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WO2010060096A2 - Compositions et procédés pour augmenter la production de cellulose - Google Patents

Compositions et procédés pour augmenter la production de cellulose Download PDF

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Publication number
WO2010060096A2
WO2010060096A2 PCT/US2009/065766 US2009065766W WO2010060096A2 WO 2010060096 A2 WO2010060096 A2 WO 2010060096A2 US 2009065766 W US2009065766 W US 2009065766W WO 2010060096 A2 WO2010060096 A2 WO 2010060096A2
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Prior art keywords
plant
srf
expression
srf7
cell
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WO2010060096A3 (fr
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Zhenbiao Yang
Stephen Karr
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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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
    • C12N15/8245—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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1059—Cellulose synthases (2.4.1.12; 2.4.1.29)

Definitions

  • This disclosure relates to methods and compositions for genetically altering cellulose biosynthesis.
  • Cellulose is a major building block of plant cell walls and provides mechanical strength and rigidity.
  • Wood contains 30 to 50% cellulose, 20 to 30% lignin and 20 to 30% hemicellulose
  • the disclosure shows that loss-of-function mutations of SRF6 (Atlg53730) and SRF7 (At3gl4350) (srf6-l and srf7-1) reduced cellulose synthase A (CesA) gene expression, had reduced hypocotyl elongation in the dark similar to the CesA6 mutant PROCUSTEl (prcl- I 1 At5g64740) and had reduced cellulose deposition as observed by Fourier Transform Infrared (FT-IR) microspectroscopy .
  • FT-IR Fourier Transform Infrared
  • the disclosure provides a recombinant plant cell comprising a heterologous polynucleotide resulting in overexpression of an SRF-6, SRF-7 or homologs thereof, wherein the recombinant host cell comprises increased cellulose production compared to a wild-type cell.
  • the recombinant host cell comprises increased cellulose synthase expression (e.g., a CesA) .
  • the heterologous polynucleotide comprises a heterologous regulatory element that increases expression of an SRF-6, SRF-7 or homolog thereof.
  • the heterologous polynucleotide comprises an expression vector comprising SRF-6, SRF-7 or homolog thereof.
  • the disclosure also provides use of the recombinant host cell in the production of a plant or tree comprising increased cellulose content compared to a wild-type plant or tree.
  • the disclosure also provides a method of producing a transgenic plant comprising growing the plant cell into a plant.
  • the disclosure provides a transgenic plant, wherein the plant comprises overexpression of an SRF-6, SRF-7 or homolog thereof, wherein the plant comprises increased cellulose production compared to a wild-type plant.
  • the plant comprises a tree.
  • the disclosure also provides an isolated polynucleotide encoding a polypeptide lacking all or a fragment of the extracellular domain of an SRF-6 polypeptide.
  • the polynucleotide comprises a sequence of SEQ ID NO: 53.
  • the disclosure provides an isolated polynucleotide encoding a polypeptide lacking all or a fragment of the extracellular domain of an SRF-7 polypeptide (e.g., SEQ ID NO:54).
  • the disclosure also provides an isolated polynucleotide encoding a polypeptide lacking all or a fragment of the C-terminal domain of an SRF-6 polypeptide or SRF-7 polypeptide.
  • the disclosure provides polypeptides encoded by the polynucleotide above, vectors comprising such polynucleotides and host cells transformed with such polynucleotides and vectors.
  • Figure IA-B shows A) Gene structure of SRF6 and SRF7 and location of insertional mutations.
  • Figure 2 shows increased resistance to isoxaben of SRF knockout mutants with insertion in the extracellular domain of the receptor-like kinase compared to insertions post transmembrane domain.
  • Both srf6-1 and srf7-1, with insertions in the extracellular domain, show increased resistance to the increasing concentration of the cellulose synthesis inhibitor isoxaben.
  • srf6-2 and srf7-3 show increased sensitivity to isoxaben and contain insertions in the c-terminal domains
  • srf7-2 showed no significant difference in sensitivity compared to the wild type. Data is taken from three independent experiments.
  • FIG. 3 shows isoxaben affects on the hypocotyls growth of combinatorial and overexpression mutants of SRF.
  • the dominant negative of SRF7 exhibits increased resistance to low levels of isoxaben (0.5 and InM) but increase sensitivity at higher levels (5 and 1OnM) .
  • Expression of DN-SRF7 in srf7-1 mutants shows some resistance to isoxaben at higher levels but is less than that of just srf7-1 alone.
  • Figure 4 shows quantitative real-time PCR analysis of gene expression levels of SRF6 for SRF mutants compared to wild type gene expression in dark treated 4-day-old seedlings grown on 0% sucrose MS media.
  • Figure 5 shows quantitative real-time PCR analysis of gene expression levels of SRF7 for SRF mutants compared to wild type gene expression in dark treated 4-day-old seedlings grown on 0% sucrose MS media.
  • Four-day-old dark treated plants were first cold treated (4°C) for 4-days before harvesting and RNA isolation. This tissue was pooled from three different plates. Data analysis was done using three independent C t values for each measurement. The delta delta C t method was used for comparison of mutant SRF gene expression compared to wild type gene expression and comparison of mutant ACTIN2 gene expression compared to wild type ACTIN2 gene expression.
  • ## no detectible expression of SRF7.
  • @@ p-value ⁇ 0.001.
  • Figure 6 shows quantitative real-time PCR analysis of gene expression levels of CesA3 for SRF mutants compared to wild type gene expression in dark treated 4-day-old seedlings grown on 0% sucrose MS media.
  • Four-day-old dark treated plants were first cold treated (4°C) for 4-days before harvesting and RNA isolation. This tissue was pooled from three different plates. Data analysis was done using three independent C t values for each measurement. The delta delta C t method was used for comparison of mutant CesA3 gene expression compared to wild type gene expression and comparison of mutant ACTIN2 gene expression compared to wild type ACTIN2 gene expression.
  • * p-value ⁇ 0.05.
  • Figure 7 shows quantitative real-time PCR analysis of gene expression levels of CesA4 for SRF mutants compared to wild type gene expression in dark treated 4-day-old seedlings grown on 0% sucrose MS media.
  • Four-day-old dark treated plants were first cold treated (4°C) for 4-days before harvesting and RNA isolation. This tissue was pooled from three different plates. Data analysis was done using three independent C t values for each measurement. The delta delta C t method was used for comparison of mutant CesA4 gene expression compared to wild type gene expression and comparison of mutant ACTIN2 gene expression compared to wild type ACTIN2 gene expression.
  • * p-value ⁇ 0.05.
  • Figure 8 shows and example of FT-IR data before and after baseline correction and normalization.
  • Figure 9 shows a principal component analysis (PCA) for srf7-1, DN-srf7 and wild type. Baseline corrected and normalized data was used for the PCA, using SAS software.
  • PCA principal component analysis
  • Figure 10A-D shows gene expression data from the diurnal experiment (A and C) and isoxaben experiment (B and D) , looking at either RLK (A and B) or cellulose synthase A (C and D) gene expression. Data was obtained from the Genevestigator database using the digital northern tool.
  • Figure 11A-E show an alignment of SRFs and a relationship diagram (an SRF-6 and -7; SEQ ID NO : 2 and 4 are shows in Figure 11A-B) .
  • SRFs-6 and -7 and related homologs are shows in Figures HC-D, SEQ ID NO:6, 8, 10, 12, 14, 16, 18, and 20).
  • Plant biomass represents a useful and valuable resource as a fermentation substrate for highly valuable organic fuels and chemicals. Plant biomass generally consists of about 25% lignin and about 75% carbohydrate polymers including cellulose and hemicellulose . The latter represents one fifth to one half of the total carbohydrates in the biomass. Cellulose is a heteropolymer of hexose and pentose sugars, with glucose and xylose as two major constituents .
  • Cellulose synthesis is a critical process in plants for both the structural integrity of the developing cell and also for the overall structure and rigidity of the plant. The mechanisms underlying the synthesis of cellulose are becoming clearer but the regulation of this important process remains unknown.
  • Cellulose is the most abundant biopolymer on earth. It is an integral component of the plant cell wall and is responsible for most of the rigidity and strength of the cell. Cellulose is currently being investigated as a new fuel source. Because of its chemical structure, a polymer of ⁇ -linked glucose residues, cellulose would make an excellent feedstock for ethanol production, which may relieve some of the pressure of dwindling fossil fuel sources.
  • the polynucleotides of the disclosure that produce more cellulose are from a superfamily of genes called receptor-like kinases (RLKs) .
  • RLKs receptor-like kinases
  • the superfamily of RLKs consists of over 600 genes; many of these are of unknown function (Shiu and Bleecker, 2001) .
  • the Strubbelig Receptor Family contains nine subfamily members and belongs to the leucine-rich repeat (LRR) class of receptor-like kinases (Eyuboglu et al . , 2007) . It has been reported that SRF4 was a positive regulator of leaf size, and that the Strubbelig Receptor Family is characterized by functional diversity when reverse genetics and bioinformatic data mining was used to determine the functions of this receptor gene family
  • the disclosure demonstrates that two members of the Strubbelig Receptor Family are not only coexpressed with cell wall synthesis genes but also have similar dark grown phenotypes to mutants affected in cellulose synthesis as well as altered CesA gene expression. Knockout mutants of SRF6 and SRF7 have shorter dark grown hypocotyls similar to the primary cell wall cellulose synthase CesA6 (prc1-1) . Using FT-IR microspectroscopy and Real- Time RT-PCR there was a coordinate relationship of CesA3 and CesA4 gene expression and the amount of cellulose specific bonding. The overexpression of SRF7 demonstrated a large increase in CesA3 and CesA4 gene expression and also in cellulose composition.
  • the disclosure provides methods and compositions for increasing cellulose content and biomass of a plant or cellulose producing microorganism.
  • the method includes transforming a plant cell or host cell with a vector the increase expression of a SRF-6 and/or SRF-7 polynucleotide or homolog thereof or comprising transforming a plant or microorganism with a mutant SRF-6 or SRF-7 that encodes a truncated SRF-6 or -7 polypeptide the promotes increased expression of a cellulase synthase gene.
  • a cellulose promoting polypeptide of the disclosure includes SRF-6 or -7 polypeptide as well as homologs thereof (collectively referred to herein as SRF-6 or -7 polypeptides, unless the context clearly indicates otherwise) .
  • an SRF-6 or -7 polypeptide comprises any of the polypeptides of SEQ ID NOs : 2 or 4.
  • polypeptides having at least 1-50 (e.g., 1-40, 1-30, 1-20, or 1-10) conservative amino acid substitutions and encoding a polypeptide that promotes increase cellulosic production in a plant.
  • the disclosure provides SRF-6 or SRF-7 polypeptides having at least 58, 60, 70, 80, 90, 95, 98, or 99% identity to any of the SRF polypeptide set forth in Figure 11 and having the ability to increase cellulosic material production in a plant.
  • polynucleotides encoding the polypeptide of any of the foregoing using skill available in the art (e.g., molecule biology cloning strategies) and with reference to SEQ ID NO:1 and 3.
  • Strubbelig Receptor Family 6 and 7 (or homologs or variants thereof) control cellulose synthase A (CesA) gene expression and affects cellulose deposition and quantity.
  • the disclosure provides DNA whose expression varies during plant cell wall component biosynthesis and wood fiber cell morphogenesis.
  • the disclosure provides methods and compositions for generating increased cellulose material in plants.
  • the methods include increasing the expression of a SRF-6 or -7 polypeptide or homolog thereof or transforming a plant cell with a mutant SRF-6 or -7 lacking a C-terminal portion of the polypeptide.
  • the disclosure also provides transgenic plants that overexpress an SRF-6 and/or SRF-7 or a homolog thereof or which express a mutant SRF-6 or SRF- 7, wherein the transgenic plant produces an increased amount of cellulose compared to a wild-type plant.
  • the disclosure provides recombinant host cells and transgenic plants that comprise a modified expression of an SRF6 and/or SRF7 or homolog thereof wherein the host cell comprises increased expression of cellulose synthase genes and wherein the transgenic plant comprise increased cellulose content compared to a plant (e.g., a plant of the same parental species) lacking a change in SRF6, SRF7 or homolog thereof expression.
  • a plant e.g., a plant of the same parental species
  • the terms "host cells” and “recombinant host cells” are used interchangeably and refer to cells (for example, plant cells) into which the compositions of the presently disclosed subject matter (for example, an expression vector comprising an SRF6, or -7 polynucleotide or homolog thereof) can be introduced.
  • the terms refer not only to the particular plant cell into which an expression construct is initially introduced, but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny might not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • An example of a useful polynucleotide for production of cellulosic material comprises an SRF-6 or -7 polynucleotide, variants, mutants and fragment thereof, wherein the variants, mutants and fragments stimulate CesA gene expression.
  • An SRF-6 or -7 polynucleotide includes homologs and variants that are capable of hybridization under stringent conditions with a DNA consisting of a nucleotide sequence described in GenBank accession no. AY518291 or GenBank accession no. AY518292.
  • Stringent hybridization conditions comprise allowing to stand overnight at 60 °C in 0. IxSSC solution, or conditions yielding stringencies similar to these.
  • Useful fragments of SRF-6 or -7 includes those lacking a function C-terminal end of the wild-type polypeptide.
  • a heterologous promoter e.g., a tissue specific, constitutive or inducible promoter
  • the disclosure contemplates a polynucleotide that encodes an polypeptide that induces expression of CesA polypeptides.
  • the polynucleotide encodes a polypeptide comprising at least 50% identity to an SRF-6 or -7.
  • the polynucleotide encodes a mutant SRF-6 or -7 polypeptide lacking a function C-terminal portion of SRF-6 or -7.
  • Homologs of an SRF-6 or -7 can be identified and isolated using techniques known in the art including, for example, hybridization reactions to isolate such DNAs under stringent conditions.
  • Stringent hybridization conditions can include, for example, conditions such as 6 M urea, 0.4% SDS, and 0.5xSSC, and those conditions yielding similar stringencies to these.
  • DNAs with higher homology are expected to be isolated when hybridization is performed under more stringent conditions, for example, 6 M urea, 0.4% SDS, and 0. IxSSC.
  • DNAs thus isolated are thought to have high homology, at an amino acid level, with amino acid sequences encoded by DNAs that hybridize under stringent conditions to DNAs comprising any one of the nucleotide sequences described in Genbank accession no.
  • high homology means an identity over the entire amino acid sequence of at least 50% or above, more preferably 70% or above, even more preferably 80% or above, still more preferably 90% or above, even still more preferably 95% or above, and most preferably 98% or above.
  • DNAs comprise degenerative variants of the DNAs that hybridize under stringent conditions with the DNAs an SRF-6 or -7 as set forth in Genbank accession no. AY518291 or GenBank accession no. AY518292.
  • Useful variants of an SRF-6 or -7 can be identified by introducing mutations by site-directed mutagenesis, directed evolution, shuffling, and the like (Kramer, W. & Fritz, H J., Methods Enzymol, 1987, 154, 350) .
  • the mutant polynucleotide can then be screened to determine if it modulates cesA expression, wherein an increase in CesA is indicative that the mutant SFR-6 or -7 can promote cellulose formation.
  • An SRF-6 or -7 polynucleotide or homolog thereof refers to a polynucleotide comprising SEQ ID NO : 1 or 3, polynucleotide having at least 80%, 90%, 95%, 98% or 99% identity to a sequence consisting of SEQ ID NO : 1 or 3, fragments of the foregoing wherein the fragments encode a polypeptide that promotes cellulase synthase expression, polynucleotide that are complementary to any of the foregoing and polynucleotides that comprise a U instead of T in their sequence.
  • Polynucleotides useful in the methods of the disclosure include naturally occurring polynucleotides, recombinant polynucleotides and chemically synthesized polynucleotides.
  • polynucleotides of the disclosure are capable of encoding polypeptides useful for modulating cellulose production (e.g., through modulating expression of cesA) and include genomic DNA, cDNA, chemically synthesized DNA, and the like.
  • Genomic DNAs may be prepared by conducting PCR (Saiki et al . , Science, 1988, 239, 487) using as a template genomic DNA prepared according to a method described in literature (Rogers and Bendich, Plant MoI.
  • cDNA may be prepared according to the standard method (Maniatis et al . , “Molecular Cloning", Cold Spring Harbor Laboratory Press) , by preparing mRNA from plants, performing reverse transcription, and conducting PCR using primers similar to those described above.
  • Genomic DNA and cDNA may also be prepared by constructing a genomic DNA library or a cDNA library according to the standard method, and screening this library using a probe, for example, one synthesized based on the a nucleotide sequence of a DNA of the disclosure.
  • the DNA thus obtained may be easily sequenced using, for example, the "Sequencer Model 373" (ABI) .
  • ABSI Stemcell Model 373
  • the terms “complementarity” and “complementary” refer to a nucleic acid that can form one or more hydrogen bonds with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of interactions.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, in some embodiments, ribonuclease activity. Determination of binding free energies for nucleic acid molecules is well known in the art. See e.g., Freier et al . , 1986; Turner et al . , 1987.
  • percent complementarity refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary) .
  • the terms “100% complementary”, “fully complementary”, and “perfectly complementary” indicate that all of the contiguous residues of a nucleic acid sequence can hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • the term “gene” refers to a nucleic acid sequence that encodes an RNA.
  • the term “gene” also refers broadly to any segment of DNA associated with a biological function.
  • the term “gene” encompasses sequences including, but not limited to, a coding sequence, a promoter region, a transcriptional regulatory sequence, a non-expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof.
  • a gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation from one or more existing sequences .
  • a gene typically comprises a coding strand and a non-coding strand.
  • coding strand and “sense strand” are used interchangeably, and refer to a nucleic acid sequence that has the same sequence of nucleotides as an mRNA from which the gene product is translated.
  • the coding/sense strand includes thymidine residues instead of the uridine residues found in the corresponding mRNA.
  • the coding/sense strand can also include additional elements not found in the mRNA including, but not limited to promoters, enhancers, and introns .
  • the terms “template strand” and “antisense strand” are used interchangeably and refer to a nucleic acid sequence that is complementary to the coding/sense strand.
  • gene expression generally refers to the cellular processes by which a biologically active polypeptide is produced from a DNA sequence and exhibits a biological activity in a cell.
  • gene expression involves the processes of transcription and translation, but also involves post- transcriptional and post-translational processes that can influence a biological activity of a gene or gene product. These processes include, but are not limited to RNA syntheses, processing, and transport, as well as polypeptide synthesis, transport, and post- translational modification of polypeptides. Additionally, processes that affect protein-protein interactions within the cell can also affect gene expression as defined herein.
  • heterologous gene refers to a sequence that originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example by mutagenesis or by isolation from native transcriptional regulatory sequences. The terms also include non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.
  • transgenic plant or host cell can comprise, for example, a heterologous promoter the promotes transcription of an SRF-6 or -7, or homologs thereof in a desired plant cell or host cell.
  • isolated refers to a molecule substantially free of other nucleic acids, proteins, lipids, carbohydrates, and/or other materials with which it is normally associated, such association being either in cellular material or in a synthesis medium.
  • isolated polynucleotide or “isolated nucleic acid” refers to a ribonucleic acid molecule or a deoxyribonucleic acid molecule (for example, a genomic DNA, cDNA, mRNA, and the like) of natural or synthetic origin or some combination thereof, which (1) is not associated with the cell in which the "isolated polynucleotide” is found in nature, or (2) is operatively linked to a polynucleotide to which it is not linked in nature.
  • isolated polypeptide refers to a polypeptide, in some embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • isolated when used in the context of an “isolated cell”, refers to a cell that has been removed from its natural environment, for example, as a part of an organ, tissue, or organism.
  • the term “modulate” refers to an increase, decrease, or other alteration of any, or all, chemical and biological activities or properties of a biochemical entity, e.g., a wild type or mutant nucleic acid molecule.
  • the term “modulate” can refer to a change in the expression level of a gene or a level of an RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits; or to an activity of one or more proteins or protein subunits that is upregulated or downregulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • the term “modulate” can mean “increasing” or "promoting”, but the use of the word “modulate” is not limited to this definition.
  • naturally occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including bacteria) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. It must be understood, however, that any manipulation by the hand of man can render a "naturally occurring" object an “isolated” object as that term is used herein.
  • polynucleotide or “nucleic acid molecule” refer to any of deoxyribonucleic acid (DNA) , ribonucleic acid (RNA) , oligonucleotides, fragments generated by the polymerase chain reaction (PCR) , and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PCR polymerase chain reaction
  • Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides) , or analogs of naturally occurring nucleotides (e.g., alpha-enantiomeric forms of naturally occurring nucleotides), or a combination of both.
  • Modified nucleotides can have modifications in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.
  • the term also includes so-called "peptide nucleic acids", which comprise naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
  • operably linked and "operatively linked” are used interchangeably.
  • each term refers to a juxtaposition wherein the regions are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence can be ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences, such as when the appropriate molecules (e.g., inducers and polymerases) are bound to the control or regulatory sequence (s) .
  • the phrase "operably linked” refers to a promoter connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that promoter.
  • Techniques for operably linking a promoter to a coding sequence are well known in the art; the precise orientation and location relative to a coding sequence of interest is dependent, inter alia, upon the specific nature of the promoter.
  • operably linked can refer to a promoter region that is connected to a nucleic acid sequence in such a way that the transcription of that nucleic acid sequence is controlled and regulated by that promoter region.
  • a nucleic acid sequence is said to be under the "transcriptional control" of a promoter to which it is operably linked.
  • Techniques for operably linking a promoter region to a nucleotide sequence are known in the art.
  • a nucleotide sequence comprises a coding sequence and/or an open reading frame.
  • operably linked can also refer to a transcription termination sequence that is connected to a nucleotide sequence in such a way that termination of transcription of that nucleotide sequence is controlled by that transcription termination sequence.
  • the disclosure provides vectors and host cells comprising an SRF-6 and/or -7 (or homolog thereof) polynucleotide operably linked to a promoter for expression (e.g., overexpression) of the polynucleotide in the plant or cell.
  • more than one of these elements can be operably linked in a single molecule.
  • multiple terminators, coding sequences, and promoters can be operably linked together.
  • regulatory sequence is a generic term used throughout the specification to refer to polynucleotide sequences, such as initiation signals, enhancers, regulators, promoters, and termination sequences, which are necessary or desirable to affect the expression of coding and non-coding sequences to which they are operatively linked.
  • Exemplary regulatory sequences are described in Goeddel, 1990, and include, for example, the early and late promoters of simian virus 40 (SV40), adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • SV40 simian virus 40
  • adenovirus or cytomegalovirus immediate early promoter the lac system
  • control sequences can differ depending upon the host organism.
  • such regulatory sequences generally include promoter, ribosomal binding site, and transcription termination sequences.
  • the term "regulatory sequence" is intended to include, at a minimum, components the presence of which can influence expression, and can also include additional components the presence of which is advantageous, for example, leader sequences and fusion partner sequences.
  • transcription of a polynucleotide sequence is under the control of a promoter sequence (or other regulatory sequence) that controls the expression of the polynucleotide in a cell-type in which expression is intended.
  • the polynucleotide can be under the control of regulatory sequences that are the same or different from those sequences which control expression of the naturally occurring form of the polynucleotide.
  • the phrase "functional derivative” refers to a subsequence of a promoter or other regulatory element that has substantially the same activity as the full length sequence from which it was derived.
  • a “functional derivative” of a seed-specific promoter can itself function as a seed-specific promoter.
  • Termination of transcription of a polynucleotide sequence is typically regulated by an operatively linked transcription termination sequence (for example, an RNA polymerase III termination sequence) .
  • transcriptional terminators are also responsible for correct mRNA polyadenylation .
  • the 3' non-transcribed regulatory DNA sequence includes in some embodiments about 50 to about 1,000, and in some embodiments about 100 to about 1,000, nucleotide base pairs and contains plant transcriptional and translational termination sequences.
  • Appropriate transcriptional terminators and those that are known to function in plants include the cauliflower mosaic virus (CaMV) 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3 ' end of the protease inhibitor I or II genes from potato or tomato, although other 3' elements known to those of skill in the art can also be employed.
  • a gamma coixin, oleosin 3, or other terminator from the genus Coix can be used.
  • promoter or “promoter region” each refers to a nucleotide sequence within a gene that is positioned 5' to a coding sequence and functions to direct transcription of the coding sequence.
  • the promoter region comprises a transcriptional start site, and can additionally include one or more transcriptional regulatory elements.
  • a method of the presently disclosed subject matter employs a RNA polymerase III promoter.
  • a "minimal promoter” is a nucleotide sequence that has the minimal elements required to enable basal level transcription to occur.
  • minimal promoters are not complete promoters but rather are subsequences of promoters that are capable of directing a basal level of transcription of a reporter construct in an experimental system.
  • Minimal promoters are often augmented with one or more transcriptional regulatory elements to influence the transcription of an operatively linked gene.
  • cell- type-specific or tissue-specific transcriptional regulatory elements can be added to minimal promoters to create recombinant promoters that direct transcription of an operatively linked nucleotide sequence in a cell-type-specific or tissue-specific manner .
  • promoters have different combinations of transcriptional regulatory elements. Whether or not a gene is expressed in a cell is dependent on a combination of the particular transcriptional regulatory elements that make up the gene's promoter and the different transcription factors that are present within the nucleus of the cell. As such, promoters are often classified as “constitutive”, “tissue-specific”, “cell-type- specific”, or “inducible”, depending on their functional activities in vivo or in vitro.
  • a constitutive promoter is one that is capable of directing transcription of a gene in a variety of cell types (in some embodiments, in all cell types) of an organism. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyl transferase
  • DHFR dihydrofolate reductase
  • PGK phosphoglycerate kinase
  • pyruvate kinase phosphoglycerate mutase
  • beta-actin promoter see e.g., Williams et al . , 1993
  • tissue-specific or “cell-type- specific” promoters direct transcription in some tissues or cell types of an organism but are inactive in some or all others tissues or cell types.
  • tissue-specific promoters include those promoters described in more detail hereinbelow, as well as other tissue-specific and cell-type specific promoters known to those of skill in the art.
  • a tissue-specific promoter is a seed-specific promoter, leaf specific, root specific promoter.
  • transcriptional regulatory sequence or “transcriptional regulatory element”, as used herein, each refers to a nucleotide sequence within the promoter region that enables responsiveness to a regulatory transcription factor. Responsiveness can encompass a decrease or an increase in transcriptional output and is mediated by binding of the transcription factor to the DNA molecule comprising the transcriptional regulatory element.
  • a transcriptional regulatory sequence is a transcription termination sequence, alternatively referred to herein as a transcription termination signal.
  • Coding sequences intended for expression in transgenic plants can be first assembled in expression cassettes operably linked to a suitable promoter expressible in plants.
  • the expression cassettes can also comprise any further sequences required or selected for the expression of the transgene .
  • Such sequences include, but are not limited to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the transgene-encoded product to specific organelles and cell compartments.
  • These expression cassettes can then be easily transferred to the plant transformation vectors disclosed below. The following is a description of various components of typical expression cassettes.
  • the selection of the promoter used in expression cassettes can determine the spatial and temporal expression pattern of the transgene in the transgenic plant.
  • Selected promoters can express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves, flowers, or seeds, for example) and the selection can reflect the desired location for accumulation of the transgene.
  • the selected promoter can drive expression of the gene under various inducing conditions. Promoters vary in their strength; i.e., their abilities to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The following are non-limiting examples of promoters that can be used in expression cassettes.
  • Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower-Binet et al . , 1991; maize-Christensen & Quail, 1989; and Arabidopsis-Callis et al . , 1990) .
  • the Arabidopsis ubiquitin promoter is suitable for use with the nucleotide sequences of the presently disclosed subject matter.
  • the ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons.
  • Suitable vectors are derivatives of pAHC25 or any of the transformation vectors disclosed herein, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences.
  • Construction of the plasmid pCGN1761 is disclosed in the published patent application EP 0 392 225, which is hereby incorporated by reference.
  • pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone.
  • a derivative of pCGN1761 is constructed which has a modified polylinker that includes Notl and Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761 ENX.
  • pCGN1761 ENX is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants.
  • the entire 35S promoter- coding sequence-tml terminator cassette of such a construction can be excised by Hindlll, Sphl, Sail, and Xbal sites 5' to the promoter and Xbal, BamHI and BgII sites 3' to the terminator for transfer to transformation vectors such as those disclosed below.
  • the double 35S promoter fragment can be removed by 5' excision with Hindlll, Sphl, Sail, Xbal, or Psfl, and 3' excision with any of the polylinker restriction sites (EcoRI, Notl or Xhol) for replacement with another promoter.
  • modifications around the cloning sites can be made by the introduction of sequences that can enhance translation. This is particularly useful when overexpression is desired.
  • pCGN1761ENX can be modified by optimization of the translational initiation site as disclosed in U.S. Pat. No. 5,639,949, incorporated herein by reference .
  • actin promoter can be used as a constitutive promoter.
  • the promoter from the rice Actl gene has been cloned and characterized (McElroy et al . , 1990).
  • a 1.3 kilobase (kb) fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts.
  • expression vectors based on the Acti promoter have been constructed (McElroy et al., 1991) .
  • the promoter expression cassettes disclosed in McElroy et al., 1991 can be easily modified for gene expression. For example, promoter-containing fragments are removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors.
  • the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. , 1993) .
  • the double 35S promoter in pCGN1761ENX can be replaced with any other promoter of choice that will result in suitably high expression levels.
  • one of the chemically regulatable promoters disclosed in U.S. Pat. No. 5,614,395, such as the tobacco PR-la promoter can replace the double 35S promoter.
  • the Arabidopsis PR-I promoter disclosed in Lebel et al., 1998 can be used.
  • the promoter of choice can be excised from its source by restriction enzymes, but can alternatively be PCR- amplified using primers that carry appropriate terminal restriction sites .
  • a promoter inducible by certain alcohols or ketones, such as ethanol can also be used to confer inducible expression of a coding sequence of the presently disclosed subject matter.
  • a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et a., 1998) .
  • the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AIcR transcription factors in presence of the chemical inducer.
  • the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter are replaced by a coding sequence of the presently disclosed subject matter to form an expression cassette having the coding sequence under the control of the alcA gene promoter.
  • This is carried out using methods known in the art.
  • Induction of expression of a nucleic acid sequence of the presently disclosed subject matter using systems based on steroid hormones is also provided.
  • a glucocorticoid- mediated induction system can be used and gene expression is induced by application of a glucocorticoid, for example, a synthetic glucocorticoid, for example dexamethasone, at a concentration ranging in some embodiments from 0.1 mM to 1 mM, and in some embodiments from 10 mM to 100 mM.
  • a glucocorticoid for example, a synthetic glucocorticoid, for example dexamethasone
  • a suitable root promoter is the promoter of the maize metallothionein-like (MTL) gene disclosed in de Framond, 1991, and also in U.S. Pat. No. 5,466,785, each of which is incorporated herein by reference.
  • This "MTL" promoter is transferred to a suitable vector such as pCGN 1761 ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene- terminator cassette to a transformation vector of interest.
  • Wound-inducible promoters can also be suitable for gene expression. Numerous such promoters have been disclosed (e.g. Xu et al., 1993; Logemann et al .
  • a maize gene encoding phosphoenol carboxylase has been disclosed by Hudspeth and Grula, 1989. Using standard molecular biological techniques, the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants .
  • transcriptional terminators are available for use in expression cassettes. These are responsible for termination of transcription and correct mRNA polyadenylation .
  • Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the octopine synthase terminator, and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
  • a gene's native transcription terminator can be used.
  • intron sequences have been shown to enhance expression, particularly in monocotyledonous cells.
  • the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild type gene under its cognate promoter when introduced into maize cells.
  • Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al . , 1987) .
  • the intron from the maize bronzel gene had a similar effect in enhancing expression.
  • Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
  • Promoters for constant expression are exemplified by the 35S promoter of cauliflower mosaic virus (Odell et al . , Nature, 1985, 313, 810), the actin promoter of rice (Zhang et al . , Plant Cell, 1991, 3, 1155), the ubiquitin promoter of corn (Cornejo et al., Plant MoI. Biol., 1993, 23, 567), etc.
  • promoters for inductive expression are exemplified by promoters that are expressed by extrinsic factors such as infection and invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drought, ultraviolet irradiation, spraying of particular compounds, and the like.
  • Such promoters are exemplified by the chitinase gene promoter of rice (Xu et al., Plant MoI. Biol., 1996, 30, 387.) and tobacco PR protein gene promoter
  • leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.
  • TMV Tobacco Mosaic Virus
  • MCMV Maize Chlorotic Mottle Virus
  • AMV Alfalfa Mosaic Virus
  • leader sequences known in the art include, but are not limited to, picornavirus leaders, for example, EMCV (encephalomyocarditis virus) leader (5' noncoding region; see Elroy-Stein et al . , 1989); potyvirus leaders, for example, from Tobacco Etch Virus (TEV; see Allison et al., 1986); Maize Dwarf Mosaic Virus (MDMV; see Kong & Steinbiss 1998); human immunoglobulin heavy-chain binding polypeptide (BiP) leader (Macejak & Sarnow, 1991); untranslated leader from the coat polypeptide mRNA of alfalfa mosaic virus (AMV; RNA 4; see Jobling & Gehrke, 1987); tobacco mosaic virus (TMV) leader (Gallie et al., 1989); and Maize Chlorotic Mottle Virus (MCMV) leader (Lommel et al., 1991). See also Della-C 10 ppa et al., 1987.
  • EMCV
  • transcription factor generally refers to a protein that modulates gene expression by interaction with the transcriptional regulatory element and cellular components for transcription, including RNA Polymerase, Transcription Associated Factors (TAFs) , chromatin-remodeling proteins, and any other relevant protein that impacts gene transcription.
  • TAFs Transcription Associated Factors
  • percent identity and percent identical in the context of two nucleic acid or protein sequences, refer to two or more sequences or subsequences that have in some embodiments at least 60%, in some embodiments at least 70%, in some embodiments at least 80%, in some embodiments at least 85%, in some embodiments at least 90%, in some embodiments at least 95%, in some embodiments at least 98%, and in some embodiments at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the percent identity exists in some embodiments over a region of the sequences that is at least about 50 residues in length, in some embodiments over a region of at least about 100 residues, and in some embodiments the percent identity exists over at least about 150 residues. In some embodiments, the percent identity exists over the entire length of a given region, such as a coding region.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence (s) relative to the reference sequence, based on the designated program parameters.
  • a "reference sequence” is a defined sequence used as a basis for a sequence comparison.
  • a reference sequence can be a subset of a larger sequence, for example, as a segment of a full- length nucleotide, or amino acid sequence, or can comprise a complete sequence.
  • a reference sequence is at least 200, 300, or 400 nucleotides in length, frequently at least 600 nucleotides in length, and often at least 800 nucleotides in length.
  • two proteins can each (1) comprise a sequence ⁇ i.e., a portion of the complete protein sequence) that is similar between the two proteins, and (2) can further comprise a sequence that is divergent between the two proteins
  • sequence comparisons between two (or more) proteins are typically performed by comparing sequences of the two proteins over a "comparison window" (defined hereinabove) to identify and compare local regions of sequence similarity.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm described in Smith & Waterman, 1981, by the homology alignment algorithm described in Needleman & Wunsch, 1970, by the search for similarity method described in Pearson & Lipman, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG WISCONSIN PACKAGE, available from Accelrys, Inc., San Diego, Calif., United States of America), or by visual inspection. See generally, Ausubel et al . , 1989.
  • One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., 1990.
  • HSPs high scoring sequence pairs
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See e.g., Karlin & Altschul 1993.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in some embodiments less than about 0.1, in some embodiments less than about 0.01, and in some embodiments less than about 0.001.
  • polypeptide As used herein, the terms “polypeptide”, “protein”, and “peptide”, which are used interchangeably herein, refer to a polymer of the 20 protein amino acids, or amino acid analogs, regardless of its size or function. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies.
  • polypeptide as used herein refers to peptides, polypeptides and proteins, unless otherwise noted.
  • protein As used herein, the terms “protein”, “polypeptide”, and “peptide” are used interchangeably herein when referring to a gene product.
  • polypeptide encompasses proteins of all functions, including enzymes.
  • exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.
  • Modification of amino acids in proteins can include conservative and non-conservative amino acid substitutions and may further include deletions, rearrangements or additions.
  • an SRF-6 or -7 polypeptide contains from about 1-50 amino acid substitutions either all conservative substitutions or some conservative and some non-conservative substitutions.
  • polypeptide fragment or fragment when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both.
  • Fragments typically are at least 5, 6, 8, or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40, or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500, or more amino acids long.
  • a fragment can retain one or more of the biological activities of the reference polypeptide. Further, fragments can include a sub- fragment of a specific region, which sub-fragment retains a function of the region from which it is derived. For example, a useful SRF-6 or -7 fragment is capable of inducing cesA expression.
  • the term "primer” refers to a sequence comprising in some embodiments two or more deoxyribonucleotides or ribonucleotides, in some embodiments more than three, in some embodiments more than eight, and in some embodiments at least about 20 nucleotides of an exonic or intronic region. Such oligonucleotides are in some embodiments between ten and thirty bases in length.
  • the term "purified” refers to an object species that is the predominant species present ⁇ i.e., on a molar basis it is more abundant than any other individual species in the composition) .
  • the term “transfection” refers to the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell, which in certain instances involves nucleic acid-mediated gene transfer.
  • the term “transformation” refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous nucleic acid. For example, a transformed cell can express a recombinant form of a polypeptide of the presently disclosed subject matter.
  • the transformation of a cell with an exogenous nucleic acid can be characterized as transient or stable.
  • the term “stable” refers to a state of persistence that is of a longer duration than that which would be understood in the art as "transient”. These terms can be used both in the context of the transformation of cells (for example, a stable transformation) , or for the expression of a transgene (for example, the stable expression of a vector-encoded nucleic acid sequence comprising a trigger sequence) in a transgenic cell.
  • a stable transformation results in the incorporation of the exogenous nucleic acid molecule (for example, an expression vector) into the genome of the transformed cell.
  • stable expression relates to expression of a nucleic acid molecule (for example, a vector-encoded nucleic acid sequence comprising a trigger sequence) over time.
  • a nucleic acid molecule for example, a vector-encoded nucleic acid sequence comprising a trigger sequence
  • stable expression requires that the cell into which the exogenous DNA is introduced express the encoded nucleic acid at a consistent level over time. Additionally, stable expression can occur over the course of generations.
  • the expressing cell divides at least a fraction of the resulting daughter cells can also express the encoded nucleic acid, and at about the same level.
  • stable expression requires only that the nucleic acid molecule of interest be stably expressed in tissue (s) and/or location (s) of the plant in which expression is desired.
  • stable expression of an exogenous nucleic acid is achieved by the integration of the nucleic acid into the genome of the host cell.
  • vector refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked.
  • Agrobacterium binary vector i.e., a nucleic acid capable of integrating the nucleic acid sequence of interest into the host cell (for example, a plant cell) genome.
  • Other vectors include those capable of autonomous replication and expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids .
  • expression vector refers to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to transcription termination sequences. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the construct comprising the nucleotide sequence of interest can be chimeric.
  • the construct can also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the nucleotide sequence of interest including any additional sequences designed to effect proper expression of the nucleotide sequences, can also be referred to as an "expression cassette".
  • an expression cassette comprising one or more elements operably linked in an isolated nucleic acid.
  • the expression cassette comprises one or more operably linked promoters, coding sequences, and/or promoters.
  • recombinant vectors comprising an expression cassette according to the embodiments of the presently disclosed subject matter.
  • plant cells comprising expression cassettes according to the present disclosure, and plants comprising these plant cells.
  • the expression cassette is expressed in a specific location or tissue of a plant.
  • the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof.
  • the presently disclosed subject matter further provides a method for modifying (i.e. increasing or decreasing) the concentration or composition of a polypeptide of the presently disclosed subject matter having an effect on cellulose content in a plant or part thereof.
  • the method comprises in some embodiments introducing into a plant cell an expression cassette comprising a nucleic acid molecule of the presently disclosed subject matter as disclosed above to obtain a transformed plant cell or tissue (also referred to herein as a "transgenic" plant cell or tissue) , and culturing the transformed plant cell or tissue.
  • the nucleic acid molecule can be under the regulation of a constitutive or inducible promoter, and in some embodiments can be under the regulation of a tissue--or cell type-specific promoter.
  • a plant or plant part having modified expression of a nucleic acid molecule of the presently disclosed subject matter can be analyzed and selected using methods known to those skilled in the art including, but not limited to, Southern blotting, DNA sequencing, and/or PCR analysis using primers specific to the nucleic acid molecule and detecting amplicons produced therefrom.
  • a host cell transformed with a vector or polynucleotide of the disclosure can be analyzed for cellulose synthase (e.g., CesA) expression compared to a non-transformed cell. Cells that have increased cellulose synthase expression are indicative of a cell transformed with a polynucleotide of the disclosure .
  • CesA cellulose synthase
  • compositions and methods can result in an increase in cesA expression or cellulose content of a plant by at least 5%, in some embodiments at least 10%, in some embodiments at least 20%, in some embodiments at least 30%, in some embodiments at least 40%, in some embodiments at least 50%, in some embodiments at least 60%, in some embodiments at least 70%, in some embodiments at least 80%, and in some embodiments at least 90% relative to a native control plant, plant part, or cell lacking the expression cassette.
  • transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation art, and the genes pertinent to the presently disclosed subject matter can be used in conjunction with any such vectors.
  • the selection of vector will depend upon the selected transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers might be employed. Selection markers used routinely in transformation include the nptil gene, which confers resistance to kanamycin and related antibiotics (Messing & Vieira, 1982; Bevan et al . , 1983); the bargene, which confers resistance to the herbicide phosphinothricin (White et al . , 1990; Spencer et al .
  • hph gene which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, 1984); the dhfr gene, which confers resistance to methotrexate (Bourouis & Jarry, 1983); the EPSP synthase gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642); and the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).
  • vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as PBIN19 (Bevan, 1984) . Below, the construction of two typical vectors suitable for Agrobacterium transformation is disclosed.
  • Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector, and consequently vectors lacking these sequences can be utilized in addition to other vectors that contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake ⁇ e.g. polyethylene glycol (PEG) and electroporation) , and microinjection. The choice of vector depends largely on the species being transformed.
  • PEG polyethylene glycol
  • nucleic acid sequence of the presently disclosed subject matter is transformed into a plant cell.
  • the expression cassettes of the presently disclosed subject matter can be introduced into the plant cell in a number of art-recognized ways. Methods for regeneration of plants are also well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles . In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells.
  • Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium.
  • Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation-mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are disclosed in Paszkowski et al., 1984; Potrykus et al . , 1985; and Klein et al . , 1987. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
  • Agrobacterium-mediated transformation is a useful technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species.
  • Agrobacterium transformation typically involves the transfer of a binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain which can depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally .
  • Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
  • Various techniques can be used to introduce an aforementioned expression vector into host plant cells.
  • these techniques include transformation of plant cells by T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transformation factor, direct introduction into a protoplast (by a method such as electroporation in which a DNA is introduced into plant cells by treating protoplasts with an electric pulse, fusion of protoplasts with liposomes and so forth, microinjection, and the use of polyethylene glycol), and the use of a particle gun.
  • a desired gene can be introduced into a plant, by using a plant virus as vector.
  • a plant virus that can be used is cauliflower mosaic virus. Namely, after first preparing a recombinant by inserting the virus genome into a vector derived from E. coli and so forth, the desired gene is inserted into the virus genome. Such desired genes can then be introduced into a plant by cutting out the virus genome modified in this manner from the recombinant with a restriction enzyme, and inoculating into the plant (Hohn, et al . (1982), Molecular Biology of Plant Tumors (Academic Press, New York), p. 549, U.S. Pat. No. 4,407,956) .
  • the technique for introducing a vector into plant cells or a plant is not limited to these, and includes other possibilities as well.
  • a simple plasmid such as a pUC derivative can be used.
  • Other DNA sequences may be required depending on the method used to introduce the desired gene into plant cells.
  • at least the sequence on the right end, and typically the sequences on both ends, of the T- DNA region of Ti and Ri plasmids must be connected so as to become an adjacent region of the gene to be introduced.
  • a gene to be introduced needs to be cloned into a special plasmid, namely an intermediate vector or a binary vector.
  • Intermediate vectors are not replicated in Agrobacterium species.
  • Intermediate vectors are transferred into Agrobacterium species by helper plasmids or electroporation . Since intermediate vectors have a region that is homologous with the T-DNA sequence, they are incorporated within the Ti or Ri plasmid of Agrobacterium species by homologous recombination. It is necessary for the Agrobacterium species used for the host to comprise a vir region. Normally, Ti or Ri plasmids comprise a vir region, and due to its function, T-DNA can be transferred into plant cells.
  • the disclosure provides transgenic plants that have been redifferentiated from the aforementioned transgenic plant cells, transgenic plants that are progenies or clones of the transgenic plants, and breeding material of the transgenic plants.
  • cell wall components include various quantitative and qualitative changes to create plants high in cellulose, low in lignin, having thick cell walls, thin cell walls, long and short fiber lengths, etc.
  • cell morphology alterations include, but are not limited to, changes in cell elongation and cell size (quantitative changes in volume) .
  • Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Pat. Nos . 4,945,050; 5,036,006; and 5,100,792; all to Sanford et al . Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the desired gene.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e.g., dried yeast cells, dried bacterium, or a bacteriophage, each containing DNA sought to be introduced
  • the methods and compositions of the disclosure can be used in Eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, yew, rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower, cotton, orange, grape, peach, pear, apple, tomato, Chinese cabbage, cabbage, radish, carrot, squash, cucumber, melon, parsley, orchid, chrysanthemum, lily, and saffron.
  • some microorganisms produce various types of cellulosic material.
  • the methods and compositions of the disclosure can be used in the generation of recombinant microorganism for the production of cellulosic material. Such microorganisms and plants may be useful for the production of biofuels and the like.
  • the disclosure provides transgenic plant cells into which a vector of the disclosure has been introduced.
  • the cells into which a vector of the disclosure is introduced include the cells of rice, corn, wheat, barley, rye, potato, tobacco, sugar beet, sugar cane, rapeseed, soybean, sunflower, cotton, orange, grape, peach, pear, apple, tomato, Chinese cabbage, cabbage, radish, carrot, squash, cucumber, melon, parsley, orchid, chrysanthemum, lily, and saffron; however, trees such as Eucalyptus, pine, acacia, poplar, cedar, cypress, bamboo, and yew are preferable.
  • plant cells of the disclosure comprise cultured cells, as well as cells present in a plant.
  • protoplasts, shoot primordia, multiple shoots, and hairy roots are also included.
  • a transgenic plant of the disclosure is useful as a plant having a novel value such as increased plant growth as a result of increasing cell wall biosynthesis, altered fiber cell morphology, or increased amounts of useful components in agricultural crops.
  • it is also useful as a plant having a novel value in developing new materials by controlling cell wall biosynthesis, increasing the digestion and absorption efficiencies of feed crops, changing fiber cell morphology, and the like.
  • a "transgenic plant” refers to a plant having the aforementioned transgenic plant cells, and includes, for example, a transgenic plant regenerated from the aforementioned transgenic cells.
  • the methods used to regenerate individual plants from transformed plant cells vary according to the type of plant cell, an example of a method used in rice plants is the method of Fujimura et al. (Fujimura et al . , Plant Tissue Culture Lett., 2, 74, 1995), the method of Shillito et al. (Shillito et al . , Bio/Technology, 7, 581, 1989) in corn plants, the method of Visser et al . (Visser et al . , Theor.
  • the disclosure includes a process of producing a plant from a plant seed by introducing into a host a gene expressed by a plant during cell wall formation and/or specifically expressed during cellulose biosynthesis, a homolog thereof, or an expression vector comprising a promoter region that is contiguous with these genes to obtain transgenic cells, regenerating a transgenic plant from said transgenic cells, and obtaining a plant seed from the resulting transgenic plant.
  • a process of obtaining a plant seed from a transgenic plant refers to a process in which, for example, a transgenic plant is acquired from a rooting medium, replanted in a pot containing moist soil, and grown at a constant temperature to form flowers, and finally seeds.
  • a process of producing a plant from a seed refers to a process in which, for example, once a seed formed in a transgenic plant has matured, the seed is isolated, sowed on moist soil, and then grown at a constant temperature and luminosity, to produce a plant.
  • the exogenously introduced DNA or nucleic acid in a transformed plant can be confirmed by known methods, such as PCR or Southern hybridization, or by analyzing the nucleotide sequence of the plant's nucleic acid.
  • known methods such as PCR or Southern hybridization, or by analyzing the nucleotide sequence of the plant's nucleic acid.
  • To extract DNA or nucleic acid from a transformed plant the known method of J. Sambrook et al . may be used (Molecular Cloning, 2nd edition, Cold Spring Harbor laboratory Press, 1989) .
  • an amplification reaction is carried out using, as a template, nucleic acid extracted from the regenerated plant.
  • Amplification reaction may be carried out in a reaction mixture containing, as primers, synthesized oligonucleotides comprising nucleotide sequences appropriately selected according to the nucleotide sequence of a DNA of the disclosure.
  • An amplified DNA fragment comprising a DNA sequence of the disclosure may be obtained by repeating several dozen cycles of the denaturation, annealing, and extension steps of the DNA amplification reaction.
  • the respective amplified DNA fragments can be separated by, for example, electrophoresing the reaction solution containing the amplified products on agarose gel. DNA fragments corresponding to a DNA of the disclosure can then be confirmed.
  • a stable supply of biomass mainly cellulose
  • biomass can be provided by cultivating a transgenic plant of the disclosure on a larger scale using clone planting.
  • fossil resources are used in large amounts in industrial productions as raw materials and fuel (energy) .
  • energy energy
  • a more effective approach would be possible by converting the biomass into a more user-friendly form (such as alcohol, and specifically ethyl alcohol) .
  • One of the objectives is to use gasoline mixed with ethanol refined from biomass.
  • a specific example is "gasohol" (a 10% blend of ethanol in gasoline) made from corn.
  • Arabidopsis thaliana ecotype Columbia-0 (CoI-O) was used. Before plating seeds were surface sterilized. First, the seeds were washed in 95% ethanol for 10 minutes, which was removed then the sterilization solution was added (20% bleach, 0.05% Tween- 20 (Sigma) and double distilled water) and shaken for 10 minutes. The sterilization solution was removed and the seeds were washed three times with sterile distilled water. The seeds were cold treated for 4 days at 4°C after plating them on the plates. Two different growth media were prepared for these experiments.
  • mutants from the Arabidopsis Biological Resource Center (ARBC) , they were first plated onto MS media containing 50 ⁇ g/ml Kanamycin to select for insertions containing the NTPII (Kanamycin resistance) gene. In the case this resistance is lost the seeds were also sown onto regular MS media containing no antibiotic and then transferred to soil after one week. Plants were then grown on soil for 2-3 weeks after transfer from the plates and they were examined for status of the T-DNA insertion. Only homozygous T 3 and T 4 knockout mutants were used for the following experiments.
  • ARBC Arabidopsis Biological Resource Center
  • RIKEN cDNA clone (pdaO6938, SRF7) was used as a template for polymerase chain reaction (PCR) amplification of extracellular and transmembrane portion of the receptor of SRF7.
  • PCR product was gel eluted using Qiagen's QIAquick gel extraction kit using the manufacturer's protocol .
  • GGAAGTCGACCGAGAGAGATAGAGAAAGTGAGACAAGG-3' SEQ ID NO: 27
  • DN-SRF7 REV(Notl): 5'-ATATGCGGCCGCCCTTCACCGAGAAGATTATCTACGCTG-3' SEQ ID NO: 28
  • Eluted DNA was subsequently ligated into Promega's pGEM- Teasy PCR vector. Gene fragment inserts were confirmed by DNA sequencing using the T7 and S6 sites in the pGEM-Teasy vector. Confirmed vectors were then restriction digested using the PCR introduced restriction sites (Sail and Notl) . The restriction digest was run on a 1% agarose (Invitrogen) gel and the cut insert was removed using the QIAquick kit. The fragment was then ligated into a TAP tagged entry vector that was made my taking the pENTR-lA vector and introducing a 6x His and T7 epitope DNA sequence into the EcoRV restriction site in the pENTR-lA vector.
  • This vector was designated pENTR-TAP2.
  • the 3' ends of the PCR fragment was designed to go into frame with the TAP sequence.
  • the pENTR-TAP2 vector containing the SRF7 extracellular and transmembrane domain was then introduced into the final destination vector that contains the 35S promoter, pGWB2 (Invitrogen, Nakagawa) .
  • This construct was introduced into Arabidopsis (CoI-O) via the floral dip method (Bechtold et al . , 1993) .
  • SRF single knockout mutant selection The TAIR website was used to locate insertional mutants of SRF6 and SRF7. Two mutants were found for SRF6, SALK_077702 and SALK_035476, named srf6-1 and srf6-2 respectively. Three mutants were found for SRF7 : SALK_039120 (srf7-1), SALK_115238 (srf7-2) and SALK_110007 (srf7- 3) . Each T 4 SALK line was examined using PCR to confirm the insertion and genotype. Only homozygous mutants were used for further experiments. Primers for insertion detection were generated using the T-DNA primer design tool
  • GAGTGTACAATGCGTGAAGGG-3' (SEQ ID NO:37); SALK_110007 (srf7-3) RP: 5'-GCATGAAGTTTGCTCACCATC-3' (SEQ ID NO:38).
  • SRF overexpression construction Construction of the overexpression of full length SRF7 also utilized Gateway technology and was constructed in a similar manner as the dominant negative SRF mutant using the pENTR TAP2 entry vector and the pGWB2 35S binary vector.
  • RNA and Real-Time RT-PCR Analysis were collected from four-day-old dark grown seedlings using Qiagen' s RNeasy Kit following the manufacture's protocol. Two hundred nanograms of total RNA was used in a reverse transcriptase (Superscript II, Invitrogen) reaction in a 20 ⁇ l reaction volume. The cDNA was subsequently diluted to a concentration of 5ng/ ⁇ l and 5 ⁇ l (25ng cDNA) was used per each real-time reaction (25 ⁇ l total reaction volume: 0.125 ⁇ l each primer (10OpM) , 12.5 ⁇ l Bio-Rad SYBR green master mix, and sterile/DEPC ddH 2 O) . Primers for real-time PCR were designed in all circumstances to span an intron and to be a final size of 300 base pairs (+/- 10 base pairs) .
  • CESA3 At5g05170 FWD: 5'-ATTGTTCCGCAGACTTGCCAG-3' (SEQ ID NO:43); CESA3 (At5g05170) REV: 5'-CACGAGTAAGATGCCAACCAAGC-3' (SEQ ID NO:44); CESA4
  • the real-time PCR protocol was: 95°C for 5 minutes, followed by 40 cycles of 95°C for 45 seconds and 60 0 C for 45 seconds with the fluorescence quantification at the end of every 60 0 C step.
  • the fold change was found using the delta delta C t method using the ACTIN2 (At3gl8780) gene expression as the control for relative gene expression values.
  • FT-IR Fourier Transform Infrared
  • Plant material for SEM imaging by embedding the etiolated hypocotyls in tissue embedding medium in the cryostat and then sectioning away material until the approximate center of the hypocotyl is reached and then viewed at 100-1,00OX on the SEM (Hitachi TM-1000).
  • This pellet was then prepared for drying by adding 35ml acetone and resuspending the pellet. This was centrifuged for 10 minutes and the process was repeated for a total of three washes. On the final wash the cap is removed and the vial is covered with a single layer of Kimwipe that was kept in place with a rubber band. The vial was then placed into a vacuum desiccator without desiccant and attached to a vacuum line and vacuum was applied for around 10 hours. After the initial drying the samples were moved to a vacuum desiccator with desiccant and a vacuum was applied for 24 hours.
  • the sugar standards were prepared.
  • the sugar standards consist of 10OnM of each sugar with 10OnM inositol added to also act as an internal control. Then the samples and standards were removed from the N 2 stream and placed in a vacuum desiccator until completely dry.
  • sample contained amino sugars then 20 ⁇ l of methanol was added followed by 20 ⁇ l of pyridine and 20 ⁇ l acetic anhydride and the vials sat at room temperature for 15 minutes. The samples were then evaporated under a N 2 stream until dry. The to all samples 30 ⁇ l of trimethylsilylating reagent was added and allowed let to sit for 15 minutes at room temperature. Then the samples were again evaporated under N 2 but not for more then a few minutes as it may drive off some of the more volatile sugars like arabinose . Finally, 200-250 ⁇ l of isooctane is added to the sample and they are now ready to be injected (l ⁇ l) into the gas chromatograph .
  • the first Excel spreadsheet calculates the glycosyl composition (mole percent) of a specimen based on the integrated areas of the sugar peaks in the gas chromatogram of the specimen and the gas chromatogram of a mixture of standard sugars (100 nmoles each), with all peak areas referenced to the area of inositol (100 nmoles), an internal standard added to all specimens.
  • the second Excel spreadsheet combines the glycosyl composition results from the analyses of two whole cell wall specimens, one prepared by sulfuric acid preswelling and hydrolysis prior to methanolysis, and one prepared with methanolysis alone. The resulting combined glycosyl composition shows the additional amounts of sugars detected due to the sulfuric acid cleavage. These additional sugar amounts are predominantly glucose, plus a much smaller amount of mannose, which was derived from the cleavage of cellulose and tightly associated polymers that due to the near crystalline structure of cellulose were resistant to cleavage by methanolysis alone.
  • BRIl was used as a well known diurnally expressed RLK
  • SERKl was used as a control RLK that does not show a diurnal fluctuation
  • SRF3 is a subfamily member related to SRF6 and SRF7 that were both being examined for diurnal fluctuations.
  • These genes were also examined for their expression levels when exposed to a primary cellulose synthesis inhibitor called isoxaben. All ten cellulose synthase A genes (CesAl-10) were also examined for gene expression during diurnal cycle and for isoxaben treatment.
  • SRF6 and SRF7 are co-expressed with cellulose synthesis genes and their proteins have homologues in diverse plant species .
  • Table 1 Top ten genes coexpressed with SRF6 and SRF7. Using the ATTED II website (Arabidopsis thaliana trans-factor and cis-element prediction database II, (June 2007) http: ⁇ www.atted.bio.titech.ac.jp) genes coexpressed with SRF6
  • SRF7 shows coexpression with all of the primary cell wall cellulose synthases while SRF6 is coexpressed with arabinogalactan proteins, which associated with cell wall and coexpressed with ROP2, which also may be involved with cell wall organization or signaling.
  • Table 2 Many species of land plants and algae contain homologues to SRF6 and SRF7 supporting their role as important genes for cell wall development and cellulose regulation. Homologous genes to SRF7 can be found in many other land plants. There is a high amount of protein identity to Isatis tinctoria a member if the Brassicaceae family, a close relative to Arabidopsis. The lowest identity is to the more primitive land plant like the Liver Wort and the aquatic algae. Information was gathered from the TAIR database using the protein sequence of SRF7 to BLAST the protein database of green plants .
  • Figure IA shows the insertion location of the SRF mutants obtained and studied in this experiment.
  • Both srf6- 1 and srf7-1 were found to be null mutations by quantitative real- time RT-PCR using gene specific primers. No expression was found for these two genes.
  • Expression of SRF6 in srf6-2 is like that of wild type plants, as is SRF7 gene expression levels in the srf7-2 mutants.
  • the insertion being after the kinase domain, shows a four-fold increase in SRF7 gene expression level compared to the wild type plant ( Figure 5) .
  • FIG. 1 Normal hypocotyl lengths in the srf7-1 mutation were restored by overexpressing the SRF7 gene; this shows that SRF7 being knocked out is responsible for the reduced hypocotyl length of srf7-1 ( Figure 1) .
  • the srf6-2 mutant grows to a similar length as the wild type in the dark ( Figure IB) , but shows an increase in sensitivity to isoxaben (p ⁇ 0.05) ( Figure 2) .
  • srf6-2 also has no change in CesA3 or CesA4 gene expression ( Figures 6 and 7) .
  • the srf7-2 mutant dark grown hypocotyl is similar to the wild type
  • Isoxaben is a specific inhibitor of cellulose synthesis by affecting the function of the CesA proteins involved in primary cell wall synthesis, such as CesA3 and CesA6 (Scheible et al., 2001; Persson et al . , 2007) . Because SRF7 in particular is co- expressed with these same CesA genes the effects of isoxaben on SRF mutants was examined. The mutants with the insertion in the extracellular domain (srf6-l (SEQ ID NO: 53) and srf7-1) were significantly more resistant to isoxaben up to lO ⁇ M (p ⁇ 0.05)
  • the primary cell wall synthesis gene, CesA3, expression for mutants of SRF6 was about the same as wild type ( Figure 6) .
  • the extracellular domain insertion of SRF7, srf7- 1 showed a five-fold reduction in CesA3 gene expression ( Figure 4.6) while the other SRF7 mutations showed an increase in CesA3 gene expression: srf1-2 (2.45 fold), srf7-3 (1.59 fold) and SRF7
  • the procustel-1 (prcl-1) mutant was used in the experiment as a primary cell wall deficient control because it is a substitution mutation in the CesA6 cellulose synthase gene that along with CesAl and CesA3 are responsible for primary cell wall cellulose synthesis.
  • This mutant exhibits a short thick dark grown hypocotyl that ectopically accumulates lignin (Hematy et al, 2007) .
  • the murusl0-2 (murl0-2) mutant was used as secondary cell wall specific CesA control.
  • the murl0-2 mutation is a substitution mutation in CesA7, that along with CesA4 and CesA8 are responsible for secondary cell wall cellulose synthesis (Persson et al., 2007).
  • FT-IR microspectroscopy reveals differences in cellular composition of SRF mutants .
  • Fourier-Transform Infrared (FT-IR) mircospectroscopy was used as a means to determine the relative (to wild type) amounts of the cell wall components, pectin and cellulose, in the various SRF mutants. All plant materials were grown for 4-days in darkness on MS media without sucrose.
  • Monosaccharide analysis of DN-srf7 reveals differences in cell wall components. Utilizing the dominant negative SRF7 line the monosaccharide composition of the cell wall was examined. There are three principal fractions isolated in this experiment and they are: imidizole soluble, sodium hydroxide soluble, and the sodium hydroxide insoluble fraction. These respectively contain the pectin, hemicellulose and cellulose components of the initial cell wall. Table 4 has the molar percent (mol%) of the monsaccharides determined in each fraction for both the DN and wild type plants. Because cellulose is primarily made from beta linked glucoses

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Abstract

La présente invention concerne des procédés et des compositions pour modifier génétiquement la biosynthèse de cellulose.
PCT/US2009/065766 2008-11-24 2009-11-24 Compositions et procédés pour augmenter la production de cellulose Ceased WO2010060096A2 (fr)

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