TITLE OF THE INVENTION

"CONSTRUCTS FOR MODULATING TRANSPIRATION IN PLANTS AND USES THEREFOR"

[0001] This application claims priority to Australian Provisional Application No. 2012903070 entitled "Constructs for Modulating Transpiration in Plants and Uses Therefor" filed 18 July 2012, and to Australian Complete Application No. 2013205472 entitled

"Constructs for Modulating Transpiration in Plants and Uses Therefor" filed 12 April 2013, the contents of each of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] 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.

BACKGROUND OF THE INVENTION

[0003] Most land plants have the ability to regulate gas exchange and transpiration by opening and closing of the stomatal aperture. The turgor pressure-mediated movement of a pair of special epidermal cells or guard cells, controls the size of the stomatal aperture and so regulates the extent of water loss via transpiration and also regulates C0 ₂ uptake into the leaf for photosynthetic carbon fixation.

[0004] Plants tightly regulate stomatal closure to prevent excess water loss that would be detrimental to growth and development. While uncontrolled water loss would be deleterious, the controlled manipulation of stomatal closure could be used to increase water loss and improve crop plants in at least three distinct ways:

[0005] First, it could be used to speed up the drying process for crops such as cotton and grain, and could decrease the transport and processing costs associated with crops such as sugar cane. An efficient drying off process prior to harvest is extremely important in crops such as cotton and grain, and controlling stomatal closure may have advantages over the currently used harvest aids for this purpose. Sugar cane is approximately 70% water at harvest, and consequently a substantial amount of energy is required to remove this water during the milling process used to extract sucrose. Further expense is incurred in the transport of this water-laden plant material from the field to the mill. Decreasing the water content of the sugar cane at harvest has the potential to substantially reduce the transport and milling costs. Other crops, such as tobacco and dried fruits such as raisins and prunes, are dried immediately post-harvest. Thus, it would be advantageous to be able to accelerate or control the rate of crop drying.

[0006] 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.

[0007] Thirdly, controlled increases in transpiration could be used to increase the sugar content of crops such as sugar cane, sweet sorghum, and sugar beet. Plants naturally respond to drought stress by increasing sugar levels, which act to protect cells from

desiccation. Therefore, increased sugar production is expected to occur concomitantly with a decrease in plant water content.

[0008] 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 sensitivity.

[0009] Several negative regulators of the ABA signaling pathway are also known. For example, reduced activity of the ABA insensitive 1 (ABIl) and ABA insensitive 2 (ABI2) type 2C protein phosphatases leads to enhanced responsiveness to ABA. Also, 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. In addition, dominant positive mutants of H(+)-ATPase 1 AHA1 (also known as open stomata 2 (OST2)), which have constitutive ATPase activity, are known to inhibit stomatal closure.

[0010] Accordingly, it may be possible to increase water loss in plants by modulating the expression of genes that code for the above proteins so as to inhibit stomatal closure. However, unrestrained inhibition of stomatal closure may be detrimental to plant growth and development due to the uncontrolled loss of water and the disruption of abscisic acid signaling pathways that are involved in plant development. By utilizing an inducible gene switch, however, it may be possible to regulate expression of genes encoding these proteins for a defined period close to harvesting time. This may enable a reduction in water content and increased sugar content without any negative effects on plant biomass. [0011] Unfortunately, most inducible gene switches currently available are leaky with consequential unwanted gene expression. Additionally, there is a lack of inducible gene switches that are effective in monocotyledonous plants such as sugar cane and sweet sorghum for giving robust, tightly regulated gene expression.

SUMMARY OF THE INVENTION

[0012] 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.

[0013] Accordingly, in one aspect, the present invention provides constructs for inhibiting stomatal closure. These 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. Suitably, 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. [0014] In some embodiments, 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. In illustrative examples of this type, the at least one nucleotide sequence is represented by the nucleotide sequence

nvGCGGNNCCGCn _y [SEQ ID NO:2], wherein 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.

[0015] In some embodiments, the 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) is selected from the group consisting of the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2,

ATGCATGCGGAACCGCACGAGG [SEQ ID NO:3], GGCCATGCGGAGCCGCACGCGT [SEQ ID NO:4], ACAAGAGCGGCTCCGCTTGACC [SEQ ID NO:5];

TACGTAGCGGAACCGCTGCTCC [SEQ ID NO:6]; TACCATGCGGAACCGCACGTCC [SEQ ID NO:7], ATGCATGCGGTGCCGCACGAGG [SEQ ID NO:8] and

TACGTTGCGGAACCGCAGCTCC [SEQ ID NO:9], in any combination, in any orientation, and/or in any order.

[0016] In some embodiments, 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, and antibodies that are immuno-interactive with 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 ). In other embodiments, 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 ).

[0017] In another aspect, 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.

[0018] Another aspect of the present invention provides transgenic plant cells (e.g., plant guard cells) that comprise a construct or construct system as broadly described above and elsewhere herein. [0019] In yet another aspect, 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.

[0020] 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.

[0021] Suitably, 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).

[0022] 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) and which interacts with the exacting element to induce expression of the nucleic acid sequence encoding the expression product that inhibits stomatal closure.

[0023] In a further aspect, 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.

[0024] In some embodiments, 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. In illustrative examples of this type, the methods comprise exposing the plant, plant part or plant organ to the compound prior to harvesting the plant, plant part or plant organ. In other illustrative examples, 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. In still other illustrative examples, the methods comprise exposing the plant, plant part or plant organ to the compound after harvesting the plant, plant part or plant organ. ,

[0025] In some embodiments, 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%).

[0026] In some embodiments of any of the methods described above, 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. In other embodiments, the plant is a

dicotyledonous plant (e.g., tobacco, cotton, dried fruits such as raisins and prunes, nuts, coffee, tea, cocoa, and ornamental goods).

[0027] In some embodiments, the plant is selected from energy crops,

representative examples of which include: Miscanthus, Erianthus, Pennisetum, Arundo,

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. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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).

[0029] 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.

[0030] Figure 3 is a graphical representation showing ethanol inducible expression from promoters containing various modified inverted repeat AlcR binding sites. [0031] 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.

[0032] Figure 5 is a graphical representation showing ethanokinducible expression in the TO and T0V 1 transgenic plants.

[0033] Figure 6 is a schematic representation of the 35S-abil binary construct used for constitutive expression of abil in N. benthamiana.

[0034] 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.

[0035] Figure 8 is a schematic representation of the eFMV e35S-ZmUbil -scoabil construct used for constitutive expression of abil in sugar cane.

[0036] Figure 9 is a schematic representation of the palcA I-scoabil construct used for ethanol inducible expression of abil in sugar cane.

[0037] 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.

[0038] 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 .

[0039] Figure 12 is a graphical representation showing relative water content of N. benthamiana leaves following ethanol inducible expression of abil. * indicates a statistically significant difference (P<0.05) within each time point relative to the control at that time point, n = number of independent clones analyzed for each of the abil transgenic events. For the control plants, data from the clones of three independent events containing the vector backbone only were combined. Ethanol treatment consisted of a single 2% ethanol root drench and aerial spray. Relative water content refers to the amount of water present in the leaves compared to the fully hydrated state.

[0040] 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.

[0041] 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-Zm6¾H -scoABII, and the pUKN vector alone.

[0042] Figure 15 is another graphical representation showing stomatal conductance in transgenic sugar cane possessing constitutive expression of scoabil. Stomatal conductance was assessed in well watered control (vector backbone only) and transgenic scoabil plants, n = number of independenet transgenic plants analyzed.

[0043] 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).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Figure 20 is a photographic representation showing expression of either scoAAPK ^K43A or scoabil in tobacco using the guard cell-preferred promoter. pGCl.

Expression was assessed using RT-PCR for 3 independent scoAAPK ^K43A and 5 independent scoabil transgenic plants growing in soil. The positive controls consist of vector DNA containing either the scoAAPK ^K43A gene or the scoabil gene. For the negative control, water was used to replace the DNA template. The expected PCR product sizes are 817 bp

(scoAAPK ^K43A ) and 724 bp (scoabil). [0048] Figure 21 is a graphical representation showing stomatal conductance in well watered wild type and two independent transgenic tobacco plants possessing the construct pGCl-scoabil. Data shown represents the mean±SEM of two independent measurements taken on different days (for the wild type n=6 independent plants).

[0049] Some figures and text contain color representations or entities. Color illustrations are available from the Applicant upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

DETAILED DESCRIPTION OF THE INVENTION

/. Definitions

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0051] The articles "a" and "an" are used herein to refer to one or to more than one

(i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Thus, for example, the term "cw-acting sequence" also includes a plurality of s-acting sequences.

[0052] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or). [0053] Further, the term "about," as used herein when referring to a measurable value such as an amount of a compound or agent, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0:1% of the specified amount.

[0054] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0055] The term "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.

[0056] The term "antisense" refers to a nucleotide sequence whose sequence of nucleotide residues is in reverse 5' to 3' orientation in relation to the sequence of

deoxynucleotide residues in a sense strand of a DNA duplex. 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." Thus 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. The complementarity of an 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. In addition, as used herein, 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. [0057] The terms "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. Those skilled in the art will be aware that 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.

[0058] The term "chimeric construct" as used herein 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. Further, 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. In specific embodiments, 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.

[0059] By "coding sequence" is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.

[0060] As used herein, "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. For example, the 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. The terms "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. As used herein, 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. The terms "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.

[0061] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. Thus, the term "consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising." Thus, 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.

[0062] As used herein 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. Thus, for example, when applied to an ATPase, 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.

[0063] The term "construct" refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. As used herein, the term "expression construct," "recombinant construct" or "recombinant DNA 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. In this manner, for example, 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. For the practice of the present invention, conventional 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.

[0064] By "corresponds to" or "corresponding to" is meant 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, 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 similarity or identity to all or a portion of the reference amino acid sequence).

[0065] "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.

[0066] 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. [0067] As used herein, the terms "encode," "encoding" and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, 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. Thus, 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. [0068] The term "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

transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a coding sequence results from transcription and translation of the coding sequence. Conversely, expression of a non-coding sequence results from the transcription of the non-coding sequence. [0070] As used herein, the terms "fragment" or "portion" 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.

[0071] As used herein, 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.

[0072] "Genome" as used herein includes the nuclear and/or plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast genome. [0073] 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.

[0074] "Guard cell-specific promoter" as used herein 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.

[0075] "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.

[0076] The term "heterologous" as used herein with reference to nucleic acids 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. Thus, 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. In addition, 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.

present in a different copy number, and/or under the control of different regulatory sequences than that found in the native state of the nucleic acid molecule. The term "heterologous" 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. For instance, 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. Similarly, 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).

[0077] As used herein the term "homology" 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." The term homolog includes homologous sequences from the same and other species and orthologous sequences from the same and other species. Homology also refers to the concept of similar functional properties among different nucleic acids, amino acids, and/or proteins. [0078] 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.

[0079] "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. Where more than one nucleic acid molecule is to be introduced 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. Accordingly, 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. Thus, the term "transformation" as used herein 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. By "stably introducing" or "stably introduced" in the context of a polynucleotide introduced into a cell, it is intended that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide. "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.

[0080] An "isolated" 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). However, the 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. [0081] Thus, 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.

Accordingly, in some embodiments, 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. It also includes a recombinant nucleic acid that is part of a hybrid nucleic acid molecule encoding an additional polypeptide or peptide sequence. The term "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). Moreover, 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.

Accordingly, "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. For example, 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

i

separated from the coexisting materials of its natural state is "isolated," as the term is employed herein. For example, with respect to polynucleotides, the term 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. In representative embodiments of the invention, 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. In other embodiments, 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.

[0082] The term, "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. The term "miRNA" includes processed sequences as well as corresponding long primary transcripts (pri-miRNAs) and processed precursors (pre-miRNAs). Genetic and biochemical studies have indicated that 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.

[0083] The term "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. Thus, the term "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. As used herein, the term "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. [0084] As used herein, the term "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. The terms "nucleotide 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

Organization (WIPO) Standard ST.25.

. [0085] The term "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. For example, a control sequence (e.g., a promoter)

"operably linked" to a coding sequence 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. Thus, for example, 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. Likewise, "operably connecting" 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.

[0086] As used herein, "plant" means any plant and thus includes, for example, 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.

[0087] As used herein, the term "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.

[0088] As used herein, "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.

[0089] The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant.

[0090] As used herein , the terms "polynucleotide," "polynucleotide sequence," "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "nucleic acid sequence"s and the like refer 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. 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. For example, 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. Other

modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.

[0091] "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. Thus, these terms apply to 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.

[0092] As used herein, the term "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.

[0093] As used herein, the term "post-transcriptional gene silencing" (PTGS) refers to a form of gene silencing in which the inhibitory mechanism occurs after transcription. This can result in either decreased steady-state level of a specific R A target or inhibition of translation (Tuschl et al. (2001) ChemBiochem 2: 239-245). In the literature, the terms RNA interference (RNAi) and posttranscriptional co-suppression are often used to indicate posttranscriptional gene silencing.

[0094] As used herein, the term "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.

[0095] "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.

[0096] The term "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.

However, it shall be understood that the term "recombinant" does not refer to alterations of polynucleotides that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis followed by selective breeding. [0097] As used herein, the terms "RNA interference" and "RNAi" refer to a sequence-specific process by which a target molecule (e.g., a target gene, protein or RNA) is downregulated via downregulation of expression. Without beiiig bound to a specific mechanism, as currently understood by those of skill in the art, RNAi involves degradation of RNA molecules, e.g. , mRNA molecules within a cell, catalyzed by an enzymatic, RNA- induced silencing complex (RISC). 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. As practiced as a technology, 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).

[0098] As used herein, the terms "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.

[0099] As used in connection with the present invention, 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. [0100] The term "sequence identity" as used herein 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. Thus, 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. For the purposes of the present invention,

"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. 20894; see 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.

[0101] "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.

TABLE A: EXEMPLARY CONSERVATIVE AMINO ACID SUBSTITUTIONS

Ala Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn Glu Asp

Gly Pro

His Asn, Gin

He Leu, Val

Leu He, Val

Lys Arg, Gin, Glu

Met Leu, He,

Phe Met, Leu, Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp, Phe

Val He, Leu

[0102] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window", "sequence identity," "percentage of sequence identity" and "substantial identity". A

"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. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al, 1997, Nucl. Acids /?es.25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994- 1998, Chapter 15.

[0103] As used herein, 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. In some embodiments, 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.

[0104] The term "transgene" as used herein, refers to any nucleotide sequence used in the transformation of a plant, animal, or other organism. Thus, 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.

[0105] As used herein, the term "5' untranslated region" or "5' UTR" refers to a sequence located upstream (i.e., 5') of a coding region. Typically, a 5' UTR is located downstream (i.e., 3') to a promoter region and 5 ' of a coding region downstream of the promoter region. Thus, such a sequence, while transcribed, is upstream of the translation initiation codon and therefore is generally not translated into a portion of the polypeptide product.

[0106] The term "3' untranslated region" or "3' UTR" 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. [0107] The terms "wild-type," "natural," "native" and the like with respect to an organism, polypeptide, or nucleic acid sequence, that the organism polypeptide, or nucleic acid sequence is naturally occurring or available in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man. [0108] As used herein, 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. For example, "ABU" shall mean the ABI1 gene, whereas "ABU" shall indicate the protein product of the "ABU" gene.

[0109] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

2. Stomata closure-modulating constructs

[0110] 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. Thus, when operably linked to this cis-acting element, 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. In accordance with the present invention, the resulting inducible promoter is useful for expressing polynucleotides that comprise a nucleic acid sequence encoding an expression product that inhibits stomatal closure. 2.1 Car-acting element

[0111] In some embodiments, 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.

[0112] 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). Thus, SEQ ID NO:l provides the nucleotide sequence GCGGNNCCGC (inverted repeat underlined), which suitably represents the minimal cis- acting sequence.

[0113] 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.

[0114] , Additional cw-acting sequences are as follows:

ATGCATGCGGAACCGCACGAGG [SEQ ID NO:3], GGCCATGCGGAGCCGCACGCGT [SEQ ID NO:4], ACAAGAGCGGCTCCGCTTGACC [SEQ ID NO:5];

TACGTAGCGGAACCGCTGCTCC [SEQ ID NO:6]; TACCATGCGGAACCGCACGTCC [SEQ ID NO:7], ATGCATGCGGTGCCGCACGAGG [SEQ ID NO:8] and

TACGTTGCGGAACCGCAGCTCC TSEQ ID NO:91.

[0115] In some embodiments, the cw-acting elements can comprise one or more nucleotides (i.e., bases) between the inverted repeats {e.g., an intervening sequence). Thus, in certain embodiments, the number of nucleotides, comprising the intervening sequence can be two ( . e. , N=2) (e.g. , SEQ ID NO: 1 = n _x GCGGNNCCGCn _y (intervening sequence bolded and underlined)). Non-limiting examples of the intervening sequence include TT, AA, GG, CC, TA, TG, TC, AT, AG, AC, GT,GC, GA, CA, CT, or CG, and the like. Thus, any 2-mer can be used as an intervening sequence in SEQ ID NO:l or 2, respectively.

[0116] In some embodiments, 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 ). The flanking sequences (i.e., n _x or n _y ) can be of any length and/or composition of nucleotides, wherein each nucleotide can be independently adenine, mymine, guanine and/or cytosine (i.e., wherein n=A, T, G, and/or C), in any combination and/or in any order. Thus, in some embodiments of the invention, 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, guanine and/or cytosine, in any combination and/or in any order. As one of ordinary skill in the art would appreciate, flanking sequences of any length and composition can be produced that are included within SEQ ID NO:2 according to art-known methods.

[0117] In some embodiments, 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. Thus, in some embodiments, 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

combination, in any orientation, and/or in any order. Thus, in some embodiments, 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.

[0118] In some embodiments, 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.

[0119] In some embodiments, 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 sequence to about twenty nucleotide sequences, from about two nucleotide sequences to about five nucleotide sequences, from about two nucleotide sequences to about seven nucleotide sequences, from about two nucleotide sequences to about nine nucleotide sequences, from about two nucleotide sequences to about ten nucleotide sequences, from about two nucleotide sequences to about eleven nucleotide sequences, from about two nucleotide sequences to about twelve nucleotide sequences, from about two nucleotide sequences to about thirteen nucleotide sequences, from about two nucleotide sequences to about fourteen nucleotide sequences, from about two nucleotide sequences to about fifteen nucleotide sequences, from about two nucleotide sequence to about sixteen nucleotide sequences, from about two nucleotide sequence to about seventeen nucleotide sequences, from about two nucleotide sequence to about eighteen nucleotide sequences, from about two nucleotide sequence to about nineteen nucleotide sequences, from about two nucleotide sequence to about twenty nucleotide sequences, from about three nucleotide sequences to about five nucleotide sequences, from about three nucleotide sequences to about seven nucleotide sequences, from about three nucleotide sequences to about nine nucleotide sequences, from about three nucleotide sequences to about ten nucleotide sequences, from about three nucleotide sequences to about eleven nucleotide sequences, from about three nucleotide sequences to about twelve nucleotide sequences, from about three nucleotide sequences to about thirteen nucleotide sequences, from about three nucleotide sequences to about fourteen nucleotide sequences, from about three nucleotide sequences to about fifteen nucleotide sequences, from about three nucleotide sequence to about sixteen nucleotide sequences, from about three nucleotide sequence to about seventeen nucleotide sequences, from about three nucleotide sequence to about eighteen nucleotide sequences, from about three nucleotide sequence to about nineteen nucleotide sequences, from about three nucleotide sequence to about twenty nucleotide sequences, from about four nucleotide sequences to about nine nucleotide sequences, from about four nucleotide sequences to about ten nucleotide sequences, from about four nucleotide sequences to about eleven nucleotide sequences, from about four nucleotide sequences to about twelve nucleotide sequences, from about four nucleotide sequences to about thirteen nucleotide sequences, from about four nucleotide sequences to about fourteen nucleotide sequences, from about four nucleotide sequences to about fifteen nucleotide sequences, from about four nucleotide sequence to about sixteen nucleotide sequences, from about four nucleotide sequence to about seventeen nucleotide sequences, from about four nucleotide sequence to about eighteen nucleotide sequences, from about four nucleotide sequence to about nineteen nucleotide sequences, from about four nucleotide sequence to about twenty nucleotide sequences, from about five nucleotide sequences to about nine nucleotide sequences, from about five nucleotide sequences to about ten nucleotide sequences, from about five nucleotide sequences to about eleven nucleotide sequences, from about five nucleotide sequences to about twelve nucleotide sequences, from about five nucleotide sequences to about thirteen nucleotide sequences, from about five nucleotide sequences to about fourteen nucleotide sequences, from about five nucleotide sequences to about fifteen nucleotide sequences, from about five nucleotide sequence to about sixteen nucleotide sequences, from about five nucleotide sequence to about seventeen nucleotide sequences, from about five nucleotide sequence to about eighteen nucleotide sequences, from about five nucleotide sequence to about nineteen nucleotide sequences, from about five nucleotide sequence to about twenty nucleotide sequences, from about six nucleotide sequences to about nine nucleotide sequences, from about six nucleotide sequences to about ten nucleotide sequences, from about six nucleotide sequences to about eleven nucleotide sequences, from about six nucleotide sequences to about twelve nucleotide sequences, from about six nucleotide sequences to about thirteen nucleotide sequences, from about six nucleotide sequences to about fourteen nucleotide sequences, from about six nucleotide sequences to about fifteen nucleotide sequences, from about six nucleotide sequence to about sixteen nucleotide sequences, from about six nucleotide sequence to about seventeen nucleotide sequences, from about six nucleotide sequence to about eighteen nucleotide sequences, from about six nucleotide sequence to about nineteen nucleotide sequences, from about six nucleotide sequence to about twenty nucleotide sequences, from about seven nucleotide sequences to about nine nucleotide sequences, from about seven nucleotide sequences to about ten nucleotide sequences, from about seven nucleotide sequences to about eleven nucleotide sequences, from about seven nucleotide sequences to about twelve nucleotide sequences, from about seven nucleotide sequences to about thirteen nucleotide sequences, from about seven nucleotide sequences to about fourteen nucleotide sequences, from about seven nucleotide sequences to about fifteen nucleotide sequences, from about seven nucleotide sequence to about sixteen nucleotide sequences, from about seven nucleotide sequence to about seventeen nucleotide sequences, from about seven nucleotide sequence to about eighteen nucleotide sequences, from about seven nucleotide sequence to about nineteen nucleotide sequences, from about seven nucleotide sequence to about twenty nucleotide sequences, from about eight nucleotide sequences to about ten nucleotide sequences, from about eight nucleotide sequences to about eleven nucleotide sequences, from about eight nucleotide sequences to about twelve nucleotide sequences, from about eight nucleotide sequences to about thirteen nucleotide sequences, from about eight nucleotide sequences to about fourteen nucleotide sequences, from about eight nucleotide sequences to about fifteen nucleotide sequences, from about eight nucleotide sequence to about sixteen nucleotide sequences, from about eight nucleotide sequence to about seventeen nucleotide sequences, from about eight nucleotide sequence to about eighteen nucleotide sequences, from about eight nucleotide sequence to about nineteen nucleotide sequences, from about eight nucleotide sequence to about twenty nucleotide sequences, from about nine nucleotide sequences to about eleven nucleotide sequences, from about nine nucleotide sequences to about twelve nucleotide sequences, from about nine nucleotide sequences to about thirteen nucleotide sequences, from about nine nucleotide sequences to about fourteen nucleotide sequences, from about nine nucleotide sequences to about fifteen nucleotide sequences, from about nine nucleotide sequence to about sixteen nucleotide sequences, from about nine nucleotide sequence to about seventeen nucleotide sequences, from about nine nucleotide sequence to about eighteen nucleotide sequences, from about nine nucleotide sequence to about nineteen nucleotide sequences, from about nine nucleotide sequence to about twenty nucleotide sequences, and the like.

[0120] In some embodiments of the present invention, 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. In other embodiments of the present invention, 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. In still other embodiments, 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. In additional embodiments, 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

combination, in any orientation, and/or in any order. In further embodiments, 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. In still further embodiments, 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

combination, in any orientation, and/or in any order. In other embodiments, 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.

[0121] As discussed above, 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. Thus, in some embodiments 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.). In other embodiments, 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. In additional embodiments, 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.

[0122] Thus, 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.). Further non-limiting examples of 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:2)(SEQ ID NO:5)(SEQ ID NO:3)(SEQ ID NO:4) ₂ ; (SEQ ID NO:2) ₂ (SEQ ID

NO:6) ₂ (SEQ ID NO:3) ₂ ; (SEQ ID NO:2) ₂ (SEQ ID NO:3) ₃ (SEQ ID NO:9); (SEQ ID

NO:3)(SEQ ID NO:4) ₃ ; (SEQ ID NO:2)(SEQ ID NO:5) ₅ (SEQ ID NO:4); (SEQ ID

NO:2)(SEQ ID NO:4)(SEQ ID NO:5)(SEQ ID NO:8)(SEQ ID NO:9); (SEQ ID NO:5) ₃ (SEQ ID NO:8); (SEQ ID NO:5) ₂ (SEQ ID NO:6)(SEQ ID NO:7)(SEQ ID NO:8) ₂ ; (SEQ ID

NO:6) ₄ ; (SEQ ID NO:5)(SEQ ID NO:4)(SEQ ID NO:6)(SEQ ID NO:9)(SEQ ID NO:8)(SEQ ID:NO:2); (SEQ ID NO:8) ₄ (SEQ ID NO:9) ₂ ; (SEQ ID NO:3)(SEQ ID NO:5) ₂ ; (SEQ ID NO:6)(SEQ ID NO:9); (SEQ ID NO:3)(SEQ ID NO:4) ₂ (SEQ ID NO:7) ₂ ; (SEQ ID NO:9) ₆ ; (SEQ ID NO:3) ₃ ; (SEQ ID NO:5) ₃ (SEQ ID NO:9) ₄ ; (SEQ ID NO:3)(SEQ ID NO:7) ₃ ; (SEQ ID NO:l)(SEQ ID NO:4) ₅ (SEQ ID NO:9) ₄ ; (SEQ ID NO:3) ₈ , and the like.

[0123] In some embodiments, 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). The spacer sequences can be of any length and composition of nucleotides, wherein each nucleotide can be independently adenine, thymine, guanine and/or cytosine (i.e., wherein n=A, T, G, or C), in any order and/or in any combination, and x and y are each independently 0-100 nucleotides or more. In some embodiments, the spacer sequences are the same as (i.e., equivalent to) the flanking sequences (i.e., n _x , n _y ) described herein. Thus, in some embodiments, 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. A non-limiting example of a multimer of the present invention comprising a spacer sequence is the following:

[0124] nnnnnGCGGNNCCGCnnnnnnnnnnnnnnnnGCGGNNCCGCnnnnnnnnn nnnnnnGCGGNNCCGCnnnnn; (SEQ ID NO:2) ₃ , wherein the flanking sequences are in order from left to right: x=5; y+6=16; y+x=15; and y=5. In this example, 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:

[0125] (1) nnnnnGCGGNNCCGCnnnnnnnnnnGCGGNNCCGCnnnnnnnnnnGC GGNNCCGCnnnnn; (SEQ ID NO:2) ₃ , wherein the flanking sequences are in order from left to right: x= ; y+6= 10; y+x= 10; and y=5 ; and

[0126] (2) nnnnnGCGGNNCCGCnnnnnnnnnnnnnnGCGGN CCGCnnnnnnnnn nGCGGNNCCGCnnnnn; (SEQ ID NO:2) ₃ , wherein the flanking sequences are in order from left to right: x=5 ; y+6= 14; y+x= 10; and y=5.

[0127] In the above two examples, 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).

2.2 Promoters

[0128] In accordance with the present invention, 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. This generally requires operably connecting a cw-acting element with a promoter (e.g., 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), to thereby make the promoter inducible by contact with a chemical compound that can induce the expression of the alcohol

dehydrogenase system of A. nidulans.

[0129] Any promoter can be made inducible using the ds-acting elements described above and elsewhere herein. In some embodiments, 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

constitutively, those that drive expression when induced, and those that drive expression in a tissue- or developmentally-specific manner, as these various types of promoters are known in the art and can be made inducible by operably linking thereto the c/s-acting elements described above and elsewhere herein.

[0130] In particular embodiments, 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. Thus, 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. Thus, the smallest fragment of a promoter which still functions as a promoter is also considered a minimal promoter.

[0131] The promoter may be endogenous to the plant. Alternatively, a heterologous promoter may be employed. For example, 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. Alternatively, 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.

[0132] The choice of 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. Where continuous expression is desired throughout the cells .of a plant in addition to inducibility via the chemicals/compounds of the present invention that induce expression of the alcohol dehydrogenase system of A. nidulans, a constitutive promoter can be chosen.

[0133] 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. (1987) Proc. Natl. Acad, Sci. USA 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144- 4148), and the ubiquitin promoter.

[0134] Illustrative examples of tissue-specific promoters include those encoding the seed storage proteins (such as β-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). Thus, the promoters associated with these tissue-specific nucleic acids can be used in the present invention.

Additional examples of 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. 12:3983-4000), S-adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115), 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. 5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore, "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. ( 1989), supra), petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBOJ. 7:1257-1263), bean glycine rich protein 1 promoter (Keller 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 (Kriz et al. (1987) Mol. Gen. Genet. 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872), a-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet. 215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol. 12:579-589), R gene complex-associated promoters (Chandler et al. (19S9) Plant Cell 1 :1175-1183), and chalcone synthase promoters (Franken e/ al. (1991) EMBOJ. 10:2605-2612). Particularly useful for seed-specific expression is the pea vicilin promoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; as well as US Patent No.

5,625,136). Other useful promoters for expression in mature leaves are those that are switched on at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al. (1995) S ience 270:1986-1988).

[0135J In specific embodiments, the promoter that is operably linked to the cis- acting element is one that is specifically or preferentially operable in a plant guard cell. Non- limiting examples of guard cell-specific or guard cell-preferential promoters include:

Arabidopsis trehalase gene promoter (EP 1111051), potato KST1 promoter (Plesch et al.

(2001) Plant Journal 28 (4): 455-464), Arabidopsis pGCl (Atlg22690) promoter (Yang et al, (2008) Plant Methods 4:6) 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

Signaling and Behavior 3, 684-686) and Arabidopsis At5g58580 promoters designated pSUH305S, pSUH305, pSUH305GB, pSUK132, pSUK134, pSUK136, pSUK342, pSUK344, pSUK132GB, pSUK134GB, pSUK136GB, pSUK342GB, pSUK344GB (U.S. Pat. Appl. Pub. 20060117408). [0136] In addition, 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).

[0137] In some instances, 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. Examples of 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. Other non-limiting examples of 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. 6:141-150), the glyceraldehyde-3- phosphate dehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol. 29:1293-1298; Martinez et al. (1989) J. Mol. Biol. 208:551-565; and Quigley et al. (1989) J. Mol. Evol. 29:412-421) the benzene sulfonamide-inducible promoters (U.S. Patent No. 5,364,780) and the glutathione S- transferase promoters. Likewise, one can use any appropriate inducible promoter described in Gatz (1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108.

[0138] 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. Suitably _s 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. nidulans, 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. nidulans, 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

recombination and cloning techniques (e.g., In Maniatis T, Fritsch E F and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience; Gelvin et al. (Eds) (1990) Plant Molecular Biology Manual; Kluwer Academic Publisher, Dordrecht, The Netherlands). The cw-acting element may be upstream or downstream of the promoter. In specific embodiments, the cw-acting element is upstream of the promoter. In some

embodiments, 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. Suitably, the distance between the m-acting element and the promoter is less than 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550 nt.

2.3 Inducer compounds

[0139] Non-limiting examples of chemical compounds that can induce the promoters of the present invention (e.g., 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 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.

[0140] An inducer compound can be provided in any concentration that is not toxic to the plant. Thus, in some embodiments, the concentration of the inducer compound is about 0.01 % to about 10% (v/v) or more. In other embodiments of the present invention, the concentration of the inducer compound is about 0.1 % to about 20% (v/v). In still other embodiments of the invention, the concentration of the inducer compound is about 0.1 % to about 5% (v/v). In further embodiments of the present invention, the concentration of the inducer compound is about 1% to about 2%. Thus, for example, 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%. about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% (v/v), and the like, and any combination thereof.

[0141] 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. In some embodiments of the invention, when the inducer compound is provided in the form of a vapor, the concentration of the inducer compound can be, for example, in a concentration of about 95% to about 100% (v/v). Thus, for example, 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. In some embodiments of the present invention, the inducer compound is provided in more than one form. Thus, for example, 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. Thus, for example, a foliar spray and a root drench can be provided at the same and/or a different concentration than one another.

2.4 Expression products for modulating stomatal closure

[0142] The constructs of the present invention also comprise an operably connected nucleic acid sequence encoding an expression product that inhibits stomatal closure. In some embodiments, the expression product inhibits or abrogates the activity or function of an endogenous polypeptide of the plant, which stimulates or otherwise facilitates stomatal closure. In other embodiments, the expression product is a negative regulator of stomatal closure. Non-limiting examples of 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. 49, 199-222.), OST1 (Mustilli 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. 67, 885-894), 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) and ATHB6 (Himmelbach et al. (2002) EMBO J. 21, 3029-3038; Grill E. (2002) EMBO J. 21 :3029-38).

[0143] 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.

[0144] The present invention contemplates the use of any suitable stomatal closure- modulating polypeptide and polynucleotide in the practice of the invention.

[0145] For example, non-limiting ABI1 polypeptides comprise the amino acid sequence:

[0146] MEEVSPAIAGPFRPFSETQMDFTGIRLGKGYCNNQYSNQDSENGDL

MVSLPETSSCSVSGSHGSESRKVLISRINSPNLNMKESAAADIVVVDISAGDEINGS DIT SEKKMISRTESRSLFEFKSVPLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDP QSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKA LFNSFLRVDSEIESVAPETVGSTSWAVVFPSHIFVANCGDSRAVLCRGKTALPLSVD HKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLB PSIIPDPEVTAVKR VKEDDCLILASDGVWDVMTOEEACEMARKRILLWHKKNAVAGDASLLADERRKEG KDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLKPRRKLKSKPLN [SEQ ID NO:l 1], as set forth for example in GenPept Accession No. 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. [0147] 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. In illustrative examples, an ABI1 nucleic acid sequence comprises the nucleotide sequence:

[0148] gaagcaattgttgcattagcctacccatttcctccttctttctctcttc ctgtgaacaaggcacattagaactct tcttr caacttttttaggtgtatatagatgaatctagaaatagttttatagttgg

tcaagaggtcctaacgaattacccacaatccaggaaacccttattgaaattc

tt gggtatatgtctctctgtttttgcW^

aaatctctcgaau^catt ttgttccattggagcMcttatagatcacaaccagagaaaaagatcaaatctttaccgtta atggaggaagt atctccggcgatcgcaggtcctttcaggccattctccgaaacccagatggatttcaccgg gatcagattgggtaaaggttactgcaataa ccaatactcaaatcaagattccgagaacggagatctaatggtttcgttaccggagacttc atcatgctctgtttctgggtcacatggtt atctaggaaagttttgatttctcggatcaattctcctaatttaaacatgaaggaatcagc agctgctgatatagtc^

ggagatgagatcaacggctcagatattactagcgagaagaagatgatcagcagaaca gagagtaggagtttgtttgaattcaagagtgt gcctttgtatggttttacttcgatttgtggaagaagacctgagatggaagatgctgtttc gactataccaagattccttcaat^ cgatgttagatggtcggmgatcctcaatccgccgctcatttcttcg

gagagaggatgcatttggctttggcggaggagatagctaaggagaaaccgatgctct gcgatggtgatacgtggctggagaagtgga agaaagctctmcaactcgttcctgagagttgactcggagattgagtcagttgcgccggag acggttgggtcaacgtcggtggttgc^ ttgtmcccgtctcacatcttcgtcgctaactgcggtga

taaaccggatagagaagatgaagctgcgaggattgaagccgcaggagg

ctcgccatgtcgagatccattggcgatagatacttgaaaccatccatcattcctgat ccggaagtgacggctgtgaagagagtaaaaga agatgattgtctgatmggcgagtgacggggmgggatgtaatgacggatgaagaagcgtgt gagatggcaaggaagcggattctctt gtggcacaagaaaaacgcggtggctggggatgcatcgttgctcgcggatgagcggagaaa ggaagggaaagatcctgcggcgatg tccgcggctgagtamgtcaaagctggcgatacagagaggaagcaaagacaacataagtgt ggtggtggttgatttgaagcctcgga ggaaactcaagagcaaacccttgaactgaggcagagagggtccttttt^

tactattattaatttgtgcttatttttttaactaacaagt^

ctaaaaagccccttgtatttttcttcccgggctra

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.

[0149] Non-limiting ABI2 polypeptides comprise the amino acid sequence:

[0150] MDEVSPAVAVPFRPFTDPHAGLRGYCNGESRVTLPESSCSGDGAMK

DSSFEINTRQDSLTSSSSAMAGVDISAGDEINGSDEFDPRSMNQSEKKVLSRTESRS LF EFKCVPLYGVTSICGRRPEMEDSVSTIPRFLQVSSSSLLDGRVTNGFNPHLSAHFFGVY DGHGGSQVANYCRERMHLALTEEIVKEKPEFCDGDTWQEKWK ALFNSFMRVDSEI ETVAHAPETVGSTSVVAVVFPTHIFVANCGDSRAVLCRGKTPLALSVDHKPDRDDEA ARIEAAGGK VIRWNGARVFG VLAMSRSIGDRYL PS VIPDPEVTS VRRVKEDDCLILA SDGLWDVMTOEEVCDLARKRILLWHKKNAMAGEALLPAEKRGEGKDPAAMSAAE YLSKMALQKGSKDNISVWVDLKGIRKFKSKSLN [SEQ ID NO: 13], as set forth for example in GenPept Accession No. 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.

[0151] 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. In illustrative examples, an ABI2 nucleic acid sequence comprises the nucleotide sequence:

[0152] tttttgttaaagttcaagaaagttctttm^

attcagaccattcactgaccctcacgccggacttagaggctattgcaacggtgaatc tagggttactttaccggaaagttcttgttct^ acggagctatgaaagattcttcctttgagatcaatacaagacaagattcattgacatcat catcatctgctatggcaggtgtggatatctcc gccggagatgaaatcaacggttcagatgagtttgatccgagatcgatgaatcagagtgag aagaaagtacttagtagaacagagagta gaagtctgtttgagttcaagtgtgttcctttatatgga^

ctagattccttcaagtttcttctagttcgttgctt^

ggccatggcggttctcaggtagcgaattattgtcgtgagaggatgcatctggctttg acggaggagatagtgaaggagaaaccggagt tttgtgacggtgacacgtggcaagagaagtggaagaaggctttgttc tgctccggaaactgttgggictacctcggtggttgcgg ^

gtgtcgcggcaaaacgccactcgcgttgtcggttgatcacaaaccggatagggatga tgaagcggcgaggatagaagctgccggtg ggaaagtaatccggtggaacggggctcgtgtamggtgttctcgcaa^

tccggatccagaagtgacttcagtgcggcgagtaaaagaagatgattgtctcatctt agcaagtgatggtctttgggatgtaatgacaaa cgaagaagtgtgcgatttggctcggaaacggattttactatggcataagaagaacgcgat ggccggagaggctttgcttccggcggag aaaagaggagaaggaaaagatcctgcagcaatgtccgcggcagagtatttgtcgaagatg gctttgcaaaaaggaagcaaagacaat ataagtgtggtagtggttgatttgaagggaataaggaaattcaagagcaaatccttgaat t

aaaaaagttttgatggtgggtaaaaattctctttagtgaaaaaagaaagataaa^^

taaatttgttatttactttctcaaaaa [SEQ ID NO: 14], as set forth for example in GenBank Accession No. Y08965, or a complement thereof;

tccaacttcaatttctctcctttctcttcccaactttgattcctgatttgggttttt gttaaagttcaagaa^

acgaagtttctcctgcagtcgctgttccattcagaccattcactgaccctcacgccg gacttagaggctattgcaacggtgaatctagggt tactttaccggaaagttcttgttctggcgacggagctatgaaagattcttcctttgagat caatacaagacaagattcattgacatcatcatc atctgctatggcaggtgtggatatctccgccggagatgaaatcaacggttcagatgagtt tgatccgagatcgatgaatcagagtgaga agaaagtacttagtagaacagagagtagaagtctgtttgagttcaagtgtgttcctttat atggagtgacttcgatttgtggtagacgacca gagatggaagattctgtctcaacgattcctagattccttcaagtttcttctagttcgttg cttgatggtcgagtcactaatggatttaatcctca cttgagtgctcatttctttggtgtttacgatggccatggcggttctcaggtaatgaatcg aWggmcga gatatgatcggaaactgca aaaacttggtttttgacatttgtttttgtgtgt aggtagcgaattattgtcgtgagaggatgcatctggcttt^

gagaagccggagtmgtgacggtgacacgtggcaagagaagtggaagaaggctttgtt caactcttttatgagagttgactcggagatt gaaactgtggctcatgctccggaaactgttgggtctacctcggtggttg^^

ctctagggcggttttgtgtcgcggcaaaacgccactcgcgttgte^

aatcatatattcattaggacttgcggttttttgttatggtgttaccaatcatagcat gmctatagatta

acatttatgtgtagccggatagggatgatgaagcggcgaggatagaagctgccggtg ggaaagtaatccggtggaacggggctcgtg tatttggtgttctcgcaatgtcaagatccattggtaattattt^

caacccgtcgcgaacaggcgatagataccttaaaccgtcagtaattccggatccaga agtgacttcagtgcggcgagtaaaagaagat gattgtctcatcttagcaagtgatggtctttgggatgtaatgacaaacgaagaagtgtgc gamggctcggaaacggattttactatggca taagaagaacgcgatggccggagaggctttgcttccggcggagaaaagaggagaaggaaa agatcctgcagcaatgtccgcggca gagtafflgtcgaagatggcmgcaaaaaggaagcaaagacaatataagtgtggtagtggt tgatttgaagggaataaggaaattcaag agcaaatccttgaattgaaaaagaaggtttggaagaaaagtgaaaaaaaaagttttgatg gtggg [SEQ ID NO: 15], as set forth for example in GenBank Accession No. 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.

[0153] An illustrative OST1 polypeptide comprises the amino acid sequence:

[0154] MDRPAVSGPMDLPIMHDSDRYELVKDIGSGNFGVARLMRDKQSNE LVAV YIERGEKIDEWKREIINHRSLRHPNIVRFKEVILTPTHLAIVMEYASGGELFER ICNAGRFSEDEARFFFQQLISGVSYCHAMQVCHRDLKLENTLLDGSPAPRLKICDFGY SKSSVLHSQPKSTVGTPAYIAPEVLL KEYDGKVADVWSCGVTLYVMLVGAYPFED PEEPKNFRKTIHRILNVQYAIPDYVHISPECRHLISRIFVADPAKRISIPEIRNHEWFLK N LPADLMNDNTMTTQFDESDQPGQSIEEIMQIIAEATVPPAGTQNLNHYLTGSLDIDDD MEEDLESDLDDLDIDSSGEIVYAM [SEQ ID NO: 16], as set forth for example in GenPept Accession No. 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. [0155] 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. In representative examples, an OSTJ nucleic acid sequence comprises the nucleotide sequence:

[0156] atggatcgaccagcagtgagtggtccaatggatttgccgattatgcacgatagtgatagg tatgaactcgtca aggatattggctccggtaattttggagttgcgagattgatgagagacaagcaa^

tgagaaggteagtttattttcttcttgtr^

gtgttcatcatccaatgagagatgtgtgmggttactttatggrtatgaatgg

accaaatttgctctgctttgctttatgacttatgtt

ggacttrttgtatttgtacagatagatgaaaatgtaaaaag

agaggtttgttttcaactctcttttaagctgttttcttattatta^

gttatattaacaccaacccatttagccattgttatgga

cgaagacgaggttgttctctctttttttt^

gaggtttttcttccagcaactcatttcaggagttagttactgtcatgctatggtaat gaaaaat^^ gaaattcagctgacttaagaagttaaattttatgttgtagca

ggcccctcgtctaaaga gtgatttcggatattcaaaggtatctttgaaaacaWcagaaactctgagttagttagt^

gtmcttmcagtcatcagtgttacattcgcaaccaaaatcaactgttggaactcctgc ttacatcgctcctgaggttttactaaagaaagaa tatgatggaaaggtactccattttcatagttcccaaactagtatgataaccatatcttat agaaagaacaatctttgtatttttatcctc agataggggaaacatgctttctcttgatgaaagctcacacaaataaaacaatcttggctc ttcaagaattttggtgaagaaagctattaaga gtctgatttgtaaactgataattcttgagttttggttgaattagtcaaatgcatcctaat gttcttctttt^

gggttactctgtatgtcatgctgg^ggagcatatcctttcgaagatcccgaggaacc aaagaatttcaggaaaactatacatgtgagcctt tcactttcttcatgcttcaatagttgaaaaatgtaattatggattttattacttgctagc taaactatctgttctcttgtgaaaatatttgctcagag aatcctgaatgttcagtatgctattccggattatgttcacatatctcctgaatgtcgcca tttgatctccagaatatttgttgctg aggtaaaggaaggatcatgaaagctgcatttgttgatttatttgtgaattttctttatag tatgactgaaaagagaacW^

ttggttggtttcttggcagaggatatcaattccagaaataaggaaccatgaatggtt tctaaagaatctaccggcagatctaatgaacgata acacgatgaccactcagtttgatgaatcggatcaaccgggccaaagcatagaagaaatta tgcagatcattgcagaagcaactgttcct cctgcaggcactcagaatctgaaccattacctcacaggtgagacaacacaaaacataaac ttttcgatttcttgtcttttttaatgcttctcaa tgtttgaaaaacttatcattaatggataaacaggaagcttggacatagatgacgatatgg aggaagacttagagagcgaccttgatgatct tgacatcgacagtagcggagagattgtgtacgcaatgtga [SEQ ID NO: 17], as set forth for example in GenBank Accession No. 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.

[0157] Representative AAPK polypeptides comprises the amino acid sequence:

[0158] MDMPPPIMHDSDRYDFVRDIGSGNFGVARLMTDKLTKDLVAVKYI ERGDKIDENVKREIINHRSLRHPNIVRFKEVILTPTHLAIVMEYASGGEMSDRISKAGR FTEDEARFFFQQLISGVSYCHSMQVCHRDL LENTLLDGDPALHLKICDFGYSKSSVL HSQPKSTVGTPAYIAPEVLLKQEYDGKIADVWSCGVTLYVMLVGSYPFEDPDNPKDF RKTIQRVLSVQYSVPDFVQISPECRDIISRIFVFDPAERITIPEIMKNEWFRKNLPADLV N ENIMDNQFEEPDQPMQSMDTDViQIISEATVPAAGSYYFDEFIEVDEDMDEIDSDYELD VDSSGEIVYAI [SEQ ID NO:58], as set forth for example in GenPept Accession No.

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. [0159] 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. In representative examples, an AAPK nucleic acid sequence comprises the nucleotide sequence:

[0160] cggcacgagattaaaaaggccacaatgttgcttactctccaacaacaaccgtaatcctct cggaatctccact acgacgccgtttacttccgatctctctccccgccggagcagcagccatggatatgccgcc gccgatcatgcacgacagtgaccgttac gacttcgttcgtgatatcggatcgggaaatttcggcgtcgctagactcatgactgataaa ctcaccaaagaccttgttgctgtcaagte cgaacgtggagataagattgatgaaaatgttaagagagaaataatcaatcacaggtctct aagacatcctaatattgttaggttteaggag gtcattttaacacctactcatctggccattgtaatggaatatgca^

ctgaggatgaggctcgtttcttctttcaacaactcatatccggggtcagctattgtc attcaatgcaagtatgtcatcgagatctgaagttgg aaaacacgttgttggatggagacccagcacttcatctgaagatttgtgattttggatact ccaaatcttcggtgcttcattcacagccaaagt caactgtgggaactcctgcttatattgctccagaagtacttctgaagcaagagtatgatg gaaagattgccgatgtctggtcatgtggtg^ accttatacgtgatgctagtggggtcatatccttttgaagatcccgataatccgaaggat ttccggaagacaattcagagggttctcagtgt ccagtattccgtaccagactttgttcaaa ctcctgaatgtcgcgacatta caagaatctttgttttt

attccagaaataatgaagaacgaatggttccgaaagaatcttcctgctgacttggtg aatgaaaatataatggataaccaatttgaagagc cagatcagcctatgcagagtatggatacgatcatgcagataatttcagaagctaccgtac cagcagctgggagctattatmgacgagtt cgaagtggatgaagaMggatgaaatagactctgactatgaacttgatgtagatagcagtg gtgagattgtatatgccatataatttaa tcatcatagaggtcacatattgaaaaggaagcaccttatattgagctttatggctttctc agcctcaaagctaaaaaaataaatattctgaga ctatmctgcagactggatgatgcacgaagttcatcatgt^

atcacttttgtgagttgaggcaacatgtmcgaarttgtagggatcttcrttartcct taaaaaaagttccacaacttc

ggcataatrttagaacgtggcatggcataattgagattttatatgcatgaaatatgg taacgagctcttgatttctffl

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.

[0161] A non-limiting AHA1 polypeptide comprises the amino acid sequence: [0162] MSGLEDIKNETVDLEKIPIEEVFQQLKCTREGLTTQEGEDRIVIFGPN KLEEKKESKILKFLGFMWWLSW MEAAALMAIALANGDNRPPDWQDFVGIICLLVI NSTISFIEENNAGNAAAALMAGLAPKTKVLRDGKWSEQEAAILVPGDIVSIKLGDIIPA DARLLEGDPLKVDQSALTGESLPVTKHPGQEVFSGSTCKQGEIEAVVIATGVHTFFGK AAHLVDSTNQVGHFQ VLTSIGNFCICSIAIGIAIEIVVMYPIQHRKYRDGIDNLLVLLI GGIPIAMPTVLSVTMAIGSHRLSQQGAITKRMTAIEEMAGMDVLCSD TGTLTLNKLS VDKNLVEVFCKGVEKDQVLLFAAMASRVENQDAIDAAMVGMLADPKEARAGIREV HFLPFNPVD RTALTYIDSDGNWHRVSKGAPEQILDLANARPDLRKKVLSCID YAE RGLRSLAVARQVVPEKTKESPGGPWEFVGLLPLFDPPRHDSAETIRRALNLGVNVKM ITGDQLAIGKETGRRLGMGTNMYPS AALLGTDKDSNI ASIPVEELIEKADGF AGVFPE HKYEIVKKLQERKHIVGMTGDGVNDAPALKKADIGIAVADATDAARGASDIVLTEPG LSVIISAVLTSRAIFQRMKNYTIYAVSITIRIVFGFMLIALIWEFDFSAFMVLIIAILND GT IMTISKDRVKPSPTPDSWKLKEIFATGIVLGGYQAIMSVIFFWAAHKTDFFSDKFGVRS IRDNNDELMGAVYLQVSIISQALIFVTRSRSWSFVERPGALLMIAFVIAQLVATLIAVY ADWTFAKVKGIGWGWAGVIWIYSIVTYFPQDILKFAIRYILSGKAWASLFDNRTAFTT KKDYGIGEREAQWAQAQRTLHGLQPKEDVNIFPEKGSYRELSEIAEQAKRRAEIARL RELHTLKGHVESVAKLKGLDIDTAGHHYTV [SEQ ID NO: 18], as set forth for example in GenPept Accession No. 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.

[0163] 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. In illustrative examples, an AHA1 nucleic acid sequence comprises the nucleotide sequence:

[0164] caaccattgatgatcaccaaatcataatcaacggtcgaaatgaatgaaaaataaaagtct gaaacggtggag agtttcgtcgttttgagtggttataaaaagagaaggcgtatatctcctctgcaacagaag ctcgcrttctcte^

gtgatcgagtggtgaaacgacagagaggggcgtcgtmgttgamcttctgggtgaaga tgtcaggtctcgaagatatcaag accgttgatctggaaaaaattccgattgaggaagttttccagcagctaaaatgtacaagg gaaggattgacaacgcaggaaggggaag acaggattgtgatatttggccccaacaagctcgaagagaagaaggaaagcaaaattctga agtttctggggttcatgtggaatccgcttt catgggttatggaagctgcagctctcatggccattgctttggctaatgg¾^

gtctgcttgttatcaactccacaatcagtttcattgaagaaaacaacgccgg

accaaggttcttagggatggaaaatggagtgaacaagaggctgctatccttgtccca ggtgatattgttagcattaaacttggagacatta tcccagccgatgcccgtcttcttgaaggagatcctttaaaggttgatca^

tggtcaagaagttttctctggttcaacttgtaaacaaggagaaatcgaagcggttgt tatagccactggagttcacaccttctttggtaaag ctgctcaccttgtggacagcactaaccaagttgggcacttccagaaag^

tatagcgattgaaatagtcgtcatgtaccctatccaacaccgaaagtacagagatgg aattgacaatctcttggtcctcttgatcggtggta tccccattgc gcccacggtcttgtctgtgactatggc cgggtctcacaggttgtctcagcaaggtgctatcaccaaacgtatgaca gccattgaagaaatggcgggaatggatgtccmgcagtgacaaaaccgggacactaaccct taataaattgagtgtggataaaaacttg gttgaggttttctgcaagggtgtggagaaagatcaagttctactatttgcagctatggct tctagggtggagaaccaggacgctattgatg cagccatggttggaatgcttgctgatccgaaagaggcccgagctggaatcagagaggttc acttccttccattcaaccctgtggataag agaactgctttgacttacatcgactctgatggtaactggcacagagtcagcaaaggtgct cccgagcagatccttgaccttgccaatgcc aggcctgaccttaggaagaaggtactctcttgtattgacaagtacgctgagcgcggtctt aggtcgttggcagtagctcgtcaggtggta cccgagaaaacaaaagaaagcccaggtggaccatgggaatttgttggcttgttgcctctt tttgaccctccaagacacgacagtgccga aaccattcgtagggcgttgaatctaggtgttaatgtgaagatgatcactggtgatcaact tgctattggtaaggaaaccggtcgcaggctt ggaatgggaaccaatatgtatccatctgcggctcttctcggtaccgacaaggactcgaac attgcatccatccctgttgaggagttgatc gagaaggctgatgggtttgccggcgtctttccagagcacaaatatgaaattgtgaaaaag ctgcaggagaggaagcacattgttggtat gaccggtgatggtgttaatgatgcacccgctttgaagaaagcagatatcggtattgctgt ggccgatgctacagatgctgctcgtggtgc ttcagatatcgtcctcacggaacctgggctgagtgtcatcatcagtgccgttctcactag cagagctatcttccagagaatgaagaactac accatttatgcagtctcaatcacaatccgtattgtgtttggtttcatgcttattgcmgat atgggaatttgacttctcagcg^

tattgccatccttaatgatggtactatcatgacaatctcaaaggacagagtcaagcc atctcccacacctgatagctggaagctcaaaga aattttcgccactggaattgtgctgggaggctaccaagccattatgagtgttattttctt ctgggctgctcacaagaccgact^ caagttcggtgtgaggtcaatcagggacaataacgatgaactaatgggtgctgtgtatct acaagttagtatcatcagtcaagctctaatct ttgtcaccaggjcaaggagttggtcatttgtcgaacgtcctggggcgctt^^

tcgcagtgtatgccgactggacamgcaaaggtgaagggtatcggttggggatgggca ggtgtgatttggatttacagtatcgtaacat acttcccacaggacattttgaagtttgccattcggtatatcttgagtggaaaggcttggg ccagcttgtttgacaacaggaccgctttcaca accaagaaagattacggtattggagaaagagaagctcaatgggcacaagctcaaaggaca ttgcacggtctgcagccaaaagaagat gttaatatcttcccagagaaaggaagttacagagagctgtctgagatcgcagagcaagcc aagagaagggccgagatagctaggctt agggagcttcacacattgaagggacatgtggaatcagtcgcaaagctaaagggattggac attgatacagcaggacatcactacactg tgtagttggagttgcacaacaacacaaacatttaccgaaaaccaaccccatcatgactca tcttttgitttgtcttcacaa^

gtcattacagtagagggaagagaactcttgtgtattgccataactcttc^

ctactcgatatgagctttgcaaatttgcctttgaagaaacaaggccctttgcctttg aagagacaaggcactttgctttttaatggcctaatgt gattattc cactcttttgtctgtttgatcaam^ [SEQ ID NO: 19], as set forth for example in GenBank Accession No. 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.

[0165] Illustrative vSNAREs AtVAMP711-14 polypeptides comprise the amino acid sequence:

[0166] MAILYALVARGTVVLSEFTATSTNASTIAKQILE VPGDNDSNVSYS

QDRYVFHVKRTDGL LCMAEETAGRRIPFAFLEDIHQRFVRTYGRAVHTALAYAM NEEFSRVLSQQIDYYSNDPNADRINRIKGEMNQVRGVMIENIDKVLDRGERLELLVD KTANMQGNTFRFRKQARRFRSNVWWRNCKLTVLLILLLLVIIYIAVAFLCHGPTLPSC

I [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.

[0167] 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. In illustrative examples, a vSNAREs AtVAMPl 11 nucleic acid sequence comprises the nucleotide sequence:

[0168] ggcgaaaaggtctccagctccagctaaactccaagaggagagagagagaaagagattcaa ttatatgcag agagaaaaagaagccaattggttctctctctctctctctctctctctgattgattgatcc agcgattcg ctggctggctcg

aaaaccttcttcttcgttgaccttacacgagtttt^

tcgtggcacggtggttctttctgagttcaccgccacctctacgaatgcgagcaccat cgccaaacagatcctcgagaaggtccctggag acaacgacagcaacgtctcctactctcaggatcgttacgtcttccacgttaaacgcaccg atggcctcaccgttctctgtatggccgaag aaaccgccggaaggagaattcctmgccttmggaggatattcaccagagattcgtacggac ttatggcagggctgttcatacagcact agcttatgcaatgaatgaggaattctctagagttctcagtcagcagattgactattactc taatgatcctaatgccgataggattaa aagggtgaaatgaatcaggtgcggggtgtcatgatagaa^

aaaaccgccaatatgcaggggaatacattccggttcagaaagcaagctcgtcgtttt agaagcaacgtctggtggagaaactgcaa^ tcacggtcctcttaatactactactactggtgatcatatac^

catctgtctcttaaggaatccatcccgattctgcgcgttgccacgactttttatctc ctcatctgattgtaaaccttgtgttttcc atgtggcttaaaaagcttatgtttccaactcacgtatatgaaaaagtatacttatttata gagcaatgtaaaagctcattgctttgcgaagtgt gtgaatctttgttgacatcatgttctataaattacatacata^

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.

[0169] A non-limiting GPA1 polypeptide comprises the amino acid sequence:

[0170] MGLLCSRSRHHTEDTDENTQAAEIERRIEQEAKAEKHIRKLLLLGAG ESGKSTIFKQIKLLFQTGFDEGELKSYVPVIHANVYQTIKLLHDGTKEFAQNETDSAK YMLSSESIAIGEKLSEIGGRLDYPRLTKDIAEGIETLWKDPAIQETCARGNELQVPDCT KYLMENLKRLSDINYIPTKEDVLYARVRTTGVVEIQFSPVGENKKSGEVYRLFDVGG QRNERRKWIHLFEGVTAVIFCAAISEYDQTLFEDEQKNRMMETKELFDWVLKQPCFE KTSFMLFLNKFDIFEKKVLDVPLNVCEWFRDYQPVSSGKQEIEHAYEFVKKKFEELY YQNTAPDRVDRVF IYRTTALDQKLVKKTFKLVDETLRRRNLLEAGLL [SEQ ID NO:22], as set forth for example in GenPept Accession No. 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. [0171] 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. In illustrative examples, a GPA1 nucleic acid sequence comprises the nucleotide sequence: [0172] gttaacttaatagtatataaaaiaaaaatgcatataggttccgtaattaatcttctttat cgtcacgagaggcacat cttttttcaacatttgaccactctctctctct^

caaatattaaaaatatatccatttttattttatttttaattaaattcataatttgca tttlat

agtgaagcaaaaacattaaagcggaaagaaagtggtaaaacaataatagaaacagga gaagcagaagtactacttcttcttcttctgc ctcttctcagaccttgttttgtactttcttcttcttcttcttcttcttgtttgcgaactc cgatatcttcttcactacc^

caggtgtaggcattgtcttgttatgagaagcaactgtagctggaagctcaag^

acttctatgttattacctgtggggatatagaaacaatcatgggcttact^^^

acacaggctgctgaaatcgaaagacggatagagcaagaagcaaaggctgaaaagcat attcggaagcttttgctacttggtgctgggg aatctggaaaatctacaatttttaagcagataaaacttctattccaaacgggatttgatg aaggagaactaaagagctatgttccagtcattc atgccaatgtctatcagactataaaattattgcatgatggaacaaaggagtttgctcaaa atgaaacagattctgctaaatatatgttatcttc tgaaag^ttgcaattggggagaaactatctgagattggtggtaggttagactatccacgt cttaccaaggacatcgctgagggaate^ aacactatggaaggatcctgcaattcaggaaacttgtgctcgtggtaatgagctt^

ttgaagagactatcagatataaattatattccaactaaggaggatgtactttatgca agagttcgcacaactggtgtcgtggaaatacagtt cagccctgtgggagagaataaaaaaagtggtgaagjg accgattg^

tcatctgtttgaaggtgtaacagctgtgatattttgtgctgccatcagcgagtacga ccaaacgctctttgaggacgagcagaaaaacag gatgatggagaccaaggaattattcgactgggtcctgaaacaaccctgttttgagaaaac atccttcatgctgttcttgaacaagttcgac atatttgagaagaaagttcttgacgttccgttgaacgtttgcgagtggttcagagattac caaccagtttcaagtgggaaacaagagattg agcatgcatacgagtttgtgaagaagaagtttgaggagttatattaccagaacacggcgc cggatagagtggacagggtattcaaaatc tacaggacgacggctttggaccagaagcttgtaaagaaaacgttcaagctcgtagatgag acactaagaaggagaaatttactggagg ctggccttttatgaccttattattacatatctctagtaaattacctctccttattattat aagaaaaactcgaaaactg

ctttcgggacaaaagacttagcgattcaaaatctaatgtgtctcgatggctacgact agmctattttatcattgtttttgttaacattcctctgt ctttgacttcttatttttttttctcate^

[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.

[0173] An illustrative AtABCG22 polypeptide comprises the amino acid sequence:

[0174] MSMEKPPLASGLARTRSEQLYETVAADIRSPHGSMDANGVPATAPA

AVGGGGTLSRKSSRRLMGMSPGRSSGAGTHIRKSRSAQLKLELEEVSSGAALSRASS

ASLGLSFSFTGFAMPPEEISDSKPFSDDEMIPEDIEAGKKKPKFQAEPTLPIFLKFR DVT

YK IKKLTSSVEKEILTGISGSVNPGEVLALMGPSGSGKTTLLSLLAGRISQSSTGGS VTYNDKPYSKYLKSKIGFVTQDDVLFPHLTV ETLTYAARLRLPKTLTREQKKQRAL DVIQELGLERCQDTMIGGAFVRGVSGGERKRVSIGNEIIINPSLLLLDEPTSGLDSTTAL RTILMLHDIAEAGKTVITTIHQPSSRLFHRFDKLILLGRGSLLYFGKSSEALDYFSSIGC S PLIAMNPAEFLLDLANGNINDISVPSELDDRVQVGNSGRETQTGKPSPAAVHEYLVEA YETRVAEQEKKKLLDPVPLDEEAKAKSTRLKRQWGTCWWEQYCILFCRGLKERRHE YFSWLRVTQVLSTAVILGLLWWQSDIRTPMGLQDQAGLLFFIAVFWGFFPVFTAIFAF PQERAMLN ERAADMYRLSAYFLARTTSDLPLDFILPSLFLLWYFMTGLRISPYPFFL SMLTVFLCIIAAQGLGLAIGAILMDLKKATTLASVTVMTFMLAGGFFVKASPLFLDFL CF [SEQ ID N0.24], as set forth for example in GenPept Accession No. 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.

[0175] 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. In illustrative examples, an AtABCG22 nucleic acid sequence comprises the nucleotide sequence:

[0176] ttccccaaaggtatcgattctatatcctaagaaaaaatcatacccatctte

tattttatgtctgttcamgtttatgttccatatatacatacatataagttctatata t^^

attatgattmggtcgacagttmgaaaaggtcaaaat

cagctatatgagacggttgcagcagacataaggtcacctcacggctccatggacgct aatggtgtgcctgcgacggctccagcagcc gttggaggaggaggaacgttgtcgaggaaatcaagccggaggttgatggggatgtctccg gggaggagtagcggcgccggaacac acataaggaagtctaggagcgctcagcttaagctcgagctagaggaagtgagtagcggcg cagctttgagccgtgcgtctagcgcat cgctcggtctttcatmccttcaccgggtttgctatgccgccggaggaaatctccgactct aaaccgttcagcgacgacgagatgatacc cgaagatattgaagcgggaaagaagaagcctaagtttcaagcagaaccaacattgcccat ctttctcaagttcagggatgttacatacaa agtggtgatcaagaaattgacttcatctgtggagaaagagatattaa^

atgggaccctcagggagtggcaaaacaactcttcttagcttacttgctggtcgaatc tctcaatcctctactggaggctctgttactte cgacaagccttactctaaatacttgaaaagcaagattgggWgtgactc^

aacctatgctgctcgtctgcgctecccaagactcttacgagagagc

agagagatgccaagacactatgattggtggagcattcgtgcgtggtgtatcaggtgg agagaggaaaagagmctattggaaacgag atcatcattaatccttctctattacttcttgatgaaccaa^

gccgaggcggggaaaaccgtgatcacaacgatacatcagccctcgagtaggctcttc cataggtttgacaagctgattctactaggaa gaggaagtcttctctactttggaaaatcatcagaagctttagattacttctcttccattg gatgctctcctcttatcgccatgaatcctgcaga gttcttgctcgatcttgccaacggtaacatcaacgatatctctg^

aactcaaactggcaagccatctcctgctgctgttcatgagtatctagtggaggccta cgagactagggttgcagaacaggagaagaag aaactattggatcctgtgccactcgatgaagaagctaaggccaaaagtacgcgtctaaag cgccaatggggaacgtgctggtgggag caatattgcatactattctgcagaggactcaaagaacggcgacacgaatacttcagttgg ttgcgtgttacgcaagttctttccacagctgt cattttaggtcttctctggtggcagtcggacattaggactccaatgggactacaagatca ggctggtttgctcttcttcatagcagttttttgg ggattcttccctgttttcacagcgatctttgcgtttccgcaagagcgagcgatgttaaat aaggagagagcagcggatatgtacagattaa gcgcatatttcctagctcgaaccacgagtgatctccctctcgactttattctaccttctc tcttccttcttgtcgtctatttc

gatcagcccatatcccttcttcttgagcatgctcacagttttcctttgcatcatcgc agctcagggactcggacttgcaattggtgccatttta atggatttaaagaaggctacgactttggcttcagtaactgtcatgac^^

cttgamcctctgtttttaacttcccctgtttcttgatttcccctgttttactgattt ccttttctgtgtctacac

ggatacgttatctatctttcaattaccacacctacaagcttcttcttaaagtacaat atcaggacttcgctgtgtccatcaacgggatgagaat agacaacggactaactgaagtagccgcactcg^gtcatgatattcggttatcgcctcctc gcgtatctgtctctaaggcaaatgaagatc gtaacataacccattttccacacgaagaaatcaaataacatagaagaagcataaaaagag tgcatcagatcttgatgatcttgacacgac caatccttgacaatgattaagagattggtcctaagattattcttgcttaattacaaaggt gttgtgagtttgataatgattggatggtgatagat gtcttgattggagataaatatatatgttcaagtttgtaattgtagttcgaatctaaaatt ggttaaagtttattaacaagaagaacctt^ gatctgatcttcttaaattccaatccaattattcttagtat [SEQ ID NO:25], as set forth for example in GenBank Accession No. 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.

[0177] Non-limiting AtABCG40 polypeptides comprise the amino acid sequence:

[0178] MEGTSFHQASNSMRRNSSVWKKDSGREIFSRSSREEDDEEALRWAA

LE LPTFDRLRKGILTASHAGGPINEIDIQKLGFQDTi KLLERLIKVGDDEHEKLLWKL

KKRIDRVGIDLPTIEVRFDHLKVEAEVHVGGRALPTFVNFISNFAD FLNTLHLVPNR KKKFTILNDVSGIVKPGRMALLLGPPSSGKTTLLLALAGKLDQELKQTGRVTYNGHG

MNEFVPQRTAAYIGQNDVHIGEMTVRETFAYAARFQGVGSRYDMLTELARREKEAN

IKPDPDIDIFMKAMSTAGEKTNVMTDYILKILGLEVCADTMVGDDMLRGISGGQKXR

VTTGEMLVGPSRALFMDEISTGLDSSTTYQIVNSLRNYVHIFNGTALISLLQPAPET FN LFDDIILIAEGEIIYEGPRDHWEFFETMGFKCPPPvKGVADFLQEVTSKKDQMQYWAR RDEPYRFIRVREFAEAFQSFHVGRRIGDELALPFDKTKSHPAALTTKKYGVGIKELVK TSFSREYLLMKRNSFVYYFKFGQLLVMAFLTMTLFFRTEMQKKTEVDGSLYTGALFF ILMMLMFNGMSELSMTIAKLPVFYKQRDLLFYPAWVYSLPPWLLKIPISFMEAALTTF ITYYVIGFDPNVGRLFKQYILLVLMNQMASALFKMVAALGRNMIVANTFGAFAMLV FFALGGWLSRDDIKKWWIWGYWISPIMYGQNAILANEFFGHSWSRAVENSSETLGV TFLKSRGFLPHAYWYWIGTGALLGFVVLFNFGFTLALTFLNSLGKPQAVIAEEPASDE TELQSARSEGWEAGANKKRGMVLPFEPHSITFDNWYSVDMPQEMIEQGTQEDRLV LLKGVNGAFRPGVLTALMGVSGAGKTTLMDVLAGRKTGGYIDGNITISGYPK QQT F ARISGYCEQTDIHSPHVTV YESLV YS A WLRLPKEVDKNKRKIFIEEVMELVELTPLR QALVGLPGESGLSTCQRKRLTIAVELVANPSIIFMDEPTSGLDARAAAIVMRTVRNTV DTGRTVVCTIHQPSIDIFEAFDELFLLKRGGEEIYVGPLGHESTHLINYFESIQGINKIT E GYNPATWMLEVSTTSQEAALGVDFAQVYKNSELYKRNKELIKELSQPAPGSKDLYFP TQYSQSFLTQCMASLWXQHWSYWRNPPYTAVRFLFTIGIALMFGTMFWDLGGKTKT RQDLSNAMGSMYTAVLFLGLQNAAS VQPVVNVERTVFYREQAAGMYS AMP YAFA QVFIEIPYVLVQAIVYGLIVYAMIGFEWTAVKFFWYLFFMYGSFLTFTFYGMMAVAM TPNHHIASVVSSAFYGIWNLFSGFLIPRPSMPVWWEWYYWLCPVAWTLYGLIASQFG DITEPMADSNMS VKQFIREF YGYREGFLGVVAAMNVIFPLLF A VIF AIGIKSFNFQKR

[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.

[0179] 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. In illustrative examples, mAtABCG40 nucleic acid sequence comprises the nucleotide sequence:

[0180] a catcmcatctacaatttctctcttgagtttcttt^

tcatctgacacaaacaaaaaaagagagaagaaaaaaaagaagaagactttgatttct tggatacaaaatggagggaactagttttcacc aagcgagtaatagtatgagaagaaactcatcggtgtggaagaaagattcaggaagggaga ttttctcgaggtcatctagagaagaaga cgatgaagaagctttgagatgggctgctcttgagaagcttcccact^

aggacccatcaacgagatcgatattcagaagcttgggtttcaagatactaagaaact gctagagaggctcatcaaagtcggtgacgatg agcatgagaaactcctctggaaactcaagaaacgtatcgatagagttggaatcgatcttc cgacaatagaagttcggtttgatcatctaaa agttgaagcagaggttcatgttggaggcagagctttacctacgttcgtcaatttcatctc caattttgctgataagttcctgaatactctgcat cttgttccgaaccgaaagaagaagttcactatactcaacgacgtcagcggaatcgicaag cctggcaggatggctctgcttttgggtcct ccaagttctgggaaaacgaccctcttgcttgccttggcgggaaagcttgatcaagaacta aagcaaactggaagagtgacatacaatg gtcatggaatgaacgagtttgtgccacaaagaacagctgcatatatcggccaaaacgatg ttcatatcggtgagatgactgttcgtgaga cttttgcttacgcagctcgcttccaaggtgttggttcgcgttatgacatgttgacagagt tggcaagaagagagaaagaagcaaacatca aacctgaccctgatattgatatattcatgaaggcgatgtcaacagcaggtgaaaaaacaa atgtgatgacagattatatcctcaagatctt aggacttgaggtctgtgcagacactatggtcggcgatgatatgttgagaggcatctccgg aggacaaaagaagcgtgtcactactggt gaaatgctggttggaccgtctagggctctgttcatggatgagatatcgactggtttagat agttcaacgacttaccagatagtgaactccct cagaaactatgttcatatcttcaatgggacagctctgatctctctccttcagcctgcgcc agagacattcaatctcttcgatgatatcattctc attgcagaaggcgagatcatctacgagggccctcgtgatcacgttgtggagttctttgag accatgggattcaaatgtcctccaagaaaa ggcgttgctgatttccttcaagaagtgacatcaaagaaagaccaaatgcagtactgggca cgacgtgatgagccttacaggttcattaga gtgagagagtttgcagaggcgtttcaatcattccacgttggccggagaatcggagatgag cttgctttgccctttgacaagacaaagag ccatccggctgctctaaccaccaagaaatacggagttgggattaaagaacttgtcaagac cagcttctcaagagaatacttactcatgaa aagaaactcctttgtttactacttcaagtttggacaactgctggt^

aagactgaggttgatgggagtctctacactggagccttgttcttcatccttatgatg ctcatgttcaatggaatgtctgaactttcaatgacca tagcaaaacttcctgtgitttacaaacaaagagatctcctctte^

aagcttcatggaagccgctctcacaacattcatcacttactatgtca^

cctcgtgctcatgaaccaaatggcttcagcattgtttaagatggtggcagcattggg aagaaacatgatcgttgcaaatacatttggtgca tttgcgatgctcgtcttctttgccttgggtggtgiggtacW^

ataatgtatggacagaacgcgatcctagccaatgagttctttggacacagctggagt cgagctgtcgaaaactcgagcgaaacacttgg agttactttccttaagtctcgtgggttcttaccccatgcatac¾

tggtttcacgctggctctgacgittctgaactccttgggaaagcctcaagctgttat tgcagaagagcctgcgagtgatgagacagaactt cagtctgctaggtcagaaggtgtagttgaagctggtgccaataagaaaagagggatggtg cttccatttgagccacattcaattaccttc gacaatgttgtatactcagttgacatgccccaggaaatgatagagcaaggcacacaagaa gacagacttgtcctgttgaaaggtgtgaa tggtgcattcaggccaggcgtgctcacggctctcatgggtgtctctggagctggcaaaac cactctgatggatgttcttgccggaagga aaaccggtggttatattgatggcaacatcaccatttccggttaccctaagaatcaacaaa catttgcccgtatctcaggatactgtgaacaa actgatatccattccccacatgtcactgtttacgagtcct¾

aaagatattcatagaggaagtgatggagctggtggagttaacgccgctgaggcaagc actggttggactacctggtgagagcggtttg tcaacagagcaaagaaagagactgaccattgcggtggagctggttgcaaatccttccatc atattcatggatgaacctacttcaggattg gatgcacgagctgctgccatcgttatgaggactgtaaggaacacagttgacactggtaga acagtcgtctgcaccattcaccagcctag catcgacatctttgaagcctttgatgagttgttcctacttaagcgtggaggtgaggagat atacgttggacctcttggccacgaatcaacc catttgatcaactattttgagagtattcaaggaatcaacaagatcacagaaggatacaac ccagcaacctggatgcttgaagtctcaacc acatctcaagaagcggctttaggagtcgamcgcccaagtctacaaaaattcagaacttta caagagaaacaaggagctaatcaagga gctaagccagccagctccaggatcaaaagatttatatttcccaacacaatactctcaatc gttcttgacacaatgtatggcttctctatggaa acaacactggtcctactggagaaatcctccttacacagccgtgagattcctcttcacaat cggcattgctcttatgttcggcacaatgtt ggaccttggaggcaaaacgaaaacgagacaggatttatcgaatgcaatgggttcaatgta cacagctgttctcttcctcggattacaaaa cgcagcttcagtgcaaccagtcgtcaacgtcgaaagaactgtcttttaccgagaacaagc cgccggaatgtactccgccatgccttatg ctttcgctcaggttttcatcgagatcccatacgttctcgtgcaagcgatagtgtacggtc tcatagtgtacgctatgataggattcgagtgg acggcggtgaagttcttctggtacctcttctttatgtacggatcattcttaactttcacc ttctacggaatgatggctgtagctatga^ accaccacatcgcctccgtcgtctcctccgctttctacggcatctggaatctcttctccg gcttcctcatccctcgtcccagtatgcc^ ggtgggaatggtactactggctttgcccagttgcatggacattgtatggattaatcgcat cacagttcggtgatattacagaacctatggca gatagtaatatgagtgtgaagcaattcattagagaattctatggatatagagaaggtttc ttgggtgtggttgccgccatgaacgtcatcttt cctttgctcmgccgttatctttgctatcggaatcaagagtttcaamccaaaaacgataga cagtttatagttttgcattctatttcatgtaac acaaataaaaagagactttttgtttatatgctattctttctatttttgtaatgccgtatt gatattaataaaaggatgatcaacaacactggattag aatg [SEQ ID NO:27], as set forth for example in GenBank Accession No. 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.

[0181] An illustrative AtMRP4 polypeptide comprises the amino acid sequence:

[0182] MWLLSSSPWLSELSCSYSAVVEHTSSVPVPIQWL FVLLSPCPQRAL FSAVDFIFLLCFALHKLFSSPSSSSEINGHAEIRKPLIGIRGRTPTRTTAW KTTVAVTVL LSFCSVVLCVLAFTG RRTQRPWNLIDPLFWLIHAVTHLVL\VLVLHQKRFAALNHPL SLRIYWISSFVLTSLFAVTGIFHFLSDAATSLRAEDVASFFSFPLTAFLLIASVRGITGL V TAETNSPTKPSDAVSVEKSDNVSLYASASVFSKTFWLWMNPLLSKGYKSPLTLEQVP TLSPEHKAERLALLFESSWPKPSENSSHPIRTTLLRCFWKEILFTAILAIVRLGVMYVGP VLIQSFVDFTSG RSSPWQGYYLVLILLVAKFVEVLTTHQFNFDSQKLGMLIRSTLITA LYKKGLKLTGSARQNHGVGQIVNYMAVDAQQLSDMMLQLHAIWLMPLQVTVALV LLYGSLGASVITAVIGLTGVFVFILLGTQRNNGYQFSLMGNRDSRMKATNEMLNYM

RVIKFQA\^NHFNiOULKFRDMEFG\\^SKFLYSIAGNIIVLWSTPVLISALTFATA LAL

GVKLDAGTVFTTTTIFKILQEPIRTFPQSMISLSQAMISLGRLDSYMMSKELSEDAV ER

ALGCDGNTAVEVRDGSFSWDDEDNEPALSDINFKVKKGELTAIVGTVGSGKSSLLAS VLGEMHRISGQVRVCGSTGYVAQTSWIENGTVQDNILFGLPMVRE YNKVLNVCSL EKDLQMMEFGDKTEIGERGINLSGGQ QRIQLARAVYQECDVYLLDDVFSAVDAHT GSDIFKKCVRGALKG TVLLVTHQVDFLHNVDCILVMRDGKIVESGKYDELVSSGLD FGELVAAHETSMELVEAGADSAAVATSPRTPTSPHASSPRTSMESPHLSDLNDEHIKS FLGSHIVEDGSKLIKEEERETGQVSLGVYKQYCTEAYGWWGIVLVLFFSLTWQGSLM ASDYWLAYETSAK AISFDASVFILGYVIIALVSrVLVSIRSYYVTHLGLKTAQIFFRQI LNSILHAPMSFFDTTPSGRILSRASTDQTNVDILIPFMLGLVVSMYTTLLSIFIVTCQYA WTTAFFVIPLG XNIWYRNYYLASSRELTRMDSITKAPIIHHFSESIAGVMTIRSFRKQ ELFRQENVKRVNDNLRMDFHNNGSNEWLGFRLELVGSWLCISALFMVLLPSNVIRP ENVGLSLSYGLSLNSVLFFAIYMSCFVENKMVSVERIKQFTDIPSESEWERKETLPPSN WPFHGNVHLEDLKVRYRPNTPLVLKGITLDIKGGEKVGWGRTGSGKSTLIQVLFRL VEPSGGKIIIDGIDISTLGLHDLRSRFGIIPQEPVLFEGTVRSNIDPTEQYSDEEIWKSL ER CQL DVVATKPEKLDSLVVDNGENWSVGQRQLLCLGRVMLKRSRLLFLDEATASV DSQTDAVIQ IIREDFASCTIISIAHRIPTVMDGDRVLVIDAGKAKEFDSPARLLERPSL FAALVQEYALRSAGI [SEQ ID NO:28], as set forth for example in GenPept Accession No. 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.

[0183] 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. In illustrative examples, an AtMRP4 nucleic acid sequence comprises the nucleotide sequence:

[0184] ccacttcaaacaaaccgataattcagaggaattttctcttcrt^

atgtggttgctttcgtcttctccatggctctctgagctctcatgttcatattcggct gttgtagaacatacgtcttcagtt

atggctcagatttgttttactctctccttgccctc^

tttcttctccttcttcttcttccgaaatca^^

accgcatggttcaaaacgacggtcgcagtcaccgttctattgte^

gactcagagaccatggaacctcatagacccgctcttttggcttattcacgccgttac acacctagtcatcgccgttctcgtcctccacca aagagattcgctgctctaaatcatcccttatctctacgaatctactggatttccagtttc gtcctcacgtctctcttcgccgt^ ccattttctctccgacgccgccacgagtctgagagcagaagacgtcgcttcattcttctc cttccctttaaccgcctttcttctcatc gtcagaggaatcaccggcctcgtcacagcggagaccaacagtcctacgaaaccatccgac gccgtttcggtggagaaatccgataac gtctctctctacgcgtctgcttctgttttctcgaaaacgttctggttatggatgaatcct ttactcagcaaaggctacaaatctccactgacgc tcgaacaagtccccacgctttctccagagcacaaagcagagaggctcgcgcttctcttcg aatcgagttggcccaaaccgtcggagaa ttctagccaccctatccgtacgactctactccgatgtttctggaaggagatcctcttcac cgcgattctagccatcgtccgtctcggcgtca tgtacgttggtcccgttctcatccagagcttcgtcgatttcacctccggcaagagatcct ccccgtggcaaggttattacctcgtcctcatc ctccttgttgccaaattcgtcgaggtcttgacgacgcatcagttcaatttcgattcccag aagcttgggatgcttataaggtcaactctaatc actgcactctacaagaaaggtttaaagctcacaggctctgcgcgtcagaaccacggcgta ggacaaatcgtgaattacatggccgtag atgcacaacagctctctgacatgatgcttcagctccacgcaatctggctcatgcctttgc aagtcactgttgcactagtgcttctctacggg agcctaggcgcgtctgttataaccgcggttattgggctgactggagtgttcgtcttcatc ctcctggggactcagagaaacaacggatac caattcagcttgatgggaaaccgagattctcggatgaaggccaccaacgagatgctcaat tacatgcgagtcatcaagtttcaggcttgg gagaatcattttaacaagaggatcctcaaattcagggacatggagtttggttggctatcc aagtttctttactccattgctggcaatatte tcctctggagcacgccagtgcttatctctgctctcaccttcgccaccgcccttgccttgg gagtcaagcttgacgctgggactgtgttcac caccacaaccattttcaagatcctgcaagaacccatcaggacgtttcctcagtctatgat ttctctctcgcaggcaatgatctctcttggga gactggactcatacatgatgagcaaagagctgtcggaagatgctgtggagagagccctgg gttgtgatggtaatactgccgtggaggt cagagatggaagctttagttgggatgatgaggacaacgaacctgctctcagtgatatcaa cttcaaggttaagaaaggtgagctcactg cgatagttggaaccgttggttcagggaaatcttctctgttagcttcggtte^

gggagcacaggttatgtagctcagacgtcgtggattgaaaacgggacggttcaagac aacatcttgtttggicttcc^tggttagagag aagtacaacaaagttctcaatgtctgttctcttgaaaaagacctacaaatgatggagttt ggagataagactgagattggagaacgcgga atcaacctcagcggagggcagaagcaacgtatacagctcgcacgtgctgtctatcaggaa tgcgatgtatacttgctcgacgatgttttt agcgcagtggatgctcataccggttcagatatattcaagaaatgtgtaagaggagctctg aaaggcaagaccgtattactcgttacccat caagtggatttcttgcacaacgtggattgcatcttggtgatgcgggatggaaagattgtt gaatcaggaaaatatgacgaattagtcagct ccggattggattttggggaacttgtggctgcacatgagacgtcaatggagctggttgaag ccggtgcagactctgcagcagtcgccac atccccaagaacaccaacgtctccccatgcaagctctccgagaacgtcaatggagtctcc tcacttaagtgatctaaacgatgagcatat caaatcatttctcggttctcacatcgtagaagatggctcgaagctcatcaaagaagaaga aagggaaaccggacaggttagcttagga gtttacaaacagtactgcactgaggcttatggctggtggggaattgtgcttgttctgttc ttctctctgacgtggcagggatctctaatggcc agcgattactggcttgcatacgaaacatcagccaaaaatgcaatatcatttga^

ttccatcgttttggtgagcatccggtcatattacgtcacccacttgggactcaagac ggctcagatctttttccgacagattcttaatagtatc ttacacgctcccatgtcattctttgacaccacgccatcgggaagaattctcagtcgggca tcgactgatcagaccaatgtcgatatccttat tccgtttatgctcggacttgtggtctcaatgtacaccact^

tgattccccttggctggcttaacatctggtaccggaactattacctcgc^^

ccatcatccaccatttctctgaaagtatagctggagtgatgacaatccgatcattca ggaagcaggagttgtttagacaagagaatgtaaa acgtgtaaatgataatctcaggatggacttccacaacaatggctccaacgaatggctcgg gtttcggctggagctggttgggagctggg tgctctgcatctcggctttgtt ggtattgttacc^

tgaactcggttctgttctttgccatatacatgagctgcttt^

ctcagaatccgagtgggagagaaaagaaacccttccaccttcgaattggcccttcca tggcaatgtacatctcgaagacctcaaggtgc gctacagaccgaacactccacttgtgctcaaggggatcactcttgacatcaaaggaggag agaaggttggtgtggttggacggacgg gaagcgggaaatcgacattgatccaagtcttgttcaggcttgtagaaccatcaggaggga agataatcatagacgggattgatataagc actctagggctacatgatctcaggtcaagattcggaatcattccgcaagaacctgtcctc tttgaaggaaccgtgagaagcaacatcgac ccgacagagcaatactctgacgaagaaatctggaagagcctggaacggtgtcaactcaag gatgttgtagctaccaagcctgagaag ctcgattcmggtggflgataatggggagaactggagcgtagggcagaggcagcttctatg cttaggcagggttatgttgaaacgcagc agacttctcttcctagacgaagcaactgcatccgttgattcccaaaccgacgccgtgatt cagaagatcatcagagaagactttgcgtcg tgcaccatcatcagcatcgcccaccggattcctacagtgatggacggcgatcgagtcctt gtcattgatgctgggaaagcgaaagagtt cgatagcccggctcgcttgctggagaggccgtctctgtttgcggcgctggtgcaagagta cgctctccgatctgccggaatatgaatctt ttacgccggcggggactgaaaatattttaaaaccttcttcaaatgagamgtatcaaagaa aattcctataatctcacatgttttagattcaa aaacgacatcgtaattttagtgcagcaagcaacacagtaaattttta^

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.

[0185] A non-limiting RBOHD polypeptide comprises the amino acid sequence:

[0186] MKMRRGNSSNDHELGILRGANSDTNSDTESIASDRGAFSGPLGRPK RASKKNARFADDLPKRSNSVAGGRGDDDEYVEITLDIRDDSVAVHSVQQAAGGGGH LEDPELALLTKKTLESSLNNTTSLSFFRSTSSRIKNASRELRRVFSRRPSPAVRRFDRTS SAAIHALKGLKFIATKTAAWPAVDQRFDKLSADSNGLLLSAKFWECLGMNKESKDF ADQLFRALARRNNVSGDAITKEQLRIFWEQISDESFDAKLQVFFDMVDKDEDGRVTE EEVAEIISLSASANKLSNIQKQAKEYAALIMEELDPDNAGFIMIENLEMLLLQAPNQSV RMGDSRILSQMLSQ IRPAKESNPLVRWSEKIKYFILDNWQRLWIMMLWLGICGGLF TYKFIQYKNKAAYGVMGYCVCVAKGGAETLKFNMALILLPVCRNTITWLRNKTKLG T PFDDSLNFH VIASGIVVGVLLHAGAHLTCDFPRLIAADEDTYEPME YFGDQPT

SYWWFVKGVEGWTGIVMVVLMAIAFTLATPWFRRNKLNLPNFLKKLTGFNAFWYT

HHLFirVYALLIVHGIKLYLTKIWYQKTTWMYLAVPILLYASERLLRAFRSSIKPVK MI

KVAVYPGNVLSLHMTKPQGFKYKSGQFMLVNCRAVSPFEWHPFSITSAPGDDYLSV HIRTLGDWTRKLRTVFSEVCKPPTAGKSGLLRADGGDGNLPFPKVLIDGPYGAPAQD YKKYDVVLLVGLGIGATPMISIL DIINNMKGPDRDSDffiN>nsiSNNNSKGFKTRKAYF YWVTREQGSFEWFKGIMDEISELDEEGIIELHNYCTSVYEEGDARVALIAMLQSLQHA KNGVDVVSGTRVKSHFAKPNWRQVYKJGAVQHPGKRIGVFYCGMPGMIKELKNLA LDFSRKTTTKFDFHKENF [SEQ ID NO:30], as set forth for example in GenPept Accession No. 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. [0187] 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. In illustrative examples, a RBOHD nucleic acid sequence comprises the nucleotide sequence:

[0188] atacacaaaaatcaaacaccttttgagagcggttarrtmctctatcaactaatacagtaa ccttacgggtgtttat ttgtatagatctctgtggtmcttggccaaatctagtgaga

accatgaacttgggattctacgaggagctaactcggacaccaactcggacacggaga gcatcgctagcgaccgtggtgcctttagcg gtccgcttggccggcctaaacgtgcgtccaagaaaaacgcaagattcgccgacgatcttc ccaagagaagcaatagtgttgctggcg gccgtggtgatgacgatgagtacgtggagatcacgctagacatcagggacgactcggtgg ccgtccatagtgtccaacaagcagctg gaggtggaggccacctggaggacccggagctagcccttcttacgaagaagactctcgaga gcagcctcaacaacaccacctccttat ctttcttccgaagcacctcctcacgcatcaagaacgcctcccgcgagctccgccgcgtgt tctctagacgtccctccccggccgtgcgg cggtttgaccgcacgagctccgcggccatccacgcactcaaaggtctcaagttcattgcc accaagacggccgcatggccggccgtc gaccaacgtttcgataaactctccgctgattccaacggcctcttac^^

agacttcgctgaccagctctttagagcattagctcgccggaataacgtctccggcga tgcaatcacaaaggaacagcttaggatattctg ggaacagatctcagacgaaagctttgatgccaaactccaagtcttttttgacatggtgga caaagatgaagatgggcgag aagaggtggctgagattattagtcttagtgcttctgcaaacaagctctcaaatattcaaa agcaagccaaagaatatgcggcactgataat ggaagagttggacccagacaatgctgggtttattatgatcgaaaacttggaaatgttgct attacaagcaccaaaccagtc gggagacagcaggatacttagtcagatgttaagtcagaagcttagaccggca

aatcaaatatttcatacttgacaattggcagagactatggataatg^

agtacaagaacaaagctgcc ggtgtcatgggttattgcgtttgtgtcgccaaaggaggcgccgagactctcaaattcaac a tcatattgttgcctgtttgtcgaaacaccatcacttggcttag

cacaaggttattgcaagcgggatagtcgtcggtgttttactccatgcgggtgcccat ttaacgtgtgattttccacgttta^ gaggacacctatgagccgatggaaaaatactttggggatcaaccgactagctactggtgg tttgtgaaaggagtggaaggatggactg gcattgtgatggttgtgctaatggctatagcctttacactcgcgacgccttggttccgac giaacaagcttaacttacctaacttccte agcttaccggtttcaacgccttttggtacacccaccatttgttcatcattgtttatgctc ttctcattgtccatggtatcaagctctaccte agatttggtatcagaaaacgacatggatg^tcttgctgtacccatccttctatatgcatc ggagaggctgctccgtgctttcagatcaagc atcaaaccggttaagatgatcaaggtggctgtttaccccgggaacgtgttgtctctacac atgacgaagccacaaggattcaaatacaaa agtggacag tcatgttggtgaactgccgagccgtatctccattcgaatggcatcctttctcaatcacat cagctcccggagacgattacc tgagcgtacatatccgcactctcggtgactggacacgtaagctcaggaccgttttctccg aggtitgcaaacctcctaccgccggtaa^ gcggtcttctccgagcagacggaggagatggaaacctcccgttcccgaaggtccttatcg acggtccatacggtgctcccgcacaag actacaagaaatacgacgtggtactcctcgtaggtctcggcattggagccacgcctatga tcagtatccttaaggacatcatcaacaaca tgaaaggtcctgaccgcgacagcgacattgagaacaataacagtaacaacaatagtaaag ggtttaagacaaggaaagcttatttctac tgggtgactagggaacaaggatcattcgagtggttcaagggaataatggacgagatttcg gagttagacgaggaaggaatcatcgag cttcacaattattgcacgagtgtgtacgaggaaggtgatgcaagagtggctctcattgcc atgcttcagtcgttgcaacacgctaagaac ggtgtggatgttgtgtcgggtacacgtgtcaagtcccacttcgctaaacctaactggaga caagtctacaagaagatcgctgttcaacat cccggcaaaagaataggagtcttctactgtggaatgccaggaatgataaaggaattaaaa aatctagctttggatttttctcgaaagacaa ctaccaagtttgacttccacaaagagaacttctagattaattatatacgttgtagaaaaa taaaacaagaaacaactatacaaataaatottt attttaaattctmcatttttatgtaaaattatctgagttatcttttmgttcttc^

gccaaattaagtataagatagtagaagtttatatagttacagctttggtgttgtaaa catgtaatcatggagttatct

atattcgaatattaacaactaaagatagttttg [SEQ ID N0:31 ], as set forth for example in GenBank Accession No. NM 124165, 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:31, or to a complement thereof; or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to SEQ ID NO:31 , or to a complement thereof.

[0189] An illustrative RBOHF polypeptide comprises the amino acid sequence:

[0190] MKPFSKNDRRRWSFDSVSAGKTAVGSASTSPGTEYSINGDQEFVEV

TIDLQDDDTIVLRSVEPATAINVIGDISDDNTGMTPVSISRSPTMKRTSSNRFRQFS QE L AEAVAKAKQLSQEL RFSWSRSFSGNLTTTSTAANQSGGAGGGLVNSALEARAL

RKQRAQLDRTRSSAQRALRGLRFISNKQKNVDGWNDVQSNFEKFEKNGYIYRSDFA

QCIGMKDSKEFALELFDALSRRRRLKVE INHDELYEYWSQINDESFDSRLQIFFDIVD

KNEDGRITEEEVKEIIMLSASANKLSRLKEQAEEYAALIMEELDPERLGYIELWQLE TL LLQKDTYLNYSQALSYTSQALSQNLQGLRGKSRIHRMSSDFVYIMQENWKRIWVLSL WTMINflGLFLwT FFQYKQKDAFHVMGYCLLTAKGAAETLKFNMALILFPVCRNTIT WLRSTRLS YF VPFDDNINFH TIAG AI V V AVILHIGDHL ACDFPRTVRATE YD YNR YLF HYFQT QPTYFDLVKGPEGITGILMVILMIISFTLATRWFRRNLVKLPKPFDRLTGFNA FWYSHHLFVrVYILLILHGIFLYFAKPWYVRTTWMYLAVPVLLYGGERTLRYFRSGS YSVRLLKVAIYPGNVLTLQMSKPTQFRYKSGQYMFVQCPAVSPFEWHPFSITSAPED DYISIHIRQLGDWTQEL RVFSEVCEPPVGGKSGLLRADETTKKSLPKLLIDGPYGAP AQDYRKYDVLLLVGLGIGATPFISILKDLLN IV MEEHADSISDFSRSSEYSTGSNGD TPRRKRILKTTNAYFYWVTREQGSFDWFKGVMNEVAELDQRGVIEMHNYLTSVYEE GDARS ALITMVQALNHAKNGVDIVSGTRVRTHF ARPNWKKVLTKLSSKHCNARIGV FYCGVPVLG ELSKLCNTFNQKGSTKFEFHKEHF [SEQ ID NO:32], as set forth for example in GenPept Accession No. 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.

[0191] 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. In illustrative examples, a RBOHF nucleic acid sequence comprises the nucleotide sequence:

[0192] atgcataaactagaatgaccaacaaagatactctcaaagtctcacttgtctaaaaaacag ataaaaatcatttcc aattttgagaaaaggaaaaaaaacaactccaataatactttttgattttctttto

ctattataagttaaaaaaactcaaaaaaaaaaaaaagtatattccg caacttfflctcagtccggttcaatcatctccatcatcgttgatctct ctctctccgtcaacgttctctctataaagcagagagtttcacagcgcgtgaaaaatggct tttcccacttagccacaacgacgcgtttcaat tctccatctctccctctctctctctctttcttcaaagattccaccaacctatacatatac ttatatataatctatgctacgtgtatatacttccgatat ccttcaaccaactctttgaattccgactttggatctatgaaaccgttctcaaagaacgat cggcgacggtggtcattt^

ggaaaaaccgccgtcggaagtgcatcaacttcaccgggaactgaatactccattaac ggtgatcaagagttcgttgaagtcacaatcga tcttcaagacgatgacacaatcgttcttcgtagcgtcgagccagcaaccgccattaatgt catcggagatatctccgacgacaacaccg gaataatgactccggtttcgatttcgagatctccgacgatgaaacga

cgaagctgtggcgaaagcgaaacagt ctcaggagttgaaacgattctcatggtctcgtt^ ccgccgctaatcaaagcggcggtgctggtggtggmggtgaactcggc^

agatcggactcggtctagtgctcaaagagctcttcgtggtttgagattcattagcaa taagcaaaagaacgttgatggttggaacgatg^ caatcaaatttcgaaaaattcgaaaaaaatggttacatctatcgctccgatttcgctcaa tgcataggaatgaaagattcgaaagaatttgc attggaactgttcgatgcattgagtagaagaagaagattaaaagtagagaaaatcaatca cgatgagctttatgagtattggtcacaaatc aacgacgagagttttgattctcgtctccagatcttcttcgacatagtggacaagaatgaa gatgggagaattacagaagaggaagtaaaa gagataataatgttgagtgcatctgcaaataagctatcaagattaaaggaacaagcagag gaatatgcagctttgattatggaagagtta gatcctgaaagacttggctacatagagctatggcaactagagactttgcttctacaaaaa gacacatacctcaattacagtcaagcattga gctatacgagccaagcattgagccaaaaccttcaagggttaaggggaaagagtcgaatac atagaatgagttcggatttcgtctacatta tgcaagagaattggaaaagga gggtlltatccttatggatcatgatcatgatcggattattcttgtggaaattcttccaat acaagcaa^ aagatgcatttcatgtgatgggatattgtttactcacagccaaaggagcagctgaaacac ttaaattcaacatggctctaatacttttcccag tttgcagaaacaccattacttggcttagatccacaagactctcttacttcgttccttttg atgataatatcaacttccacaagacaattgctgga gccattgtagtagctgtgatccttcatattggagaccatcttgcttgtgatttccctaga attgttagagccaccgaatacgattacaatcggt atctgtttcattactttcaaacaaaacagccaacatacttcgacctcgttaagggacctg aaggaatcactgggattttaatggtcattttgat gattamcattcacattagcaacaagatggtttaggcgtaacctagtcaagcttcctaagc camgatcgactaaccggttttaacgcctttt ggtattcgcatcamgttcgtcattgtttatatcttgcttattcttcatggtatcttcctc tatttcgccaagccttggte^

atgtatcttgcagtaccagttttactctatggtggagaaagaacacttagg^

tatatatcctggtaatgttctaacgctacaaatgtcgaaaccaactcaatttcgtta caaaagcggacaatacatgtttgtccaa^ ggtttcgccattcgagtggcatccattctcaattacttccgcacctgaagatgattatat cagcattcacattagacaacttggtgattggact caagaactcaaaagagtattctctgaagtttgtgagccaccggttggcggtaaaagcgga cttctcagagccgacgaaacaacaaaga aaagtttgccaaagctattgatagatggaccgtacggtgcaccagcacaagattatagga a^

attggtgcaactccatttatcagtatcttgaaagatttgcttaacaacattgttaaa atggaagagcatgcggattcgatctcggatttcag^ gatcatcagaatacagcacaggaagcaacggtgacacgccaagacgaaagagaatactaa aaaccacaaatgcttatttctactgggt cacaagagaacaaggctcttttgattggttcaaaggtgtcatgaacgaagttgcagaact tgaccaacggggtgtgatagagatgcata actatttaacaagtgtgtatgaagaaggtgatgctcgttctgctctcattacaatggttc aagctcttaatcatgccaaaaatggtgtcgaca ttgtctctggcactagggtcagaacacactttgcaagacctaattggaagaaggttctca caaagctaagttccaagcattgcaatgcaa gaateggagtgttttattgcggagtaccggttttagggaaggagcttagcaaactatgca acacattcaatcaaaaaggttcaaccaagtt tgaatttcacaaggagcatttctaaaagacaagaaggaagaagccaaaagccctctagat tctttaatatctcaaatttagccacttatagt ateaaggcaatctcttcactatttaattcaaagtgattaaacgttaacacactgtcaaaa gtgagtgtgttaacgtttagctccacacg^ ggtttatatacaccgaggcatacgtgtaaatatacgagacagaagaaattcaagggggtt tgatagaagcatatagtaaaattttaaaattt cttgtatagtaagcaaatgagatggagactctagaagagaggtgaggtttggtgagggat agcgacagtaatacgacgtcgtattcgatt gtagaggaagtgcaatacagctgcagggaagataaatgatggaagcaaataatgcgttga taaggtccatgtgattaggaagatgaca ctcttmgggggatattttmctattttttttttttagtggag cggtgaaagagagactagag

agggagttataatattmgtatcattgtattatataactaatgtaaggtgtacaaagc ataaaatttgatagttcctttcttct [SEQ ID NO:33], as set forth for example in GenBank Accession No. 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;.

[0193] Non-limiting PLDalphal polypeptides comprise the amino acid sequence:

[0194] MAQHLLHGTLHATIYEVDALHGGGVRQGFLGKILANVEETIGVGKG ETQLYATIDLQKARVGRTRKIKNEPKNPKWYESFHIYCAHLASDIIFTVKDDNPIGATL IGRAYIPVDQVINGEE VDQ WVEILDNDRNPIQGGSKIHVKLQ YFHVEEDRNWNMGIK SAKFPGVPYTFFSQRQGCKVSLYQDAHIPDNFVPRIPLAGGKNYEPQRCWEDIFDAIS NAKHLIYITGWSVYAEIALVRDSRRPKPGGDVTIGELLKKKASEGVRVLLLVWDDRT SVDVL KDGLMATHDEETENFFRGSDVHCILCPRNPDDGGSIVQSLQISTMFTHHQKI VVVDSEMPSRGGSEMRRTVSFVGGIDLCDGRYDTPFHSLFRTLDTVHHDDFHQPNFT GAAITKGGPREPWHDIHSRLEGPIA WDVMYNFEQRWSKQGGKDILVKLRDLSDIIITP SPVMFQEDHDVWNVQLFRSIDGGAAAGFPESPEAAAEAGLVSGKDNIIDRSIQDAYIH AIRRAKDFIYVENQYFLGSSFAWAADGITPEDINALHLIPKELSLKIVS IE GEKFRVY WVPMWPEGLPESGSVQAILDWQRRTMEMMYKDVIQALRAQGLEEDPRNYLTFFCL GNREVKKDGEYEPAEKPDPDTDYMRAQEARRFMIYVHTKMMIVDDEYIIIGSANINQ RSMDGARDSEIAMGGYQPHHLSHRQPARGQIHGFRMSLWYEHLGMLDETFLDPSSL ECIEKVNRISDKYWDFYSSESLEHDLPGHLLRYPIGVASEGDITELPGFEFFPDTKARIL GTKSDYLPPILTT [SEQ ID NO:34], as set forth for example in GenPept Accession No. 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.

[0195] 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. In illustrative examples, an PLDalphal nucleic acid sequence comprises the nucleotide sequence:

[0196] aaagagcttccatcacggaccagatcccgaattcttcttctgaccaccgaacgattgagt ttctccgatcagat ctcagtttctgggaataattcgaagtgaaaaaatggcgcagcatctgttgcacg^

catggtggtggtgttaggcaaggcttccttggcaagattctg^

gtatgcgacgattgatctgcaaaaagctagagttgggagaaccaggaagatcaaaaa tgaacctaagaacccaaagtggtatgagtcg tttcatatttactgtgctcacttggcttctga catcttcactgttaaagatgataatcccattggagctaccc^

cctgttgatcaagtcattaacggcgaggaagtggatcagtgggttgagatcttggat aatgacagaaaccctattcagggaggatcaa^ gattcatgtcaagcttcaatatttccatgttgaggaggatcgtaactggaacatgggtat caaaagtgccaagttccc¾^ acattcttctcgragagacaaggctgcaaagtttctctgtacca^^

gggaagaactatgagcctcaaagatgttgggaggatatttttgatgctattagcaat gcaaaacacttgatctacattactggtt tacgctgagattgctttagtgagggactcgaggaggcctaagcctggaggtgatgtgacc attggtgagctactcaagaagaaggcta gtgaaggtgtcagggttcttttgcttgWgggatgacagaactt^

gagaccgagaatttcttcaggggaagtgatgtccattgtattctgtgccctcgtaac ccggatgacggtggtagcatagtccaaagtttgc agatctctactatgttcacgcatcatcagaaaatcgttgttgtggacagcgagatgccaa gcagaggaggatcagaaatgaggagaatt gtgagttttgttggcggfattgatctttgtgatggaa^

gacttccatcaacctaacttcactggtgctgc cactaaaggtggtccaagggagccttggcatgacattcactcccgtcttgaaggtc caattgcttgggatgtcatgtacaacttcgagcagagatggagcaagcagggt^

atattattatcaccccttctcctgttatgttccaagaggaccacgatgtgtggaatg tccaattgtttaggtccatt^

ctgggtttcccgagtcgcctgaagctgctgcggaagccgggcttgtaagtgggaaag ataacatcattgataggagtatccaagatgct tacattcatgcaatcagacgtgctaaggatttcatctacgttgaa

ctcctgaggacatcaatgccctgcacttaatcccaaaagagttgtcgctgaagatag ttagcaagattgagaaaggagagaagttcagg gtctatgttgtggttccaatgtggccagaaggtctcccagagagtggatcagtgcaagct atattagactggcagaggaggaccatgga gatgatgtacaaggatgtgattcaggctctcagggcccagggtcttgaggaagatccaag aaactatctgacattcttctgtctt^ cgtgaggtcaagaaagatggagagtatgagcctgctgagaaaccagaccccgacactgat tacatgagggcgcaagaagcacgcc gmcatgatttacgtccacaccaaaatgatgatcgttgacgatgaatacattatcattggg tctgctaacatcaaccagaggtcaatggac ggigcaagagactctgagatagcaatgggaggttatcaaccacatcacttgtcccataga caaccagctcgtggccagatccatgggtt tcgtatgtcactctggtacgaacacctgggaatgctcgatgaaaccttcctcgatccatc aagcttggaatgcattgagaaagttaaccgc atttctgacaagtattgggacttttactcaagtgagtcactcgaacatgaccttcctggt cacttgctccgc^

cgaaggcgacatcactgagcttccaggatttgaattcttcccggacacaaaggcccg tatcctcggcaccaaatcagactacctgcctc caatccnacaacctaatctcactaagcatgtcaagtaatgatctctctctccctctctgc tttgctgctgttgtagctttgaataaaacttgagt gtctaccrttagaattaagaagtcaaatggttgttatgatga¾^

tgtagctttgtgatcttgtWgttgttgt gta^^ [SEQ ID NO:35], as set forth for example in GenBank Accession No. NM l 12443, 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: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.

[0197] Non-limiting examples ofPKS3 polypeptides comprise the amino acid sequence:

[0198] MEKKGSVLMLRYEVGKFLGQGTFAKVYHARHLKTGDSVAIKVIDK EMLKVGMTCQIKREISAMRLLRHPNIVELHEVMATKSKIYFVMEHVKGGELFN VST GKLREDVARKYFQQLVRAVDFCHSRGVCHRDLKPENLLLDEHGNLKISDFGLSALSD SRRQDGLLHTTCGTPTYCAPEVISRNGYDGFKADVWSCGVILFVLLAGYLPFRDSNL MELYKKIGKAEVKFPNWLAPGAKRLLKRILDPNPNTRVSTEKIMKSSWFRKGLQEEV KESVEEETEVDAEAEGNASAEKEKKRCINLNAFEIISLSTGFDLSGLFE GEEKEEMRF TSNREASEITEKLVEIGKDLKMKVRKKEHEWTRVKMSAEA VEAEVFEIAPSYHMV VLKKSGGDTAEYKRVMKESIRPALIDFVLAWH [SEQ ID NO:60], as set forth for example in GenPept Accession No. 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.

[0199] 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. In illustrative examples, an PKS3 nucleic acid sequence comprises the nucleotide sequence:

[0200] atggagaagaaaggatctgtgttgatgctccgttatgaggttgggaagmctcggtcaagg tacctttgctaa ggtataccatgctaggcatttgaaaactggtgatagtgtagc

gcagattaagcgagagatctctgccatgagactcttgaggcatcccaacatcgttga gctccatgaagtcatggccaccaaatctaaaat ctacttcgtcatggaacatgttaagggtggtgagctcttc tcagcagcttgtacgcgctgttgacttctgtcacagccgtggagtatgccacagggacct gaagccggagaatctcttgttggatgagc atgggaatcttaagatctctgattttggtctcagcgctctttctgactctagaaggcaag acgggttgctgcatactacatgcgg^^ acatattgtgcaccggaggtgataagcaggaacgggtatgatgggtttaaagcggat^

tcgctggatatcttcctttccgtgattccaatctgatggagctgtataagaagatag gcaaagctgaagtcaagttccccaactggcttgc^ ccgggggcaaagagattgctcaagaggatcttggatcctaaccccaacacaagggtatca actgagaaaataatgaagagctcttggt tccgtaaaggcctacaagaggaggtgaaagaatcagttgaggaagagacagaagtggacg cagaggcagagggaaacgcaagtg cagagaaggaaaagaagcggtgtatcaacctgaacgcgtttgagatcatatctctgtcca cggggtttgatctctcgggactgttcgaga agggagaggagaaggaggagatgaggtttacatcaaacagagaggcatctgagataacag agaagctggtggagattgggaagga ccttaagatgaaagtgaggaagaaggaacacgaatggagggtgaaaatgtcggctgaggc tacagtggtggaagcggaagtgtttg agattgcgccgagctatcacatggtggtgctaaagaagagcggtggagatactgctgagt ataagagagtcatgaaggagagtataag accggctttgatcgactttgtattagcttggcactga [SEQ ID N0:61], as set forth for example in GenBank Accession No. 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.

[0201] An illustrative ATHB6 polypeptide comprises the amino acid sequence:

[0202] MMKRLSSSDSVGGLISLCPTTSTDEQSPRRYGGREFQSMLEGYEEEE EArVEERGHVGLSE KR LSINQVKALEKNFELENKLEPERKVKLAQELGLQPRQVA VWFQNRRARWXT QLEKDYGVLKTQYDSLRHNFDSLRRDNESLLQEISKLKTKLNG GGGEEEEEENNAAVTTESDISVKEEEVSLPEKITEAPSSPPQFLEHSDGLNYRSFTDLR DLLPLKAAASSFAAAAGSSDSSDSSALLNEESSSNVTVAAPVTVPGGNFFQFVKMEQ TEDHEDFLSGEEACEFFSDEQPPSLHWYSTVDHWN [SEQ ID NO:36], as set forth for example in GenPept Accession No. 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.

[0203] 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. In illustrative examples, an ATHB6 nucleic acid sequence comprises the nucleotide sequence:

[0204] gttcatatggaaaacgctcatcaacctcaacaatctctctcctctctctctctgtatata gaagaatctccattgtc tttaafflctctccatttctctttctctcttc

gagaagttattaaaaatttcgaaagtaattaaagattgttgatgatgaagagattaa gtagttcagattcagtggg^

cctacaacttccacagatgagcagagtccgaggagatacggtgggagagagtttcag tcgatgcttgaaggatacgaggaagaagaa gaagctatag agaagaaagaggacacgtgggcttgtcggagaagaagagaaggttaagca

aatWgagttagagaataagcttgagcctgagaggaaagttaag^

caaaaccgtcgtgctcggtggaagacaaaacagcttgagaaagattacggtgttct^

attccctccgccgtgacaatgaatctctccttcaagagattagtaaactgaaaacga agcttaatggaggaggaggagaagaagaaga agaagagaacaacgcggcggtgacaacggagagtgatatttcggtcaaggaggaagaagt ttcgttgccggagaagattacagag caccgtcgtctcctccacagtttcttgaacattctgatggtcttaattaccggagtttca cagatctacgtgatctt^

ggcttcttcattcgccgccgcagctggatcttcagacagtagcgattcaagcgctct gctgaatgaagaaagcagctctaatgtcactgt ggcggctccggtgacggttccaggaggtaamcttccagmgtgaaaatggagcagacggag gatcatgaggactttctgagtggag aagaagcttgtgaattcttttccgatgaacaaccgccgtctctacactggtactccaccg ttgatcattggaattga^^

ccggaagagattttggattggattagatgctctmcttctrt^

ggtgaaagggcaattaaggaagggttaagtctgggcgggaataatgatttagggtga atttgtaatta^

cacttcttgtaattaaggatcatcagatcaaagagaaattgagaaggggt^

acaattgacccttgaaacctaaattataccacttctgacccttaaatcaatttttga atcataaaac^ [SEQ ID

NO:37], as set forth for example in GenBank Accession No. NM l 27808, 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:37, or to a complement thereof.

[0205] In some embodiments, 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. ,

AAPK ^Lys43Ala ). While not limiting the invention to any one mechanism, these mutant proteins compete with their wild-type counterparts for interacting proteins in the transgenic plant, or poison multimeric complexes that normally recruit the wild-type counterparts. [0206] In other embodiments, the encoded expression product is an antibody that is immuno-interactive with the endogenous polypeptide that stimulates or otherwise facilitates stomatal closure. In non-limiting examples of this type, 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. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art. In illustrative examples, 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, 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). Knowledge of the antigen-binding regions (e.g.,

complementarity-determining regions) of such antibodies can be used to prepare synthetic antibodies as described for example above.

[0207] In still other embodiments, 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. In illustrative examples of this type, 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. Non-limiting examples of such target genes include AAPK, OST1, v-SNAREs AtVAMP711-14, GPA1, AtABCG22, AtABCG40, AtMRP4, RBOHD, RBOHF, and

PLDalphal.

[0208] In certain embodiments, the targeting sequence displays 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% identity to a nucleotide sequence of the target gene. In other embodiments, 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. Reference herein to 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. 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) 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. Desirably, the targeting sequence hybridizes to a nucleotide sequence of the target gene under physiological conditions.

[0209] Other stringent conditions are well known in the art. A skilled artisan will recognize that various factors can be manipulated to optimize the specificity of the hybridization/Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al, supra at pages 2.10.1 to 2.10.16 and Sambrook et al, supra at sections 1.101 to 1.104.

[0210] Suitably, the targeting region has sequence identity with the sense strand or antisense strand of the target gene. In certain embodiments, the RNA molecule is

unpolyadenylated, which can lead to efficient reduction in expression of the target gene, as described for example by Waterhouse et al in U.S. Patent No. 6,423,885. [0211] Typically, 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.

Generally, 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.

[0212] 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. Generally, 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.

[0213] The RNA molecule may further comprise a reverse complement of the targeting region. Typically, in these embodiments, 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. Typically, the spacer sequence is a non-coding sequence, which in some instances is an intron. In embodiments in which the spacer sequence is a non-intron spacer sequence, 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'. Alternatively, in

embodiments in which 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. 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.

[0214] Alternatively, 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. In this embodiment, the reverse complement of the targeting region hybridizes to the targeting region of the RNA molecule transcribed from the second polynucleotide.

[0215] In another example, 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. In this embodiment, 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.

[0216] In another example, PTGS of the target gene is achieved using the strategy by Glassman et al described in U.S. Patent Application Publication No 2003/0036197. In this strategy, 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. Thus, in some embodiments of the present invention, the RNA molecule further comprises two

complementary RNA regions which are unrelated to any endogenous RNA in the host cell and which are in proximity to the targeting region. In other embodiments, 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. In the above embodiments, one of the complementary RNA regions can be located upstream of the targeting region and the other downstream of the targeting region. Alternatively, both the complementary regions can be located either upstream or downstream of the targeting region or can be located within the targeting region itself.

[0217] In some illustrative examples, 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).

[0218] In other embodiments, 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.

[0219] In still other embodiments, the expression product that inhibits stomatal closure corresponds to an expression product of a negative regulator of the ABA signaling pathway. In illustrative examples of this type, the negative regulator is ATHB6. In some of these examples, 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 οϊ&ΑΤΗΒό coding sequence is represented the following sequence:

[0220] atgatgaagagattaagtagttcagattcagtgggtggtctcatctctttatgtcctaca acttccacagatgagc agagtccgaggagatacggtgggagagag ttcagtcgatgcttgaaggatacgaggaagaagaagaagctatagtagaagaaaga ggacacgtgggc gtcggagaagaagagaagg taagcattaaccaagttaaagctttggagaagaatttt

tgagcctgagaggaaagttaagttagctcaagaacttggtcttcaacctcgtcaagt tgctgtttggtttca

agacaaaacagcttgagaaagattacggtgttcrtaaaacccagtaGgattctctcc gtcataactttgattccctccgccgtgacaatgaa tctctccttcaagagartagtaaactgaaaacgaagcttaatggaggaggaggagaagaa gaagaagaagagaacaacgcggcggt gacaacggagagigatatttcggtcaaggaggaagaagmcgttgccggagaagattacag aggcaccgtcgtctcctccacagtttc ttgaacattctgatggtcttaattaccggagtttcacagatctacgtgatcttcttccat taaaggcggcggctt^

ctggatcttcagacagtagcgattcaagcgctctgctgaatgaagaaagcagctcta atgtcactgtggcggctccggtgacggttcca ggaggtaatttcttccagtttgtgaaaatgga

atgaacaaccgccgtctctacactggtactccaccgttgatcattggaattga [SEQ ID NO: 38], as for example set out in GenBank Accession NM 127808, 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 that coding sequence or a nucleotide sequence that hybridizes under at least medium stringency or high stringency conditions to that coding sequence.

[0221] Alternatively, 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.

[0222] In other embodiments, 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.

[0223] In still other embodiments, 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 ^{Trp875 u} . [0224] In still other embodiments, 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).

2.5 Other construct elements

[0225] In addition to the operably linked cw-acting elements, promoters and nucleic acid sequence encoding an expression product that inhibits stomatal closure described above, the constructs of the present invention, which are suitably expression constructs, can also include other regulatory sequences. As used herein, "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.

[0226] A number of non-translated leader sequences derived from viruses are known to enhance gene expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "Ω-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). Other 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. (1986), supra); human immunoglobulin heavy-chain binding protein (BiP) leader (Macejak & Samow (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of AMV (AMV RNA 4; Jobling & Gehrke (1987) Nature 325:622- 625); tobacco mosaic TMV leader (Gallie et al. (1989) Molecular Biology of RNA 237-256); and MCMV leader (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. In some embodiments, 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).

[0227] An expression construct also can optionally include a transcriptional and/or translational termination region (i. e. , termination region) that is functional in plants. A variety of transcriptional terminators are available for use in expression constructs and are

responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation. 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. In addition, 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. In this manner, 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.

[0228] 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. As used herein, "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). Of course, many examples of suitable selectable markers are known in the art and can be used in the expression constructs described herein.

[0229] Examples of 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. 6:915-922); 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. 5,767,378 and 5,994,629); a 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.

[0230] Additional selectable markers include, but are not limited to, a nucleotide sequence encoding β-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 β-lactamase, an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); a nucleotide sequence encoding xylE that encodes a catechol dioxygenase (Zukowsky et al. ( 1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding β-galactosidase, an enzyme for which there are chromogenic substrates; a nucleotide sequence encoding luciferase (lux) that allows for bioluminescence detection (Ow et al.

(1986) Science 234:856-859); a nucleotide sequence encoding aequorin which may be employed in calcium-sensitive bioluminescence detection (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126:1259-1268); or a nucleotide sequence encoding green fluorescent protein (Niedz et al. ( 1995) Plant Cell Reports 14:403-406). One of skill in the art is capable of choosing a suitable selectable marker for use in an expression construct of this invention.

[0231] 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

transformation. If stacked by genetically transforming the plants, the nucleotide sequences of interest can be combined at any time and in any order. For example, 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

simultaneously in a co-transformation protocol with a nucleotide sequence, nucleic acid molecule, nucleic acid construct, and/or composition of this invention, provided by any combination of expression constructs. For example, if two nucleotide sequences will be introduced, they 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.

[0232] In addition to the nucleic acid encoding an expression product that inhibits stomatal closure, 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. 5,569,823; 5,304,730; 5,495,071 ; 6,329,504; and 6,337,431. 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

precipitation), or one that allows identification of a plant exhibiting a trait of interest (e.g., a selectable marker, seed coat color, etc.). Various polypeptides of interest, as well as methods for introducing these polypeptides into a plant, are described, for example, in US Patent Nos. 6,084,155; 6,329,504 and 6,337,431; as well as US Patent Publication No. 2001/0016956. See also, on the World Wide Web at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/. Nucleotide sequences conferring resistance/tolerance to an herbicide that inhibits the growing point or meristem, such as an imidazalinone or a sulfonylurea can also be suitable in some

embodiments of the invention. Exemplary 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. 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).

[0233] In specific embodiments, 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. In these embodiments, 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. 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. In non-limiting examples, the ethanol receptor comprises the amino acid sequence:

[0234] MADTRRRQNHSCDPCRKGKRRCDAPENRNEANENGWVSCSNCKR WTsIKDCTFNWLSSQRSKAKGAAPRARTT KARTATTTSEPSTSAATff

VINSHDALPSWTQGLLSHPGDLFDFSHSAIPANAEDAANVQSDAPFPWDLAIPGDFS M GQQLEKPLSPLSFQAVLLPPHSPNTDDLIRELEEQTTDPDSVTDTNSVQQVAQDGSLW SDRQSPLLPENSLCMASDSTARRYARSmTKNLMRIYHDSMENALSCWLTEHNCPYS DQISYLPPKQRAEWGPNWSNRMCIRVCRLDRVSTSLRGRALSAEEDKAAARALHLAI VAFASQWTQHAQRGAGLNVPADIAADERSIRRNAWNEARHALQHTTGIPSFRVIFAN IIFSLTQSVLDDDEQHGMGARLDKLLENDGAPVFLETANRQLYTFRHKFARMQRRGK AFNRLPGGSVASTFAGIFETPTPSSESPQLDPVVASEEHRSTLSLMFWLGIMFDTLSAA MYQRPLVVSDEDSQISSASPPRRGAETPINLDCWEPPRQVPSNQEKSDVWGDLFLRTS DSLPDHESHTQISQPAARWPCTYEQAAAALSSATPVKVLLYRRVTQLQTLLYRGASP ARLEAAIQRTLYVYNHWTAKYQPFMQDCVANHELLPSRIQSWYVILDGHWHLAAM LLADVLESIDRDSYSDINHIDLVTKLRLDNALAVSALARSSLRGQELDPGKASPMYRH FHDSLTEVAFLVEPWTWLIHSFAKAAYILLDCLDLDGQGNALAGYLQLRQNCNYCI RALQFLGRKSDMAALVAKDLERGLNGKVDSFL [SEQ ID NO:56] ; or

[0235] 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:56.

[0236] Exemplary AlcR-encoding polynucleotides may be selected from:

[0237] atggctgacagcatccagaccgcacgtttttggacttctgataccatctatacagatatc actgtcgatattctcc tgcacacagcatggcagatacgcgccgacgccagaatcatagctgcgatccctgtcgcaa gggcaagcgacgctgtgatgccccgg aaaatagaaacgaggccaatgaaaacggctgggtttcgtgttcaaattgcaagcgttgga acaaggattgtaccttcaattg cccaacgctccaaggcaaaaggggctgcacctagagcgagaacaaagaaagccaggaccg caacaaccaccagtgaaccatcaa cttcagctgcaacaatccctacaccggaaagtgacaatcacgatgcgcctccagtcataa actctcacgacgcgctcccgagctggac tcaggggctactctcccaccccggcgaccttttcgatttcagccactctgctattcccgc aaatgcagaagatgcggccaacgtgcagtc agacgcaccttttccgtgggatctagccatccccggtgatttcagcatgggccaacagct cgagaaacctctcagtccgctcagttttca agcagtccttcttccgccccatagcccgaacacggatgacctcattcgcgagctggaaga gcagactacggatccggactcggttacc gatactaatagtgtacaacaggtcgctcaagatggatcgctatggtctgatcggcagtcg ccgctactgcctgagaacagtctgtgcatg gcctcagacagcacagcacggcgatatgcccgttccacaatgacgaagaatctgatgcga atctaccacgatagtatggagaatgcac tgtcctgctggctgacagagcacaattgtccatactccgaccagatcagctacctgccgc ccaagcagcgggcggaatggggcccga actggtcaaacaggatgtgcatccgggtgtgccggctagatcgcgtatctacctcattac gcgggcgcgccctgagtgcggaagagg acaaagccgcagcccgagccctgcatctggcgatcgtagcttttgcgtcgcaatggacgc agcatgcgcagaggggggctgggcta aatgttcctgcagacatagccgccgatgagaggtccatccggaggaacgcctggaatgaa gcacgccatgccttgcagcacacgac agggattccatcattccgggttatatttgcgaatatcatcttttctctcacgcagagtgt gctggatgatgatgagcagcacggtatgggtg cacgtctagacaagctactcgaaaatgacggtgcgcccgtgttcctggaaaccgcgaacc gtcagctttatacattccgacataagtttg cacgaatgcaacgccgcggtaaggctttcaacaggctcccgggaggatctgtcgcatcga cattcgccggtattttcgagacaccgac gccgtcgtctgaaagcccacagcttgacccggttgtggccagtgaggagcatcgcagtac attaagccttatgttctggctagggatcat gttcgatacactaagcgctgcaatgtaccagcgaccactcgtggtgtcagatgaggatag ccagatatcatcggcatctccaccaaggc gcggcgctgaaacgccgatcaacctagactgctgggagcccccgagacaggtcccgagca atcaagaaaagagcgacgtatggg gcgacctcttcctccgcacctcggactctctcccagatcacgaatcccacacacaaatct ctcagccagcggctcgatggccctgcacc tacgaacaggccgccgccgctctctcctctgcaacgcccgtcaaagtcctcctctaccgc cgcgtcacgcagctccaaaccctcctcta tcgcggcgccagccctgcccgccttgaagcggccatccagagaacgctctacgtttataa tcactggacagcgaagtaccaaccattt atgcaggactgcgttgctaaccacgagctcctcccttcgcgcatccagtcttggtacgtc attctagacggtcactggcatctagccgcg atgttgctagcggacgttttggagagcatcgaccgcgattcgtactctgatatcaaccac atcgaccttgtaacaaagctaaggctcgata atgcactagcagttagtgcccttgcgcgctcttcactccgaggccaggagctggacccgg gcaaagcatctccgatgtatcgccatttc catgattctctgaccgaggtggcattcctggtagaaccgtggaccgicgttcttattcac tcgtttgccaaagctgcg^tatctt^ tgtttagatctggacggccaaggaaatgcactagcggggtacctgcagctgcggcaaaat tgcaactactgcattcgggcgctgcaatt tctgggcaggaagtcggatatggcggcgctggttgcgaaggatttagagagaggmgaatg ggaaagttgacagctm

[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.

[0238] Thus, expression of the 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. Generally, 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. Use of 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). Alternatively, 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.

3. Transgenic plants, plant parts, plant organs and plant cells

[0239] The present invention further encompasses plant cells, plant parts, plant organs and plants in accordance with the embodiments of this invention. Thus, in some embodiments, 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.

[0240] In some embodiments, the plants are selected from monocotyledonous plants. Non-limiting examples of 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. In some embodiments, 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. As used herein, "sugar cane" and "Saccharum spp." mean any of six to thirty-seven species (depending on taxonomic interpretation) of tall perennial grasses of the genus Saccharum. In particular, the plant can be Saccharum aegyptiacum, Saccharum esculentum, Saccharum arenicol, 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.

[0241] Further non-limiting examples of 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.

[0242] In some embodiments, the plants are selected from energy crops, representative examples of which include:

[0243] Miscanthus (e.g., Miscanthus ffinis, M. boninensis, M. brevipilus, M.

capensis, M. changii, M. chejuensis, M. chinensis, M. chrysander, M. condensatus, M.

coreensis, M. cotulifer, M. depauperatus, M. ecklonii, M. eulalioides, M. flavidus, M.

floribundus, M. floridulus, M. formosanus, M. fuscus, M. gossweileri, M. hackelii, M.

hidakanus, M. intermedius, M. ionandros, M. japonicus, M. jucundum, M. junceus, M.

kanehirai, M. kokusanensis, M. littoralis, M. longiberbis, M. lutarioriparius, M. luzonensis, M. matsudae, M. matsumurae, M. miser, M. nakaianus, M. neo-coreanus, M. nepalensis, M. nudipes, M. ogiformis, M. oligostachyus, M. oligostachyus, M. paniculatus, M. polydactylos, M. purpurascens, M. py otocephalus, M. ridleyi, M. rufipilus, M. ryukyuensis, M.

saccariflorus, M. sacchaliflorus, M. saccharifer, M. sacchariflorus, M. sieboldi, M. sinensis, M. sorghum, M. szechuanensis, M. tanakae, M. taylorii, M. teretifolius, M. tincrorius, M. tinctorius, M. transmorrisonensis, M. violaceus, M. wardii, M. yunnanensis, M. zebrinus Hybrid: M. x giganteus);

[0244] 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.

cuspidatus, E. decus-sylvae, E. deflorata, E. divaricatus, E. dohrni, E. ecklonii, E. elegans, E. elephantinus, E. erectus, E. fallax, E. fastigiatus, Efilifolius, E.fischerianus, E. flavescens,

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.

jamaicensis, E. japonicus, E. junceus, E. kajkaiensis, E. kanashiroi, E. lancangensis, E. laxus,

E. longesetosus, E. longifolius, E. longisetosus, E. longisetus, E. lugubris, E. luzonicus, E. mackinlayi, E. macratherus, E. malcolmi, E. manueli, E. maximus, E. mishmeensis, E. mollis, E. monstierii, E. munga, E. munja, E. nepalensis, E. nipponensis, E. nudipes, E. obtusus, E. orientalis, E. pollens, E. parviflorus, E. pedicellaris, E. perrieri, E. pictus, E. pollinioides, E. procerus, E. pungens, E. purpurascens, E. purpureus, E. pyramidalis, E. ravennae, E. rehni,

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);

[0245] 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.

atrichum, P. aureum, P. bambusiforme, P. baojiense, P. barbatum, P. barteri, P. basedowii,

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. compressum, P. cornucopiae, P. corrugatum, P. crinitum, P. crus-galli, P. cupreum, P. cylindricum, P. cynosuroides, P. dalzielii, P. darfuricum, P. dasistachyum, P. dasystachyum, P. davyi, P. densiflorum, P. depauperatum, P. dichotomum, P. dilloni, P.

dioicum, P. dispiculatum, P. distachyum, P. distylum, P. divisum, P. domingense, P. dowsonii,

P. durum, P. echinurus, P. elatum, P. elegans, P. elymoides, P. erubescens, P. erythraeum, P. exaltatum, P. exiguum, P. exile, P. fallax, P. fasciculatum, P. felicianum, P. flaccidum, P. flavescens, P. flavicomum, P. flavisetum, P. flexile, P. flexispica, P. foermerianum, P.

franchetianum, P.frutescens, P. gabonense, P. gambiense, P. geniculatum, P. germanicum, P. gibbosum, P. giganteum, P. glabrum, P. glaucifolium, P. glaucocladum, P. glaucum, P.

gossweileri, P. gracile, P. gracilescens, P. grandiflorum, P. griffithii, P. haareri, P.

hamiltonii, P. helvolum, P. henryanum, P. hirsutum, P. hohenackeri, P. holcoides, P.

hordeiforme, P. hordeoides, P. humboldtianum, P. humile, P. identicum, P. imberbe, P.

implicatum, P. inclusum, P. incomptum, P. indicum, P. intectum, P. intertextum, P. italicum,

P. jacquesii, P. japonicum, P. javanicum, P. kamerunense, P. karwinskyi, P. kirkii, P.

kisantuense, P. lachnorrhachis, P. laevigatum, P. lanatum, P. lanuginosum, P. latifolium, P. laxior, P. laxum, P. lechleri, P. ledermanni, P. leekei, P. leonis, P. linnaei, P. longifolium, P. longisetum, P. longissimum, P. longistylum, P. macrochaetum, P. macropogon, P.

macrostachyon, P. macrostachys, P. macrostachyum, P. macrourum, P. maiwa, P.

malacochaete, P. marquisense, P. massaicum, P. megastachyum, P. merkeri, P. mexicanum,

P. mezianum, P. mildbraedii, P. molle, P. mollissimum, P. mongolicum, P. monostigma, P. montanum, P. multiflorum, P. mutilatum, P. myosuroides, P. myurus, P. natalense, P.

nemorum, P. nepalense, P. nervosum, P. nicaraguense, P. nigricans, P. nigritarum, 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. pallidum, P. panormiianum, P. pappianum, P. parisii, P. parviflorum, P. paucisetum, P. pauperum, P. pedicellatum, P. pennisetiforme, P. pentastachyum, P. persicum, P.

perspeciosum, P. peruvianum, P. petiolare, P. petraeum, P. phalariforme, P. phalaroides, P. pilcomayense, P. pirottae, P. plukenetii, P. plumosum, P. polycladum, P. polygamum, P.

polystachion, P. polystachyon, P. preslii, P. prieurii, P. pringlei, P. procerum, P. prolificum,

P. proximum, P. pruinosum, P. pseudotriticoides, P. pumilum, P. pungens, P. purpurascens, P. purpureum, P. pycnostachyum, P. qianningensis, P. quartinianum, P. ramosissimum, P. ramosum, P. rangei, P. refractum, P. respiciens, P. reversum, P. richardii, P. rigidum, P. riparioides, P. riparium, P. robustum, P. rogeri, P. rueppelianum, P. rufescens, P. rupestre,

P. ruppellii, P. sagittatum, P. sagittifolium, P. salifex, P. sampsonii, P. scaettae, P. scandens, P. schimperi, P. schliebenii, P. schweinfurthii, P. sciureum, P. sclerocladum, P. scoparium, P. secundiflorum, P. sericeum, P. setaceum, P. setigerum, P. setosum, P. shaanxiense, P.

sichuanense, P. sieberi, P. sieberianum, P. siguiriense, P. simeonis, P. sinaicum, P. sinense,

P. snowdenii, P. somalense, P. sordidum, P. spectabile, P. sphacelatum, P. spicatum, P.

squamulatum, P. stapfianum, P. stenorrhachis, P. stenostachyum, P. stolzii, P. stramineum, P. subangustum, P. subeglume, P. swartzii, P. tempisquense, P. teneriffae, P. tenue, P.

tenuifolium, P. tenuispiculatum, P. thulinii, P. thunbergii, P. tiberiadis, P. togoense, P.

trachyphyllum, P. triflorum, P. trisetum, P. tristachyon, P. tristachyum, P. triticoides, P.

typhoides, P. typhoideum, P. uliginosum, P. uniflorum, P. unisetum, P. vahlii, P. validum, P. variabile, P. versicolor, P. verticillatum, P. villosum, P. violaceum, P. viride, P. vulcanicum, P. vulpinum, P. weberbaueri, P. yemens);

[0246] Saccharum {e.g., as described above including S. ravennae and S.

sponteneum);

[0247] Arundo {Arundo donax, A. formosana, A. mediterranea, A. pliniana);

[0248] Sorghum {e.g., Sorghum abyssinicum, S. aethiopicum, S. album, S.

andropogon, S. ankolib, S. annuum, S. anomalum, S. arctatum, S. arduini, S. arenarium, S. argenteum, S. arunidinaceum, S. arvense, S. asperum, S. aterrimum, S. australiense, S.

avenaceum, S. balansae, S. bantuorum, S. barbatum, S. basiplicatum, S. basutorum, S.

bicorne, S. bipennatum, S. bourgaei, S. brachystachyum, S. bracteatum, S. brevicallosum, S. brevicarinatum, S. brevifolium, S. burmahicum, S. cabanisii, S. cqffrorum, S. campanum, S. campestre, S. camporum, S. candatum, S. canescens, S. capense, S. capillare, S. carinatum, S. castaneum, S. caucasicum, S. caudatum, S. centroplicatum, S. cernum, S. cernuum, S.

chinense, S. Chinese, S. cirratum, S. commune, S. compactum, S. condensatum, 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. dochna, 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. flavescens, S.flavum, S. friesii, S. fulvum, S.fiiscum, S. gambicum, S. giganteum, S. glabrescens, S. glaucescens, S. glaziovii, S. glomeratum, S. glycychylum, S. gracile, S. gracilipes, S. grandes, S. guineence, S. guineense, S. guinense, S. halapense, S. halenpensis, S. halepensis, S. hallii, S. hewisonii, S. hirse, S. hirtiflorum, S. hirtifolium, S. hirtum, S. hybrid, S. incompletum, S. japonicum, S. junghuhnii, S. lanceolatum, S. laterale, S. laxum, S. leicladum, S. leptocladum, S. leptos, S. leucostachyum, S. liebmanni, S. liebmannii, S. lithophilum, S. longiberbe, S. macrochaeta, S. malacostachyum, S. margaritiferum, S. medioplicatum, S. mekongense, S. melaleucum, S. melanocarpum, S. mellitum, S. membranaceum, S. micratherum, S. miliaceum, S, miliiforme,

S. minarum, S, mixture, S. mjoebergii, S. muticum, S. myosurus, S. nankinense, S. negrosense,

S. nervosum, S. nigericum, S. nigricans, S. nigrum, S. niloticum, S. nitens, S. notabile^ S.

nubicum, S. nutans, S. orysoidum, S. pallidum, S. panicoides, S. papyrascens, S. parviflorum,

S. pauciflorum, S. piptatherum, S. platyphyllum, S. pogonostachyum, S. pohlianum, S.

provinciale, S. pugionifolium, S. purpureo-sericeu , S. pyramidale, S. quartinianum, S.

repens, S. riedelii, S. rigidifolium, S. rigidum, S. rollii, S. roxburghii, S. rubens, S. rufum, S. ruprechtii, S. saccharatum, S. saccharoides, S. salzmanni, S. sativum, S. scabriflorum, S. schimperi, S. schlumbergeri, S. schottii, S. schreberi, S. scoparium, S. secundum, S.

semiberbe, S. serratum, S. setifolium, S. simulans, S. somaliense, S. sorghum, S. spathiflorum,

S. splendidum, S. spontaneum, S. stapfii, S. striatum, S. subglabrescens, S. sudanense, S.

tataricum, S. technicum, S. technicus, S. tenerum, S. ternatum, S. thonizzi, S. trichocladum, S. trichopus, S. tropicum, S. truchmenorum, S. usambarense, S. usorum, S. verticillatum, S.

verticilliflorum, S. vestitum, S. villosum, S. virgatum, S. virginicum, S. vogelianum, S. vulgare,

S. wrightii, S. zeae, S. zollingeri Hybrid: S. x almum, S. x almum Parodi, S. bicolor x sudanense, S. ^χ derzhavinii, S. ^χ drummondii, S. x randolphianum);

[0249] Poplars (e.g., Populus P. acuminata, P. adenopoda, P. alba, P. afghanica,

P. alaschanica, P. amurensis, P. angustifolia, P. baicalensis, P. balsamifera, P. beijingensis, P. candicans, P. cathayana, P. charbinensis, P. ciliata, P. davidiana, P. deltoides, P.

dimorpha, P. euphratica, P. flexibilis, P. fremontii, P. grandidentata, P. heterophylla, P.

incrassata, P. koreana, P. lasiocarpa, P. laurifolia, P. maximowiczii, P. moskoviensis, P. nigra, P. petrowskiana, P. pruinosa, P. purdomii, P. rasumowskiana, P. sargentii, P.

sieboldii, P. simonii, P. suaveolens, P. szechuanica, P. tomentosa, P. tremula, P. tremuloides, P. trichocarpa, P. tristis, P. vernirubens, P. wilsonii, P. woobstii, P.

yunnanensisUNothospecies: P. x acuminata, P. x berolinensis, P. x brayshawii, P. x canadensis, P. x canescens, P. x generiosa, P. hinckleyana, P. ^χ jackO); [0250] wheat (e.g., Triticum abyssinicum, T. accessorium, T. acutum, 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.

alpinum, T. amyleum, T. amylosum, T. angustifolium, T. angustum, T. antiquorum, T.

apiculatum, T. aragonense, T. aralense, T. araraticum, T. arenarium, T. arenicolum, T. arias, T. aristatum, T. arktasianum, T. armeniacum, T. arras, T. arundinaceum, T. arvense, T.

asiaticum, T. asperrimum, T. asperum, T. athericum, T. atratum, T. attenuatum, T. aucheri, T. baeoticum, T. barbinode, T< barbulatum, T. barrelieri, 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.

bromoides, T. brownei, T. bucharicum, T. bulbosum, T. bungeanum, T. buonapartis, T.

burnaschewi, T. caeruleum, T. caesium, T. caespitosum, T. campestre, T. candissimum, T. caninum, T. capense, T. carthlicum, T. caucasicum, T. caudatum, T. cereale, T. cerulescens, T. cevallos, T. chinense, T. cienfuegos, T. ciliare, T. ciliatum, T. cinereum, T. clavatum, T. coarctatum, T. cochleare, T. comosum, T. compactum, T. compositum, T. compressum, T. condensatum, T. crassum, T. cretaceum, T. creticum, T. crinitum, T. cristatum, T. curvifolium, T. cylindricum, T. cynosuroides, T. czernjaevi, T. dasyanthum, T. dasyphyllum, 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. 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.

geminatum, T. geniculatum, T. genuense, T. giganteum, T. glaucescens, T. glaucum, T.

gmelini, T. gracile, T. halleri, T. hamosum, T. hebestachyum, T. heldreichii, T. hemipoa, T. hieminflatum, T. hirsutum, T. hispanicum, T. hordeaceum, T. hordeiforme, T. hornemanni, T. horstianum, T. hosteanum, T. hybernum, T. ichyostachyum, T. imbricatum, T. immaturatum, T. infestum, T. inflatum, T. intermedium, T. ispahanicum, T. jakubzineri, T. juncellum, T. junceum, T. juvenale, T. kiharae, T. kingianum, T. kirgianum, T. koeleri, T. kosanini, T.

kotschyanum, T. kotschyi, T. labile, T. lachenalii, T. laevissimum, T. lasianthum, T. latiglume, T. latronum, T. laxiusculum, T. laxum, T. leersianum, T. ligusticum, T. linnaeanum, T.

litorale, T. litoreum, T. littoreum, T. loliaceum, T. lolioides, T. longearistatum, T. longisemineum, T. longissimum, T. lorentii, T. lutinflatum, T. luzonense, T. macha, T.

macrochaetum, T. macrostachyum, T. macrourum, T. magellanicum, T. maritimum, T.

markgrafii, T. martius, T. maturatum, T. maurorum, T. maximum, T. mexicanum, T.

miguschovae, 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.

ovatum, 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.

pruinosum, T. pseudo-agropyrum, T. pseudocaninum, T. puberulum, T. pubescens, T.

pubiflorum, T. pulverulentum, T. pumilum, T. pungens, T. pycnanthum, T. pyramidale, T. quadratum, T. ramificum, T. ramosum, T. rarum, T. recognitum, T. rectum, T. repens, T. reptans, T. requlenii, T. richardsonii, T. rigidum, T. rodeti, T. roegnerii, T. rossicum, T.

rottboellia, T. rouxii, T. rufescens, T. rufinflatum, T. rupestre, T. sabulosum, T. salinum, T. salsuginosum, T. sanctum, T. sardinicum, T. sartarii, T. sativum, T. savignionii, T. savignonii,

T. scaberrimum, T. scabrum, T. schimperi, T. schrenkianum, T. scirpeum, T. secale, T.

secalinum, 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.

squarrosum, T. striatum, T. strictum, T. strigosum, T. subaristatum, T. subsecundum, T.

subtile, T. subulatum, T. sunpani, T. supinum, T. sylvaticum, T. sylvestre, T. syriacum, T. tanaiticum, T. tauri, T. tauschii, T. tenax, T. tenellum, T. tenue, T. tenuiculum, T. teretiflorum, T. thaoudar, T. tiflisiense, T. timococcum, T. timonovum, T. timopheevi, T. timopheevii, T. tomentosum, T. tournefortii, T. trachycaulon, T. trachycaulum, T. transcaucasicum, 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.

velutinum, T. ventricosum, T. venulosum, T. villosum, T. violaceum, T. virescens, T. volgense,

T. vulgare, T. youngii, T. zea, T. zhufavskyi); [0251] rice {e.g., Oryza abnensis, O. abromeitiana, O. alta, O. angustifolia, O. aristata, O. australiensis, O. barthii, O. brachyantha, O. breviligulata, O. carinata, O.

caudata, O. ciliata, O. clandestine!, O. coarctata, O. collina, O. communissima, O. cubensis,

O. den data, O. dewildemani, O. eichingeri, O. elongata, O.fatua, O. filiformis, O.

formosana, O. glaberrima, O. glaberi, O. glaberrima, O. glaberrina, O. glauca, O.

glumaepatula, O. glutinosa, O. grandiglumis, O. granulata, O. guineensis, O. hexandra, O. hybrid, O. indandamanica, O. jeyporensis, O. latifolia, O. leersioides, O. linnaeus, O.

longiglumis, O. longistaminata, O. madagascariensis, O. malampuzhaensis, O. manilensis, O. marginata, O. meijeriana, O. meridionalis, O. meridonalis, O. mexicana, O. meyeriana, O. mezii, O. minuta, O. monandra, O. montana, O. mutica, O. neocaledonica, O. nepalensis, O. nigra, O. nivara, O. officinalis, O. oryzoides, O. palustris, O. paraguayensis, O. parviflora,

O. perennis, O. perrieri, O. platyphyla, O. plena, O. praecox, O. prehensilis, O. pubescens, O. pumila, O. punctata, O. repens, O. rhizomatis, O. ridleyi, O. rubra, O. rubribarbis, O.

rufipogon, O. sativa, O. schlechteri, O. schweinfurthiana, O. segetalis, O. sorghoidea, O. sorghoides, O. spontanea, O. stapfii, O. stenothyrsus, O. subulata, O. sylvestris, O. tisseranti,

O. tisserantii, O. triandra, O. triticoides, O. ubanghensis);

[0252] 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,

A. arenaria, A. argaea, A. argentea, A. argentoideum, A. ariguensis, A. aristelliformis, A. aristidioides, A. aristidoides, A. armeniaca, A. arundinacea, A. arvensis, A. aspera, A.

atheranthera, A. atlantica, A. atropurpurea, A. aurata, A. australis, A. azo-carti, A. barbata,

A. baregensis, A. baumgartenii, A. beguinotiana, A. bellardi, A. benghalensis, A. besseri, A. bifida, A. bipartita, A. blavii, A. bolivaris, A. borbonia, A. bornmuelleri, A. breviaristata, A. brevifolia, A. brevis, A. bromoides, A. brownei, A. brownii, A. bruhnsiana, A. bulbosa, A. burnoufii, A. byzantina, A. caespitosa, A. cqffra, A. calicina, A. callosa, A. calycina, A.

canariensis, A. candollei, A. canescens, A. canina, A. cantabrica, A. capensis, A. capillacea,

A. capillaris, A. carmeli, A. caroliniana, A. carpatica, A. caryophyllea, A. cavanillesii, A. cernua, A. chinensis, A. chlorantha, A. ciliaris, A. cinerea, A. clarkei, A. clauda, A. coarctata,

A. colorata, A. compacta, A. compressa, A. condensata, A. convoluta, A. coquimbensis, A. coronensis, A. corymbosa, A. crassifolia, A. cristata, A. cupaniana, A. cuspidata, A.

daenensis, A. dahurica, A. damascena, A. decora, A. delavayi, A. depauperata, A. desertorum,

A. deusta, A. deyeuxioides, A. discolor, A. dispermis, A. distans, A. disticha, A. distichophylla,

A. dubia, A. dufourei, A. dura, A. editissima, A. elata, A. elatior, A. elegans, A. elephantina, A. elongata, A. eriantha, A. fallax, A. fatua, A. fedtschenkoi, A. fertilis, A. festucaeformis, A. festucoides, A. filifolia, A. filiformis, A. flaccida, A. flava, A. flavescens, A. flexuosa, A.

forskolei, A. forsteri, A. fragilis, A. freita, A. fiisca, A. fuscoflora, A. gallecica, A. gaudiana, A. gaudiniana, A. geminiflora, A. georgiana, A. georgica, A. gigantea, A. glabra, A. glabrata, A. glabrescens, A. glacialis, A. glauca, A. glomerata, A. glumosa, A. gonzaloi, A. gracilis, A. gracillima, A. grandis, A. hackelii, A. haussknechtii, A. heldreichii, A. heteromalla, A.

hexantha, A, hideoi, A. hirsuta, A. hirta, A. hirtifolia, A. hirtula, A. hispa ica, A. hispida, A. hookeri, A. hoppeana, A. hostii, A. hugeninii, A. hungarica, A. hybrida, A. hydrophila, A. insubrica, A. insularis, A. intermedia, A. involucrata, A. involuta, A. jahandiezii, A. japonica,

A. junghuhnii, A. koenigii, A. kotschyi, A. lachnantha, A. laconica, A. laeta, A. laevigata, A. laevis, A. lanata^ A. lanuginosa, A. lasiantha, A. latifolia, A. leiantha, A. lejocolea, A.

lendigera, A. leonina, A. leptostachys, A. letourneuxii, A. levis, A. lodunensis, A. loejjflingiana,

A. loeflingiana, A. longa, A. longepedicellata, A. longepilosa, A. longespiculata, A. longifolia,

A. longiglumis, A. longipilosa, A. lucida, A. ludoviciana, A. lupulina, A. lusitanica, A. lutea,

A. macilenta, A. macra, A. macrantha, A. macrocalycina, A. macrocalyx, A. macrocarpa, A. macrostachya, A. magellanica, A. magna, A. malabarica, A. malzevii, A. mandoniana, A. marginata, A. maroccana, A. matritensis, A. maxima, A. mediolanensis, A. melillensis, A. meridionalis, A. micans, A. michelii, A. micrantha, A. mirandana, A. mollis, A. mongolica, A. montana, A. montevidensis, A. mortoniana, A. multiculmis, A. muralis, A. muricata, A.

muriculata, A. murphyi, A. mutica, A. myriantha, A. mysorensis, A. nana, A. neesii, A.

neglecta, A. nemoralis, A. nervosa, A. neumeyeriana, A. newtonii, A. nigra, A. nitens, A.

nitida, A. nodipilosa, A. nodosa, A. noeana, A. notarisii, A. nuda, A. nudibrevis, A. nutans, A. nutkaensis, A. occidentalis, A. odorata, A. oligostachya, A. opulenta, A. orientalis, A. ovata,

A. ovina, A. palaestina, A. pallens, A. pallida, A. palustris, A. panicea, A. panormitana, A. papillosa, A. paradensis, A. paradoxa, A. parlatorei, A. parlatorii, A. parviflora, A.

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.

praecox, A. praegravis, A. praeusta, A. pratensis, A. precatoria, A. preslii, A. prostrata, A. provincialis, A. pruinosa, A. pseudolucida, A. pseudosativa, A. pseudoviolacea, A. puberula,

A. pubescens, A. pulchella, A. pumila, A. pungens, A. purpurascens, A. purpurea, A. pusilla,

A. quadridentula, A. quadriseta, A. quinqueseta, A. racemosa, A. radula, A. redolens, A.

riabushinskii, A. rigida, A. rotae, A. rothii, A. roylei, A. rubra, A. rufescens, A. rupestris, A. ruprechtii, A. sarracenorum, A. sativa, A. saxatilis, A. scabriuscula, A. scabrivalvis, A.

schelliana, A. scheuchzeri, A. secalina, A. secunda, A. sedenensis, A. segetalis, A.

sempervirens, A. sensitiva, A. septentrionalis, A. serrulatiglumis, A. sesquiflora, 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.

subulata, A. subvillosa, A. suffusca, A. sulcata, A. sylvatica, A. symphicarpa, A. syriaca, A. tatarica, A. taygetana, A. tenorii, A. tentoensis, A. tenuiflora, A. tenuis, A. thellungii, A.

thorei, A. tibestica, A. tibetica, A. toluccensis, A. tolucensis, A. torreyi, A. trabutiana, A.

triaristata, A. trichophylla, A. trichopodia, A. triseta, A. trisperma, A. triticoides, A. truncata,

A. tuberosa, A. turgidula, A. turonensis, A. uniflora, A. unilateralis, A. valesiaca, A. varia, A. vasconica, A. vaviloviana, A. velutina, A. ventricosa, A. versicolor, A. vilis, A. villosa, A. virescens, A. viridis, A. volgensis, A. wiesiii, A. wilhelmsi);

[0253] 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);

[0254] soybean (i.e., Glycine max); barley (i.e., Hordeum vulgare); sugar beet (i.e., Beta vulgaris); hay and fodder crops.

[0255] 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,

cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation,, sonication, infiltration, PEG-mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into the plant cell, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and

Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)). [0256] Thus, in some particular embodiments, 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.

[0257] 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. coli strain that carries a plasmid that is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, 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.

[0258] 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. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the nucleic acid of interest. Alternatively, 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, a dried bacterium or a bacteriophage, each containing one or more nucleic acids sought to be introduced) also can be propelled into plant tissue. Thus, in particular embodiments of the present invention, 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. I (1984), and Vol. II (1986)). Methods of selecting for transformed, transgenic plants, plant cells and/or plant tissue culture are routine in the art and can be employed in the methods of the invention provided herein. Likewise, the genetic properties engineered into the transgenic seeds and plants, plant parts, and/or plant cells of the present invention described above can be passed on by sexual reproduction or vegetative growth and therefore can be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as harvesting, sowing or tilling. A nucleotide sequence therefore can be introduced into the plant, plant part and/or plant cell in any number of ways that are well known in the art. 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. Where more than one nucleotide sequence is to be introduced, 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. Accordingly, 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. In some embodiments of this invention, 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).

Alternatively, the introduced construct may be present on an extra-chromosomal non- replicating vector and be transiently expressed or transiently active. Whether present in an extra-chromosomal non-replicating vector or a vector that is integrated into a chromosome, the nucleic acid molecule can be present in a plant expression construct.

4. Uses of the constructs to control transpiration in plants

[0259] 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. In most of these cases failure to adequately dry the crop due to weather or other conditions results in substantial losses. In other cases including but not limited to tobacco, dried fruits such as raisins and prunes, nuts, coffee, tea, cocoa, and many ornamental goods, the produce needs to be dried immediately after harvest prior and to use. In these cases again, the mutants of this invention may be of tremendous value to growers who could accelerate or control the rate of crop drying.

[0260] 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

dehydrogenase (ADH1) system of Aspergillus nidulans, as described for example above so as to inhibit stomatal closure and thereby increase transpiration in the plant, plant part, plant organ or plant leaf. 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 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. [0261] Generally, 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%).

[0262] In some embodiments, 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.).

[0263] Alternatively, or in addition, 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. 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).

[0264] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples.

EXAMPLES

EXAMPLE 1

PRODUCTION OF NON-OPTIMIZED CONSTRUCTS AND TRANSFORMATION INTO SUGAR CANE

[0265] 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

5'-CCCGGGATCCTAAACCATGGTCCGTCCTGTAGAAACCC-3* [SEQ ID NO: 39] and reverse primer 5*-TCATTGTTTGCCTCCCTGCTG-3' [SEQ ID NO:40] and KAPAHiFi DNA polymerase (Geneworks). The resulting PCR product was cloned into pGEM-T

(Promega) and sequence verified. The 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.

- 97 - r [0266] 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

5 ^, -TTACTTCTGCAGCCCTAAACCATGGCAGATACGCGCCGACGCCAG3 ^, [SEQ ID NO:41] and reverse primer

5 -TGTTTGAACGATCCCCTACAAAAAGCTGTCAACTTTCCCA -3' [SEQ ID NO:42] and KAPAHiFi DNA polymerase. The resulting PCR product was cloned into the Smal site of ZmUbi-nos/pBS using the Clontech In Fusion ^® PCR Cloning System to generate

ZmUbi- AlcR, and the PCR product was sequence verified. To generate ZmUbi-AlcR AlcA- GS, the AlcA-GS-nos sequence was subcloned from 35S-AlcR Ale A-GS/pUC into ZmUbi- AlcR using Hindlll.

[0267] The ZmUbi-GS and ZmUbi-AlcR AlcA-GS constructs were transformed into sugar cane using microprojectile bombardment of callus material. To generate the callus, sugar cane (cultivar Q 117) "tops" were obtained from The Bureau of Sugarcane Experimental Stations (BSES) LTD Meringa Queensland, Australia. Calli were initiated as described by Franks and Birch (1991, Australian J. Plant Physiol. 18: 471-480) using MSC ₃ media consisting of: 4.43g/L MS basal salts with vitamins (PhytoTechnology Laboratories®, Shawnee

Mission, KS, USA ), 500mg/L casein hydrolysate (Merck), 13.6μΜ 2,4- Dichlorophenoxyacetic acid (2, 4-D; Phytotechnology laboratories), lOOml/L young coconut juice ("Cock" brand, Thailand), 3% (w/v) sucrose and 8 g/L agar (Research organics 10020). Petri dishes used for tissue culture media were 90mm x 25mm high vented type and sealed with micropore surgical tape. Calli were maintained for nine weeks in the dark at 26°C and subcultured every 14 days.

[0268] Microprojectile bombardment of callus was performed according to the method of Bower et al (Molecular Breeding 1996 2: 239-249). For callus transformation, 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) was co-bombarded with each of the GS expression constructs to allow for selection of transformed cells using geneticin. For microprojectile bombardment, a 2 μί aliquot of a 1 : 1 mixture of pUKN (1 μg/μL) and the

GS expression construct DNA (1 μg/μL) was added to approximately 3mg of 1 μπι 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 μί of supernatant was removed. The remaining DNA coated gold solution was mixed and 5 μΐ, 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 μπι was positioned approximately 1.5 cm above the target tissue within the PIG chamber. 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

[0269] 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 μΜ 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. After ten weeks of regeneration with BAP, the plants were transferred to rooting medium with selection (the same as regeneration medium, however BAP is replaced with 10.7 μΜ 1 -Naphthalene Acetic Acid (NAA; Sigma). The plants were grown until roots of approximately 1 cm in length had developed after which the plants were transferred to soil for acclimatization in a growth cabinet under the above mentioned lighting and temperature conditions. After approximately six weeks the plants were transferred to soil in 20cm pots, and grown in the greenhouse.

EXAMPLE 2

CHARACTERIZATION OF TRANSGENIC PLANTS CONTAINING NON-OPTIMIZED

CONSTRUCTS.

[0270] 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.

(1987) EMBO J 6: 3901-3907) within four hours of collection. Histochemical analysis was carried out by the addition of 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. After 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.

[0271] No ethanol inducible gene expression was detected in any of the leaf samples taken from 21 independent transgenic sugar cane plants containing the ethanol switch construct ZmUbi-AlcR AlcA-GS. In the transgenic sugar cane plants containing the ZmUbi- GS positive control construct, leaf samples from six of 13 independent events showed visible GUS staining in both the uninduced and induced leaf samples.

[0272] Ten days after the plants had been treated with 10% ethanol, the transgenic plants were retreated with 2% ethanol using both a root drench and aerial spray until runoff. Leaf samples consisting of five standard hole punches were taken at three and five days post treatment for GUS histochemical staining. Again, none of the leaf samples taken from the 21 transgenic sugar cane plants containing the ethanol switch construct ZmUbi-AlcR AlcA-GS showed any detectable GUS staining.

EXAMPLE 3

PRODUCTION OF OPTIMIZED ETHANOL SWITCH CONSTRUCTS

[0273] To create ethanol switch constructs that may be capable of giving reliable ethanol inducible gene expression in sugar cane, the following modifications were made to the original constructs:

[0274] The AlcR and GUS coding sequences were optimized for expression in sugar cane (Geneart optimization). A different Kozak sequence, 5'-gcggccgcc-3' [SEQ ID NO:42] was placed immediately upstream of the scoAlcR and scoGUS coding sequences. The TMV Ω translational enhancer sequence

5'-gtatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattactat tlacaattac [SEQ ID NO:43] was placed immediately upstream of the ozak sequence. 12 different AlcA promoter variants were created as follows:

[0275] ( 1 ) 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).

[0276] (2) 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).

[0277] (3) The SC 14 construct consists of an Aspergillus AlcA promoter including the upstream direct repeat AlcR binding site (-400 to -112 relative to the

translational start site) fused to the original CaMV 35S minimal sequence (-32 to +3).

[0278] (4) 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).

[0279] (5) 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.

[0280] (6) 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. [0281] (7) 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.

[0282] (8) 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.

[0283] (9) 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.

[0284] ( 10) 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.

[0285] (11) 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.

[0286] (12) The SC23 construct consists of the native Aspergillus AlcA promoter

(-400 to -64 relative to the translational start site).

[0287] The sequences for the AlcA promoter variants, sugar cane optimized GUS (scoGUS; with TMV Ω and Kozak) with nos terminator, sugar cane optimized AlcR

(scoAlcR; with TMV Ω 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 Ω . To generate the final ethanol switch constructs (SCI 2, SCI 3, and SCI 6-20), 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.

[0288] An optimized, constitutive 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.

[0289] 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).

EXAMPLE 4

AGROBACTERIUM-MEDLATED TRANSFORMATION OF SUGAR CANE

Plant source and material:

[0290] 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)

Infection and co-cultivation:

[0291] Callus tissue was heat shocked at 45°C for 5 minutes by adding 50 mL of pre- warmed 1/2 strength MS medium (without sucrose) and then maintaining the callus in a water bath at 45° C. MS medium was then drained from the callus tissue, and 25 mL of the Agrobacterium inoculation suspension was added to each vessel and mixed gently. The diX l Agrobacterium mixture was vacuum-infiltrated by placing it into a vacuum chamber for 10 min at -27.5 mmHg of vacuum. The callus/ 'Agrobacterium mixture was then rested for 5- 10 min the dark. 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 ^χ 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®, MICROPORE™ tape (3M; Minneapolis, MN) or similar material. The dishes were incubated in the dark at 22° C for 2-3 days.

Post-transformation:

[0292] After co-cultivation, 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

50 mg/L of geneticin and 200 mg/L of timentin for 14-15 days in the dark at 25° C.

Regeneration and rooting:

[0293] 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.

[0294] Shoot formation was seen between 2-4 weeks. When the first leaves appeared, the shoots were transferred to MS 4 medium (see below) until the plants grew to 4- 5 cm in height. Transformed plants were initially moved from tissue culture and placed in seedling trays containing soil and incubated in a growth chamber. Plants were initially characterized for ethanol inducible GUS expression at 4-7 weeks of age. At approximately eight weeks of age, the plants were moved to 20 cm diameter pots and maintained in a greenhouse. At approximately seven months of age, plants were transferred into 30cm pots until maturity.

Media:

[0295] The components within the media referred to above are as follows: [0296] EM3: MS salts and vitamins; 0.5 g/L casein hydrolysate; 100 ml/L coconut water; 20 g/L sucrose and 3 mg/1 2,4-D.

[0297] LB basic: 10 g/L NaCl; 5 g/L yeast extract; and 10 g/L tryptone.

[0298] LB solid: LB basic with 15 g/L of agar. [0299] 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.

[0300] MS basic: MS medium salts and vitamins, with 25 g/L sucrose.

[0301] MS 1: MS basic supplemented with 3.0 mg/L 2,4- D and 200 mg/L

Timentin.

[0302] MS2: MS basic supplemented with 3.0 mg/L 2,4- D and 50 mg/L Geneticin and 200mg/L Timentin.

[0303] 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.

[0304] 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.

[0305] CoCult: Media co-cultivation media as described for banana in Khanna et al. (2004) Molecular Breeding 14(3): 239-252.

EXAMPLE 5

CHARACTERIZATION OF TRANSGENIC PLANTS CONTAINING OPTIMIZED CONSTRUCTS

[0306] 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.

[0307] After the transgenic sugar cane plants had been in soil for 4-7 weeks, a leaf sample of approximately 3cm in length was taken from each plant just prior to ethanol treatment and analyzed for GUS expression by histochemical staining as described above. After sampling, the plants were treated with 2% ethanol using a daily root drench and aerial spray for four days. 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.

[0308] Prior to ethanol treatment, only a small number of the ethanol switch plants showed detectable GUS expression while three out of seven ZmUbi-scoGUS plants were found to be GUS positive (Table ί).

[0309] Following the 2% ethanol treatment, GUS expression was detected for 11 of the 12 optimized ethanol switch constructs with between 6% and 83% of the plants containing these constructs showing detectable GUS expression (Table 1). Visual inspection of the intensity of staining suggested that constructs SC 15, SC 18, SC 19, SC20, and SC22 gave the highest ethanol inducible expression.

Table 1:

Results from the histochemical GUS staining of transgenic sugar cane plants containing the different ethanol switch constructs in response to the continuous 2% ethanol treatment.

pUbi-scoGUS 7 ^' 3 3 NA

[0310] An independent group of 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.

[0311] Prior to ethanol treatment, leaky GUS expression was visible in a subset of the plants for some of the various constructs (Table 2). Following the 1% ethanol treatment, GUS expression was detected for six of the 12 optimized ethanol switch constructs with between 4% and 65% of the plants containing these constructs showing detectable GUS expression (Table 2). Plants containing constructs SC 18, SC 19 and SC20 had the highest proportion of inducible plants, and visual inspection of the intensity of staining suggested that these plants also had the highest ethanol inducible expression.

Table 2:

Results for the histochemical GUS staining of transgenic sugar cane plants containing the different ethanol switch constructs in response to the 1% ethanol treatment.

[0312] For the quantitative analysis of ethanol inducible expression in more mature plants, only the single copy plants for each construct were selected and transferred to the greenhouse. When the transgenic sugar cane plants had been in soil for approximately six months they were assessed to verify that there was no residual GUS expression from the previous ethanol treatment that was carried out at 4-7 weeks of age. To do this, a tissue sample was taken from the first fully unfurled leaf of each plant and analyzed for GUS expression by histochemical staining. No residual GUS expression from the original ethanol treatment was detected in these transgenic plants.

[0313] Subsequently, these plants were reanalyzed for ethanol inducible expression.

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. Each plant was sampled in duplicate from the same first unfurled leaf at each timepoint. Samples were frozen at - 80°C, freeze-dried, and GUS expression was subsequently quantitated by ELISA. For the GUS ELISA, high-binding 96-well plates (Nunc Maxisorp®) were coated at 4° C overnight with 2 μg mL rabbit anti-GUS IgG (Sigma G5545) in 25 mM borate, 75 mM NaCl, pH 8.5 (100 μΙΛνεΙΙ). Plates were washed three times with 10 mM Tris, pH 8.0 containing 0.05% Tween-20 and 0.2% NaN3. Samples or standards (GUS Type VII- A, Sigma G7646) were added to the plate (100 μΙ,ΛνβΙΙ), incubated for lhr at room temperature with shaking, and washed five times. 100 μΙΛνεΙΙ of 2 μg/mL HRP-labeled rabbit anti-GUS IgG (Invitrogen A5790 conjugated to HRP) was then added to the plate, incubated for lhr at room temperature with shaking, and washed as before. The HRP-conjugated antibody was detected by adding 100 μΙ-Λνβ11 tetramethylbenzidine (TMB, Sigma T0440) and developing for 30min at room temperature. The reaction was stopped by the addition of 100 μΤΛνεΙΙ of 0.1N HC1. The absorbance was measured at 450 nm with 620 as a reference using a microplate reader (Tecan Sunrise™, Research Triangle Park, NC). The GUS standard curve uses a 4-parameter curve fit. The curve is plotted linear vs. log with a range from 0 to 320 ng/mL. Results from the GUS ELIS A indicated that there was no detectable GUS expression in any of the transgenic sugar cane plants prior to ethanol treatment. Following ethanol treatment, the highest, most consistent ethanol inducible expression was detected from those plants containing the constructs with multiple copies of the inverted repeat AlcR binding site (SCI 8, SCI 9, and SC20; Fig. 1). Little or no ethanol inducible expression was detected in plants containing the other nine constructs. These results indicate that multiple copies of the inverted repeat AlcR binding site can substantially improve ethanol inducible gene expression. EXAMPLE 6

PRODUCTION AND TESTING OF ADDITIONAL INDUCIBLE PROMOTER CONSTRUCTS WITH VARYING NUMBERS OF THE INVERTED REPEAT ALCR BINDING SITE.

[0314] Nucleic acid constructs having one copy or nine copies of the inverted repeat AlcR binding site were generated as follows:

[0315] (1) 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).

[0316] (2) 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.

[0317] The 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

H/ndlll and Ascl. 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.

[0318] 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. After sampling, the plants were treated with 2% ethanol using a single root drench (800 mL/seedling tray) and aerial spray until runoff. At four days post treatment, 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. Prior to ethanol treatment, little or no GUS expression was detected in the leaves of the transgenic sugar cane plants (Table 3, Figure 2). Following ethanol treatment, robust ethanol inducible expression was detected from those plants containing the constructs with either five or nine copies of the inverted repeat AlcR binding site (SC20 and SC36; Table 3 and Fig. 2). Only low levels of ethanol inducible expression were detected from plants containing the construct with one copy of the inverted repeat AlcR binding site (SC35; Table 3 and Fig. 2). Furthermore, a substantially higher percentage of the transgenic plants containing the constructs with multiple inverted repeat AlcR binding sites (SC20 and SC36) showed ethanol inducible expression compared to those plants containing a single copy of the inverted repeat AlcR binding site (SC35; Table 3).

Table 3:

Ethanol inducible expression from promoters containing either 1, 5, or 9 copies of the inverted repeat AlcR binding site.

EXAMPLE 7

PRODUCTION AND TESTING OF ADDITIONAL INDUCIBLE PROMOTER CONSTRUCTS IN SUGAR

CANE

[0319] To identify the minimal sequence within the inverted repeat AlcR binding site that is necessary for ethanol inducible expression, the following modified inverted repeat AlcR binding sites were generated:

[0320] (1) The SC38 construct modifies the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] to

"5'-tacgtagcggaaccgctgctcc-3 ^m [SEQ ID NO:6].

[0321] (2) The SC39 construct modifies the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] to

"S'-tacgttgcggaaccgcagctcc^"' [SEQ ID NO:9].

[0322] (3) The SC41 construct modifies the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] to

"5'-atgcatgcggtgccgcacgagg-3"' [SEQ ID NO:8].

[0323] (4) The SC42 construct modifies the inverted repeat AlcR binding site region sequence "5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] to

"S'-atgcatgcggaatgcaaccgcacgagg-S"' [SEQ ID NO: 10].

[0324] The above sequences were synthesized as pentamers fused with the long

35S minimal sequence. These modified promoter variants were cloned as described above

- I l l - (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.

[0325] Prior to ethanol treatment, little or no GUS expression was detected in the leaves of the transgenic sugar cane plants (Table 4, Fig. 3). Following ethanol treatment, robust ethanol inducible expression was detected from those plants containing the constructs with the various point mutations to the inverted repeat AlcR binding site (SC38, SC39, and SC41 ; Table 4 and Fig. 3). In addition, 100% of the transgenic plants containing these constructs were found to exhibit ethanol inducible expression (Table 4). No ethanol inducible expression was detected from transgenic plants containing the construct that alters the length of the 2 bp spacer between the inverted repeat AlcR binding sites (SC42; Table 4 and Fig. 3). From this data, the sequence of "GCGGnnCCGC" [SEQ ID NO: 1 ] (with n representing any nucleotide) can be defined as a minimal sequence necessary for ethanol inducible expression.

Table 4:

Ethanol inducible expression from promoters containing various modified inverted repeat AlcR binding sites.

EXAMPLE 8

PREPARATION AND TESTING OF A MAIZE PROMOTER IN COMBINATION WITH THE ALCR

"B" INVERTED REPEAT BINDING SITES.

[0326] 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:

[0327] 5'-ccacaggcggccaaaccgcaccctccttcccgtcgtttcccatctcttcctccttta gagctaccactatat aaatcagggctcatWctcgctcctcacaggctcatctcgcmgga

gatgatttgggattctgttcgaagatttgcggaggggggca-3 ¹ +106 [SEQ ID NO:48].

[0328] The maize Adhl minimal promoter sequence was fused with five tandem repeats of the inverted repeat AlcR binding site region sequence

"5'-atgcatgcggaaccgcacgagg-3"' [SEQ ID NO:3] (replacing the long 35S minimal promoter sequence as described in Example 3, construct SC20). Ethanol inducibility of this promoter (SC37) was compared to that of construct SC20 using GUS ELISA as described above.

[0329] Prior to ethanol treatment, little or no GUS expression was detected in the leaves of the transgenic sugar cane plants (Table 5 and Fig. 4). Following ethanol treatment, robust ethanol inducible expression was detected from those plants containing the construct with the five copies of the inverted repeat AlcR binding sites fused to the maize Adhl minimal promoter (Table 5 and Fig. 4).

Table 5:

Ethanol inducible expression from promoters containing five copies of the inverted repeat AlcR binding sites fused to different minimal promoters.

Construct Uninduced Induced Plants showing detectable

Expression (ng/mg Expression expression (% of TaqMan® ID

protein) (ng mg protein) positive plants showing

expression)

SC20 0 36.1±11.8 5/5 (100%)

SC37 2.5±4.3 107.9±19.4 11 (69%) EXAMPLE 9

CHARACTERIZATION OF ETHANOL INDUCIBLE EXPRESSION IN THE VEGETATIVE PLANTINGS OF THE PRIMARY TRANSGENIC SUGAR CANE PLANTS

[0330] Single-bud setts from stems of selected mature, TO transgenic plants were planted in soil and maintained in a glasshouse. After the T0V1 transgenic sugar cane plants were in soil for approximately four months (at which time they were comparable in size to the six month old TO plants describe above), 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. After sampling, the TOV 1 plants were treated with 5% ethanol using a single root drench (700 mL/pot) and aerial spray until runoff. At four days post treatment, 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.

[0331] Prior to ethanol treatment, little or no GUS expression was detected in the leaves of the TOV 1 transgenic sugar cane plants (data not shown). Following ethanol treatment, robust ethanol inducible expression was detected for all of the T0V1 transgenic plants (Fig. 5), indicating that ethanol inducibility from these constructs is maintained in the vegetatively-propagated material.

EXAMPLE 10

PRODUCTION OF TRANSGENE CONSTRUCTS FOR NICOTIANA BENTHAMIANA

[0332] To clone the ABI1 and abil genes, 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. The forward primer, 5 '-

CCCCGGATCCCAACAATGGAGGAAGTATCTCCGGC-3' [SEQ ID NO:49] incorporates a BamHl restriction enzyme site and the ozak sequence CAACA. The reverse primer,

5'-CCCCGTCGACTCAGTTCAAGGGTTTGCTCT-3' [SEQ ID NO:50] 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.

Constitutive promoter constructs

[0333] 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.

[0334] 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.

ale eene switch promoter constructs

[0335] To improve plant expression of the AlcR transcription factor that is required for functionality of the ale gene switch, an optimized alcR gene (scoalcR) was synthesised by Geneart (Regensburg, Germany). To generate the ale gene switch constructs for

transformation into 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.

[0336] 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).

[0337] The palcA O sequence was cloned from the construct AlBl-scoGUS- scoALCR-nptll using PCR with the forward primer 5 -

CCCCCC ATGGCTGC AGGC ATGC AAGCTTAG-3 ' [SEQ ID NO: 51] and the reverse primer 5'-CCCCGGATCCAATACCTGCAGGTCCTCTC-3' [SEQ ID NO: 52] that incorporate an Ncol and BamHl restriction enzyme site, respectively. 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.

[0338] 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

promoters).

[0339] 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

5'-CCCCGGATCCTGCAGGTCCTCTCCAAATG-3' [SEQ ID NO: 55] that incorporate an Ncol and BamHl restriction enzyme site, respectively. 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.

[0340] 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.

EXAMPLE 11

PRODUCTION OF TRANSGENE CONSTRUCTS FOR SUGAR CANE

[0341] 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]. Constitutive promoter constructs

[0342] 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

(gtatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattacta tte driving expression of scoabil. The scoabil gene with tNos was sub-cloned into the plasmid eFMVe35 S-Zm£/oi7- scoGUS using the restriction enzyme sites Notl and Asel, replacing the scoGUS gene and tNos with scoabil and tNos, and generating the plasmid eFMVe35S-Zwi/b i -scoabil (Fig. 8) used for transformation of sugar cane.

[0343] 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. ale sene switch promoter constructs

[0344] 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.

[0345] 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. [0346] 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.

EXAMPLE 12 PRODUCTION OF TRANSGENIC N. BENTHAMIANA

[0347] 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.

benthamiana 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 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 μg/mL) and Timentin (200 μg/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 μg/mL, respectively. When well defined stems were visible, the emerging shoots were excised and transferred to 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.

EXAMPLE 13

PRODUCTION OF TRANSGENIC SUGAR CANE

[0348] Transgenic sugar cane plants were regenerated from sugar cane callus that was transformed by microprojectile bombardment (MPB) as described by Finer et al. ( 1992,

Plant Cell Reports 11 :323-328) and Bower et al. (1996, Molecular Breeding 2:239-249). To generate the callus, sugar cane (cultivar KQ228) "tops" were obtained from The Bureau of

Sugar cane experimental stations (BSES) LTD Meringa Queensland. Calli were initiated as described by Franks and Birch (1991, Australian Journal of Plant Physiology 18:471-480) using MSC ₃ media consisting of 4.43 g/L MS basal salts with vitamins (Phytotechnology laboratories Shawnee Mission, KS, USA ), 500 mg/L Casein Hydrolysate (Merck), 13.6 μΜ

2, 4-Dichlorophenoxyacetic acid (2, 4-D; Phytotechnology laboratories), 100 ml L young coconut juice ("Cock" brand, Thailand), 3 % (w/v) sucrose and 8 g/L agar (Research organics). Calli were maintained for seven weeks in the dark at 26 °C and subcultured every 14 days.

[0349] 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. For MPB, a 2 μΙ_, aliquot of a 1 : 1 mixture of pUKN (1 μg/μL) and the experimental construct DNA (1 μg/μL) was added to approximately 3 mg of 1 μπι gold particles (Bio-Rad). The solution was mixed briefly and 25 μί of 2.5 M CaC^and 5 μΐΐ, 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 μί of supernatant was removed. The remaining DNA-coated gold solution was mixed and 5 μΐ, was used per bombardment.

[0350] 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 μιη 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. After four hours the callus was transferred to MSC ₃ medium for 4-6 days before being transferred to selection media consisting of MSC3 and 50 mg/L G418 (Geneticin) (Roche). Non-transformed callus used for the regeneration of wild- type plants was transferred to MSC ₃ without selection.

[0351] Following MPB, the callus remained on selection media for four weeks in the dark with fortnightly subculturing after which it was transferred to regeneration medium with selection, consisting of MSC ₃ with the 2,4-D replaced by 4.4 μΜ 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. After 10 weeks of regeneration with BAP, the plants were transferred to rooting medium with selection (the same as regeneration medium, however BAP is replaced with 10.7 μΜ α-Naphthalene Acetic Acid (NAA; Sigma)). The plants were grown until roots of approximately 1 cm in length had developed, after which the plants were transferred to soil for acclimatization in a growth cabinet under the above mentioned lighting and temperature conditions.

EXAMPLE 14

CHARACTERIZATION OF TRANSGENIC N. BENTHAMIANA

[0352] Plants are verified to contain the transgene constructs using PCR to screen for the presence of either the ABI1 or abil transgene. Confirmed transgenic plants are subsequently screened using reverse transcriptase PCR (RT-PCR) to identify plants with detectable levels of ABIllabil transgene expression. RT-PCR is carried out using cDNA generated from leaf RNA. For transgenic plants possessing the ale gene switch constructs, leaf 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 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 (along with the relevant control plants) are characterized for any phenotypic effects that are known to be associated with reduced stomatal closure, in particular a wilty phenotype.

[0353] To further assess whether expression of abil is reducing stomatal closure in N. benthamiana, stomatal conductance is measured using the LI-6400XT portable

photosynthesis system (LI-COR biosciences). Stomatal conductance is measured in plants grown under well-watered conditions, in drought-stressed plants, and in plants treated with the hormone abscisic acid. Transgenic plants are also characterized for relative water content and for the levels of sugar accumulation using High Performance Liquid Chromatography (HPLC). Transgenic plants with constitutive expression of abil (and the relevant control plants) 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.

[0354] Transgenic N. benthamiana plants displaying ethanol inducible expression of abil were identified (Fig. 10). The expression of abil in these plants _x was readily detected following ethanol treatment, while little or no expression was detected prior to ethanol treatment (Fig. 10). Constitutive expression of abil is known to be detrimental to N.

benthamiana growth and development, with plants displaying stunted growth and smaller leaves, as well as a strong propensity to wilt when removed from tissue culture and placed in soil (Armstrong et al. , Proc. Natl. Acad. ScL USA. 92:9520-9524 (1995)). Plants containing the ethanol inducible abil construct did not display these aberrant phenotypes and looked similar to the wild type (Fig. 11, 0 timepoint).

[0355] Ethanol induced expression of abil resulted in visible wilting (Fig. 11) and significant water loss (Fig. 12) in the leaves following ethanol treatment. The ethanol treatment used had no detectable effect on control plants (Figs. 11 and 12). These data demonstrate that the regulation of abil expression with the ale gene switch can be used to control water loss in plants. The data also show that the ale gene switch can be used to generate healthy transgenic plants possessing genes that are detrimental to plant growth and development when under the control of a constitutive expression system. EXAMPLE 15

CHARACTERIZATION OF TRANSGENIC SUGAR CANE

[0356] Plants are verified to contain the transgene constructs using PCR to screen for the presence of either the scoABll or scoabil transgene. Confirmed transgenic plants are subsequently screened using reverse transcriptase PCR (RT-PCR) to identify plants with detectable levels of scoABll I scoabil transgene expression. RT-PCR is carried out using cDNA generated from leaf RNA. Leaf samples are taken from the first fully unfurled leaf of each transgenic plant and frozen until use. For transgenic plants possessing the ale gene switch constructs, leaf samples are taken just prior to ethanol induction and at 4-48 hours post ethanol treatment. Ethanol treatment is carried out using a single 4-5% ethanol root drench and aerial spray. RNA is extracted from sugar cane leaf samples using Tri Reagent (Sigma) according to the manufacturer's instructions.

[0357] 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. To further assess whether expression of scoabil is reducing stomatal closure in sugar cane, stomatal conductance is measured using the LI-6400XT portable photosynthesis system (LI-COR biosciences).

Stomatal conductance is measured in plants grown under well-watered conditions, in drought- stressed plants, and in plants treated with the hormone abscisic acid. 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. Sugar accumulation and water content in the stem of the ethanol inducible scoabil transgenic sugar cane (along with the relevant control plants) is assessed in approximately 6-9 month old plants at approximately 12-24 hours or 1-5 weeks after the start of the ethanol treatment. Ethanol treatment is carried out using either a single treatment or multiple treatments of a 4- 5% root drench and aerial spray. Characterization of the transgenic plants is carried out under well watered conditions and under varying levels of drought stress.

[0358] 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.

[0359] Transgenic sugar cane plantlets displaying ethanol inducible expression (2% ethanol root drench and aerial spray) of scoabil were also identified, demonstrating that the ale gene switch can be used for inducible expression of abil in sugar cane (Fig. 17).

EXAMPLE 16

PRODUCTION OF TRANSGENE CONSTRUCTS FOR N. TABACCUM {TOBACCO)

[0360] A sugar cane-optimized Vicia faba AAPt? ⁴³⁴ (scoAAPK ^{K 3A} ) 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 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

5 -AAGCTTATGGTTGCAACAGAGAGGATG-3' [SEQ ID NO:58] and the reverse primer 5'-CCATGGTTCTTGAGTAGTGATTTTGAAGTAG-3' [SEQ ID NO:59], introducing a HmdIII restriction enzyme site at the 5' end and an Ncol restriction enzyme site at the 3 ^* end. Guard cell-preferred promoter constructs

[0361] 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 . Subsequently, 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 . Next, 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).

[0362] 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. Next, the pGCl- scoabil sequence with nopaline synthase terminator were subcloned into the binary vector pBINplus using the restriction enzyme sites HmdIII and Ascl to generate the pGCl- scoabil vector used for transformation into tobacco (Fig. 18). ale eene switch promoter constructs

[0363] The ethanol inducible palcA I-abil binary construct (Fig. 7) previously described for N. benthamiana was also used for transformation into N. tabaccum.

EXAMPLE 17

PRODUCTION OF TRANSGENIC N. TABACCUM (TOBACCO)

[0364] 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

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 μg/mL) and Timentin (200 μg/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 μg/mL, respectively. When well defined stems were visible, the emerging shoots were excised and transferred to 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.

EXAMPLE 18

CHARACTERIZATION OF TRANSGENIC TABACCUM(TQBACCQ)

[0365] Plants are verified to contain the transgene constructs using PCR to screen for the presence of the scoAAPK ^K43A , scoabil or abil transgene. Confirmed transgenic plants are subsequently screened using reverse transcriptase PCR (RT-PCR) to identify plants with detectable levels of scoAAPK ^K43A I scoabil I abil transgene expression. RT-PCR is carried out using cDNA generated from leaf RNA. For transgenic plants possessing the ale gene switch constructs, leaf 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 (along with the relevant control plants) are characterized for any phenotypic effects that are known to be associated with reduced stomatal closure, in particular a wilty phenotype.

[0366] 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).

[0367] 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).

[0368] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0369] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0370] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.