WO2011002519A2 - Matériau et procédés pour réguler l'allocation de carbone et la croissance biomassique - Google Patents

Matériau et procédés pour réguler l'allocation de carbone et la croissance biomassique Download PDF

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WO2011002519A2
WO2011002519A2 PCT/US2010/001892 US2010001892W WO2011002519A2 WO 2011002519 A2 WO2011002519 A2 WO 2011002519A2 US 2010001892 W US2010001892 W US 2010001892W WO 2011002519 A2 WO2011002519 A2 WO 2011002519A2
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plant
gene
cpgl3
expression
seq
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PCT/US2010/001892
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WO2011002519A3 (fr
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Matias Kirst
Evandro Novaes
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University Of Florida Research Foundation, Inc.
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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
    • C12N15/8242—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
    • C12N15/8255—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 involving lignin biosynthesis
    • 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
    • C12N15/8245—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 involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
    • 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
    • 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

  • Woody stems are composed of secondary xylem tissue, which in angiosperms is primarily vessel and fiber cells with thickened secondary walls.
  • Existing data suggests that carbon is competitively partitioned into the major cell wall components, lignin, cellulose and hemicelluloses (Higuchi 1997; Sjostrom 1993).
  • Lignin biosynthesis requires the formation of monolignol precursors from phenylalanine with methylation from S- adenosyl methionine (SAM).
  • Phenylalanine is derived from the shikimate pathway, which initiates with the formation of 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) from erythrose-4-phosphate and phosphoenolpyruvate, through the action of DAHP synthetase (EC:2.5.1.54).
  • DAHP 3-deoxy-D-arabinoheptulosonate 7-phosphate
  • EC:2.5.1.54 3-deoxy-D-arabinoheptulosonate 7-phosphate
  • Cellulosics are derived from sucrose and hexoses which are transferred from the phloem (Flugge 1999).
  • Lignin is the main obstacle for the efficient bioconversion of wood cellulosics to renewable fuels. Lignin content is directly and inversely proportional to fermentable sugar yield for biofuel production (Chen and Dixon 2007). Lignin content is also highly negatively correlated with biomass productivity in several woody species. This negative correlation is evident in studies with Populus (Novaes et al. 2009) and Eucalyptus (Kirst et al. 2004), and in breeding populations (Yu et al. 2006) and a natural mutant of loblolly pine (Wu et al. 1999). In these and other studies (Hu et al. 1999; Li et al.
  • the subject invention concerns materials and methods for modulating lignin levels, cellulose content, and/or growth rates in plants.
  • the invention concerns materials and methods for decreasing lignin levels and/or increasing cellulose content and/or increasing growth rates in plants.
  • a method of the invention comprises inhibiting or decreasing expression of a carbon partitioning and growth locus in chromosome XIII (cpg!3) gene (or a homolog thereof that provides for substantially the same activity), or inhibiting or decreasing expression or activity of the protein encoded by a cpgl3 gene thereof, in a plant, wherein inhibition of expression of the cpgl3 gene or inhibition of expression or activity of the protein encoded by a cpgl3 gene results in decreased lignin levels and/or increased cellulose levels in the plant (relative to lignin and cellulose levels of the plant not having inhibition o ⁇ cpgl3).
  • the cpgl3 gene encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4, or a fragment or variant thereof having substantially the same activity, hi a specific embodiment, the cpgl3 gene comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:3.
  • a method of the invention provides for increased expression of a cpgl3 gene of the invention (or a homolog thereof that provides for substantially the same activity), or increased expression or activity of a protein encoded by the cpgl3 gene.
  • multiple copies of a cpgl3 gene of the invention, or a protein encoding portion thereof, are incorporated in a plant.
  • the cpgl3 gene encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4, or a fragment or variant thereof having substantially the same activity, hi a specific embodiment, the cpgl3 gene comprises the nucleotide sequence shown in SEQ ED NO:1 or SEQ ID NO:3.
  • Figure 2 shows a QTL scan of cpgl3 gene expression, stem cellulose and lignin content, and total plant biomass (tbiomass). Traits were measured in 396 genotypes of Family 52-124, and overlapping peaks were identified for all the traits on linkage group XIII.
  • Figures 3A-3D show that expression of cpgl3 is highest in tissues undergoing secondary cell wall formation.
  • Figures 3A and 3B show relative expression level measured for cpg!3 in five vegetative tissues - fold-change determined relative to the signal detected in a set of eighty negative control genes (figure and data from Quesada et ai, 2008).
  • Figures 3C and 3D show gene expression signal detected for cpgl3 in five stem sections, representing different stages of wood formation (figure and data from Schrader et al., 2004).
  • Figures 4 A and 4B depict the map localization of all 12 QTL identified on LGXIII. Colored bars indicate map regions where the LOD profile is above statistical threshold and black arrows the position of each QTL peak.
  • Figure 4B contains the LOD peak, grand-parental origin of allele increasing mean of the traits, as well as the percentage of the phenotypic and heritable variance explained by each QTL.
  • Figure 5 is a graph showing the LOD profile of gwl .41.566.1 on LGXIII region.
  • Figures 6A and 6B show co-expression network analysis with all 97 genes that have xylem eQTL on the pleiotropic QTL region of LGXIII.
  • Red circle indicates the candidate gene gwl .41.566.1 in the networks. Blue edges indicate positive correlation, and red negative correlation.
  • Diamond nodes indicate genes physically located on the LGXIII interval (cis-regulated). Yellow nodes represent the phenotypic traits, blue O- methyltransferases, red caffeic acid O-methyltransferase, green calmodulin-domain protein kinase, white unknown function proteins, and grey proteins with several different functional annotations.
  • Figure 6A Network constructed with gene-gene correlation threshold of
  • Figure 6B Network constructed with gene-gene correlation threshold of
  • Figure 7 is a diamond plot showing the distribution of Log 2 transformed microarray intensities for gwl.41.566.1 probe hybridized with cDNA from leaf, root and xylem tissues.
  • SEQ ID NO:1 is a nucleotide sequence of the coding region of a cpgl3 gene of the present invention that encodes the amino acid sequence of SEQ ID NO:2.
  • SEQ ID NO:2 is an amino acid sequence of a protein (including an N-terminal signal sequence) encoded by a cpg!3 gene having the nucleotide sequence of SEQ ID NO:1.
  • SEQ ID NO:3 is a nucleotide sequence of the coding region (without the N- terminal signal sequence) of a cpgl3 gene of the present invention that encodes the amino acid sequence of SEQ ID NO:4.
  • SEQ ID NO:4 is an amino acid sequence of a protein (without the N-terminal signal sequence) encoded by a cpg!3 gene having the nucleotide sequence of SEQ ID NO:3.
  • a method of the invention comprises inhibiting or decreasing expression of a cpgl3 gene (or a homolog thereof that provides for substantially the same activity), or the protein encoded by a cpgl3 gene thereof, in a plant, wherein inhibition of expression of the cpgl3 gene results in decreased lignin levels and/or increased cellulose levels in the plant (relative to lignin and cellulose levels of the plant not having inhibition of cpgl 3).
  • the cpgl3 gene encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4, or a fragment or variant thereof having substantially the same activity as a full-length sequence.
  • the cpgl 3 gene comprises the nucleotide sequence shown in SEQ ED NO:1 or SEQ ID NO:3.
  • a method of the invention provides for increased expression of a cpgl 3 gene of the invention (or a homolog thereof that provides for substantially the same activity), or a protein encoding portion thereof.
  • multiple copies of a cpgl3 gene of the invention, or a protein encoding portion thereof, are incorporated in a plant.
  • the cpgl 3 gene encodes a protein comprising the amino acid sequence shown in SEQ ED NO:2 or SEQ ED NO:4, or a fragment or variant thereof having substantially the same activity as a full-length sequence.
  • the cpgl3 gene comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
  • any method available in the art for modulating expression of a cpgl3 gene and/or cpgl3 gene product is contemplated within the scope of the present invention.
  • one or more mutations are introduced into a cpgl3 gene of a plant that results in decreased transcription of the cpgl3 gene, or decreased translation of cpgl3 mRNA, and/or that results in a cpgl3 protein exhibiting decreased activity or function.
  • a mutation is introduced in the cpgl3 gene upstream of the transcription start site and/or downstream of the transcription start site. Mutations include one or more nucleotide insertions, deletions, or substitutions.
  • inhibition or reduction of expression of a cpgl3 gene of the invention can be obtained by introducing an antisense construct in a plant that provides for a sequence that is antisense to a cpgl3 polynucleotide, or a portion thereof.
  • expression of a cpgl3 gene is inhibited by providing in the plant a ribozyme that can cleave cpgl3 RNA and thereby lead to inhibition of endogenous cpgl3 gene expression.
  • a cpgl3 gene in a plant is inhibited by providing the plant with small interfering RNA (siRNA) that target cpgl3 polynucleotides.
  • cpgl3 gene expression can be inhibited by engineering a plant to contain a mutation in the cpgl3 gene that results in the insertion of one or more premature stop codons or nonsense mutations in the cpgl3 gene transcript.
  • inhibition or reduction of cpgl3 gene expression can be achieved by eliminating or non- functionalizing one or more endogenous cpgl3 genes in the plant, for example, by homologous recombination.
  • a heterologous cpgl3 encoding polynucleotide is incorporated into a plant and the polynucleotide expressed therein.
  • the cpgl3 protein encoded by the polynucleotide comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4, or a fragment or variant thereof having substantially the same activity as a full-length sequence.
  • the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
  • a method of the invention comprises introducing a polynucleotide into a plant wherein the polynucleotide, or the expression product thereof, provides for decreased expression of a cpgl3 gene or protein relative to a plant wherein the polynucleotide has not been introduced (e.g., a wild type plant).
  • a polynucleotide can be introduced that increases degradation of cpg 13 gene transcripts or gene product.
  • a polynucleotide can be introduced that encodes a cpg 13 protein that exhibits decreased activity (for example, via decreased resistance to inhibition of enzyme activity).
  • a polynucleotide can be introduced that encodes a protein having cpg 13 activity, wherein the polynucleotide comprises regulatory elements that provide for decreased expression of the polynucleotide and/or the protein encoded thereby.
  • Plants containing the polynucleotide, or progeny thereof, optionally can be screened for decreased expression of cpg 13 gene and/or protein, or decreased activity of the protein.
  • Antisense technology can be used to inhibit expression of a target gene involved in lignin biosynthesis in a plant.
  • a nucleic acid that hybridizes with a nucleotide sequence of an mRNA of a target gene is provided in a plant cell.
  • Nucleic acid constructs that when expressed provide the nucleic acid that hybridizes with the mRNA can be incorporated (e.g., stably) in the genome of a plant.
  • the antisense nucleic acid can hybridize to an entire coding strand of a target sequence, or to a portion thereof, or to a non-coding portion of a target sequence or to both a coding and non- coding portion of a target sequence.
  • Antisense constructs can have, for example, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, 97%, 98%, or 99% sequence identity, or up to 100% sequence identity to the portion of the mRNA that the antisense nucleic acid hybridizes with.
  • Antisense nucleic acids can comprise any suitable number of nucleotides.
  • an antisense nucleic acid construct of the invention can comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides.
  • the antisense nucleic acid comprises at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 150, or at least about 200, or at least about 250, or at least about 300, or at least about 350, or at least about 400, or at least about 450, or at least about 500, or at least about 550, or at least about 600 or more nucleotides.
  • the antisense construct is selectively expressed in leaf cells and/or tissue of the plant, e.g., by use of a leaf-specific promoter. Antisense methods for down-regulating or inhibiting expression of a target gene are known in the art. Plants comprising and expressing the antisense nucleic acid constructs can be grown from cells transformed with and/or incorporating the nucleic acid construct.
  • Cosuppression or post-transcriptional gene silencing (PTGS) technology can also be used to inhibit expression of a target gene involved in lignin biosynthesis in a plant.
  • a nucleic acid sequence corresponding to and having sequence homology with a target gene sequence is provided in a plant cell in a sense orientation and in a construct suitable for expression of the nucleic acid (e.g., a construct comprising the nucleic acid operably linked to a promoter sequence capable of driving transcription in a plant cell).
  • the nucleic acid can have, for example, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96%, 97%, 98%, or 99% sequence identity, or up to 100% sequence identity to the target gene sequence, hi one embodiment, the nucleic acid construct is selectively expressed in leaf cells and/or tissue of the plant, e.g., by use of a leaf-specific promoter. Plants comprising and expressing the nucleic acid constructs can be grown from cells transformed with and/or incorporating the nucleic acid construct.
  • RNA interference (RNAi) technologies can also be used to inhibit expression of a target gene involved in lignin biosynthesis in a plant, hi RNAi, a double-stranded RNA molecule that is complementary to all or a portion of an expressed RNA of a target gene is provided in a plant cell.
  • the double-stranded RNA molecule is processed into smaller RNA molecules which are then processed into a silencing complex which results in inhibition of expression of the target gene, such as by cleavage of target gene mRNA.
  • the RNAi molecule has 100 or more nucleotides, and more typically has 200 or more nucleotides.
  • RNAi molecules can be provided by introduction and expression in a cell of a nucleic acid construct that results in transcription and production of the RNAi molecule, hi one embodiment, RNA interference via expression of a nucleic acid that provides for micro RNA (miRNA) is contemplated within the scope of the invention. miRNAs are generally 19 to 23 nucleotide RNAs that have been processed from a longer precursor RNA comprising hairpin structures. In another embodiment, RNA interference via expression of a nucleic acid that provides for short interfering RNA (siRNA) is contemplated with the scope of the invention. siRNAs are generally 20 to 25 nucleotide RNAs having 3' overhangs and that have been processed from a longer precursor double- stranded RNA.
  • miRNA micro RNA
  • siRNAs are generally 20 to 25 nucleotide RNAs having 3' overhangs and that have been processed from a longer precursor double- stranded RNA.
  • RNAi molecules comprising and expressing RNAi molecules, including miRNAs and siRNAi can be grown from cells transformed with and/or incorporating polynucleotide molecules that provide for the RNAi molecules.
  • Methods and materials for RNA interference have been described, for example, in U.S. Patent Nos. 7,056,704; 7,078,196; 7,365,058; 7,232,086; 6,506,559; 7,282,564; and 7,538,095 and reviewed in Milhavet et al. (2003); Agrawal et al. (2003); Kusaba (2004); and Doran and Helliwell (2009).
  • the RNAi molecules are selectively expressed in leaf cells and/or tissue of the plant, e.g., by use of a leaf-specific promoter.
  • Ribozyme technology can also be used to inhibit expression of a target gene involved in lignin biosynthesis in a plant.
  • Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene.
  • Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Ribozyme encoding nucleotide sequences can be introduced into plant cells and incorporated into the plant genome through gene-delivery mechanisms known in the art.
  • Plants comprising and expressing the ribozyme encoding sequences can be grown from cells transformed with and/or incorporating the ribozyme encoding sequences.
  • a ribozyme having specificity for cpgl3 can include one or more sequences complementary to the nucleotide sequence of at least a portion of one or more cpg!3 mRNA, and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Patent No. 5,093,246 or Haselhoff et al. 1988).
  • a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the cpgl3 mRNA (see, e.g., U.S. Patent No. 4,987,071 ; and U.S. Patent No. 5,116,742).
  • cpgl3 mRNA encoding a cpgl3 protein can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel et al. 1993).
  • the ribozymes are selectively expressed in leaf cells and/or tissue of the plant, e.g. , by use of a leaf-specific promoter.
  • the subject invention also contemplates mutations in target genes, or wherein mutant genes can be provided for in a plant cell wherein target gene expression or gene product levels or activity is decreased or inhibited.
  • a mutant cpgl3 gene is incorporated into the genome of a plant wherein the mutant cpgl3 gene exhibits decreased or no expression of gene transcripts or translation thereof.
  • a mutation is introduced into a cpgl3 gene of a plant that results in decreased transcription of the cpg!3 gene, or decreased translation of mRNA, and/or that results in a protein exhibiting decreased enzymatic activity.
  • one or more mutations are introduced in the protein coding region of a cpgl3 gene.
  • a mutation is introduced in a cpgl3 gene upstream of the transcription start site and/or downstream of the transcription start site.
  • a mutation is introduced into or near a regulatory sequence of a cpgl3 gene, e.g., in a promoter sequence. The mutation may block or inhibit transcription of the cpgl3 gene sequence, e.g., by blocking or inhibiting binding of transcription factors or polymerase to the cpgl3 nucleic acid sequence.
  • a mutation in the cpgl3 gene is selectively introduced into leaf cells and/or leaf tissue of the plant.
  • Mutations can also include one or more nucleotide or amino acid insertions, deletions, and/or substitutions that inhibit or decrease functional activity ⁇ e.g., enzymatic) of a cpgl3 polypeptide.
  • Methods for creating and introducing mutations are known in the art.
  • the mutation is introduced into one or more wild-type cpgl3 genes in a plant, hi another embodiment, a mutant cpgl3 gene replaces one or more wild-type cpgl3 genes in a plant, hi one embodiment, mutant cpgl3 genes are selectively expressed in leaf cells and/or tissue of the plant.
  • a polynucleotide encoding a cpgl3 protein comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or a fragment or variant thereof having substantially the same activity as a full-length sequence, replaces one or more wild-type cpgl3 genes.
  • the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:3.
  • a nucleic acid encoding an antibody, or an antigen binding fragment thereof, that binds to and inhibits activity ⁇ e.g., enzymatic activity) of a protein can be incorporated and expressed in a cell of a plant.
  • a plant comprising and expressing a nucleic acid encoding an antibody, or an antigen binding fragment thereof, can be grown from cells transformed with and/or incorporating the nucleic acid.
  • the antibody is a monoclonal antibody, or an antigen binding fragment thereof.
  • Antigen binding fragments include, but are not limited to, F(ab') 2 , Fab 1 , Fab, and Fv, and can be prepared using standard methods known in the art.
  • the antibody can be derived from any animal capable of producing antibodies to a target protein epitope, and include, for example, human, primate, mouse, rat, goat, sheep, pig, and cow. In a specific embodiment, the antibody binds to a cpgl3 protein.
  • the cpgl3 protein is encoded by a cpgl3 gene.
  • the cpgl3 gene comprises all or a part of the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
  • the cpgl3 gene encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4, respectively, or a fragment or variant thereof.
  • the antibody binds to a cpgl3 protein comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or a fragment or epitope thereof.
  • the nucleic acid encoding the antibody is selectively expressed in leaf tissue of the plant, e.g., by using a leaf specific promoter.
  • the activity (e.g., enzymatic) of proteins encoded by target genes involved in lignin biosynthesis can also be inhibited by expressing and/or contacting the target protein with an aptamer that binds to a specific target protein.
  • Aptamers are oligonucleotides or peptides that can be selected for binding to a target molecule (see, for example, Ellington and Szostak (1990) and Hoppe-Seyler and Butz (2000) and U.S. Patent Nos. 5,582,981; 5,270,163; 5,595,877; 5,817,785; 6,344,318; 6,933,116; 7,368,236; and 7,700,759).
  • a nucleic acid encoding an aptamer that binds to a protein involved in lignin biosynthesis is incorporated and expressed in a cell of a plant.
  • a plant comprising and expressing a nucleic acid encoding an aptamer can be grown from cells transformed with and/or incorporating the nucleic acid.
  • the aptamer binds to and inhibits a cpgl3 protein.
  • the cpgl3 protein is encoded by a cpgl3 gene of the invention.
  • the cpgl3 gene comprises all or a part of the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
  • the cpgl3 gene encodes a polypeptide having the amino acid sequence shown in SEQ ID NOs:2 or SEQ ID NO:4, respectively, or a fragment or variant thereof.
  • the aptamer binds to a cpgl3 protein comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or a fragment or epitope thereof.
  • the nucleic acid encoding the aptamer is selectively expressed in leaf tissue of the plant, e.g., by using a leaf specific promoter.
  • the subject invention also concerns isolated polynucleotides encoding the gene product of a cpgl3 gene of Populus, or a homolog thereof having substantially the same activity.
  • the polynucleotide encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4, or a fragment or variant thereof having substantially the same activity.
  • the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
  • the subject invention also concerns plants, plant tissue, and plant cells of the invention that exhibit increased or decreased expression of a cpgl3 gene (or homolog thereof) or the protein encoded thereby, or that expresses a mutant cpgl3 polynucleotide or a mutant cpgl3 protein.
  • the plant, plant tissue, or plant cell is a woody tree.
  • Plants contemplated within the scope of the present invention include, but are not limited to, plants of the genus Abies, Acacia, Acer, Aesculus, Ailanthus, Alnus, Amelanchier, Arbutus, Arctostaphylos, Artemisia, Asiminia, Atriplex, Aucuba, Berberis, Betula, Buddleia, Buxus, Calocedrus, Camellia, Campsis, Carpinus, Carya, Castanea, Catalpa, Ceanothus, Cedrus, Celastrus, Celtis, Cephalanthus, Cercidium, Cercis, Chaenomeles, Chamaecyparis, Chilopsis, Chionanthus, Chrysothamnus, Cistus, Cladrastis, Clematis, Coleogynia, Cornus, Corylus, Cotinus, Cotoneaster, Cowania, Crataegus, Crataegus, Cupressus, Cytis
  • the plant, plant tissue, or plant cell of the invention can be a hybrid plant or from a hybrid plant.
  • Plants of the invention can have increased or decreased lignin content and/or increased or decreased cellulose content and/or increased or decreased growth rates depending on how cpgl3 gene expression is modulated, hi one embodiment, a plant comprises one or more mutations introduced into a cpgl3 gene of a plant that results in decreased transcription of the cpgl3 gene, or decreased translation of cpg!3 mRNA, and/or that results in a cpgl3 protein exhibiting decreased activity or function, hi a further embodiment, a plant comprises an antisense construct in the plant that provides for a sequence that is antisense to a cpgl3 polynucleotide, or a portion thereof.
  • expression of a cpgl3 gene is inhibited by providing in the plant a ribozyme that can cleave cpg!3 RNA and thereby lead to inhibition of endogenous cpgl3 gene expression.
  • RNA interference RNA interference
  • siRNA small interfering RNA
  • cpgl3 gene expression can be inhibited by engineering a plant to contain a mutation in the cpgl3 gene that results in the insertion of one or more premature stop codons or nonsense mutations in the cpgl3 gene transcript, hi another embodiment, inhibition or reduction of cpgl3 gene expression can be achieved by eliminating or non-functionalizing one or more endogenous cpgl3 genes in the plant, for example, by homologous recombination, hi one embodiment, a plant, plant tissue, or plant cell comprises a heterologous polynucleotide that encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4,
  • the plants disclosed herein may further exhibit one or more agronomic traits that primarily are of benefit to a seed company, a grower, or a grain processor, for example, herbicide resistance, virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, and fungal resistance. See, e.g., U.S. Patent Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431.
  • Such trait may also be one that increases plant vigor or yield (including traits that allow a plant to grow at different temperatures, soil conditions and levels of sunlight and precipitation), or one that allows identification of a plant exhibiting a trait of interest (e.g., selectable marker gene, seed coat color, etc.).
  • a trait of interest e.g., selectable marker gene, seed coat color, etc.
  • Expression constructs of the invention generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed.
  • Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.
  • expression construct refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence.
  • operably linked refers to a juxtaposition of the components described wherein the components are in a relationship that permits them to function in their intended manner. In general, operably linked components are in contiguous relation.
  • An expression construct of the invention can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a polypeptide of the invention. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct of the invention. In a preferred embodiment, a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.
  • plant viral promoters such as, for example, a cauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35S promoter (see, for example U.S. Patent No. 5,106,739)) or a CaMV 19S promoter or a cassava vein mosaic can be used.
  • CaMV cauliflower mosaic virus
  • Other promoters that can be used for expression constructs in plants include, for example, prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA 1'- or 2'-promoter of A.
  • tumefaciens polygalacturonase promoter, chalcone synthase A (CHS-A) promoter from petunia, tobacco PR- Ia promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al, 1993), maize Wipl promoter, maize trpA gene promoter (U.S. Patent No. 5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter (U.S. Patent No. 5,034,322) can also be used.
  • CHS-A chalcone synthase A
  • Tissue-specific promoters for example xyl em-specific promoters, such as the promoter of Cald5H, SAD, XCPl, CAD, CesAl, CesA2, CesA3, tubulin gene (TUB) promoter, lipid transfer protein gene (LTP) promoter, or coumarate-4-hydroxylase gene (C4H) promoter (see, for example, Lu et al, 2008; Funk et al, 2002; Sibout et al, 2005; published U.S. application no. 2008/0196125) can be used.
  • xyl em-specific promoters such as the promoter of Cald5H, SAD, XCPl, CAD, CesAl, CesA2, CesA3, tubulin gene (TUB) promoter, lipid transfer protein gene (LTP) promoter, or coumarate-4-hydroxylase gene (C4H) promoter
  • xyl em-specific promoters such as the promoter of Cald5H
  • Leaf-specific promoters that can be used in a nucleic acid construct of the invention include Cabl promoter (Brusslan and Tobin, 1992), Cab 19 promoter (Bassett et al, 2007), PPDK promoter (Matsuoka et al, 1993), and ribulose biphosphate carboxylase (RBCS) promoter (Matsuoka et al (1994) and U.S. Patent No. 7,723,575).
  • Other plant leaf-specific promoters that can be used with an expression construct of the invention include, but are not limited to, the Actl promoter (U.S. Published Application No. 20090031441), AS-I promoter (U.S. Patent No. 5,256,558), RBC-3A promoter (U.S. Patent No.
  • the CaMV 35S promoter (Odell et al, 1985), the enhanced CaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter (Richins et al, 1987), the mannopine synthase (mas) promoter, the octopine synthase (ocs) promoter, or others such as the promoters from CaMV 19S (Lawton et al, 1987), nos (Ebert et al, 1987), Adh (Walker et al, 1987), sucrose synthase (Yang et al, 1990), a-tubulin, ubiquitin, actin (Wang et al, 1992), cab (Sullivan et al, 1989), PEPCase (Hudspeth et al, 1989) or those associated with the R gene complex (Chandler et al, 1989).
  • CaMV 35S promoter (Odell et al, 1985), the enhanced CaMV 35S promoter, the
  • promoters that direct expression in the xylem of plants include the 4-coumarate Co-enzyme A ligase (4CL) promoter of Populus described in U.S. Patent No. 6,831,208.
  • Seed-specific promoters such as the promoter from a ⁇ -phaseolin gene (for example, of kidney bean) or a glycinin gene (for example, of soybean), and others, can also be used.
  • Endosperm-specific promoters include, but are not limited to, MEGl (EPO application No. EP1528104) and those described by Wu et al.
  • Root-specific promoters such as any of the promoter sequences described in U.S. Patent No. 6,455,760 or U.S. Patent No. 6,696,623, or in published U.S. patent application Nos. 20040078841; 20040067506; 20040019934; 20030177536; 20030084486; or 20040123349, can be used with an expression construct of the invention.
  • Constitutive promoters such as the CaMV, ubiquitin, actin, or NOS promoter
  • developmentally-regulated promoters such as those promoters than can be induced by heat, light, hormones, or chemicals
  • inducible promoters such as those promoters than can be induced by heat, light, hormones, or chemicals
  • RNA can be transcribed by reverse transcriptase to produce a cDNA, and the cDNA can be used to isolate clones containing the full-length genes.
  • the cDNA can also be used to isolate homeologous or homologous promoters, enhancers or terminators from the respective gene using, for example, suppression PCR. See also U.S. Patent No. 5,723,763.
  • Expression constructs of the invention may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements.
  • Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination.
  • a signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment.
  • Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct.
  • Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35 S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element.
  • CMV cytomegalovirus
  • Intron- mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent. Examples include the maize shrunken-1 enhancer element (Clancy and Hannah, 2002).
  • DNA sequences which direct polyadenylation of mRNA transcribed from the expression construct can also be included in the expression construct, and include, but are not limited to, an octopine synthase or nopaline synthase signal.
  • the expression constructs of the invention can also include a polynucleotide sequence that directs transposition of other genes, i.e., a transposon.
  • Polynucleotides of the present invention can be composed of either RNA or DNA. Preferably, the polynucleotides are composed of DNA.
  • the subject invention also encompasses those polynucleotides that are complementary in sequence to the polynucleotides disclosed herein. Polynucleotides and polypeptides of the invention can be provided in purified or isolated form.
  • polynucleotide sequences can encode polypeptides of the present invention.
  • a table showing all possible triplet codons (and where U also stands for T) and the amino acid encoded by each codon is described in Lewin (1985).
  • U also stands for T codons
  • references to "essentially the same" sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions which do not materially alter the functional activity of the polypeptide encoded by the polynucleotides of the present invention. Allelic variants of the nucleotide sequences encoding a cpgl3 protein of the invention are also encompassed within the scope of the invention.
  • amino acids other than those specifically exemplified or naturally present in a polypeptide of the invention are also contemplated within the scope of the present invention.
  • non-natural amino acids can be substituted for the amino acids of a cpgl3 polypeptide, so long as the polypeptide having the substituted amino acids retains substantially the same functional activity as the polypeptide in which amino acids have not been substituted.
  • non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodo tyrosine, 2,4-diaminobutyric acid, a -amino isobutyric acid, 4-aminobutyric acid, 2- amino butyric acid, ⁇ -amino butyric acid, e-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3 -amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C-methyl amino acids
  • Non-natural amino acids also include amino acids having derivatized side groups.
  • any of the amino acids in the protein can be of the D (dextrorotary) form or L (levorotary) form.
  • Allelic variants of a protein sequence of a polypeptide of the present invention are also encompassed within the scope of the invention.
  • Amino acids can be generally categorized in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby a polypeptide of the present invention having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the polypeptide having the substitution still retains substantially the same functional activity as the polypeptide that does not have the substitution. Polynucleotides encoding a polypeptide having one or more amino acid substitutions in the sequence are contemplated within the scope of the present invention. Table 1 below provides a listing of examples of amino acids belonging to each class. Table 1.
  • the subject invention also concerns variants of the polynucleotides of the present invention that encode functional polypeptides of the invention.
  • Variant sequences include those sequences wherein one or more nucleotides of the sequence have been substituted, deleted, and/or inserted.
  • the nucleotides that can be substituted for natural nucleotides of DNA have a base moiety that can include, but is not limited to, inosine, 5-fluorouracil, 5- bromouracil, hypoxanthine, 1 -methylguanine, 5-methylcytosine, and tritylated bases.
  • the sugar moiety of the nucleotide in a sequence can also be modified and includes, but is not limited to, arabinose, xylulose, and hexose.
  • the adenine, cytosine, guanine, thymine, and uracil bases of the nucleotides can be modified with acetyl, methyl, and/or thio groups. Sequences containing nucleotide substitutions, deletions, and/or insertions can be prepared and tested using standard techniques known in the art.
  • Fragments and variants of a polypeptide of the present invention can be generated as described herein and tested for the presence of function using standard techniques known in the art. Thus, an ordinarily skilled artisan can readily prepare and test fragments and variants of a polypeptide of the invention and determine whether the fragment or variant retains functional activity relative to full-length or a non-variant polypeptide.
  • Polynucleotides and polypeptides contemplated within the scope of the subject invention can also be defined in terms of more particular identity and/or similarity ranges with those sequences of the invention specifically exemplified herein.
  • the sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%.
  • the identity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.
  • the subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the polynucleotide sequences exemplified herein so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis et al., 1982).
  • stringent conditions for hybridization refers to conditions wherein hybridization is typically carried out overnight at 20-25 C below the melting temperature (Tm) of the DNA hybrid in 6x SSPE, 5x Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA.
  • Tm melting temperature
  • the melting temperature, Tm is described by the following formula (Beltz et al., 1983):
  • Tm 81.5 C+16.6 Log[Na+]+0.41(%G+C)-0.61(% formamide)-600/length of duplex in base pairs.
  • Washes are typically carried out as follows:
  • nucleic acid and “polynucleotide” refer to a deoxyribonucleotide, ribonucleotide, or a mixed deoxyribonucleotide and ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides.
  • the polynucleotide sequences include the DNA strand sequence that is transcribed into RNA and the strand sequence that is complementary to the DNA strand that is transcribed.
  • polynucleotide sequences also include both full- length sequences as well as shorter sequences derived from the full-length sequences. Allelic variations of the exemplified sequences also fall within the scope of the subject invention.
  • the polynucleotide sequence includes both the sense and antisense strands either as individual strands or in the duplex.
  • the subject invention also concerns methods for producing a plant that exhibits increased or decreased cpgl3 content and/or protein functional activity relative to a wild type plant.
  • a polynucleotide encoding a cpgl3 or a mutant cpgl3 protein of the present invention is introduced into a plant cell and the polypeptide(s) encoded by the polynucleotide(s) is expressed, or wherein a polynucleotide that is antisense to a cpgl3 polynucleotide sequence is introduced into a plant cell, or wherein an siRNA (or a polynucleotide that provides the siRNA) that targets a cpgl3 polynucleotide is introduced into a plant cell.
  • the polynucleotide or polynucleotides is incorporated into the genome of the plant cell and a plant is grown from the plant cell.
  • the plant grown from the plant cell stably expresses the incorporated polynucleotide or polynucleotides.
  • the subject invention also concerns methods and materials for selecting for plants having increased or decreased lignin or cellulose levels.
  • a cpgl3 gene or polynucleotide that exhibits decreased transcription and/or translation, or that encodes a cpgl3 enzyme having decreased activity relative to a wild type or non-mutated cpgl3 is utilized as a genetic marker.
  • the cpgl3 protein comprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or a fragment or variant thereof having substantially the same activity as the full-length sequence
  • the cpgl3 gene or polynucleotide comprises a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or a fragment or variant thereof.
  • Methods of the invention comprise determining whether a plant, plant tissue, or plant cell contains a cpgl3 gene or polynucleotide of the invention, and/or determining whether a plant, plant tissue, or plant cell comprises or expresses a cpgl3 protein of the present invention.
  • the presence of a cpgl3 gene or polynucleotide is determined by screening nucleic acid from the plant, plant tissue, or plant cell for hybridization with a nucleic acid probe ⁇ e.g., an oligonucleotide of the invention) that hybridizes with a cpgl3 gene or polynucleotide of the invention, hi another embodiment, the presence of a cpgl3 gene or polynucleotide is determined by restriction fragment length polymorphism (RFLP) analysis, by polymerase chain reaction (PCR) amplification of specific cpgl3 nucleic acid sequences, or by sequencing cpgl 3-encoding nucleic acid from the plant, plant tissue, or plant cell and identifying whether the gene or polynucleotide comprises a sequence that provides for decreased cpgl 3 mRNA levels or decreased cpgl 3 activity.
  • RFLP restriction fragment length polymorphism
  • PCR polymerase chain reaction
  • the subject invention also concerns methods for marker assisted selection and breeding in plants using a gene or polynucleotide that provides for modulated expression (increased or decreased) of cpgl3 or the gene product thereof for selecting for plants, plant tissue, or plant cells that exhibit a phenotypic characteristic of interest, e.g., increased or decreased lignin or cellulose content, etc.
  • Methods for marker assisted selection are known in the art.
  • a method uses a cpg!3 gene or polynucleotide of the invention that encodes a cpgl 3 enzyme that is non-functional or that exhibits decreased activity relative to a non-mutant cpgl 3 protein, or wherein the cpgl 3 gene or polynucleotide provides for decreased or no expression of gene transcripts or translation of gene transcripts.
  • the subject invention also concerns oligonucleotide probes and primers, such as polymerase chain reaction (PCR) primers, that can hybridize to a coding or non-coding sequence of a polynucleotide of the present invention.
  • Oligonucleotide probes of the invention can be used in methods for detecting and quantitating nucleic acid sequences encoding a polypeptide of the invention.
  • Oligonucleotide primers of the invention can be used in PCR methods and other methods involving nucleic acid amplification.
  • a probe or primer of the invention can hybridize to a polynucleotide of the invention under stringent conditions.
  • Probes and primers of the invention can optionally comprise a detectable label or reporter molecule, such as fluorescent molecules, enzymes, radioactive moiety (e.g., H, 35 S, 125 I, etc.), and the like.
  • Probes and primers of the invention can be of any suitable length for the method or assay in which they are being employed. Typically, probes and primers of the invention will be 10 to 500 or more nucleotides in length. Probes and primers that are 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, 91 to 100 or more nucleotides in length are contemplated within the scope of the invention.
  • Probes and primers of the invention can have complete (100%) nucleotide sequence identity with the polynucleotide sequence, or the sequence identity can be less than 100%.
  • sequence identity between a probe or primer and a sequence can be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or any other percentage sequence identity so long as the probe or primer can hybridize under stringent conditions to a nucleotide sequence of a polynucleotide of the invention.
  • a probe or primer of the invention has 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% to 100% sequence identity with a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or the complement thereof.
  • a polypeptide of the invention has an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:4, or functional fragment or variant thereof that exhibits substantially the same activity as a full-length amino acid sequence.
  • a polypeptide of the invention can be purified using standard techniques known in the art.
  • a polynucleotide of the invention encoding a cpgl3 polypeptide is incorporated into a microorganism, such as E. coli, and the polypeptide expressed in the microorganism and then isolated therefrom.
  • Polypeptides of the invention can be used to generate antibodies that bind specifically to a polypeptide of the invention, and such antibodies are contemplated within the scope of the invention.
  • the antibodies of the invention can be polyclonal or monoclonal and can be produced and isolated using standard methods known in the art.
  • an antibody of the invention binds specifically to a polypeptide that comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4, or a fragment or variant thereof.
  • Antigen binding fragments (such as Fab or Fab 2 or Fv fragments) of antibodies of the invention can be routinely prepared and are also contemplated within the scope of the invention.
  • Fragments of a polypeptide of the invention can be obtained by cleaving the polypeptides of the invention with a proteolytic enzyme (such as trypsin, chymotrypsin, or collagenase) or with a chemical reagent, such as cyanogen bromide (CNBr).
  • a proteolytic enzyme such as trypsin, chymotrypsin, or collagenase
  • CNBr cyanogen bromide
  • polypeptide fragments can be generated in a highly acidic environment, for example at pH 2.5.
  • Polypeptide fragments can also be prepared by chemical synthesis or using host cells transformed with an expression vector comprising a polynucleotide encoding a fragment of a polypeptide of the invention, for example, a polypeptide that is a fragment of the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4.
  • Fragments of a polypeptide of the invention also contemplated herein include fragments of the polypeptide
  • the subject invention also concerns cells transformed with a polynucleotide of the present invention encoding a cpgl3 polypeptide of the invention, or that exhibit increased or decreased expression of a cpgl3 encoding polynucleotide or the protein encoded by the polynucleotide, or that expresses a mutant cpgl3 polynucleotide or a mutant cpgl3 protein that is characterized by increased or decreased expression or activity or function, or a fragment or variant thereof.
  • the cell is transformed with a polynucleotide sequence comprising a sequence encoding the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4, or a functional fragment or variant thereof.
  • the cell is transformed with a polynucleotide sequence shown in SEQ ED NO:1 and/or SEQ ID NO:3, or a sequence encoding a functional fragment or variant of SEQ ID NO:2 and/or SEQ ID NO:4, or a sequence that is antisense to a sequence of SEQ ID NO:1 or SEQ ID NO:3, or an siRNA that targets a sequence of SEQ ID NO:1 or SEQ ID NO:3.
  • the polynucleotide sequence is provided in an expression construct of the invention.
  • the transformed cell can be a prokaryotic cell, for example, a bacterial cell such as E. coli or B.
  • subtilis or the transformed cell can be a eukaryotic cell, for example, a plant cell, including protoplasts, or an animal cell.
  • Plant cells include, but are not limited to, dicotyledonous, monocotyledonous, and gymnosperm cells, such as conifer cells.
  • the plant cell is a cell from a Populus plant.
  • the plant cell can be a cell from a hybrid plant, e.g., a poplar hybrid.
  • Animal cells include human cells, mammalian cells, avian cells, and insect cells. Mammalian cells include, but are not limited to, COS, 3T3, and CHO cells.
  • the subject invention also concerns plant tissue and plant parts, including, but not limited to, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as branches, kernels, ears, cobs, husks, root tips, anthers, seeds, roots, embryos, hypocotyls, cotyledons, pollen, ovules, anthers, shoots, stalks, stems, leaves, fruits, and flowers, derived from a plant of the invention.
  • the subject invention also concerns cuttings produced from a plant of the invention. In one embodiment, the cutting is a rootstock or a scion.
  • the cutting is a stem or branch from a young plant of the invention.
  • the stem is from a poplar plant comprising a cpgl3 gene, or the protein encoding portion thereof, or a sequence that is antisense to a sequence of SEQ ID NO:1 or SEQ ID NO:3, or an siRNA that targets a sequence of SEQ ID NO:1 or SEQ ID NO:3.
  • the poplar plant stem or branch is from a hybrid poplar plant.
  • the subject invention also encompasses plants and plant tissue that are bred from or otherwise derived from a plant of the present invention comprising a polynucleotide encoding a cpgl3 polypeptide of the invention, or a fragment or variant thereof that provides for substantially the same activity, or a sequence that is antisense to a sequence of SEQ ID NO:1 or SEQ ID NO:3, or an siRNA that targets a sequence of SEQ ID NO:1 or SEQ ID NO:3.
  • Seeds encompassed within the scope of the invention include hybrid seeds produced from a cross of a plant of the invention with another plant, such as an inbred plant.
  • the plant of the invention and/or the other plant is a homozygous inbred line.
  • the other plant can be one that exhibits desirable agronomic traits and/or fruit quality.
  • the other plant is one that exhibits resistance to one or more plant pathogens, diseases, or herbicides.
  • the subject invention also concerns hybrid plants grown from hybrid seed or cuttings of the invention.
  • the subject invention also concerns plants on which plant tissue of the subject invention has been grafted.
  • an “antisense” nucleic acid sequence can include a nucleotide sequence that is complementary to a "sense" nucleic acid sequence encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to at least a portion of a cpg!3 gene.
  • the antisense nucleic acid sequence can be complementary to an entire coding strand of a target sequence, or to only a portion thereof.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence within the gene.
  • An antisense oligonucleotide can be, for example, about 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • An antisense nucleic acid sequence can be designed such that it is complementary to the entire gene, but can also be an oligonucleotide that is antisense to only a portion of the gene.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the target mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest.
  • an antisense nucleic acid sequence of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleic acid sequence also can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid sequence will be of an antisense orientation to a target nucleic acid sequence of interest, described further in the following subsection).
  • Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme encoding nucleotide sequences can be introduced into cells through gene-delivery mechanisms known in the art.
  • a ribozyme having specificity for cpgl3 RNA can include one or more sequences complementary to the nucleotide sequence of at least a portion of one or more cpgl3 mRNA, and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Patent No. 5,093,246 or Haselhoff et al. 1988).
  • a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the cpgl3 mRNA (see, e.g., U.S. Patent No.
  • cpg!3 mRNA encoding a cpg!3 protein can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel et al. 1993).
  • RNA interference refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.
  • small interfering RNA or “short interfering RNA”
  • RNA refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference.
  • RNAi target-specific RNA interference
  • siRNA having a "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the siRNA has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.
  • mRNA messenger RNA
  • transcript each refer to single-stranded RNA that specifies the amino acid sequence of one or more polypeptides. This information is translated during protein synthesis when ribosomes bind to the mRNA.
  • the inventors have identified a major gene involved in the regulation of carbon partitioning to lignin and cellulose, and whole plant biomass productivity, by applying a genetical genomic approach to the analysis of a P. deltoides and P. trichocarpa hybrid population.
  • a major locus for carbon partitioning and growth which explains 41% and 65% of the heritable variation in lignin and cellulose content, respectively, and 56% of the ratio between lignin and cellulose, was detected in linkage group XIII (Novaes et al. 2009). The same locus also explains 20-25% of the phenotypic variance for several biomass productivity traits, including whole plant, root and shoot biomass, and stem diameter.
  • cpgl3 carbon partitioning and growth locus in chromosome XIII
  • Plants carrying the P. trichocarpa allele for cpgl3 have about 10 to 20% less lignin, and a proportionate increase in cellulose content and biomass productivity when compared to those plants carrying the P. deltoides allele.
  • Cpgl3 and its closest homologue in Arabidopsis are currently annotated as genes with unknown function. In both species, they are preferentially expressed in tissues undergoing vascular development (Brown et al. 2005; Hertzberg et al. 2001; Schrader et al. 2004). Cpgl3 regulates the metabolic competition for carbon, affecting growth, cellulose biosynthesis and lignification.
  • Cpgl3 regulates the competitive partitioning of carbon to cellulose and lignin in a pseudo-backcross of poplar.
  • the inventors recently reported a locus with major pleiotropic effects on carbon partitioning and biomass productivity located on linkage group XIII (carbon partitioning and growth locus, cpgl3, Figure 2) (Novaes et al. 2009).
  • This quantitative trait locus (QTL) explains 56% of the heritable variation in carbon partitioning between lignin and cellulose and 20 to 25% of the heritable variance for several biomass productivity traits, including stem diameter and biomass accumulation in the stem.
  • This QTL does not control syringyl to guaiacyl (S/G) ratio in lignin (Novaes et al. 2009).
  • SFP single-feature polymorphism
  • GEM gene expression markers
  • cpgl3 has the strongest correlations with genes coding for enzymes from the shikimate/phenylpropanoid pathways (Table 2).
  • a co-expression network constructed with 97 genes expressed in xylem and controlled by the QTL region (in cis- or trans-) identified cpgl3 in the hub of this network.
  • Cpgl3 protein sequence contains a signal peptide and a domain of unknown function present in all land plants.
  • a sequence analysis of cpgl 3 was carried out based on the full-length cDNA generated previously (Ralph et al. 2008).
  • Cpgl3 is predicted to encode a protein with 304 amino acids (aa) (SEQ ID NO:2), with an amino-terminal 31 aa signal peptide for the secretory pathway (TargetP 1.1 and SignalP 3.0) and one potential N-linked glycosylation site near the C-terminus.
  • This specific DUF is found in several genes of land plants: 10 genes in Arabidopis, 10 in Populus, 8 in Vitis (grape), 6 in Or ⁇ za (rice), 2 in Selaginella (lycophyte) and 1 in Physcomitrella (moss). All these genes have unknown function and encode proteins with similar structure to that of cpgl 3, i.e., they contain a relatively short N-terminus and the DUF in the C-terminus. The level of conservation suggests that the DUF domain modulates an important molecular function that has yet to be discovered in plant species.
  • Transgenic plants will be screened by PCR and a set of 20 lines that cover a spectrum of transcript abundance variation in cpgl 3 ( ⁇ 10 over, and 10 under-expressing cpgl 3) will be selected in each species to be characterized phenotypically. Wild-type controls with the T-DNA vector alone are being processed concurrently with cpgl 3 transgenic trees. Plants rooted in vitro will be acclimated in a greenhouse and after 3-4 months of growth, shoots from axillary buds will be harvested and rooted in miniplugs in a misthouse. Three rooted cuttings of similar size (cloned biological replicates) will be selected and planted in separate barcoded pots for each plant.
  • Plants will be grown using an ebb-and-flow flood bench system to ensure daily and uniform supply of a complete nutrient solution for each plant as previously described (Novaes et al. 2009), and will be arranged in a complete block design, with each biological replicate on a separate bench. After the plants have grown for 5-6 months, we will collect the vegetative material for gene expression, metabolite, and wood chemistry and structure analysis.
  • Example 1 Evidences supporting the role of cpsl 3 (gwl.41.566.1) in wood formation
  • Cis eQTL for genes within LGXIII region.
  • a microarray chip was designed to contain all Populus gene models predicted in the genome sequence (Tuskan et al. 2006). This chip was hybridized with cDNA from xylem, leaf and root tissues of a representative sample (100 - 200 genotypes) from family 52-124 progeny. Hybridization intensities for each gene were 1Og 2 transformed, quantile normalized and subsequently treated as quantitative traits in a QTL analysis. This analysis allowed identification of expression QTL (eQTL), i.e. genomic loci that control part of the transcript variation of a gene.
  • QTL eQTL
  • the causative gene also has eQTL in the QTL region of LGXIII.
  • Polymorphisms in cis-regulatory sequences of a gene would drive detection of a cis eQTL, i.e. an eQTL detected where the gene is physically located, that could than affect the trait and cause the QTL.
  • Table 3 Top 10 pair-wise correlation between lignin biosynthesis genes and genes with eQTL on LGXlII QTL region.
  • RNAi- mediated suppression of p-coumaroyl-CoA 3 '-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism. Proceedings of the National Academy of Sciences of the United States of America 105: 4501-4506. Cormen, T.H. 2001. Introduction to algorithms. MIT Press, Cambridge, MA.
  • RNA interference Methods for plants and animals
  • the Arabidopsis xylem peptidase XCPl is a tracheary element vacuolar protein that may be a papain ortholog. Plant Physiology, 128:84-94.
  • ER-to-Golgi transport by COPII vesicles in Arabidopsis involves a ribosome-excluding scaffold that is transferred with the vesicles to the Golgi matrix.
  • CINNAMYL ALCOHOL DEHYDROGENASE-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis.
  • Maize Brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity. Plant Physiology 145: 1444-1459. Sjostrom, E. 1993. Wood chemistry. Academic Press, San Diego.

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Abstract

La présente invention concerne des matériaux et des procédés permettant de moduler les teneurs de lignine, la teneur de cellulose, et/ou les vitesses de croissance des plantes. Selon un aspect, l'invention concerne des matériaux et des procédés permettant de réduire les teneurs de lignine et/ou d'augmenter la teneur de cellulose et/ou d'augmenter les vitesses de croissance des plantes. Dans un mode de réalisation, un procédé selon l'invention comprend l'inhibition ou la réduction de l'expression d'un gène cpgl3 (ou d'un homologue de celui-ci manifestant essentiellement la même activité), ou l'inhibition ou la réduction de l'expression ou de l'activité de la protéine codée par un gène cpgl3, chez une plante, ladite inhibition de l'expression du gène cpgl3 ou ladite inhibition de l'expression ou de l'activité de la protéine codée par un gène cpgl3 entraînant une réduction des teneurs de lignine et/ou une augmentation des teneurs de cellulose dans la plante (par rapport aux teneurs de lignine et de cellulose de ladite plante quand il n'y a pas inhibition du cpgl3).
PCT/US2010/001892 2009-07-02 2010-07-02 Matériau et procédés pour réguler l'allocation de carbone et la croissance biomassique WO2011002519A2 (fr)

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