WO2014012145A1 - Constructions pour moduler la transpiration chez les plantes et leurs utilisations - Google Patents

Constructions pour moduler la transpiration chez les plantes et leurs utilisations Download PDF

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WO2014012145A1
WO2014012145A1 PCT/AU2013/000799 AU2013000799W WO2014012145A1 WO 2014012145 A1 WO2014012145 A1 WO 2014012145A1 AU 2013000799 W AU2013000799 W AU 2013000799W WO 2014012145 A1 WO2014012145 A1 WO 2014012145A1
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plant
seq
sequence
nucleotide sequence
nucleic acid
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Mark David KINKEMA
Ian Mark O'hara
Manuel Benito Sainz
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University of Queensland UQ
Queensland University of Technology QUT
Syngenta Participations AG
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University of Queensland UQ
Queensland University of Technology QUT
Syngenta Participations AG
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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8217—Gene switch
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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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    • 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
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    • 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
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    • 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
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    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention is directed generally to the field plant engineering. Specifically, this invention relates to constructs for controlling stomatal closure, as well as plants, plant parts and plant cells comprising those constructs.
  • a second way of improving plants through increased stomatal opening is to enable plants to take up and store more carbon dioxide in the form of stored carbohydrates such as starch and simple sugars.
  • Stomatal closure is generally controlled by the hormone abscisic acid (ABA) in response to drought stress.
  • Proteins involved in ABA-mediated stomatal regulation include the Ca -independent ABA-activated protein kinase (AAPK), protein kinase open stomata 1 (OSTl), respiratory burst oxidase homologs (Rboh) RBOHD and RBOHF NADPH oxidases, the vacuolar trafficking pathway v-SNAREs AtVAMP711-14, heterotrimeric GTP- binding (G) protein alpha subunit gene (GPA1), ATP-binding cassette (ABC) transporter AtABCG22, ABC transporter AtABCG40, ABC transporter AtMRP4, and phospholipase D alpha 1 (PLDalphal). Reducing the expression of, or introducing loss of function mutations in, the endogenous genes that code for the above proteins is known to inhibit stomatal closure, resulting in uncontrollable water loss and drought
  • ABA insensitive 1 ABS1
  • ABS2 ABA insensitive 2
  • ABA insensitive 2 ABS2
  • ectopic expression of the Arabidopsis homeobox-leucine zipper transcription factor ATHB6 and dominant positive mutants of the Arabidopsis SOS2-like protein kinase PKS3 is known to inhibit stomatal closure.
  • dominant positive mutants of H(+)-ATPase 1 AHA1 also known as open stomata 2 (OST2)
  • OST2 open stomata 2
  • the present invention is predicated in part on the development of an improved inducible gene switch that has enhanced sensitivity and inducibility, as well as being operable in monocotyledonous plants such as sugar cane.
  • the present inventors propose using this gene switch to control the expression of stomatal aperture-modulating genes such as those noted above in order to inhibit or reduce stomatal closure and thereby accelerate or control the rate of crop drying and/or improve the water and sugar content of crops, as described hereafter.
  • the present invention provides constructs for inhibiting stomatal closure.
  • constructs generally comprise in operable connection: (1) a cw-acting element comprising, consisting or consisting essentially of a nucleotide sequence represented by the sequence GCGGNNCCGC [SEQ ID NO: 1 ] ; (2) a promoter that is operable in a plant cell (e.g., a plant guard cell); and (3) a nucleic acid sequence encoding an expression product that inhibits stomatal closure.
  • the construct is a chimeric construct and in illustrative examples of this type, the cw-acting element is heterologous with respect to the promoter and/or the expression product-encoding nucleic acid sequence.
  • the cw-acting element comprises, consists or consists essentially of at least one nucleotide sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) as set forth in SEQ ID NO: 1.
  • the at least one nucleotide sequence is represented by the nucleotide sequence
  • N or n can be independently any nucleic acid base (A, G, C, or T) and wherein x and y can be independently any number.
  • the at least one nucleotide sequence (e.g., 1, 2, 3, 4,
  • TACGTTGCGGAACCGCAGCTCC [SEQ ID NO:9] in any combination, in any orientation, and/or in any order.
  • the expression product that inhibits stomatal closure is a stomatal closure-inhibiting polypeptide, illustrative examples of which include negative regulators of stomatal closure (e.g. , ATHB6, ABI 1 , ABI2), mutant forms of ABI 1 , ABI2,
  • AAPK, PKS3 and AHA1 which have reduced ABA sensitivity and/or which inhibit stomatal closure
  • antibodies that are immuno-interactive with a polypeptide that stimulates stomatal closure or that inhibits stomatal opening e.g., OST1, AAPK, v-SNAREs
  • the expression product that inhibits stomatal closure is a stomatal closure-inhibiting RNA molecule (e.g., siRNA, shRNA, microRNAs, antisense RNA etc.) that inhibits expression of an endogenous nucleotide sequence encoding a polypeptide that stimulates stomatal closure or that inhibits stomatal opening (e.g., OST1, AAPK, v-SNAREs AtVAMP711-14, GPA1, AtABCG22, AtABCG40, ABC transporter AtMRP4, RBOHD and RBOHF and PLDalphal ).
  • a stomatal closure-inhibiting RNA molecule e.g., siRNA, shRNA, microRNAs, antisense RNA etc.
  • the present invention provides a construct system for inhibiting stomatal closure.
  • the construct system generally comprises, consists or consists essentially of a construct as broadly described above (“first construct") and a second construct comprising a nucleotide sequence encoding a transcription factor (e.g., AlcR), which activates in the presence of a compound that induces expression of the alcohol dehydrogenase (ADH1) system of Aspergillus nidulans (e.g., a primary alcohol such as ethanol) and which interacts with the cw-acting element of the first construct to induce expression of the nucleic acid sequence encoding the expression product that inhibits stomatal closure.
  • ADH1 alcohol dehydrogenase
  • transgenic plant cells e.g., plant guard cells
  • the present invention provides transgenic plants, plant parts or plant organs (e.g. , plant leaves) comprising plant cells (e.g. , plant guard cells) as broadly described above and elsewhere herein.
  • Still another aspect of the present invention provides methods for increasing transpiration in a plant, plant part or plant organ (e.g. plant leaf). These methods generally comprise expressing in a cell (e.g., a guard cell) of the plant, plant part or plant organ a polynucleotide that comprises a nucleic acid sequence encoding an expression product that inhibits stomatal closure, where the nucleic acid sequence is under the control of a cis-acting element as broadly defined above and elsewhere herein to thereby increase transpiration in the plant, plant part or plant organ.
  • a cell e.g., a guard cell
  • a polynucleotide that comprises a nucleic acid sequence encoding an expression product that inhibits stomatal closure, where the nucleic acid sequence is under the control of a cis-acting element as broadly defined above and elsewhere herein to thereby increase transpiration in the plant, plant part or plant organ.
  • the methods comprise inducing expression of the polynucleotide in the presence of a compound that induces expression of the alcohol dehydrogenase (ADHl) system of Aspergillus nidulans (e.g. , a primary alcohol such as ethanol).
  • ADHl alcohol dehydrogenase
  • the methods suitably comprise co-expressing in the cell a nucleotide sequence encoding a transcription factor (e.g. , AlcR), which activates in the presence of a compound that induces expression of the alcohol dehydrogenase (ADHl) system of
  • Aspergillus nidulans e.g., a primary alcohol such as ethanol
  • Aspergillus nidulans e.g., a primary alcohol such as ethanol
  • the present invention provides methods for increasing transpiration in a plant, plant part or plant organ (e.g., plant leaf) that comprises a construct or construct system as broadly defined above and elsewhere herein.
  • These methods generally comprise exposing the plant, plant part or plant organ to a compound that induces expression of the alcohol dehydrogenase (ADHl) system of Aspergillus nidulans (e.g., a primary alcohol such as ethanol) so as to inhibit stomatal closure and thereby increase transpiration in the plant, plant part or plant organ.
  • ADHl alcohol dehydrogenase
  • Aspergillus nidulans e.g., a primary alcohol such as ethanol
  • the methods comprise exposing the plant, plant part, plant organ (e.g., plant leaf) to the compound around the time of harvesting the plant, plant part or plant organ.
  • the methods comprise exposing the plant, plant part or plant organ to the compound prior to harvesting the plant, plant part or plant organ.
  • the methods comprise exposing the plant, plant part or plant organ to the compound at the time of harvesting the plant, plant part or plant organ.
  • the methods comprise exposing the plant, plant part or plant organ to the compound after harvesting the plant, plant part or plant organ.
  • the methods further comprise permitting increased transpiration in the plant, plant part or plant organ over a time and under conditions sufficient for the water content of the plant, plant part or plant organ to reduce by at least about 5% (e.g., at least about 6%, 7%, 8%, 9%, 10%, 15% 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%).
  • at least about 5% e.g., at least about 6%, 7%, 8%, 9%, 10%, 15% 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%.
  • the plant is a monocotyledonous plant, illustrative examples of which include sugar cane, corn, barley, rye, oats, wheat, rice, flax, millet, sorghum, grasses (e.g., switch grass, giant reed etc.), banana, onion, asparagus, lily, coconut, and the like.
  • the plant is a monocotyledonous plant, illustrative examples of which include sugar cane, corn, barley, rye, oats, wheat, rice, flax, millet, sorghum, grasses (e.g., switch grass, giant reed etc.), banana, onion, asparagus, lily, coconut, and the like.
  • the plant is a monocotyledonous plant, illustrative examples of which include sugar cane, corn, barley, rye, oats, wheat, rice, flax, millet, sorghum, grasses (e.g., switch
  • dicotyledonous plant e.g., tobacco, cotton, dried fruits such as raisins and prunes, nuts, coffee, tea, cocoa, and ornamental goods.
  • the plant is selected from energy crops
  • Sorghum, Poplars, wheat, rice, oats, willows e.g., Salix species
  • switch grass i.e., Panicum virgatum
  • alfalfa i.e., Medicago sativa
  • prairie bluestem e.g.,Andropogon gerardii
  • maize i.e., Zea mays
  • soybean i.e., Glycine max
  • barley i.e., Hordeum vulgare
  • sugar beet i.e., Beta vulgaris
  • hay and fodder crops e.g., Salix species
  • switch grass i.e., Panicum virgatum
  • alfalfa i.e., Medicago sativa
  • prairie bluestem e.g.,Andropogon gerardii
  • maize i.e., Zea mays
  • soybean i.e., Glycine max
  • barley
  • Figure 1 is a graphical representation showing the results for ethanol inducible GUS expression at two days, four days and seven days post treatment of six month old transgenic sugar cane plants containing the different ethanol switch constructs.
  • n number of independent, single copy transgenic plants analyzed for each construct. Data with different letters are significantly different (**P ⁇ 0.01; ***P ⁇ 0.001).
  • Figure 2 is a graphical representation showing ethanol inducible expression from promoters containing either 1, 5, or 9 copies of the inverted repeat AlcR binding site.
  • Figure 3 is a graphical representation showing ethanol inducible expression from promoters containing various modified inverted repeat AlcR binding sites.
  • Figure 4 is a graphical representation showing ethanol inducible expression from promoters containing five copies of the inverted repeat AlcR binding sites fused to different minimal promoters.
  • Figure 5 is a graphical representation showing ethanokinducible expression in the TO and T0V 1 transgenic plants.
  • Figure 6 is a schematic representation of the 35S-abil binary construct used for constitutive expression of abil in N. benthamiana.
  • Figure 7 is a schematic representation of the palcA-I-abil binary construct used for ethanol inducible expression of abil in N. benthamiana and tobacco.
  • Figure 8 is a schematic representation of the eFMV e35S-ZmUbil -scoabil construct used for constitutive expression of abil in sugar cane.
  • Figure 9 is a schematic representation of the palcA I-scoabil construct used for ethanol inducible expression of abil in sugar cane.
  • Figure 10 is a photographic representation showing ethanol inducible expression of abil in transgenic N. benthamiana. Expression of abil was assessed using RT- PCR for5 independent transgenic plants prior to ethanol treatment and at 12 hours post ethanol treatment. The positive control consists of vector DNA containing the abil gene. For the negative control, water was used to replace the DNA template. The expected PCR product size is 1311 bp.
  • Figure 11 is a photographic representation showing ethanol inducible expression of abil in N. benthamiana. Representative images are shown for transgenic control (vector backbone only) and ethanol inducible abil plants. Plants were photographed prior to ethanol treatment and at 12, 24, and 36 hours post treatment (h.p. ). Ethanol treatment consisted of a single 2% ethanol root drench and aerial spray .
  • Figure 13 is a photographic representation showing constitutive expression of either scoABII or scoabil in transgenic sugar cane. Expression was assessed using RT- PCR for 8 independent scoABII and 7 independent scoabil transgenic plants growing in soil. The expected PCR product size is 460 bp.
  • Figure 14 is a graphical representation of stomatal conductance in wild type sugar cane and transgenic sugar cane possessing the constructs eFMVe35S-ZmUbil -scoabil, eFMVe35S-Zm63 ⁇ 4H -scoABII, and the pUKN vector alone.
  • Figure 16 is a photographic representation showing wilty phenotype of a transgenic sugar cane plant possessing constitutive expression of scoabil (i.e. , containing eFMVe35S-ZmUbil -scoabil) compared to a control plant (vector backbone only).
  • scoabil i.e. , containing eFMVe35S-ZmUbil -scoabil
  • Figure 17 is a photographic representation showing ethanol inducible expression of scoabil in sugar cane. Plantlets were treated using a 2% ethanol root drench and aerial spray. Expression of scoabil was assessed using RT-PCR for one transgenic control (vector backbone only; UKN9) and 15 independent transgenic plantlets prior to ethanol treatment and at 14 hours post ethanol treatment. Uil and Ui3 represent transgenic plantlets possessing the ethanol inducible promoter combined with the 5' half of the rice polyubiquitin- 2 1 st intron. The positive control consists of vector DNA containing the scoabil gene. For the negative control, water was used to replace the DNA template. The expected PCR product size is 400 bp.
  • Figure 18 is a schematic representation of the pGCl-scoAAPK K43A and pGCl -scoabil constructs used for guard cell-preferred expression of scoAAPK K43A and scoabil in tobacco.
  • Figure 19 is a photographic representation showing ethanol inducible expression of abil in tobacco. Representative images are shown for wild type and transgenic ethanol inducible abil plants. Plants were photographed prior to ethanol treatment and at 24 hours post treatment. Ethanol treatment consisted of a single 2% ethanol root drench and aerial spray.
  • Figure 20 is a photographic representation showing expression of either scoAAPK K43A or scoabil in tobacco using the guard cell-preferred promoter. pGCl.
  • an element means one element or more than one element.
  • the term “cw-acting sequence” also includes a plurality of s-acting sequences.
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • antisense refers to a nucleotide sequence whose sequence of nucleotide residues is in reverse 5' to 3' orientation in relation to the sequence of
  • a "sense strand” of a DNA duplex refers to a strand in a DNA duplex which is transcribed by a cell in its natural state into a “sense mRNA.”
  • an "antisense' sequence is a sequence having the same sequence as the non-coding strand in a DNA duplex.
  • the term “antisense RNA” refers to 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 by interfering with the processing, transport and/or translation of its primary transcript or mRNA.
  • antisense RNA may be with any part of the specific gene transcript, in other words, at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
  • antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression.
  • Ribozyme refers to a catalytic RNA and includes sequence- specific endoribonucleases.
  • Antisense inhibition refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein.
  • as-acting element As-acting sequence or “ay-regulatory region” are used interchangeably herein to mean any sequence of nucleotides which modulates transcriptional activity of an operably linked promoter and/or expression of an operably linked nucleotide sequence.
  • a a ' s-sequence may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of any nucleotide sequence, including coding and non-coding sequences.
  • chimeric construct refers to construct of two or more nucleic acid sequences of different origin assembled into a single nucleic acid molecule.
  • the term chimeric construct refers to any construct that contains ( 1 ) DNA sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined.
  • a chimeric construct may comprise cis- acting sequences, promoters and/or and stomatal closure-modulating nucleic acid sequences that are derived from different sources, or comprise as-acting sequences, promoters and/or and stomatal closure-modulating nucleic acid sequences derived from the same source, but arranged in a manner different from that found in nature.
  • a chimeric construct of the present invention comprises an expression cassette comprising a s-acting sequence, a promoter that is heterologous with respect to the as-acting sequence and a stomatal closure-modulating nucleic acid sequence that is heterologous with respect to the as- acting sequence, or to the promoter, or to both the as-acting sequence and the promoter.
  • coding sequence is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene.
  • non-coding sequence refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.
  • complementary polynucleotides are those that are capable of hybridizing via base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
  • G:C guanine paired with cytosine
  • A:T thymine
  • A:U adenine paired with uracil
  • sequence "A-G-T” binds to the complementary sequence "T-C-A.” It is understood that two polynucleotides may hybridize to each other even if they are not completely or fully complementary to each other, provided that each has at least one region that is substantially complementary to the other.
  • complementary or “complementarity,” as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • Complementarity between two single-stranded molecules may be "partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules either along the full length of the molecules or along a portion or region of the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • the terms “substantially complementary” or “partially complementary” mean that two nucleic acid sequences are complementary at least at about 50%, 60%, 70%, 80% or 90% of their nucleotides. In some embodiments, the two nucleic acid sequences can be complementary at least at about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of their nucleotides.
  • substantially complementary and
  • “partially complementary” can also mean that two nucleic acid sequences can hybridize under high stringency conditions and such conditions are well known in the art.
  • the term “consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”
  • the term “consisting essentially of (and grammatical variants), as applied to a nucleic acid sequence of this invention means a polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of twenty or less (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) additional nucleotides on the 5' and/or 3' ends of the recited sequence such that the function of the polynucleotide is not materially altered.
  • the total of twenty or less additional nucleotides includes the total number of additional nucleotides on both ends added together.
  • the term “constitutively active” refers to a protein that is always active, i.e., the physiological effect of the protein is always obtained even in the absence of an activator of that protein.
  • the term “constitutively active” refers to an ATPase that has the ability to catalyze the hydrolysis of ATP to ADP in the absence of an activator of the ATPase.
  • construct refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources.
  • expression construct refers to any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear ; or circular single-stranded or double- stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked.
  • An "expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest.
  • plant promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in a plant, plant part, plant organ and/or plant cell.
  • Methods are known for introducing constructs into a cell in such a manner that a transcribable polynucleotide molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product.
  • Constructs may also be made to be capable of expressing inhibitory RNA molecules in order, for example, to inhibit translation of a specific RNA molecule of interest.
  • compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3.sup.rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.
  • a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to all or a portion of the reference nucleic acid sequence) or an amino acid sequence that displays substantial sequence similarity or identity to a reference amino acid sequence (e.g., at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
  • Dominant negative refers to a gene product that adversely affects, blocks or abrogates the function of a normal, wild-type gene product when co-expressed with the wild type gene product within the same cell even when the cell is heterozygous (wild-type and dominant negative). Expression of the dominant negative mutant generally results in a decrease in normal function of the wild-type gene product.
  • the term "dominant positive” refers to a gene product that partially or fully mimics the function of a normal, wild-type gene product (and thus possesses the same activity) when co-expressed with the wild type gene product within the same cell even when the cell is heterozygous (wild-type and dominant positive). Expression of the dominant positive mutant generally results in an increase in normal function of the wild-type gene product.
  • the terms "encode,” “encoding” and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide.
  • a nucleic acid sequence is said to "encode" a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide.
  • Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence.
  • the terms "encode,” "encoding” and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.
  • endogenous refers to any polynucleotide or polypeptide which is present and/or naturally expressed within a plant or a cell thereof. For example, an
  • endogenous nucleic acid refers to a nucleic acid molecule or nucleotide sequence that is naturally found in the cell into which a construct of the invention is introduced. [0069] The term "expression" with respect to a gene sequence refers to
  • expression of a coding sequence results from transcription and translation of the coding sequence.
  • expression of a non-coding sequence results from the transcription of the non-coding sequence.
  • fragment when used in reference to a nucleic acid molecule or nucleotide sequence will be understood to mean a nucleic acid molecule or nucleotide sequence of reduced length relative to a reference nucleic acid molecule or nucleotide sequence and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence.
  • Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • the term "gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, siRNA, shRNA, miRNA, and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g. , introns, regulatory elements, promoters, enhancers, termination sequences and 5' and 3' untranslated regions).
  • a gene may be "isolated” by which is meant a nucleic acid molecule that is substantially or essentially free from components normally found in association with the nucleic acid molecule in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid molecule.
  • Gene as used herein includes the nuclear and/or plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast genome.
  • the term “guard cell” refers to specialized epidermal cells that regulate the aperture (i.e. , the opening and closing) of stomata and by this control the bulk of gas exchange as well as transpiration. These pairs of bean-like shaped cells are characterized by their highly regulated turgor (i.e., pressure-dependent shape), which causes the stomata to close or to open at states of low or high turgor, respectively.
  • Guard cells derive from epidermal cells and are evenly spaced in the epidermis, i.e., the outermost cell layer of plant organs. Guard cells differ from their surrounding epidermal cells not only by shape but also by their ability to photosynthesize.
  • Guard cell-specific promoter refers to a promoter that transcribes an operably connected nucleic acid sequence in a way that transcription of the nucleic acid sequence in guard-cells contribute to more than 90%, 95%, 99% of the entire quantity of the RNA transcribed from said nucleic acid sequence in the entire plant during any of its developmental stages.
  • Guard cell-preferential promoter in the context of this invention refers to a promoter that transcribes an operably connected nucleic acid sequence in a way that transcription of the nucleic acid sequence in guard-cells contribute to more than 50%, preferably more than 70%, more preferably more than 80% of the entire quantity of the RNA transcribed from said nucleic acid sequence in the entire plant during any of its developmental stages.
  • heterologous refers to a nucleic acid molecule or nucleotide sequence that either originates from another species or is from the same species or organism but is modified from either its original form or the form primarily expressed in the cell.
  • a nucleotide sequence derived from an organism or species different from that of the cell into which the nucleotide sequence is introduced is heterologous with respect to that cell and the cell's descendants.
  • a heterologous nucleotide sequence includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g.
  • nucleic acid when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • a nucleic acid may be recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a protein from one source and a nucleic acid encoding a peptide sequence from another source.
  • a “heterologous" protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • homologs refers to the level of similarity between two or more nucleotide sequences and/or amino acid sequences in terms of percent of positional identity (/ ' . e. , sequence similarity or identity). Different nucleotide sequences or polypeptide sequences having homology are referred to herein as "homologs.”
  • homolog includes homologous sequences from the same and other species and orthologous sequences from the same and other species.
  • homologology also refers to the concept of similar functional properties among different nucleic acids, amino acids, and/or proteins.
  • Reference herein to "immuno-interactive" includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
  • "Introducing" in the context of a plant cell, plant part and/or plant organ means contacting a nucleic acid molecule with the plant, plant part, and/or plant cell in such a manner that the nucleic acid molecule gains access to the interior of the plant cell and/or a cell of the plant and/or plant part.
  • these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs.
  • these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, e.g. , as part of a breeding protocol.
  • transformation refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient. "Transient transformation” in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.
  • stable transformation or “stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. Stable transformation as used herein can also refer to a nucleic acid molecule that is maintained extrachromosomally, for example, as a minichromosome.
  • nucleic acid molecule or nucleotide sequence or nucleic acid construct or double stranded RNA molecule of the present invention is generally free of , nucleotide sequences that flank the nucleic acid of interest in the genomic DNA of the organism from which the nucleic acid was derived (such as coding sequences present at the 5' or 3' ends).
  • nucleic acid molecule of this invention can include some additional bases or moieties that do not deleteriously or materially affect the basic structural and/or functional characteristics of the nucleic acid molecule.
  • an "isolated nucleic acid molecule” or “isolated nucleotide sequence” is a nucleic acid molecule or nucleotide sequence that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated nucleic acid includes some or all of the 5' noncoding (e.g., promoter) sequences that are immediately contiguous to a coding sequence.
  • the term therefore includes, for example, a recombinant nucleic acid that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences.
  • isolated can further refer to a nucleic acid molecule, nucleotide sequence, polypeptide, peptide or fragment that is substantially free of cellular material, viral material, and/or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized).
  • an "isolated fragment” is a fragment of a nucleic acid molecule, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found as such in the natural state. "Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.
  • isolated refers to a nucleic acid molecule, nucleotide sequence, polypeptide, peptide or fragment that is altered "by the hand of man” from the natural state; i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a naturally occurring polynucleotide or a polypeptide naturally present in a living organism in its natural state is not “isolated,” but the same polynucleotide or polypeptide
  • isolated means that it is separated from the chromosome and/or cell in which it naturally occurs.
  • a polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs in and is then inserted into a genetic context, a chromosome and/or a cell in which it does not naturally occur.
  • an "isolated" nucleic acid molecule, nucleotide sequence, and/or polypeptide is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% pure (w/w) or more.
  • an "isolated" nucleic acid, nucleotide sequence, and/or polypeptide indicates that at least about a 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold or more enrichment of the nucleic acid (w/w) is achieved as compared with the starting material.
  • microRNA or "miR As” refer to small, noncoding R A molecules that have been found in a diverse array of eukaryotes, including plants. miR A precursors share a characteristic secondary structure, forming short 'hairpin' RNAs.
  • miRNA includes processed sequences as well as corresponding long primary transcripts (pri-miRNAs) and processed precursors (pre-miRNAs).
  • miRNAs are processed to their mature forms by Dicer, an RNAse III family nuclease, and function through RNA -mediated interference (RNAi) and related pathways to regulate the expression of target genes (Harmon (2002) Nature 418, 244-251 ; Pasquinelli, et al. (2002) Annu. Rev. Cell. Dev. Biol. 18, 495-513).
  • miRNAs may be configured to permit experimental manipulation of gene expression in cells as synthetic silencing triggers 'short hairpin RNAs' (shRNAs) (Paddison et al. (2002) Cancer Cell 2, 17-23). Silencing by shRNAs involves the RNAi machinery and correlates with the production of small interfering RNAs (siRNAs), which are a signature of RNAi.
  • non-coding refers to sequences of nucleic acid molecules that do not encode part or all of an expressed protein.
  • Non-coding sequences include but are not limited to introns, enhancers, promoter regions, 3' untranslated regions, and 5' untranslated regions.
  • 5'-non-coding region shall be taken in its broadest context to include all nucleotide sequences which are derived from the upstream region of a gene. Such regions may include an intron, e.g., an intron.
  • 3' non-coding region refers to nucleic acid sequences located downstream of a coding sequence and include polyadenylation recognition sequences (normally limited to eukaryotes) and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal (normally limited to eukaryotes) is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • nucleotide sequence refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded or double stranded.
  • nucleic acid sequence “nucleic acid,” “nucleic acid molecule,” “oligonucleotide” and “polynucleotide” are also used interchangeably herein to refer to a heteropolymer of nucleotides.
  • Nucleic acid sequences provided herein are presented herein in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR 1.821 - 1.825 and the World Intellectual Property
  • operably connected or “operably linked” as used herein refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence e.g., a promoter
  • operably linked refers to positioning and or orientation of the control sequence relative to the coding sequence to permit expression of the coding sequence under conditions compatible with the control sequence.
  • the control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct the expression thereof.
  • intervening untranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • a cw-acting sequence to a promoter encompasses positioning and/or orientation of the exacting sequence relative to the promoter so that (1) the c/s-acting sequence regulates (e.g., inhibits, abrogates, stimulates or enhances) promoter activity.
  • plant means any plant and thus includes, for example, angiosperms (monocots and dicots), gymnosperms, bryophytes, ferns and/or fern allies.
  • angiosperms monocots and dicots
  • gymnosperms bryophytes
  • ferns and/or fern allies Non- limiting examples of monocot plants of the present invention include sugar cane, corn, barley, rye, oats, wheat, rice, flax, millet, sorghum, grasses, banana, onion, asparagus, lily, coconut, and the like.
  • plant part includes but is not limited to embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plant cells including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant cell tissue cultures, plant calli, plant clumps, and the like.
  • plant cell refers to a structural and physiological unit of the plant, which comprises a cell wall and also may refer to a protoplast.
  • a plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue or a plant organ.
  • plant organ refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant.
  • polynucleotide refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof.
  • the term also encompasses RNA/DNA hybrids.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of RNA or DNA.
  • dsRNA When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing.
  • less common bases such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others
  • polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression.
  • modifications such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.
  • Polypeptide “peptide,” “protein” and “proteinaceous molecule” are used interchangeably herein to refer to molecules comprising or consisting of a polymer of amino acid residues and to variants and synthetic analogues of the same.
  • amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • This term also includes within its scope two or more complementing or interactive polypeptides comprising different parts or portions (e.g. , polypeptide domains, polypeptide chains etc.) of a polypeptide of the present invention, wherein the individual complementing polypeptides together reconstitute the activity of the different parts or portions to form a functional polypeptide.
  • polypeptide that inhibits stomatal closure refers to polypeptides that interfere, impair, reduce or otherwise inhibit stomatal closure (e.g., ABA- induced stomatal closure), or that stimulate or enhance stomatal opening.
  • RNA interference RNA interference
  • posttranscriptional co-suppression RNA interference
  • promoter refers to a region of a nucleotide sequence that incorporates the necessary signals for the expression of a coding sequence operably associated with the promoter. This may include sequences to which an RNA polymerase binds, but is not limited to such sequences and can include regions to which other regulatory proteins bind, together with regions involved in the control of protein translation and can also include coding Sequences. Furthermore, a “promoter” of this invention is a promoter (e.g., a nucleotide sequence) capable of initiating transcription of a nucleic acid molecule in a cell of a plant.
  • Promoter activity refers to the ability of a promoter to drive expression of a nucleic acid sequence operably linked to the promoter. Promoter activity of a sequence can be assessed by operably linking the sequence to a reporter gene, and determining expression of the reporter.
  • recombinant polynucleotide refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, or polynucleotides, that are linked or joined to heterologous sequences.
  • RNA interference refers to a sequence-specific process by which a target molecule (e.g., a target gene, protein or RNA) is downregulated via downregulation of expression.
  • a target molecule e.g., a target gene, protein or RNA
  • RNAi involves degradation of RNA molecules, e.g.
  • RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs) triggered by dsRNA fragments cleaved from longer dsRNA which direct the degradative mechanism to other RNA sequences having closely homologous sequences.
  • RNAi can be initiated by human intervention to reduce or even silence the expression of target genes using either exogenously synthesized dsRNA or dsRNA transcribed in the cell (e.g., synthesized as a sequence that forms a short hairpin structure).
  • small interfering RNA and “short interfering RNA” (“siRNA”) refer to a short RNA molecule, generally a double-stranded RNA molecule about 10-50 nucleotides in length (the term “nucleotides” including nucleotide analogs), preferably between about 15-25 nucleotides in length. In most cases, the siRNA is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. Such siRNA can have overhanging ends (e.g., 3 '-overhangs of 1, 2, or 3 nucleotides (or nucleotide analogs). Such siRNA can mediate RNA interference.
  • the term “shRNA” refers to an RNA molecule having a stem-loop structure.
  • the stem-loop structure includes two mutually complementary sequences, where the respective orientations and the degree of complementarity allow base pairing between the two sequences.
  • the mutually complementary sequences are linked by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. , A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) 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 (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g. , A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Tip, Lys, Arg
  • sequence identity will be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo et al. (Applied Math 48:1073(1988)). More particularly, preferred computer programs for determining sequence identity include but are not limited to the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md.
  • BLAST Basic Local Alignment Search Tool
  • BLAST Manual Altschul et al, NCBI, NLM, NIH; (Altschul et al, J. Mol. Biol. 215:403-410 (1990)); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and for polynucleotide sequence BLASTN can be used to determine sequence identity.
  • Similarity refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table A below. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids ResearchU 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • reference sequence is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two
  • polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al
  • BESTFIT Pearson FASTA
  • FASTA Pearson's Alignment of sequences
  • TFASTA Pearson Sequence Protocol for polypeptidet al
  • the terms “transformed” and “transgenic” refer to any plant, plant cell, callus, plant tissue, or plant part that contains all or part of at least one isolated or recombinant (e.g., heterologous) polynucleotide.
  • all or part of the isolated or recombinant polynucleotide is stably integrated into a chromosome or stable extra- chromosomal element, so that it is passed on to successive generations.
  • transgene refers to any nucleotide sequence used in the transformation of a plant, animal, or other organism.
  • a transgene can be a coding sequence, a non-coding sequence, a cDNA, a gene or fragment or portion thereof, a genomic sequence, a regulatory element and the like.
  • a "transgenic" organism such as a transgenic plant, transgenic microorganism, or transgenic animal, is an organism into which a transgene has been delivered or introduced and the transgene can be expressed in the transgenic organism to produce a product, the presence of which can impart an effect and/or a phenotype in the organism.
  • 5' untranslated region refers to a sequence located upstream (i.e., 5') of a coding region.
  • a 5' UTR is located downstream (i.e., 3') to a promoter region and 5 ' of a coding region downstream of the promoter region.
  • sequence while transcribed, is upstream of the translation initiation codon and therefore is generally not translated into a portion of the polypeptide product.
  • 3' untranslated region refers to a nucleotide sequence downstream (i.e., 3') of a coding sequence. It generally extends from the first nucleotide after the stop codon of a coding sequence to just before the poly(A) tail of the corresponding transcribed mRNA.
  • the 3' UTR may contain sequences that regulate translation efficiency, mRNA stability, mRNA targeting and/or polyadenylation.
  • underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated in the absence of any underscoring or italicizing.
  • ABU shall mean the ABI1 gene
  • ABU shall indicate the protein product of the "ABU” gene.
  • the present invention is based in part on a novel cw-acting element that confers a pattern of inducibility on a promoter, which is similar to that of the alcohol dehydrogenase (ADH1) system of ⁇ . nidulans.
  • ADH1 alcohol dehydrogenase
  • a promoter that is not already inducible by contact with a chemical compound that can induce the expression of the alcohol dehydrogenase system of A. nidulans is made so inducible.
  • the resulting inducible promoter is useful for expressing polynucleotides that comprise a nucleic acid sequence encoding an expression product that inhibits stomatal closure.
  • the m-acting element comprises one or more nucleotide sequences selected from the group consisting of a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 in any combination, in any orientation, and/or in any order, including but not limited to multiples of the same nucleotide sequence.
  • the cw-acting element e.g. , the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, comprises, consists or consists essentially of, inverted repeats of the alcR inverted repeat binding sites, or variants thereof, of the A. nidulans alcohol dehydrogenase system (ADH1).
  • SEQ ID NO:l provides the nucleotide sequence GCGGNNCCGC (inverted repeat underlined), which suitably represents the minimal cis- acting sequence.
  • SEQ ID N0:2 provides the nucleotide sequence of n y GCGGNNCCGCn y .
  • N or n can be independently any nucleic acid base (A, G, C, or T), and x and y can be independently any number, as set forth below.
  • the cw-acting elements can comprise one or more nucleotides (i.e., bases) between the inverted repeats ⁇ e.g., an intervening sequence).
  • the intervening sequence include TT, AA, GG, CC, TA, TG, TC, AT, AG, AC, GT,GC, GA, CA, CT, or CG, and the like.
  • any 2-mer can be used as an intervening sequence in SEQ ID NO:l or 2, respectively.
  • SEQ ID NO:2 (ngGCGGNNCCGCii j , (flanking sequences bolded and underlined)) of the present invention can comprise zero to 100 or more nucleotides (i.e., bases) that flank the left (i.e., flanking sequence, n x ) and/or right side of the inverted repeats (i.e., flanking sequence, n y ).
  • x and y are each independently zero to 100 nucleotides.
  • the number of nucleotides in a flanking sequence can suitably be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, ' 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more, wherein each nucleotide in a flanking sequence can be independently adenine, thymine, gu
  • the cw-acting element can comprise, consist or consist essentially of one or more of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9.
  • the cw-acting element can comprise, consist or consist essentially of, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty etc., nucleotide sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, in any
  • the exacting element can comprise a multimer of any one or more of the nucleotide sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, wherein the nucleotide sequences of the multimer (e.g. , the m-acting element) can be the same and/or different from one another, in any combination, in any orientation, and/or in any order.
  • the nucleotide sequences of the multimer e.g. , the m-acting element
  • a cw-acting element comprising one nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and or SEQ ID NO:9, does not comprise only one nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
  • the number of nucleotide sequences in a cw-acting element can be in a range from one nucleotide sequence to about nine nucleotide sequences, from one nucleotide sequence to about ten nucleotide sequences, from one nucleotide sequence to about eleven nucleotide sequences, from one nucleotide sequence to about twelve nucleotide sequences, from one nucleotide sequence to about thirteen nucleotide sequences, from one nucleotide sequence to about fourteen nucleotide sequences, from one nucleotide sequence to about fifteen nucleotide sequences, from one nucleotide sequence to about sixteen nucleotide sequences, from one nucleotide sequence to about seventeen nucleotide sequences, from one nucleotide sequence to about eighteen nucleotide sequences, from one nucleotide sequence to about nineteen nucleotide sequences, from one nucleotide
  • the cw-acting element comprises, consists or consists essentially of at least two nucleotide sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, and/or SEQ ID NO:9, in any combination, in any orientation, and/or in any order.
  • the cis-acting element comprises, consists or consists essentially of at least three nucleotide sequences of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, in any combination, in any orientation, and/or in any order.
  • the c/s-acting element comprises, consists or consists essentially of at least five nucleotide sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, in any combination, in any orientation, and/or in any order.
  • the exacting element comprises, consists or consists essentially of about two to about nine nucleotide sequences of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, in any
  • the cw-acting element comprises, consists or consists essentially of about three to about nine nucleotide sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, and/or SEQ ID NO: 10, in any combination, in any orientation, and/or in any order.
  • the exacting element comprises, consists or consists essentially of about five to about nine nucleotide sequences of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, in any
  • the cw-acting element comprises, consists or consists essentially of about five to about fifteen nucleotide sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, in any combination, in any orientation, and/or in any order.
  • the c/s-acting element comprising multimers of the nucleotide sequences can comprise, consist or consist essentially of multimers of any of the nucleotide sequences of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, in any number of copies of a particular nucleotide sequence of the invention and/or in any combination, in any orientation, and/or in any order of the nucleotide sequences of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9.
  • the isolated nucleic acid molecule can comprise, consist essentially of, or consist of multiple copies of the same nucleotide sequence (e.g., 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, 10 copies, 11 copies, 12 copies, 13 copies, 14 copies, fifteen copies, sixteen copies, seventeen copies, eighteen copies, nineteen copies, twenty copies etc.).
  • the cw-acting element can comprise, consist or consist essentially of multiple nucleotide sequences as defined above each of which are different from one another.
  • the cz ' s-acting element can comprise, consist or consist essentially of multimers of the nucleotide sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, wherein some of the nucleotide sequences are the same (i.e., particular sequences are present in multiple copies) and some of the nucleotide sequences are different from one another, in any combination, in any orientation, and/or in any order.
  • non-limiting examples of a multimer of the nucleotide sequences defined above includes (SEQ ID NO:l) a (SEQ ID NO:2) b (SEQ ID NO:3) c (SEQ ID NO:4) d (SEQ ID NO:5) e (SEQ ID NO:6) f (SEQ ID NO:7) g (SEQ ID NO:8) h (SEQ ID NO:9) i5 wherein a, b, c, d, e, f, g, h, i are each independently 0 to 9 or more (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.).
  • multimers of the nucleotide sequences of the present invention include (SEQ ID NO:2) 4 ; (SEQ ID NO:5)(SEQ ID NO:3)(SEQ ID NO:9); (SEQ ID NO:2)(SEQ ID NO:5)(SEQ ID NO:3)(SEQ ID NO:4); (SEQ ID NO:2) 2 (SEQ ID NO:6) 4 (SEQ ID NO:3) 3 ; (SEQ ID NO:2) 2 (SEQ ID NO:6)(SEQ ID NO:3)(SEQ ID NO:4) 2 ; (SEQ ID NO:5) 3 (SEQ ID NO:3)(SEQ ID NO:4); (SEQ ID NO:2)(SEQ ID NO:5) 2 (SEQ ID NO:3) 2 ; (SEQ ID NO:6) 4 (SEQ ID NO:4) 2 ; (SEQ ID NO:2)(SEQ ID NO:6) 2 (SEQ ID NO:6) 2 (SEQ ID NO:4) 2 ; (SEQ ID NO:2)(SEQ ID NO:6) 2 (SEQ ID NO
  • the c/s-acting elements comprising multimers of the nucleotide sequences of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9, can further comprise one or more nucleotides (i.e., bases) between each of the inverted repeats (i.e., between the nucleotide sequences of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9) (e.g., a spacer sequence).
  • nucleotides i.e., bases
  • the spacer sequences are the same as (i.e., equivalent to) the flanking sequences (i.e., n x , n y ) described herein.
  • the cw-acting element comprises, consists or consists essentially of spacer sequences (i.e., flanking sequences, n x , n y ) that can be the same as one another (i.e., the same as other spacer sequences of the nucleic acid molecule) and/or different than one another, or any combination thereof.
  • spacer sequences i.e., flanking sequences, n x , n y
  • a non-limiting example of a multimer of the present invention comprising a spacer sequence is the following:
  • the spacer sequences are bolded and underlined and are shown to be of different lengths.
  • Further non-limiting examples of a multimer comprising a spacer sequence are the following:
  • the bolded and underlined nucleotides represent both the spacer sequences and the flanking sequences of the nucleotide sequences that comprise the multimer (/. e. , in these examples, the spacer sequences are equivalent to the flanking sequences).
  • the cw-acting elements described above and elsewhere herein are useful for conferring a pattern of inducibility on a promoter, including a promoter that is operable in a plant cell (e.g. , a guard cell), which pattern is similar to that of the alcohol dehydrogenase (ADH1) system of A. nidulans.
  • ADH1 alcohol dehydrogenase
  • promoters that can be made inducible with the subject m-acting elements can include chemically inducible promoters that are not naturally or endogenously inducible by the same compounds/chemicals that induce the alcohol dehydrogenase system of A. nidulans. Additionally, promoters useable with the present invention can include those that drive expression of a nucleotide sequence
  • a promoter that can be made inducible with the cw-acting elements described above and elsewhere herein includes a minimal promoter.
  • a minimal promoter is a promoter having only the nucleotides/nucleotide sequences from a selected promoter that are required for binding of the transcription factors and transcription of a nucleotide sequence of interest that is operably associated with the minimal promoter including but not limited to TATA box sequences. These portions or sequences from a promoter are generally placed upstream ⁇ i.e., 5') of a nucleotide sequence to be expressed.
  • nucleotides/nucleotide sequences from any promoter useable with the present invention can be selected for use as a minimal promoter.
  • Any promoter may be altered to generate a minimal promoter by progressively removing nucleotides from the promoter until the promoter ceases to function in order to identify the minimal promoter.
  • the smallest fragment of a promoter which still functions as a promoter is also considered a minimal promoter.
  • the promoter may be endogenous to the plant.
  • a heterologous promoter may be employed.
  • a promoter can be heterologous when it is operably linked to a polynucleotide from a species different from the species from which the polynucleotide was derived.
  • a promoter can be heterologous to a selected nucleotide sequence if the promoter is from the same/analogous species from which the polynucleotide is derived, but one or both (/ ' . e. , promoter and/or polynucleotide) are modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.
  • promoters useable with the present invention can be made among many different types of promoters. This choice generally depends upon several factors, including, but not limited to, cell- or tissue-specific expression, desired expression level, efficiency, inducibility and/or selectability. For example, where expression in a specific tissue or organ is desired in addition to inducibility, a tissue-specific promoter can be used (e.g., a guard cell specific promoter). In contrast, where expression in response to a stimulus is desired in addition to inducibility via chemical compounds that induce the expression of the alcohol dehydrogenase system of A. nidulans, a promoter inducible by other stimuli or chemicals can be used.
  • a constitutive promoter can be chosen.
  • Non-limiting examples of constitutive promoters include cestrum virus promoter (cmp) (U.S. Patent No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as US Patent No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA 84:5745- 5749), Adh promoter (Walker et al.
  • tissue-specific promoters include those encoding the seed storage proteins (such as ⁇ -conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g. , Kridl et al. (1991) Seed Sci. Res. 1 :209-219; as well as EP Patent No. 255378).
  • the promoters associated with these tissue-specific nucleic acids can be used in the present invention.
  • tissue-specific promoters include, but are not limited to, the root- specific promoters RCc3 (Jeong et al. Plant Physiol. 153:185-197 (2010)) and RB7 (U.S. Patent No. 5459252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res.
  • SAMS S-adenosyl-L-methionine synthetase
  • corn light harvesting complex promoter Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658
  • com heat shock protein promoter O'Dell et al. (1985) EMBOJ. 5:451-458; and Rochester et al. (1986) EMBOJ.
  • RuBP carboxylase promoter Ceashmore, "Nuclear genes encoding the small subunit of ribulose-l,5-bisphosphate carboxylase" 29-39 In: Genetic Engineering of Plants (Hollaender ed., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), Ti plasmid mannopine synthase promoter (Langridge «?/ al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopaline synthase promoter (Langridge et al.
  • petunia chalcone isomerase promoter van Tunen et al. (1988) EMBOJ. 7:1257-1263
  • bean glycine rich protein 1 promoter Kerman et al. (1989) Genes Dev. 3 : 1639- 1646
  • truncated CaMV 35S promoter O'Dell et al. (1985) Nature 313:810-812)
  • potato patatin promoter Wenzler et al. (1989) Plant Mol. Biol. 13:347-354
  • root cell promoter Yamampto et al. (1990) Nucleic Acids Res. 18:7449
  • maize zein promoter Yama et al. (1987) Mol.
  • the promoter that is operably linked to the cis- acting element is one that is specifically or preferentially operable in a plant guard cell.
  • guard cell-specific or guard cell-preferential promoters include:
  • Arabidopsis trehalase gene promoter (EP 1111051), potato KST1 promoter (Plesch et al.
  • Arabidopsis pGCl Arabidopsis KATl (At5g46240) potassium channel promoter (Nakumura et al. (1995) Plant Physiol. 109, 371-374), Arabidopsis AtMYB60 (Atlg08810) promoter (U.S. Pat. Appl. Pub. No. 20080064091), gcPepC promoter (Kopka et al. (1997) Plant J ' 11, 871-882), Arabidopsis AtCYP86A2 promoter (Francia et al. (2008) Plant
  • At5g58580 promoters designated pSUH305S, pSUH305, pSUH305GB, pSUK132, pSUK134, pSUK136, pSUK342, pSUK344, pSUK132GB, pSUK134GB, pSUK136GB, pSUK342GB, pSUK344GB (U.S. Pat. Appl. Pub. 20060117408).
  • promoters functional in plastids can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5' UTR and other promoters disclosed in U.S. Patent No. 7,579,516.
  • Other promoters useful with the present invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the unitz trypsin inhibitor gene promoter (Kti3).
  • inducible promoters that are not inducible by the same compounds that induce expression of the alcohol dehydrogenase system of A. nidulans are useable with cw-acting sequence described above and elsewhere herein.
  • inducible promoters useable with the present invention include, but are not limited to, tetracycline repressor system promoters, Lac repressor system promoters, copper-inducible system promoters, salicylate-inducible system promoters ⁇ e.g., the PRla system),
  • glucocorticoid-inducible promoters (Aoyama et al. (1997) Plant J. 11 :605- 612), and ecdysone-inducible system promoters.
  • inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoid glycosyltransferase promoter (Ralston et al. (1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al. (1994) Plant J.
  • the m-acting elements described herein are operably linked to a promoter so as to control the activity of the promoter.
  • the activity or strength of a promoter may be measured in terms of the amount of mRNA or protein accumulation it specifically produces, relative to the total amount of mRNA or protein.
  • an operably linked cis-acting element as described herein is placed at a distance from the promoter so that: (1) in the absence of a compound that induces expression of the alcohol dehydrogenase system of A.
  • the promoter suitably expresses an operably linked nucleic acid sequence at a level no more than 1%, 0.1%, 0.01%, 0.001%, 0.0001% or 0.00001% of the total cellular RNA or protein; and (2) in the presence of a compound that induces expression of the alcohol dehydrogenase system of A.
  • the promoter suitably expresses an operably linked nucleic acid sequence at a level greater than 0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% (w/w) of the total cellular RNA or protein. Positioning of the cw-acting element relative to the promoter can be determined by the skilled person using customary
  • the cw-acting element may be upstream or downstream of the promoter. In specific embodiments, the cw-acting element is upstream of the promoter.
  • the distance between the cw-acting element and the promoter is no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 nt.
  • the distance between the m-acting element and the promoter is less than 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550 nt.
  • Non-limiting examples of chemical compounds that can induce the promoters of the present invention include a primary alcohol, a primary monoamine, a ketone, a C3 to C ketone, a methyl ketone, a hydrolysable ester, an aliphatic aldehyde, ethanol, allyl alcohol acetaldehyde, ethyl methyl ketone, acetone, emylamine, cyclohexanone, butan-2-ol, 3-oxobutyric acid, propan-2-ol, propan-l-ol, butan-2- ol, threonine, and/or any combination thereof.
  • inducer compound include a primary alcohol, a primary monoamine, a ketone, a C3 to C ketone, a methyl ketone, a hydrolysable ester, an aliphatic aldehyde, ethanol, allyl alcohol acetaldehyde, ethyl methyl
  • An inducer compound can be provided in any concentration that is not toxic to the plant.
  • the concentration of the inducer compound is about 0.01 % to about 10% (v/v) or more.
  • the concentration of the inducer compound is about 0.1 % to about 20% (v/v).
  • the concentration of the inducer compound is about 0.1 % to about 5% (v/v).
  • the concentration of the inducer compound is about 1% to about 2%.
  • the concentration of the inducer compound can be about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%. about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.6%.
  • the inducer compound can be provided as a root drench, a spray, a mist, a suspension, an emulsion, a powder, a granule, an aerosol, a foam, a paste, a dip, a vapor, a paint, and the like, and combinations thereof.
  • the concentration of the inducer compound can be, for example, in a concentration of about 95% to about 100% (v/v).
  • the inducer compound provided in a concentration of about 95% to about 100% (v/v), can be placed in proximity to the plant, plant part, plant organ or plant leaf (e.g., in a container such as a tube, a dish, and the like, or on a cloth, paper, beads, and the like, that is soaked in the inducer compound), thereby exposing the plant, plant part, plant organ or plant leaf to a vapor comprising the inducer compound.
  • the inducer compound is provided in more than one form.
  • the inducer compound can be provided as a foliar spray and as a root drench.
  • the concentration of the inducer compound when applied in more than one form can be the same or can be different in the different forms provided.
  • a foliar spray and a root drench can be provided at the same and/or a different concentration than one another.
  • constructs of the present invention also comprise an operably connected nucleic acid sequence encoding an expression product that inhibits stomatal closure.
  • the expression product inhibits or abrogates the activity or function of an endogenous polypeptide of the plant, which stimulates or otherwise facilitates stomatal closure.
  • the expression product is a negative regulator of stomatal closure.
  • endogenous polypeptides include ABI1, ABI2 (Himmelbach e/ fl/. (1998) Philos. Trans. R. Soc. Lond. B Biol. Sci. 353, 1439-1444; Leung et al. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol.
  • OST1 Mostilli et al. (2002) The Plant Cell 14, 3089-3099; U.S. Patent No. 7,211,436), AAPK (related to OST1) (Li et al. (2000) Science 287(5451), 300-303), AHA1 (also referred to as OST2) (Merlot et al, (2007) EMBO J. 26, 3216-3226), v-SNAREs AtVAMP711-14 (Leshem et al., (2010) J. Exp. Bot. 61, 2615-2622), GPA1 (Wang et al. (2001) Science 292, 2070-2072), AtABCG22 ( uromori et al. (2011) Plant J.
  • AtABCG40 ang et al. , (2010) Proc. Natl. Acad. Sci. USA 107, 2355-2360
  • AtMRP4 Klein et al. (2004) Plant J. 219-236
  • RBOHD and RBOHF Kwak et al. (2003) EMBO J. 22, 2623-2633
  • PLDalphal Zhang et al. (2004) Proc. Natl. Acad. Sci. USA 101, 9508-9513
  • PKS3 Guo et al. (2002) Developmental Cell 3, 233-244
  • ATHB6 Himmelbach et al. (2002) EMBO J. 21, 3029-3038; Grill E. (2002) EMBO J. 21 :3029-38.
  • Amino acid sequences corresponding to the above endogenous polypeptides as well as nucleic acid sequences corresponding to genes that code for these polypeptides are useful for modulating stomatal closure, as described below.
  • the present invention contemplates the use of any suitable stomatal closure- modulating polypeptide and polynucleotide in the practice of the invention.
  • non-limiting ABI1 polypeptides comprise the amino acid sequence:
  • NP l 94338 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO: 11.
  • a representative ABI1 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 11, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:l 1, a complement of that nucleotide sequence.
  • an ABI1 nucleic acid sequence comprises the nucleotide sequence:
  • gaagcaattgttgcattagcctacccatttcctccttcttctctcttc cttc ctgtgaacaaggcacattagaactct tcttr caactttttaggtgtatatagatgaatctagaatagttttatagttgg
  • actttacatac [SEQ ID NO: 12], as set forth for example in GenBank Accession NM l 18741, or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:12, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO: 12 or to a complement thereof.
  • Non-limiting ABI2 polypeptides comprise the amino acid sequence:
  • CAA70162 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO: 13.
  • a representative ABI2 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 13, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO: 13, or a complement of that nucleotide sequence.
  • an ABI2 nucleic acid sequence comprises the nucleotide sequence:
  • Y08966, or a complement thereof or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 14 or 15, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO : 14 or 15 , or to a complement thereof.
  • An illustrative OST1 polypeptide comprises the amino acid sequence:
  • CAC87047 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO: 16.
  • a non-limiting OS77 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 16, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO: 16, or a complement of that nucleotide sequence.
  • an OSTJ nucleic acid sequence comprises the nucleotide sequence:
  • AJ316009 or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 17, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO: 17, or to a complement thereof.
  • AAPK polypeptides comprises the amino acid sequence:
  • AAF27340 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:58.
  • Illustrative AAPK nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 58, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:58, or a complement of that nucleotide sequence.
  • an AAPK nucleic acid sequence comprises the nucleotide sequence:
  • aaaa [SEQ ID NO:59], as set forth for example in GenBank Accession No. API 86020, or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:59, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:59, or to a complement thereof.
  • a non-limiting AHA1 polypeptide comprises the amino acid sequence: [0162] MSGLEDIKNETVDLEKIPIEEVFQQLKCTREGLTTQEGEDRIVIFGPN KLEEKKESKILKFLGFMWWLSW MEAAALMAIALANGDNRPPDWQDFVGIICLLVI NSTISFIEENNAGNAAAALMAGLAPKTKVLRDGKWSEQEAAILVPGDIVSIKLGDIIPA DARLLEGDPLKVDQSALTGESLPVTKHPGQEVFSGSTCKQGEIEAVVIATGVHTFFGK AAHLVDSTNQVGHFQ VLTSIGNFCICSIAIGIAIEIVVMYPIQHRKYRDGIDNLLVLLI GGIPIAMPTVLSVTMAIGSHRLSQQGAITKRMTAIEEMAGMDVLCSD TGTLTLNKLS VDKNLVEVFCKGVEKDQVLLFAAMASRVENQDAIDAAMVGMLADPKEARAGIREV HFLPFNPVD RTALTYIDSD
  • NP l 79486 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO: 18.
  • a representative AHA1 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 18, , or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO: 18, , or a complement of that nucleotide sequence.
  • an AHA1 nucleic acid sequence comprises the nucleotide sequence:
  • NM_127453j or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 19, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO: 19, or to a complement thereof.
  • AtVAMP711-14 polypeptides comprise the amino acid sequence:
  • SEQ ID NO:20 as set forth for example in GenPept Accession No. NP_194942, or an amino acid sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:20.
  • a representative vSNAREs AtVAMPl 11-14 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:20, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:20, or a complement of that nucleotide sequence.
  • a vSNAREs AtVAMPl 11 nucleic acid sequence comprises the nucleotide sequence:
  • cttc [SEQ ID N0:21], as set forth for example in GenBank Accession No. NM_119367, or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:21, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:21, or to a complement thereof.
  • a non-limiting GPA1 polypeptide comprises the amino acid sequence:
  • NP l 80198 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:22.
  • a non-limiting GPA1 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:22, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:22, or a complement of that nucleotide sequence.
  • a GPA1 nucleic acid sequence comprises the nucleotide sequence: [0172] gttaacttaatagtatataaaaiaaaatgcatataggttccgtaattaatcttctttatcgtcacgagaggcacat cttttttcaacatttgaccactctctctctct ⁇
  • SEQ ID NO:23 as set forth for example in GenBank Accession No. NM_128187, or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:23, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:23, or to a complement thereof.
  • An illustrative AtABCG22 polypeptide comprises the amino acid sequence:
  • NP_001031843 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:24.
  • a representative AtABCG22 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 24, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:24, a complement of that nucleotide sequence.
  • an AtABCG22 nucleic acid sequence comprises the nucleotide sequence:
  • NM 001036766 or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:25, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:25, or to a complement thereof.
  • Non-limiting AtABCG40 polypeptides comprise the amino acid sequence:
  • VTTGEMLVGPSRALFMDEISTGLDSSTTYQIVNSLRNYVHIFNGTALISLLQPAPETFN LFDDIILIAEGEIIYEGPRDHWEFFETMGFKCPPPvKGVADFLQEVTSKKDQMQYWAR RDEPYRFIRVREFAEAFQSFHVGRRIGDELALPFDKTKSHPAALTTKKYGVGIKELVK TSFSREYLLMKRNSFVYYFKFGQLLVMAFLTMTLFFRTEMQKKTEVDGSLYTGALFF ILMMLMFNGMSELSMTIAKLPVFYKQRDLLFYPAWVYSLPPWLLKIPISFMEAALTTF ITYYVIGFDPNVGRLFKQYILLVLMNQMASALFKMVAALGRNMIVANTFGAFAMLV FFALGGWLSRDDIKKWWIWGYWISPIMYGQNAILANEFFGHSWSRAVENSSETLGV TFLKSRGFLPHAYWYWIGTGALLGFVVLFNFGFTL
  • SEQ ID NO:26 as set forth for example in GenPept Accession No. NP l 73005, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:26.
  • a non-limiting AtABCG40 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:26, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:26, or a complement of that nucleotide sequence.
  • mAtABCG40 nucleic acid sequence comprises the nucleotide sequence:
  • NM 101421, or a complement thereof or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:27, or to a complement thereof, or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:27, or to a complement thereof.
  • AtMRP4 polypeptide comprises the amino acid sequence:
  • NP_182301 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:28.
  • a representative AtMRP4 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:28, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid; 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:28; or a complement of that nucleotide sequence.
  • an AtMRP4 nucleic acid sequence comprises the nucleotide sequence:
  • ctaccaacacgtgaatttttctc [SEQ ID NO:29], as set forth for example in GenBank Accession No. NM_130347, or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:29, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:29, or to a complement thereof.
  • a non-limiting RBOHD polypeptide comprises the amino acid sequence:
  • NP_199602 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:30.
  • a non-limiting RBOHD nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:30, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO.30, or a complement of that nucleotide sequence.
  • a RBOHD nucleic acid sequence comprises the nucleotide sequence:
  • RBOHF polypeptide comprises the amino acid sequence:
  • NP 564821 or an amino acid sequence having at least 70%, ' 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:32.
  • a representative RBOHF nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:32, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:32, or a complement of that nucleotide sequence.
  • a RBOHF nucleic acid sequence comprises the nucleotide sequence:
  • NM l 05079 or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:33, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:33, or to a complement thereof;.
  • Non-limiting PLDalphal polypeptides comprise the amino acid sequence:
  • NP 188194 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:34.
  • a non-limiting PLDalphal nucleic acid sequence comprises a nucleotide sequence encoding the sequence set forth in SEQ ID NO:34, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:34, or a complement of that nucleotide sequence.
  • an PLDalphal nucleic acid sequence comprises the nucleotide sequence:
  • nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:35> or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:35, or to a complement thereof.
  • Non-limiting examples ofPKS3 polypeptides comprise the amino acid sequence:
  • AAK26842 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:60.
  • a representative PKS3 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:60, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:60, or a complement of that nucleotide sequence.
  • an PKS3 nucleic acid sequence comprises the nucleotide sequence:
  • AF339144 or a complement thereof; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:37, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:61, or to a complement thereof.
  • An illustrative ATHB6 polypeptide comprises the amino acid sequence:
  • NP 565536 or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:36.
  • a representative ATHB6 nucleic acid sequence comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 36, or a complement of the nucleotide sequence; or a nucleotide sequence encoding an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:36, or a complement of that nucleotide sequence.
  • an ATHB6 nucleic acid sequence comprises the nucleotide sequence:
  • nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:37, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:37, or to a complement thereof.
  • the encoded expression product is a dominant negative form of a polypeptide that stimulates or otherwise facilitates stomatal closure, illustrative examples of which include dominant negative forms of AAPK ⁇ e.g. ,
  • the encoded expression product is an antibody that is immuno-interactive with the endogenous polypeptide that stimulates or otherwise facilitates stomatal closure.
  • the endogenous polypeptide is selected from, OST1, AAPK, v-SNAREs AtVAMP711-14, GPA1, AtABCG22, AtABCG40, AtMRP4, RBOHD, RBOHF and PLDalphal .
  • Exemplary antibodies for use in the practice of the present invention include monoclonal antibodies, Fv, Fab, Fab' and F(ab')2
  • immunoglobulin fragments as well as synthetic antibodies such as but not limited to single domain antibodies (DABs), synthetic stabilized Fv fragments, e.g., single chain Fv fragments (scFv), disulfide stabilized Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv or engineered human equivalents.
  • DABs single domain antibodies
  • scFv single chain Fv fragments
  • dsFv disulfide stabilized Fv fragments
  • dAbs single variable region domains
  • minibodies combibodies and multivalent antibodies such as diabodies and multi-scFv or engineered human equivalents.
  • antibodies can be made by conventional immunization (e.g., polyclonal sera and hybridomas) with isolated, purified or recombinant peptides or proteins corresponding to at least a portion of an endogenous polypeptide, or as recombinant fragments corresponding to at least a portion of an endogenous polypeptide, or as recombinant fragments corresponding to at least a portion of an endogenous polypeptide, or as recombinant fragments corresponding to at least a portion of an
  • endogenous polypeptide usually expressed in Escherichia coli, after selection from phage display or ribosome display libraries (e.g., available from Cambridge Antibody Technology, Biolnvent, AfFitech and Biosite).
  • phage display or ribosome display libraries e.g., available from Cambridge Antibody Technology, Biolnvent, AfFitech and Biosite.
  • complementarity-determining regions of such antibodies can be used to prepare synthetic antibodies as described for example above.
  • the expression product inhibits by RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) the expression of a target gene, which encodes a polypeptide that stimulates or otherwise facilitates stomatal closure.
  • RNAi RNA interference
  • PTGS post-transcriptional gene silencing
  • the expression product is a RNA molecule (e.g. , siRNA, shRNA, miRNA, dsRNA etc.) that comprises a targeting region corresponding to a nucleotide sequence of the target gene and that attenuates or otherwise disrupts the expression of the target gene.
  • target genes include AAPK, OST1, v-SNAREs AtVAMP711-14, GPA1, AtABCG22, AtABCG40, AtMRP4, RBOHD, RBOHF, and
  • the targeting sequence displays at least 60, 61 , 62,
  • the targeting sequence hybridizes to a nucleotide sequence of the target gene under at least low stringency conditions, more suitably under at least medium stringency conditions and even more suitably under high stringency conditions.
  • low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C.
  • Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0 4 (pH 7.2), 5% SDS for washing at room temperature.
  • BSA Bovine Serum Albumin
  • Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42° C.
  • Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2x
  • BSA Albumin
  • 1 mM EDTA 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0 4 (pH 7.2), 5% SDS for washing at 42° C.
  • High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42° C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42° C.
  • High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, lmM EDTA, 40 mM NaHP0 4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C.
  • the targeting sequence hybridizes to a nucleotide sequence of the target gene under physiological conditions.
  • the targeting region has sequence identity with the sense strand or antisense strand of the target gene.
  • the RNA molecule is
  • the length of the targeting region may vary from about 10 nucleotides (nt) up to a length equaling the length (in nucleotides) of the target gene.
  • the length of the targeting region is at least 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nt, usually at least about 50 nt, more usually at least about 100 nt, especially at least about 150 nt, more especially at least about 200 nt, even more especially at least about 500 nt. It is expected that there is no upper limit to the total length of the targeting region, other than the total length of the target gene. However for practical reason (such as e.g., stability of the targeting constructs) it is expected that the length of the targeting region should not exceed 5000 nt, particularly should not exceed 2500 nt and could be limited to about 1000 nt.
  • the RNA molecule may further comprise one or more other targeting regions (e.g., from about 1 to about 10, or from about 1 to about 4, or from about 1 to about 2 other targeting regions) each of which has sequence identity with a nucleotide sequence of the target gene.
  • one or more other targeting regions e.g., from about 1 to about 10, or from about 1 to about 4, or from about 1 to about 2 other targeting regions
  • the targeting regions are identical or share at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with each other.
  • the RNA molecule may further comprise a reverse complement of the targeting region.
  • the RNA molecule further comprises a spacer sequence that spaces the targeting region from the reverse complement.
  • the spacer sequence may comprise a sequence of nucleotides of at least about 100-500 nucleotides in length, or alternatively at least about 50-100 nucleotides in length and in a further alternative at least about 10-50 nucleotides in length.
  • the spacer sequence is a non-coding sequence, which in some instances is an intron.
  • transcription of the nucleic acid sequence will produce an RNA molecule that forms a hairpin or stem-loop structure in which the stem is formed by hybridization of the targeting region to the reverse complement and the loop is formed by the non-intron spacer sequence connecting these 'inverted repeats'.
  • the spacer sequence is an intron spacer sequence
  • the presence of intron/exon splice junction sequences on either side of the intron sequence facilitates the removal of what would otherwise form a loop structure and the resulting RNA will form a double-stranded RNA (dsRNA) molecule, with optional overhanging 3' sequences at one or both ends.
  • dsRNA double-stranded RNA
  • Such a dsRNA transcript is referred to herein as a "perfect hairpin”.
  • the RNA molecules may comprise a single hairpin or multiple hairpins including "bulges" of single- stranded RNA occurring adjacent to regions of double-stranded RNA sequences.
  • a dsRNA molecule as described above can be conveniently obtained using an additional polynucleotide from which a further RNA molecule is producible, comprising the reverse complement of the targeting region.
  • the reverse complement of the targeting region hybridizes to the targeting region of the RNA molecule transcribed from the second polynucleotide.
  • a dsRNA molecule as described above is prepared using a second polynucleotide that comprises a duplex, wherein one strand of the duplex shares sequence identity with a nucleotide sequence of the target gene and the other shares sequence identity with the complement of that nucleotide sequence.
  • the duplex is flanked by two promoters, one controlling the transcription of one of the strands, and the other controlling the transcription of the complementary strand. Transcription of both strands produces a pair of RNA molecules, each comprising a region that is complementary to a region of the other, thereby producing a dsRNA molecule that inhibits the expression of the target gene.
  • RNA molecule further comprises two nucleic acid sequences and their reverse complement.
  • suitable nucleic acid sequences and their reverse complement can be used to alter the expression of any homologous, endogenous target RNA (i.e. , comprising a transcript of the target gene) which is in proximity to the suitable nucleic acid sequence and its reverse complement.
  • the suitable nucleic acid sequence and its reverse complement can be either unrelated to any endogenous RNA in the host or can be encoded by any nucleic acid sequence in the genome of the host provided that nucleic acid sequence does not encode any target mRNA or any sequence that is substantially similar to the target RNA.
  • the RNA molecule further comprises two
  • the RNA molecule further comprises two complementary RNA regions which are encoded by any nucleic acid sequence in the genome of the host provided that the sequence does not have sequence identity with the nucleotide sequence of the target gene, wherein the regions are in proximity to the targeting region.
  • one of the complementary RNA regions can be located upstream of the targeting region and the other downstream of the targeting region.
  • both the complementary regions can be located either upstream or downstream of the targeting region or can be located within the targeting region itself.
  • the RNA molecule is an antisense molecule that is targeted to a specific region of R A encoded by the target gene, which is critical for translation.
  • the use of antisense molecules to decrease expression levels of a pre-determined gene is known in the art.
  • Antisense molecules may be designed to correspond to full-length RNA transcribed from the target gene, or to a fragment or portion thereof. This gene silencing effect can be enhanced by transgenically over-producing both sense and antisense RNA of the target gene coding sequence so that a high amount of dsRN A is produced as described for example above (see, for example, Waterhouse et al. (1998) Proe Natl Acad Sci USA 95:13959 13964).
  • the expression product that inhibits stomatal closure corresponds to an expression product of the endogenous target gene targeted for repression. In many cases, this "co-suppression" results in the complete repression of the native target gene as well as the transgene.
  • the expression product that inhibits stomatal closure corresponds to an expression product of a negative regulator of the ABA signaling pathway.
  • the negative regulator is ATHB6.
  • the nucleic acid sequence encoding the expression product that inhibits stomatal closure comprises a nucleotide sequence corresponding to the coding sequence of ATHB6.
  • a non-limiting example ⁇ & ⁇ coding sequence is represented the following sequence:
  • any other nucleotide sequence that codes for the amino acid sequence of ATHB6 may be used.
  • a non-limiting example of a ATHB6 amino acid sequence is represented by [SEQ ID NO:36], or an amino acid having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:36, as described for example above.
  • the negative regulator is selected from ABI1 , ABI2 or mutant forms of ABI1 (e.g., ABIl G,yl80Asp ) or ABI2 (e.g., ABI2 G,yl68Asp ), which result in reduced ABA sensitivity and/or which inhibit stomatal closure. While not limiting the invention to any one mechanism or mode of operation, these mutant proteins may have reduced susceptibility to ABA inhibition than the wild-type counterparts, or compete with their wild-type counterparts for interacting proteins in the transgenic plant, or poison multimeric complexes that normally recruit the wild-type counterparts.
  • ABI1 e.g., ABIl G,yl80Asp
  • ABI2 e.g., ABI2 G,yl68Asp
  • the negative regulator is a dominant positive AHAl mutant (e.g., a constitutively active AHAl polypeptide), illustrative examples of which include AHAl Ttp875Leu ; AHAl 8861 ; AHAl ul69Phe ; AHAl Gly867Ser ; AHAl Glul0Asp ;
  • AHAl mutant e.g., a constitutively active AHAl polypeptide
  • the negative regulator is a dominant positive
  • PKS3 mutant an non-limiting example of which includes the dominant positive PKS3 deletion mutant disclosed by Guo et al. (2002, supra).
  • promoters and nucleic acid sequence encoding an expression product that inhibits stomatal closure described above can also include other regulatory sequences.
  • regulatory sequences means nucleotide sequences located upstream (5' non-coding sequences), within or downstream (3 1 non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, enhancers, introns, translation leader sequences and polyadenylation signal sequences.
  • leader sequences derived from viruses are known to enhance gene expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the " ⁇ -sequence"), Maize Chlorotic Mottle Virus (MCMV) and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (Gallie et al. (1987) Nucleic Acids Res. 15 :8693-8711 ; and Skuzeski et al. ( 1990) Plant Mol. Biol. 15 :65-79).
  • 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 such as an encephalomyocarditis (EMCV) 5' noncoding region leader (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders such as a Tobacco Etch Virus (f EV) leader (Allison et al. (1986) Virology 154:9-20); Maize Dwarf Mosaic Virus (MDMV) leader (Allison et al.
  • picornavirus leaders such as an encephalomyocarditis (EMCV) 5' noncoding region leader (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders such as a Tobacco Etch Virus (f EV) leader (Allison et al. (1986) Virology 154:9-20); Maize Dwarf
  • translational enhancers are employed such as the overdrive-sequence containing the 5'-untranslated leader sequence from tobacco mosaic virus enhancing the polypeptide per RNA ratio (Gallie et al. (1987) Nucleic Acids Research 15:8693-8711).
  • An expression construct also can optionally include a transcriptional and/or translational termination region (i. e. , termination region) that is functional in plants.
  • a transcriptional and/or translational termination region i. e. , termination region
  • a variety of transcriptional terminators are available for use in expression constructs and are
  • the termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the plant host, or may be derived from another source (i. e. , foreign or heterologous.to the promoter, the nucleotide sequence of interest, the plant host, or any combination thereof).
  • Appropriate transcriptional terminators include, but are not limited to, the CAMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcs E9 terminator. These can be used in both monocotyledons and dicotyledons.
  • a coding sequence's native transcription terminator can be used.
  • a signal sequence can be operably linked to a nucleic acid molecule of the present invention to direct the nucleic acid molecule into a cellular compartment.
  • the expression construct will comprise a nucleic acid molecule of the present invention operably linked to a nucleotide sequence for the signal sequence.
  • the signal sequence may be operably linked at the N- or C- terminus of the nucleic acid molecule.
  • Exemplary polyadenylation signals can be those originating from Agrobacterium tumefaciens t-DNA such as the gene known as octopine synthase of the Ti- plasmid pTiACH5 (Gielen et al. (1984) EMBOJ. 3:835) or functional equivalents thereof, but also all other terminators functionally active in plants are suitable.
  • the expression construct also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed plant, plant part and/or plant cell.
  • selectable marker means a nucleotide sequence that when expressed imparts a distinct phenotype to the plant, plant part and/or plant cell expressing the marker and thus allows such transformed plants, plant parts and/or plant cells to be distinguished from those that do not have the marker.
  • Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g. , the R-locus trait).
  • a selective agent e.g., an antibiotic, herbicide, or the like
  • screening e.g. , the R-locus trait
  • selectable markers include, but are not limited to, a nucleotide sequence encoding neo or nptll, which confers resistance to kanamycin, G418, and the like (Potrykus et al. (1985) Mol. Gen. Genet. 199:183-188); a nucleotide sequence encoding bar, which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5- enolpyruvylshikimate-3 -phosphate (EPSP) synthase, which confers resistance to glyphosate (Hinchee et al. (1988) Biotech.
  • a nucleotide sequence encoding neo or nptll which confers resistance to kanamycin, G418, and the like
  • a nucleotide sequence encoding bar which confers resistance to phosphinothricin
  • a nucleotide sequence encoding a nitrilase such as bxn from Klebsiella ozaenae that confers resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP
  • Patent Application No. 154204 a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263:12500-12508); a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleotide sequence encoding a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI)) that confers an ability to metabolize mannose (US Patent Nos.
  • DHFR methotrexate-resistant dihydrofolate reductase
  • PMI phosphomannose isomerase
  • nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5 -methyl tryptophan and/or a nucleotide sequence encoding hph that confers resistance to hygromycin.
  • One of skill in the art is capable of choosing a suitable selectable marker for use in an expression construct of this invention.
  • Additional selectable markers include, but are not limited to, a nucleotide sequence encoding ⁇ -glucuronidase or uidA (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus nucleotide sequence that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., "Molecular cloning of the maize R-nj allele by transposon-tagging with Ac” 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); a nucleotide sequence encoding ⁇ -lactamase, an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc.
  • GUS ui
  • nucleotide sequence encoding ⁇ -galactosidase an enzyme for which there are chromogenic substrates
  • nucleotide sequence encoding luciferase (lux) that allows for bioluminescence detection
  • An expression construct of the present invention also can include nucleotide sequences that encode other desired traits.
  • Such nucleotide sequences can be stacked with any combination of nucleotide sequences to create plants, plant parts or plant cells having the desired phenotype. Stacked combinations can be created by any method including, but not limited to, cross breeding plants by any conventional methodology, or by genetic
  • nucleotide sequences of interest can be combined at any time and in any order.
  • a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation.
  • the additional nucleotide sequences can be introduced
  • nucleotide sequence can be incorporated in separate cassettes (trans) or can be incorporated on the same cassette (cis).
  • expression of the nucleotide sequences can be driven by the same promoter or by different promoters. It is further recognized that nucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, e.g., Int'l Patent Application Publication Nos. WO 99/25821 ; WO 99/25854; WO 99/25840; WO 99/25855 and WO 99/25853.
  • the expression construct can include a coding sequence for one or more polypeptides for agronomic traits that primarily are of benefit to a seed company, grower or grain processor.
  • a polypeptide of interest can be any polypeptide encoded by a nucleotide sequence of interest.
  • Non-limiting examples of polypeptides of interest that are suitable for production in plants include those resulting in agronomically important traits such as herbicide resistance (also sometimes referred to as "herbicide tolerance"), virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, and/or fungal resistance. See, e.g., U.S. Patent Nos.
  • the polypeptide also can be one that increases plant vigor or yield (including traits that allow a plant to grow at different temperatures, soil conditions and levels of sunlight and
  • nucleotide sequences in this category code for mutant ALS and AHAS enzymes as described, e.g., in U.S. Patent Nos. 5,767,366 and 5,928,937.
  • U.S. Patent Nos. 4,761,373 and 5,013,659 are directed to plants resistant to various imidazalinone or sulfonamide herbicides.
  • U.S. Patent No. 4,975,374 relates to plant cells and plants containing a nucleic acid encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that are known to inhibit GS, e.g., phosphinothricin and methionine sulfoximine.
  • GS glutamine synthetase
  • U.S. Patent No. 5,162,602 discloses plants resistant to inhibition by
  • cyclohexanedione and aryloxyphenoxypropanoic acid herbicides The resistance is conferred by an altered acetyl coenzyme A carboxylase (ACCase).
  • a polynucleotide comprising a nucleotide sequence encoding a transcription factor is expressed in the same cell in which the nucleic acid sequence encoding the expression product that inhibits stomatal closure is expressible.
  • the transcription factor activates in the presence of a compound that induces expression of the alcohol dehydrogenase (ADH1) system of Aspergillus nidulans (e.g., as broadly described above) and interacts with the c/s-acting element to induce expression of the stomatal closure-inhibiting nucleic acid sequence.
  • ADH1 alcohol dehydrogenase
  • Illustrative transcription factors comprise an amino acid sequence corresponding to the amino acid sequence of the AlcR transcription factor, as set forth for example in GenPept Accession No. AAQ06627.
  • the ethanol receptor comprises the amino acid sequence:
  • amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity or sequence identity to SEQ ID NO:56.
  • Exemplary AlcR-encoding polynucleotides may be selected from:
  • SEQ ID NO: 57 as set forth for example in GenBank Accession No. AF496548; or a nucleotide sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:57, or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:57.
  • transcription factor-encoding nucleotide sequence in the host cell permits chemical compounds that induce the expression of the alcohol dehydrogenase system of A. nidulans to activate expression of the stomatal closure-inhibiting nucleic acid sequence.
  • the transcription factor-encoding nucleotide sequence is operably linked to a promoter that is operable in a plant cell to form a separate construct ("second construct") relative to the construct that comprises the cw-acting element ("first construct").
  • the promoter of the second construct is suitably selected from constitutive promoters and cell or tissue specific/preferential promoters, as described for example above.
  • a cell or tissue specific/preferential promoter to drive expression of the transcription factor-encoding nucleotide sequence facilitates chemically inducible expression of the stomatal closure-inhibiting nucleic acid sequence in specific cell or tissues or preferentially in specific cells or tissues (e.g. , guard cells).
  • use of a constitutive promoter to drive expression of the transcription factor-encoding nucleotide sequence facilitates chemically inducible expression of the stomatal closure-inhibiting nucleic acid sequence throughout the plant.
  • the first and second constructs may be present on the same vector or on separate vectors.
  • the present invention further encompasses plant cells, plant parts, plant organs and plants in accordance with the embodiments of this invention.
  • the present invention provides a transformed plant cell, plant part, plant organ and/or plant comprising a nucleic acid molecule, a nucleic acid construct, a nucleotide sequence, a promoter, and/or a composition of this invention.
  • Representative plants include, for example, angiosperms (monocots and dicots), gymnosperms, bryophytes, ferns and/or fern allies.
  • the plants are selected from monocotyledonous plants.
  • monocot plants include sugar cane, corn, barley, rye, oats, wheat, rice, flax, millet, sorghum, grasses (e.g., switch grass, giant reed, turf grasses etc.), banana, onion, asparagus, lily, coconut, and the like.
  • the monocot plants of the invention include plants of the genus Saccharum (i.e., sugar cane, energy cane) and hybrids thereof, including hybrids between plants of the genus Saccharum and those of related genera, such as Miscanthus, Erianthus, Sorghum and others.
  • saccharum spp mean any of six to thirty-seven species (depending on taxonomic interpretation) of tall perennial grasses of the genus Saccharum.
  • the plant can be Saccharum aegyptiacum, Saccharum esculentum, Saccharum arenicol, Saccharum
  • Saccharum arundinaceum Saccharum barberi, Saccharum bengalense, Saccharum biflorum, Saccharum chinense, Saccharum ciliare, Saccharum cylindricum, Saccharum edule, Saccharum elephantinum, Saccharum exaltatum, Saccharum fallax, Saccharum fallax, Saccharum floridulum, Saccharum giganteum, Saccharum hybridum, Saccharum japonicum, Saccharum koenigii, Saccharum laguroides, Saccharum munja, Saccharum narenga, Saccharum officinale, Saccharum officinarum, Saccharum paniceum, Saccharum pophyrocoma,
  • Saccharum purpuratum Saccharum ravennae, Saccharum robustum, Saccharum roseum, Saccharum sanguineum, Saccharum sara, Saccharum sinense, Saccharum spontaneum, Saccharum tinctorium, Saccharum versicolor, Saccharum violaceum, Saccharum violaceum, and any of the interspecific hybrids and commercial varieties thereof.
  • plants of the present invention include soybean, beans in general, Brassica spp., clover, cocoa, coffee, cotton, peanut, rape/canola, safflower, sugar beet, sunflower, sweet potato, tea, vegetables including but not limited to broccoli, brussel sprouts, cabbage, carrot, cassava, cauliflower, cucurbits, lentils, lettuce, pea, peppers, potato, radish and tomato, fruits including, but not limited to, apples, pears, peaches, apricots and citrus, avocado, pineapple and walnuts; and flowers including, but not limited to, carnations, orchids, roses, and any combination thereof.
  • the plants are selected from energy crops, representative examples of which include:
  • Miscanthus e.g., Miscanthus ffinis, M. boninensis, M. brevipilus, M.
  • M. cotulifer M. depauperatus
  • M. ecklonii M. eulalioides
  • M. flavidus M.
  • Erianthus e.g., Erianthus acutecarinatus, E. acutipennisJL. adpressus, E. alopecuroides, E. angulatus, E. angustifolius, E. armatus, E. articulatus, E. arundinaceus, E. asper, E. aureus, E. bakeri, E. balansae, E. beccarii, E. bengalensis, E. biaristatus, E. bifidus, E. birmanicus, E. bqlivari, E. brasilianus, E. brevibarbis, E. capensis, E. chrysothrix, E. ciliaris, E. clandestinus, E. coarctatus, E. compactus, E. contortus, E. cumingii, E.
  • Erianthus e.g., Erianthus acutecarinatus, E. acut
  • E. flavipes E. flavoinflatus, E. floridulus, E. formosanus, E. formosus, E. fruhstorferi, E. fulvus, E. giganteus, E. glabrinodis, E. glaucus, E. griffithii, E. guttatus, E. hexastachyus, E. hookeri, E. hostii, E. humbertianus, E. inhamatus, E. irritans, E. jacquemontii, E.
  • E. repens E. rocMi, E. roxburghii, E. rufipilus, E. rufus, E. saccharoides, E. sara, E.
  • scriptorius E. sesquimetralis, E. sikkimensis, E. smallii, E. sorghum, E. speciosus, E. strictus, E. sukhothaiensis, E. sumatranus, E. teretifolius, E. tinctorius, E. tonkinensis, E. tracyi, E. trichophyllus, E. trinii, E. tristachyus, E. velutinus, E. versicolor, E. viguieri, E. villosus, E. violaceus, E. vitalisi, E. vulpinus, E. wardii, E. williamsii);
  • Pennisetum e.g., Pennisetum adoense, P. advena, P. alapecuroides, P. albicauda, P. alopecuroides, P. alopecuros, P. americanum, P. amethystinum, P. amoenum, P. ancylochaete, P. angolense, P. angustifolium, P. annuum, P. antillarum, P. araneosum, P. aristidoides, P. arnhemicum, P. articulare, P. arvense, P. asperifolium, P. asperum, P.
  • Pennisetum adoense e.g., Pennisetum adoense, P. advena, P. alapecuroides, P. albicauda, P. alopecuroides, P. alopecuros, P. americanum, P. amethy
  • P. beckeroides P. benthami, P. blepharideum, P. borbonicum, P. brachystachyum, P. breve, P. breviflorum, P. c ffrum, P. calyculatum, P. caninum, P. carneum, P. catabasis, P. cauda- ratti, P. cenchroides, P. centrasiaticum, P. cereale, P. chevalieri, P. chilense, P. chinense, P. chudeaui, P. ciliare, P. ciliares, P. ciliatum, P. cinereum, P. clandestinum, P. cognatum, P. complanatum, P.
  • niloticum P. nitens, P. nodiflorum, P. notarisii, P. nubicum, P. numidicum, P. obovatum, P. occidentale, P. ochrops, P. orientate, P. orthochaete, P. ovale, P. oxyphyllum, P. pallescens,
  • P. ruppellii P. sagittatum, P. sagittifolium, P. salifex, P. sampsonii, P. scaettae, P. scandens, P. schimperi, P. sch Kunststoffenii, P. schweinfurthii, P. sciureum, P. sclerocladum, P. scoparium, P. secundiflorum, P. sericeum, P. setaceum, P. setigerum, P. setosum, P. shaanxiense, P.
  • trachyphyllum P. triflorum, P. trisetum, P. tristachyon, P. tristachyum, P. triticoides, P.
  • Saccharum ⁇ e.g., as described above including S. ravennae and S.
  • Sorghum ⁇ e.g., Sorghum abyssinicum, S. aethiopicum, S. album, S.
  • consanguineum S. conspicuum, S. contortum, S. controversum, S. coriaceum, S. crupina, S. cubanicus, S. cubense, S. deccanense, S. decolor, S. decolorans, S. dimidiatum, S. bathna, S. dora, S. dubium, S. dulcicaule, S. durra, S. elegans, S. elliotii, S. elliottii, S. elongatum, S. eplicatum, S. exaratum, S. exsertum, S. fastigiatum, S. fauriei, S.
  • nubicum nubicum
  • S. nutans nubicum
  • S. orysoidum nubicum
  • S. pallidum S. panicoides
  • S. papyrascens S. parviflorum
  • Poplars e.g., Populus P. acuminata, P. adenopoda, P. alba, P. afghanica,
  • yunnanensisUNothospecies P. x acuminata, P. x berolinensis, P. x brayshawii, P. x canadensis, P. x canescens, P. x generiosa, P. hinckleyana, P. ⁇ jackO); [0250] wheat (e.g., Triticum abyssinicum, T. accessorium, T. acutum, T.
  • wheat e.g., Triticum abyssinicum, T. accessorium, T. acutum, T.
  • T. aegilapoides T. aegilopoides, T. aegilops, T. aesticum, T. aestivum, T. aethiopicum, T. qffine, T. afghanicum, T. agropyrotriticum, T. alatum, T. album, T. algeriense, T. alpestre, T.
  • T. batalini T. bauhini, T. benghalense, T. bicorne, T. bifaria, T. biflorum, T. biunciale, T. bonaepartis, T. boreale, T. borisovii, T. brachystachyon, T. brachystachyum, T. breviaristatum, T. brevisetum, T. brizoides, T.
  • T. crassum T. cretaceum, T. creticum, T. crinitum, T. cristatum, T. curvifolium, T. cylindricum, T. cynosuroides, T. czernjaevi, T. dasyanthum, T. dasyphyllum, T.
  • T. dasystachys T. dasystachyum, T. densiflorum, T. densiusculum, T. desertorum, T. dichasians, T. dicoccoides, T. dicoccon, T. dicoccum, T. distachyon, T. distans, T. distertum, T. distichum, T. divaricatum, T. divergens, T. diversifolium, T. donianum, T. dumetorum, T. duplicatum, T. duriusculum, T. duromedium, T. durum, T. duvalii, T. elegans, T. elongatum, T. elymogenes, T.
  • T. elymoides T. emarginatum, T. erebuni, T. erinaceum, T.farctum, T.farrum, T.fastuosum, T.festuca, T. festucoides, T.fibrosum, T.filiforme, T.firmum, T. flabellatum, T.flexum, T. forskalei, T. fragile, T. freycenetii, T. fuegianum, T. fungicidum, T. gaertnerianum, T.
  • T. missuricum T. molle
  • T.. monococcum T. monostachyum
  • T. multiflorum T. murale
  • T. muricatum T. nardus
  • T. neglectum T. nigricans
  • T. nodosum T. nubigenum, T. obtusatum, T. obtusiflorum, T. obtusifolium, T. obtusiusculum, T. olgae, T. orientale, T.
  • T. palaeo-colchicum T. palmovae, T. panarmitanum, T. paradoxum, T. patens, T. patulum, T. pauciflorum, T. pectinatum, T. pectiniforme, T. percivalianum, T. peregrinum, T. persicum, T. peruvianum, T. petraeum, T. petropavlovskyi, T. phaenicoides, T. phoenicoides, T. pilosum, T. pinnatum, T. planum, T. platystachyum, T. poa, T. poliens, T. polonicum, T. poltawense, :. m polystachyum, i. nticum, T. pouzolzii, T. proliferum, T. prostratum, T.
  • T. pseudo-agropyrum T. pseudocaninum
  • T. puberulum T. pubescens
  • T. secundum T. segetale
  • T. semicostatum T. sepium
  • T. sibiricum T. siculum
  • T. siliginum T. silvestre
  • T. simplex T. sinaicum
  • T. sinskajae T. solandri
  • T. sparsum T. spelta
  • T. speltaeforme T. speltoides
  • T. sphaerococcum T. spinulosum
  • T. spontaneum T.
  • T. squarrosum T. striatum, T. strictum, T. strigosum, T. subaristatum, T. subsecundum, T.
  • subtile subtile, T. subulatum, T. sunpani, T. supinum, T. sylvaticum, T. sylvestre, T. syriacum, T. tanaiticum, T. tauri, T. tauschii, T. tenax, T. tenellum, T. ubenelletiflorum, T. thaoudar, T. tiflisiense, T. timococcum, T. timonovum, T. timopheevi, T. timopheevii, T. tomentosum, T. tournamentfortii, T. trachycaulon, T. trachycaulum, T. transcaucasicum, T.
  • T. triaristatum T. trichophorum, T. tricoccum, T. tripsacoides, T. triunciale, T. truncatum, T. tumonia, T. turanicum, T. turcomanicum, T. turcomanieum, T. turgidum, T. tustella, T.
  • umbellulatum T. uniaristatum, T. unilaterale, T. unioloides, T. urartu, T. vagans, T. vaginans, T. vaillantianum, T. variabile, T. variegatum, T. varnense, T. vavilovi, T. vavilovii, T.
  • T. ventricosum T. venulosum
  • T. villosum T. violaceum
  • T. virescens T. volgense
  • oats e.g., Avena abietorum, A. abyssinica, A. adsurgens, A. adzharica, A. aemulans, A. aenea, A. ffinis, A. agadiriana, A. agraria, A. agraria-mutica, A. agraria- sesquialtera, A. agropyroides, A. agrostidea, A. agrostoides, A. airoides, A. alba, A. albicans, A. albinervis, A. algeriensis, A. almeriensis, A. alopecuros, A.
  • alpestris A. alpina, A. alta, A. altaica, A. altior, A. altissima, A. ambigua, A. americana, A. amethystina, A. anathera, A. andropogoides, A. andropogonoides, A. anglica, A. anisopogon, A. antarctica, A. arduensis,
  • Atheranthera A. atlantica, A. atropurpurea, A. aurata, A. australis, A. azo-carti, A. barbata,
  • daenensis A. dahurica, A. damascena, A. decora, A. delavayi, A. depauperata, A. desertorum,
  • A. loeflingiana A. longa, A. longepedicellata, A. longepilosa, A. longespiculata, A. longifolia,
  • pauciflora A. paupercula, A. pendula, A. penicillata, A. pennsylvanica, A. pensylvanica, A. persarum, A. persica, A. peruviana, A. phleoides, A. pilosa, A. planiculmis, A. podolica, A. polonica, A. polyneura, A. ponderosa, A. pourretii, A. praecocioides, A. praecoqua, A.
  • A. quadridentula A. quadriseta, A. quinqueseta, A. racemosa, A. radula, A. redolens, A.
  • sesquitertia A. setacea, A. setifolia, A. sexaflora, A. sexflora, A. shatilowiana, A. sibirica, A. sibthorpii, A. sicula, A. sikkimensis, A. smithii, A. solida, A. spica-venti, A. spicaeformis, A. spicata, A. splendens, A. squarrosa, A. sterilis, A. stipaeformis, A. stipoides, A. striata, A. stricta, A. strigosa, A. subalpestris, A. subcylindrica, A. subdecurrens, A. subspicata, A.
  • triaristata A. trichophylla, A. trichopodia, A. triseta, A. trisperma, A. triticoides, A. truncata,
  • willows e.g., Salix species
  • switch grass i.e., Panicum virgatum
  • alfalfa i.e., Medicago sativa
  • prairie bluestem e.g., Andropogon gerardii
  • maize i.e., Zea mays
  • soybean i.e., Glycine max
  • barley i.e., Hordeum vulgare
  • sugar beet i.e., Beta vulgaris
  • hay and fodder crops i.e., hay and fodder crops.
  • Procedures for transforming plants are well known and routine in the art and are described throughout the literature.
  • Non-limiting examples of methods for transformation of plants include transformation via bacterial-mediated nucleic acid delivery (e.g., via
  • Agrobacteria viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium-phosphate-mediated transformation,
  • the introducing into a plant, plant part, plant organ and/or plant cell is via bacterial-mediated transformation, particle bombardment transformation, calcium-phosphate-mediated transformation, cyclodextrin- mediated transformation, electroporation, liposome-mediated transformation, nanoparticle- mediated transformation, polymer-mediated transformation, virus-mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, sonication, infiltration, polyethylene glycol-mediated transformation, any other electrical, chemical, physical and/or biological mechanism that results in the introduction of nucleic acid into the plant, plant part and/or cell thereof, or a combination thereof.
  • Agrobacterium-mcdiated transformation is a commonly used method for transforming plants, in particular, dicot plants, because of its high efficiency of transformation and because of its broad utility with many different species.
  • Agrobacterium- ediated transformation typically involves transfer of the binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain that may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (Uknes et al. (1993) Plant Cell 5:159-169).
  • the transfer of the recombinant binary vector to Agrobacterium can be accomplished by a triparental mating procedure using Escherichia coli carrying the recombinant binary vector, a helper E.
  • the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation (Ho gen & Willmitzer (1988) Nucleic Acids Res. 16:9877).
  • Transformation of a plant by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissue is regenerated on selection medium carrying an antibiotic or herbicide resistance marker between the binary plasmid T-DNA borders.
  • Another method for transforming plants, plant parts and plant cells involves propelling inert or biologically active particles at plant tissues and cells. See, e.g., U.S. Patent Nos. 4,945,050; 5,036,006 and 5,100,792. Generally, this method involves propelling inert or biologically active particles at the plant 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 nucleic acid of interest.
  • a cell or cells 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.
  • a dried yeast cell also can be propelled into plant tissue.
  • a plant cell can be transformed by any method known in the art and as described herein and intact plants can be regenerated from these transformed cells using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue culture and/or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co.New York (1983)); and Vasil I. R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol.
  • the methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into a plant, only that they gain access to the interior of at least one cell of the plant.
  • the respective nucleotide sequences can be assembled as part of a single nucleic acid construct/molecule, or as separate nucleic acid constructs/molecules, and can be located on the same or different nucleic acid constructs/molecules.
  • the nucleotide sequences can be introduced into the cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol.
  • the introduced nucleic acid molecule may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosome(s).
  • the introduced construct may be present on an extra-chromosomal non- replicating vector and be transiently expressed or transiently active.
  • the nucleic acid molecule can be present in a plant expression construct.
  • the constructs of the present invention are useful for controlling stomatal closure, and therefore can be used to control transpiration in transgenic plants containing the constructs.
  • the constructs taught in this invention are particularly valuable in that expression of the stomatal closure-inhibiting nucleic acid sequence of the construct is regulated effectively. In specific embodiments, the expression of the stomatal closure-inhibiting nucleic acid sequence is found only in guard cells.
  • Such constructs will therefore be particularly useful in crop and horticultural varieties in which reduction of moisture content is important. Examples of such crops include but are not limited to cereal grains such as corn, wheat, rye, oats, barley, and rice, soybeans and other beans, as well as other products such as hay and commercial seed.
  • Expression of the stomatal closure-inhibiting nucleic acid sequence of the construct can be achieved by exposing a plant, plant part, plant organ or plant leaf, which comprises the construct, to a compound that induces the expression of the alcohol
  • ADH1 dehydrogenase
  • the plant, plant part, plant organ or plant leaf may be exposed to the compound prior to harvesting (e.g., no more than one month, three weeks, two weeks, one week, six, days, five days, four days, three days, two days, one day prior to harvesting), at the time of harvesting or after harvesting (e.g., no more than one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month after harvesting), the plant, plant part, plant organ or plant leaf.
  • the compound prior to harvesting e.g., no more than one month, three weeks, two weeks, one week, six, days, five days, four days, three days, two days, one day prior to harvesting
  • the time of harvesting or after harvesting e.g., no more than one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month after harvesting
  • the compound may be applied to the plant, plant part, plant organ or plant leaf using any suitable technique including vapor, dipping, spraying, spray drenching and the like.
  • the time and duration of exposing the plant, plant part, plant organ or plant leaf to the compound are chosen to permit increased transpiration in the plant, plant part, plant organ or plant leaf so that the water content of the plant, plant part, plant organ or plant leaf reduces by at least about 5% (e.g., at least about 6%, 7%, 8%, 9%, 10%, 15% 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%).
  • the reduced water content results in other beneficial phenotypes, including for example increased stored carbohydrates such as starch and simple sugars (e.g., sucrose, fructose, glucose etc.).
  • increased stored carbohydrates such as starch and simple sugars (e.g., sucrose, fructose, glucose etc.).
  • the increased transpiration provided by the present invention can be used to dry out the growth medium (e.g. , soil) in which transgenic plants of the invention are grown.
  • the growth medium e.g. , soil
  • Illustrative applications of these embodiments include drying out sporting fields with transgenic turf grass, and drying out fields containing transgenic crops (e.g. , to permit more facile harvesting of crops).
  • a ZmUbi-GS construct was constructed comprising the maize ubiquitin promoter (ZmUbi), the GUS reporter gene containing a synthetic intron (GS), and the nopaline synthase (nos) terminator.
  • the GS sequence consists of a 232 bp 1 st exon, 84 bp synthetic intron, and 1580 bp 2 nd exon.
  • the GS sequence was amplified from p35S-GS by polymerase chain reaction (PCR) using the forward primer
  • GS sequence was excised from pGEM-T using Smal and Notl, treated with T4 DNA polymerase (Promega) to blunt the Notl ends and then ligated into the Smal site of a pBluescript (pBS) vector containing the maize Ubil promoter and nos terminator (ZmUbi-nos/pBS) to generate ZmUbi-GS.
  • pBS pBluescript
  • a ZmUbi- AlcR AlcA-GS construct was constructed comprising the AlcR coding sequence and nos under the control of ZmUbi, as well as GS with nos under the control of the AlcA promoter.
  • the AlcR sequence was amplified from 35S-AlcR-AlcA Rep/pUC by PCR using the forward primer
  • Microprojectile bombardment of callus was performed according to the method of Bower et al (Molecular Breeding 1996 2: 239-249).
  • sugar cane calli were transferred from MSC 3 media to MSO media (MSC 3 media with the addition of 190mM sorbitol (Sigma) and 190 mM mannitol (Sigma) and arranged in a 3cm diameter circle four hours prior to microprojectile bombardment.
  • the plasmid pUKN ZmUbi driving expression of the neomycin phosphotransferase II gene
  • GS expression construct DNA (1 ⁇ g/ ⁇ L) was added to approximately 3mg of 1 ⁇ gold particles (Bio-Rad). The solution was vortexed briefly and 25 ih of 1 M CaC ⁇ and 5 xL of 0.1 M spermidine were added simultaneously. The mixture was iced and vortexed for 15 seconds every minute for a total of 5 minutes. The mixture was then allowed to settle on ice for 10 minutes, after which 22 ⁇ of supernatant was removed. The remaining DNA coated gold solution was mixed and 5 ⁇ , was used per bombardment. A particle inflow gun (PIG) was used to deliver the DNA to the target tissue. A baffle utilizing stainless steel mesh screen with an aperture of 500 ⁇ was positioned approximately 1.5 cm above the target tissue within the PIG chamber.
  • PIG particle inflow gun
  • the microflight distance of the DNA from the tip of the swinny to the leaf explant was 10.5 cm.
  • the PIG chamber was vacuum evacuated to -90 kPa and a 10 ms pulse of helium at 1500 psi was used to accelerate the microprojectiles. The vacuum was released immediately following bombardment and each sample plate was rotated 180 degrees and subjected to a second bombardment. 1
  • the callus Following bombardment, the callus remained on MSO media for four hours. The callus was subsequently transferred to MSC3 medium for 4-6 days before being transferred to selection media consisting of MSC3 and 50 mg/L G418 (Geneticin; Roche). Following microprojectile bombardment, the callus remained on selection media for four weeks in the dark with fortnightly subcultunng after which it was transferred to regeneration medium with selection, consisting of MSC3 with the 2,4-D replaced by 4.4 ⁇ 6- Benzylaminopurine (BAP; Sigma). The callus was maintained at 27° C, under a 16 hour light, and 8 hour dark cycle with fortnightly subculturing. Individual plants were separated and one plant from each clump of callus was retained.
  • Plants were verified to contain the ZmUbi-GS and ZmUbi-AlcR AlcA-GS constructs by TaqMan® analysis for the GUS transgene. Approximately 15 week-old transgenic plants were analyzed for ethanol inducible GUS reporter gene expression. Leaf samples consisting of five standard hole punches were taken from the first fully unfurled leaf of transgenic plants just prior to ethanol induction. Ethanol treatment was carried out by using a 10% ethanol root drench and aerial spray until runoff. After treatment, the plants were enclosed using plastic sheeting to maximize their exposure to the ethanol vapor. After ethanol treatment, first unfurled leaf samples were taken at three and six days post treatment. All leaf samples were collected on ice and used for GUS histochemical analysis (Jefferson et al.
  • GUS staining buffer 50 mM NaP04 pH 7, 0.1 % Triton, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM EDTA, and 1 mM X-Gluc
  • vacuum infiltration of the tissue for 45 minutes, and incubation at 37° C for 48 hours.
  • the GUS staining buffer was removed and replaced with 100% ethanol to clear the tissue of chlorophyll. Visual inspection of the cleared leaf discs was used to assess GUS expression.
  • the SC 12 construct has an unmodified AlcA promoter sequence identical to the sequence present in ZmUbi-AlcR AlcA-GS.
  • This AlcA promoter sequence is identical to the original AlcA promoter sequence used in plants as reported in Caddick et al. ( 1998, Nature Biotechnology 6: 177- 180), and consists of the Aspergillus AlcA promoter (from -349 to -112 relative to the translational start site and lacking the upstream direct repeat AlcR binding site) fused to the CaMV 35S minimal promoter (-32 to +3 relative to the CaMV 35S transcriptional start site) at the TATA box (the TATA box is identical in the AlcA and CaMV 35S promoters).
  • the SC 13 construct consists of the Aspergillus AlcA promoter sequence (-349 to - 25 112) fused to a longer CaMV 35S minimal promoter element (-73 to +7 relative to the CaMV 35S transcriptional start site).
  • the SC 14 construct consists of an Aspergillus AlcA promoter including the upstream direct repeat AlcR binding site (-400 to -112 relative to the
  • the SC 15 construct consists of an Aspergillus AlcA promoter including the upstream direct repeat AlcR binding site (-400 to -112) fused to the longer CaMV 35S minimal sequence (-73 to +7).
  • the SCI 6 construct consists of an Aspergillus AlcA promoter (-400 to -112) in which the upstream direct repeat AlcR binding site region has been changed from "5'-cgtccgcatcggcatccgcagc-3*" [SEQ ID NO:44] to "5'-tatccgcatgggtatccgcatg-3"' [SEQ ID NO:45] and fused to the original CaMV 35S minimal sequence.
  • the SC 17 construct consists of an Aspergillus AlcA promoter (-400 to -112) in which the upstream direct repeat AlcR binding site region has been changed from "5'-cgtccgcatcggcatccgcagc-3"' [SEQ ID NO:46] to "5 * -tatccgcatgggtatccgcatg-3"' [SEQ ID NO: 47] and fused to the longer CaMV 35S minimal sequence.
  • the SC 18 construct consists of five tandem repeats of the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] at the 5' end of the Aspergillus AlcA promoter including the upstream direct repeat AlcR binding site fused to the original CaMV 35S minimal sequence.
  • the SCI 9 construct consists of five tandem repeats of the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] at the 5' end of the Aspergillus AlcA promoter including the upstream direct repeat AlcR binding site fused to the longer CaMV 35S minimal sequence.
  • the SC20 construct consists of five tandem repeats of the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] (without any additional AlcA promoter sequence) fused to the longer CaMV 35S minimal sequence.
  • the SC21 construct consists of the Aspergillus AlcA promoter including the upstream direct repeat AlcR binding site fused to the original CaMV 35S minimal sequence with the addition of the maize Adhl intron (Callis et al. (1987) Genes & Dev. 1:1183-1200) at the 3' end.
  • the SC22 construct consists of the Aspergillus AlcA promoter including the upstream direct repeat AlcR binding site fused to the long CaMV 35S minimal sequence with the addition of the maize Adhl intron at the 3' end.
  • the SC23 construct consists of the native Aspergillus AlcA promoter
  • scoAlcR (scoAlcR; with TMV ⁇ and Kozak), and the Adhl intron (with 15 bp of 5' exon and 6 bp of 3' exon) were made synthetically and included restriction enzyme sites at each end for cloning.
  • the scoAlcR was excised using the flanking, engineered Hpal sites and cloned into the Smal site of ZmUbi-nos/pBS to generate ZmUbi-scoAlcR.
  • ZmUbi-scoAlcR was digested with EcoRV and Spel, and the ZmUbi-AlcR-nos region was subcloned into the Avrll (blunted using Klenow) and Spel sites of the binary construct UbinptllNos(S) to generate
  • scoAlcRnptll The scoGUS gene with nos was subcloned into pBluescript using Pstl and SacII to generate scoGUS/pBS. All of the AlcA promoter variants were subsequently cloned into scoGUS/pBS using HmdIII and Pstl. For constructs SC21 and SC22, the Adhl intron was cloned into the Pstl site located between the AlcA promoter and TMV ⁇ .
  • the AlcA promoter variant, scoGUS, and nos sequences were subcloned into scoAlcRnptll using HmdIII and Ascl.
  • the final ethanol switch constructs (SC14, SC15, and SC21-23) were made by subcloning the AlcA promoter variant, scoGUS, and nos sequences into scoAlcRnptll using Ascl alone.
  • ZmUbi-scoGUS expression construct was generated as follows: ZmUbi was PCR amplified (adding a HmdIII site at the 5' end and a Pstl site at the 3' end), cloned into pGEM-T, and sequence verified. ZmUbi was subsequently subcloned into scoGUS/pBS using HmdIII and Pstl to generate ZmUbi-scoGUS. The ZmUbi- scoGUS and nos sequence was subcloned into the binary construct UbinptllNos(S) using HmdIII and Ascl.
  • the binary constructs were transferred into Agrobacterium strain AGL1 using a standard heat shock transformation method.
  • Agrobacterium containing each of the binary constructs were used to transform sugar cane using following methods (see, Example 4).
  • Leaf whorl material from field grown sugar cane plants was collected and initiated on EM3 medium (see below). Transverse sections (approximately 20) of immature leaf whorl between 1-3 mm in thickness were taken from just above the meristem and placed in the top-up orientation. Cultures were maintained in the dark at 25° C for 28 to 42 days. Callus utilized for transformation was within 4-10 days of the last subculture. Callus was selected on morphological characteristics such as compact structure and yellow color. Yellow embryogenic calli were selected wherever possible, as they provided good regeneration, consistent transformation, and fragmented in small clusters (2-4 mm)
  • the Agrobacterium inoculation suspension was then drained from the callus, and the remaining callus culture was blotted dry to remove excess Agrobacterium inoculation suspension. Plant tissues were blotted on filter paper such as Whatman Grade 1 paper, until the Agrobacterium inoculation suspension was substantially removed. The callus was then transferred for co-cultivation to 90 ⁇ 25 mm petri dishes containing no co-culture medium or containing dry filter papers or filter papers wet with sterile water, and sealed with
  • NESCOFILM®, MICROPORETM tape (3M; Minneapolis, MN) or similar material The dishes were incubated in the dark at 22° C for 2-3 days.
  • the callus tissue was transferred to MS 1 medium (see below) containing with 200 mg L of timentin ("resting" medium) and kept in the dark at 25°C for 4 days.
  • the first selection step was made in MS 2 medium (see below) containing
  • Regeneration was conducted on MS 3 medium (see below) supplemented with 50 mg/L of geneticin and 200 mg/L of timentin at 25°C in 16 hr. light. Gradual increases in light intensity were required. For the first week, the culture was left on a laboratory bench under normal room lighting, and for the next 3 weeks, the culture was grown at moderate light intensity.
  • EM3 MS salts and vitamins
  • 0.5 g/L casein hydrolysate 100 ml/L coconut water
  • 20 g/L sucrose 20 g/L sucrose and 3 mg/1 2,4-D.
  • LB basic 10 g/L NaCl; 5 g/L yeast extract; and 10 g/L tryptone.
  • LB solid LB basic with 15 g/L of agar.
  • AB The following salts were autoclaved and added: 2g/L (NH4)2S0 4 ; 6 g/L
  • Na2HP04 3 g/L KH2P04; and 3 g/L NaCl.
  • the following compounds were filter sterilized: O.l mM CaC12; 1.0 raM MgCl 2 0.003 mM FeCl 3 ; and 5 g/L glucose.
  • MS basic MS medium salts and vitamins, with 25 g/L sucrose.
  • MS 1 MS basic supplemented with 3.0 mg/L 2,4- D and 200 mg/L
  • MS2 MS basic supplemented with 3.0 mg/L 2,4- D and 50 mg/L Geneticin and 200mg/L Timentin.
  • MS3 MS basic supplemented with 40 ml of coconut water filter sterilized and 1.0- 2.0 mg/L BAP (cultivar dependent, thus not required for all cultivars) and 50mg/L Geneticin and 200mg/L Timentin.
  • MS4 MS basic supplemented with 1.0 g/L charcoal and 1.0 mg IB A (indole-3- butyric acid, not required for all cultivars and 50 mg/L Geneticin.
  • CoCult Media co-cultivation media as described for banana in Khanna et al. (2004) Molecular Breeding 14(3): 239-252.
  • Plants were screened for the presence of the nptll, scoAlcR, and scoGUS genes using TaqMan® analysis. Plants that contained at least one copy of each gene of interest were subsequently characterized for ethanol inducible expression using GUS histochemical staining.
  • transgenic sugar cane plants were subsequently analyzed for ethanol inducible expression using a less robust ethanol treatment. After these transgenic plants had been in soil for six weeks, a leaf sample of approximately 3 cm in length was taken from each plant just prior to ethanol treatment and analyzed for GUS expression by histochemical staining. After sampling, the plants were treated with 1% ethanol using a single root drench (800ml/seedling tray) and aerial spray until runoff. At five days post treatment, a leaf sample of approximately 3cm in length was taken from each plant and analyzed for GUS expression by histochemical staining.
  • a tissue sample roughly equivalent to one standard hole punch was taken from the top, middle, and bottom of the first fully unfurled leaf of each plant. These three leaf samples were combined and placed into one well of a 96 well sample block on ice. Each plant was sampled in duplicate from the same first unfurled leaf. Samples were then frozen at -80° C and freeze dried. After sampling, the plants were treated with 5% ethanol using a single root drench (700 mL / pot) and aerial spray until runoff. At two, four, and seven days post treatment, a tissue sample roughly equivalent to one standard hole punch was taken from the top, middle, and bottom of the same first fully unfurled leaf of each plant. These three leaf samples were combined and placed into one well of a 96 well sample block on ice.
  • the SC35 construct consists of one copy of the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] (without any additional AlcA promoter sequence) fused to the longer CaMV 35S minimal sequence (-73 to +7 relative to the CaMV 35S transcriptional start site).
  • the SC36 construct consists of nine tandem repeats of the inverted repeat AlcR binding site region sequence "S'-atgcatgcggaaccgcacgagg-S"' [SEQ ID NO:3] (without any additional AlcA promoter sequence) fused to the longer CaMV 35S minimal sequence.
  • SC35 and SC36 promoter sequences were made synthetically and included restriction enzyme sites at each end for cloning. These sequences were cloned into scoGUS/pBS using Hindlll and Pstl. To generate the final ethanol switch constructs the promoter variant, scoGUS, and nos sequences were subcloned into scoAlcRnptll using
  • the binary SC35 and SC36 constructs were transferred into Agrobacterium strain AGL1 using a standard heat shock transformation method. Agrobacterium containing each of the binary constructs were used to transform sugar cane as described above.
  • Plants were screened for the presence of the nptll, scoAlcR, and scoGUS genes using TaqMan® analysis. Plants that contained at least one copy of each gene of interest were subsequently characterized for ethanol inducible expression using GUS ELISA as described above. After the transgenic sugar cane plants were in soil for approximately six weeks, a leaf sample of approximately 3 cm in length was taken from the first fully unfurled leaf of each plant just prior to ethanol treatment and placed into one well of a 96 well sample block on ice. Each plant was sampled in duplicate from the same first unfurled leaf. Samples were frozen at -80° C and freeze dried.
  • the SC38 construct modifies the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] to
  • the SC39 construct modifies the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] to
  • the SC41 construct modifies the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] to
  • the SC42 construct modifies the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] to
  • Example 5 The binary SC38, SC39, SC41 and SC42 constructs were transferred into Agrobacterium strain AGL1 using a standard heat shock transformation method. Agrobacterium containing each of the binary constructs were used to transform sugar cane as described above. Ethanol inducibility from these promoters was compared to that of construct SC20 using GUS ELISA as described above.
  • the minimal maize Adhl promoter as described by Walker et al. (1987, Proc. Natl. Acad. Sci. USA 84: 6624-6628), can be tested for its ability to be made inducible using the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and/or SEQ ID NO:9.
  • the Adhl nucleotide sequence (SEQ ID NO:48), below, was identified by successive 5' deletions of the Adhl promoter, and shown to give only background levels of expression:
  • a leaf sample of approximately 3 cm in length was taken from the same leaf previously sampled, and placed into one well of a 96 well sample block on ice. Each plant was sampled in duplicate from the same first unfurled leaf. Samples were frozen at -80° C, freeze-dried, and GUS expression was subsequently quantitated by ELISA as described above.
  • PCR was carried out on cDNA made from RNA that was isolated from leaves of wild-type and abil mutant Arabidopsis plants, respectively.
  • the PCR primers were designed to the ABI1 GenBank sequence with the accession identifier, AY 142623.1.
  • 5'-CCCCGTCGACTCAGTTCAAGGGTTTGCTCT-3' incorporates a Sail restriction enzyme site.
  • the engineered BamHl and Sail restriction enzyme sites were used for sub-cloning the ABI1 and abil genes into the plant expression constructs.
  • the 35S-abil plant expression construct consists of the constitutive CaMV 35S promoter driving expression of the Arabidopsis abil gene.
  • the abil gene was sub-cloned into the 35S-GS construct using the restriction enzyme sites BamHl and Sail, replacing the GUS syntron (GS) coding sequence with abil, and generating the plasmid 35S-abil.
  • the 35S- abil-35S cassette was excised from the 35S-abil plasmid using EcoRI, and cloned into the EcoRI site of the binary vector pBINPLUS to generate the 35S-abil binary (Fig. 6) construct used for transformation of N. benthamiana.
  • the 35S-ABI1 plant expression construct consists of the constitutive CaMV 35S promoter driving expression of the Arabidopsis wild type ABU gene.
  • the ABU gene was sub-cloned into the 35S-GS construct using the restriction enzyme sites BamHl and Sail, replacing the GUS syntron (GS) coding sequence vAihABIl, and generating the plasmid 35S- ABIL
  • the 35S-,4Z?H-35S cassette was excised from the 35S-ABI1 plasmid using Asel and HmdIII, and cloned into the Asel and Hwdlll sites of the binary vector pBINPLUS to generate the 35S-ABI1 binary construct used for transformation of N. benthamiana.
  • the scoalcR gene was excised from construct 0919814- scoALCR-pMK-RQ using the restriction enzymes Notl and Asel, and the DNA ends were blunted using Klenow DNA polymerase.
  • the scoalcR was subsequently sub-cloned into the blunted BamHl site of the 35S-GS plasmid to generate the 35S-scoalcR plasmid.
  • the 35S- scoalcR-35S cassette was excised from the 35S-scoalcR plasmid using EcoRI, and cloned into the EcoRI site of the binary vector pBINPLUS to generate the 35S-scoalcR binary construct.
  • the 35S-scoalcR binary construct was used to create the ale gene switch constructs described below.
  • the palcA O-abil plant expression construct consists of the original ale gene switch promoter (palcA O) driving expression of the Arabidopsis abil gene. This palcA
  • O promoter sequence is identical to the original alcA promoter sequence used in plants by Caddick etal. (Nature Biotechnology 1998 16:177-180), and consists of the Aspergillus alcA promoter (from -349 to -112 relative to the translational start site and lacking the upstream direct repeat AlcR binding site) fused to the CaMV 35S minimal promoter (-32 to +3 relative to the CaMV 35S transcriptional start site) at the TATA box (the TATA box is identical in the alcA and CaMV 35S promoters).
  • the palcA O sequence was cloned from the construct AlBl-scoGUS- scoALCR-nptll using PCR with the forward primer 5 -
  • the palcA O PCR product was amplified using KAPAHiFi DNA polymerase (Geneworks) and A-tailed using Taq polymerase. The resulting PCR product was cloned into pGEM-T (Promega) to generate palcA O-pGEM-T, and the palcA O promoter was sequence verified.
  • the palcA O promoter was excised from palcA O-pGEM-T using Ncol and BamHl, and cloned into the Ncol and ⁇ BamHl sites of the 35S-abil plasmid to generate the plasmid pale A O-abil.
  • the pale A O- abil -35S cassette was excised from the pale A O-abil plasmid using HzVidlll, and cloned into the HmdIII site of the 35S-scoalcR binary construct to generate the palcA O-abil binary construct used for transformation of N. benthamiana.
  • the palcA I-abil plant expression construct consists of an improved ale gene switch promoter (palcA I) driving expression of the Arabidopsis abil gene.
  • This palcA I promoter sequence consists of five tandem repeats of the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:53] at the 5 * end of the
  • Aspergillus alcA promoter (which includes the upstream direct repeat AlcR binding site) fused to the CaMV 35S minimal promoter (-32 to +3 relative to the CaMV 35S transcriptional start site) at the TATA box (the TATA box is identical in the ale A and CaMV 35S
  • the palcA I sequence was cloned from the construct pAlcA AlB4-scoGUS using PCR with the forward primer 5'-CCCCCCATGGGTATCGATAAGCTTAGCTAGC-3' [SEQ ID NO:54] and the reverse primer
  • the palcA I PCR product was amplified using KAPAHiFi DNA polymerase (Geneworks) and A-tailed using Taq polymerase. The resulting PCR product was cloned into pGEM-T (Promega) to generate palcA 7-pGEM-T, and the palcA / promoter was sequence verified.
  • the palcA / promoter was excised from palcA /-pGEM-T using Ncol and BamHl, and cloned into the Ncol and BamHl sites of the 35S-abil plasmid to generate the plasmid palcA I-abil.
  • the palcA I-abil-35S cassette was excised from the palcA I-abil plasmid using Hwdlll, and cloned into the Hwdlll site of the 35S-scoalcR binary construct to generate the palcA I-abil binary construct (Fig. 7) used for transformation of N. benthamiana.
  • the palcA I-ABIl plant expression construct consists of the palcA I promoter driving expression of the Arabidopsis wild type ABIl gene.
  • the ABIl gene was excised from the 35S-ABU plasmid using BamHl and Sail, and sub-cloned into the BamHl and Sail restriction enzyme sites of the palcA I-abil plasmid (thejreby replacing abil with ABIl) to generate the palcA I-ABU plasmid.
  • the palcA I-ABI1-35S cassette was excised from the palcA I-ABIl plasmid using Asel and HmdIII, and cloned into the Asel and Hwdlll sites of the 35S-scoalcR binary construct to generate the pale A I-ABU binary construct used for transformation of N. benthamiana.
  • a sugar cane-optimized Arabidopsis abil scoabil sequence (including the nopaline synthase terminator, tNos) was synthesised by Geneart (Regensburg, Germany), introducing a Notl restriction enzyme site at the 5' end and an Asel restriction enzyme site at the 3' end. To generate a sugar cane-optimized sequence of the ABU wild-type gene
  • scoABU scoabil was used in a PCR site-directed mutagenesis to change nucleotide 554 from an "A” to "G” using the forward primer 5 '- ATGGCC ACGGTGGTTCC-3 1 [SEQ ID NO:56] and the reverse primer 5'-GGAACCACCGTGGCCAT-3' [SEQ ID NO:57].
  • the eFM ⁇ e35 -ZmUbil-scoabil plant expression construct consists of the figwort mosaic virus/CaMV 35S dual enhancer (eFMVe35S), and the maize polyubiquitin-1 promoter (ZmUbil) with TMV omega translational enhancer sequence
  • the e ⁇ M ⁇ e35S-ZmUbil-scoABH plant expression construct consists of the eFMVe35S dual enhancer and ZmUbil promoter with TMV omega translational enhancer sequence driving expression of scoABIl.
  • the scoABIl gene and tNos was sub-cloned into the plasmid eFMV e35S-ZmUbil -scoGUS using the restriction enzyme sites NotI and AscI, replacing the scoGUS gene and tNos with scoABIl and tNos, and generating the plasmid eFMVe35S-ZmUbil -scoABIl used for transformation of sugar cane.
  • the palcA I-scoabil plant expression construct consists of the pale A I promoter with TMV omega translational enhancer driving expression of scoabil, and the ZmUbil promoter with TMV omega translational enhancer driving expression of scoalcR.
  • the scoabil gene and tNos were excised from the 1107897_abil-tNos_pMK-RQ plasmid (Geneart) using NotI and AscI, and sub-cloned into the NotI and AscI restriction enzyme sites of the pAlcA A2B4-scoGUS plasmid (thereby replacing scoGUS and tNos with scoabil and tNos) to generate the palcA I-scoabil intermediate plasmid.
  • the ZmUbil -scoAlcR-tNos cassette in the AlBl-scoGUS-scoAlcR-nptll binary vector was excised using the restriction enzyme Kpnl, and cloned into the Kpnl restriction enzyme site in the intermediate plasmid palcA I-scoabil to generate the palcA I-scoabil construct (Fig. 9) used for transformation of sugar cane.
  • the palcA I-scoABIl plant expression construct consists of the palcA I promoter with TMV omega translational enhancer driving expression of scoABIl, and the ZmUbil promoter with TMV omega translational enhancer driving expression of scoalcR.
  • the scoABIl gene and tNos were excised from the scoABIl -pGEM-T plasmid using NotI and AscI, and sub-cloned into the NotI and AscI restriction enzyme sites of the pAlcA A2B4- scoGUS plasmid (thereby replacing scoGUS and tNos with scoABIl and tNos) to generate the palcA I-scoABIl intermediate plasmid.
  • the ZmUbil -scoAlcR-HNos cassette in the A1B1- scoGUS-scoAlcR-nptll binary vector was excised using the restriction enzyme Kpnl, and cloned into the Kpnl restriction enzyme site in the intermediate plasmid palcA I-scoABIl to generate the pale A I-scoABIl construct used for transformation of sugar cane.
  • the pale A I-scoabil -tNos cassette will be subcloned into the scoalcRnptll binary vector using HmdIII and Ascl to generate the palcA I-scoabil binary construct used for AgrobacteriUm-mediated transformation of sugar cane.
  • the binary constructs were transferred into Agrobacterium strain LBA4404 using electroporation.
  • Agrobacterium containing each of the binary constructs were used to transform N. benthamiana using Agrobacterium-mediated transformation as described by Horsch et al. (1985, Science 227:1229-1231).
  • Leaf explants were harvested from N.
  • the explants were blotted dry and maintained on selection-free, shoot-induction media consisting of MS salts plus vitamins (Phytotechnology Laboratories), sucrose (30 g L), 6-benzylaminopurine (BAP) (1 mg/mL), naphthalene acetic acid (NAA) (0.1 mg/mL) and 0.8 % agar for two to three days.
  • the explants were then transferred to media containing Kanamycin (200 ⁇ g/mL) and Timentin (200 ⁇ g/mL) and subcultured twice weekly.
  • the concentration of BAP and NAA in the media was reduced to 0.25 mg mL and 0.025 ⁇ g/mL, respectively.
  • MS media consisting of MS salts plus vitamins, sucrose (30 g/ L) and 0.8 % agar. Soon after plants had visible roots, they were transferred to soil and acclimatized at 25° C with a 16 hr. photoperiod.
  • Transgenic sugar cane plants were regenerated from sugar cane callus that was transformed by microprojectile bombardment (MPB) as described by Finer et al. ( 1992,
  • the plasmid pUKN (possessing a selectable marker for geneticin resistance) was co-bombarded with each of the constructs when transforming callus to allow for selection of transformed cells.
  • a 2 ⁇ _, aliquot of a 1 : 1 mixture of pUKN (1 ⁇ g/ ⁇ L) and the experimental construct DNA (1 ⁇ g/ ⁇ L) was added to approximately 3 mg of 1 ⁇ gold particles (Bio-Rad).
  • the solution was mixed briefly and 25 ⁇ of 2.5 M CaC ⁇ and 5 ⁇ , of 0.1 M spermidine were added simultaneously.
  • the mixture was iced and mixed for 15 seconds every minute for a total of five minutes.
  • the mixture was then allowed to settle on ice for 10 minutes, after which 22 ⁇ of supernatant was removed.
  • the remaining DNA-coated gold solution was mixed and 5 ⁇ , was used per bombardment.
  • a particle inflow gun (PIG) was used to deliver the DNA to the target tissue.
  • a screen utilizing stainless steel mesh with an aperture of 500 ⁇ was positioned approximately 1.5 cm above the target tissue within the PIG chamber.
  • the distance of the DNA-coated particles to the leaf explant was 10.5 cm.
  • the PIG chamber was vacuum evacuated to -90 kPa and a 10 ms pulse of helium at 1500 psi was used to accelerate the DNA-coated particles. The vacuum was released immediately following the MPB and each sample plate was rotated 180 degrees and subjected to a secorid MPB. The callus remained on MSO media for four hours post MPB.
  • RNA is extracted from N. benthamiana leaf samples using Tri Reagent (Sigma) according to the manufacturer's instructions.
  • Transgenic plants showing either constitutive or ethanol inducible expression of abil are characterized for any phenotypic effects that are known to be associated with reduced stomatal closure, in particular a wilty phenotype.
  • Transgenic plants are also characterized for relative water content and for the levels of sugar accumulation using High Performance Liquid Chromatography (HPLC).
  • HPLC High Performance Liquid Chromatography
  • Transgenic plants with constitutive expression of abil are assessed at various times over the course of development. Analysis of transgenic plants showing ethanol inducible abil expression are also carried out at various times over the course of development with data collected just prior to ethanol treatment and approximately 12-24 hours or 1-4 weeks following ethanol treatment. Ethanol treatment is carried out using either a single treatment or multiple treatments of a 2% root drench and aerial spray.
  • Characterization of the transgenic plants is carried out under well watered conditions and under varying levels of drought stress.
  • RNA is extracted from sugar cane leaf samples using Tri Reagent (Sigma) according to the manufacturer's instructions.
  • Transgenic plants showing constitutive and ethanol inducible expression of scoabil are characterized for any phenotypic effects that are known to be associated with reduced stomatal closure, in particular a wilty phenotype.
  • stomatal conductance is measured using the LI-6400XT portable photosynthesis system (LI-COR biosciences).
  • Transgenic plants (scoabil and the relevant control plants) are also characterized for the levels of sugar accumulation in their stem using HPLC, as well as leaf and stem water content.
  • Transgenic sugar cane plants with constitutive expression of scoabil are assessed at various times over the course of development (along with the relevant control plants), except for sugar accumulation which is characterized in approximately 6-9 month old plants.
  • Analysis of stomatal closure in transgenic plants showing ethanol inducible scoabil expression is also carried out at various times over the course of development, with data collected just prior to ethanol treatment and for up to approximately four weeks following ethanol treatment.
  • Transgenic sugar cane plants displaying constitutive expression of either scoABIl or scoabil were identified (Fig. 13). Characterization of transgenic sugar cane having constitutive expression of scoabil revealed that abil is able to reduce stomatal closure in sugar cane (Figs. 14 and 15). Constitutive scoabil expression significantly (P ⁇ 0.05) increased the stomatal conductance (Fig. 15) and could generate a wilty phenotype (Fig. 16) in transgenic sugar cane grown under well-watered conditions. These data demonstrate that expression of abil is able to control stomatal function in monocotyledonous plants like sugar cane in addition to dicotyledonous plants.
  • a sugar cane-optimized Vicia faba AAPt? 434 (scoAAPK K 3A ) sequence was synthesised by Geneart (Regensburg, Germany), introducing a Notl restriction enzyme site at the 5' end and an Ascl restriction enzyme site at the 3' end.
  • the guard cell-preferred promoter pGCl from Arabidopsis was PCR amplified from Arabidopsis genomic DNA using the forward primer
  • the pGCl- scoAAPt? 43A plant expression construct consists of the guard cell-preferred promoter pGCl driving expression of scoAAP ⁇ 43A .
  • the scoAAP ⁇ 43A gene with tNos was sub-cloned into the plasmid eFMVe35S-ZmUbil-scoABIl using the restriction enzyme sites Notl and Ascl, replacing the scoABIl gene and tNos with scoAAPK K43A and tNos and generating the plasmid eFMVe35S-Zm£/Z>/7- scoAAPl 43A .
  • the PCR- amplified pGCl promoter was sub-cloned into the plasmid eFMVe35S-Zrw(7&i7- scoAAPK K43A using the restriction enzyme sites HmdIII and Ncol, replacing the eFMVe35S- ZmUbil with pGCl to generate the plasmid pGCl- scoAAPK 43A .
  • the pGCl- scoAAPK K43A sequence with nopaline synthase terminator were subcloned into the binary vector pBINplus using the restriction enzyme sites Hind l and Ascl to generate the pGCl- scoAAP ⁇ 43A vector used for transformation into tobacco (Fig. 18).
  • the pGCl-scodbil plant expression construct consists of the guard cell preferred promoter pGCl driving expression of scoabil.
  • the PCR-amplified pGCl promoter was sub-cloned into the plasmid e ⁇ MVe35S-ZmUbil- scoabil using the restriction enzyme sites HmdIII and Ncol, replacing the eFMVe35S-Z/wi7oz7 with pGCl to generate the plasmid pGCl - scoabil.
  • the binary constructs were transferred into Agrobacterium strain LB A4404 using electroporation.
  • Agrobacterium containing each of the binary constructs were used to transform N. tabaccum using Agrobacterium-raediated transformation as described by Horsch et al. (1985, Science 227: 1229-1231).
  • Leaf explants were harvested from N. tabaccum and infected via immersion and incubation with transformed Agrobacterium. After infection of the leaf explants with Agrobacterium, the explants were blotted dry and maintained on selection- free, shoot-induction media consisting of MS salts plus vitamins (Phytotechnology
  • sucrose (30 g/L), 6-benzylaminopurine (BAP) (1 mg/mL), naphthalene acetic acid (NAA) (0.1 mg/mL) and 0.8 % agar for two to three days.
  • BAP 6-benzylaminopurine
  • NAA naphthalene acetic acid
  • the explants were then transferred to media containing Kanamycin (200 ⁇ g/mL) and Timentin (200 ⁇ g/mL) and subcultured twice weekly. After four to six weeks, the concentration of BAP and NAA in the media was reduced to 0.25 mg mL and 0.025 ⁇ g/mL, respectively.
  • MS media consisting of MS salts plus vitamins, sucrose (30 g/ L) and 0.8 % agar. Soon after plants had visible roots, they were transferred to soil and acclimatized at 25° C with a 16 hr. photoperiod.
  • RNA samples are taken just prior to ethanol induction and at 4-48 hours post ethanol treatment. Ethanol treatment is carried out using a single 2% ethanol root drench and aerial spray until runoff.
  • RNA is extracted from tobacco leaf samples using Tri Reagent (Sigma) according to the manufacturer's instructions.
  • Transgenic plants showing either constitutive or ethanol inducible expression of scoAAP ⁇ 43A I scoabil labil are characterized for any phenotypic effects that are known to be associated with reduced stomatal closure, in particular a wilty phenotype.
  • Ethanol treatment of transgenic tobacco possessing the ethanol inducible abil construct resulted in visible wilting of the leaves following ethanol treatment (Fig. 19).
  • the ethanol treatment used had no detectable effect on control plants (Fig. 19).
  • Transgenic tobacco plants displaying expression of either scoAAPK K43A or scoabil from the guard cell-preferred promoter pGCl were identified (Fig. 20). Characterization of transgenic tobacco having expression oi coabil from the pGCl promoter revealed that abil is able to reduce stomatal closure in tobacco (Fig. 21).

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Abstract

L'invention concerne des constructions génétiques comprenant une commutation du gène alc agissant en cis qui régule la fermeture des stomates, ainsi que des plantes, des parties de plantes et des cellules de plantes comprenant de telles constructions. La présente invention concerne également des procédés pour augmenter la transpiration chez les plantes et les parties de plantes utilisant ces constructions.
PCT/AU2013/000799 2012-07-18 2013-07-18 Constructions pour moduler la transpiration chez les plantes et leurs utilisations Ceased WO2014012145A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018024883A1 (fr) 2016-08-05 2018-02-08 Biogemma Constructions et procédés pour commander la fermeture stomatique chez les plantes
CN113545293A (zh) * 2021-08-19 2021-10-26 云南省农业科学院园艺作物研究所 一种毛枝京梨猕猴桃的组培增殖方法
CN116287372A (zh) * 2022-12-14 2023-06-23 赣南师范大学 一种鉴定圆果杜英的dna条形码、鉴定方法与应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994013820A1 (fr) * 1992-12-10 1994-06-23 Gist-Brocades N.V. Production de proteines homologues dans des champignons filamenteux
WO2011003800A1 (fr) * 2009-07-07 2011-01-13 Basf Plant Science Company Gmbh Plantes ayant un partitionnement de carbone modulé et procédé pour les fabriquer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994013820A1 (fr) * 1992-12-10 1994-06-23 Gist-Brocades N.V. Production de proteines homologues dans des champignons filamenteux
WO2011003800A1 (fr) * 2009-07-07 2011-01-13 Basf Plant Science Company Gmbh Plantes ayant un partitionnement de carbone modulé et procédé pour les fabriquer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GAROOSI, G. A. ET AL.: "Characterization of the ethanol-inducible alc gene expression system in tomato", JOURNAL OF EXPERIMENTAL BOTANY, vol. 56, 2005, pages 1635 - 1642 *
XIONG, T. C. ET AL.: "An inducible, modular system for spatio-temporal control of gene expression in stomatal guard cells", JOURNAL OF EXPERIMENTAL BOTANY, vol. 60, 2009, pages 4129 - 4136 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018024883A1 (fr) 2016-08-05 2018-02-08 Biogemma Constructions et procédés pour commander la fermeture stomatique chez les plantes
US12180493B2 (en) 2016-08-05 2024-12-31 Limagrain Europe Constructs and methods for controlling stomatal closure in plants
CN113545293A (zh) * 2021-08-19 2021-10-26 云南省农业科学院园艺作物研究所 一种毛枝京梨猕猴桃的组培增殖方法
CN116287372A (zh) * 2022-12-14 2023-06-23 赣南师范大学 一种鉴定圆果杜英的dna条形码、鉴定方法与应用
CN116287372B (zh) * 2022-12-14 2024-01-09 赣南师范大学 一种鉴定圆果杜英的dna条形码、鉴定方法与应用

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