (1 ⁇ L per MagentaTM box, Bedoukian Research Inc., Danbury, CT). Control plants were treated with the same volume of water. Following treatments with SA or MeJa, seedlings were harvested at 0, 4, 8, 12 and 24 hr, frozen in liquid nitrogen and extracted for RNA.
  • RNAse I (Roche Molecular Biochemicals)
  • DNAse I was heat inactivated at 95° C for 5 min, and the RNA was precipitated with ethanol.
  • cDNA was synthesized using Moloney Murine Leukemia Virus reverse transcriptase as recommended (New England Biolabs). An aliquot (2 ⁇ L) of the RT-reaction was used for the polymerase chain reaction that was performed with ExTaqTM DNA Polymerase (PanVera) and the following oligonucleotides: for AtST ⁇ a: 5'-CTTTCTTACACAAGGAGAAGTCTCAAGA-3' (SEQ ID NO: 15) and 5'-AACAGAAGGACCAGAATAAAACACATTCA-3' (SEQ ID NO: 16); for AtST ⁇ b: 5'-CTTCACAAGGAAAGGACAGAGCT-3' (SEQ ID NO: 17)
  • primers exonl 5'-CTGGTGATG GTGTGTCTCACAC-3' (SEQ ID NO: 19) and exon2, 5'-GTTGTCTCA TGGATTCCAGGAG-3' (SEQ ID NO: 20) were designed from the sequence of the gene ACT1 (GenBankTM ace. No. 439449).
  • sulfotransferase-coding sequences were retrieved from The Arabidopsis Information Resource database using the flavonol 3-ST of Flaveria chloraefolia as a query sequence.
  • the coding sequences of the putative STs were amplified by PCR from Col-0 genomic DNA and cloned in the bacterial expression vector pQE30TM.
  • AtST ⁇ a, AtST ⁇ b and AtST ⁇ c were the only members to exhibit specificity for DSGs.
  • AtST ⁇ a and AtST ⁇ b are clustered on chromosome I with an intergenic distance of 434 nucleotides.
  • the three deduced protein sequences contain all the regions involved in the binding of PAPS (Fig. 3).
  • AtST ⁇ a and AtST ⁇ b share 78% amino acid sequence identity and 89% similarity.
  • AtST ⁇ a and AtST ⁇ c share 73% amino acid sequence identity and 84% similarity and 79% nucleotide sequence identity; and
  • AtST ⁇ b and AtST ⁇ c share 77% amino acid sequence identity and 87% similarity and 79% nucleotide sequence identity.
  • the variations between the three sequences are mostly observed in the two subdomains known to be involved in ST acceptor substrate recognition (Fig. 3).
  • the AtST ⁇ a and AtST ⁇ b proteins share 4 ⁇ % sequence identities with the flavonol STs of Flaveria species, 40% identities with the other A. thaliana STs and approximately 2 ⁇ % identities with the mammalian cytosolic STs.
  • AtST ⁇ a In order to characterize the ST activities of AtST ⁇ a, AtST ⁇ b and AtST ⁇ c the purified recombinant proteins were tested using a wide range of substrates, including steroids, flavonoids and phenolic acids.
  • the three enzymes were found to exhibit strict specificity for DSGs. They did not accept structurally related oxime precursors, such as 3-indolyacetaldoxime and ⁇ -hydroxypentanaldoxime, indicating a strict requirement for the presence of the thioglucose moiety.
  • DSGs with different side-chain structures were tested to determine the substrate preferences of the three enzymes (Fig. 1). Low specific activities were obtained with AtST ⁇ a and AtST ⁇ c as compared with AtST ⁇ b.
  • the three enzymes accept DSGs with different side chain structures including some that are not naturally occurring in A. thaliana, such as methylsulfonylpropyl DSG. However, differences in substrate preference were observed between the three enzymes (Table I). AtST ⁇ b accepts all substrates tested at ⁇ ⁇ M, with the highest preference for 3-methylsulfonylpropyl DSG. At 1 ⁇ M substrate concentration, AtST ⁇ b exhibits the highest activity with desulfobenzyl glucosinolate. If we consider only the naturally occurring Arabidopsis glucosinolates, 4- methylthiobutyl- and 2-phenylethyl DSGs are the preferred substrates among the aliphatic and aromatic ones, respectively.
  • AtST ⁇ c also exhibits a broad specificity with preference for 4-methylthiobutyl-, 3-indolylmethyl- and 2-phenylethyl-DSGs.
  • AtST ⁇ a exhibits very low activity with the aliphatic substrates at both concentrations, with a preference for the 3-indolylmethyl derivative among the naturally occurring aromatic substrates.
  • AtST ⁇ b has a high affinity for all of the substrates tested, except for 3-methylsulfinylpropyl DSG.
  • the catalytic efficiency of AtST ⁇ b is ⁇ 80-fold higher with 3-methylthiobutyl- as compared with 3-methylsulfinylpropyl DSG.
  • AtST ⁇ b exhibits similar affinity for the aromatic and indolyl substrates tested, a 4- to 8- fold lower catalytic activity is observed with 3-indolylmethyl DSG.
  • AtST ⁇ c has kinetic properties similar to AtST5b for the substrates tested.
  • AtST ⁇ a exhibits the highest affinity and catalytic efficiency with 3-indolymethyl DSG with a Km value 1 ⁇ - to 3 ⁇ -fold lower than those obtained with other DSG tested.
  • AtST ⁇ a, AtST ⁇ b and AtST ⁇ c are constitutively expressed in Arabidopsis thaliana
  • RT-PCR experiments were conducted to study the pattern of expression of AtST ⁇ a and AtST ⁇ b. Except for AtST ⁇ a which is not expressed in ⁇ -days old seedlings, the two genes are expressed constitutively at all stages of development (Fig. 4). The transcripts of the two genes are also present in flowers and siliques. It has been proposed that glucosinolates are playing a role in plant protection against herbivores and pathogen infection. In support of this hypothesis, previous experiments demonstrated that Brassica napus responds to MeJa treatment by increasing the accumulation of indole glucosinolates while similar treatments with SA led to increased accumulation of 2-phenylethyl glucosinolate.
  • promoter gus reporter gene fusion indicates that AtST ⁇ a, AtST ⁇ b and AtST ⁇ c are expressed in vascular tissues of the leafs, stems, sepals and anthers, and also in pollen grains.

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Abstract

L'invention concerne des séquences d'acides nucléiques et d'acides aminés codant pour des désulfoglucosinolate sulfotransférases (DSG-ST). Les DSG-ST sont des enzymes intervenant dans la biosynthèse de glucosinolate dans les plantes. L'invention concerne également des méthodes de modulation de la biosynthèse de glucosinolate ainsi que des plantes génétiquement modifiées présentant un taux endogène de glucosinolate modulé.
PCT/CA2002/001144 2001-07-24 2002-07-24 Desulfoglucosinolate sulfotransferases, sequences codant pour celles-ci et utilisations associees pour moduler la biosynthese de glucosinolate dans les plantes WO2003010318A2 (fr)

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