WO2010081005A1 - Method for controlling flower development in plants - Google Patents

Abstract

The present invention provides a method of controlling the sexuality of a plant comprising treating the plant with a composition comprising a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof.

Description

Methods for Controlling Flower Development in Plants

BACKGROUND OF Ti ll: INVHNTlON

Most flowering plants produce perfect flowers containing both the male organs (stamens) and female organs (pistils). In maize, which has physically separated male and female inflorescences, floral meristems become unisexual through sex determination (Dellaporta & Calderon-Urrca, 1994, Science 266: 1501 ; Irish,

1996, Bioessays 18:363). The basic unit of the maize inflorescence, called a spikclet, contains one upper and one lower flower (known as florets in grasses). Hach floret initiates a series of floral organs including three stamen primordia and a central pistil primordium (Bonnet, 1940, J. Agric. Res. 60:25; Kiesselbach, "The Structure and Reproduction of Corn," Univ. of Nebraska Press, Lincoln, NH, 1949). These initially bisexual florets become exclusively staminate in the tassel (by abortion of pistil primordia) and exclusively pistillate in the car (by arrest of developing stamens) (Bonnet, 1940, J. Agric. Res. 60:25; Cheng el al., 1983, Am. Bot. 70:450). Each ear spikelct produces a solitary functional pistil in the upper floret due to abortion of the pistil in the lower floret (Bonnet. 1940, J. Agric. Res. 60:25; Kiesselbach. "The Structure and Reproduction of Corn," Univ. of Nebraska Press. Lincoln. NH, 1949; Cheng et al., 1983, Am. Bot. 70:450).

Mutations altering the sexual fate of florets in maize indicate that sex determination is under genetic control. The non-homeotic iasselseed (is) mutations is I and is2 result in the conversion of the tassel inflorescence from staminale to pistillate (Emerson, 1920, J. Hered. 1 1 :65; Nickerson & Dale, 1955, Ann. Mo. Bot. Gard. 42: 195). Both IsI and is2 are required to eliminate pistil primordia through cell death (Calderon-Urrea & Dellaporta, 1999, Development 126:435; Kim ct al.. 2007, Genetics 177:2547). The Is2 gene encodes a short-chain dehydrogenase/reductase (DcLong et al., 1993, Cell 74:757) with broad activity, which has complicated the discovery of its natural substrate (Wu et al.. 2007, FHBS J. 274:1 172). It is unknown how is genes mediate pistil cell death, although it has been suggested that the dehydrogenase/reductase activity of is2 may produce a pro- apoptotic signal or metabolize a substrate required for cell viability (Calderon-Urrca & Dellaporta, 1999, Development 126:435; Wu et al., 2007. Fl-BS J. 274: 1 172). Even less is known about the tsl gene. TS2 transcripts arc low or undetectable in is I mutant tassels, which suggests that tsl may act upstream of ts2 by regulating ts2 RNΛ levels and possibly other sex determination genes (Calderon-Urrca & Dellaporta, 1999, Development 126:435).

Chemicals have been used to modulate and modify sexual differentiation in plants. The plant hormone ethylene has been observed to promote feminization in cucumber (Yamasaki et al., 2005, Vitam. Norm. 72:79). Recent genetic and biochemical evidence has confirmed the role of ethylene in sex determination of melon, a related species (Boualem et al., 2008. Science 321 :836). Conversely, gibberellin has masculinizing effects in cucumber but promotes feminization in maize (Bensen et al., 1995, Plant Cell 7:75), and auxin also has opposing effects in cucumber and Mercurial is annua (Yamasaki et al., 2005, Vitam. Horm. 72:79). However, little is known at this point about the role that chemicals play in sexual development and maturation of plants.

There is thus a great interest in identifying chemical compounds that modulate sexual differentiation in plants. Such compounds would be useful in suppressing or enhancing specific sexual phenotypes in plants, allowing the control of vast groups of plants without the need for time-consuming mechanical manipulation of the plants. The present invention fulfills these needs.

SUMMARY OF Tl IE INVENTION

The invention includes an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof. In one aspect, the derivative is a jasmonic acid ester. In another aspect, the derivative is jasmonic acid methyl ester or methyl jasmonate.

The invention also includes a method of modulating sexuality in a plant. The method comprises the step of administering to the plant an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof.

The invention also includes a method of suppressing complete feminization or restoring male sexuality in a plant with a tsl or ts2 mutation. The method comprises the step of administering to the plant an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof.

The invention also includes a method of creating homozygous stock in a plant with a IsI or ts2 mutation. The method includes the step of administering to the plant an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof, wherein the progeny of the plant is homozygous for the mutation and is male-sterile.

In one aspect, the derivative is jasmonic acid methyl ester. In one aspect, the plant is grass-related. In another aspect, the plant is maize or rice. In yet another aspect, the plant is maize.

In one aspect, the method of the invention further comprises the step of administering to the plant at least one additional compound useful for controlling plant sexuality. In another aspect, the at least one additional compound is selected from the group consisting of ethylene, gibberelin and auxin.

BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumental ities of the embodiments depicted in the drawings.

Figure 1 is a series of images illustrating the genetic and physical map of the SsI locus in maize chromosome 2.

Figure 2, comprising Figures 2A-2C, is a series of images illustrating the structure of the tsl gene and the tsl mutant alleles. In Figure 2Λ, hollow boxes at left and right are 5' and 3' untranslated regions (UTRs), respectively; darkened boxes are exons and angled lines are introns. Mutations in eight IsI mutant alleles are positioned above the corresponding exons. Insertions are represented by inverted triangles and a single deletion by a triangle. In Figure 2B, TSl protein features include a predicted chloroplast transit peptide (cTP, on the left), the PLA 17LH2 beta- barrel (in the middle), and the lipoxygenase domain (on the right) as well as five conserved residues (H501, H583, H770, N774, 1918) necessary for iron binding and the phenylalanine (F636) residue predicting 13-LOX regiospecificity. In Figure 2C, Bayesian and maximum parsimony consensus tree of predicted type 2 13- lipoxygenases in angiosperms is displayed. The arrowhead indicates the position of the maize Λv/-encoded lipoxygenase. Posterior probabilities from Baycsian inference and bootstrap support from maximum parsimony analysis less than 100% are displayed below internal nodes to the IcH and right of a slash, respectively. This subclade is part of a more extensive phylogenetic analysis shown in Figure 5.

Figure 3 is an image of the Southern blot hybridization of a is I probe to Kpnl-digested genomic DNA from inbred lines W22 and B73 and is I mutant lines tsl-Mu92 and tsl-MuOI along with plasmid DNA from BAC bO4781 O4. Both is I and islb arc detected in the inbred lines and the mutant ts!-Mu92, while only islb is present in the is! full-deletion mutant tsl-MuOl.

Figure 4 is a representation of the alignment of the TSl and TS I b proteins to potato lipooxygenase H3 (StLOXS) and soybean L-I (GmLOXl ). StL0X5 is the closest TS l relative that has been biochemically characterized, while GmLOXl is one of the best studied plant lipoxygenases. Sequences were aligned with Clustal W2 and the gray similarity shading style in the background was applied with Jalview with the BLOSUM62 color scheme. The is! CDS was predicted to encode a protein of 918 amino acids with a theoretical mass of 103 kDa. Predicted domains are shown surrounded by colored boxes: the chloroplast transit peptide (c TP. row 1), the PLΛT/LH2 domain (rows 1-3) and the lipoxygenase domain (rows 3-10). The predicted cTP has different lengths in TS 1 , TS 1 b and StL0X5, while GmLOX 1 , a type 1 lipoxygenase, docs not possess a cTP. Asterisks indicate the five conserved residues necessary for iron binding. The black arrowhead marks the phenylalanine residue predicting 13-LOX regiospccificity.

Figure 5 is a representation of the phylogenetic analysis of TS l and selected plant lipoxygenases by Bayesian and Maximum Parsimony inference. Nearly identical tree topologies were generated by Bayesian and Maximum Parsimony (MP). When values are lower than 100%, posterior probabilities from Bayesian inference and bootstrap support from MP analysis were displayed below internal nodes to the left and right of a slash sign, respectively. The fourth box from the top surrounds the type 2 13- LOX cladc, which includes Λv/ -encoded LOX (arrow). The clade groups lipoxygenases from both monocotyledons and dicotyledons. Several lipoxygenases from this clade have been experimentally shown to display 13-LOX regiospccificity (second box from the top) and/or to localize to chloroplasts (fourth box from the top). The other clades in the tree contain type 1 lipoxygenases from: monocotyledons (third box from the top) with 9-LOX, 13-LOX or mixed regiospecificity; dicotyledons with 13-LOX regiospccificity (second box from the top); and dicotyledons with 9-LOX regiospecificity (first box from the top).

Figure 6, comprising Figures 6A-6I, is a series of images relating to expression of tsl, tslb and ts2 in maize. Figure 6A illustrates the expression profile of tsl, is Ib, and ts2 in different maize tissues by quantitative RT-PCR on three biological replicates for each tissue. Results were plotted as the ratio to the lowest detected level {tslb in root) ± SE. The y axis is in logarithmic scale. Figures 6B to 6E illustrate RNA in situ hybridization targeting the 3'UTR of tsl (dark purple) in developing inflorescences. Scale bars, 200 mm. Figures 6B and 6C illustrate wild- type hcterozygote male inflorescences (tassels) of 1.6 and 1.5 cm, respectively. Figure 6D illustrates wild-type female inflorescence (car) of 1.5 cm. Figure 6E illustrates homozygous ts J-MuOl deletion mutant tassel showing no hybridization signal. Figures 6F to 61 illustrate co-localization of TS 1 :mChcrry and bcSnt:GFP fusion proteins in plastids of transfected onion epidermal cells. Scale bars, 50 mm. Figure 6F illustrates TSl :mCherry red fluorescence (shown as gray color). Figure 6G illustrates RbcSnt:GFP green fluorescence (shown as gray color). Figure 6H illustrates the merge of Tsl :mCherry and RbcSnt:GFP plus two additional channels: 4',6'-diamidino-2-phcnylindole (blue fluorescence, shown as large grey spots) for distinguishing nuclei, and differential interference contrast (DIC) for displaying cellular morphology. Figure 61 illustrates the scattcrplol of pixel gray value frequencies for RbcSnt:GFP (x axis) and Tsl :mCherry (y axis) channels. Frequencies were displayed using a rainbow lookup table (bottom, units between 0 and 255). Region 3 (upper right) contains pixels with signal above background in both channels, and a linear correlation in this region is a qualitative indicator of co-localization.

Figure 7, comprising Figures 7Λ-7E, illustrate the determination of linoleic acid oxidation products in maize. Figure 7A illustrates the partial gas chromatography-MS chromatograms displaying linoleic acid oxidation products generated by crude extracts of wild-type W22 tassels (light line) but not tsl-rcf tassels (dark line). HPLC analysis of oxidation products (inset) indicated that the lipid hydroperoxide (HOD) peak was a mixture of 9-hydroxy-10,12-octadecadicnoic acid (9-HOD) and 13-hydroxy-9, 1 1-octadecadienoic acid ( 13-HOD). Figure 7B is a series of box plots summarizing the distribution of jasmonic acid in three tassel sets. Circles represent individual measurements. Diamonds show the 95% confidence interval of the mean (horizontal blue line). +/+ corresponds to inbred line W22. Figure 6C illustrates the blank-treated mutant tsl tassel. Figure 6D illustrates JΛ-treated tsl tassel. Figure 6E illustrates JA-treated ts2 tassel.

Figure 8 illustrates the biosynthesis of jasmonic acid through the octadecanoid pathway.

Figure 9, comprising Figures 9A-9D, illustrates additional phenotypes of JA-treated tsl and ts2 mutant tassels. Figure 9A illustrates tsl bisexual spikelets contained both anthers (dark arrows) and pistils (light arrows) while glumes display numerous trichomes and anthocyanins ring at the base. Anthers emerging from IsI (Figure 9B) and Is2 (Figure 9C) rescued spikelets. Figure 91) illustrates blank-treated tsl spikelets with short, glabrous glumes without anthocyanin ring at base.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the discovery that certain compounds are useful for controlling the sexuality of plants. In one aspect, these compounds act by restoring plant masculinization. In another aspect, these compounds suppress floral sexuality mutations and restore plant masculinization.

In one aspect, the invention provides an agriculturally compatible composition comprising a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof.

In another aspect, the invention provides a method of modulating sexuality in a plant. The method comprises the step of administering to the plant an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof.

In yet another aspect, the invention provides a method of suppressing complete feminization or restoring male sexuality in a plant with a tsl or ts2 mutation. The method comprises the step of administering to the plant an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof

In yet another aspect, the invention provides a method of creating homozygous stock in a plant with a tsl or ts2 mutation. The method includes the step of administering to the plant an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, ajasmonic acid derivative, and a salt thereof, wherein the progeny of the plant is homozygous for the mutation and is male-sterile.

In one embodiment, the derivative is ajasmonic acid ester. In another embodiment, the derivative is methyl jasmonate or jasmonic acid methyl ester. In one embodiment, the plant is grass-related. In another embodiment, the plant is maize or rice. In yet another embodiment, the plant is maize.

In one embodiment, the method of the invention further comprises the step of administering to the plant at least one additional compound useful for controlling plant sexuality. In another aspect, the at least one additional compound is selected from the group consisting of ethylene, gibberelin and auxin.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well known and commonly employed in the art. As used herein, the articles "a" and "an" 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.

As used herein, the term "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, the teπn "jasmonic acid derivative" refers to a derivative of jasmonic acid, such as but not limited to. ajasmonic acid ester. The class of jasmonic acid esters includes, but is not limited to. ajasmonic acid alkyl ester. jasmonic acid aryl ester, jasmonic acid hcteroaryl ester, jasmonic acid arylakyl ester. and jasmonic acid hetcroaryl ester. The term "jasmonic acid derivative" also refers to chemical compounds that give rise to jasmonic acid or other jasmonic acid derivative by chemical or microorganism-based decomposition, regardless whether the decomposition takes place under controlled conditions or not.

As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides may be synthesized, for example, using an automated polypeptide synthesizer. As used herein, the term "protein" typically refers to large polypeptides. As used herein, the term "peptide" typically refers to short polypeptides. Conventional notation is used herein to represent polypeptide sequences: the left- hand end of a polypeptide sequence is the amino-terminus, and the right-hand end of a polypeptide sequence is the carboxyl-terminus.

As used herein, the polypeptides include natural peptides, recombinant peptides, synthetic peptides or a combination thereof. A peptide that is not cyclic will have an N-terminus and a C-terminus. The N-terminus will have an amino group, which may be free (i.e., as a NHi group) or appropriately protected (for example, with a BOC or a Fmoc group). The C-terminus will have a carboxylic group, which may be free (i.e., as a COOM group) or appropriately protected (for example, as a benzyl or a methyl ester). A cyclic peptide does not necessarily have free N- or C-termini, since they are covalently bonded through an amide bond to form the cyclic structure. The term "peptide bond" means a covalcnt amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid.

As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated below:

Full Name Three- Letter Code One-Letter Code

Aspartic Acid Asp D

Glutamic Acid GIu E

Lysine Lys K

Arginine Arg R

Histidine Mis M

Tyrosine Tyr Y

Cysteine Cys C

Asparagine Λsn N

Glutaminc GIn Q

Serine Ser S

Threonine Thr T Glycine GIj Ci

Alanine Λla Λ

Valine VaI V

Leucine Leu L

Isolcucine lie I

Methionine Met M

Proline Pro P

Phenylalanine Phe I-

Tryptophan Trp

Λ "polynucleotide" means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.

The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, which arc generally not greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNΛ sequence (i.e., Λ, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5¹- end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction.

The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNΛ strand having the same sequence as an mRNA is referred to as the "coding strand;" sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as "upstream sequences;" sequences on the DNA strand which arc 3' to a reference point on the DNA are referred to as "downstream sequences."

A "portion" of a polynucleotide means at least about twenty sequential nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide may include every nucleotide residue of the polynucleotide.

"Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers arc useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

"Probe" refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. Λ probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or cukaryote, or which exists as a separate molecule (e.g, as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNΛ) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNΛ corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

As used herein, the term "plant" refers to a living organism belonging to the kingdom Planiae. Examples of plants are grasses, such as switchgrass, rice, oats, wheat, barley, sorghum, millet, rye, triticalc, buckwheat, fonio. quinoa. IcH^', wild rice, amaranth, kaniwa, spelt, cinkorn. cmmer, durum, and maize (corn). Preferably, the plant is rice or maize. Most preferably, the plant is maize.

As used herein, the term "effective amount" refers to a non-toxic but sufficient amount of an agent to provide the desired biological result. That result can be modulation of sexual differentiation in plants, suppression or enhancement of specific sexual phenotypes, or any other desired alteration of a plant phcnotype. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. As used herein, the term "'agriculturally acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to a plant without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term "agriculturally acceptable composition" refers to a mixture of at least one compound useful within the invention with agriculturally acceptable chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or cxcipients. The agriculturally acceptable composition facilitates administration of the compound to a plant. Multiple techniques of administering an agriculturally acceptable composition exist in the art including, but not limited to: watering, spraying, fumigation, acrolizalion, injecting, and dusting. As used herein, the language "acceptable salt" refers to a salt of the administered compounds prepared from agriculturally acceptable non-toxic acids including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof. The compounds useful within the invention may form salts with acids or bases, and such salts are included in the present invention. The term "salts" embraces addition salts of free acids or free bases of the compounds useful within the invention. Preferred salts are formed from cationic and anionic countcrions that have been approved or validated for agricultural applications. Unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, purification or formulation of compounds useful within this invention.

As used herein, the "instructional material" includes a publication, a recording, a diagram, or any other medium of expression that may be used to communicate the usefulness of the compounds described herein. In some instances, the instructional material may be part of a kit useful for effecting the alleviating or treating the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of controlling plant sexuality. The instructional material of the kit may, for example, be affixed to a container that contains the compounds useful within the invention or be shipped together with a container that contains the compounds. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. For example, the instructional material is for use of a kit; instructions for use of the compound; or instructions for use of a formulation of the compound.

Compounds of The Invention

The compounds useful within the invention may be synthesized using techniques well-known in the art of organic synthesis or may be isolated from natural sources. In one aspect, the compound is jasmonic acid ((lR,2R)-3-oxo-2-(2Z)- 2-pentenylcyclopentaneacctic acid). In another aspect, the compound is a jasmonic acid ester. In yet another aspect, the compound is a jasmonic acid alkyl ester, jasmonic acid aryl ester, jasmonic acid heteroaryl ester, jasmonic acid arylakyl ester, and jasmonic acid heteroaryl ester. Non-limiting examples of jasmonic acid esters are methyl jasmonate (or jasmonic acid methyl ester), ethyl jasmonate, n-propyl jasmonate, isopropyl jasmonate, n-butyl jasmonate, sec-butyl jasmonate. t-butyl jasmonate, mcthoxyethyl jasmonate, pentyl jasmonate, phenyl jasmonate, 4-chloro jasmonate, 4-tluoro jasmonate, naphtyl jasmonate, benzyl jasmonate, pyridinyl jasmonate, phenylethyl jasmonate and so on. In yet another aspect, the compound is methyl jasmonate (methyl (1 R,2R)-3-oxo-2-(2Z)-2-pentenylcyclopcntancacetate).

jasmonic acid

jasmonic acid ester (R= alkyl, aryl, heteroaryl, arylalkyl)

methyl jasmonate

Acceptable base addition salts of compounds useful within the present invention include, for example, metallic salts and non-metallic salts. Metallic cationic counterions include alkali metal, alkaline earth metal and transition metal ions such as, for example, aluminum, bismuth, calcium, lithium, magnesium, neodymium, potassium, rubidium, sodium, strontium and zinc. Non-metallic cationic counterions include organic basic amines such as, for example, ammonium, benethamine [N- benzylphenelhylamine], benzathine [N,N'-dibenzylethylenediamine], betaine [(carboxymethyl)trimethylammonium hydroxide], carnitine, clemizole [ 1-p- chlorobcnzyl-2-pyrrolidin-l '-ylmethylbenzimidazole], chlorcyclizine [ l -(4- chlorobenzhydryl)-4-methylpiperazine], choline, dibenylamine, diethanolamine, diethylaminc, dicthylammonium, diolamine, cgluminc [N-ethylglucamine], erbuminc [t-butylamine], ethylenediamine, heptaminol [6-amino-2-mcthylhcptan-2-ol ], hydrabamine [N,N'-di(dihydroabictyl)ethylenediamineJ, hydroxyethylpyrrolidone, imadazole, meglumine [N-methylglucamine], olamine, piperazine, 4- phenylcyclohexylamine, procaine, pyridoxine, triethanolamine, and tromethamine [tris(hydroxymethyl)aminomethane]. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, and phosphoric. Appropriate organic acids may be selected, for example, from aliphatic, aromatic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, camphorsulfonic, citric, fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric. para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic (besylate), stearic, sulfanilic, alginic, galacturonic, and the like.

The compounds useful within the present invention may also be useful in combination with at least one additional compound useful for controlling plant sexuality. These additional compounds may comprise compounds described in the present invention or compounds, e.g., commercially available compounds, known to modify, modulate or alter plant sexuality.

In non-limiting examples, the compounds of the invention may be used in combination with at least one of the following compounds: ethylene, gibberelin and auxin.

A synergistic effect may be calculated, for example, using suitable methods such as. for example, the Sigmoid-E_nm equation (1 Iolford & Schcincr.

19981, Clin. Pharmacokinet. 6: 429-453), the equation of Locwc additivity (Locwc &

Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 1 14: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22: 27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the compound combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

Administration/Dosage/Formulations Routes of administration of any of the compositions of the invention include, but are not limited to, watering, spraying, fumigation, aerolization, injecting, or dusting. Administration may involve, in non-limiting examples, direct surface application to an intact or cut portion of the plant, microinjection into a tissue or cell thereof, or micro-bombardment, preferably under low pressure. The regimen of administration may affect what constitutes an effective amount. The formulations of the invention may be administered to the plant at any stage of its development. Preferably, the formulations of the invention may be administered to the plant at the time of inflorescence development, during which the sex determination process is taking place. In the case of most lines of maize, this time corresponds to the period when the developing inflorescence reaches approximately 1.0 cm in height. At this stage of floral development, most maize plants possess 6-8 fully expanded leaves. This precise inflorescence height and expanded leaf number vary according to variety of maize and environmental growth conditions. Further, several divided dosages, as well as staggered dosages, may be administered daily or sequentially, or the dose may be continuously administered. Further, the dosages of the formulations may be proportionally increased or decreased as indicated by the exigencies of the situation.

Administration of the compositions of the present invention to a plant, preferably a grass-related plant, more preferably maize, may be carried out using known procedures, at dosages and for periods of time effective to modulate the plant sexuality. An effective amount of the compound necessary to achieve the desired sexuality modulation may vary according to factors such as the nature of the plant, its age, status and location; and the ability of the compound to modulate plant sexuality. Dosage regimens may be adjusted to provide the optimum response. For example. several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the situation. A non-limiting example of an effective dose range for a compound useful within the invention is from about 0.001 to 1 ,000 mg/kg of plant weight/per day. In a non-limiting example, an individual plant is treated with 1 mL of a 1 mM solution of a jasmonic acid derivative, and the treatment is performed three times at 48-hour intervals. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the compound without undue experimentation.

Actual dosage levels of the active ingredients in the compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired response for a particular plant, without being toxic to the plant.

A plant specialist, e.g., botanist or agricultural technician, having ordinary skill in the art may readily determine and prescribe the effective amount of the composition required. For example, the plant specialist could start doses of the compounds useful within the invention at levels lower than that required in order to achieve the desired effect and gradually increase the dosage until the desired effect is achieved.

In one embodiment, the compositions of the invention are formulated using one or more agriculturally acceptable excipients or carriers. In one embodiment, the compositions of the invention comprise an effective amount of a compound of the invention and an agriculturally acceptable carrier.

In one embodiment, the compositions of the invention are administered to the plant in dosages that range from one to live times at two-day intervals. In another embodiment, the compositions of the invention are administered to the plant in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. In another embodiment, the composition of the invention is applied once to the plant as a slow- release preparation. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from plant to plant, depending on many factors including, but not limited to. age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any plant will be determined by the plant specialist based on the evaluation of the plant in question. In one embodiment, the present invention is directed to a packaged agriculturally acceptable composition comprising a container holding an effective amount of a compound of the invention, alone or in combination with a second agricultural agent; and instructions for using the compound to modulate plant sexuality.

The term "container" includes any receptacle for holding the agriculturally acceptable composition. For example, in one embodiment, the container is the packaging that contains the agricultural composition. In other embodiments, the container is not the packaging that contains the agricultural composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged agricultural composition or unpackaged agricultural composition and the instructions for use of the agricultural composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the agricultural composition may be contained on the packaging containing the agricultural composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., modulating plant sexuality.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction limes, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, arc within the scope of the present application.

It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, arc also contemplated by the present application.

The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein. EXAMPLKS

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

Materials and Methods

Genetic stocks and mutants Wild-type maize inbred lines W22, B73 and Mo 17 were used. The

was previously described (Emerson, 1920, J. Hered. 1 1 :65; Emerson et al., 1935, Cornell Univ. Agric. Exp. Stn. Memoir 180). tsl-69-Alex-Mol 7 was identified as a spontaneous mutant in Mo 17; and tsl -0174 was discovered in an unknown genetic background. These three alleles were obtained from the Maize Genetics Cooperation Stock Center, Maize COOP (University of Illinois,

Urbana/Champaign, IL). The isl -MuSII and ts I -SI 104 alleles were obtained from Plant Gene Expression Center, United States Department of Agriculture - Agricultural Research Service and the University of California, Albany, California). The tsl-Mu92 and tsI-Mu93 alleles were recovered in progeny of self-pollinated maize plants known to contain active Mutator (Mu) elements (Monsanto, Crcve Coeur, MO).

The remaining alleles, tsl-Mu01 and tsI-MuO2, were isolated by gene tagging over the course of several generations of testcrosses

to Mutator lines. In brief, /.v/-re/mutant plants were crossed to a W22 line carrying actively transposing Mu elements. Since tsl-ref is a recessive mutation, only wild- type plants are expected from such a cross unless a new tsl mutant allele is recovered in any gametes of the /V/w-active W22 line. About 40,000 individuals of this cross were grown during the summer seasons of 2001 and 2002. Plants showing a tasselseed phcnotype, potentially containing new tsl mutant alleles, were outcrossed to the W22 inbred line for at least two generations in order to segregate the new alleles and to reduce the number of active Mu elements in the genome. Additionally, these Fl plants were crossed to tsl-ref muXanls to confirm allelism. The /.v/-linked hlml marker was used to distinguish the new isl mutant alleles from the original /Λ7- rcf allele in the first outcrossed generation. Homozygotes for the new allele were usually obtained in the third generation by self-pollinating heterozygous F2 plants.

lsl mapping populations Since the lsl mutant plants arc completely feminized, the is J -rcf stock was maintained by sib mating is 1 -refits 1-ref wiih is 1 -ref/Ts I . These sib matings were used to generate the original mapping population (n = 93), which was selected for plants phenotypically scored as tsl/lsl mutants. Any lsl -refits 1-refi p\ar\\ carrying one or more of the linked molecular markers in a heterozygote state were deemed to be recombinant between the heterozygous marker and the tsl locus. Once IsI tightly- linked flanking markers were identified, they were subsequently used to screen three additional testcross populations containing both heterozygote wild-type plants and plants homozygous for the mutant tsl- re/allelc. Two of these populations (n = 1 19) also come from the sib mating tsl-ref/tsl-refx tsl-ref/Tsl , while another mapping population (n = 208) carried a wild-type Tsl allele from the inbred W22 {tsl-ref/tsl- refx tsl -ref/Ts 1-W22).

Molecular marker development

Candidate marker sequences were selected through genetic, physical and rice-maize synteny mapping. These sequences usually corresponded to (a) maize BAC ends or ESTs deposited in GenBank (Nil I; Benson ct al., 2008, Nucleic Acids Research 36(Database issuc):D25-30) ; or (b) rice genes annotated in the regions syntenic to maize intervals at Gramcne (Cold Spring Harbor Laboratory, www.gramene.org). Candidate sequences were analyzed by BLAST through the TIGR Maize Database. Maize sequences corresponding to repetitive DNA were not further considered for marker design. Non-repetitive DNA sequences were selected for marker development and in some cases these sequences were extended by alignment with AZM sequences (TIGR).

Candidate marker sequences were PCR-amplified from genomic DNA of W22, tsl-ref/Tsl and lsl -refits 1 -re/ plants. In a few instances, the PCR products had size polymorphisms easily resolved by agarose gel electrophoresis. In most cases, however, the PCR products had the same apparent size. In these cases, the PCR products were sequenced and examined for the presence of SNPs. If present, SNPs that represented differences in restriction enzyme recognition sites were used as CAPS (Cleaved Amplified Polymorphic Sequences) markers (Konieczny & Λusubel, 1993, Plant J. 4:403) to distinguish between the different alleles in the mapping population.

New markers were initially evaluated in 20 non-recombinant individuals of known phcnotype to confirm co-segregation of the marker with the appropriate is] allele. This was necessary to prevent the use of markers corresponding to a duplicate region of the isl interval in chromosome 10 or to repeal sequences that have "escaped" filtering in the repeal database. Table 1 displays lhe features of the molecular markers defining lhe is J genetic region.

fable 1. Molecular markers used for mapping the is I locus

* CAPS, Cleaved Amplified Polymorphic Sequence; restriclion enzymes used for CAPS marker analysis arc indicated in parenthesis. SSR, Simple Sequence Repeat (Microsatcllitc) t Size corresponds to the allele found in inbred line B73

% Previously reported SSR

Evaluation of PCR-based molecular markers

PCR primers were designed with Primcr3 (Rozcn & Skalctsky, 2000, Methods MoI. Biol. 132:365) and synthesized by the W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University, New Haven, CT). PCR-based markers were routinely amplified with Taq DNA polymerase (Qiagcn, Valencia, CA, USA) in 20 μl reactions containing IX PCR buffer and I X Q-Solution supplied by the manufacturer, 200 μM of each dNTP, 500 nM of each primer and 2.5 ng/μl genomic template DNA. Q-Solution contains the chemical betaine, which improves the amplification of DNA by reducing the formation of secondary structure in GC-rich regions. The addition of Q-Solution to the PCR reaction was mandatory for amplification of most maize sequences, which have high GC contents. Q-solution was excluded only for target sequences with <50 % GC content. PCR cycling conditions were as follows: 95 ⁰C (3 minutes); 35 cycles of 95 °C (30 seconds), 59- 63 ⁰C (30 seconds), 72 ⁰C (1 minute/ 1 kb); 72 ⁰C (10 minutes). Annealing temperature was variable, adjusted to 3 to 5 ⁰C below the Primer3-calculated Tm of the primers used in each assay.

Southern blot analysis

Southern blot hybridization was performed with a published protocol (Dellaporta & Moreno, in "The Maize Handbook". M. Freeling. V. Walbot. Kds., Springer Verlag, New York, 1993, pp. 569-572). except that the hybridization solution consisted of 0.25 M sodium phosphate, pH 7.2 and 7% SDS, as suggested by the manufacturer of the Zcta-Probc GT blotting membranes (Biorad, Hercules, CA, USA). The hybridization probe consisted of a 624 bp fragment from the isl-W22 gene (bases 2590-3213, spanning the end of exon 5 through the beginning of exon 7) and bore sufficient similarity to the duplicate islb gene (87-90 %) so that both IsI and tslb were detected.

Phylogemtic analysis

Sixty-four plant lipoxygenase amino acid sequences (Table 2), including that of TSl and TSIb, were aligned with ClustalW2 (Chenna ct al., 2003, Nucleic Acids Res. 31 :3497). Two different algorithms, maximum parsimony and Bayesian inference, were employed to estimate phylogenetic relationships of TSl and related proteins. Maximum parsimony was implemented in PAUP* 4.ObIO (Swofford, "PAUP*, Phylogenetic Analysis Using Parsimony (* and other methods)". Sinauer Associates, Sunderland, MA, 1998) where the heuristic search option was applied with default values. Bootstrap values for the maximum parsimony tree were obtained by resampling 1 ,000 replicates under the full heuristic search method.

Bayesian inference was performed with MrBayes 3.1.2 (Huelsenbcck & Ronquist, 2001, Bioinformatics 17:754) with a mixed amino acid substitution model, four independent chains run for 5,000,000 generations, and sampling every 1000th tree. Convergence was estimated when the standard deviation of split frequencies reached a plateau approaching zero, and the consensus tree was determined with a burn-in of 25 % (1250 trees).

Table 2. Plant lipoxygenase sequences used in TSl phylogenetic analysis

- 99 -

Quantitative RT-PCR analysis (qRT-PCR)

Maize plants from inbred line W22 were grown in the greenhouse. AU tissue samples used for qRT-PCR assays were quickly dissected and immediately frozen in liquid nitrogen. Approximately 100 mg of frozen tissue were ground in a mortar and pestle and quickly re-suspended in 1 ml of Trizol® Reagent (Invitrogen. Carlsbad, CA, USA). Developing inflorescences between 0.8 and 3 cm in length were directly placed in 1.5 ml microcentrifuge tubes, resuspended in 1 ml of Trizol® reagent and ground with a plastic pestle attached to a table top drill press. Total RNA was isolated according to manufacturer's recommendations and re-suspended in water previously treated with diethylpyrocarbonate (DEPC; Sigma, St. Louis, MO, USA) containing 20 units of Protector RNase Inhibitor (Roche, Indianapolis, IN, USA).

Prior to cDNA synthesis, 1 μg of total RNA was treated with 1 unit of DNase I, Amplification Grade (Invitrogen) in a 10 μl reaction containing IX DNase I buffer supplied by the manufacturer. The reaction proceeded for 15 minutes at room temperature and the enzyme was inactivated by adding 1 μl of 25 mM EDTA and heating at 65 ⁰C for 10 min. The DNase-treated RNA (1 μg) was directly used in cDNA synthesis with the Superscript® III First-Strand Synthesis SuperMix for qRT- PCR (Invitrogen) following the manufacturer's instructions. The 2X RT Reaction Mix included both oligo(dT)20 and random hcxamers to prime the reverse transcription reaction. The cDNA was diluted to 100 μl with IX TE and stored at -8O⁰C. PCR reactions were performed in optical 96-well plates with a 7500 Fast Real-Time PCR System sequence detection system (Applied Biosystems, Foster City, CA, USA). Reactions contained IX Power SYBR Green Master Mix reagent (Applied Biosystems), 300 nM of each gene-specific primer (Table 3) and 1 μl of diluted cDNA in a final volume of 25 μl. The standard thermal profile recommended by the manufacturer of the PCR master mix was followed. Three technical (PCR) replicates were set up for each one of the three biological replicates of each tissue sample. qRT-PCR data were normalized with aclinl as a reference gene.

Table 3. Primers used for quantitative qRT-PCR analysis

* The specificity for tsl and tslb is achieved with the reverse primers P 1894 and P2648, respectively t P2648 is specific for the tsl-W22 allele

In situ hybridization

Primers Pl 930 (5^t-CCTCTCΛGTACCGACAGACAGC-3¹; SEQ ID NO: 15) and P 1931 (5'-CCATrCAGTTCCTCACAGTCTTGC-S'; SEQ ID NO: 16) were used to amplify a 217 bp fragment of the tsl gene corresponding to part of the 3" UTR. The PCR product was cloned into the pCRII-TOPO® vector (Invitrogen) generating pYU1672, the plasmid used for synthesis of the tsl in situ probe. The ts2 in situ hybridization probes have been previously described (Del.ong ct al.. 1993. Cell 74:757) and are contained in the plasmids pYU59 and pYU60.

In situ hybridizations were performed as described (D. Jackson, in "Plant Molecular Pathology: A Practical Approach," S. J. Gurr, M. J. McPherson, D. J. Bowles, Eds., Oxford University Press, Oxford, 1992, vol. I, pp. 163-174) with modifications as described (Bortiri ct al., 2006, Plant Cell 18:574). Three additional modifications were adopted: (a) no RNase treatment was performed after hybridizations; (b) the anti-DIG antibody was diluted 1/1000 and incubated for 2 hr at room temperature; (c) the buffer used for color detection included 10% polyvinyl alcohol (PVA) to increase reaction sensitivity.

Construction of a ts 1. mCherry fusion gene

The mCherry gene was PCR amplified with PfuUltra™ High-Fidelity DNA Polymerase (Stratagene, La Jolla, CA, USA) from the pREST-B mCherry vector (S 12) with primers P2672 (5¹- cggggtaccccATGGTGΛGCAAGGGCGΛGGAGGΛT-3¹; SEQ ID NO: 17) and P2673 (5'-ctagtctagatggatccCTTGTACAGCTCGTCCATGCCGCC-3': SEQ ID NO: 18). which added Kpnl, Ncol and Baml ll, Xbal sites respectively (lower case letters in primer sequences). The PCR product did not contain the endogenous stop codon. Instead, P2673 provided a new stop codon in the Xbal site downstream of the Baml ll site. The PCR product was digested with Kpnl and Xbal and the 733 bp Kpnl-Xbal mCherry fragment was gel purified. Plasmid pYU 1721 was derived from the plant expression vector pRTL2 (Restrepo ct al., 1990, Plant Cell 2:987) and contained the full-length tsi CDS without the stop codon (tø/ΔSTOP) fused in frame with the gene encoding the monomer Red Fluorescent Protein (mRFPl ) (construction details are available upon request). pYU1721 was digested with Kpnl and Xbal to release the mRFPl gene. The remaining -6.5 kb plasmid containing pRTL2 plus the /A- /ASTO P CDS was gel purified and ligated to the 733 bp Kpnl-Xbal mCherry gene. The resulting plasmid, pYUI 743, was shown by sequencing to contain an in-frame N- terminus fusion of the tsl ASTOP CDS to the mCherry gene. Biolistic experiments in onion epidermal cells

One microgram of plasmid DNA was precipitated on gold particles (1.0 μm, Biorad) essentially as described (Kleinet et al., 1987, Nature 327:70). Onion (Allium cepa L.) bulbs were cut in small pieces. The epidermal cell layers were carefully peeled and transferred to the surface of Petri dishes containing Murashige and Skoog basal medium (MS from Invilrogen, or Sigma-Aldrich, St. Louis, MO USA) solidified with 3.5% Phytagel (Sigma-Aldrich). Epidermal cell layers were bombarded with a Biolistic PDS 1000/He Particle Delivery System (Biorad) with 1350 psi rupture discs. After bombardment the plates were incubated at 27°C in darkness for 8-20 h. The epidermal cell layers were then stained for 10 minutes in 1 μg/ml DAPI dissolved in IX PBS then washed for 10 minutes in IX PBS and mounted in glass slides with 70% glycerol in IX PBS.

Epifluorescence microscopy, photography and image analysis Transformed cells were examined by epifluorescence microscopy with an Axioplan 2 microscope (Carl Zeiss Microimaging, Thornwood. NY, USA) with the appropriate excitation/emission filters. Differential Interference Contrast (DIC or Nomarski microscopy) was used to visualize cells under transmitted light. Digital images were captured with a Zeiss Axiocam with several Image Acquisition Modules of the Zeiss Axiovision software. The Multichannel Fluorescence module allowed the sequential acquisition of DAPI, GFP, mCherry and DIC images for each sample. The onion epidermal cell layer is very thick and not all cell features appear in the same focal plane. Therefore, a series of images over a defined z-focus range were acquired with the Z-Stack module of Axiovision. This module automatically calculated the distance between individual images of the z-stack to achieve the maximum axial resolution of the objective used. The Z-stack was reconstructed with the 3D Deconvolution module where the Regularized Inverted Filter (RIF) method was used. Final image display was completed by applying a maximum intensity projection (MIP) over the entire image volume within the context of the orthogonal slice view (Cut View). The Colocalization module was used to quantitatively assess the colocalization of the TSl :mCherry red fluorescent signal along with the RbcSnt:GFP green fluorescent signal. Example 1. Positional cloning and mapping of tsl.

The molecular marker html was isolated from the Hanking sequence of a Mu4 clement that was identified as tightly linked to the ts!-Mu92 mutation. The hlml marker was mapped approximately 1 cM from tsl in a small tcstcross population (n = 93) segregating for the /.y/-re/allele. The maize ZMMBBb BΛC library (CUGl) was probed with him 1 and six BΛC clones from contig 78 of the current maize physical map (Arizona Genomics Institute) were identified. Λ marker within this contig. umc2251, was tested for linkage and found to map distal to both tsl and him] - 8 and 9 cM respectively (Figure 1 ). Therefore, the genetic interval containing /Λ / was initially defined as a 9 cM region by the proximal him I and the distal umc2251 markers. To further refine this interval, BΛC end sequences in the physical region proximal to umc225J were analyzed for potential low copy sequences. A molecular marker designed from the end sequence of BAC b0148G01 (CC760183) was tested and found to map 1 cM distal to tsl (Figure 1). A larger mapping population (total n = 420) was analyzed with CC760183 and him I and a total of 3 distal and 4 proximal recombinants to tsl were detected. This analysis placed tsl within a ~1.6 cM genetic interval defined by the proximal him I and the distal CC760183 markers, which spanned a physical region of -500 kb (Figure 1 ).

A genomic sequence (AZM4 1 15428) corresponding to him I was identified in the TIGR AZM 4.0 assembly of the maize methyl-filtered and high-Cot genomic libraries which consists of sequences highly enriched for coding regions (Palmer et al.. 2003, Science 302:21 15; Whitelaw et al., 2003. Science 302:21 18). Orthologs of CC760183 and AZM4 1 15428 were annotated in the rice genome delineating a 62 kb syntenic region that contained 9 predicted genes.

Example 2. Predicted function of TS l protein.

The tsl gene was thus located in a region with extensive syntcny with rice (Salse et al., 2004, Plant J. 38:396). Out of the nine genes contained in the tsl syntenic interval within the sequcnced genome of rice, no maize orthologs were found for four of these genes, and another three were mapped to locations unlinked to the tsl locus in maize. Maize homologs of the remaining two rice genes, one encoding a putative glutamate decarboxylase and the other encoding a putative lipoxygenase, were confirmed to be contained within the tsl physical interval (Figure 1 ). Sequencing showed that the gene encoding glutamate decarboxylase was monomorphic, whereas the gene encoding lipoxygenase in the tsl-ref Vine showed an 864-base pair (bp) insertion in the predicted first exon with complete linkage with the tsl phenotype in mapping populations. To confirm that the lipoxygenase corresponded to the tsl gene, eight tsl mutant alleles were analyzed. Each contained an independent mutation in the gene encoding lipoxygenase (Figure 2A, and Table 3). Complementary DNA sequence analysis showed that the IsI gene contains seven exons with a coding sequence of 2757 bp (Figure IA). A closely related gene was also identified in the database of the TlGR AZM 4.0 assembly and by Southern blot analysis (Figure 3). This gene, named IsIb, has an identical cxon- intron structure to that of IsI and shares 93% nucleotide similarity. The is Ih gene is located on maize chromosome 10S, a segmental duplication of chromosome 2S (Gaut.

2001, Genome Res. 1 1 :55). The TSl protein displays 38 to 60% similarity to plant lipoxygenases and contains two conserved domains characteristic of this family: a beta-barrel (cdOl 751 ) and a catalytic helical bundle (pfam00305) (Shibata & Λxelrod. 1995, J. Lipid Mediat. Cell Signal. 12:213) (Figure 2B, and Figure 4).

Lipoxygenases are non-hemc iron-containing fatty acid dioxygenascs that catalyze the peroxidation of polyunsaturated fatty acids such as linolcic acid, α- linolenic acid, and arachidonic acid. They arc classified according to the positional specificity of linolcic acid oxygenation, which occurs at carbon 9 of the hydrocarbon backbone for the 9-LOX types and at carbon 13 for the 13-LOX types; a further subdivision (classes 1 and 2) has been recognized for 13-lipoxygenases without or with a putative chloroplast transit peptide (cTP), respectively (Fcussncr &Wasternack,

2002, Annu. Rev. Plant Biol. 53:275). According to ChloroP. a neural network-based method for predicting cTPs, the N-terminal 48 amino acids of the TS 1 protein contain a cTP (Emanuelsson et al., 1999, Protein Sci. 8:978) (Figure 2B, and Figure 4). Additionally, TSl contains a conserved phenylalanine (Phe636) previously identified as a determinant of 13-LOX regiospecificity (Hornung et al., Proc. Natl. Acad. Sci. U.S.A. 96:4192; Liavonchanka & Fcussner, 2006, J. Plant Physiol. 163:348). Therefore, the primary structure of TSl suggests that it is a member of the class 2 plastid-localized 13-lipoxygenases. This prediction was supported by Bayesian and maximum parsimony phylogenetic analyses of plant lipoxygenases, which placed TSl and TSI b in a cladc including characterized and predicted class 2 13-lipoxygenase (Figure 2C, and Figure 5).

Table 3. Characterization of nine IsI mutant alleles

Example 3. Tissue-specific expression of is J and is Ib genes.

The tissue-specific expression of both IsJ and is Ib was established by quantitative reverse transcription polymerase chain reaction (R T-PCR) analysis of root, stem, leaf, tassel, and ear transcripts. The tsl RNΛ was detected in all maize tissues examined, whereas islb RNΛ was detected at very low levels (less than that of tsl by a factor of 90 to 500) (Figure 6). The low expression of is Ib may explain why it does not also appear to be a component of sex determination in tsl mutant plants. The broad expression of tsl was unexpected because its mutant phenotype suggests a sex-specific function. Although no alterations in other tissues have been reported, it is possible that additional phenotypcs for the tsl mutation may be uncovered by more careful analyses. The ts2 gene was expressed almost as broadly as tsl, except in stem tissue, where expression was less than that of is I by a factor of -35. In situ hybridization showed that TSI transcripts form stripes following the borders of the central inflorescence axis and projecting toward the spikelet attachment points (Figures 6B and 6I)). In spikelet adaxial views, TSI expression domains surround the spikelets, delineating their base (Figure 6C). None of these expression patterns were observed in a homozygous tsl -MuOl deletion mutant line containing a functional Is Ib gene (Figure 6E). These observations indicate that 7'.SV transcripts subtend maize spikelets at their junction with the central inflorescence axis (rachis). This expression domain suggests a function for TSl, as metabolites synthesized through the lipoxygenase encoded by tsl could act as diffusible signals affecting floral development in a non-ccll-autonomous fashion. The ChloroP-based prediction that TS l localizes in plastids was confirmed with a fluorescent-tagged TSl protein (TS l :mCherry) and a plastid- localized RbcSnt:GFP protein (Lcc et al., 2002, MoI. Cells 14:388). To quantitatively assess the colocalization of RbcSnt:GFP and TSl :mChcrry fluorescent signals, correlation analysis of the intensity values of color (green and red) pixels in the dual- channel image was performed. Specifically, the Colocalization module in the

Axiovision software (Carl Zeiss Microimaging, Thornwood, NY, USA) plotted the pixel grey values of fluorescent intensity of the x-axis and y-axis channels against each other (Figure 61). Then, correlation coefficients were calculated to measure the strength of the linear relationship between the two variables (Bolte & Cordeliercs. 2006, J. Microsc. 224:213; Mandcrs et al., 1992. J. Cell Sci. 103:857). The images in Figures 6E and 6F showed a Manders¹ overlap coefficient of 0.986 strongly indicating colocalization between the two signals. Additionally, weighted co-localization coefficients Ml and M2, which are independent of differences in fluorescence intensity between the two channels, were calculated. For TSl :mChcrry, M2 = 0.906. indicating that a high proportion of red signal coincided with a signal in the green channel over its total intensity (Bolte & Cordclieres. 2006. J. Microsc. 224:213: Manders ct al., 1992, J. Cell Sci. 103:857). A similar conclusion was drawn for the weighted co-localization coefficient of RbcSnt:GFP (Ml=O.903), indicating that TSl :mCherry protein is targeted to the same subcellular compartment as RbcSnt:GFP. TS 1 is thus targeted to plant plastids.

Example 4. Analysis of lipoxygenase activity in crude protein extracts from tassel inflorescences

Frozen tassels were ground in liquid nitrogen and/or homogenized in a 1.5-ml microcentrifuge tube with 1 to 2 volumes of ice-cold 0.1 M Tris buffer, pl l 7.5, containing 0.1 M NaCl, 5 mM EDTA, 0.1% β-mercaptoethanol, 0.6% Triton X- 100, 1 mM PMSF and EDTA-free Protease Inhibitors (Roche, Indianapolis, IN, USA). The mixture was clarified by centrifugation at 14,000 x g for 30 minutes at 4⁰C. The supernatant was transferred to a new tube and total protein concentration estimated with the Micro BCA Protein Assay Kit (Pierce, Rockford, IL, USA).

Aliquots of crude protein extracts were added to 3 ml of potassium phosphate buffer, pH 6.0 containing 150 μM linoleic acid and stirred at 23⁰C for 15 min. Peroxidation products were reduced to hydroxides, MOD) by adding 12 ml of a solution of 5 mg/ml SnCl₂ in ethanol and incubating for another 5 minutes at 23 ⁰C. Products isolated by extraction with diethyl ether were methyl-esterified and analyzed by GC-MS and HPLC. For all analyses, reference oxylipins of high chemical purity (Larodan Fine Chemicals, Malmo, Sweden) were used. Material to be analyzed by GC-MS was derivatized by treatment with trimethylchlorosilane/hexarnethyldisilazane/pyridine (2: 1 :2, v/v/v) at room temperature for 15 min. Excess reagent and solvent were removed in vacuo and the residue was resuspended in hexane. GC-MS was carried out with a mass-selective detector (Hewlett-Packard model 5970B, Avondale, PA, USA) connected to a gas chromatograph (Hewlett-Packard model 5890) equipped with a capillary column of 5 % phenylmethylsiloxane (12 m, 0.33 μm film thickness). Helium was used as the carrier gas, and the column temperature was raised from 120 ⁰C to 300 ⁰C at 10 °C/min.

SP-HPLC of methyl-esterified incubation products was carried out with a column of Nucleosil 50-5 (250 x 4.6 mm) purchased from Macherey-Nagel, Dϋren, Germany, and a solvent system of 0.6% 2-propanol/hexane at a flow rate of 2 ml/min. The absorbance (234 nm) and radioactivity of HPLC effluents were determined on-line with a Spectromonitor 111 ultraviolet detector (Laboratory Data Control, Riviera Beach, FL, USA) and a liquid scintillation counter (IN/US Systems, Tampa, FL, USA), respectively. Under the conditions used, the elution order of hydroxyoctadecadienoates were: methyl 13- hydroxy-9(Z),l l (E)-octadccadicnoate (first), methyl 13-hydroxy-9(E),l 1(E)- octadecadienoatc, methyl 9-hydroxy- 10(E),12(Z)-octadecadienoate and methyl 9- hydroxy- 10(E), 12(E)-octadecadienoatc (last).

Biochemical analysis of protein extracts from developing tassels for activity on the lipoxygenase substrate linoleic acid suggests that TSl is capable of lipid peroxidation. Protein extracts from wild-type tassels catalyzed hydroperoxidation of linoleic acid, whereas no such activity was detected in mutant

tassels (Figure 7). Mass spectrometry (MS) and high- performance liquid chromatography (HPLC) analyses showed that the products of this lipoxygenase activity are a mixture of 9-and 13-hydroperoxides in a 50:50 ratio (Figure 7A); primary structure analysis had suggested that I S l was a lipoxygenase with 13-regiospecificity. Therefore, it is possible that TS l possesses dual 9- and 13- regiospecificity — which has not previously been described for a plastid-localizcd lipoxygenase — or that TSl function promotes the action of a separate 9-lipoxygcnasc.

Example 5. Quantification of iasmonic acid and other metabolites in tassel inflorescences

Class 2 13-lipoxygenases participate in the biosynthesis of the plant hormone jasmonic acid (JA) (Wasternack, 2007, Ann. Bot. 100:681 ) (Figure 8). The involvement of TS l in JA biosynthesis was evaluated by measuring endogenous JA levels in developing wild-type and tsl-ref/tsl -ref mutant tassels. As shown in Figure 8, the first dedicated step in jasmonate biosynthesis is the peroxidation of α-linolenic acid ( 18:3) by 13-lipoxygcnase to form (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT). This is the putative function of TS 1. 13-1 IPOT is transformed into the specific stereoisomer cis-(+)- 12- oxophytodienoic acid (OPDA) through the sequential action of allenc oxide synthase (yielding (13S)- 12,13-cpoxy-octadecatrienoic acid (12,13-EO T)) and allenc oxide cyclase. These steps in JA biosynthesis occur in plant plastids, where the corresponding enzymes arc localized. Subsequent reactions occur in the peroxisomes. First, the cyclopcntenone ring of OPDA is reduced to 12-oxophytocnoic acid (OPC-8) by OPDA reductase. Next, three β-oxidation cycles are proposed to shorten the carboxylic side chain of OPC-8 to produce the 12-carbon JA. A β-oxidation cycle is a set of four enzymatic reactions: oxidation, hydration, oxidation, and thiolysis. Not all enzymes acting on β-oxidation during JA biosynthesis have been identified. Because the oxidation in the third step is normally performed by a dehydrogenase activity, it is possible that TS2 may participate in this step of JA biosynthesis.

Maize plants of W22, Is 1-ref/ls J -ref and tsl-ref/+ were field-grown during the summer of 2007. Developing tassel inflorescences between 0.8 and 3 cm in length were quickly dissected, placed in 1.5 ml microcentrifuge tubes and rapidly frozen in liquid nitrogen. Tissue samples were stored at -80 ⁰C prior to solvent extraction. Jasmonic acid quantification was performed with vapor phase extraction for sample preparation and chemical ionization gas chromatography/mass spectrometry (CI-GC/MS) as described (Schmelz et al., 2004, Plant J. 39:790). Descriptive and comparative statistics were obtained with Λnalyse-it® Standard edition (Analyse-it Software, Ltd, Leeds. England), an add-in for Microsoft Rxccl.

The average concentration of JA in wild-type and tsl-rejh heterozygotes was 44.2 T 13.9 ng per gram of fresh weight (ng/g FW) and 40.3 T 20.2 ng/g FW, respectively (Figure 7B). Homozygous ts I -ref/lsl-ref \asse\s showed an average JA concentration of 4.3 T 2.1 ng/g FW (Figure 7B), significantly below that of the wild type in a Kruskal-Wallis test and pairwise comparisons with a Bonfcrroni correction (P < 0.0001). The tsl mutation thus appears to reduce JA levels by a factor of -10, indicating a role for the hormone in the pistil cell death process. JA levels of wild-type and mutant tsl tassels are similar to those of wounded and nonwounded maize seedlings, respectively (Engelberth et al., 2007, MoI. Plant Microbe Interact. 20:707), which supports the notion that JA is actively synthesized during normal tassel development.

Example 6. Chemical treatment of maize plants

Maize plants were grown under greenhouse conditions. Two seeds were planted in 2-gallon reusable pots containing Super-Fine Germinating Mix (Fafard, Agawam, MA, USA) or Redi-earth Professional Growing Mix (Sun Gro. Bellevue, WA, USA). Fertilization was performed with controlled release Osmocote Plus 15-9-12 (Scotts, Marysville, OH, USA) following the manufacturer recommendations. Additional watering was performed with 4% ammonium iron citrate (Sigma-Aldrich) every 2 weeks to prevent chlorosis. Greenhouse average temperatures were 28°C (day) and 21⁰C (night). Supplemental lightning was provided to achieve a 16:8 hour photoperiod yearlong. Tassels of about 1 cm were considered to be at an ideal stage to initiate chemical treatments. Since a thick leaf whorl covers the maize tassel at this time of development, only a destructive dissection of the plant permits to assess with certainly the stage of tassel development (Bonnet, 1940, J. Agricult. Res. 60:25; Bonnet, 1948, Ann. Mo. Bot. Gard. 35:269). Additionally, the sex determination phase was reached within a wide time window, between 28 and 46 days post-planting, depending on the growing conditions. Leaf number, node number or internode distance were not always reliable criteria to predict the tassel developmental stage.

Seedlings from a family segregating 1 : 1 for wild-type (tsl-ref/Tsl) and tsl mutant (tsl-reβtsl-rej) plants were gcnotyped for the Tsl and tsl-ref alleles with a PCR-based assay. Forty-six days after planting, one tsl and one heterozygote plant showing 6-8 fully expanded leaves were dissected and their tassels were shown to be approximately 1.0 cm in height. This was used as a rough estimate indicating that similar plants, as judged by height and leaf number, were at the right stage to receive chemical treatments. Jasmonic acid (JA, Sigma-Aldrich) was dissolved at a concentration of 200 mM in absolute ethanol and stored at -20°C. Prior to plant treatment, the JA stock solution was diluted to a concentration of 1 mM in deionized water. Control plants were treated with a 0.005% ethanol solution ("blank" treatment or negative control). One ml of the corresponding solution was applied into the apical leaf cavity of each plant. Treatments were performed three times at 48-hour intervals. The developmental timing of the pistil abortion process occurs in tassel inflorescences when they are 1.0 to 3.0 cm in length (Irish & Nelson, 1993, Am. J. Bot. 80:292), which was also the stage at which tsl expression occurred in the subtending glumes (Figures 6B to 6D). A 0.005% ethanol solution, with or without 1 mM JA, was applied to tassels of ~1 cm in wildtype (ts!-ref/+) or tsl mutant (tsl- ref/tsl-ref) sibling plants. In tsl mutant plants, JA application reversed feminization, as evidenced by the presence of staminate spikelets mostly in the mid- to apical regions (Figure 7D). Wildtype rescue in tsl mutants was observed in the appearance of subtending floral bracts (glumes) about 3 to 4 weeks after treatment. JΛ-treated glumes in tsl mutants were elongated, were covered with numerous trichomcs, and had a ring of anthocyanin deposited at the base (Figure 7D). all three characteristics of wild-type staminate spikelets. Later in floral development, stamens emerged from JΛ-treated tsl mutant spikelets (Figure 9B).

Staminate florets from rescued JA-treated l.sl/t.sl plants produced viable pollen, which was used for both self-pollination and test crosses to untreated tsl/tsl mutant sibs. All test cross progeny (n > 100) were homozygous for the tsl-ref allele and displayed a complete tsl mutant phenotypc. The JΛ-rcscucd phenotypc of the tassel inflorescence was incomplete in that some spikelets were bisexual (Figure 9A), containing both pistils and stamens, and others (mainly those located at the base of the inflorescence) were pistillate. These effects, however, may have been due to the timing of JA treatments, because the stage of floral maturation differs in a positional Iy dependent fashion throughout the inflorescence. Rescued staminate spikelets were never observed in blank-treated is 1 -reβls I -refplanls (Figure 7C; Figure 9D, and Table 4), nor did JA treatment affect heterozygous tsl-ref/ ^ sibs (Table 4). The similar phenotype of tsl and ts2 mutations indicates that both genes may act in the same metabolic pathway. Therefore, JA was also applied to mutant ts2-ref/ts2-ref and ts2-ref/+ plants, which responded in the same manner as the JA- treated tsl mutants (Figure 7E; Figure 9C; and Table 4). These results indicate that JA can restore the wild-type phenotype in both tsl and ls2 mutant plants. Moreover. TS2 may have an unexpected role in JA biosynthesis, perhaps as one of the yct- unidentifled enzymes catalyzing a series of β-oxidations in this metabolic pathway (Figure 4).

Genes regulating mcristcm dctcrminacy early in maize inflorescence development are expressed at the boundary of the meristcm and the inflorescence axis rather than within the mcristem itself. These genes, such as rαmosαl, rαmυsiβ, and barren stalkl (Gallovatti et al, 2004, Nature 432:630; Satoh-Nagasawa et al., 2006. Nature 441 :227; Vollbrecht et al., 2005, Nature 441 :227). probably act non-cell- autonomously by producing a diffusible signal at the base of the mcristcm (Vollbrecht et al., 2005, Nature 441 :227). Analogously, tsl expression at the boundary of developing spikelet initials and inflorescence axis produce the hormone JA, which may diffuse within the spikelet to regulate sexual development. This situation parallels JA-mediated anther dehiscence in ArabiJopsis, where JA biosynthetic genes are highly expressed in the anther filament where it signals development both in the filament and within the anther (Ishiguro ctal., 2001 , Plant Cell 13:2191 ; Sanders et al., 2000, Plant Cell 12: 1041). The expression o( ts2 is known to be reduced in is I mutants (Calderon-Urrea & Dellaporta, 1993, Development 126:435). The finding that ts2 may be involved in the same biosynthetic pathway as IsI is not necessarily at odds with previous observation, as most genes encoding enzymes of the JA biosynthetic pathway are transcriptionally up-regulated by JA in a characteristic positive feedback loop (Wasternack, 2007, Ann. Bot. 100:681).

JA signals plant responses to biotic and abiotic stresses (Wasternack, 2007, Ann. Bot. 100:68) and regulates plant developmental processes such as root growth (Staswicknet al., 1992, PNAS U.S.A. 89:6837) and mechanotransduction

(Weiler et al., 1993, Phytochemistry 32:591). In Arabitlopsis, JA is required for male fertility because pollen maturation and anther dehiscence arc blocked in mutations that impair JA biosynthesis (Ishiguro elal., 2001, Plant Cell 13:2191 ; Sanders ct al., 2000, Plant Cell 12: 1041). JA may promote anther dehiscence by signaling degeneration of the stomium, a group of specialized cells that run along the length of the anther and are necessary for dehiscence (Sanders et al., 1999, Sex, Plant Reprod. 1 1 :297). The present results imply a role for JA in maize sex determination, wherein JA is necessary for signaling the lasselseed-medialed pistil abortion and the acquisition of the male characteristics of staminatc spikclets. The diverse mechanisms of hormonal control in plant sex determination support the notion that the systems have evolved independently multiple times (Ainswortyh et al., 1998, Curr. Top. Dev. Biol. 38: 167).

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed:

1. Λn agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof.

2. The composition of claim 1 , wherein said compound is jasmonic acid.

3. The composition of claim I , wherein said compound is a jasmonic acid ester.

4. The composition of claim 3. wherein said compound is jasmonic acid methyl ester.

5. A method of modulating sexuality in a plant, comprising the step of administering to said plant an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof.

6. The method of claim 5, wherein said compound is jasmonic acid or jasmonic acid methyl ester.

7. The method of claim 5, wherein said plant is grass-related.

8. The method of claim 7, wherein said plant is mai/.e or rice.

9. The method of claim 8, wherein said plant is mai/.e.

10. The method of claim 5, further comprising the step of administering to said plant at least one additional compound useful for controlling plant sexuality.

1 1. The method of claim 10, wherein said at least one additional compound is selected from the group consisting of ethylene, gibberelin and auxin.

12. A method of suppressing complete feminization or restoring male sexuality in a plant with a isl or is2 mutation, comprising the step of administering to said plant an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof.

13. The method of claim 12, wherein said compound is jasmonic acid or jasmonic acid methyl ester.

14. The method of claim 12, wherein said plant is grass-related.

15. The method of claim 14, wherein said plant is maize or rice.

16. The method of claim 15, wherein said plant is maize.

17. The method of claim 12. further comprising the step of administering to said plant at least one additional compound useful for controlling plant sexuality.

18. The method of claim 17, wherein said at least one additional compound is selected from the group consisting of ethylene, gibberelin and auxin.

19. Λ method of creating homozygous stock in a plant with a is I or ts2 mutation, comprising the step of administering to said plant an agriculturally compatible composition comprising an effective amount of a compound selected from the group consisting of jasmonic acid, a jasmonic acid derivative, and a salt thereof, wherein the progeny of said plant is homozygous for said mutation and is male-sterile.

20. The method of claim 19, wherein said compound is jasmonic acid or jasmonic acid methyl ester.

21. The method of claim 19, wherein said plant is grass-related.

22. The method of claim 21, wherein said plant is maize or rice.

5 23. The method of claim 22, wherein said plant is maize.

24. The method of claim 19, further comprising the step of administering to said plant at least one additional compound useful for controlling plant sexuality.

I O

25. The method of claim 24, wherein said at least one additional compound is selected from the group consisting of ethylene, gibberelin and auxin.