WO2001038551A1 - Regulation de l'expression d'un gene du groupe polycomb destinee a augmenter la taille des graines chez les plantes - Google Patents

Regulation de l'expression d'un gene du groupe polycomb destinee a augmenter la taille des graines chez les plantes Download PDF

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WO2001038551A1
WO2001038551A1 PCT/US2000/031428 US0031428W WO0138551A1 WO 2001038551 A1 WO2001038551 A1 WO 2001038551A1 US 0031428 W US0031428 W US 0031428W WO 0138551 A1 WO0138551 A1 WO 0138551A1
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mea
seed
gene
expression
construct
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Ueli Grossniklaus
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Cold Spring Harbor Laboratory
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • This invention relates to a method of increasing seed size in plants. Specifically, the invention relates to the functional regulation of Polycomb group gene expression to provide for an increase in seed size over those seeds without such regulatory manipulation. BACKGROUND OF THE INVENTION
  • Homeotic genes specify the identity of serially repeated units such as the whorls of flowers or the segments of insects (Coch, E.S., et al., "The war of the whorla genetic interactions controlling flower development", Nature, 353:31-37 (1991); Lewis, "A gene complex controlling segmentation in Drosophila, Nature 276:565-570 (1978)).
  • Coch E.S., et al., "The war of the whorla genetic interactions controlling flower development", Nature, 353:31-37 (1991); Lewis, “A gene complex controlling segmentation in Drosophila, Nature 276:565-570 (1978)).
  • Coch E.S., et al., "The war of the whorla genetic interactions controlling flower development", Nature, 353:31-37 (1991); Lewis, “A gene complex controlling segmentation in Drosophila, Nature 276:565-570 (1978)).
  • Coch E.S., et al., "The war of the whorla genetic interactions controlling flower development
  • Pc-G Polycomb group genes and trithorax group in Drosophila encode transacting factors that are responsible for preventing the transcription of homeotic selector (HOM-C) genes outside of their appropriate expression domains through positive and negative regulation (Duncan and Lewis, 1982; Struhl and Akam, 1985; Paro, 1990).
  • HOM-C homeotic selector
  • Enhancer ofzeste has been classified as a Pc-G gene (Jones and Gelbart, 1990; Phillips and Shearn, (1990). A loss-of-function allele of the E(z) gene was recovered in a screen for late larval/pupal recessive lethal mutations that cause imaginal disc abnormalities (Shearn et al., 1971). It also however exhibits several properties of the trithorax group as well (LaJeunesse and Shearn 1996).
  • the homeotic AGAMOUS (AG) gene is required to specify stamen and carpal identity in whorls 3 and 4 respectively.
  • Molecular isolation of the AG gene indicates that it encodes a protein belonging to the MADS box family of transcription factors, and that its RNA is confined to its domain of function in whorls 3 and 4 (Yanofsky, M.F., et al., "The protein encoded by the Arabidopsis homeotic gene AGAMOUS resembles transcription factors", Nature, 346:35-39 (1990); Drews, G.N., "Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product", Cell, 63: 991-1002 (1991)).
  • the plant life cycle alternates between a diploid and a haploid generation, the sporophyte and the gametophyte. Unlike in animals where meiotic products differentiate directly into gametes, the plant spores undergo several divisions to form a multi cellular organism. Differentiation of the gametes occurs later in gametophyte development.
  • the mature female gametophyte In most flowering plants, the mature female gametophyte consists of seven cells: three antipodals, two synergids, the egg cell, and a binucleate central cell whose nuclei fuse prior to fertilization
  • the delivery of two sperm cells into the multicellular female gametophyte ensures fertilization of both the egg cell and the binucleate central cell, the precursors of the embryo and the triploid endosperm, respectively.
  • Viable seed formation depends on the coordinated development of the embryo, the endosperm, and the maternal seed coat. Although these interactions are poorly understood, seed morphogenesis requires maternal gene activity in the haploid as well as in the diploid tissues of the developing ovule.
  • the seed specific Polycomb group gene MEDEA (MEA) regulates cell proliferation by exerting a gametophytic maternal control during seed development. Grossniklaus et al. (1998), Maternal control of embryogenesis by MEDEA, a Polycomb group gene in Arabidopsis '. Science 280:446-450.
  • mea seeds seeds derived from embryo sacs carrying a mutant mea-X allele (hereinafter referred to as mea seeds) abort after delayed morphogenesis with excessive cell proliferation in the embryo and reduced free nuclear divisions in the endosperm.
  • the mea mutation affects an imprinted gene expressed maternally in cells of the female gametophyte and after fertilization only from maternally inherited MEA (wild-type) alleles.
  • MEA encodes a SET domain protein with homology to members of the Polycomb and trithorax group, which are believed to maintain active or repressed states of gene expression during development by modulating higher order chromatin structure. (Kennison, 1995; Orlando and Paro, 1995; Pirotta, 1997). The nature of the maternal effect on seed development in mea mutants is not completely understood. Because the endosperm inherits two maternal copies but only one paternal copy of the genome, it is believed that mea could affect a dosage-sensitive gene required for endosperm development.
  • the mutation could disrupt a maternally produced gene product stored in the egg and/or central cell, which is subsequently required for seed development.
  • the mutation could affect an imprinted gene that is transcribed exclusively from the maternally inherited alleles after fertilization.
  • the present invention relates to the spatial and/or temporal regulation of Polycomb group gene expression to provide for larger seed production by plants. According to the invention, the timing of seed specific Polycomb group gene expression is critical and may be manipulated according to the teachings herein.
  • the invention comprises manipulation of seed specific Polycomb group gene expression such that wild-type gene expression is absent, inhibited, or suppressed during early seed development or so that the gene product is inactivated, during this period. Active gene product then is allowed to occur in late seed development.
  • the scheme is summarized in the following table:
  • Gene expression can be manipulated to any of a number of techniques well known to those of skill in the art and described in detail herein and in the materials incorporated by reference, including but not limited to gene activation by use of tissue specific, time specific or inducible promoters with an appropriate transgenic expression system; or inactivation to manipulate Polycomb group gene expression: such as screening for identification or inactive mutants, or transgenic protocols such as inactivation by mutation, expression of a second site modifier gene, sense co-suppression anti-sense co-suppression, homologous recombination, or double stranded RNA interference (dsRNA).
  • dsRNA double stranded RNA interference
  • a mutation in the MEA modifier gene has been identified which is a regulator of genomic imprinting which can be used to regulate Polycomb expression to produce larger seed size in plants.
  • This mutant modifier gene, DDM1 is required for the maintenance, but not the establishment, of the imprint at the mea locus in Arabidopsis.
  • mutations at the decrease in DNA methylationl (ddmX) locus are able to rescue mea seeds during seed development. Quite unexpectedly, these rescued seeds have the desirable trait of being larger than the wild type seeds.
  • Figures 2A-2J are photographs depicting the localization of MEA mRNA in ovules and developing seeds of wild-type Arabidopsis. Magnification: bar is 17 ⁇ m in (A); 22 ⁇ m in (B); 4.3 ⁇ m in (C); 35 ⁇ m in (D) to (G); 51 ⁇ m in (H) to (J).
  • Figure 2A is an ovule (ov) containing an 8-nucleated non-cellularized female gametophyte (fg).
  • the MEA transcript is present in the developing female gametophyte.
  • Figure 2B is a mature ovule with unfertilized female gametophyte; MEA mRNA is present in the cytoplasm of the synergids (sy), the egg cell (ec), and the central cell containing a homo-diploid fused polar nucleus (fpn).
  • Figure 2C is detail of the synergids (sy) and the fused polar nucleus (fpn) shown in (B); the transcript is localized in the cytoplasm of the synergids, but appears closely associated with the fused polar nucleus.
  • Figure 2D is MEA mRNA localized in the globular embryo (e) and in the free nuclear endosperm (fne) of a developing seed; artifactual staining in the endothelium (en) is seen in sense and anti-sense experiments.
  • Figure 2E is a globular embryo hybridized with a sense MEA probe.
  • Figure 2F is a heart stage embryo hybridized with an anti-sense probe; the MEA transcript is localized in the embryo(e) and the free nuclear endosperm (fne).
  • Figure 2G is a heart stage embryo hybridized with a sense MEA probe.
  • Figure 2H is a developing seed with early torpedo embryo (e), cellularized endosperm, and seed coat (sc); MEA mRNA is absent from the cellularized endosperm.
  • Figure 21 is a developing seed with cotyledonary embryo.
  • Figure 2J is a developing seed showing cellularized endosperm (ce), and the seed coat (sc); MEA mRNA is localized in the free endosperm (fne).
  • Figure 3 are photographs depicting the segregation of mea alleles in a duplex tetraploid.
  • c which is the frequency at which the alleles of two sister chromatids are recovered in the same gamete.
  • Small boxes represent haploid genomes from mutant mea-X (red) or wild-type MEA (yellow) alleles.
  • c 0.1.
  • Figures 4A-4E are photographs depicting MEA transcription in the central cell before and after fertilization. Magnification: bar is approximately 0.5 ⁇ m in (A); 1.2 ⁇ m in (B); 1.4 ⁇ m in (C); 1.5 ⁇ m in (D); 1.7 ⁇ m in (E).
  • Figure 4A are two haploid polar nuclei in the unfertilized central cell; a single nuclear dot is localized in each nucleus.
  • Figure 4B is a tetraploid nucleus (resulting from the fusion of two diploid polar nuclei in a tetraploid ovule) showing four nuclear dots prior to fertilization.
  • Figure 4C is a diploid polar nucleus in the central cell of a diploid ovule prior to fertilization showing two nuclear dots.
  • Figure 4D is a triploid nuclei resulting from the division of the primary endosperm nucleus, each showing two nuclear dots after fertilization.
  • Figure 4E are two nuclear dots in the triploid endosperm nuclei located at the chalazal pole of the embryo sac.
  • FIG. 5 is photograph depicting the expression of the mea-1 allele during early seed development.
  • the two panels show amplification of mea-1 and ACTIN-11 (ACTll) as a control for cDNA synthesis.
  • RNA was isolated from siliques derived from self- pollinated mea-1 homozygous pistils (mea x mea), self-pollinated wild-type pistils (wt x wt), a cross between a homozygous mea-1 female and wild-type male (mea x wt), and a cross between wild-type female and homozygous mea-X male (wt x mea).
  • Primers that specifically amplify the mea-X allele under these conditions were used for RT-PCR.
  • (M) indicates the marker lane and (G) genomic DNA as a control.
  • Figure 6 is photograph depicting size comparison of mea- l m /MEA p ; ddml-2/ddml-2 and wild-type seeds and embryos.
  • A Rescued mea-l m /MEA p ; ddml-2/ddml-2 (top) are larger than their sibling wild-type seeds (bottom).
  • Figure 7 is a photograph depicting the morphology of mea- l m /MEA p ; ddml-2/ddml- 2 seeds.
  • A-D Diagrams illustrating the three different classes of rescued seeds;
  • F-I Morphological analysis of rescued embryos belonging to class depicted in (B).
  • A Wild- type seed;
  • B Rescued seed with giant embryo and partial endosperm;
  • C Rescued seed with partially bent embryo and massive endosperm;
  • D Rescued seed with T-shaped embryo and large volume of endosperm.
  • E General morphology of a rescued seed belonging to class depicted in (B); the arrowhead indicates an abnormal region of cell proliferation in the hypocotyl.
  • F Sagital section of a wild-type cotyledon.
  • MEA Polycomb group gene isolated from Arabidopsis has previously been identified and is involved in cell proliferation associated with seed development. See, Grossniklaus, et al., 1998. "Maternal Control of Embryogenesis by MEDEA - a Polycomb group gene in Arabidopsis", Science, 280:446-450. If the MEA gene product is missing, embryos overproliferate and the endosperm underproliferates. Thus, the MEA gene and protein product can regulate proliferation both negatively and positively depending on the tissue. The wild type protein restricts embryo growth but promotes endosperm proliferation. A role in the control of cell proliferation either positively or negatively has also been shown for some of the animal homologs and this aspect of SET domain protein function appears to be conserved.
  • MEA (SEQ ID NO:l, Figure 1) encodes a SET domain protein with homology to members of the Polycomb and trithorax group, which are believed to maintain active or repressed states of gene expression during development by modulating higher order chromatin structure. (Kennison, 1995; Orlando and Paro, 1995; Pirotta, 1997). Other mutants such as fertilization-independent endosperm (fie) and fertilization-independent seed2 (fis2) have been implicated in the control of seed development and also show a gametophytic maternal effect on seed formation and autonomous endosperm development. Ohad et al. (1999), Mutations in FIE, a WD Polycomb group gene, allow endosperm development without fertilization. Plant Cell 11 :407-416.
  • FIE encodes another member of the Polycomb group
  • FIS2 encodes a protein with a Zn-finger motif. Ming et al. (1999), Genes controlling fertilization-independent seed development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 96:296-301.
  • MEA is a member of a large gene family in Arabidopsis. There are about 8 other SET domain proteins in the art. MEA is the only one with a known function in seed development, and is at the very least likely to be present in other angiosperm genomes, including the closely-related species Brassica napus, a species in which the seeds are used to obtain canola oil. This is particularly true since its structure and function is conserved even in animals.
  • the invention herein in its broadest sense contemplates the discovery that seed specific Polycomb group genes, one non-limiting example of which is MEA, can be regulated to provide for increased seed size in plants.
  • MEA gene activity must be absent or gene products produced must in inactive, during early seed development (from fertilization to late heart or torpedo stage) and present during late seed development (after heart or torpedo stage).
  • RNAi double stranded RNA interference
  • Antisense RNA has been used to inhibit plant target genes in a tissue-specific manner, van der Krol et al., Biotechniques 6:958-976 (1988). Antisense inhibition has been shown using the entire cDNA sequence as well as a partial cDNA sequence.
  • Double stranded RNAi constructs produce a single transcript that encodes part of the gene to be silenced, a spacer, and the same part of the gene in antisense orientation. After transcription of this engineered gene, the RNA folds back on itself and forms a panhandle structure containing a piece of double stranded RNA. This method also works well if the spacer is an intron and in principle could get spliced out just leaving the dsRNA. See, Sharp, PA 1999, Genes and Development. "RNAi: and Double-Strand RNA", Jan 15; 13(2) pgs. 139-41.
  • MEA or another homologous gene can be turned on or off in a predictable way; the same is true for its regulators such as ddml .
  • the state early MEA OFF, late MEA ON can be achieved in a variety of ways using such general methods: A)expression of MEA or variants thereof under specific promoters, B)regulating expression of MEA by second site mutations such as ddml or Qchemically inducing MEA expression.
  • protocols useful for the invention include: Expression of wild-type MEA activity in a mea mutant (or preferably a mea deficient) background caused by mutation of gene silencing using a promoter that is specifically activated later during seed development only; expression of a gene silencing construct to inactivate MEA (e.g. sense, antisense or dsRNAi construct) under a promoter active only early during seed development; expression of wild-type MEA activity under an inducible promoter in a mea deficient background; (the inducing conditions are only applied late in seed development); expression of a gene silencing construct to inactivate MEA under an inducible promoter that is only induced early during seed development.
  • a gene silencing construct to inactivate MEA e.g. sense, antisense or dsRNAi construct
  • protocols useful for the invention include: in seeds inheriting an inactive mea allele from the mother, the paternally inherited wild-type MEA allele can be activated by a lack of activity of DDM1 (through mutation or gene silencing); other genes acting as second site modifiers will act similarly and are easily identified using routine screening techniques disclosed herein, ddm2, which is a mutation in the gene encoding DNA methyl transferase 1 , has a similar effect;
  • protocols useful for the invention include: Drugs which demethylate DNA, for instance 5' azacytidine (5AC) and S- adenosyl-homocysteine (SAH).
  • DDM1 may act each of which can be manipulated through pharmacological drugs. Since ddml reduces DNA methylation levels to 30%, it is likely that the MEA gets reactivated by a change in its methylation pattern; Drugs known to affect chromatin organization, such as trichostatin A, sodium butyrate and others, a second mode of action is likely to go via changes in chromatin structure since DDM1 encodes a chromatin remodeling factor.
  • the mea mutation affects an imprinted gene expressed maternally in cells of the female gametophyte after fertilization only from maternally inherited MEA alleles, while paternally inherited MEA alleles are transcriptionally silent in both the embryo and the endosperm, at least early during seed development.
  • Mutations at the ddml locus are able to rescue mea seeds by functionally reactivating paternally inherited MEA alleles during seed development. Rescued seeds are larger than the wild type and show some of the abnormalities found in aborting mea seeds.
  • the term "MEDEA” or “MEA” shall be interpreted to include any seed specific Polycomb group gene identified by the teachings herein or references incorporated herein and shall not be limited to any plant type. Such sequences will by substantially equivalent to the amino acid and nucleic acid sequence depicted in figure 1.
  • the term “substantially equivalent” as used herein means that the peptide is a substance having an amino acid with at least 30%-50% homology with at least one form of the protein as disclosed herein. 80% homology is preferred and 90% homology is most preferred especially including conservative substitutions.
  • the term substantially equivalent means that the sequence will encode a protein or peptide that is substantially equivalent.
  • homology is calculated by standard methods which involve aligning two sequences to be compared so that maximum matching occurs, and calculating the percentage of matches.
  • Substantially equivalent substances to these include those wherein one or more of the residues of the native sequence is deleted, substituted for, or inserted by a different amino acid or acids.
  • substitutions are those which are conservative, i.e., wherein a residue is replaced by another of the same general type.
  • naturally occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar.
  • three of the encoded amino acids are aromatic. It is generally preferred that peptides differing from the native MEA sequence contain substitutions which are from the same group as that of the amino acid replaced.
  • the basic amino acids Lys and Arg are interchangeable; the acidic amino acids aspartic and glutamic are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gin, and Asn are interchangeable; the nonpolar aliphatic acids Gly, Ala, Val, He, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, He and Leu are more closely related), and the aromatic amino acids Phe, Trp, and Tyr are interchangeable.
  • proline is a nonpolar neutral amino acid, it represents difficulties because of its effects on conformation, and substitutions by or for proline are not preferred, except when the same or similar conformational results can be obtained.
  • Polar amino acids which represent conservative changes include Ser, Thr, Gin, Asn; and to a lesser extent, Met.
  • Ala, Gly, and Ser seem to be interchangeable, and Cys additionally fits into this group, or may be classified with the polar neutral amino acids.
  • a "structural gene” is a DNA sequence that is transcribed into messenger RNA (mRNA) which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • an “antisense oligonucleotide” is a molecule of at least 6 contiguous nucleotides, preferably complementary to DNA (antigene) or RNA (antisense), which interferes with the process of transcription or translation of endogenous proteins so that gene products are inhibited.
  • a “promoter” is a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene, proximal to the transcriptional start site of a structural gene.
  • expression refers to biosynthesis of a gene product. Structural gene expression involves transcription of the structural gene into mRNA and then translation of the mRNA into one or more polypeptides.
  • Co-suppression is a method of inhibiting gene expression in plants wherein a construct is introduced to a plant.
  • the construct has one or more copies of sequence which is identical to or which shares nucleotide homology with a resident gene.
  • homologous recombination is another method of inhibiting gene function by introducing a disruption construct to a plant cell under conditions which facilitate recombination of endogenous genetic material with the construct.
  • a "cloning vector” is a DNA molecule such as a plasmid, cosmid, or bacterial phage that has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance, kanamycin resistance, basta resistance, hygromycin resistance or ampicillin resistance.
  • An “expression vector” is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory elements including promoters, tissue specific regulatory elements, and enhancers. Such a gene is said to be “operably linked to” the regulatory elements.
  • a “recombinant host” may be any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the clone genes in the chromosome or genome of the host cell.
  • a “transgenic plant” is a plant having one or more plant cells that contain an expression vector.
  • Plant tissue includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and culture such as single cells, protoplasm, embryos, and callus tissue.
  • the plant tissue may be in plant or in organ, tissue, or cell culture.
  • the invention in one aspect comprises expression constructs comprising a DNA sequence which encodes upon expression a seed specific Polycomb group gene product or DDM1 regulatory gene product operably linked to a promoter to direct expression of the protein. These constructs are then introduced into plant cells using standard molecular biology techniques.
  • the invention can be also be used for hybrid plant or seed production, once transgenic inbred parental lines have been established.
  • the invention involves the inhibition of a Polycomb group gene product in plants through introduction of a construct designed to inhibit the same gene product.
  • the design and introduction of such constructs based upon known DNA sequences is known in the art and includes such technologies as antisense RNA or DNA, co-suppression, double stranded RNA interference or any other such mechanism.
  • antisense RNA or DNA co-suppression
  • double stranded RNA interference any other such mechanism.
  • the methods of the invention described herein may be applicable to any species of plant.
  • Production of a genetically modified plant tissue either expressing or inhibiting expression of a structural gene combines the teachings of the present disclosure with a variety of techniques and expedients known in the art. In most instances, alternate expedients exist for each stage of the overall process. The choice of expedients depends on the variables such as the plasmid vector system chosen for the cloning and introduction of the recombinant DNA molecule, the plant species to be modified, the particular structural gene, promoter elements and upstream elements used. Persons skilled in the art are able to select and use appropriate alternatives to achieve functionality. Culture conditions for expressing desired structural genes and cultured cells are known in the art.
  • a number of both monocotyledonous and dicotyledonous plant species are transformable and regenerable such that whole plants containing and expressing desired genes under regulatory control of the promoter molecules according to the invention may be obtained.
  • expression in transformed plants may be tissue specific and/or specific to certain developmental stages. Truncated promoter selection and structural gene selection are other parameters which may be optimized to achieve desired plant expression or inhibition as is known to those of skill in the art and taught herein.
  • constructs, promoters or control systems used in the methods of the invention may include a tissue specific promoter, an inducible promoter or a constitutive promoter.
  • CaMV cauliflower mosaic virus 35S. It has been shown to be highly active in many plant organs and during many stages of development when integrated into the genome of transgenic plants including tobacco and petunia, and has been shown to confer expression in protoplasts of both dicots and monocots.
  • Organ-specific promoters are also well known.
  • the E8 promoter is only transcriptionally activated during tomato fruit ripening, and can be used to target gene expression in ripening tomato fruit (Deikman and Fischer, EMBO J. (1988) 7:3315; Giovannoni et al., The Plant Cell (1989) 1 :53).
  • the activity of the E8 promoter is not limited to tomato fruit, but is thought to be compatible with any system wherein ethylene activates biological processes.
  • the Lipoxegenase (“the LOX gene") is a fruit specific promoter.
  • Leaf specific promoters include as the AS-1 promoter disclosed in US Patent 5,256,558 to Coruzzi and the RBCS-3 A promoter isolated from pea the RBCS-3 A gene disclosed in US Patent 5,023,179 to Lam et al.
  • root specific promoters include the Cam 35 S promoter disclosed in US Patent 391,725 to Coruzzi et al; the RB7 promoter disclosed in US patent 5,459,252 to Conking et al and the promoter isolated from Brassica napus disclosed in US Patent 5,401, 836 to Bazczynski et al. which give root specific expression.
  • promoters include maternal tissue promoters such as seed coat, pericarp and ovule. Promoters highly expressed early in endosperm development are most effective in this application. Of particular interest is the promoter from the a' subunit of the soybean ⁇ -conglycinin gene [Walling et al., Proc. Natl. Acad. Sci. USA 83:2123-2127 (1986)] which is expressed early in seed development in the endosperm and the embryo.
  • Further seed specific promoters include the Napin promoter described in united States Patent 5,110,728 to Calgene, which describes and discloses the use of the napin promoter in directing the expression to seed tissue of an acyl carrier protein to enhance seed oil production; the DC3 promoter from carrots which is early to mid embryo specific and is disclosed at Plant Physiology. Oct. 1992 100(2) p. 576-581, "Hormonal and Environmental Regulation of the Carrot Lea-class Gene Dc 3, and Plant Mol. Biol.. April 1992, 18(6) p.
  • phaseolin promoter described in United States Patent 5,504,200 to Mycogen which discloses the gene sequence and regulatory regions for phaseolin, a protein isolated from P. vulgaris which is expressed only while the seed is developing within the pod, and only in tissues involved in seed generation.
  • Other organ-specific promoters appropriate for a desired target organ can be isolated using known procedures. These control sequences are generally associated with genes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, Phil, Trans. R. Soc. London (1986) B314-343.
  • mRNAs are first isolated to obtain suitable probes for retrieval of the appropriate genomic sequence which retains the presence of the natively associated control sequences.
  • An example of the use of techniques to obtain the cDNA associated with mRNA specific to avocado fruit is found in Christoffersen et al., Plant Molecular Biology (1984) 3:385. Briefly, mRNA was isolated from ripening avocado fruit and used to make a cDNA library. Clones in the library were identified that hybridized with labeled RNA isolated from ripening avocado fruit, but that did not hybridize with labeled RNAs isolated from unripe avocado fruit. Many of these clones represent mRNAs encoded by genes that are transcriptionally activated at the onset of avocado fruit ripening.
  • the promoter used in the method of the invention may be an inducible promoter.
  • An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of a DNA sequence in response to an inducer. In the absence of an inducer, the DNA sequence will not be transcribed.
  • the protein factor that binds specifically to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer.
  • the inducer may be a chemical agent such as a protein, metabolite (sugar, alcohol etc.), a growth regulator, herbicide, or a phenolic compound or a physiological stress imposed directly by heat, salt, toxic elements etc. or indirectly through the action of a pathogen or disease agent such as a virus.
  • a plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell such as by spraying, watering, heating, or similar methods.
  • inducible promoters include the inducible 70 kd heat shock promoter of D. melanogaster (Freeling, M., Bennet, D.C., Maize ADN 1, Ann.
  • the inducible promoter may be in an induced state throughout seed formation or at least for a period which corresponds to the transcription of the DNA sequence of the recombinant DNA molecule(s).
  • an inducible promoter is the chemically inducible gene promoter sequence isolated from a 27 kd subunit of the maize glutathione-S-transferase (GST II) gene.
  • a number of other potential inducers may be used with this promoter as described in published PCT Application No. PCT/GB90/00110 by ICI.
  • inducible promoter is the light inducible chlorophyll a/b binding protein (CAB) promoter, also described in published PCT Application No. PCT/GB90/00110 by ICI.
  • CAB chlorophyll a/b binding protein
  • inducible promoters have also been described in published Application No. EP89/103888.7 by Ciba-Geigy.
  • PR protein genes especially the tobacco PR protein genes, such as PR- la, PR- lb, PR-lc, PR-1, PR- A, PR-S, the cucumber chitinase gene, and the acidic and basic tobacco beta-l,3-glucanase genes.
  • inducers for these promoters as described in Application No. EP89/103888.7.
  • the preferred promoters may be used in conjunction with naturally occurring flanking coding or transcribed sequences of the seed specific Polycomb genes or with any other coding or transcribed sequence that is critical to Polycomb formation and/or function. It may also be desirable to include some intron sequences in the promoter constructs since the inclusion of intron sequences in the coding region may result in enhanced expression and specificity. Thus, it may be advantageous to join the DNA sequences to be expressed to a promoter sequence that contains the first intron and exon sequences of a polypeptide which is unique to cells/tissues of a plant critical to seed specific Polycomb formation and/or function.
  • regions of one promoter may be joined to regions from a different promoter in order to obtain the desired promoter activity resulting in a chimeric promoter.
  • Synthetic promoters which regulate gene expression may also be used.
  • the expression system may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer elements. OTHERREGULATORY ELEMENTS
  • an expression cassette or construct should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region or polyadenylation signal may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., EMBO J. (1984) 3:835-846) or the nopaline synthase signal (Depicker et al., Mol. and Appl. Genet. (1982) 1 :561-573). MARKER GENES
  • Recombinant DNA molecules containing any of the DNA sequences and promoters described herein may additionally contain selection marker genes which encode a selection gene product which confer on a plant cell resistance to a chemical agent or physiological stress, or confers a distinguishable phenotypic characteristic to the cells such that plant cells transformed with the recombinant DNA molecule may be easily selected using a selective agent.
  • selection marker gene is neomycin phosphotransferase (NPT II) which confers resistance to kanamycin and the antibiotic G-418.
  • Cells transformed with this selection marker gene may be selected for by assaying for the presence in vitro of phosphorylation of kanamycin using techniques described in the literature or by testing for the presence of the mRNA coding for the NPT II gene by Northern blot analysis in RNA from the tissue of the transformed plant. Polymerase chain reactions are also used to identify the presence of a transgene or expression using reverse transcriptase PCR amplification to monitor expression and PCR on genomic DNA. Other commonly used selection markers include the ampicillin resistance gene, the tetracycline resistance and the hygromycin resistance gene. Transformed plant cells thus selected can be induced to differentiate into plant structures which will eventually yield whole plants. It is to be understood that a selection marker gene may also be native to a plant.
  • a recombinant DNA molecule whether designed to inhibit expression or to provide for expression containing any of the DNA sequences and/or promoters described herein may be integrated into the genome of a plant by first introducing a recombinant DNA molecule into a plant cell by any one of a variety of known methods.
  • the recombinant DNA molecule(s) are inserted into a suitable vector and the vector is used to introduce the recombinant DNA molecule into a plant cell.
  • Cauliflower Mosaic Virus (Howell, S.H., et al, 1980, Science, 208:1265) and gemini viruses (Goodman, R.M., 1981, J. Gen Virol. 54:9) as vectors has been suggested but by far the greatest reported successes have been with Agrobacteria sp. (Horsch, R.B., et al, 1985, Science 227:1229-1231). Methods for the use of Agrobacterium based transformation systems have now been described for many different species. Generally strains of bacteria are used that harbor modified versions of the naturally occurring Ti plasmid such that DNA is transferred to the host plant without the subsequent formation of tumors.
  • CaMV Cauliflower Mosaic Virus
  • a plant cell be transformed with a recombinant DNA molecule containing at least two DNA sequences or be transformed with more than one recombinant DNA molecule.
  • the DNA sequences or recombinant DNA molecules in such embodiments may be physically linked, by being in the same vector, or physically separate on different vectors.
  • a cell may be simultaneously transformed with more than one vector provided that each vector has a unique selection marker gene.
  • a cell may be transformed with more than one vector sequentially allowing an intermediate regeneration step after transformation with the first vector.
  • it may be possible to perform a sexual cross between individual plants or plant lines containing different DNA sequences or recombinant DNA molecules preferably the DNA sequences or the recombinant molecules are linked or located on the same chromosome, and then selecting from the progeny of the cross, plants containing both DNA sequences or recombinant DNA molecules.
  • Expression of recombinant DNA molecules containing the DNA sequences and promoters described herein in transformed plant cells may be monitored using Northern blot techniques and/or Southern blot techniques or PCR-based methods known to those of skill in the art.
  • a large number of plants have been shown capable of regeneration from transformed individual cells to obtain transgenic whole plants. For example, regeneration has been shown for dicots as follows: apple, Malus pumila (James et al., Plant Cell Reports (1989) 7:658); blackberry, Rubus, Blackberry/raspberry hybrid, Rubus, red raspberry, Rubus (Graham et al., Plant Cell.
  • Banana hybrid Musa (Escalant and Teisson, Plant Cell Reports (1989) 7:665); bean, Phaseolus vulgaris (McClean and Grafton, Plant Science (1989) 60:117); cherry, hybrid Prunus (Ochatt et al., Plant Cell Reports (1988) 7:393); grape, Vitis vinifera (Matsuta and Hirabayashi, Plant Cell Reports.
  • the regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner. After the expression or inhibition cassette is stably incorporated into regenerated transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. It may be useful to generate a number of individual transformed plants with any recombinant construct in order to recover plants free from any position effects. It may also be preferable to select plants that contain more than one copy of the introduced recombinant DNA molecule such that high levels of expression of the recombinant molecule are obtained.
  • plants may be self-fertilized, leading to the production of a mixture of seed that consists of, in the simplest case, three types, homozygous (25%), heterozygous (50%) and null (25%) for the inserted gene.
  • homozygous 25%
  • heterozygous 50%)
  • null 25%)
  • Transgenic homozygous parental lines make possible the production of hybrid plants and seeds which will contain a modified protein component.
  • Transgenic homozygous parental lines are maintained with each parent containing either the first or second recombinant DNA sequence operably linked to a promoter. Also incorporated in this scheme are the advantages of growing a hybrid crop, including the combining of more valuable traits and hybrid vigor.
  • ISH in situ hybridization
  • the MEA transcript In contrast to the synergrids where the signal is present in the cytoplasm, the MEA transcript is in close association with the nuclei of the egg cell and the central cell. MEA mRNA is not detected in any floral organs, including sepals, petals, stamens, or carpels at any stage of development. No signal could be detected in developing or mature pollen grains. After fertilization, MEA mRNA was detected in all cells of the suspensor and the embryo proper (Fig. ID, E). The transcript persists at a high level to the heart and torpedo stage (Fig. 1F-H), but gradually becomes weaker as embryos reach the cotyledonary stage (Fig. II).
  • MEA mRNA is abundant in dense regions of free nuclei, accumulating at the micropylar and chalazal poles of the embryo sac (Fig. ID, J). The transcript becomes undetectable as the free nuclei start to cellularize at the periphery of the central cell.
  • MEA mRNA is maternally transcribed in the female gametophyte, and MEA mRNA is detectable in both the embryo and the endosperm after fertilization.
  • the high levels of MEA mRNA detected in late heart and torpedo stage embryos (Fig. IH) and in free nuclear endosperm prior to cellularization (Fig. 1 J) cannot be accounted for by maternal expression in the egg and central cell only, suggesting that MEA is zygotically transcribed.
  • mea is not a haplo-insufficient locus by adding an extra wild-type MEA allele to both embryo and endosperm using a tetraploid pollen donor.
  • wild-type MEA product i.e. four or more wild-type MEA copies have to be present in the hexaploid endosperm, it would be expected that 31.5% of the developing seeds to be normal.
  • mea maternal effect depends on the maternal gametophytic genotype only, i.e. at least one wild-type MEA allele has to be inherited from the mother, 78.8% of the developing ovules are expected to form normal seeds.
  • ISH ISH-imprinting
  • nuclear dots of intense staining have not previously been reported in plants but they have been observed in mammalian and Drosophila nuclei where they were shown to be tightly associated with nascent transcripts of actively expressed genes. Because the nuclear dots are associated with a transcribed genomic locus, they allow an analysis of the transcriptional state of this locus at any given time in development. It was confirmed that the nuclear dots represented nascent transcription sites associated with a genomic locus by hybridizing MEA riboprobes to ovules of a tetraploid plant.
  • the number of nuclear dots was doubled in the nuclei of a tetraploid plant as compared to a diploid one (Fig. 3B) confirming that the nuclear dots are correlated with the number of MEA loci present in these nuclei.
  • the absence of nuclear dots in sections that were incubated with RNAse prior to hybridization indicates that the signal is due to the presence of nascent RNA and not to hybridization to chromosomal DNA (data not shown).
  • an analysis of nuclear dots allows a determination of the transcriptional state of all mea loci present in the central cell nucleus at a specific time in development.
  • Nascent transcripts could only be visualized in the polar nuclei of the central cell.
  • Nuclear dots were not observed in the smaller nuclei of the egg cell and synergids.
  • MEA expression by reverse transcription-polymerase chain reaction was examined on RNA isolated from developing siliques derived from reciprocal crosses between wild-type and mea plants approximately 54 hours after pollination (HAP) when the embryos have reached the mid-globular stage.
  • HAP pollination
  • MEA is expressed both maternally and zygotically, it is not clear whether parent-specific expression after fertilization is relevant to the phenotype observed in mea mutants. It is possible that MEA is only required during a short time before fertilization and that a lack of MEA activity in the female gametophyte causes seed abortion later in development. Alternatively, post-fertilization expression of MEA, which is under the control of genomic imprinting, may be responsible for the mea phenotype. To distinguish between these possibilities, it was attempted to manipulate MEA expression in the developing seed. For instance, suppression of the mea seed abortion phenotype by post- fertilization expression of MEA would suggest that zygotic MEA activity is sufficient for normal seed development. Ecotype hybrids gave a first indication that this might be the case since some hybrids produced a small percentage of wild-type seeds.
  • DDM1 which reduces genomic DNA methylation to 30%, has been shown to encode a chromatic remodeling factor of the SW12/SNF2 family. Jeddeloh et al. (1999), Maintenance of genomic methylation requires a SWI2/SNF2-like protein. Nature Genetics 22:94-97.
  • One class showed an abortion frequency (47.5%) close to the one expected for mea-1/MEA (50%), the other had a significantly lower abortion rate (39.7%).
  • the two classes segregated in a 1 :1 ratio suggesting that ddml acts as a suppresser of mea seed abortion. All plants produced slightly more normal seeds than expected which we attribute to a weak suppression of mea seed abortion due to a genetic background effect.
  • the ddml -2 allele is in the Columbia (Col) ecotype whereas mea-1 is in Landsberg erecta (Ler).
  • Plants heterozygous for the ddml -2 allele did not only show a decrease in the frequency of aborted seeds but also produced seeds that were considerably larger than their wild-type siblings ( Figure 6). They were still green when the wild-type seeds had lost their chlorophyll pigmentation suggesting a delay in seed development. These enlarged seeds were observed at a frequency of 5.1% (Table 2) which suggests that they were homozygous for ddml-2.
  • Table 2 which suggests that they were homozygous for ddml-2.
  • the predicted genotype (mea-l m /MEA p ; ddml -2/ ddml -2) was confirmed of the enlarged seeds by scoring kanamycin resistance associated with the mea-1 allele and by confirming homozygosity for ddml by Southern analysis or scoring a CAPS marker linked to the ddml-2 allele (data not shown).
  • ddml acts as a zygotic modifier of mea, i.e. seeds carrying a mutant maternal mea-1 allele survive to maturity if they are also homozygous for ddml.
  • MEA activity is likely to be provided by the paternally inherited MEA allele that gets re-activated during seed development due to a lack of DDM1 activity. This is supported by the fact that all 15 plants derived from enlarged seeds that we tested were heterozygous for mea-1 and, thus, had inherited a wild-type MEA allele from the father. If the effect of ddml were bypassing MEA rather than reactivating the paternal copy, it would be expected that half the plants to inherit a mutant mea-1 allele from the father and twice more mea seeds should survive than observed. These findings strongly suggest that zygotic MEA activity provided from a reactivated paternal allele is sufficient to support seed development. Thus, post-fertilization expression of MEA, which is subject to genomic imprinting, is responsible for the mea phenotype. Table 2
  • the size of the tissue and its degree of cellularization appear to be variable. Embryos have a normal morphology with enlarged tissue sectors in the hypocotyl or the cotyledons; however, growth of the bent cotyledons appears to be limited and the embryos only reach the "walking stick" stage (Bowman, 1994) of development. The region which is not occupied by the cotyledons is filled with persistent endosperm.
  • the mea-1 mutant used in this study was described by Grossniklaus et al. (1998).
  • the duplex tetraploid plants carrying two copies of mea-1 were obtained in the progeny of a self-pollinated simplex mea-1 plant described previously.
  • the ddml-2 mutant was kindly provided by Eric Richards.
  • the ecotypes used in this study were obtained from the Arabidopsis Biological Resource Center at Ohio State University (stock numbers in parenthesis).
  • RNA probes were hydrolyzed as described, and 3% to 6% of each labeling reaction (400 to 800 ng of RNA) were mixed with 40 ⁇ l 50% formamide, added to 200 ⁇ l of hybridization at 55°C, the slides were washed twice with gentle agitation in 0.2X SSC for 1 hour at 55°C, followed by two rinses at room temperature (25°C) in NTE (0.5 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA), and treated with 20 mg/ml of Rnase in the same buffer at 37°C for 30 minutes. They were subsequently rinsed in fresh NTE and washed again in 0.2X SSC for 1 hour at 55°C.
  • NTE 0.5 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA
  • slides were incubated twice (45 and 30 minutes) with gentle agitation in 0.5% blocking agent (Bohehringer-Mannheim) in TBS (100 mM Tris-HCl pH 7.5, 150 mM NaCl), followed by 45 minutes in 1% BSA, 0.3% Triton X-100 in TBS. This was followed by a 2 hour incubation in anti-digoxygenin conjugated antibody diluted 1 :1250 in 1% BSA, 0.3% Triton X-100 in TBS, and four washes of 20 minutes in the same buffer.
  • 0.5% blocking agent Bohehringer-Mannheim
  • the slides were washed twice for 15 minutes in buffer C (100 mM Tris pH 9.5, 50 mM MgCl , 100 mM NaCl), and incubated for 2 to 5 days in 0.34 mg/ml nitroblue tetrazolium salt (NBT) and 0.175 mg/ml 5-bromo-4-chloro-3-indyl phosphate toluidinium salt (BCIP) in buffer C containing 7.6 mM levamisole (SIGMA).
  • buffer C 100 mM Tris pH 9.5, 50 mM MgCl , 100 mM NaCl
  • RNA preparation young siliques were harvested between 52 and 56 HAP in liquid nitrogen.
  • control siliques of self-fertilized Ler wild-type plants and mea-1 homozygous plants (mea-1 /mea-1) were emasculated and hand pollinated in the same way as the reciprocal crosses between Ler and mea-1 /mea-1.
  • RNA was prepared using the Trizol LS reagent (GIBCO-BRL).
  • Trizol LS reagent GABCO-BRL
  • RT-PCR approximately 5 ⁇ g of total RNA were treated with 5 units of RNAse-free DNAse (Boehringer-Mannheim) in IX PCR buffer (GIBCO-BRL) containing 2.5 mM MgCl 2 at 37°C for 30 minutes.
  • PCR was performed in IX PCR buffer (Perkin Elmer) containing 2 mM MgCl 2 , 0.2 mM of each dNTP, 1 unit of Taq DNA polymerase (Perkin-Elmer/Cetus), TaqStart antibody (Clontech) in a molar ratio of 28:1 relative to Taq DNA polymerase, and 20 pmoles of each gene specific primer for 30 cycles at an annealing temperature of 58°C.
  • the primers used for amplification of ACTll were as described.
  • the primers which specifically amplify the mea-1 allele under these conditions were: meaS12 (5'-CTCATGATGAAGCTAATGAGC-3')(SEQ ID NO:2) and meaASl 1 (5'GCATGTTCTGGTCCATAGC-3')(SEQ ID NO:3). Histological Analysis
  • Grossniklaus U., and K. Schneitz. 1998. The molecular and genetic basis of ovule and megagametophyte development. Sem. Cell and Dev. Biol. 9: 227-238. Grossniklaus, U., and J-P. Dahlle-Calzada. 1998. Response: parental conflict and infanticide during embryogenesis. Trends in PI. Sci. 3: 328. Grossniklaus, U., J-P. Dahlle-Calzada, M.A. Hoeppner, and W.B. Gagliano. 1998. Maternal control of embryogenesis by MEDEA, a Polycomb group gene in Arabidopsis. Science 280: 446-450. Haig, D., and C. Graham. 1991.
  • Arabidopsis thaliana studied by means of a chlorophyll mutant with a distinct simplex phenotype.
  • Arabidopsis Inf. Serv. 10 11-12.

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Abstract

La présente invention concerne une méthode de régulation de l'expression d'un gène du groupe Polycomb permettant aux plantes de produire des graines de plus grande taille. Les déposants ont découvert que l'absence de l'expression d'un gène du groupe Polycomb spécifique des graines à un stade précoce du développement des graines (de la fécondation au stade avancé coeur, torpille ou au stade de la cellularisation), puis l'expression dudit gène à une stade avancé du développement des graines (à n'importe quel moment après ces stades de développement) permet d'obtenir des graines de taille plus importante. L'invention concerne également des méthodes et des compositions utilisées dans le cadre de cette manipulation.
PCT/US2000/031428 1999-11-29 2000-11-15 Regulation de l'expression d'un gene du groupe polycomb destinee a augmenter la taille des graines chez les plantes WO2001038551A1 (fr)

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