WO2005017114A2 - Procede permettant d'ameliorer le metabolisme et la tolerance au stress d'une plante - Google Patents

Procede permettant d'ameliorer le metabolisme et la tolerance au stress d'une plante Download PDF

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WO2005017114A2
WO2005017114A2 PCT/US2004/026159 US2004026159W WO2005017114A2 WO 2005017114 A2 WO2005017114 A2 WO 2005017114A2 US 2004026159 W US2004026159 W US 2004026159W WO 2005017114 A2 WO2005017114 A2 WO 2005017114A2
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seq
plant
promoter
plants
dna
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WO2005017114B1 (fr
WO2005017114A3 (fr
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Gary J. Lee
Jindong Sun
Daniel J. Tennessen
Jingrui Wu
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Monsanto Technology Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • circadian rhythms can be set by light and dark transition, but these rhythms can persist even in the absence of such external time cues.
  • the importance of such rhythms in plant development was long recognized, however no circadian clock components were recognized until the recent past (Wang and Tobin, Cell 98: 1207- 1217, 1997).
  • Current work in this area is directed towards the understanding of circadian clocks as a function of gene or genes.
  • One such gene is Circadian Clock Associated-1 (CCAl).
  • CCAl encodes a MYB -related transcription factor protein involved in the phytochrome induction of a light-harvesting chlorophyll a/b-protein (Lhcb) gene.
  • the CCAl transcript In its native state, the CCAl transcript is transiently induced by phytochrome and oscillates with a circadian rhythm. It has been shown that overexpression of the CCAl protein in transgenic Arabidopsis plants abolished the circadian rhythm of other genes with dramatically different phases. As evidenced by the fact that CCAl protein level was similar to peak levels in wild-type plants, it was determined that overexpression of CCAl in Arabidopsis alters the circadian rhythm of other genes (such as Lhcbl*l, CCR2 and CAT3) leading to late flowering and elongated hypocotyls in Arabidopsis plants.
  • other genes such as Lhcbl*l, CCR2 and CAT3
  • Described herein is a method of providing an improved or new agriculturally desirable trait to a plant by ectopically expressing a transcription factor capable of providing one or more of such traits to the plant.
  • the transcription factor is the CCAl protein as described herein, its homologs, orthologs, analogs or variants thereof and such protein is expressed in the plant at least during its vegetative growth phases.
  • Another embodiment of the present invention provides a method for improving the agronomic characteristics of a plant by transforming a plant cell with a plant expressible DNA construct comprising a DNA polynucleotide that encodes a protein selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18, or a protein substantially homologous thereto to obtain a transformed plant cell and regenerating such transformed plant cell into a transgenic plant containing the DNA construct.
  • a DNA polynucleotide that encodes a protein selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18, or a protein substantially homologous thereto to obtain a transformed plant cell and
  • transgenic plants are then screened for the desired agronomic characteristics as compared to a plant of the same species as said transgenic plant that does not contain said DNA construct and a transgenic plant containing at least one or more of said improved agronomic characteristics is selected.
  • the DNA construct comprising a DNA molecule that encodes a protein substantially homologous to a protein selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18, may be inserted into a plant cell and used to produce a fertile transgenic plant containing the DNA molecule; wherein the fertile transgenic plant exhibits at least one of the traits selected from the group consisting of a higher rate of photosynthesis, a higher rate of higher carbon assimilation , and increased tolerance to environmental stress, as compared to a plant of a same plant species not transformed to contain the said DNA molecule.
  • a DNA construct contains a promoter that is plant promoter, wherein said promoter drives transcription of an operably linked DNA molecule selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19 or DNA molecules substantially homologous thereto.
  • transgenic plant, seed and/or hybrid seed containing a DNA molecule of the present invention are provided, wherein the transgenic plant, seed or hybrid seed exhibit at least one of the traits of a higher rate of • photosynthesis, higher carbon assimilation, and increased tolerance to environmental stress, particularly drought stress.
  • the present invention also provides a transformed plant comprising in its genome a polynucleic acid molecule with a 5' non-coding DNA sequence that functions in the cell to cause the production of an mRNA molecule; and that is operably linked to a structural polynucleotide molecule isolated from a plant, wherein .the structural polynucleotide molecule encodes a polypeptide with an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to a member selected from group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18; and that is operably linked to a 3' non-translated DNA molecule that functions in said cell to cause termination of transcription.
  • Figure 1 shows a plasmid map for plant transformation vector pMON 10098.
  • Figure 2 shows a plasmid map for plant transformation vector pMON CCA-1.
  • Figure 3 shows a plasmid map for plant transformation vector pMON 67289.
  • Figure 4 shows a plasmid map for plant transformation vector pMON 67293.
  • Figure 5 shows a plasmid map for plant transformation vector pMON 57231.
  • Figure 6 shows a plasmid map for plant transformation vector pMON 56622.
  • Figure 7 shows a plasmid map for plant transformation vector pMON 67278.
  • Figure 8 shows a plasmid map for plant transformation vector pMON 67294.
  • Figure 9 shows a plasmid map for plant transformation vector pMON 67288.
  • Figure 10 shows a plasmid map for plant transformation vector pMON 67292.
  • Figure 11 shows a plasmid map for plant transformation vector pMON 41124.
  • Figure 12 shows a plasmid map for plant transformation vector PCGN 10947.
  • Figure 13 shows a plasmid map for plant transformation vector pMON 73975.
  • Figure 14 shows a plasmid map for plant transformation vector pMON 73979.
  • Figure 15 shows a plasmid map for plant transformation vector pMON 41162.
  • Figure 16 shows a plasmid map for plant transformation vector pMON 67250.
  • Figure 17 shows a plasmid map for plant transformation vector pMON 67299.
  • Figure 18 shows a plasmid map for plant transformation vector pMON 65154.
  • Figure 19 shows a plasmid map for plant transformation vector pMON 72469.
  • Figure 20 shows a plasmid map for plant transformation vector pMON 72472.
  • Figure 21 shows a
  • transgenic plants expressing the transcription factor CCA-1 or DNA molecules substantially homologous thereto provide a higher rate of photosynthesis, increased carbon assimilation and/or increased tolerance to stress to the plant and thereby lead to increased yield of the plant, particularly grain yield.
  • This property can also be used to enhance the quality and quantity of forage production of forage plants by regulating the ratio of vegetative to reproductive growth in a manner to increase the amount of vegetative biomass accumulate by the plant.
  • the present invention is based, in part, on the identification of polynucleic acid molecules encoding polypeptides of the present invention from crop plants including maize, soybean, rice, canola, cotton, alfalfa and wheat and utilizing these molecules to improve agronomic characteristics of plants such as elevated rate of photosynthesis, increased metabolite content and increased stress tolerance by expression of polypeptides of the invention leading to enhancement in yield.
  • One aspect of the present invention relates to isolated polynucleic acid molecules comprising a nucleotide sequence or complement thereof that encode a transcription factor or CCA-1 like proteins. These proteins when expressed in a plant can impart a significant yield increase characteristic to the plant when grown under field conditions.
  • Polynucleotide or polypeptide molecules of the invention are plant transcription factors having CCA-1 like properties and their homologs, orthologs or paralogs. These molecules are identified by comparing SEQ ID NO:l or SEQ ID NO:2 with other nucleic acid or polypeptide sequences of plant cDNA molecules and developing an evolutionary relationship among them. Nucleic acid analysis to identify homologs, orthologs or paralogs and building phylogenetic trees there from is well known in the art.
  • these molecules will encode a polypeptide from a crop plant having an amino acid sequence that has at least 70% sequence identity, or at least 75% or 80% sequence identity, or at least 85% or 90% sequence identity, or at least 95% sequence identity, or at least 98% sequence identity to a member selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.
  • transgenic plants and seeds have incorporated into their genome, or transformed into their chloroplast or plastid genomes, a polynucleic acid molecule that comprises at least a structural nucleotide sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.
  • polynucleic acid molecule as used herein means a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule.
  • Both DNA and RNA molecules are constructed from nucleotides linked end to end, wherein each of the nucleotides contains a phosphate group, a sugar moiety, and either a purine or a pyrimidine base.
  • Polynucleic acid molecules can be single or double-stranded polymers of nucleotides read from the 5' to the 3' end. Polynucleic acid molecules may also optionally contain synthetic, non-natural or altered nucleotide bases that permit correct read through by a polymerase and do not alter expression of a polypeptide encoded by that polynucleic acid molecule.
  • an isolated polynucleic acid molecule means a polynucleic acid molecule that is no longer accompanied by those materials with which it is associated in its natural state, or to a polynucleic acid molecule for which the structure of which is not identical to that of any of naturally occurring polynucleic acid molecule. It is also contemplated by the inventors that the isolated polynucleic acid molecules of the present invention also include known types of modifications.
  • nucleotide sequence as used herein means the linear arrangement of nucleotides to form a polynucleotide of the sense and complementary strands of a polynucleic acid molecule as either individual single strands or in the duplex.
  • a coding sequence and "a structural polynucleotide molecule” mean a polynucleotide molecule that is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory molecules. The boundaries of the coding sequence are determined by a translation start codon at the 5 '-terminus and a translation stop codon at the 3'- terminus.
  • a coding sequence can include, but is not limited to, genomic DNA, cDNA, and recombinant polynucleotide sequences.
  • recombinant DNAs as used herein means DNAs that contains a genetically engineered modification through manipulation via mutagenesis, restriction enzymes, and the like.
  • synthetic DNAs as used herein means DNAs assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art.
  • polypeptide and protein as used herein, mean a polymer composed of amino acids connected by peptide bonds. An amino acid unit in a polypeptide (or protein) is called a residue.
  • polypeptide and protein also applies to any amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to any naturally occurring amino acid polymers.
  • amino acid sequence means the sequence of amino acids in a polypeptide (or protein) that is written starting with the amino-terminal (N-terminal) residue and ending with the carboxyl-terminal (C-terminal) residue.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (that does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical when used as reference to two polypeptide sequences or two polynucleotide sequences, means that one polypeptide sequence or one polynucleotide sequence has at least 70% sequence identity compared to the other polypeptide sequence or polynucleotide sequence as a reference sequence using the Gap program in the WISCONSIN PACKAGE version 10.0-UNIX from Genetics Computer Group, Inc. based on the method of Needleman and Wunsch (J. Mol. Biol.
  • Polypeptides that are "substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • Constant amino acid substitutions mean substitutions of one or more amino acids in a native amino acid sequence with another amino acid(s) having similar side chains, resulting in a silent change. conserveed substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
  • phenylalanine-tyrosine phenylalanine-tyrosine
  • lysine-arginine alanine-valine
  • aspartic acid-glutamic acid and asparagine-glutamine.
  • Substantial identity of nucleotide sequences for these purposes normally means sequence identity of at least 70%.
  • codon degeneracy means divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide.
  • the skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for ectopic expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of codon usage of the host cell as observed in a codon usage table.
  • the polynucleic acid molecules encoding a polypeptide of the present invention may be combined with other non-native, or "heterologous" sequences in a variety of ways.
  • heterologous sequences it is meant any sequence that is not naturally found joined to the nucleotide sequence encoding polypeptide of the present invention, including, for example, combinations of nucleotide sequences from the same plant that are not naturally found joined together, or the two sequences originate from two different species.
  • operably linked as used in reference to a regulatory molecule and a structural polynucleotide molecule, means that the regulatory molecule causes regulated expression of the operably linked structural polynucleotide molecule.
  • “Expression” means the transcription and stable accumulation of sense or antisense RNA derived from the polynucleic acid molecule of the present invention. Expression may also refer to translation of mRNA into a polypeptide.
  • RNA transcript means RNA transcript that includes the mRNA and so can be translated into polypeptide or protein by the cell.
  • Antisense RNA means a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Patent No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-translated sequence, introns, or the coding sequence.
  • RNA transcript means the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence.
  • RNA transcript When- the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post- transcriptional processing of the primary transcript and is referred to as the mature RNA. It is understood that to practice the present invention it is essential to introduce the selected polynucleotide molecule in a form that is capable of producing an active polypeptide molecule in a desired plant. Exogenous polynucleic acid molecules are transferred into a crop plant cell by use of a recombinant DNA construct (or vector) designed for such purpose.
  • the DNA construct of the present invention can, in one embodiment, contain a promoter which causes the over expression of the polypeptide of the present invention, where "overexpression” means the expression of a polypeptide either not normally present in the host cell, or present in said host cell at a higher level than that normally expressed from the endogenous gene encoding said polypeptide.
  • Promoters which can cause the overexpression of the polypeptide of the present invention, are generally known in the art.
  • the DNA construct of the present invention can, in another embodiment, contain a promoter which causes the ectopic expression of the polypeptide of the invention, where "ectopic expression” means the expression of a polypeptide in a cell type other than a cell type in which the polypeptide is normally expressed; at a time other than a time at which the polypeptide is normally expressed; or at a expression level other than the level at which the polypeptide normally is expressed.
  • Promoters which can cause ectopic expression of the polypeptide of the present invention, are generally known in the art.
  • the expression level or pattern of the promoter of the DNA construct of the present invention may be modified to enhance its expression.
  • enhancing elements for example, sub-domains of the CaMV 35S promoter, Benfey et. al, 1990 EMBO J. 9: 1677-1684
  • enhancing elements may be added to create a promoter, which encompasses the temporal and spatial expression of the native promoter of the gene of the present invention, but have quantitatively higher levels of expression.
  • tissue specific expression of the promoter can be accomplished through modifications of the 5' region of the promoter with elements determined to specifically activate or repress gene expression (for example, pollen specific elements, Eyal et al, 1995 Plant Cell 7: 373-384).
  • the isolated polynucleic acid molecules of the present invention can also be used for gene suppression e.g. in antisense or RNA technology to suppress endogenous native gene expression.
  • a polynucleic acid molecule derived from a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19 is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The construct is then transformed into plants and the antisense strand of RNA is produced.
  • the polynucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous gene or genes of the present invention to be repressed.
  • the polynucleic acid sequence need not be perfectly identical to inhibit expression.
  • the recombinant vectors of the present invention can be designed such that the inhibitory effect applies to other genes within a family of genes exhibiting homology or substantial homology to the target gene.
  • the term "a gene" means the segment of DNA that is involved in producing a polypeptide. Such segment of DNA may include regulatory molecules preceding (5' non-coding DNA molecules) and following (3' non-coding DNA molecules) the coding region, as well as intervening sequences (introns) between individual coding segments (exons).
  • a “native gene” means a gene as found in nature with its own regulatory DNA sequences.
  • “Chimeric gene” means any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • “Endogenous gene” means a native gene in its natural location in the genome of an organism.
  • a “foreign gene” means a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure resulting in a transgenic organism.
  • "Regulatory sequences” means polynucleotide molecules located upstream (5' non-coding sequences), within, or downstream (3' non-translated sequences) of a structural polynucleotide sequence, and that influence the transcription, RNA processing or stability, or translation of the associated structural polynucleotide sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • promoter sequence means a polynucleotide molecule that is capable of, when located in cis to a structural polynucleotide sequence encoding a polypeptide, functions in a way that directs expression of one or more mRNA molecules that encodes the polypeptide.
  • promoter regions are typically found upstream of the trinucleotide, ATG, at the start site of a polypeptide coding region.
  • Promoter molecules can also include DNA sequences from which transcription of transfer RNA (tRNA) or ribosomal RNA (rRNA) sequences are initiated. .
  • the promoter sequence typically consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an "enhancer” is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
  • Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that are known or are found to cause transcription of DNA in plant cells can be used in the present invention. Such promoters may be obtained from a variety of sources such as plants and plant viruses.
  • promoters including constitutive promoters, inducible promoters and tissue-specific promoters, that are active in plant cells have been described in the literature. It is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of a polypeptide to cause the desired phenotype.
  • promoters that are known to cause transcription of DNA in plant cells
  • other promoters may be identified for use in the current invention by screening a plant cDNA library for genes that are selectively or preferably expressed in the target tissues and then determine the promoter regions.
  • the term "constitutive promoter" means a regulatory sequence that causes expression of a structural nucleotide sequence in most cells or tissues at most times.
  • Constitutive promoters are active under most environmental conditions and states of development or cell differentiation.
  • a variety of constitutive promoters are well known in the art. Examples of constitutive promoters that are active in plant cells include but are not limited to the nopaline synthase (NOS) promoters; the cauliflower mosaic virus (P-CaMV) 19S and 35S (U.S. Patent No. 5,858,642); the figwort mosaic virus promoter (P-FMV, U.S. Patent No. 6,051,753); and actin promoters, such as the rice actin promoter (P-Os.Actl, U.S. Patent No. 5,641,876).
  • NOS nopaline synthase
  • P-CaMV cauliflower mosaic virus
  • U.S. Patent No. 5,858,642 the cauliflower mosaic virus
  • P-FMV figwort mosaic virus promoter
  • actin promoters such as the rice actin promoter
  • inducible promoter means a regulatory sequence that causes conditional expression of a structural nucleotide sequence under the influence of changing environmental conditions or developmental conditions.
  • inducible promoters include but are not limited to the light-inducible promoter from the small subunit of ribulose- 1,5 -bis-phosphate carboxylase (ssRUBISCO); the drought-inducible promoter of maize (Busk et al., Plant J. 11 : 1285-1295, 1997), the cold, drought, and high salt inducible promoter from potato (Kirch, Plant Mol. Biol.
  • tissue-specific promoter means a regulatory sequence that causes transcriptions or enhanced transcriptions of DNA in specific cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues.
  • tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots, leaves or stems, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistils, flowers, or any embryonic tissue.
  • Reproductive tissue specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific, pollen-specific, petal-specific, sepal-specific, or some combination thereof.
  • tissue-specific promoter may drive expression of operably linked DNA molecules in tissues other than the target tissue.
  • a tissue-specific promoter is one that . drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.
  • a variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers, can be used to express the polynucleic acid molecules of the present invention.
  • tuber-specific promoters include, but are not limited to the class I and II patatin promoters (Bevan et al, EMBO J. 8:1899-1906, 1986; Koster-Topfer et al., Mol Gen Genet. 219:390-396, 1989; Mignery et al., Gene. 62:27-44, 1988; Jefferson et al., Plant Mol. Biol. 14: 995-1006, 1990), the promoter for the potato tuber ADPGPP genes, both the large and small subunits; the sucrose synthase promoter (Salanoubat and Belliard, Gene. 60:47-56, 1987; Salanoubat and Belliard, Gene.
  • class I and II patatin promoters Bevan et al, EMBO J. 8:1899-1906, 1986; Koster-Topfer et al., Mol Gen Genet. 219:390-396, 1989; Mignery et al., Gene. 62:27-44,
  • leaf-specific promoters include but are not limited to the ribulose biphosphate carboxylase (RBCS or RuBISCO) promoters (see, e.g., Matsuoka et al., Plant J. 6:311-319, 1994), the light harvesting chlorophyll a/b binding protein gene promoter (see, e.g., Shiina et al., Plant Physiol. 115:477- 483, 1997; Casal et al., Plant Physiol.
  • RBCS or RuBISCO ribulose biphosphate carboxylase
  • Root-specific promoter examples include, but are not limited to the promoter for the acid chitinase gene (Samac et al., Plant Mol. Biol. 25:587-596, 1994), the root specific subdomains of the CaMV35S promoter that have been identified (Lam et al., Proc. Natl. Acad. Sci.
  • the "SHOOTMERISTEMLESS” and “SCARECROW” promoters which are active in the developing shoot or root apical meristems can be used (Di Laurenzio et al., Cell 86:423- 433, 1996; Long, Nature 379:66-69, 1996).
  • Another example of a useful promoter is that which controls the expression of 3-hydroxy-3- methylglutaryl coenzyme A reductase HMG2 gene, whose expression is restricted to meristematic and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto et al., Plant Cell.
  • a useful promoter is that which controls the expression of knl-related genes from maize and other species that show meristem-specific expression (see, e.g., Granger et al., Plant Mol. Biol. 31:373-378, 1996; Kerstetter et al., Plant Cell 6:1877-1887, 1994; Hake et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45-51, 1995).
  • Another example of a meristematic promoter is the Arabidopsis thaliana KNATl promoter.
  • KNATl transcript is localized primarily to the shoot apical meristem; the expression of KNATl in the shoot meristem decreases during the floral transition and is restricted to the cortex of the inflorescence stem (see, e.g., Lincoln et al., Plant Cell 6:1859-1876, 1994).
  • Suitable seed-specific promoters can be derived from the following genes: MAC1 from maize (Sheridan et al., Genetics 142:1009-1020, 1996; Cat3 from maize (GenBank No. L05934, Abler et al., Plant Mol. Biol. 22:10131-1038, 1993; vivparous-1 from Arabidopsis (Genbank No.
  • the egg and central cell specific MEA (FIS1) and FIS2 promoters are also useful reproductive tissue-specific promoters (Luo et al., Proc. Natl. Acad. Sci. USA, 97:10637-10642, 2000; Dahlle-Calzada, et al., Genes Dev. 13:2971-2982, 1999).
  • a maize pollen-specific promoter has been identified in maize (Guerrero et al., Mol. Gen. Genet. 224:161-168, 1990).
  • Other genes specifically expressed in pollen have been described (see, e.g., Wakeley et al., Plant Mol. Biol. 37:187-192, 1998; Ficker et al., Mol. Gen.
  • Promoters derived from genes encoding embryonic storage proteins which includes the gene encoding the 2S storage protein from Brassica napus (Dasgupta et al., Gene 133:301-302, 1993); the 2s seed storage protein gene family from Arabidopsis; the gene encoding oleosin 20kD from Brassica napus (GenBank No. M63985); the genes encoding oleosin A (GenBank No. U09118) and oleosin B (GenBank No.
  • Promoters derived from zein encoding genes can be also used.
  • the zeins are a group of storage proteins found in maize endosperm.
  • Native promoters derived from genes of present invention or promoters from genes of the same biochemical or developmental pathway, which can express the polypeptides of the present invention in a temporal and spatial manner can also be used to practice the present invention. It is recognized that additional promoters that may be utilized are described, for example, in U.S. Patent Nos.
  • tissue specific enhancer may be used (Fromm et al., The Plant Cell 1:977-984, 1989). It is further recognized that the exact boundaries of regulatory sequences may not be completely defined; DNA fragments of different lengths may have identical promoter activity.
  • the construct of the present invention may also contain a "translation leader sequence.” As used herein, "translation leader sequence” means a DNA sequence located between the promoter sequence of a gene and the coding sequence.
  • the translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence.
  • the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
  • Examples of translation leader sequences include maize and petunia heat shock protein leaders, plant virus coat protein leaders, plant rubisco gene leaders among others (Turner and Foster, Molecular Biotechnology 3:225, 1995).
  • a further feature of a construct of the present invention is a "3' non-translated sequences" or "3' termination region.”
  • a "3' non-translated sequence" or “3' termination region” means DNA sequences located downstream of a structural nucleotide sequence and include sequences encoding polyadenylation and other regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal functions in plants to cause the addition of polyadenylate nucleotides to the 3' end of the mRNA precursor.
  • the polyadenylation sequence can be derived from the natural gene, from a variety of plant genes, or from T-DNA.
  • An example of the polyadenylation sequence is the nopaline synthase 3' sequence (nos 3'; Fraley et al., Proc. Natl. Acad. Sci. USA 80: 4803-4807, 1983).
  • the use of different 3' non-translated sequences is exemplified by Ingelbrecht et al. (Plant Cell 1:671-680, 1989).
  • the laboratory procedures in recombinant DNA technology used herein are those well known and commonly employed in the art.
  • Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloning - A Laboratory Manual, 2nd. ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), herein referred to as Sambrook et al., (1989).
  • a "substantial portion" of a polynucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a polynucleic acid molecule comprising the sequence.
  • Polynucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. J Mol. Biol. 215:403-410, 1993; see also www.ncbi.nlm.nih.gov/BLAST/).
  • BLAST Basic Local Alignment Search Tool
  • Altschul et al. J Mol. Biol. 215:403-410, 1993; see also www.ncbi.nlm.nih.gov/BLAST/ a sequence of thirty or more contiguous nucleotides is necessary in order to putatively identify a nucleotide sequence as homologous to a gene.
  • gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern ,. • hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
  • short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular polynucleic acid molecule comprising the primers.
  • the skilled artisan having the benefit of the polynucleic acid molecules as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art.
  • the instant invention comprises the complete polynucleotide sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
  • Isolation of polynucleic acid molecules encoding homologous polypeptides using polynucleotide sequence-dependent protocols is well known in the art.
  • Examples of polynucleotide sequence-dependent protocols include, but are not limited to, methods of polynucleic acid molecule hybridization, and methods of DNA and RNA amplification as exemplified by various uses of polynucleic acid molecule amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
  • structural polynucleic acid molecules encoding additional polypeptides of the present invention could be isolated directly by using all or a substantial portion of the polynucleic acid molecules of the present invention as DNA hybridization probes to screen cDNA or genomic libraries from any desired plant employing methodology well known to those skilled in the art. Methods for forming such libraries are well known in the art. Specific oligonucleotide probes based upon the polynucleic acid molecules of the present invention can be designed and synthesized by methods known in the art.
  • the entire sequences of the polynucleic acid molecules can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems.
  • specific primers can be designed and used to amplify a part or all of the sequences.
  • the resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length cDNA or genomic DNAs under conditions of appropriate stringency.
  • the polynucleic acid molecules of interest can be isolated from a mixture of polynucleic acid molecules using amplification techniques.
  • the disclosed polynucleic acid molecules may be used to define a pair of primers that can be used with the polymerase chain reaction (Mullis, et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273, 1986; EP 50,424; EP 84,796, EP 258,017, EP 237,362, EP 201,184; US 4,683,202; Erlich, US 4,582,788, and US 4,683,194, all of which are herein incorporated by reference in their entireties) to amplify and obtain any desired polynucleic acid molecule directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
  • PCR and other in vitro amplification methods may also be useful, for example, to clone nucleotide sequences that encode for polypeptides to be expressed, to make polynucleic acid molecules to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • two short segments of the polynucleic acid molecules of the present invention may be used in polymerase chain reaction protocols to amplify longer polynucleic acid molecules encoding homologues of a polypeptide of the invention from DNA or RNA.
  • the skilled artisan can follow the RACE protocol (Frohman et al., Proc. Natl. Acad. Sci.
  • Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin, Techniques 1:165, 1989).
  • Polynucleic acid molecules of interest may also be synthesized, either completely or in part, especially where it is desirable to provide modifications in the polynucleotide sequences, by well-known techniques as described in the technical literature, see, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983).
  • polynucleic acid molecules of the present invention may be synthesized using a codon usage table of a selected plant host. Other modifications of the coding gene sequences may result in mutants having slightly altered activity. All or a substantial portion of the polynucleic acid molecules of the present invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired pheno types. For example, the polynucleic acid molecules of the present invention may be used as restriction fragment length polymorphism (RFLP) markers.
  • RFLP restriction fragment length polymorphism
  • Southern blots (Sambrook et al., 1989) of restriction-digested plant genomic DNA may be probed with the polynucleic acid fragments of the present invention.
  • the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al., Genomics 1:174-181, 1987), in order to construct a genetic map.
  • the polynucleic acid fragments of the present invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross.
  • Polynucleic acid probes derived from the polynucleic acid molecules of the present invention may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al., In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346).
  • polynucleic acid probes derived from the polynucleic acid molecules of the present invention may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask, Trends Genet. 7:149-154, 1991).
  • FISH direct fluorescence in situ hybridization
  • a variety of polynucleic acid amplification-based methods of genetic and physical mapping may be carried out using the nucleotide molecules of the present invention. Examples include allele-specific amplification (Kazazian et al., J. Lab. Clin. Med. 11:95-96, 1989), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al., Genomics 16:325-332, 1993), allele-specific ligation (Landegren et al., Science 241:1077-1080, 1988), nucleotide extension reactions (Sokolov et al., Nucleic Acid Res.
  • Isolated polynucleic acid molecules of the present invention may find use in the identification of loss of function mutant phenotypes of a plant, due to a mutation in one or more endogenous genes encoding polypeptides of the present invention. This can be accomplished either by using targeted gene disruption protocols or by identifying specific mutants for these genes contained in a population of plants carrying mutations in all possible genes (Ballinger and Benzer, Proc. Natl. Acad Sci USA 86:9402-9406, 1989; Koes et al., Proc. Natl. Acad. Sci. USA 92:8149-8153, 1995; Bensen et al., Plant Cell 7:75-84, 1995; all of which are incorporated herein by reference in their entirety).
  • short segments of the polynucleic acid molecules of the present invention may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which mutator transposons or some other mutation-causing DNA element has been introduced. The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding polypeptides.
  • the polynucleic acid molecules of the present invention may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adapter.
  • polypeptides of the present invention may also include fusion polypeptides.
  • a polypeptide that comprises one or more additional polypeptide regions not derived from that polypeptide is a "fusion" polypeptide.
  • Such molecules may be derivatized to contain carbohydrate or other moieties (such as keyhole, limpet, hemocyanin, etc.). Fusion polypeptides of the present invention are preferably produced via recombinant means.
  • polypeptide molecules of the present invention may also include polypeptides encoded by all or a substantial portion of polypeptide-encoding sequences set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18 or complements thereof or, fragments or fusions thereof in which conservative, non-essential, or not relevant, amino acid residues have been added, replaced, or deleted.
  • An example of such a homologue is the homologue polypeptide (or protein) from different species. Such a homologue can be obtained by any of a variety of methods.
  • one or more of the disclosed sequences all or a substantial portion of a polypeptide-encoding sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18; and complements thereof will be used to define a pair of primers that may be used to isolate the homologue-encoding polynucleic acid molecules from any desired species. Such molecules can be expressed to yield homologues by recombinant means.
  • Polynucleic acid molecules that encode all or part of the polypeptides of the present invention can be expressed, via recombinant means, to yield polypeptides that can in turn be used to elicit antibodies that are capable of binding the expressed polypeptides. It may be desirable to derivatize the obtained antibodies, for example with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, a radioisotope or an enzyme). Such antibodies may be used in immunoassays for that polypeptide. In a preferred embodiment, such antibodies can be used to screen cDNA expression libraries to isolate full-length cDNA clones of the present invention (Lemer, Adv. Immunol.
  • the isolated polynucleic acid molecules of the present invention can find particular use in creating transgenic plants in which polypeptides of the present invention are overexpressed. Overexpression of these polypeptides in a plant can enhance plant stress tolerance and thereby lead to improvement in the yield of the plant. It will be particularly desirable to enhance plant drought and osmotic stress tolerance in crop plants that undergo such stresses over the course of a normal growing season.
  • transgenic plant means a plant that contains an exogenous polynucleic acid, which can be derived from the same plant species or from a different species.
  • exogenous it is meant that a polynucleic acid molecule originates from outside the plant that the polynucleic acid molecule is introduced.
  • An exogenous polynucleic acid molecule can have a naturally occurring or non-naturally occurring nucleotide sequence.
  • an exogenous polynucleic acid molecule can be a heterologous polynucleic acid molecule derived from a different plant species than the plant into which the polynucleic acid molecule is introduced or can be a polynucleic acid molecule derived from the same plant species as the plant into which it is introduced.
  • Crop plant cell includes without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
  • the term "genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components of the cell. DNAs of the present invention introduced into plant cells can therefore be either chromosomally integrated or organelle-localized.
  • the term "genome” as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell.
  • Encoding DNAs of the present invention introduced into bacterial host cells can therefore be either chromosomally integrated or plasmid-localized.
  • Exogenous polynucleic acid molecules may be transferred into a crop plant cell by the use of a recombinant DNA construct (or vector) designed for such a purpose.
  • the present invention also provides a plant recombinant DNA construct (or vector) for producing transgenic plants; wherein the plant recombinant DNA construct (or vector) comprises a structural nucleotide sequence encoding an polypeptide of the present invention. Methods that are well known to those skilled in the art may be used to prepare the crop plant recombinant DNA construct (or vector) of the present invention.
  • a plant recombinant DNA construct (or vector) of the present invention contains a structural nucleotide sequence encoding a polypeptide of the present invention and operably linked to regulatory sequences.
  • exemplary regulatory sequences include but are not limited to promoters, translation leader sequences, introns and 3' non-translated sequences.
  • the promoters can be constitutive, inducible, native, or tissue-specific promoters.
  • DNA constructs used for transforming plants to practice current invention comprises any promoter known to function to cause the transcription in plant cells and any antibiotic or herbicide tolerance encoding polynucleotide sequence known to confer antibiotic or herbicide tolerance to plant cells.
  • the antibiotic tolerance polynucleotide sequences include, but are not limited to polynucleotide sequences encoding for proteins involved in tolerance to kanamycin, neomycin, hygromycin, and other antibiotics known in the art.
  • Antibiotic tolerance gene in such a vector can be replaced by herbicide tolerance encoding for 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS, described in U.S. Patent Nos.
  • EPSPS 5- enolpyruvylshikimate-3-phosphate synthase
  • Herbicides for which transgenic plant tolerance has been demonstrated and the method of the present invention can be applied include, but are not limited to: glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, and isoxaslutole herbicides.
  • Genetic elements of transgene DNA constructs used for plant transformation and expression of transgenes in plants include, but are not limited to: plant virus promoters, e.g., P-CaMV.35S promoter (U.S. Patent No.
  • the genetic elements of the DNA construct further comprise 5' leader polynucleotides for example, the Hsp70 non-translated leader sequence from Petunia hybrida as described in U. S. Patent No. 5,362,865, herein incorporated by reference in its entirety.
  • the genetic elements further comprise herbicide tolerance genes that include, but are not limited to, for example, the aroA:CP4 coding region for EPSPS glyphosate resistant enzyme isolated from Agrobacterium tumefaciens (AGRTU) strain CP4 as described in U. S. Patent No. 5,633,435, herein incorporated by reference in its entirety.
  • the genetic elements of the DNA construct further comprise 3' termination regions that include, but are not limited to, the E9 3' termination region of the pea RbcS gene that functions as a polyadenylation signal; the nos3' is the 3' end of the Ti plasmid nopaline synthase gene that functions as a polyadenylation signal ; or the TML is 3' the end of the Ti plasmid octopine pTil5955 synthase gene (GenBank Accession AF 242881) that functions as a polyadenylation signal .
  • the genetic elements of the DNA construct further comprise the right border (RB) and left borders (LB) of the Ti plasmid of Agrobacterium tumefaciens octopine and nopaline strains.
  • a plant recombinant DNA construct (vector) of the present invention will typically comprise a selectable marker that confers a selectable phenotype on plant cells. Selectable markers may also be used to select for plants or plant cells that contain the exogenous polynucleic acid molecules encoding polypeptides of the present invention.
  • the marker may encode biocide resistance, antibiotic resistance (e.g, kanamycin, G418, bleomycin, hygromycin, etc.), or herbicide resistance (e.g., glyphosate, glufosinate, etc.).
  • antibiotic resistance e.g, kanamycin, G418, bleomycin, hygromycin, etc.
  • herbicide resistance e.g., glyphosate, glufosinate, etc.
  • selectable markers include, but are not limited to, a neo gene (Potrykus et al., Mol. Gen. Genet.
  • a plant recombinant DNA construct (vector) of the present invention may also include a screenable marker. Screenable markers may be used to monitor expression. Exemplary screenable markers include a ⁇ -glucuronidase or uidA gene (GUS:1) that encodes an enzyme for that various chromogenic substrates are known (Jefferson, Plant Mol. Biol, Rep.
  • tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983) that encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone that in turn condenses to melanin; an - galactosidase, that will turn a chromogenic ⁇ -galactose substrate.
  • selectable or screenable marker genes are also genes that encode a secretable marker whose secretion can be detected as a means of identifying or selecting for transformed cells.
  • Examples include markers that encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes that can be detected catalytically.
  • Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA, small active enzymes detectable in extracellular solution (e.g., ⁇ -amylase, ⁇ -lactamase, phosphinothricin transferase), or proteins that are inserted or trapped in the cell wall (such as proteins that include a leader sequence such as that found in the expression unit of extension or tobacco PR-S).
  • Other possible selectable and/or screenable marker genes will be apparent to those of skill in the art. In addition to a selectable marker, it may be desirable to use a reporter gene.
  • reporter gene may be used with or without a selectable marker.
  • Reporter genes are genes that are typically not present in the recipient organism or tissue and typically encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Wising et al. Ann. Rev. Genetics, 22, 421 (1988), that is incorporated herein by reference.
  • Preferred reporter genes include the beta-glucuronidase (GUS) of the uidA locus of E. coli, the chloramphenicol acetyl transferase gene from Tn9 of E.
  • GUS beta-glucuronidase
  • the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector, e.g., a plasmid that is capable of replication in a bacterial host, e.g., E. coli.
  • a convenient cloning vector e.g., a plasmid that is capable of replication in a bacterial host, e.g., E. coli.
  • the cloning vector with the desired insert may be isolated and subjected to further manipulation, such as restriction digestion, insertion of new fragments or nucleotides, ligation, deletion, mutation, resection, etc. so as to tailor the components of the desired sequence.
  • Transforming desired constructs capable of expressing polypeptide of the present invention can produce transgenic plants.
  • Transgenic corn can be produced by particle bombardment transformation methods as described in U.S. Patent No. 5,424,412.
  • the vector DNA are digested with suitable restriction endonucleases to isolate a plant expression cassette that expresses the polypeptides of the present invention in the plant.
  • the desired expression cassette is purified by agarose gel electrophoresis, then bombarded into embryogenic corn tissue culture cells using a Biolistic® (Dupont, Wilmington, DE) particle gun with purified isolated DNA fragment.
  • Transformed cells are selected by challenging transformed cells by selection media.
  • aroA:CP4 gene is part of expression cassette
  • aroA:CP4 gene is part of expression cassette
  • glyphosate N-phosphonomethyl glycine and its salts
  • Whole plants are regenerated then grown under greenhouse conditions. Fertile seed is collected, planted and screened for selectable marker; for example plant expressing desired polypeptide of the invention along with aroA:CP4 gene product can be screened by spraying glyphosate to select glyphosate tolerant plant.
  • Pant expressing desired polypeptide of the invention is then back crossed into commercially acceptable corn germplasm by methods known in the art of corn breeding (Sprague et al., Corn and Corn Improvement 3 rd Edition, Am. Soc. Agron.
  • Transgenic corn plants can also be produced by an Agrobacterium mediated transformation method.
  • a disarmed Agrobacterium strain C58 (ABI) harboring a DNA construct can be used for all the experiments.
  • the construct is transferred into Agrobacterium by a triparental mating method (Ditta et al., Proc. Natl. Acad. Sci. 77:7347-7351).
  • Liquid cultures of Agrobacterium are initiated from glycerol stocks or from a freshly streaked plate and grown overnight at 26°C-28°C with shaking (approximately 150 rpm) to mid-log growth phase in liquid LB medium, pH 7.0 containing 50 mg/1 kanamycin, 50 mg/1 streptomycin and spectinomycin and 25 mg/1 chloramphenicol with 200 ⁇ M acetosyringone (AS).
  • the Agrobacterium cells are resuspended in the inoculation medium (liquid CM4C) and the density is adjusted to OD 66 o of 1.
  • Freshly isolated Type JJ immature HiIIxLH198 and Hill corn embryos are inoculated with Agrobacterium containing a DNA construct of the present invention and co-cultured 2-3 days in the dark at 23 °C.
  • the embryos are then transferred to delay media (N6 1-100-12/micro/Carb 500/20 ⁇ M AgN03) and incubated at 28 °C for 4 to 5 days. All subsequent cultures are kept at this temperature. Coleoptiles are removed one week after inoculation.
  • the embryos are transferred to the first selection medium (N61-0-12/Carb 500/0.5 mM glyphosate). Two weeks later, surviving tissues are transferred to the second selection medium (N61-0-12/Carb 500/1.0 mM glyphosate).
  • the present invention also provides a transgenic plant comprising in its genome a polynucleic acid that comprises: (A) a 5' non-coding sequence that functions in the cell to cause the production of a mRNA molecule; that is operably linked to (B) a structural poly nucleotide sequence encoding a polypeptide of this invention that is operably linked to (C) a 3' non-translated sequence that functions in said cell to cause termination of transcription.
  • the amino acid sequence of the polypeptide has at least 75% sequence identity, about 80% sequence identity, or about 85% or about 90% sequence identity to a member selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.
  • the polypeptide can also have one of the sequences set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18 with or without conservative amino acid substitutions.
  • Transgenic plants of the present invention have incorporated into their genome, or transformed into their chloroplast or plastid genomes, an exogenous polynucleic acid molecule that comprises at least a structural nucleotide sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.
  • Transgenic plants are also meant to comprise progeny (descendant, offspring, etc.) of any generation of such a transgenic plant.
  • a seed of any generation of all such transgenic plants wherein said seed comprises a DNA sequence encoding the polypeptide of the present invention is also an important aspect of the invention.
  • Hybrid seeds are also envisioned as being within the scope of the present invention and such hybrid seeds containing a structural nucleotide sequence of the present invention may be produced by techniques known in the art.
  • the present invention also provides a method of screening transformed plants for the presence of transformed nucleic acids of the invention or polypeptides expressed by the nucleic acid of the invention conferring desired agronomic traits. The nature of these screens will generally be chosen on practical grounds. For example, one can screen by looking for changes in gene expression by using antibodies specific for the polypeptide encoded by the gene being suppressed.
  • the transgenic plants of the present invention will have a higher rate of photosynthesis, measured as the rate of carbon assimilation due to the expression of an exogenous polynucleic acid molecule encoding a polypeptide of the present invention.
  • the transgenic plants of present invention will have higher rate of photosynthesis when compared to natural plants of same species. Natural plants of same species will be without exogenous polynucleic acid molecules encoding polypeptide of the present invention.
  • the transgenic plants of the present invention will have higher carbon assimilation, measured as an increase in metabolite content due to the expression of an exogenous polynucleic acid molecule encoding a polypeptide of the present invention.
  • Plant metabolites include carbon-based molecules, which are produced or metabolized by plants.
  • the transgenic plants of present invention will have increased tolerance to environmental stress due to the expression of an exogenous polynucleic acid molecule encoding a polypeptide of the present invention.
  • the transgenic plants of present invention will have tolerance to abiotic stresses for example variations from optimal condition to sub-optimal conditions for water, humidity, temperature, light or other radiations, organic and inorganic nutrients, or salinity.
  • Drought is defined as sub-optimal conditions for water and humidity needed for normal growth of natural plants.
  • the transgenic plants of the present invention will have higher tolerance to drought and a higher yield of agricultural products under drought conditions as compared to natural plants.
  • the DNA construct of the present invention may be introduced into the genome of a desired plant host by a variety of conventional transformation techniques that are well known to those skilled in the art. Methods of transformation of plant cells or tissues include, but are not limited to Agrobacterium mediated transformation method and the Biolistics or particle-gun mediated transformation method.
  • Suitable plant transformation vectors for the purpose of Agrobacterium mediated transformation include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella et al., Nature 303:209 (1983); Bevan, Nucleic Acids Res. 12: 8711-8721 (1984); Klee et al, Bio- Technology 3(7): 637-642 (1985); and EP 120,516.
  • Ri root-inducing
  • a plasmid expression vector suitable for the introduction of a polynucleic acid encoding a polypeptide of present invention in monocots using electroporation or particle-gun mediated transformation is composed of the following: a promoter that is constitutive, tissue-specific, tissue enhanced or native; an intron that provides a splice site to facilitate expression of the gene, such as the maize Hsp70 intron (U.S. Patent No.
  • This expression cassette may be assembled on high copy replicons suitable for the production of large quantities of DNA.
  • An example of a useful Ti plasmid cassette vector for plant transformation is pMON17227. This vector is described in U.S.
  • Patent 5,633,435 herein inco ⁇ orated by reference in its entirety, and contains a gene encoding an EPSPS enzyme with glyphosate resistance (herein referred to as aroA:CP4), that is an excellent selection marker gene for many plants.
  • the gene is fused to the Arabidopsis EPSPS chloroplast transit peptide (At. EPSPS:CTP2) and expressed from the Figwort mosaic virus (P-FMV) promoter as described therein.
  • Arabidopsis EPSPS chloroplast transit peptide Arabidopsis EPSPS chloroplast transit peptide (At. EPSPS:CTP2)
  • P-FMV Figwort mosaic virus
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences.
  • the methodology for the regeneration step is well known in the art in regard of the crop plants (Klee et al., Ann. Rev. Plant Phys. 38:467-486 1987).
  • the development or regeneration of transgenic plants containing the exogenous polynucleic acid molecule that encodes a polypeptide of interest is well known in the art.
  • the regenerated plants are self-pollinated to provide homozygous transgenic plants, as discussed above.
  • Plants of the present invention include, but are not limited to, Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus, Clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, forest trees, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, loblolly pine, mango, melon, mushroom, nut, oat, o
  • Crop plants are defined as plants, which are cultivated to produce one or more commercial product, examples of such crops or crop plants include alfalfa, soybean, canola, rape, cotton (cottonseeds), sunflower, and grains such as corn, wheat, rice, rye, and the like.
  • crops or crop plants include alfalfa, soybean, canola, rape, cotton (cottonseeds), sunflower, and grains such as corn, wheat, rice, rye, and the like.
  • the following examples are provided to better elucidate the practice of the present invention and should not be interpreted in any way to limit the scope of the present invention.
  • Those skilled in the art will recognize that various modifications, additions, substitutions, truncations, etc., can be made to the methods and genes described herein while not departing from the spirit and scope of the present " invention.
  • Arabidopsis thaliana var Columbia seeds are obtained from Lehle Seeds Co. (LEHLE SEEDS 1102 South Industrial Blvd., Suite D, Round Rock TX 78681 USA). The seeds are sown into 2 inch pots prepared with soil covered with bridal veil or a mesh screen, making sure that the soil is not packed too tightly and the mesh is in contact with the soil surface (this ensures that the germinating seedlings will be able to get through the mesh. Seeds are sown and covered with a germination dome. Seeds are vernalized for 3-4 days. Plants are grown under conditions of 16 hours light / 8 hours dark at 20-22° C, 70% humidity.
  • RNA is purified by using Trizol reagent from Life Technologies (Gibco BRL, Life Technologies, Gaithersburg, Maryland U.S.A.), essentially as recommended by the manufacturer.
  • Poly A+ RNA is purified using magnetic oligo dT beads essentially as recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake Success, New York U.S.A.). Construction of plant cDNA libraries is well known in the art and a number of cloning strategies exist. A number of cDNA library construction kits are commercially available.
  • the SuperscriptTM Plasmid System for cDNA synthesis and Plasmid Cloning (Gibco BRL, Life Technologies, Gaithersburg, Maryland U.S.A.) is used, following the conditions suggested by the manufacturer.
  • the cDNA libraries are plated on LB agar containing the appropriate antibiotics for selection and incubated at 37° for sufficient time to allow the growth of individual colonies.
  • Single selective media colonies are individually placed in each well of a 96-well microtiter plates containing LB liquid including the selective antibiotics. The plates are incubated overnight at approximately 37°C with gentle shaking to promote growth of the cultures.
  • the plasmid DNA is isolated from each clone using Qiaprep plasmid isolation kits, using the conditions recommended by the manufacturer (Qiagen Inc., Santa Clara, California U.S.A.).
  • the template plasmid DNA clones are used for subsequent sequencing.
  • a commercially available sequencing kit such as the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS, is used under the conditions recommended by the manufacturer (PE Applied Biosystems, Foster City, CA).
  • the cDNAs of the present invention are generated by sequencing initiated from the 5' end or 3' end of each cDNA clone.
  • Entire inserts or only part of the inserts are sequenced.
  • a number of DNA sequencing techniques are known in the art, including fluorescence-based sequencing methodologies. These methods have the detection, automation and instrumentation capability necessary for the analysis of large volumes of sequence data.
  • the 377 and 3700 DNA Sequencer Perkin-Elmer Corp., Applied Biosystems Div., Foster City, CA
  • fluorescent dye-labeled sequence reaction products are detected and data entered directly into the computer, producing a chromatogram that is subsequently viewed, stored, and analyzed using the corresponding software programs.
  • high frequency words are masked to prevent spurious clustering; sequence common to known contaminants such as cloning bacteria are masked; high frequency repeated sequences and simple sequences are masked; unmasked sequences of less than 100 base pairs are eliminated.
  • the thus-screened and filtered ESTs are combined and subjected to a word-based clustering algorithm that calculates sequence pair distances based on word frequencies and uses a single linkage method to group like sequences into clusters of more than one sequence, as appropriate. Clustered sequences are assembled individually using an iterative method based on PHRAP/CRAW/MAP providing one or more self-consistent consensus sequences and inconsistent singleton sequences.
  • the assembled clustered sequence files are checked for completeness and parsed to create data representing each consensus contiguous sequence (contig), the initial EST sequences, and the relative position of each EST in a respective contig.
  • the sequence of the 5' most clone is identified from each contig.
  • the initial sequences that are not included in a contig are separated out.
  • Above described databases with nucleotide and peptide sequences are queried with sequences of present invention to get following homologues, orthologs or paralogs as shown in Table 1.
  • the BLAST 2.2.1 software Altschul, et.al., Nucleic Acids Res. 25: 3389-3402 (1997), with BLOSUM62 matrix and "no Filter” options, is used in the queries.
  • RNA is isolated from appropriate crop and other desired plant species by pooling tissues of different developmental stages of all vegetative and reproductive organs. RNA is prepared from pooled plant tissue by the Trizol method (Gibco BRL, Life Technologies, Gaithersburg, Maryland U.S.A.) essentially as recommended by the manufacturer. Sequences are amplified out from total RNA by using Superscript TJ kit (Gibco BRL, Life Technologies, Gaithersburg, Maryland U.S.A.) according to the manufacturer's directions.
  • PCR primers for isolating sequences of present invention are based on the sequence information provided in the sequence listing of this disclosure. Design of primers and reaction conditions are determined as described in the art. (PCR Strategies, Edited by Michael A. Innis; David H. Gelfand; & Johm J. Sninsky; Academic Press 1995 and PCR Protocols, A Guide to Method and Applications, Edited by Michael A. Innis; David H. Gelfand; Johm J. Sninsky; & Thomas J. White Academic Press 1990). All reagents for isolating sequences of the invention can be procured form Gibco BRL, Life Technologies, Gaithersburg, Maryland U.S.A.
  • EXAMPLE 4 This example describes different plant expression vectors for transforming plant to obtain plants of present inventions. DNA constructs and corresponding backbones are provided as figure as per the following table: TABLE 2
  • the expression cassette used for transforming plants to practice the current invention comprises any one of the known promoter to function to cause the transcription of desired gene in plant cells and any one of the known antibiotic or herbicide tolerance encoding polynucleotide sequence known to confer antibiotic or herbicide tolerance to plant cells. It is essential that components of expression cassette in a DNA constructs (expression vector) of the invention are operabily linked with each other in a specific order to cause the expression of desired gene product in a plant for generating transgenic plants of the invention. Specific order of operably linked essential components of each expression vectors are shown in figures 2, 4, 6, 8, 10, 12, 14, 16, 17, 19 and 20.
  • Expression cassette is assembled in a circular DNA construct in order to generate, isolated desired amounts of DNA in E. coli and is known as vector backbone.
  • vector backbone Many vector backbones are well known in the art, one of such vector backbone is pBR 322 which can be used for generation of DNA constructs for practicing the invention.
  • the DNA constructs are double border plant transformation constructs that also contain DNA segments that provide replication function and antibiotic selection in bacterial cells, for example, an E.
  • coli origin of replication such as ori322, a broad host range origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spc/Str that encodes for Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectable marker gene.
  • aadA Tn7 aminoglycoside adenyltransferase
  • Gm, Gent gentamicin
  • the host bacterial strain is Agrobacterium tumefaciens ABI or LBA4404.
  • the polylinker regions in these DNA construct provide for multiple restriction endonuclease cut sites that digest the DNA to provide a cloning site in order to clone genes of present in invention in expression cassette.
  • cloning sites may include Bglll, Ncol, EcoRI, Sail, Notl, Xhol and other sites known to those skilled in the art of molecular biology.
  • construct may also include an epitope tag ( For example Flag® peptide catalog number F-3290, SIGMA, P.O. Box 14508 St. Louis, MO 63178 USA) at the 3' termination region of gene of interest ( Figures A, B, C).
  • the GATEWAYTM cloning technology is also used for construction of few vectors of the invention ( Figures C, D, E).
  • GATEWAYTM technology uses phage lambda base site-specific recombination for vector construction, instead of restriction endonucleases and ligases.
  • the GATEWAYTM method produces a high frequency of inserts in a plasmid in the correct orientation relative to other elements in the plasmid such as promoters, enhancers, and the such. Routine cloning of any desired DNA sequence into a vector comprising operable plant expression elements is thereby facilitated.
  • a desired DNA sequence such as a coding sequence, may be amplified by PCR with the phage lambda ⁇ ttBl sequence added to the 5' primer and the ⁇ ttB2 sequence added to the 3' primer.
  • nested primers comprising a set of ⁇ ttBl and ⁇ ttB2 specific primers and a second set of primers specific for the selected DNA sequence can be used.
  • Sequences such as coding sequences, flanked by ⁇ ttBl and ⁇ ttB2 sequences can be readily inserted into plant expression vectors using GATEWAYTM methods. Assembly of DNA constructs are done by standard molecular biology techniques as described in Sambrooks et al. EXAMPLE 5 Arabidopsis plant transformation with construct expressing gene of present invention Arabidopsis plants are transformed by any one of many available methods.
  • Arabidopsis plants may be transformed using In planta transformation method by vacuum infiltration (see, Bechtold et al., In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad. Sci. Paris Sciences de la vie/life sciences 316: 1194-1199 (1993). Plants are grown as described in Examplel. Seeds recovered from plants transformed with construct were germinated and selected on media containing antibiotic kanamycin. Surviving plants were assayed by TaqMan assay for the copy number of the insert. Several individual lines were selected that had single copy plant expression cassettes and were designated as PLANT -1, PLANT -2, PLANT -3, and PLANT -4.
  • EXAMPLE 6 This example describes how photosynthesis was measured in plants. Rate of photosynthesis was measured as by measuring rate of carbon assimilation as reflected by carbon dioxide gas exchange under different concentrations of C0 2 .
  • C0 2 carbon dioxide
  • Li-6400 Portable Photosynthesis System Li-Cor Bioscience, Lincoln, Iowa
  • Measurement was done under varying C0 2 concentration and at light intensity of 150 ⁇ E M "2 S "1 , as shown in the Table 3.
  • Carbon assimilation rates were measured as C02 gas exchange from Arabidopsis plants over expressing the CCA-1 gene by using the e35S promoter.
  • C0 2 exchange was expressed as micromole. per square meter leaf area per second ( ⁇ mol/m 2 /s).
  • EXAMPLE 7 This example describes how plants of the present invention grown under different light conditions can assimilate different amounts of carbon as reflected by carbon dioxide gas exchange.
  • C0 2 exchange was measured as indicated in example 6.
  • C0 2 gas exchange measurements indicated that CCAl over expression also increases the photosynthetic efficiency of plants grown under low light.
  • Wild type and PLANT- 1 plants were grown for 21 days under 150 micro Einstein/m Is. Half the plants were transferred to a chamber covered with cloth to reduce the light intensity to 65 micro Einstein/m2/s. The remaining plants were allowed to continued growth under 150 micro Einstein/m 2 /s.
  • This example describes how plants of the present invention grown under different light conditions can assimilate different amounts of carbon, measured as C0 2 gas exchange.
  • C0 2 gas exchange measurements indicated that CCAl over expression also increases the photosynthetic efficiency of plants grown under low light.
  • Wild type and PLANT-1 plants were grown for 21 days under 150 micro Einstein/m 2 /s of light intensity. Half the plants were transferred to a chamber covered with cloth to reduce the light intensity to 65 micro Einstein/m 2 /s. The remaining plants were allowed to continued growth under 150 micro Einstein/m 2 /s.
  • EXAMPLE 9 The following example describes the comparison of metabolite contents in plants of the present invention in comparison with natural plants. Carbohydrates are the primary products of photosynthesis. Increases in several metabolites, including many mono- and disaccharides and starch were apparent in plants transformed to express the CCAl gene product. The amount of total soluble sugar from leaves was determined by extracting total sugar by coupled enzymatic assay as described by Angelov M.N, Sun J, Byrd GT, Brown RH, and Black CC in Photosynthesis Research 38: 61-72, 1993.
  • Leaf starch present in the same leaf punches was extracted from the punches and digested to glucose by homogenizing the punches in 0.5 N NaOH, boiling for 10 min, titrating the pH to 5.5 with acetic acid, then treating with amyloglucosidase(Winder T.L., Sun J., Okita T.W., Edwards G.E., Plant Cell Physiol 39: 813-820, 1998). The resulting glucose equivalents were determined similarly to total leaf soluble sugar as described above (Angelov M.N., Sun J., Byrd G.T., Brown R.H., and Black CC, Photosynthesis Research 38: 61-72, 1993). Leaf total sugar was highly elevated in PLANT-1 plants compared to wild-type plants, particularly during daylight hours, whereas leaf starch was modestly increased in PLANT-1 plants (Table 6).
  • EXAMPLE 10 The following example describes the effect of CCA-1 gene expression on the morphology of Arabidopsis plants. Over expression of CCAl behind a constitutive promoter results in Arabidopsis plants with altered morphology. However, the altered morphology can be corrected by the expression of CCAl behind light regulated promoters. For example, Arabidopsis lines PLANT-1 and PLANT-2, which over express Arabidopsis CCAl behind the e35S promoter, have elongated hypocotyls, petioles, and leaves compared to wild-type plants.
  • Leaf total soluble sugar extracted from leaf punches was determined by coupled enzymatic assay. For the determination of leaf total soluble sugar content of pMON56622 and wild-type Arabidopsis plants, a 6 mm diameter punch was taken after 8 h in the light and extracted three times in 80% ethanol. Extracted sugar was dried at 60 C then resuspended in water. Total sugar was determined by measuring the formation of NADH at 340 nm in the presence of NAD+, ATP, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, and invertase. TABLE 8:
  • EXAMPLE 11 This example describes how to create soil based simulated drought conditions and evaluation of drought stress in Arabidopsis plants, eg. plant-1, plant-2, plant-3 or plant-4 containing genes of the invention as well as plants with empty vector.
  • Two inch pots were filled with Metromix 200 (Scotts-Sierra Horticultural Products Co., Marysville, OH) planting mix.
  • Each pot was adjusted to a weight of 37+/- lgram prior to immersing the flat in nutrient solution (100 PPM of Peters 20:20:20; Scotts-Sierra Horticultural Products Co., Marysville, OH; Stock Number 91010) for water and nutrient saturation. These pots were stored at 4 degree C for approximately three days, then transferred to 22°C/18°C, 16-hr-light 8-hr-dark, 150 ⁇ mol m "2 sec "1 with 70% relative humidity. After seed germination pots were thinned for a plant density of 1 plant/pot and allowed to grow until bolting/first flower open, which was usually 4-5 weeks after sowing.
  • experimental plants were watered with 25% of water capacity, that is approximately 25% of water that was needed for saturation of soil in the pots ( ⁇ 25 ml). Three days after watering the experimental plants, all measurements were repeated along with photographic records of experimental and control plants. All pots were saturated with nutrient solutions at this stage and the phenotype was recorded for seven days without watering.
  • Drought scoring in Arabidopsis plants was done by taking a photograph of the soil based drought assay scoring system in Arabidopsis plants, where A) were healthy plants, no difference from control plants; B) were near wilting, leaves becoming wilted; C) were wilted green recoverable plants after watering, D) were severely wilted, anthocyanic plants and not recoverable after watering. No visual difference under drought conditions was observed for wild type plants as compared to plants transformed with an empty cloning vector (see Table 9 and 10); therefore physiological measurements were done only in wild type (WT) plants and compared with plants transformed with constructs of the present invention.
  • EXAMPLE 12 The following example describes the effect of CCA-1 gene expression on water pressure in Arabidopsis plants.
  • Water pressure was used in the art to predict the movement of liquid water into or out of a plant cell.
  • the water potential difference across a membrane determines the direction of flow. Water moves spontaneously from regions of high water potential from adjoining regions of high water potential to regions of low water potential.
  • Leaf water potential was measured by sealing a sample tissue in a chamber containing a thermocouple. After an equilibration period a cooling current was applied to the thermocouple to condense water on the thermocouple junction. The amount of condensed water was proportional to the water potential of the sample tissue. Condensed water was allowed to evaporate causing a change in the thermocouple output.
  • Relative water content is another physiological measure of plant water deficit. It measures the effect of osmotic adjustment in plant water status, when a plant is under stressed conditions. RWC was measured in small leaf samples and it reflects on the mass of water held in relation to the mass that can be held at full turgor pressure. For measuring RWC, leaf samples were taken into an airtight container, weighed (W) and floated on or set in water for 4 hours, after that they are weighed again to determine turgid weight (TW). After these measurements, the leaf sample was dried in an oven at 80° C for 24 hours and weighed to determine dry matter weight (DW).
  • W airtight container
  • TW turgid weight

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Abstract

L'invention concerne un procédé et des molécules d'ADN qui, lorsqu'elles sont exprimées dans une plante, produisent des plantes transgéniques avec des caractéristiques organiques améliorées. Cette invention porte aussi sur des vecteurs d'expression de plante contenant les molécules d'ADN, ces molécules d'ADN codant des protéines qui sont liées à des facteurs de transcription de plante et des plantes contenants ces molécules d'ADN.
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US8153863B2 (en) * 2007-03-23 2012-04-10 New York University Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity
CN117402909A (zh) * 2023-10-20 2024-01-16 中国农业大学 紫花苜蓿MsMYB58基因调控植物抗旱性的应用

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US6265637B1 (en) * 1996-06-21 2001-07-24 Plant Bioscience Limited Genetic control of flowering

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US8153863B2 (en) * 2007-03-23 2012-04-10 New York University Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity
US9464296B2 (en) 2007-03-23 2016-10-11 New York University Methods of affecting nitrogen assimilation in plants
CN117402909A (zh) * 2023-10-20 2024-01-16 中国农业大学 紫花苜蓿MsMYB58基因调控植物抗旱性的应用

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