EP1501345A1 - Regulated ethylene sensitivity to control flower longevity in a plant - Google Patents

Abstract

Provided is the tissue specific manipulation of the EIN2 and/or EIN3 ethylene­signaling genes, permitting the controlled regulation of flower and/or leaf abscission in a commercially useful plant, since flower and leaf abscission are positively regulated by the plant hormone ethylene. In particular, controlled expression of the EIN2 gene, permits reduction of early-season flower abscission. The altered expression of ethylene signaling gene(s) enhances the commercial value of the plant, thereby producing a more productive and/or more efficiently harvested crop. Further provided are new insights into the mechanisms involved in the ethylene signaling pathway.

Description

REGULATED ETHYLENE SENSITIVITY TO CONTROL FLOWER LONGEVITY IN A PLANT

REFERENCE TO RELATED APPLICATIONS This application is a Continuation-in-Part of PCT Application No. PCT/US02/34566, filed October 28, 2002, which further claims priority to US Provisional Application 60/339,596, filed October 26, 2001. This application also claims priority to US Provisional Application 60/ 374,555, filed April 22, 2002, which is herein incoφorated by reference in its entirety.

BACKGROUND OF THE INVENTION

The plant hormone ethylene regulates a wide range of developmental processes, including seed germination, abscission of leaves and flowers, stem elongation, and fruit ripening. Ethylene signal transduction is controlled by a complex multicomponent pathway (Kieber, Annu. Rev. Plant Physiol. Plant Mol. Biol 48:277-296 (1997). To address the ethylene signaling mechanisms, a molecular/genetic approach has been applied using the ethylene-evoked triple response phenotype of Arabidopsis thaliana seedlings.

In Arabidopsis, the "triple response" typically involves inhibition of root and stem elongation, radial swelling of the stem and absence of normal geotropic response (diageotropism). Etiolated morphology of a plant can be dramatically altered by stress conditions that induce ethylene production, so that, for example, the ethylene-induced triple response provides a seedling with the additional strength required to penetrate compacted soils. Based upon the triple response, a dozen Arabidopsis mutants have been isolated into two classes (Ecker, Science 268:661-675 (1995); U.S. Pat. Nos. 5,367,065; 5,444,166; 5,602,322 and 5,650,553, each of which is herein incorporated by reference). One class of mutants, the ein (ethylene insensitive) mutants, showed reduced or complete insensitivity to exogenous ethylene. The other class of mutants, the constitutive hormone response mutants, display constitutive ethylene response phenotypes in the absence of exogenously applied hormones.

The first component of the ethylene signal transduction pathway to be identified was an ethylene receptor gene from Arabidopsis, ETRl. This gene encodes a histidine kinase with homology to bacterial two-component systems as reported by Chang et al, Science 262:539-544 (1993). To date, a total of five ethylene receptor genes have been cloned from Arabidopsis, ETRl, ETRl, ERS1, ERS2, and EIN 4 (Hua et al., Science 269:1712-1714 (1995); Hua et al, Plant Cell 10:1321-1332 (1998); Sakai et al, Proc. Natl. Acad. Sci. USA 95:5812-5817 (1998)). Loss-of-function mutations in multiple ethylene receptors increase sensitivity to ethylene, indicating that the receptors are negative regulators of ethylene response (Hua et al., Cell 94:261-271 (1998)).

Downstream components of the pathway have also been identified in Arabidopsis. These include CTR1 (constitutive triple response), which is homologous to the Raf family of serine/threonine kinases (Kieber et al, Cell 72: 427-441 (1993)), and which interacts with the two ethylene receptor proteins ETRl (ethylene receptor 1) and ERS1 in a yeast two- hybrid system (Clark et al, Proc. Natl. Acad. Sci. USA 95:5401-5406 (1998)). Loss-of- function mutations in C7R1 cause a constitutive ethylene signaling phenotype of severe leaf epinasty and reduced leaf expansion, indicating that, like the receptor proteins, CTR1 is a negative regulator of ethylene response. EIN2, a key component of the ethylene signal transduction pathway, is an integral membrane spanning protein with 12 membrane spanning regions. It is homologous to the Nramp metal-ion transport proteins (Alonso et al, Science 284:2148-2152 (1999)). Epistasis analyses indicate that ELN2 acts downstream of CTR1. EIN2 loss-of-function mutants are insensitive to ethylene, indicating that these proteins are positive regulators of ethylene response.

Several transcription factors that control the expression of ethylene-regulated genes have also been identified. The Arabidopsis EIN3 family contains several proteins that bind to an ethylene response element in the promoter of the Ethylene Response Factor 1 (ERF1) gene. ERF1 binds to the promoters of pathogenesis-related (PR) genes and regulates their expression (Solano et al, Genes & Development 12:3703-3714 (1998)). Loss-of-function mutations in EZN3 reduce sensitivity to ethylene. Over-expression of wild-type EIΝ3 results in constitutive ethylene response (Chao et al, Cell 89:1133-1144 (1997)). Therefore, ELN3 is a positive regulator of ethylene response.

Isolation of several components of the ethylene signal transduction pathway has made it possible to genetically engineer crop species with altered ethylene sensitivity. In tomato, antisense expression of the tomato ethylene receptor LeETR4 resulted in constitutive ethylene responses, including leaf epinasty, premature flower senescence, and accelerated fruit ripening (Tieman et al, Proc. Natl. Acad. Sci. 97:5663-5668 (2000)). Over-expression of the tomato ethylene receptor protein NR resulted in tomato plants with reduced ethylene sensitivity as evidenced by increased stem and seedling elongation and reduced necrosis in response to a bacterial pathogen (Ciardi et al., Plant Phys. 123:81-92 (2000)). Heterologous expression of mutant receptor proteins can also decrease sensitivity. For example, over- expression of the mutant Arabidopsis ethylene receptor protein ETR1-1 in both petunia and tomato resulted in delayed flower abscission, flower senescence, and fruit ripening (Wilkinson et al., Nature Biotech. 15:444-447 (1997)).

Nevertheless, until the present invention there has remained a need for an understanding of the two ethylene-regulated developmental processes in flower and/or leaf abscission. Premature flower abscission results in early flower drop in the floraculture industry, resulting in significant reduction in dollar value, or it can greatly reduce fruit set, e.g., in tomatoes and other fruit or vegetable crops. Reduced boll set in cotton potentially means a substantially loss in total yield. Thus, there has remained a need for commercially useful transgenic plants having reduced ethylene sensitivity in flowers to control and prevent premature flower abscission as well as to control the effect of senescence in other tissues.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions for controlled regulation of flower longevity or abscission in transformed plants. In a preferred embodiment, flower longevity is enhanced and flower abscission is reduced as compared with untreated wild type plants. The preferred method comprises overexpressing EIN2 and/or EIN3 in the plant, although other methods may be suitable if it results in the same level of enhanced flower longevity or other disclosed activity. In each case, whenever the term ^lEIN3 gene' is used or 'ELN3 polypeptide,' it is also intended to include EINS-like genes and EIN3-like polypeptides, respectively, as they are defined herein. Accordingly the additional phrase, specifically restating that EIN3 includes the EIN3-like genes for the puφoses of this invention or that EIN3 is intended to include ELN3-like polypeptides, is not and need not be repeated, but will be understood to be present herein at each occurrence of EIN3 or ΕΪN3.

In a particularly preferred embodiment, the methods comprise overexpression of the EIN2 and/or EIN3 genes in selected transgenic commercially useful plants, such as flower crops, including but not limited to, petunia or geranium or begonia; food crops, including but not limited to, lettuce and tomato; or in a plant in which the plant tissue is useful for other commercial puφoses, such as in the fiber (e.g., cotton) or drug or pharmaceutical industries (e.g., cone flower) or the like; preferably to enhance flower longevity by the regulation of abscission. In an alternative embodiment, the trait of inducible premature leaf abscission is selected; while in yet another embodiment the selected trait comprises enhanced leaf longevity and reduced premature leaf abscission.

In at least one embodiment of the invention, the overexpression of the EIN 2 gene is driven or controlled by a flower abscission zone-specific promoter. In another embodiment, the overexpression of the EIN3 gene is driven or controlled by an inducible promoter. In yet another embodiment in which ELN2 and ELN3 are both overexpressed, the overexpression of the EIN2 gene is driven or controlled by a flower abscission zone-specific promoter and the overexpression of the E1N3 gene is driven or controlled by an inducible promoter. It is an object of the invention to further provide a method for producing at least one cell line in which EIN2 is overexpressed comprising: transforming tissue from a first commercially useful plant with an exogenous EIN2 gene or active fragment thereof, selected to provide overexpression of EIN2 in a cell line displaying a strong phenotype of enhanced flower longevity and/or reduced flower abscission, thereby stably effecting the overexpression of EIN2 in that cell line of plants.

It is also an object to provide a method for producing at least one cell line in which EIN3 is overexpressed comprising: transforming a first commercially useful plant tissue with an exogenous EIN3 gene or active fragment thereof, selected to provide overexpression of EIN3 in a cell line displaying a strong phenotype of enhanced flower longevity and/or reduced flower abscission, thereby stably effecting the overexpression of EIN3 in that cell line of plants.

It is another object of the invention to provide a method for producing at least one cell line in which EIN2 and EIN3 together, are overexpressed comprising: transforming a first tissue from a commercially useful plant with an exogenous EIN2 gene or active fragment thereof, selected to provide overexpression of EIN2 and transforming a second tissue from a commercially useful plant with an exogenous EIN3 gene or active fragment thereof, selected to provide inducible overexpression of EIN3; then genetically crossing the EIN2 and EIN3 overexpressing transformed cell lines; and selecting at least one crossed EIN2/ELN3 overexpressing cell line displaying a strong phenotype of enhanced flower longevity and/or reduced flower abscission, thereby stably effect the overexpression of EIN2/EIN3 in that cell line of plants. In an alternative embodiment, the trait of inducible premature leaf abscission is selected; while in yet another embodiment the selected trait comprises enhanced leaf longevity and reduced premature leaf abscission. In a preferred embodiment the ELN2 or EIN3 or crossed EIN2 and/or ELN3 overexpressing cell line is in a commercially useful flowering plant, such as but not limited to petunia, geranium or begonia; or in a commercially useful food crop, such as but not limited to tomato or lettuce; or in a plant in which the plant tissue is useful for other commercial purposes, such as in the fiber (e.g., cotton) or drug industries (e.g., cone flower) or the like.

It is also an object to provide a transformed plant or plant part, in which the cells, organs, flowers, tissues, seeds or progeny, comprise the EIN2 overexpressing gene or active fragment thereof; the EIN3 overexpressing gene or active fragment thereof; or in an alternative embodiment, the combined EIN2 and EIN 3 overexpressing genes or active fragments thereof. In addition, a cotton cell line produced by the foregoing methods is provided. The transformed plant or cell line is further provided, wherein the genes or active fragments thereof, comprise recombinant nucleic acids.

It is a further object to provide a transformed plant or plant part, in which the cells, organs, flowers, tissues, seeds or progeny comprise the controlled overexpression of ΕIΝ2 or ELN3 polypeptides, or in an alternative embodiment, the combined EIN2/ELN3 polypeptides, encoded by EIN2 or EIN3 genes or active fragments thereof, or by combined EIN2 and EIN3 genes or active fragments thereof.

Additional objects, advantages and novel features of the invention will be set forth in part in the description, examples and figures which follow, and in part will become apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an amino acid sequence comparison of ELN2 proteins from petunia, tomato, lettuce, and Arabidopsis. Amino acids that are conserved among all four proteins are shown as the consensus sequence in the bottom row.

FIG. 2 is an amino acid sequence comparison of petunia ELN3-like proteins (PEIL-1, PEIL-2 and PEIL-3) and Arabidopsis EIN3 protein. Amino acids that are conserved among all four proteins are shown as the consensus sequence in the bottom row. FIGs. 3A and 3B depict two petunia flowers 8 days after treatment with 5 ppm ethylene. The flower on the left (FIG. 3 A) is overexpressing a 1.1 kb segment of the petunia EIN2 gene; the flower on the right (FIG. 3B) is wildtype. FIGs. 4A and 4B depicts two petunia flowers 9 days after pollination. The flower on the left (FIG. 4A) is wildtype; the flower on the right (FIG. 4B) is overexpressing a 1.1 kb segment of the petunia EIN2 gene.

FIG. 5 is a photograph showing delayed flower senescence in two independent lines (ELN2-1 and ELN2-2) ofEIN2 co-suppressed tomato plants and a wild type plant as labeled. Flower clusters were cut from the plant, placed in a vial of water, and treated with 10 ppm ethylene for 16 hours. Flowers are shown 48 hours after ethylene treatment.

FIG. 6 is a photograph of a gel showing overexpression of the TGV transcription factor in leaves of transgenic tomato plants. The chimeric transcription factor TGV was overexpressed in tomato under transcriptional control of a constitutive promoter. TGV expression was quantified by RT-PCR in T₀ plants from 10 independent transgenic lines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the tissue specific manipulation of several ethylene-signaling genes, permitting the controlled regulation of flower and/or leaf abscission. As a result of the present invention, new insights into the mechanisms involved in the ethylene signaling pathway are evident. In a particularly preferred embodiment of the invention, the methods comprise overexpression of the EIN2 and or EIN3 genes in selected transgenic commercially useful plants, such as flower crops, including but not limited to, petunia or geranium or begonia; food crops, including but not limited to, lettuce and tomato; or in a plant in which the plant tissue is useful for other commercial puφoses, such as in the fiber (e.g., cotton) or drug industries (e.g., cone flower) or the like; preferably to enhance flower longevity by the regulation of abscission. In an alternative embodiment, the trait of inducible premature leaf abscission is selected; while in yet another embodiment the selected trait comprises enhanced leaf longevity and reduced premature leaf abscission In wild type plants, ethylene binding to its receptor inactivates the activity of ethylene receptors (presumably causing a reduction in the histidine kinase activity), and consequently causing induction of the ethylene response through activation (de-repression) of the signaling pathway. Loss-of-function ethylene receptor mutants have been shown to function as negative regulators of the signaling pathway and show significant functional overlap. Moreover, binding of ethylene to the receptor(s) presumably inhibits biochemical activity.

A number of biological stresses are known to induce ethylene production in plants, including wounding, abscission, bacterial, viral or fungal infection, and treatment with elicitors, such as glycopeptide elicitor preparations from fungal pathogen cells. In the case of abscission, a particular layer of cells in a zone located between the base of the leaf stalk and the stem (the abscission zone) responds to a complex combination of ethylene and other endogenous plant growth regulators by a process that is, to date, not fully understood. However, the effect of "abscission" is the controlled loss of part of the plant, typically localized; the result of which is visible as a dead leaf or flower, or as softening or "ripening" of fruit, ultimately leaving the plant wounded at the point of separation. "Senescence" refers to aging of a plant part, such as a flower, but it does not necessarily involve separation of that part from the plant. Often senescence is associated with chlorophyll degradation, loss of nutrients from the tissue, and loss of turgor.

By "plant," as used herein, means any whole plant, or any part thereof, of wild type, treated, genetically manipulated or recombinant plant or plant part, including transgenic plants. The term broadly refers to any and all parts of the plant, including the plant cell, tissue, flower, leaf, stem, root, organ, and the like, and also including seeds, progeny and the like, whether such part is specifically named or not.

EIN 2. Homologs of the EIN2 gene, as it was originally identified in Arabidopsis (e.g., Alonso et al, 1999), were cloned by the inventors in three diverse species, petunia (Petunia x hybrida) lettuce (Lactuca sativa) and geranium (Pelargonium x hortorum). Full- length cDNAs of the petunia and lettuce EIN2 genes shared 56 and 57% nucleotide identity, respectively, with Arabidopsis EIN2. An isolated 450 bp region of the geranium EIN2 gene was found to share 67% nucleotide identity with Arabidopsis EIN2.

In a preferred embodiment of the invention, the petunia EIN2 gene is shown to play a critical role in regulating flower senescence. Thus, manipulating EIN2 expression, as described herein, causes significant increases in flower longevity and flower number in greenhouse and field trials in a representative plant species. The increased flower longevity was also shown to be heritable in the progeny of primary transformants. Consequently, the gene manipulation provides a stable, heritable trait.

Significantly, the methods utilizing overexpression of EIN2, as demonstrated in the preferred embodiment in petunia, has proven to be much more effective than antisense expression methods described in the prior art for increasing flower life in a commercially useful plant species by suppressing premature or early flower abscission.

EIN 3. As originally noted in Arabidopsis, the EIN3 genes comprise a small family of transcription factors that regulate the expression of ethylene-responsive genes (see, e.g. , Chao et al, 1997). Three homologs of the Arabidopsis EIN3 genes were isolated by the inventors from petunia, designated PEILl (Petunia x hybrida EIN3-like), PEIL2, and PEIL3. These genes share 50-59% nucleotide identity with Arabidopsis EIN3 and the proteins share 50-73% amino acid sequence similarity. Moreover, in a preferred embodiment of the invention, manipulating expression of one of these EIN3 homologs, PEIL2, by the methods defined herein, increases flower longevity in a commercially useful plant species by suppressing premature or early flower abscission, as exemplified by petunia.

Significantly, as demonstrated in a preferred embodiment, it has been demonstrated that only overexpression of PEIL2 is effective in increasing flower longevity. Antisense expression of PEIL2 had no effect on flower senescence and abscission. Moreover, antisense expression of the other two petunia EIN3 genes, PEILl and PEIL3, also proved to have no effect on flower longevity.

The term "homolog," meaning biological molecules that are "homologous" to each other, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. This similarity can occur either within the same species (e.g., PEILl and PEIL2 are homologs) or among different species (e.g., Arabidopsis ELN2, tomato ELN2, and petunia EIN2 are also homologs). The more formal definition is to refer to homologs between species as "orthologs," which are chromosomal DNA carrying the same genetic loci. When carried on a diploid cell there is a copy of the homolog from each parent but both terms are used. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they share identity at that position. The identity between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are the same, then the two sequences share 50% identity. If 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% identity.

"Identity" refers to the percent of bases or amino acids that are identical between two sequences, it refers to either DNA or proteins. "Similarity" is only used in reference to proteins, it refers to the percent of amino acids that are similar (i.e., have similar structures and polarity), but are not necessarily identical. The percent similarity of two proteins is always greater than or equal to the percent identity. The biological significance of identity and similarity is that they predict the probability that two genes or proteins have similar functions, i.e., the more similar the sequences are, the more similar the functions are likely to be to each other.

In an alternative embodiment of the invention, the research focussed on cotton, with two major goals: to reduce early-season flower abscission and accelerate late-season leaf abscission. Abscission of cotton flower buds ("squares") is caused by biotic and abiotic stress, and can significantly reduce cotton yields. Leaf abscission is necessary to clear the cotton plant of debris before mechanical harvesting. This is currently induced by herbicide application. Since cotton flower and leaf abscission are positively regulated by the plant hormone ethylene, these responses are, in a preferred embodiment, manipulated by altering expression of ethylene signaling genes.

Square abscission is reduced through co-suppression of the cotton EZ/V2 gene. Promoters were evaluated for driving E/N2 expression, e.g. , a constitutive promoter and a flower pedicel specific promoter from a cotton chitinase gene, although the invention is not intended to be so limited. Other promoters suitable for this puφose would also be known to one of skill in the art, and their use is encompassed by the present invention.

Preliminary work was conducted in tomato to test the efficacy of these approaches. Constitutive over-expression of the tomato E/N2 gene was found to effectively delay flower abscission by at least five- fold (5X) over that of normal wild-type plants. For example, wild-type tomato flowers treated with ethylene abscised 24 hours after treatment, while the transgenic flowers remained attached to the pedicel for at least 5 days after treatment (see FIG. 5, and as disclosed in greater detail in the examples that follow). Moreover, when progeny of the primary transformants were grown, it was confirmed that the controlled, delayed flower abscission trait is heritable in subsequent generations.

Consequently, to extend the foregoing work into cotton. The cotton EIN1 gene was cloned and the clones sequenced, to permit the construct for constitutive co-suppression to be assembled and to be transformed into Agrobαcteriu . The cotton chitinase promoter, when evaluated in tomato, has been shown to drive pedicel-specific expression of a marker gene.

Cotton leaf abscission is accelerated through over-expression of i Q Arabidopsis E/N3 (AtEIN3) gene. Plants are developed in which chemically-inducible promoters are evaluated for their ability to drive AtEIN3 expression. By determining the optimal expression pattern for AtEIN3, developmentally-regulated promoters are selected, although one of ordinary skill in the art could adapt other promoters for this puφose by following the examples that follow herein below. Since one of the promoters is inducible by ethanol, leaf abscission is accelerated in such plants by ethanol treatment, it becomes possible to control leaf removal from the plants by less expensive compositions, using methods in the field that were less toxic to the plants and the environment than herbicides. Using a tomato model system, a glucocorticoid-inducible gene system was developed for regulating leaf abscission. In plants produced in which a glucocorticoid-inducible transcription factor (such as, but not limited to, TGV) was over-expressed (FIG. 6), no negative effects on plant growth and/or development was detected. Screening of these plants by recognized reverse transcriptase-polymerase chain reaction (RT-PCR) techniques, identified a series of transgenic lines having high levels of overexpression, which when crossed with a second set of transgenic plants containing the AtEIN3 gene under transcriptional control of a glucocorticoid-inducible promoter, demonstrate the controlled regulation of leaf abscission in accordance with a preferred embodiment of the invention. In yet another alternative embodiment, the construct comprises the transcription factor and AtEIN3 over-expression cassette on the same transfer DNA (tDNA).

In sum, preferred embodiments of the invention should be construed to include nucleic acid comprising isolated EIN2 having > 50% identity to Arabidopsis EIN2, which when placed under the control of a promoter acceptable to the selected plant species, results in the over-expression of EIN2 in the selected plant species as demonstrated by delayed flower abscission. Also included is nucleic acid embodiment comprising isolated EIN3 (or EIN3-like gene), having > 50% identity to Arabidopsis EIN3, which when placed under the control of a promoter acceptable to the selected plant species, results in the over-expression of ELN3 in the selected plant species as demonstrated by inducible leaf abscission.

In another preferred embodiment, the resulting plant lines are crossed, resulting in the combined effect of the over-expression of both ELN2 and ELN3, which produces plants having delayed flower abscission and inducible leaf abscission. Further included within the present invention is any mutant, derivative, or homologue of the foregoing, or fragment thereof, which encodes the regulated ELN2-controlled delayed flower abscission or the regulated EIN3 -controlled inducible leaf abscission, or in a preferred embodiment, the combination thereof.

In accordance with the present invention, nucleic acid sequences include, but are not limited to DNA, including and not limited to cDNA and genomic DNA; RNA, including and not limited to mRNA and tRNA, and may include chiral or mixed molecules. Preferred nucleic acid sequences include, for example, those set forth in petunia EIN2 (SEQID NO:l), lettuce EIN2 (SEQID NO:2), tomato ELN2 (SEQID NO:3), geranium partial EIN2 (SEQID NO:4), begonia partial EIN2 (SEQID NO:5), and cotton EIN2 (SEQID NO:6), as well as modifications in those nucleic acid sequences, including alterations, insertions, deletions, mutations, homologues and fragments thereof encoding the regulatory protein, ELN2 in the ethylene response pathway resulting in plants exhibiting EIN2-controlled delayed flower abscission. Also provided are homologs of the Arabidopsis EIN3 genes, as represented by those EIN3-like genes isolated from petunia, and designated PEILl (Petunia x hybrida EIN3-like) (SEQID NO:7), PEIL2 (SEQID NO:8), and PEIL3 (SEQID NO:9), as well as those isolated nucleic acids encoding a combination of regulatory proteins, EIN2/EIN3. The PEILl, PEIL2, PEIL3, and petunia, begonia and geranium ELN2 full length or nearly full length sequences herein are novel, although partial sequences were available for tomato, cotton, and lettuce EIN2 in at least one public database, such information was previously incomplete or contained errors. A "fragment" of a nucleic acid is included within the present invention if it encodes substantially the same expression product as the isolated nucleic acid, or if it encodes peptide(s) disclosed herein having the desired regulatory effect(s) on flower and/or leaf abscission.

The invention should also be construed to include peptides, polypeptides or proteins comprising ELN2 and/or ELN3 (or ELN3-like, e.g., as defined by those encoded by PEILl, 2, or 3) alone or in EIN2/ELN3 (or ELN3-like) combination, as encoded by the foregoing defined nucleic acid sequences (SEQID Nos: 1-9) or any mutant, derivative, variant, analog, homolog or fragment thereof, having flower (and/or leaf) abscission controlling activity in the ethylene signaling pathway. The terms "protein," "peptide," "polypeptide," and "protein sequences" are used interchangeably within the scope of the present invention, and include, but are not limited to the amino acid sequences corresponding to nucleic acid SEQID NOs: 1-9, as well as those sequences representing mutations, derivatives, analogs or homologs or fragments thereof having having flower and/or leaf abscission controlling activity in the ethylene response pathway. The invention also provides for analogs of proteins, peptides or polypeptides encoded by E/N2 or E/N3 (or ΕLΝ3-like, e.g., as defined by those encoded by PEILl, 2, or 3) alone or in ELN2/EIN3 (or ELN3-like) combination. "Analogs" can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. "Homolog" as previously defined refers to the subunit sequence similarity between two polymeric molecules, e.g., between two polypeptide molecules.

For example, conservative amino acid changes may be made, which although they alter the primary sequence of the peptide, do not normally alter its function. Conservative amino acid substitutions of this type are known in the art, e.g., changes within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; or phenylalanine and tyrosine. Modifications (which do not normally affect the primary sequence) include in vivo or in vitro chemical derivatization of the peptide, e.g., acetylation or carbonation. Also included are modifications of glycosylation, e.g., modifications made to the glycosylation pattern of a polypeptide during its synthesis and processing, or further processing steps. Also included are sequences in which amino acid residues are phosphorylated, e.g., phosphotyrosine, phosphoserine or phosphothreonine.

Also included in the invention are polypeptide embodiments which have been modified using ordinary molecular biology techniques to improve their resistance to proteolytic degradation or to optimize solubility or to render them more effective as a regulatory agent. Analogs of such peptides include those containing residues other than the naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic molecules. However, the polypeptides of the present invention are not intended to be limited to products of any specific exemplary process defined herein.

"Derivative" is intended to include both functional and chemical derivatives, including fragments, segments, variants or analogs of a molecule. A molecule is a "chemical derivative" of another, if it contains additional chemical moieties not normally a part of the molecule. However for the puφoses of this invention, the derivative molecule must still demonstrate ELN2 or EIN3 activity or a combination thereof. Nevertheless, such moieties may improve the molecule's solubility, absoφtion, biological half-life, and the like, or they may decrease toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 18th Edition (1990), Martin ed., Mack Publishing Co., Pa. Procedures for coupling such moieties to a molecule are well known in the art. Included within the meaning of the term "derivative," as used in the present invention, are "alterations," "insertions," and "deletions" of either nucleotides or peptides, polypeptides or the like. A "variant" or "allelic or species variant" of a protein refers to a molecule substantially similar in structure and biological activity to the protein. Thus, if two molecules possess a common activity and may substitute for each other, it is intended that they are "variants," even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical. A fragment of a polypeptide is included within the present invention if it retains substantially the same activity as the purified peptide, or if it has EIN2 or EIN3 (or EIN3-like, e.g., as defined by those encoded by PEILl, 2, or 3) alone or combined controlled EIN2/EIN3 (or ELN3-like) activity resulting in delayed flower abscission and/or with inducible leaf abscission.

In accordance with the invention, the EIN2 or EIN3 (or ΕIN3-like, e.g., as defined by those encoded by PEILl, 2, or 3) nucleic acid sequences employed in certain embodiments may be exogenous sequences. Exogenous or heterologous, as used herein, denotes a nucleic acid sequence which is not obtained from and would not normally form a part of the genetic makeup of the plant, cell, organ, flower or tissue to be transformed, in its untransformed state.

Transformed plant cells, tissues and the like, comprising nucleic acid sequences of EIN2 and or EIN3 (or ELΝ3-like, e.g., as defined by those encoded by PEILl, 2, or 3), such as, but not limited to, the nucleic acid sequence of SEQID NOs: 1-9, are within the scope of the invention. Transformed cells of the invention may be prepared by employing standard transformation techniques and procedures, such as, but not limited to, those set forth in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

By the term "nucleic acid encoding" the resulting plant cell and the like having controlled flower and/or leaf abscission activity, as used herein, is meant a gene encoding a polypeptide capable of controlling the abscission as described above. The term is meant to encompass DNA, RNA, and the like.

As described in the following Examples, EIN2 and E7N3 genes (including EIN3-like genes, e.g., as defined by PEILl, 2, or 3) encode proteins having specific domains located therein, for example, terminal extensions, transmembrane spans, TMl and TM2, nucleotide binding folds, a putative regulatory domain, and the C-terminus. A mutant, derivative, homolog or fragment of the subject gene is, therefore also one in which selected domains in the expressed protein share significant identity (at least about 50% identity to that of Arabidopsis) with the same domains in the preferred embodiment of the present invention so long as the activity of the expression product is that of EIN2 or EIN3, including EIN3-like protein as it is herein defined. It will be appreciated that the definition of such a nucleic acid encompasses those gene(s) having at least about 45-50% identity to corresponding gene(s) in Arabidopsis, in any of the described domains contained therein. In addition, when the term "identity" is used herein to refer to the domains of these proteins, it should be construed to be applied to identity at both the nucleic acid and the amino acid levels. Significant identity between similar domains in such nucleic acids or their protein products is considered to be at least about 45-50%, preferably the identity between domains is at least about 50%, more preferably at least about 60%, more preferably, at least about 70%, even more preferably, at least about 80%, yet more preferably, at least about 90% and most preferably the identity is about 99%, or in the protein expression products thereof.

According to the present invention, preferably, the isolated nucleic acid encoding the EIN2 or ELN3 polypeptide(or ELN3-like,polypeptide, e.g., as encoded by PEILl, 2, or 3) alone or as combined, or fragment thereof, is full length or of sufficient length to effect controlled regulation over flower longevity and abscission, as well as in some embodiments, leaf abscission, in resulting plant(s). In one embodiment the nucleic acid is at least about 500, in another it is at least about 1000 nucleotides in length. More preferably, it is at least 2000 nucleotides, even more preferably, at least 3500 nucleotides, yet more preferably, at least 4000 nucleotides, and even more preferably, at least 4900 nucleotides in length. In another embodiment, preferably, the putative or purified preparation of the isolated polypeptide(s) having abscission-controlling activity in the ethylene signal system is at least about 160 amino acids in length, in another it is at least 300 amino acids in length. More preferably, it is at least 500 amino acids, even more preferably, at least 1000 amino acids, yet more preferably, at least 1200 amino acids, and even more preferably, at least 1600 amino acids in length. In an additional embodiment the polypeptide encodes the full length EIN2 protein (e.g., as encoded by SEQID Nos: 1-6) or ELN3 protein (including ELN3-like protein, e.g., as encoded by PEILl, 2, or 3) or a regulated combined EIN2/EIN3 version thereof.

The invention further includes a vector or vectors comprising a gene encoding EIN2 and/or EIN 3 (or EIN3-like, e.g., as defined by those encoded by PEILl, 2, or 3). DNA molecules composed of a protein gene or a portion thereof, can be operably linked into an expression vector and introduced into a host cell to enable the expression of these proteins by that cell. Alternatively, a protein may be cloned in viral hosts by introducing a "hybrid" gene operably linked to a promoter into the viral genome. The protein may then be expressed by replicating such a recombinant virus in a susceptible host. A DNA sequence encoding a protein molecule may be recombined with vector DNA in accordance with conventional techniques. When expressing the protein molecule in a virus, the hybrid gene may be introduced into the viral genome by techniques well known in the art. Thus, embodiments of the present invention encompass the expression of the desired proteins in either prokaryotic or eukaryotic cells, or viruses, which replicate in prokaryotic or eukaryotic cells. Procedures for generating a vector for delivering the isolated nucleic acid or a fragment thereof, are well known, and are described for example in Sambrook et al, supra. Suitable vectors include, but are not limited to, disarmed Agrobacterium tumor inducing (Ti) plasmids (e.g., pBIN19) containing a target gene under the control of a vector, such as the cauliflower mosaic (CaMV) 35S promoter (Lagrimini et al, Plant Cell 2:7-18 (1990)) or its endogenous promoter (Bevan, Nucl. Acids Res. 72:8711-8721(1984)), tobacco mosaic virus and the like.

Once the vector or DNA sequence containing the constructs has been prepared for expression, the DNA constructs may be introduced or transformed into an appropriate host. Various techniques may be employed, such as protoplast fusion, calcium phosphate precipitation, electroporation, or other conventional techniques. As is well known, viral sequences containing a "hybrid" protein gene may be directly transformed into a susceptible host, or first packaged into a viral particle, and then introduced into a susceptible host by infection. After the cells have been transformed with the recombinant DNA (or RNA) molecule, or the virus or its genetic sequence is introduced into a susceptible host, the cells are grown in media and screened for appropriate activities. Expression of the sequence results in the production of the protein of preferred embodiments of the present invention.

Procedures for generating a plant cell, tissue, flower, organ or a fragment thereof, are well known in the art, and are described, for example, in Sambrook et al, supra. Suitable cells include, but are not limited to, cells from yeast, bacteria, mammal, baculovirus-infected insect, and plants, with applications either in vivo, or in tissue culture. Also included are plant cells transformed with the gene of interest for the puφose of producing cells and regenerating plants having modulated flower and/or leaf abscission capability. Suitable vector and plant combinations will be readily apparent to those skilled in the art and can be found, for example, in Maliga et al, 1994, Methods in Plant Molecular Biology: A Laboratory Manual, Cold Spring Harbor, New York).

Transformation of plants may be accomplished, e.g., using grobαcter/wm-mediated leaf disc transformation methods described by Horsch et al, 1988, Leaf Disc Transformation: Plant Molecular Biology Manual) or other methods known in the art. Numerous procedures are known in the art to assess whether a transgenic plant comprises the desired DNA, and need not be reiterated at this point.

The expression of the desired protein in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, but are not limited to, the SV40 early promoter (Benoist et al, Nature (London) 290:304-310 (1981)); the yeast gal4 gene promoter (Johnston et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982)) and the exemplified pYES3 PGK1 promoter. In addition, inducible promoters are used as described below. As is widely known, translation of eukaryotic mRNA is initiated at the codon encoding the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the desired protein does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The desired protein encoding sequence and one or more operably linked promoters may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the desired protein may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome. For expression of the desired protein in a virus or plant, the hybrid gene operably linked to a promoter is typically integrated into the viral genome, be it RNA or DNA. Cloning into plants is well known and thus, one of skill in the art will know numerous techniques to accomplish such cloning. Cells that have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more reporter genes or markers which allow for selection of host cells which contain the expression vector. The reporter gene or marker, such as kanamycin resistance, may complement an auxotrophy in the host (such as leu2, or ura3, which are common yeast auxo trophic markers), biocide resistance, e.g., antibiotics, or the effect can be seen as a physical response, such as flower or leaf abscission or the like. A selectable marker gene, such as kanamycin resistance, can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection.

Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. The cDNA expression vectors incoφorating such elements include those described by Okayama, H., Mol. Cell. Biol. 5:280 (1983), and others.

In another embodiment, the introduced sequence is incoφorated into a plasmid or viral vector capable of autonomous replication in the recipient host cell. Any of a wide variety of vectors may be employed for this puφose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. The invention further defines methods for manipulating the nucleic acid in a plant to permit the regulation, control or modulation of abscission, flower or leaf senescence, flower maturation, fruit ripening, or response to stress. In a preferred embodiment the method initiates or enhances one or more of the above responses; whereas, in another preferred embodiment the method inhibits or prevents one or more of the above responses. Thus, methods of the present invention define embodiments in which controlled flower abscission activity is prevented or inhibited. By "prevention" is meant the cessation of flower drop for a period of time beyond which ethylene pathway-controlled flower abscission (or in the alternative leaf abscission) normally occurs in the selected plant species. By "inhibition" is meant a statistically significant reduction in flower abscission activity (or in the alternative leaf abscission), as compared with plants, plant cells, organs, flowers or tissues grown without the inhibitor or disclosed method of inhibition. Preferably, the inhibitor reduces flower abscission by at least 20 %, more preferably by at least 50%, even more preferably by 80% or greater, and also preferably, in a dose-dependent manner. Once inhibitors satisfying these requirements are identified, the utilization of assay procedures to identify the manner in which flower abscission is inhibited are particularly useful.

Similarly, methods of the present invention are defined in which leaf abscission activity is "induced," "initiated," "stimulated" or "enhanced" if there is a statistically significant increase in the amount of controlled leaf abscission activity, as compared with plants, plant cells, organs, flowers or tissues grown without the enhancer or disclosed method of enhancement. Preferably, the enhancer increases controlled flower and/or leaf abscission by at least 20 %, more preferably by at least 50%, even more preferably by 80% or greater, and also preferably, in a dose-dependent manner. Once enhancers satisfying these requirements are identified, the utilization of assay procedures to identify the manner in which controlled leaf abscission is enhanced are particularly useful. In a preferred embodiment of the invention the enhanced leaf abscission is under the control of an inducing composition. For example, encompassed are E/N3 cDΝA sense and antisense constructs (including EIN3-like cDΝA as defined herein), wherein inducible promoters, such as a glucocorticoid-inducible promoter (Bohner et al., Plant J. 19:87-95 (1999)) or an ethanol-inducible promoter (Salter et al, Plant J. 16: 127-132 (1998)), or an ecdysone agonist-inducible promoter (Martinez et al, Plant J. 19:97-106 (1999)) or others, are incoφorated to regulate the expression of the gene. Over-expression of EIN3 in Arabidopsis was shown to induce several ethylene responses, including inhibition of leaf expansion. Since growth was severely reduced in EIN3 over-expressing Arabidopsis lines, an inducible promoter was used to prevent undesirable pleiotropic effects of the transgene. When leaves were treated, for example in the glucocorticoid inducible promoter model, treatment involves the application of a solution of glucocorticoid dexamethasone, and the result is severe leaf epinasty of the transgenic plants.

A selected glucocorticoid-inducible transcription factor is TGV, which is essential for the operation of ΕIΝ3. TGV (a chimeric gene comprising a tetracylene repressor, a glucocorticoid receptor, and the transcriptional activator VPI 6) allows chemical induction of ELN3 expression, which in turn accelerates leaf abscission. The DNA samples from 10 independent transgenic lines (a portion of the TGV gene that was amplified from RNA isolated from leaves of the transgenic tomato plants by RT PCR), were run in a 1% agarose gel in TBE buffer at 120 V for 1 hour as shown in FIG. 6. The ladder is a 1 kb DNA ladder from Invitrogen (Carlsbad, CA).

Similar inducible effects were seen for the other constructs, such as when the selected transgenic plants were sprayed with ethanol solution or the ecdysone agonist, muristeroneA. However, for cost and efficiency puφoses, the ethanol-inducer is a good selection for field use. By comparison, treatment of wild type plants with each of the chemical inducers had no visible effects on growth or development.

Selected embodiments of the invention further contain constructs comprising other regulatory genes in a sense or antisense direction, in addition to EIN2 and/or ELN3, alone or in EIN2/EΓN3 combination. For example, when the constructs further contained an antisense copy of CTR 1 (a negative regulator of ethylene response), the transcriptional control of the inducible promoters, the ethylene response and resulting leaf abscission was as previously noted. However, the effect of treatment of the CTR1 antisense plants with dexamethasone, ethanol, and muristeroneA resulted in a rate of leaf abscission that was less rapid as compared with that which was seen in plants in which ELN3 was over-expressed. The invention further features an isolated preparation of a nucleic acid that is antisense in orientation to a portion or all of a plant gene, such as is described for constructs comprising the antisense CTRl gene. The antisense nucleic acid should be of sufficient length as to inhibit expression of the target gene of interest. The actual length of the nucleic acid may vary, depending on the target gene, and the region targeted within the gene.

Typically, such a preparation will be at least about 15 contiguous nucleotides, more typically at least 50 to even more than 500 contiguous nucleotides in length.

As used herein, a sequence of nucleic acid is considered to be antisense when the sequence being expressed is complementary to, and essentially identical to the non-coding DNA strand of the selected gene, but which does not encode the expression product of the gene, such as CTRl . "Complementary" refers to the subunit complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are said to be complementary to each other. Thus two nucleic acids are considered to be complementary when a substantial number (at least 50%) of the corresponding positions in each of the molecules are occupied by nucleotides which normally base-pair with each other (e.g., A:T and G:C nucleotide pairs).

By "transgenic plant" as used herein, is meant a plant, plant cell, tissue, flower, organ, including seeds, progeny and the like, or any part of a plant, which comprise a gene inserted therein, which gene has been manipulated to be inserted into the plant cell by recombinant DNA technology. The manipulated gene is designated a "transgene." The "non-transgenic," but substantially homozygous "wild type plant," as used herein, means a non-transgenic plant from which the transgenic plant was generated. The transgenic transcription product may also be oriented in an antisense direction as describe above. The generation of transgenic plants comprising modified or exogenous sense or antisense DNA encoding EIN2 and/or ELN3 of the ethylene signaling pathway, may be accomplished by transforming the plant with a plasmid, liposome, or other vector encoding the desired DNA sequence. Such vectors would, as described above, include, but are not limited to the disarmed Agrobacterium tumor-inducing (Ti) plasmids containing a sense or antisense strand placed under the control of a strong constitutive promoter, such as 35CaMV 35S or under an inducible promoter. Methods of generating such constructs, plant transformation and plant regeneration methods are well known in the art once the sequence of the gene of interest is known, for example as described in Ausubel et al, 1993, Current Protocols in Molecular Biology, Greene & Wiley, New York. In accordance with the present invention, plants included within its scope include both higher and lower plants of the Plant Kingdom. Mature plants, including rosette stage plants, and seedlings are included in the scope of the invention. A mature plant, therefore, includes a plant at any stage in development beyond the seedling. A seedling is a very young, immature plant in the early stages of development. Transgenic plants are also included within the scope of the present invention, having a phenotype characterized by EIN2- and/or EIN3-controlled flower and/or leaf abscission. Preferred plants of the present invention include, but are not limited to, high yield crop species for which cultivation practices have already been perfected (including monocots and dicots), or engineered endemic species.

Preferred plants in which the ELN2-control of flower abscission is exhibited include any commercially useful or valuable home-grown flowering species, e.g., roses, carnations, or chrysanthemums, and many others, or leafy ornamental plants, such a geranium and many others. Preferred plants in which the EIN3-control of leaf abscission is exhibited include any commercially valuable or home-grown leafy green ornamental plant, such as Ficus, palms, and the like, in which longevity of the leaf stem on the plant (delayed abscission) is of particular relevance, as are harvested plants in which inducing premature leaf abscission facilitates mechanical or other means of harvesting. Delayed flowering in such plants may also be advantageous. However, a particularly preferred advantage of the present invention is seen in plants, particularly including commercially valuable flowering plants, such as cotton and the like, in which longevity of the flower on the stem (delayed abscission) is of particular relevance, e.g., harvested flowers or flower parts, such as in food crops, e.g., broccoli, cauliflower, etc. or certain flowering herbs or spices, and wherein harvesting, such as by a mechanical harvester, is facilitated by the early removal of plant leaves and plant debris by inducing premature leaf abscission.

The present invention is further described in the following examples. These examples are not to be construed as limiting the scope of the appended claims.

EXAMPLES

Example 1 - Homologs of Arabidopsis EIN2 in commercially useful species. To isolate homologs of the Arabidopsis EIN2 gene in commercially useful plant species, nucleotide sequences of EIN2 genes from several different species were compared to identify conserved regions. RN4 extraction and cDNA synthesis: RΝA was extracted from petunia and lettuce leaves according to the method of Reuber and Ausubel (Plant Cell 8:241-249 (1996); see also Ausubel et al., eds, 1987; Sambrook et al., 1989, and the various references cited therein 1989). Briefly, approximately 500 mg of tissue was pulverized in ESPE buffer (ESPE buffer = 100 mM Tris, pH 8.0; 50 mM EDTA; 250 mM ΝaCl; 1% (w/v) SDS) by rapid mixing with glass beads. CTAB buffer (CTAB buffer = 200 mM Tris, pH 8.0;50 mM EDTA; 2 M ΝaCl; 2% (w/v) CTAB available from Sigma, St. Louis, MO) was added and the mixture was then extracted with equal volumes of phenol and chloroform. Nucleic acids were ethanol precipitated, and RNA was then isolated by precipitation in 2M LiCl. RNA was ethanol precipitated, washed with 80% ethanol, and then resuspended in water.

Concentration and quality of the RNA was determined by spectrophotometry and denaturing gel electrophoresis as outlined by Maniatis et al, 1989 and Sambrook et al, 1989 and the various references cited therein.

First strand cDNA synthesis was performed using a "SUPERSCRIPT" Preamplification System in accordance with manufacturer's instruction (Invitrogen, Carlsbad,CA). RNA was isolated from geranium by extraction in SDS-phenol and purification by LiCl precipitation (Kneissl and Deikman, Plant Physiol. 112: 537-547 (1996)). Geranium cDNA was synthesized by the Advantage RT for PCR Kit (Clontech, Palo Alto, CA). Primer design for cloning ofEIN2 genes: To identify sequences with homology to

Arabidopsis EIN2, the GENBANK nucleotide sequence database was searched with the amino acid sequence of the ELN2 carboxyl terminus. The carboxy-terminal sequence of ELN2 was used to avoid isolating Nramp proteins that share strong identity with the ELN2 amino-terminus. A number of sequences with homology to the deduced amino acid sequence of Arabidopsis EIN2 were identified, including genes from soybean (Glycine max), loblolly pine (Pinus radiata), maize (Zea mays), tomato (Lycopersicon esculentum), petunia (Petunia hybrida), lotus (Lotus japonicus), and cotton (Gossypium hirsutum). These sequences were aligned using a clustal program, and areas of strong homology between sequences were identified. Nucleotide sequences of these homologous regions were compared to isolate the areas of greatest nucleotide identity. Degenerate primers were then designed to hybridize to these areas of strong nucleotide identity. A total of 38 degenerate primers were used. Once the petunia and lettuce EIN2 cDNAs were isolated and sequenced, new degenerate primers were designed for amplification of geranium EIN2 homologs. These primers were designed by identifying regions of strong homology among the lettuce, petunia, tomato (Lycopersicon esculentum) and Arabidopsis EIN2 genes.

Amplification ofEIN2: Using first strand cDNA as template, the petunia and lettuce EIN2 genes were amplified using the Expand High Fidelity PCR System (Roche, Indianapolis, IN) (see, e.g., PCR: The Polymerase Chain Reaction, (Mullis et al., eds,

1994)). Several combinations of degenerate oligos were used in the preliminary experiments to identify pairs that lead to amplification of a product with an expected size (see, e.g., Oligonucleotide Synthesis (Gait, ed., 1984)).

To determine the optimal hybridization temperature for the degenerate oligos, amplifications were performed at 12 different annealing temperatures. The PCR conditions were as follows: an initial denaturation step at 94° C for 2 minutes, followed by 40 cycles of 94° C for 30 seconds, a gradient of 47-56° C for 2 minutes, and then 72° C for 90 seconds. The reactions were run in a DNA Engine PTC 200 equipped with an alpha unit 96 well assembly (MJ Research, Incline Village, NV). Based on these experiments the degenerate primers:

YTNGAYGARTTYTGGGG (5' end)(SEQID NO: 10) and GCCTGAANGAYTGAAGAAGCT (3' end)(SEQID NO:l 1) were used to generate a 1.1 kb PCR product from petunia cDNA. For lettuce, the degenerate primers: CTWGATGARTTYTGGGG (5 ' end) (SEQID NO: 12) and

CCAHACTCCAAAGCTTATTATCAATCVGGTTTCCA (3'end)(SEQID NO:13) were used to amplify a 1.1 kb region from lettuce cDNA.

The PCR conditions for the final experiments were as follows: an initial denaturation step at 94° C for 2 minutes followed by 40 cycles of 94° C for 30 seconds, 53.5° C for 45 seconds, 72° C for 90 seconds, followed by 72° C for 10 minutes. For geranium, a total of 4 degenerate primers were evaluated. The degenerate primers:

GARCARTTTGGTGTAGC (5' end)(SEQID NO:28) and CTCHGGCCKRCTYTCCAT (3' end)(SEQID NO:29) were used to amplify 0.5 kb regions of the geranium EIN2 gene. The PCR conditions were 95° C for 7 minutes followed by 40 cycles of 95° C for 1 minute, 55° C for 1 minute, and 72° C for 1 minute, and then 72° C for 10 minutes.

PCR products were cloned into a TOPO TA cloning vector according to manufacturer's instructions (Invitrogen, Carlsbad, CA). Plasmid DNA was isolated from several positive colonies using the Perfect Plasmid Mini-prep kit (Eppendorf Scientific Inc., Westbury NY). 400 ng of purified plasmid DNA was sequenced in an automated DNA sequencer (Model # 377, PE Biosystem) using Big Dye terminator (PE Biosystems, Foster City, CA). Both strands were completely sequenced at least twice.

Identification of full-length cDNA sequences by RACE: A total of 38 degenerate primers were designed, and multiple combinations of these primers were used to amplify regions of EIN2 cDNAs from petunia and lettuce. The remaining 5' and 3' ends of the cDNAs were isolated by random amplification of cDNA ends (RACE), and full-length cDNAs were then amplified by PCR.

Once partial cDNA sequences were identified by PCR with degenerate primers, the remaining 5' and 3' cDNA sequences of the lettuce and petunia ELN2 genes were isolated by (RACE). The Gene Racer amplification system (Invitrogen, Carlsbad, CA) and EXPAND Taq polymerase (Perkin Elmer) were used for all RACE experiments. The primers used for RACE are listed in Table 1 (lettuce) and Table 2 (petunia). Once the sequence of the 5' and 3' ends of each cDNA had been determined, full-length cDNAs were isolated by RT-PCR using the primers listed in Tables 1 and 2.

Table 1. Primers used for amplification of Lettuce EIN2 cDNA

Table 2. Primers used for amplification of Petunia EIN2 cDNA

Comparison of the lettuce and petunia EIN2 genes with Arabidopsis EIN2: The petunia EIN2 gene shares 56-86 % nucleotide identity (Table 3) and 60-88% amino acid sequence similarity (Table 4 and FIG. 1) with Arabidopsis, lettuce, and tomato EIN2. The lettuce EIN2 gene is 57-58% identical to the other EIN2 genes and the lettuce EIN2 protein shares 61-66% amino acid sequence similarity with the other ELN2s. Table 3. Nucleic Acid Sequence Identity (%) of E1N2 cDNAs

Table 4. Amino Acid Sequence Similarity (%) of EIN2 Proteins

A 464 bp region near the 3 'end of the geranium EIN2 gene was isolated by RT-PCR using degenerate primers as described above. This region shares from 65-69% nucleotide identity and 71-79% amino acid sequence similarity with Arabidopsis, lettuce, tomato, and petunia EIN2.

FIG. 1 presents a consensus sequence comparing the amino acid sequence of ELN2 in petunia (SEQID NO:30), tomato (SEQID NO:31), lettuce (SEQID NO:32) and Arabidopsis (SEQID NO:33). A "consensus sequence" offers a comparison of three or more amino acid sequences either within a species or among different species. Consensus sequences are used to show regions of high similarity among several sequences. Consequently, they are used to predict similarity of function. If several different proteins share the same amino acid sequence, then it is likely that they also share the same function.

As shown in the present invention, the consensus sequences (SEQID NO:34) among the EIN2 gene expression products of the various species are strong, thus confirming that they are homologs of the Arabidopsis EIN2 genes. Even the lowest levels of nucleotide identity in the petunia EIN2 (56%) are still considered to be high, providing evidence that these genes are homologs of Arabidopsis EIN2.

Cloning of petunia EIN3 (PEIL) genes: Segments of the PEILl and PEIL2 genes were isolated by RT-PCR using the methods described above. A third gene, PEIL3, was isolated from a petunia flower cDNA library by screening with radio-labeled probes made from the Arabidopsis EIN3 gene and three tomato EIN3 homologs (Tieman et al, Plant J. 26:47-58 (2001)). A full-length PEIL2 cDΝA was also isolated through screening of this library. The remaining segments of the PEILl and PEIL3 cDΝAs were cloned by RACE as described above. Production of transgenic petunia plants: The function of ELΝ2 in regulating petunia flower senescence was analyzed by antisense expression and overexpression of a 1.1 kb region of the petunia EIN2 gene under transcriptional control of the constitutive CAMV 35S promoter. A 1.1 kb segment of the petunia EIN2 cDNA spanning from nucleotide 2824 to 3940 was cloned into a vector downstream from a cauliflower mosaic virus promoter (CAMV 35S) and upstream of the Agrobacterium nopaline synthase (nos) terminus region. Two separate constructs were made with the EIN2 cDNA fragment in either the sense or antisense orientation. For the petunia EIN3 constructs, all cDNAs were under transcriptional control of the figwort mosaic virus promoter (Richins et al., Nucleic Acid Res. 15: 8451-8466 (1987)) and were followed by the nos terminus region. Three constructs were made containing each of the three PEIL cDNAs in antisense orientation. These contained a 1.9 kb segment from the 3' end of PEILl, a full length (2.7 kb) cDNA of PEIL2, and a 1.1 kb segment from the 3' end of PEIL3. A fourth EIN3 construct was made with the PEIL2 cDNA in sense orientation.

Each of the constructs was then cloned into a transformation vector containing a gene for kanamycin resistance within the transgene. The transformation vector was transferred to Agrobacterium through triparental mating. Petunia plants (cv Mitchell Diploid) were transformed with this construct through Agrobacterium-mediated transformation.

Analysis of transgenic plants: Approximately 100 transgenic petunia lines were generated for each construct and evaluated for changes in flower longevity. All plants were grown under standard greenhouse conditions. Presence of the transgene was confirmed in T₀ plants through PCR by amplifying a segment of the kanamycin resistance gene. Two different assays were used to determine differences in ethylene sensitivity.

Using the first screening method, flowers were cut from the plant on the day before anthesis and placed in vials of water. The flowers were then sealed in a glass container and treated overnight with 2-5 ppm ethylene for 16 hours. The flowers were then placed in a growth room and the day on which the flowers completely wilted was recorded.

Using the second screening assay, flowers were pollinated on the plant in the greenhouse on the day before anthesis and the number of days to wilting was recorded. Seed was collected from plants that displayed increased flower longevity and these plants were also analyzed for presence of the transgene and flower longevity in subsequent generations.

Wildtype flowers lasted an average of 1.5 days after ethylene treatment, whereas by comparison, overexpression of EIN2 extended flower life up to 11 days (Table 5 and FIGs. 3A and 3B). Overexpression of EIN2 also extended flower longevity after pollination, increasing flower life from 2 days after pollination in wildtype plants to up to 11 days in the transgenic lines (Table 5 and FIGs. 4A and 4B).

Table 5. Increased flower longevity in petunia plants overexpressing the petunia EIN2 gene.

Comparison with the antisense method for expression EIN2: It was also found that antisense expression of EIN2 did increased flower longevity, but the effect was not as strong as in the overexpressing lines. Antisense flowers lasted up to 5 days after ethylene treatment (Table 6). A total of eight overexpressing and four antisense lines exhibited enhanced flower longevity. Therefore overexpression of EIN2 was more effective than antisense expression in extending flower life.

Table 6. Increased flower longevity in petunia plants with antisense expression of the petunia EIN2 gene.

Inheritance of the enhanced flower longevity phenotype: To study the inheritance of the enhanced flower longevity phenotype, the pollination assay was repeated on 12 plants from each line in the subsequent (TI) generation. For two of the overexpressing lines, 182 and 183, each plant that contained a transgene also displayed a phenotype, indicating that the trait was heritable (Table 7). For two of the other lines, 144 and 150, none of the 12 plants displayed increased flower longevity, although several plants in each line had inherited a transgene. In the remaining lines, some of the plants that contained a transgene also displayed a phenotype, while others did not, indicating that the transgene was silenced in some plants. In the EIN2 antisense lines, none of the TI plants exhibited a phenotype, indicating that the transgene was also silenced in these plants.

Table 7. Inheritance of flower longevity phenotype in petunia plants overexpressing the petunia EIN2 gene: TI generation.

Comparison of Petunia EIN3 homologs with Arabidopsis EIN3: Two homologs of the Arabidopsis EIN3 gene were cloned from petunia by screening a petunia flower cDNA library with Arabidopsis EIN3 and tomato EIN3 homologs. A third gene was isolated by RT-PCR. The three petunia genes share 50-59 % nucleotide identity and 50-73 % amino acid sequence similarity with Arabidopsis EIN3 (Table 8). The areas of strongest homology occurred in the amino terminal half of the protein (FIG. 2), which is the area that seems to be involved in DNA binding. Table 8. Sequence comparison of Petunia EIN3 homologs with Arabidopsis EIN3.

FIG. 2 presents a consensus sequence comparing the amino acid sequence of three petunia ELN3-like polypeptides (PEILl (SEQID NO:35), PEIL2 (SEQID NO:36) and PEIL3 (SEQID NO:37)) with Arabidopsis EIN3 (SEQID NO:38). As shown in the present invention, the consensus sequences (SEQID NO:39) among the EIN3 (and EIN3-like) genes of the various species are strong, thus confirming that they are homologs of the Arabidopsis EIN3 gene. Even the lowest levels of nucleotide identity (50%) are considered to be high, indicating a high probability that these genes are homologs of Arabidopsis EIN3.

Role of petunia EIN3 genes in regulating flower senescence: To analyze the function of the petunia EIN3 (or EIN3-like) genes in regulating flower senescence, each gene was antisensed in petunia under transcriptional control of the constitutive CAMV 35S promoter. One of the genes, PEIL2, was also overexpressed under control of the CAMV 35 S promoter. Over 70 transgenic lines were produced for each construct. Only plants that were overexpressing PEIL2 displayed an increase in flower longevity (Table 9); whereas by comparison, the antisense expression of each gene had no effect.

Table 9. Petunia plants with antisense expression or overexpression of petunia EIN3 genes.

The absence of a phenotype in the EIN2 and EIN3 antisense plants demonstrates that overexpression is more effective in altering gene expression in petunia. Overexpression of PEIL2 increased flower longevity up to 9 days after gassing and 6 days after pollination compared to 1.5 days and 2 days, respectively, for wildtype flowers (Table 10). Therefore, while effective, overexpression of PEIL2 was not quite as effective as overexpression of EIN2 in increasing flower longevity after pollination, since EIN2 overexpressing flowers lasted up to 11 days.

Table 10. Increased flower longevity in petunia plants overexpressing PEIL2.

Evaluation of field-grown petunia TI plants: TI plants with altered expression of petunia EIN2 and EIN3 genes were also evaluated in field trials. Two EIN2 overexpressing lines, 115 and 182, exhibited a three-fold increase in total flower number in the field (Table 11), likely as a result of the greater flower longevity observed in these lines in greenhouse trials. EIN2 antisense and PEIL2 sense lines displayed no difference in flower number.

Table 11. Flower number of petunia EIN2 and PEIL2 overexpressing plants grown in the field, TI generation.

In sum, as shown in variety of commercially useful plant species, the EIN2 gene plays a critical role in regulating flower senescence. Manipulating EIN2 expression resulted in significant increases in flower longevity and flower number in greenhouse and field trials, and the trait was shown to be heritable in the progeny of primary transformants.

Example 2 - Enhance flower longevity and enhanced leaf abscission in cotton.

Initial work was done in tomato to evaluate the effectiveness of each construct. Once the construct is proven to be effective, the experiment is repeated in cotton using an analogous construct.

Regulation of EIN2

Plant material. Tomato (Lycopersicon esculentum cv. Pearson) and cotton

(Gossypium hirsutum cv. Coker 312) plants were grown under standard greenhouse conditions. Flower and leaf tissue for RNA extraction was harvested, frozen in liquid nitrogen, and stored at -80° C.

Isolation of the cotton EIN2 genomic clone. The Arabidopsis EIN2 cDNA was used to screen a cotton genomic library. Several cotton genomic clones were isolated, from which

Hindlll and BamHI fragments were subcloned and sequenced. Coding regions from one of the clones was found to have 70% nucleotide identity with the Arabidopsis EIN2 cDNA.

This clone is used for transformation of cotton.

Delay of flower abscission in tomato. Flower abscission was delayed in tomato by manipulating EIN2 gene expression. Transgenic plants over-expressing a 2.1 kb partial cDNA encoding the 3' end of the EIN2 gene were produced through Agrobacterium- mediated transformation. This partial EIN2 cDNA was under transcriptional control of either the constitutive figwort mosaic virus promoter (Richins et al., 1987) or an abscission zone specific promoter from a cotton pathogenesis-related gene. Primary transformants were self-pollinated and lines that were homozygous for the transgene were selected from subsequent generations. Production of transgenic plants. Transgenic tomato (Lycopersicon esculentum cv.

Pearson) and cotton (Gossypium hirsutum cv. Coker 312) were produced using

Agrobacterium-mQdiated transformation with kanamycin resistance as a selectable marker.

Introduction and inheritance of the transgenes was confirmed by PCR using primers specific for the selectable marker or, for the crosses, primers specific to each transgene. All experiments were performed on plants that were homozygous for the transgene.

RNA isolation. Total RNA was isolated, as previously described by Ciardi et al.,

2000. For real-time quantitative PCR, RNA samples were treated with Dnase I (Ambion, Austin, TX) followed by purification with a Rneasy RNA extraction kit (Qiagen, Valencia, CA).

Lines with Delayed Flower Abscission. To identify lines with delayed flower abscission, flowers were tagged on the day of anthesis, and the number of days until abscission was recorded. Both the constitutive promoter construct and the abscission specific construct had similar effects on flower longevity. Unfertilized wildtype flowers abscised an average of 4 days after anthesis, while many of the transgenic lines still contained turgid unfertilized flowers for more than 20 days after anthesis. Fertilized wildtype flowers also abscised from the developing fruit an average of 4 days after anthesis, while flowers on the EIN2 sense lines remained attached to the fruit for at least 21 days after anthesis (FIG. 1)

Based upon quantitative real-time PCR, transgenic lines with the greatest flower longevity also exhibited the lowest EIN2 expression levels in flowers, indicating co- suppression of the native EIN2 gene. Regulation of EIN3

Induction of leaf abscission in tomato. Six different constructs are evaluated for the promotion of premature leaf abscission in tomato. The first construct contain the Arabidopsis EIN3 cDNA in sense orientation under control of a glucocorticoid-inducible promoter (Bohner et al., Plant J. 19:87-95 (1999)). The second and third constructs contain the same Arabidopsis EIN3 cDNA under control of an ethanol-inducible promoter (Salter et al, Plant J. 16:127-132 (1998)), or an ecdysone agonist-inducible promoter (Martinez et al, Plant J. 19:97-106 (1999)). Over-expression ofEIN3 in Arabidopsis is shown to induce several ethylene responses, including inhibition of leaf expansion. Since growth is severely reduced in E7N3 over-expressing Arabidopsis lines, an inducible promoter is used in tomato to prevent undesirable pleiotropic effects of the transgene.

Homozygous ELΝ3 lines are isolated as described above for EIN2, and are then evaluated for inducible leaf abscission. Leaves are treated with the synthetic glucocorticoid dexamethasone by painting the upper surface of each leaf with a 10 mg/L solution. Dexamethasone treatment of the transgenic plants result in severe leaf epinasty of the transgenic plants and leaf abscission rates ranging from 2 to 4 days after treatment. Similar effects are seen for the other constructs when the plants are sprayed with a 10% ethanol solution or a 1.5 mM solution of the ecdysone agonist muristeroneA. Treatment of wildtype plants with each, of the chemical inducers have no visible effects on growth and development. Three additional constructs are assembled containing an antisense copy of TCTRl, the tomato homologue of the Arabidopsis CTRl gene, under transcriptional control of the three promoters mentioned above. Since CTRl is a negative regulator of ethylene response, antisense expression of CTRl is not expected to increase ethylene response. Leaf abscission rates are evaluated by the methods described above.

Although treatment of TCTRl antisense plants with dexamethasone, ethanol, and muristeroneA also induced leaf abscission, abscission is not as rapid as it was for those plants in which ELN3 was over-expressed, wherein abscission occurs 5 to 7 days after treatment. Since ethanol is the least expensive and least toxic of the three chemical inducers, plants containing the ethanol-inducible promoter are the most easily adaptable to field production. Therefore, the plants over-expressing EIN3 that contain the ethanol-inducible promoter are the focus of further evaluation. Combined Regulation of EIN2 and EIN3

Combining delayed flower abscission and accelerated leaf abscission. To produce plants with delayed flower abscission and accelerated leaf abscission, the EIN2 over- expressing and ELN3 over-expressing tomato lines are crossed, and plants which are homozygous for both transgenes (EIN2 ELN3) are selected from subsequent generations. The resulting EIN2/ELN3 over-expressing plants maintain the characteristics of each line, and exhibit delayed flower abscission along with glucocorticoid-inducible leaf abscission. Delayed flower abscission in cotton. A 4.9 kb genomic clone was isolated from a cotton genomic library. It contains approximately 2.5 kb of the cotton EIN2 coding region set forth in SEQ ID NO:6, having 70% identity with the E/N2 gene of Arabidopsis, that was used for over-expression to induce co-suppression.

To delay flower abscission, a flower abscission zone specific promoter from a cotton chitinase gene is selected to drive expression of the cotton EIN2 gene. Cotton is transformed with each of these constructs and lines displaying the strongest phenotypes are selected.

Based upon the foregoing effectiveness of the controlled over-expression of ΕLΝ2, independently in tomato and in cotton, to produce plant lines characterized by greatly reduced flower abscission, the methods and constructs of the present invention are shown to be applicable to any plant in which the disclosed characteristics are desired. For example, the disclosed methods and compositions were effective to produce commercially useful plants in which one wishes to cause ELN2-controlled reduction of flower abscission.

The disclosures of each patent, patent application and publication cited or described in this document are hereby incoφorated herein by reference, in their entirety. While the foregoing specification has been described with regard to certain preferred embodiments, and many details have been set forth for the puφose of illustration, it will be apparent to those skilled in the art that the invention may be subject to various modifications and additional embodiments, and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention. Such modifications and additional embodiments are also intended to fall within the scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method for controlling abscission in a transformed, commercially-useful plant comprising effecting the overexpression of ELN2 or ELN3, or the combination thereof, in the resulting transgenic plant as compared with a non-transformed wild type plant.

2. The method of claim 1, wherein flower abscission is modulated, and the method comprises overexpressing EIN2.

3. The method of claim 2, wherein flower abscission is inhibited or reduced

4. The method of claim 2, wherein the resulting transgenic plant is any commercially useful plant species.

5. The method of claim 4, wherein the plant species is selected from the group consisting of commercially useful flowering plants, wherein the flower or the ability of the plant to produce a flower provides commercial utility to the plant; food source plants, wherein the use of the flower or any part of the plant as a food source provides commercial utility to the plant; and other commercially useful plants for the production of fibers or pharmaceuticals, wherein use of the flower or any part of the plant as a fiber or pharmaceutical is commercially useful.

6. The method of claim 1 , wherein flower or leaf abscission, or a combination thereof, is modulated, and the method comprises overexpressing ELN2 and EIN3.

7. The method of claim 6, wherein the resulting transgenic plant is any commercially useful plant species.

8. The method of claim 7, wherein the plant species is selected from the group consisting of commercially useful flowering plants, wherein the flower or the ability of the plant to produce a flower provides commercial utility to the plant; food source plants, wherein the use of the flower or any part of the plant as a food source provides commercial utility to the plant; and other commercially useful plants for the production of fibers or pharmaceuticals, wherein use of the flower or any part of the plant as a fiber or pharmaceutical is commercially useful.

9. The method of claim 2, further comprising driving overexpression of the E/N2 gene with a flower abscission zone-specific promoter.

10. The method of claim 2, wherein the overexpressed EIN2 gene has at least 50% identity to the E7N2 gene in Arabidopsis.

11. A method of producing at least one cell line in which ELN2 is overexpressed comprising: transforming a plant tissue with an exogenous EIN2 gene or active fragment thereof, selected to provide overexpression of EIN2; and selecting at least one resulting EIN2 overexpressing transformed cell line displaying a strong phenotype of combined reduced flower abscission.

12. A method of producing at least one cell line in which ELN2 and ELN3 are together overexpressed comprising: transforming a first plant tissue with an exogenous EIN2 gene or active fragment thereof, selected to provide overexpression of ELN2; and transforming a second plant tissue with an exogenous EIN3 gene or active fragment thereof, selected to provide inducible overexpression of EIN3; then genetically crossing the EIN2 and EIN3 overexpressing transformed cell lines; and selecting at least one crossed ELN2/ELN3 overexpressing cell line displaying a strong phenotype of reduced flower abscission.

13. The transformed plant or plant part, in which the cells, organs, flowers, tissues, seeds or progeny, comprise the EIN2 overexpressing gene or active fragment thereof according to claim 2.

14. The transformed plant or cell line according to claim 13, wherein the genes or active fragments thereof, comprise recombinant nucleic acids.

15. The transformed plant or plant part, in which the cells, organs, flowers, tissues, seeds or progeny comprise the controlled overexpression of an ELN2 polypeptide encoded by an EIN2 gene or active fragments thereof according to claim 13.

16. The transformed plant or plant part, in which the cells, organs, flowers, tissues, seeds or progeny, comprise the E/N2 and EIN3 overexpressing genes or active fragments thereof according to claim 6.

17. The transformed plant or cell line according to claim 16, wherein the genes or active fragments thereof, comprise recombinant nucleic acids.

18. The transformed plant or plant part, in which the cells, organs, flowers, tissues, seeds or progeny comprise the controlled overexpression of ELΝ2 and EIN3 polypeptides encoded by EIN2 and E7N3 genes or active fragments thereof according to claim 16.

19. An isolated nucleic acid from a commercially useful plant, wherein the isolated nucleic acid has at least 50% identity to Arabidopsis EIN2, and wherein the expression product of said isolated nucleic acid has ELΝ2 activity, which when overexpressed results in delayed abscission of the flowers of the plant when compared with wild type plants without EIN2 overexpression.

20. The isolated nucleic acid of claim 19, wherein the nucleic acid sequence is that of petunia EIN2.

21. The isolated nucleic acid of claim 19, wherein the nucleic acid sequence is that of lettuce EIN2.

22. The isolated nucleic acid of claim 19, wherein the nucleic acid sequence is that of tomato EIN2.

23. The isolated nucleic acid of claim 19, wherein the nucleic acid sequence is that of geranium EIN 2.

24. The isolated nucleic acid of claim 19, wherein the nucleic acid sequence is that of begonia EIN 2.

25. The isolated nucleic acid of claim 19, wherein the nucleic acid sequence is that of cotton EIN2.

26. The expression product of any one of claims 19 - 25.