WO2001057063A1 - Eto1 and related proteins, and methods of regulating ethylene biosynthesis - Google Patents

Abstract

The invention provides a negative regulator of ethylene biosynthesis in plants, which is embodied by ETHYLENE-OVERPRODUCER1 (ETO1), a recessive gene, which controls the expression of the synthesis of the plant hormone ethylene. ETO1 is a member of a novel gene family, which is highly conserved in the plant kingdom, and which is unique in that it comprises both BTB and TPR domains. Two-hybrid experiments, together with in vitro binding experiments and double mutant analyses in planta revealed direct interaction of ETO1 with 1-aminocyclopropane-1-carboxylate synthase (ACS), a key enzyme of ethylene biosynthesis. Furthermore, functional assay in E. coli showed that members of the related ETO1-LIKE (EOL) proteins also significantly inhibit ethylene biosynthesis. Thus, the invention further provides methods of controlling, regulating and modulating ethylene biosynthesis in plants, plant cells, and the like.

Description

ETOl AND RELATED PROTEINS, AND METHODS OF REGULATING ETHYLENE BIOSYNTHESIS

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Npplication 60/180,840 filed February 7, 2000.

GOVERNMENT SUPPORT This work was supported in part by grants from the United States Department of

Energy, grant number DE-FG02-93ER20104. The government may have certain rights in this invention.

FIELD OF THE INVENTION This invention relates generally to the study of the ethylene pathway in plants, to ethylene biosynthesis and the regulation thereof, and to novel genes and proteins involved in the ethylene pathway.

BACKGROUND OF THE INVENTION Ethylene is a simple gaseous molecule (C₂H₄) also known as one of plant hormones that governs many aspects of plant growth and development, including seed germination, cell elongation, defense response to pathogen attack, sex determination, wounding, nodulation, flower and leaf senescence, leaf abscission and fruit ripening (Abeles et al, (1992) In Ethylene in Plant Biology, 2^nd Ed., New York, NY: Academic Press; Ecker, Science (Weekly) 268(5211):667-675 (1995); Johnson and Ecker, Annu. Rev. Genet. 32:227-254 (1998)). These aspects are controlled by the combination of biosynthesis, perception and signaling pathway of ethylene gas.

In order to understand how plants regulate the ethylene response under such diverse conditions, physiological, biochemical, molecular and genetic approaches have been applied. A particularly powerful tool has been Arabidopsis thaliana, which is well characterized. Its so-called "triple-response" or "ethylene triple response" to stress or adverse conditions has proven to be an invaluable phenotypic marker (Guzman, and Ecker, Plant Cell 2:513-523 (1990); Ecker, 1995). Genetic approaches have established the backbone of ethylene signal perception-transduction pathway (Bleecker et al, Science 241:1086-1089 (1988); Guzman and Ecker, 1990; Roman et al, Genetics 139:1393-1409 (1995)), and molecular genetic efforts using Arabidopsis mutants have allowed isolation of many components and steps involved in the pathway.

Ethylene is perceived by a family of ethylene receptors (ETR1 (ethylene receptor 1), ETR2, EIN4 (ethylene insensitive 4), ERS 1 and ERS2) that are supplemented with copper cofactor, and which negatively regulate the ethylene responses (Chang et al, Science 262:539-44 (1993); Hua et al, Science 269:1712-1714 (1995); Sakai et al, Plant Cell 12:225-236 (2000); Hua et al, Plant Cell 10:1321-1332 (1998); Hua and Meyerowitz, Cell 94:261-271 (1998); Rodriguez et al, Science 283:996-998 (1999); Hirayama et al, Cell 97:383-393 (1999)). These receptors have similarity to the two-component regulators in prokaryotes and eukaryotes (reviewed in Chang, Trends Biochem Sci. 21 : 129-133 (1996)).

Downstream ofthe ETR1 -related receptors, CTRl (constitutive triple response) acts as a negative regulator (Kieber et al, Cell 72:427-441 (1993)). CTRl mutants display 'ethylene' phenotypes even in the presence of inhibitors of ethylene biosynthesis or receptor binding. A transmembrane protein, EIN2 mediates the signal propagation between CTRl and further downstream components, EIN3/EIL (ethylene insensitive like) family (Alonso et al, Science 284:2148-2152 (1999)).

EIN3 protein regulates the transcription of primary ethylene response genes and under these primary genes, secondary ethylene response genes containing ethylene responsive element (ERE/GCC box) in their promoters are thought to be activated (Chao et al, Cell 89:1133-44 (1997); Solano et al, Genes Dev. 12:3703-3714 (1998)). Other components, such as EΓN5/ATN1 and EIN6, are known to act downstream of CTRl (Roman et al, 1995).

The biosynthetic pathway of ethylene has been studied in detail and genes encoding key enzymes have been cloned (Sato et al, Proc. Natl. Acad. Sci. USA 86:6621-6625 (1989); Nakajima et al, Plant Cell Physiol 29:989-998 (1990); Van der Straeten et al, Proc. Natl. Acad. Sci. USA 89:9969-9973 (1992); Hamilton et al, Proc. Natl. Acad. Sci. USA 88:7434-7437 (1991); Spanu et al, EMBO J. 10:2007-2013 (1991)). The study of ethylene biosynthesis has focused on the cloning and characterization ofthe genes of two key enzymes, 1-aminocyclopropane-l-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO). ACS converts S-adenosyl-L-methionine (SAM) to ACC, and ACO produces ethylene from ACC. Generally the reaction catalyzed by ACS is a rate-limiting step (Kende, Annu. Rev. Plant Physiol. Plant Mol. Biol. 44:283-307 (1993). Both ACS and ACO are encoded by gene families in many plant species. In the case of Arabidopsis, at least eleven (11) ACS genes and at least four (4) ACO genes have been identified. ACS and A CO genes are differentially expressed in response to divergent stimuli, such as germination, leaf senescence and flower abscission, flowering signal, fruit ripening, wounding, touch and pathogen attack (Yang et al, Annu. Rev. Plant Physiol. 35:155-189 (1984); Mattoo and Suttle, The Plant Hormone Ethylene. (Boca Raton: CRC Press), 1991; Abeles et al, 1992; Samach et al, Science 288:1613-1616 (2000); Nakatsuka et al, Plant Physiol. 118:1295-1305 (1998); Peck et al, Plant Mol. Biol. 38:977-982 (1998)), although a body of evidence suggests the existence of post-transcriptional regulation of ACS genes (Nakajima et al, 1990; Spanu et al, Plant Physiol. 106:529-535 (1994); Oeticker et al, Plant Mol. Biol. 34:275-286 (1997); Vogel et al, Proc. Natl. Acad. Sci. USA 95:4766-4771 (1998); Woeste et al, Plant Physiol. 119:521-530 (1999)). From physiological and molecular biological study, involvement of phosphorylation has been suggested (Spanu et al, 1994; Liang et al, Proc. Natl. Acad. Sci. USA 89:11046-11050 (1992); Liang et al., Plant J. 10:1027-1036 (1996)). However, the mechanism by which plants regulate ethylene biosynthesis at the post-transcriptional level remained unclear.

In this regard, a series of ethylene-overproducing mutants (etol, eto2-l, eto3 and eto4) constitutively show triple response phenotypes in the absence of exogenously applied ethylene in etiolated seedlings (Guzman and Ecker, 1990; Kieber et al, 1993; Alonso and Ecker, unpublished). However, the eto mutants can be distinguished from a Ctrl mutant that also displays triple response in the absence ofthe hormone, because their phenotypes are suppressed by antagonists of ethylene biosynthesis and action.

Based on the results of genetic experiments, over-expression ofthe normal or truncated versions ofthe regulatory gene etol in transgenic plants was identified and characterized as a recessive mutation that results in approximately 10-fold overproduction of ethylene in etiolated seedlings, as compared to the wild type plant (Guzman and Ecker, 1990), while eto2-l and eto3 are dominant mutations that cause 20- and 100-fold increases, respectively, of ethylene biosynthesis also in etiolated seedlings (Kieber et al, 1993). The eto2-l mutation was found to lie in the ACS5 gene, where it was determined that a base frame-shift mutation in the C-terminal region of this gene caused an alteration ofthe C- terminal amino acid sequence (Vogel et al, 1998). These results suggested that the C- terminus of ACS 5 might be involved in the post-transcriptional regulation/processing or stability ofthe protein. There have also been recent suggestions that etol and eto3 may be involved in the post-transcriptional regulation of ACS (Woeste et al, 1999).

When combined, these studies indicated that the eto mutants are likely impaired in either the regulator, or the structural-enzymes of ethylene biosynthesis. However, prior to this invention, modulation of ethylene biosynthesis by ETOl was undefined and the mechanisms unknown. Any number of possible functions were indicated in the prior art for this gene product. For example, it could have functioned as a transcription factor or as a post-transcriptional regulator, for which there are many examples among the tetratrico- peptide repeat (TPR) proteins with known function. Ssn6 is one ofthe more well characterized TPR proteins, which functions as a co-repressor that interacts with another component ofthe co-repressor, Tu l protein via its TPR motifs (Tzamarias et al, Genes Dev. 9:821-831 (1995)). On the other hand, TPR proteins like the SPINDLY gene in Arabidopsis (Jacobsen et al, Proc. Natl. Acad. Sci. USA 93:9292-9296 (1996)), function as O-GlcNac-transferases, and presumably are post-transcriptional regulators. In fact, recent evidence suggested that treatment with low-dose of cytokinin causes induction of ethylene biosynthesis by an unknown post-transcriptional mechanism (Vogel et al, 1998).

Thus, many possibilities were available as the mechanism behind the negative regulation of ethylene biosynthesis. If ETOl had functioned as a transcription factor that repressed the expression of ACS mRNA(s), then etol mutants would theoretically have displayed a significant accumulation of ACS mRNA(s). However, steady-state rnRNA levels for each of six evaluated A CS genes were not significantly increased in the etol mutants (Woeste et al, 1999; Yoshida and Ecker, unpublished data). Additionally, the ACS activity of crude extract from etol plants was increased in contrast with little increase ofthe activity of ACC oxidase (Woeste et al, 1999). Furthermore, many ofthe known inducers of ACS gene transcription were found to act synergistically with the etol mutation, further suggesting an alternative mechanism of regulation (Woeste et al, 1999).

Because, however, there are other unexamined putative ACS genes in the Arabidopsis genome, the possibility remained that ETOl regulates the transcription of yet another ACS gene(s). Alternatively, like AC02 (Raz and Ecker, Development 126:3661- 3668 (1999)), the increase in ACS rnRNA expression in etol mutants could have been highly localized in the etiolated seedling, thereby escaping detection.

Thus, the significance of identifying the proteins that interact with the C-terminus of ACS proteins and the TPR-domain of ETOl was clear, and the inventors recognized the need to elucidate the mechanism(s) by which ETOl functions in the regulation of ethylene biosynthesis. This need has been met by the teachings ofthe present invention, which characterizes the role ofthe ETO genes and identifies key proteins involved in the biosynthesis of ethylene in plants throughout the plant kingdom, thus providing novel methods for controlling, modulating and/or regulating ethylene production.

By thus improving understanding ofthe ethylene biosynthesis, it is now possible to improve the tolerance of plants to pathogens, as well as to develop easier and more efficient methods for identifying pathogen tolerant plants. Moreover, by providing insight into plant hormones and mechanisms for their control, and by modulating and regulating their functions, it is possible to significantly improve the quality, quantity and or longevity of plant food products, such as fruits and vegetables, flowers and flowering ornamentals, arid other non-food plant products, such as commercially valuable crops, e.g., cotton or flax, or ornamental green plants, thereby providing more and better products for market in both developed and underdeveloped countries.

SUMMARY OF THE INVENTION

The invention provides methods for and insight into the mechanism(s) of regulating ethylene biosynthesis in plants, and provides isolated nucleic acid sequences, which encode related plant negative regulators of ethylene biosynthesis. The regulator family of genes encode a novel type of protein, having at least one BTB/POZ (Broad-Complex, tramtrack, and brie a brae I poxvirus and zinc finger) domain in its N-terminus, and at least one tetratricopeptide repeat (TPR) motif in its C-terminus. Both motifs have been associated with protein-protein interaction.

In the present disclosure, the negative ethylene biosynthesis regulator is embodied by an isolated and characterized ETHYLENE-OVERPROD UCER1 (ETOl) gene, and members ofthe ETOl gene family, which comprise ETO genes, as well as ETOl -Like (EOL) genes including, for example, EOL1 and EOL2, and active fragments thereof. Embodied expression products ofthe ETOl family comprise ETOl, EOL1, EOL2, purified preparations and active fragments thereof. As described below, experiments by the inventors have clearly shown that ETOl directly interacts with, and inhibits, the activity of at least one protein responsible for the biosynthesis of ethylene in plants, ACS5. In contrast, in an etol mutant, ETOl was unable to regulate ACS5, and thus, 10-fold more ethylene was produced. Also embodied within the invention are identified homologs and paralogs of ETOlin a variety of plant species, indicating the ubiquity ofthe embodied systems for regulating ethylene biosynthesis in plant kingdom, and thus providing mutants, derivatives, paralogs or homologs of ETOl, encoding an expression product having ETOl or EOL activity in a plant cell, wherein the mutant, derivative, paralog or homolog is at least 40% homologous to ETOl, or a member ofthe ETOl family of genes, including the EOL genes or nucleotide sequences encoding ETOl or and ETO or EOL peptide. Also provided by the invention are polypeptides encoded by ETOl, or a member ofthe ETOl family of genes, including ETOl or EOL peptides, as well as mutants, derivatives, homologs, paralogs and analogs thereof, having ETOl or EOL activity in a plant cell, wherein the mutant, derivative, homolog, paralog or analog is at least 40% homologous to ETOl or a member ofthe ETOl family, including EOLs or an amino acid sequence therefor.

Most mutations ofthe present invention were found embedded in the TPR domain of ETOl, and the ETOl family showed direct interaction with C-terminus ofthe ACS 5 protein in a two-hybrid system. Moreover, inhibition of ACS activity by ETOl and EOL proteins was clearly shown by the use of a functional assay in E. coli, and a double mutant analysis between etol and eto2-2, a loss-of-function mutant allele ofthe ACS5 gene.

Further, the invention provides a recombinant cell comprising the isolated nucleic acid of any member of the ETOl family, including EOL genes, and fragments thereof, having ETOl or EOL activity in a plant cell. Also provided is a vector comprising the isolated nucleic acid of any member ofthe ETOl family, including EOL genes, and fragments thereof, having ETOl or EOL activity in a plant cell.

In addition, the invention provides antibodies specific for a plant ETOl or EOL polypeptide, or to homologs, paralogs, analogs, derivatives or fragments thereof, wherein the polypeptide has ETOl or EOL activity in a plant cell. Also provided are isolated nucleic acid sequences comprising a sequence which is complementary to all or part ofthe nucleic acid sequence of one ofthe ETOl family of genes, or a portion thereof, and which inhibits the activity of such gene or gene fragment. Further provided are such nucleic acids, having antisense activity at a level sufficient to regulate, control, or modulate the ethylene biosynthesis activity of a plant, plant cell, organ, flower or tissue comprising same.

The invention also provides plants, plant cells, organs, flowers, tissues, seeds, and progeny comprising any ofthe foregoing nucleic acids selected from the ETOl gene family. Also provided are transgenic plants, the cells, organs, flowers, tissues, seeds or progeny of which comprise such ETOl family of nucleic acid, or which comprise the polypeptide expression product of such ETOl family of nucleic acids. Moreover, promoter sequences and / or reporter genes or active fragments thereof are provided when operably fused to the nucleic acids ofthe present invention in a plant cell, or in a transgenic plant or plant cell. In addition, the invention provides a method for manipulating in a plant any ofthe foregoing nucleic acids selected from the ETOl gene family to permit the regulation, control or modulation ofthe ethylene response in a plant or plant cell, organ, flower, tissue, seed or progeny comprising same. Also provided is such a method, wherein regulation, control or modulation initiates or enhances the germination, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit ripening, insect, herbicide or pathogen resistance, abscission, or response to stress, injury or pathogens in the plant or plant cell, organ, flower, tissue, seed or progeny comprising same. Also provided is such a method, wherein said regulation, control or modulation inhibits or prevents the germination, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit ripening, insect, herbicide or pathogen resistance, abscission, or response to stress, injury or pathogens in the plant or plant cell, organ, flower, tissue, seed or progeny comprising same.

Moreover, the invention provides a method of identifying a compound capable of affecting ethylene biosynthesis in a plant or plant cell comprising (i) providing a cell comprising an isolated nucleic acid encoding a polypeptide selected from the ETOl family, having a reporter sequence operably linked thereto; then (ii) adding to the cell a compound being tested; and then (iii) measuring the level of reporter gene activity in the cell, wherein a higher or lower level of reporter gene activity in the cell compared with the level of reporter gene activity in a second cell to which the compound being tested was not added is an indicator that the compound being tested is capable of affecting the ethylene biosynthesis of a plant.

In addition, the invention provides a method for generating a modified plant with modified ethylene biosynthesis activity as compared to that of comparable wild type plant comprising introducing into the cells ofthe modified plant an isolated nucleic acid encoding ETOl or a gene in the EOL family, wherein the nucleic acid ofthe ETOl or EOL gene regulates ethylene biosynthesis ofthe modified plant. Further provided are methods, wherein ethylene biosynthesis is either (i) enhanced or activated, or (ii) reduced or blocked in the modified plant. Also, the invention provides a method for manipulating ethylene biosynthesis in a plant cell comprising (i) operably fusing an ETOl or EOT gene, or an operable portion thereof to a plant promoter sequence in the plant cell to form a chimeric DNA, and then (ii) generating a transgenic plant, the cells of which comprise said chimeric DNA, whereupon controlled activation ofthe plant promoter, manipulates expression of ΕTΟ1 or EOT, which operates as a regulator of ethylene biosynthesis in the plant cell.

Additional objects, advantages and novel features ofthe 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 ofthe following, or may be learned by practice of the invention.

DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings, certain embodiment(s) which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. FIGs. 1(A)- 1(C) depicts a model showing the general process of ethylene biosynthesis in plants and as FIGs. 1(A)- 1(C) respectively, three alternate methods for the negative regulation of ethylene biosynthesis by ETOl and a related family of proteins.

FIGs. 2(A)-2(D) depict the nucleic acid sequences for the genes encoding the ETOl, EOLl and EOL2 proteins. The first codon (NTG) and the termination codon are underlined. FIG. 2(A) depicts the genomic D A sequence for ETOl (SEQ ID NO: 1), in which exons are indicated by capital letters, introns by lower case. FIG. 2(B) depicts the cDNA for ETOl (SEQ ID NO:2). FIG. 2(C) depicts the cDNA for EOLl (SEQ ID NO:3); and FIG. 2(D) depicts the cDNA for EOL2 (SEQ ID NO:4)

FIGs. 3(A)-3(B) graphically depict the mapping and cloning ofthe ETOl gene. FIG. 2(A) depicts positional cloning of ETOl, wherein ETOl was mapped to a ~60kb region (AtEml locus) at the bottom of chromosome3. Open rectangles show SSLP, CAPS or dCAPS markers. Predicted ORFs at the AtEml locus are shown by arrows according to the annotation of AtEml locus (GenBank accession no. AF049236). FIG. 3(B) depicts a schematic diagram ofthe ETOl gene. Closed boxes represent exons (coding region), open boxes represent introns (untranslated region). FIG. 4 depicts the alignment ofthe protein structures of ETOl, EOLl and EOL2. The BTB domain ofthe ETOl protein (amino acids 243-342) is predicted by SMART program and is indicated by solid boxes. Ten TRP (tetratricopeptide repeat) motifs are indicated by empty boxes. Bar graph in lower right corner of FIG. 4 shows the % of sequences conserved among the 3 proteins.

FIG. 5 photographically depicts complementation ofthe etol phenotype in 35 S:: ETOl transgenic plants. As labeled, the photographs show etiolated seedlings (3 -days after germination) representative of wild-type plants (Col-0, column 1), and T2-generation etol-435S::ET01 transgenic plants (column 2), and etol-4 plants (column 3). Each is shown as it appears grown in air (row 1) or in 10 ppm ethylene (C₂H )(row 2).

FIGs. 6(A) and 6(B) depict specific interactions of ETOl and EOL proteins with ACS5 in a yeast two-hybrid system. FIG. 6(A) depicts a plate assay in which the strong interaction between ETOl, and its homologs, EOLl and EOL2, specifically interact with ACS5, but not vector, in the cells. FIG. 6(B) depicts quantification ofthe strength ofthe interaction by β-galactosidase activity liquid assay, and shows that the strength of interaction with ACS5 is ordered: EOLl > ETOl > EOL2.

FIGs. 7(A)-7(C) depicts co-expression of ACS5 and EOL proteins in the JAde6 strain of E. coli, transformed with the constructs as indicated in chart shown in FIG. 7(C). Transformants were grown on minimal media (M9) (shown in FIG. 7(A)), or on minimal media (M9) supplemented with 3 mM ACC (shown in FIG. 7(B)).

FIG. 8 photographically shows that the eto2-2 mutation suppresses the etol phenotype. As labeled, the photographs show 3 day, dark-germinated, etiolated seedlings, representative of wild-type plants (Col-0, column 1), etol-4 plants (column 2), eto2-2 plants (column 3) and etol-4 eto2-2 double mutant plants (column 4). Each is shown as it appears grown in air (row 1) or in ethylene (row 2).

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The invention providing novel methods for controlling, modulating and/or regulating ethylene production in plants. Moreover, the inventors have characterized the role of the ETO genes and identified key proteins involved in the biosynthesis of ethylene in plants throughout the plant kingdom. Identification and characterization ofthe ETOl gene, one of only two known negative regulators of ethylene biosynthesis (the other is an as yet unpublished member ofthe ETO gene family; Alonso and Ecker, unpublished), as well as genetic, molecular and biochemical studies on the gene and its expression product, provide new insight into the molecular basis of post-translational regulation of ethylene biosynthesis in plants. Moreover, functional studies of ETOl and a family of ethylene overproductionlike proteins (EOL) demonstrate that the mechanisms controlling ethylene biosynthesis are highly conserved throughout the plant kingdom.

The ETOl Protein

ETOl was found to encode a novel protein with a BTB POZ (Broad-Complex, tramtrack, and brie a brae I poxvirus and zinc finger) domain in its N-terminus, and tetratricopeptide repeat (TPR) motifs in its C-terminus, respectively. Both motifs have been associated with protein-protein interaction. Nevertheless, the mechanism by which ETOl regulates expression of ACS remained unknown until the present invention. Because, however, there are a number of unexamined putative ACS genes in the Arabidopsis genome, the possibility remained, prior to the present invention, that ETOl regulated the transcription of more than one ACS gene. Alternatively, like AC02 (Raz and Ecker, Development 126:3661-3668 (1999)), the increase in ACS rnRNA expression in etol mutants could have been highly localized in the etiolated seedling, thereby escaping detection. However, this theory was disproved by Northern blot analysis using total seedling rnRNA. In fact, before proceeding further, several alternative models are depicted in FIG. 1 to facilitate understanding ofthe functional relationship between ACS and ETOl (although the models are not intended to in any way limit the invention).

One model depicted in FIG 1 for the action ofthe ETOl protein shows that it ^• interacts directly with the C-terminus of ACS, 'covering' the catalytic domain (active site) or modifying the structure ofthe ACS protein, inhibiting its enzymatic activity. In the alternative, it was possible that ETOl interacts with a ubiquitin/proteasome system and that it is involved in a protein degradation pathway. The catalytic domains ofthe ACS proteins are highly similar in sequence, but the carboxy-termini ofthe known and annotated Arabidopsis ACS proteins are poorly conserved and vary in length among the different isoforms. Nevertheless, C-terminal truncation of ACS5 has been shown to result in an increase in ethylene production that is not clearly related to increased rnRNA levels for this gene (Vogel et al, 1998). This unexplained increase in ethylene production and ACS enzyme activity in eto2-l (truncated ACS5), along with the hyperactivity displayed by a tomato ACS protein that was altered in the carboxyl-terminus (Li et al, J. Biol. Chem. 269:6908-6917 (1994)), support this model.

Recently it was also proposed that TPR proteins function as scaffolds for the assembly of multiprotein complexes (Das et al, EMBO J. 17: 1192-1199 (1998); Scheufler, Cell 101 :199-210 (2000); Lapouge, Mol. Cell 6:899-907 (2000)). It was further suggested that the resulting assembly may recruit individual TPR motifs to interact with distinct proteins, as in the case of Ssn6 (Tzamarias et al, 1995). In any case, truncation ofthe C- terminus of ACS5 appears to confer increased specific activity and inability to interact with ETO1/EOL in the eto2-l mutant. It appears that direct inhibition of ACS5 enzyme activity by ETOl occurs via its

TPR domain, as shown in FIG. 1. This is analogous to the case of immunophilins, which interact with Hsp90, regulation ofthe ATPase activity of Hsp90, and inhibits binding of a second protein to Hsp90. This is because both require an intact TPR domain (Ratajczak et al., J. Biol Chem. 271:2961-2965 (1996); Prodromou et α/., E 5OJ. 18:754-762 (1999)). Recently, in light ofthe knowledge that ACS 5 forms a dimer with a shared active site, as interfered by its C-terminal tail, it was proposed that ACS protein could act as a homo- or hetero-dimer with shared active sites (Tarun et al, Theologis, J Biol Chem. 273, 12509-12514 (1998B)). As a result, another possibility became apparent, that ΕTO1 may inhibit dimerization ofthe ACS5 monomers to form a shared active site, and thus negatively regulate its activity, as shown in FIG. 1. It was also possible that ΕTO1 interacts with Hsp90, or one or more other chaperone proteins, to affect folding ofthe ACS5 protein. In tomato suspension cells, protein phosphorylation dephosphorylation were reported to affect the rate of turnover of ACS proteins (Spanu et al, 1994). Consequently, phosphorylation was also implicated in the regulatory systems. Another characteristic ofthe ΕTO1 family of proteins is the BTB domain in the N- terminus. BTB is also a degenerate amino acid sequence, found in a variety of proteins involved in transcription regulation, cytoskeleton organization and development. Recently the BTB domain has also been found in plant proteins NPR1, NPH3 and RPT2 (Aravind et al, 1999; Motehoulski et al., Science 286:961-964 (1999); Sakai et al, 2000). One ofthe apparent functions ofthe BTB domain is homo/hetero-dimerization or tetramerization of proteins (Aravind et al, 1999), suggesting that ΕTO1 and ΕOL proteins act as multimer. Weak interaction between ΕTO1 and ΕOL proteins themselves detected in the yeast two- hybrid system could reflect this interaction (Yoshida and Εcker, unpublished result). The coiled-coil motif also found in the C-terminus of ETOl family provides another site for protein : protein interaction surface. This domain is also highly conserved in ETOl, and the EOL family of proteins, indicating its essential role in the function ofthe protein through protein interactions. No missense mutations were found in the course of this study in either the BTB or the coiled-coil domains. Nevertheless, conservation of these protein interaction domains, together with TPR motifs in the ETOl family suggested that the regulation of ethylene biosynthesis is controlled through the action of a multiprotein complex.

Carboxy Termini of ACS Proteins as a Common Target ofthe ETOl Family

As noted, specific interactions were demonstrated between the C-terminus of ACS5 and the ETOl family proteins. However, strong interactions were also detected between other ACS proteins and the ETOl family of proteins (Wang and Ecker, unpublished data). This indicates that the C-termini ofthe ACS proteins share certain common target sequence(s).

Actually, although the carboxy-termini ofthe known and annotated Arabidopsis ACS proteins are poorly conserved, and vary in length among the different isoforms, small consensus sequences, i.e., a basic region with an R K/H-tract - RLSF - (X)5 - EER, can be found among them (Yoshida and Ecker, unpublished data). However, they can be classified into three groups as follows: (1) those with long tails after the consensus sequences (like AtACS2), (2) those ending with the consensus (like AtACS5), and (3) those lacking the consensus (like AtACS7). These groupings suggested possible approaches in the negative regulation of ethylene synthesis. For example, the isoforms of group 1 would need processing at the C-termini combined with phosphorylation, and result in the group 2 form. The isoforms of group 3 may represent isoforms with high activity because of they escape regulation by the ETOl family. An additional exception, with long tails but lacking the consensus is found in tomato (LeACS4).

Physiological Role of ETOl Family To identify mutations in novel components ofthe ethylene gas signal transduction pathway, a screen has been established for Arabidopsis thaliana mutants that exhibit an ethylene-like triple response phenotype in response to a potent hormone antagonist. The "triple response" in Arabidopsis consists of three distinct morphological changes in dark- grown seedlings upon exposure to ethylene: 1) inhibition of hypocotyl and root elongation, 2) radial swelling ofthe stem and 3) exaggeration ofthe apical hook. 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 causes induction of the ethylene response through activation (de-repression) ofthe signaling pathway. As a result, even in the absence of ethylene, the hormone response pathway is constituitively activated.

By "plant" as used herein, is meant any plant and any part of such plant, wild type, treated, genetically manipulated or recombinant, including transgenic plants. The term broadly refers to any and all parts ofthe plant, including 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. "Modified plants" are plants in which the wild-type gene or protein character has been altered. Phenotypic alterations may enhance or inhibit a typical wild-type response in or by the plant cells; or there may be no phenotypic effect whatsoever. As one skilled in the art would recognize, absolute levels of endogenous ethylene production by a plant or plant cell will change with growth conditions. However, in "ethylene sensitive" plants, including wild type plants or plants in which the signaling cascade is complete, secondary ethylene responses are activated. Such plants or plant cells typically demonstrate a shut-down or diminution of endogenous gas production in the presence of high concentrations of exogenous ethylene.

By comparison, an "ethylene insensitive" plant or plant cell typically continues to produce endogenous ethylene, despite administration of inhibitory amounts of exogenous ethylene. An ethylene insensitive plant will produce more ethylene or produce it at a rate greater than that of a wild type plant upon administration of an inhibitory amount of exogenous ethylene. For the purposes ofthe present invention, ethylene insensitivity includes either a total or partial inability to display the triple response in the presence of increased levels of exogenous ethylene, such as would be expected if the ethylene signaling pathway in were interrupted in the plant or plant cell.

The constitutive triple response phenotype of ETOl is restricted in etiolated seedlings. Light grown seedlings, adult leaves, flowers and siliques produce almost the same levels of ethylene to wild-type, suggesting the light- and/or stage-dependent regulation (Kieber et al, 1993; Woeste et al, 1999). The paralogs of ETOl, EOL proteins appear to play important roles in differential regulation of ethylene biosynthesis. Plants produce increased amount of ethylene when they are ripening or attacked by pathogen. Under these conditions, a feedback regulation of ethylene biosynthesis is also known. However, it has been shown that ETOl and EOL proteins interact with not only ACS5, but also with other members ofthe ACS protein family (Wang and Ecker, unpublished data). So, the expression of ETOl and EOL genes appears to be coordinated developmentally, spatially and conditionally in combination with^tCS, and possibly with other ethylene biosynthetic genes. T-DNA inserted lines have been identified for both EOLl and EOL2 (Alonso, Lurin and Ecker, unpublished data).

Conserved Regulatory System of Ethylene Biosynthesis in Plant Kingdom

ETOl orthologs were found in other plant species, including fern, mono- cotyledonous and dicotyledonous (including trees) plants, which fits with a common regulatory system of ethylene biosynthesis all through the plant kingdom further supporting the significance ofthe ETOl family of proteins for regulating ethylene biosynthesis by biotechnological procedures.

In fact, the inventors have found that the ETOl family is the only protein family containing both the BTB and TPR domains. No other protein could be found in the databases of genetic sequences with this combination of domains, so the ETOl family is very unique protein family in the plant kingdom. By comparison, ethylene receptors in plants, such as homologous sequences of ETRl -related proteins, are found in cyanobacteria and its ethylene-binding activity has been shown (Rodriguez et al, 1999). However, although the cyanobacterial genome has been completely sequenced, no homologous sequence of ETOl was found in it. This is reasonable to some extent because cyanobacteria does not synthesize ethylene in the same way as a plant. This suggests that the ETO1- related regulatory system, together with ethylene synthesis system, was originally developed in plants, or it evolved from an organism other than cyanobacteria.

The ETOl and EOL Family of Genes

The gene corresponding to ETOl, has been cloned, isolated and sequenced as set forth below and the genomic sequence as well as that ofthe cDNA clone is described. Nevertheless, in view of he descriptions provided, it is understood that other alleles and variations would be available to one of ordinary skill in the art. Embodiments ofthe invention should be construed to include nucleic acid comprising ETOl, or any mutant, derivative, homolog, paralog, ortholog or fragment thereof, which encodes an ethylene overproducer protein, including ETOl, or other ethylene overproducer like protein, including other ETO proteins and members ofthe EOLl or EOL2 family, affecting ethylene biosynthesis. In accordance with additional embodiments ofthe invention, nucleic acid sequences include, but are not limited to DNA, including but not limited to cDNA and genomic DNA; RNA, including but not limited to rnRNA and tRNA, and may include chiral or mixed molecules.

Preferred nucleic acid sequences include the gene encoding ETOl, for example, comprising the genomic sequence set forth in SEQ ID NO:l (FIG. 2(A)), and the cDNA set forth in SEQ ID NO:2 (FIG. 2(B)). Also included are the sequences encoding other ETOs and members ofthe EOL family of proteins, comprising, for example, the cDNA of EOLl set forth in SEQ ID NO:3 (FIG. 2(C); and the cDNA of EOL2 set forth SEQ ID NO:4 (FIG2(D)), as well as modifications in those nucleic acid sequences, including alterations, insertions, deletions, mutations, homologs, paralogs, orthologs and fragments thereof which remain capable of encoding an active protein having ETOl or EOL function which provides negative regulatory activity affecting ethylene biosynthesis in plants, plant cells, plant parts or the like by ACS.

Also included in embodiments ofthe invention are derivatives ofthe disclosed nucleic acid sequences. "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 ofthe molecule. Such moieties, however, still encode a gene which negatively regulates ethylene biosynthesis activity, meaning a ETOl type polypeptide or fragment thereof which is capable of affecting or modulating the synthesis of ethylene in a plant, plant cell, plant part, and the like. Included within the meaning ofthe term

"derivative" as used in the preferred embodiments are "alteration(s)," "insertion(s)," and "deletion(s)" of nucleic acids or the like.

A "fragment" of a nucleic acid is embodied within the invention if it encodes substantially the same expression product as the full length isolated nucleic acid, or if it encodes a peptide having ETOl or EOL capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like by ACS.

In addition, "homologs" are chromosomal DNA carrying the same genetic loci, which would include homologous regions found in paralogs and orthologs. When carried on a diploid cell there is a copy ofthe homolog from each parent. "Paralogs" on the other hand, are technically homolog genes found within the same genome. Thus, EOLl, and EOL2 are 'paralogs.' Nevertheless, when the term "homolog" is used herein, or as claimed, it is intended to refer to homology at the nucleic acid level by methods recognized in the art for determining homology. E.g., a homologous sequence of AtETOl in another plant species, such as wheat or poplar (technically an ortholog), would have significant homology between such nucleic acids, considered to be at least about 40%, preferably, the homology between nucleic acid domains is at least about 50%, more preferably, at least about 60%, even more preferably, at least about 70%, even more preferably, at least about 80%, yet more preferably, at least about 90% and most preferably, the homology between similar nucleic acid domains is about 99%. Moreover, the homologous sequence, even if only 40% homologous, must necessarily encode a peptide having ETOl capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like by ACS, or it is not a homolog. This, the term homolog is broadly intended to encompass paralogs and orthologs in the present invention. Similarly, homologs would include homologous regions of other members ofthe ETOl and EOL families.

According to preferred embodiments ofthe invention, the isolated nucleic acid encoding the biologically active ETOl polypeptide, or other ETO or EOL, or fragment thereof is full length or of sufficient length to encode a negative regulator of ethylene synthesis by ACS. In one embodiment the nucleic acid is at least about 200 nucleotides in length. More preferably, it is at least 400 nucleotides, even more preferably, at least 600 nucleotides, yet more preferably, at least 800 nucleotides, and even more preferably, at least 1000 nucleotides in length. However, as above, the resulting sequence must necessarily encode a peptide having ETOl capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like by ACS.

Expression Products of EOT1 and/or the EOL Family of Genes

The invention should also be construed to include peptides, polypeptides or proteins comprising ETOl, or a member ofthe EOL family, any mutant, derivative, variant, analog, homolog, paralog, ortholog or fragment thereof, having ETOl or EOL capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like. The terms "protein(s)," "peptide(s)," "polypeptide(s)," and "protein sequence(s)" are used interchangeably within the scope ofthe present invention, and include, but are not limited to the expression products encoded by the nucleic acid sequences set forth in SEQ ID NOs:l- 4, the amino acid sequences corresponding substantially to nucleic acid SEQ ID NOs:l-4, as well as those sequences representing mutations, derivatives, analogs, homologs, paralogs, orthologs or fragments thereof having ETOl or EOL capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like.

Embodiments ofthe invention also provide for analog(s) of proteins, peptides or polypeptides encoded by the gene of interest, preferably etol, eto2, eo/1 or eo 2. "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. For example, conservative amino acid changes may be made, which although they alter the primary sequence ofthe 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 ofthe peptide, e.g., acetylation or carbonation. Also included are modification 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 phosphoylated, e.g., phosphotyrosine, phosphoserine or phosphothreonine.

Also embodied in the invention are polypeptides 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 therapeutic 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 ofthe present invention are not intended to be limited to products of any specific exemplary process defined herein, so long as it encodes a peptide having ETOl or EOL capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like by ACS.

"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 ofthe molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, and the like, or they may decrease toxicity ofthe molecule, eliminate or attenuate any undesirable side effect ofthe molecule, and the like. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art. Included within the meaning ofthe term "derivative" as used in the preferred embodiments are "alteration(s)," "insertion(s)," and "deletion(s)" of peptides, polypeptides or the like, so long as it encodes a peptide having ETOl or EOL capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like by ACS. 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 ofthe molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical, so long as it encodes a peptide having ETOl or EOL capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like by ACS.

A "fragment" of a polypeptide is embodied within the invention if it retains substantially the same activity as the purified peptide, or if it has ETOl or EOL activity as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like. Such fragment of a peptide is also meant to define a fragment of an antibody responsive to or capable of binding a peptide having ETOl or EOL capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like by ACS.

As described in the following Examples, ETOl and EOL genes encode proteins having specific domains located therein, for example, terminal extensions, transmembrane spans, TM1 and TM2, nucleotide binding folds, a putative regulatory domain, and the C- terminus. A mutant, derivative, homolog, paralog, ortholog or fragment ofthe subject peptide is, therefore also one in which selected domains in the related protein share significant homology (at least about 40% homology) with the same domains in the preferred embodiment ofthe present invention. It will be appreciated that the definition of such a nucleic acid encompasses those peptides genes having at least about 40% homology, in any ofthe described domains contained therein under conditions of stringency that would be appreciated by one of ordinary skill in the art. In addition, when the term "analog" or "homologous amino acid sequence" is used herein to refer to the domains of these proteins, it should be construed to be applied to homology at both the nucleic acid and the amino acid levels by methods recognized in the art for determining homology. As noted above, however, 'paralogs,' such as EOT1 and EOT2 are technically encoded by homologous genes found within the same genome.

Nevertheless, for the purpose ofthe invention, both paralogs and orthologs are encompassed by the term 'analog' or 'homologous sequence.' E.g., a homologous sequence of AtETOl in another plant species (technically an ortholog), such as wheat or poplar, would have significant homology between such amino acids, considered to be at least about 40%, preferably, the homology between amino acid domains is at least about 50%, more preferably, at least about 60%, even more preferably, at least about 70%, even more preferably, at least about 80%, yet more preferably, at least about 90% and most preferably, the homology between similar amino acid domains is about 99%. Moreover, the homologous sequence, even if only 40% homologous, must necessarily encode a peptide having ETO 1 capability as a negative regulator of ethylene biosynthesis in plants, plant cells, plant parts or the like by ACS, or it is not a homolog. Similar analogs or homologous amino acid sequences are intended for other members ofthe ETO or EOL families. According to a preferred embodiment, the isolated amino acid encoding the biologically active ETOl polypeptide, or other ETO or EOL polypeptide, or fragment thereof is full length or of sufficient length to effect negative regulation of ethylene synthesis by ACS. In one embodiment the isolated polypeptide is at least 120 amino acids, even more preferably, at least 300 amino acids, yet more preferably, at least 500 amino acids, and even more preferably, at least 700 amino acids in length. In an additional embodiment the polypeptide encodes the full-length ETOl protein, or other ETO or EOL polypeptide, or a regulated version thereof.

Expression ofthe Protein, and Methods of Use the Regulate Ethylene Biosynthesis

Embodiments ofthe invention further include a vector comprising a gene encoding ETO 1 , or other ETO or EOL polypeptide. 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 the "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 ofthe invention encompass the expression ofthe desired proteins in either prokaryotic or eukaryotic cells, or viruses that replicate in prokaryotic or eukaryotic cells.

Preferably, proteins embodied in the invention are cloned and expressed in a virus. Viral hosts for expression ofthe proteins ofthe present invention include viral particles which replicate in prokaryotic host or viral particles which infect and replicate in eukaryotic hosts. 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. 12: 8711 -8721 ( 1984)), adenovirus, bovine papilloma virus, simian virus, 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 the "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 ofthe sequence results in the production of a protein ofthe present invention.

In accordance with the invention, the ETOl amino acid sequence or other ETO sequences employed in embodiments ofthe invention, or corresponding members ofthe EOL family may be exogenous sequences. Exogenous or heterologous, as used herein, denotes a nucleic acid or amino acid sequence which is not obtained from, and which would not normally form a part ofthe genetic makeup ofthe plant, cell, organ, flower or tissue to be transformed, in its untransformed state. Plants comprising exogenous sequences for ETOl or EOLl or 2,or etol or 2, or eo/1 or 2 mutations are encoded by, but not limited to, the nucleic acid sequences of SEQ ID NOs:l-4, and/or the amino acid sequences corresponding to the nucleic acid sequences of SEQ ID NOs:l-4, including alterations, insertions, deletions, mutations, homologs, paralogs, orthologs and fragments thereof.

Transformed plant cells, tissues and the like, comprising nucleic acid sequence of ETOl or etol mutations, such as, but not limited to, the nucleic acid sequence of SEQ ID NO: 1 or 2 are within the scope ofthe invention, as are the corresponding sequences ofthe EOL family in SEQ ID NOs 3 or 4. Transformed cells ofthe invention may be prepared by employing standard transformation techniques and procedures as set forth e.g., in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). 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 purpose of producing cells and regenerating plants having the described negative regulatory effect on ethylene synthesis by ACS, thereby modulating the expression ofthe target ethylene biosynthesis elements.

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 using Agrobacterium-mediateά leaf disc transformation methods described by Horsch et αl, 1988, Leaf Disc Transformation: Plant Molecular Biology Manual A5: 1). Numerous procedures are known in the art to assess whether a transgenic plant comprises the desired DNA, and need not be reiterated.

The expression ofthe 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 αl, Nature (London) 290:304- 310 (1981)); the yeast GAL4 gene promoter (Johnston et al, Proc. Natl. Acad. Sci. (USA) 79:6911-6915 (1982)) and the exemplified ρYES3 PGK1 promoter. As is widely known, translation of eukaryotic rnRNA is initiated at the codon, which encodes 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 an operably linked promoter 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 ofthe desired protein may occur through the transient expression ofthe introduced sequence. Alternatively, permanent expression may occur through the integration ofthe introduced sequence into the host chromosome. For expression ofthe desired protein in a virus, the hybrid gene operably linked to a promoter is typically integrated into the viral genome, be it RNA or DNA. Cloning into viruses is well known and thus, one of skill in the art will know numerous techniques to accomplish such cloning. Cells which 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 may complement an auxotrophy in the host (such as leu2, or ura3, which are common yeast auxotrophic markers), biocide resistance, e.g., antibiotics, or resistance to heavy metals, such as copper, or the like. The selectable marker gene 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 rnRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. The cDNA expression vectors incorporating such elements include those described by Okayama, H., Mol. Cell. Biol. 3:280 (1983), and others.

In another embodiment, the introduced sequence will be incorporated 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 purpose. 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 ofthe 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 germination, abscission, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit ripening, insect, herbicide or pathogen resistance, or response to stress in said plant. In a preferred embodiment the method activates or enhances the above responses, whereas in another preferred embodiment the method inhibits or prevents the above responses.

Thus, methods ofthe present invention define embodiments in which the ethylene biosynthesis activity by ACS is prevented or inhibited. By "prevention" is meant the cessation of ethylene biosynthesis by ACS in plants, plant cells or the like. By "inhibition" is meant a statistically significant reduction in the amount of ethylene produced by ACS, or in the amount of expression of ACS, or of detectable ethylene as compared with plants, plant cells, organs, flowers, tissues or the like grown without ETOl or an ETO or EOL inhibitor or disclosed method of negative regulation (inhibition). Preferably, by blocking or inhibiting ACS, the ETOl, or ETO or EOL, negatively regulates (reduces) ethylene biosynthesis, thereby inhibiting or reducing ACS expression 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. Similarly, in accordance with this embodiment, total ethylene production is also inhibited or reduced by at least 20 %, more preferably by at least 50%, even more preferably by 80% or greater, also preferably, in a dose-dependent manner. The effect of such prevention or inhibition would or negatively regulate or inhibit the ethylene biosynthesis of a plant, plant cell or the like comprising such DNA or protein expression product.

In accordance with another preferred embodiment ofthe invention, once ETOl, or another ETO or EOL or inhibitor satisfying these requirements are identified, the utilization of recognized assay procedures to identify ethylene reduction or the manner in which the ACS activity and/or ethylene biosynthesis is negatively regulated or inhibited are particularly useful.

Ethylene insensitive plants are disease and pathogen tolerant. For purposes ofthe present invention, disease tolerance is the ability of a plant or plant cell to survive stress, infection or injury with minimal damage or reduction in the harvested yield of commercial product. Plants with disease tolerance may have extensive levels of infection, but little necrosis and few or no lesions. The plants or plant cells may also have reduced necrotic and water soaking responses and chlorophyll loss may be virtually absent. In contrast, resistant plants generally limit the growth ofthe pathogens and contain the infection to a localized area within multiple apparently injurious lesions.

Similarly, embodied methods ofthe invention are also defined in which negative regulation of ACS is blocked or inhibited, and the ACS activity and/or ethylene biosynthesis is initiated, stimulated or enhanced if there is a statistically significant increase in the amount of ethylene produced by ACS, or in the amount of expression of ACS, or of detectable ethylene as compared with plants, plant cells, organs, flowers, tissues or the like grown without ETOl or an ETO or EOL inhibitor or disclosed method of negative regulation (inhibition). Preferably, blocking or inhibiting the ETOl, or ETO or EOL, negative regulation of ethylene biosynthesis will effect an increase or enhancement of ACS expression 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. Similarly, in accordance with this embodiment, total ethylene production is also increased or enhanced by at least 20 %, more preferably by at least 50%, even more preferably by 80% or greater, also preferably, in a dose-dependent manner. Once enhancers (or blockers ofthe negative regulation by e.g., ETOl) satisfying these requirements are identified, the utilization of new assay procedures to identify enhanced ethylene production or the manner in which ACS activity and/or ethylene biosynthesis is enhanced are particularly useful. The invention further features an isolated preparation of a nucleic acid which is antisense in orientation to a portion or all of ETOl, or of an ETO or EOL gene encoding a negative regulator of ethylene biosynthesis by ACS in a plant, plant cell or the like. The antisense nucleic acid is of sufficient length to enhance expression of ACS or the target gene of interest. In other words, if an antisense DNA fragment of ETOl or an EOL is expressed in the plants, it will inhibit the endogenous expression of ETOl/EOL, thereby resulting in the derepression of ACS activity, as opposed to inhibiting ACS activity. The actual length ofthe 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 or even more than 50 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 ETOl, or an ETO or EOL gene, or its homolog or the like, but which does not encode ETOl, or another ETO or EOL peptide. "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 complementary when a substantial number (at least 40% or at least 50%, or preferably at least 60%, or more preferably at least 70% or 80%, or more preferably at least 90% or at least 99%) ofthe corresponding positions in each ofthe molecules are occupied by nucleotides which normally base-pair with each other (e.g., A:T and G:C nucleotide pairs.). In yet another aspect ofthe invention, antibodies are provided which are directed against the ACS-affecting, negative regulatory peptides or polypeptides, such as ETOl, which are capable of binding to ETOl, or another ETO or EOL peptide, thereby blocking or modulating their expression. Such an antibody is specific for the whole molecule, its N- or C-terminal, or internal portions. Methods of generating such antibodies are well known in the art.

In the embodiment directed to the antibody specific for a plant ETOl polypeptide, including functional equivalents ofthe antibody, the term "functional equivalent" refers to any molecule capable of specifically binding to the same antigenic determinant as the antibody, thereby neutralizing the molecule, e.g., antibody-like molecules, such as single chain antigen binding molecules .

The invention further includes a transgenic plant comprising an isolated DNA encoded ETOl, or another ETO or EOL peptide capable of controlling or modulating the expression of ACS or another gene responsible for ethylene biosynthesis in a plant, plant cell or the like. In the alternative is provided a transgenic plant comprising a mutant etol or corresponding mutant from the ETO or EOL gene family in which the negative regulatory capability has been disrupted, thereby permitting ethylene production by the plant, plant cell, etc. AC synthase system.

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 "nontransgenic," but substantially homozygous "wild type plant" as used herein, means a nontransgenic 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 sense or antisense DNA encoding the negative regulators of ethylene production as ETOl, or corresponding members ofthe ETO or EOL family, capable of activating, blocking or modulating ethylene biosynthesis (ACS target genes), 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 CaMV 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 ofthe 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 ofthe 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 ofthe present invention, having a phenotype characterized by the ETOl gene or etol mutations, or by another ETO or EOL gene or eto or eol mutations (including eto2-l, eto3 and the like) affecting the activation of, or expression of ACS or ACS-controlled ethylene biosynthesis in a plant, plant cell or the like.

Preferred plants of this invention, in which ACS gene expression or ACS-controlled ethylene biosynthesis is negatively regulated by ETOl, or by another ETO or EOL gene, or expression products thereof, or by eto or eol mutations (including etol, eto2-l, eto3 and the like) to modulate expression of an ACS gene (including the ACS5 gene) (either of which results in reduced or blocked ethylene biosynthesis), or in which control of ETOl, or another ETO or EOL gene or expression product controls or prevents the negative regulation of such ethylene biosynthesis (resulting in enhanced ethylene production), include, but are not limited to, high yield crop species for which cultivation practices have already been perfected (including monocots and dicots, e.g., alfalfa, cashew, cotton, peanut, fava bean, french bean, mung bean, pea, walnut, maize, petunia, potato, sugar beet, tobacco, oats, wheat, barley and the like), or engineered endemic species. Particularly preferred plants are those from: the Family Umbelliferae, particularly ofthe genera Daucus

(particularly the species carota, carrot) and Apium (particularly the species grαveolens dulce, celery) and the like; the Family Solαnαceα, particularly ofthe genus Lycopersicon, particularly the species esculentum (tomato) and the genus Solαnum, particularly the species tuberosum (potato) and melongena (eggplant), and the like, and the genus Capsicum, particularly the species annum (pepper) and the like; and the Family Leguminosae, particularly the genus Glycine, particularly the species max (soybean) and the like; and the Family Cruciferae, particularly ofthe genus Brassica, particularly the species campestris (turnip), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and the like; the Family Compositae, particularly the genus Lactuca, and the species satira (lettuce), and the genus Arabidopsis, particularly the species thaliana (Thale cress) and the like. Of these Families, the a preferred model species, representative ofthe response in the remainder ofthe plant species, include the leafy vegetables, for example, the Family Cruciferae, especially the genus Arabidopsis, most especially the species thaliana.

Preferred plants particularly include flowering plants, such as roses, carnations, chrysanthemums and the like, in which longevity ofthe flower on the stem (delayed abscission) is of particular relevance, and especially include ornamental flowering plants, such as geraniums. Additional preferred plants include leafy green ornamental plants, such as Ficus, palms, and the like, in which longevity ofthe leaf stem on the plant (delayed abscission) is of particular relevance. Delayed flowering in such plants may also be advantageous. Similarly, other preferred plants include fruiting plants, such as banana and orange, wherein pectin-dissolving enzymes are involved in the abscission process. The present invention will benefit plants subjected to stress. Stress includes, and is not limited to, infection as a result of pathogens such as bacteria, viruses, fungi, and conditions involving aging, wound healing and soil penetration. Bacterial infections include, and are not limited to, Clavϊbacter michiganense (formerly Coynebacterium michiganense), Pseudomonas solanacearum and Erwinia stewartii, and more particularly, Xanthomonas campestris (specifically pathovars campestris and vesicatoria), Pseudomonas syringae (specifically pathovars tomato, maculicolά).

In addition to bacterial infections, other examples of plant viral and fungal pathogens within the scope ofthe invention include, but are not limited to, tobacco mosaic virus, cauliflower mosaic virus, turnip crinkle virus, turnip yellow mosaic virus; fungi including Phytophthora infestans, Peronospora parasitica, Rhizoctonia solani, Botrytis cinerea, Phoma lingam (Leptosphaeria maculans), and Albugo Candida.

EXAMPLES The invention is further described in the following examples. These examples, however, are provided for purposes of illustration to those skilled in the art, and are not intended to be limiting unless otherwise specified. Moreover, these examples are not to be construed as limiting the scope ofthe appended claims. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result ofthe teaching provided herein.

Experimental Procedures; Strains and Growth Conditions All the alleles of etol are of ecotype Columbia (Col-0), except for eto2-2 in Wassilewskija (WS). Ecotype Lansberg erecta (Ler) was used for mapping. Seeds for eto2-2 were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, Ohio). Plant growth in air and ethylene was performed as previously described by Kieber et al, 1993.

Example 1 - Mapping of ETOl.

The ETOl gene has been genetically mapped to the bottom of chromosome 3 using visible markers (Roman et al, 1995). In the inventors' laboratory, an etol-4 mutation was fine mapped using simple sequence length polymorphism (SSLP) markers (Bell and Ecker,

Genomics 19:37-44 (1994)) and cleaved amplified polymorphic sequence (CAPS) markers (Konieczny et al, Plant J. 4:403-410 (1993)).

A homozygous etol-4 mutant was crossed to wild-type Ler, and used for generating F2 population segregating etol mutation. F2 seeds were from T. Hirayama. Templates for SSLP from etol F2 mutants were prepared as described by Klimyuk et al, Plant J. 3 :493- 494 (1993), except that frozen-and-ground powder of young leaf tissues was used instead of pieces of leaves, and 2 μl from the final of 100 μl was added to each 15 μl of reaction. For a CAPS reaction, DNA was prepared as described (Konieczny et al, 1993). After scoring 1824 recombinant chromosomes from the cross, etol was mapped between two CAPS markers, AtPK41 A and AFCl .

Using further SSLP and CAPS markers, the ETOl gene was fine mapped on 58kb region at AtEml locus (GenBank accession no. AF049236) (FIG. 2(A)) using genomic sequencing, carried out as described (Yang et al, Gene 83:347-354 (1989)) for both Col-0 and Ler ecotypes. Sequences ofthe SSLP and CAPS markers were as follows.

EmlA-f: 5'- CAATTCATCAAGGTAAAGGCTTG-3' (SEQ ID NO:5); EmlA-r: 5'-ACGCCAGATACTGCTGCGTG-3' (SEQ ID NO:6); EmlB-f: 5'-CAAGGAGACCAAATTATGATTGAG-3' (SEQ ID NO:7); EmlB-r: 5'-GTAGATCGAAGAAGCGTACGG-3' (SEQ ID NO:8); EmlH-f: 5'-GCGTCCCTTTATTCGAATAG-3' (SEQ ID NO:9); EmβlH-r: 5'-GTGTGACACCCCTTTTTTGG-3' (SEQ ID NO:10) (EmlH is to be cut with-M/fel); EmlL-f: 5'-CCATAGATCTGTCAGAATC-3 ' (SEQ ID NO: 11); EmlL-r: 5'-CGACCATCGTCTTTATCTTC-3' (SEQ ID NO: 12); EmlNl-f: 5'-GGCCAATACTTGATGTACATTTC-3' (SEQ ID NO:13); EmlNl-r: 5'-GGTGCTTTATCCTCTTCTATC-3' (SEQ ID NO:14).

Length polymorphisms were examined ofthe fifteen ORFs predicted in this region using ten alleles of etol. For checking the length polymorphisms at the predicted ORF2 of AtEml locus, two sets of primers were used as below.

ORF2a-f: 5'-CTGGTTCACTCAAACCAAGC-3' (SEQ ID NO:15); ORF2a-r: 5'-AGGATTACGAGGGTGCTTTG-3' (SEQ ID NO:16);

ORF2b-f: 5'-CAAAGCACCCTCGTAATCCT-3' (SEQ ID NO:17); ORF2b-r: 5'-CCGAGAAGAAGAAGAAGACG-3' (SEQ ID NO:18). A 50 base pairs deletion of an ORF (ORF2 at AtEml locus) was found in two X-ray alleles, etol-2 and etol-3 (FIG. 3(B), FIG. 3(C) and FIG. 2(A)). Although this deletion was found within the predicted first intron, 50 out of total of 107 base pairs ofthe intron were lost, suggesting that inefficient splicing would occur, thus resulting in an altered-and- truncated protein. Further sequencing and PCR analysis of seven (7) other alleles, including X-ray, EMS and DEB-induced alleles, enabled the identification of a single base-pair change, deletion, or large genomic rearrangement, which resulted in nonsense or missense mutations (FIG. 3(C) and 2(A)). Thus, it was confirmed that ORF2 encoded the ETOl locus.

Example 2 - Nucleic Acid Analysis.

Total RNA and DNA extraction as well as Northern and Southern analysis were performed as described by Kieber et al, 1993. DNA sequences of mutant alleles ofthe ETOl gene were determined directly by genomic sequencing as described by Yang et al, 1989, using templates from four independent PCR reactions. The predicted ETOl protein sequence was subjected to BLASTP, TBLASTN, PSI-BLAST (Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997)) and SMART (Schultz et al, Proc. Natl. Acad. Sci. USA 95:5857-64 (1998)). Multiple alignments were carried out using the CLUSTALW algorithms (Thompson et al, Nucleic Acids Research, 22:4673-4680 (1994)) and shaded with MacBoxShade Ver. 1.0.8 (Baron, Inst. for Animal Health, Pirbright, Surrey, UK). See FIG. 4.

An etiolated seedling cDNA library (Kieber et al, 1993) was screened with the PCR fragment derived from the predicted ORF2. The longest cDNA was 3595 nucleotides in length, and its longest open reading frame encoded a protein of 951 amino acids with a predicted molecular mass of approximately 107 kDa. The predicted ETOl protein contained two distinct protein : protein interaction domains. The BTB domain is on the N- terminus (FIGs. 2(A)-2(D, and FIG. 4)); and the C-terminal comprises the TPR domain, predominantly comprising 10 TPR motifs, and harboring a coiled-coil motif within it (FIGs. 2(A)-2(D) and FIG. 4).

The TPR motif has been defined as a degenerated 34 amino acids with amphipathic α-helices, and it is believed to be involved in protein : protein interactions (Goebl et al, Trends Biochem. Sci. 16:173-177 (1991); Lamb et al, Trends Biochem. Sci. 20:257-259 (1995)). It is found in many proteins of diverse functions, and it had been proposed that it functions as a scaffold for the assembly of multi-protein complexes (Das et al, 1998; Scheufler, 2000; Lapouge, 2000)). Etol -5, a DEB-induced mutation, contained a T-to-A transversion at nucleotide

1396, which affected the predicted replacement of a phenylalanine residue with an isoleucine residue at amino acid 466. The phenylalanine residue is located in a predicted TPR1, implying the importance of its role in the function of ETOl . The bulky phenylalanine (or tyrosine) may form a "knob," fitting into a hydrophobic "hole" between different α-helices, or in neighboring TPR motifs to maintain the TPR structure (Goebl et al, 1991; Das et al, 1998).

Etol-1, an EMS-induced mutation with similar extent of ethylene-overproduction as the other alleles, contained a C-to-T transition at nucleotide 2994 and is predicted to introduce a stop codon at amino acid 867, resulting in truncation of only the last two TPR motifs. This result also suggested the important role ofthe TPR domain. Furthermore, all ofthe sequences from the examined alleles, except for eto 1-6 allele which had a large genomic rearrangement in promoter and/or 5' region, lacked or altered the sequence ofthe TPR domain, strongly suggesting its indispensable role in the function ofthe ETOl protein

(Table 1).

Table 1 - Summary of etol Mutant Alleles and Effect of Mutations.

Example 3 - ETOl is a Member of Highly Conserved Plant Gene Family.

TBLASTN search ofthe predicted ETOl" protein provided two putative paralogs with both BTB and TPR domains (GenBank accession nos. AB020755, AC002330) and one pseudogene (AP002053) from Arabidopsis genomic sequences or expressed sequence tags. The two paralogs are referred to as EOLl (for ETOl -LIKE 1) to the top of chromosome 4 and EOL2 to the bottom of chromosome 5, respectively. Using Reverse-Transcription Polymerase Chain Reaction (RT-PCR), the cDNA ofthe EOL genes was cloned and sequenced (FIGs. 2(C) and 2(D)). The homology between ETOl and the predicted EOL proteins was significant, although much higher in their C-termini (76 to 77%) as compared to N-terminal regions (48 to 60%). The carboxy-termini of EOLl and EOL2 also contain 6 TPR motifs and a coiled coil motif.

The putative amino acid sequences for the polypeptides ETOl (SEQ ID NO: 19), EOLl (SEQ ID NO: 20), and EOL2 (SEQ ID NO: 21), encoded by ETOl, EOLl and EOL2 respectively were compared, as shown in FIG. 4. Both EOLl and EOL2 conserved the phenylalanine residue that is mutated in the etol-5 allele, further implicating its essential role in these proteins (FIG. 4)). Also, all ofthe amino-termini ofthe ETOl and EOL proteins contained the BTB domain, indicating its essential role in their function. A potential function of this domain is multimerization of these proteins, such as homo- or hetero-dimerization, as has been proposed for other BTB proteins (Aravind et al., J. Mol. Biol. 285:1353-1361 (1999)).

One remarkable difference between ETOl and other EOL proteins is its relatively long proline- and glycine-rich N-terminal stretch. This may implicate a difference in their respective functions.

Several putative sequences were also found for orthologs from fern, monocots (rice, corn, sorghum), dicots (tomato, cotton, soybean, Medicago truncatula, Lotus japonicus, chestnut and hybrid aspen) through TBLSTN database search. Interestingly, no orthologous sequences were found outside ofthe plant kingdom, indicating that ETOl and its homologs represent a novel gene family unique to higher and lower plants.

Example 4 - Complementation ofthe etol Mutation by the ETOl Gene.

The sequence changes found in all alleles of etol strongly indicated that this gene was responsible for the ethylene-overproducing phenotype observed in the etol mutants. To further confirm that the gene was ETOl, the etol mutation was complemented and the effect on phenotype recorded.

A 3.5kb BamΑl-Kpnl fragment from the longest cDNA clone (pcETO1.9) was cloned into BamRl-Kpnl digested pROK2 (Baulcombe et al, Nature 321 :446-449 (1989)) in the sense direction. The C58 strains harboring the constructs above were then used to transform the Arabidopsis ecotype Col-0 or etol-4 mutant using in planta vacuum infiltration (Bechtold et al, C. R. Acad. Sci. Paris Life Sci. 316:1194-1199 (1993)). Kanamycin-resistant TI plants were selected on the plate of Murashige and Skoog medium supplemented with lOOμg/ml kanamycin and transferred to soil. Similarly, antisense constructs were prepared. Transgenic plants expressing sense or antisense ETOl rnRNA under the control of

CaMV 35S promoter were made in the background of both wild type and etol-4 mutant. In 20 ofthe 50 independent transformants, the introduction ofthe ETOl cDNA into etol-4 totally restored the non-ethylene-overproducing phenotype (FIG. 5), indicating that the introduced 35S::ET01 transgene had complemented the etol mutation. Furthermore, when antisense ETOl was expressed in wild type plants, they showed etol phenotype (data not shown). Thus, it was concluded that the selected gene was the ETOl gene. Example 5 - ETOl and EOL Proteins Directly Interact with the C-terminus of ACS 5.

To explore the potential interaction between ETOl and ACS5, the yeast two-hybrid system and in vitro peptide binding assay were used.

^'The complete coding sequence of ETOl (2.8 kb Bam Hi-Sal I fragment)) was amplified from pcETOl .9 by Pyrococcus furiosus (pfu) DNA polymerase and also subcloned to pAS2. Deletion ofthe sequence for twelve (12) amino acid residues from the carboxyl terminus of ACS5 were achieved by PCR, and subsequently cloned to pACT2. In the plate assay, the HIS3 gene was used as a reporter gene in the yeast two-hybrid system. The addition of 1,2,4 aminotriazole (3-AT, analog ofthe substrate for HIS3 protein) decreased the background expression ofthe reporter gene, and also authenticated the interaction between the two hybrid proteins. Expression ofthe above hybrid clones in yeast was verified by Western blot analysis using a commercially available monoclonal antibody (Roche Diagnostics, Basel Switzerland) that recognizes the HA1 epitope tag in the hybrid constructs. Yeast manipulation and quantitative liquid assay for β-galactosidase activity were performed as described, e.g., in Guthrie & Fink (Eds.): Methods in Enzymology, Vol. 194, Guide to Yeast Genetics and Molecular Biology. Acad. Press, NY, (1991)).

Constructs to be used in the yeast two-hybrid interaction were prepared as described. First, RT-PCR was used to amplify the full-length sequences encoding EOLl, EOL2, and ACS5 from total RNA prepared as follows. Eleven-day old etiolated Arabidopsis thaliana seedlings were immersed with 150 μM of cycloheximide (CHX) for 8 hours in the dark. The CHX-treated seedlings were collected and quickly frozen in liquid nitrogen. Five (5) μg of total RNA was used to synthesize the first-strand cDNA by using the Superscript Preamplification System from GIBCO-BRL (Rockville, MD) according to the manufacturer's protocol. Gene-specific primers were used to amplify the individual cDNA with. pfu DNA polymerase (Strategene, La Jolla, CA). The resulting full-length cDNA for EOLl (2.7 kb Bam Hi-Sal I fragment), EOL2 (2.8 kb Bam Hl-Sal I fragment), and ACS5 (1.4 kb Bam Hl-Xho I fragment) were subcloned to the two-hybrid vectors, pAS2 (for EOLl and EOL2) and pACT2 (for ACS5).

The results showed that ETOl interacted strongly with ACS5 in the two-hybrid system (FIG. 6(A)). Furthermore, both EOLl and EOL2 also interacted with ACS5 to a similar degree. Quantitative data based upon β-galactosidase activity showed that EOLl interacts with ACS5 at a level that was nearly 3.5-fold stronger than EOL2, and two fold stronger than ETOl, in the yeast two-hybrid system (FIG. 6(B)). Thus, the strength of interaction with ACS5 is ordered: EOLl > ETOl > EOL2. The interactions observed in the yeast cells were specific, because alone, yeast cannot activate the reporter genes, nor do the yeast cells interact with another non-specific protein, ETHYLENE-INSENSITIVE2 (ELN2). The carboxyl terminus of either ETOl or ACS5 protein is essential for this specific interaction, because deletion of as few as 12 amino acids of ACS5 or 25 amino acids of ETOl abolished the interaction (data not shown).

The in vivo binding results clearly demonstrate that the C-terminus of ACS5 interacts specifically and strongly with ETOl, as well as with its paralogs, but not the vector, in the yeast cells. The significance ofthe C-terminus is confirmed because the truncated ACS5 construct (with a deletion at the C-terminus), lost its strong interaction with ETOl (compare FIGs. 6(A) and 6(B)).

Example 6 - ETOl Family of Proteins Have an Inhibitory Effect on ACS in vivo.

To test the direct inhibitory effect of ETOl and EOL proteins on ACS activity, a functional expression assay was conducted in a bacterial system.

ETOl, EOLl and EOL2 cDNA were cloned in a pTrc99A vector (Pharmacia Corp., Peapack, NJ). under the control of an IPTG inducible promoter. ACS5 cDNA was cloned in the same vector in which the ampicillin resistance gene was replaced by a chloramphenicol resistance gene. JAde 6 was then transformed with the different constructs as indicated in the chart shown in FIG. 7(C). JAde 6 is an Escherichia coli isoleucine auxotroph strain, that expresses ACC deaminase from Pseudomonas sp. It also metabolizes ACC synthesized when it is transformed with an ACC synthase gene (Tarun et al, Proc. Natl. Acad. Sci. USA 95:9796-801 (1998A)). The JAde 6-ETOl, -EOLl, -EOL2 and -ACS5 cDNA transformants were selected on

LB plates for expression and co-expression of ACS5 and the EOL proteins, and growth was assayed on minimal media M9 for 2 days (FIG. 7(A)). In a control experiment, bacteria were grown on M9 plates supplemented with 3 mM ACC (FIG. 7(B)). For each construct, two independent clones were plated. After expression ofthe Arabidopsis ACS5 gene, the transformed JAde 6 bacteria were able to grow on minimal media without the need for supplemental ACC (FIGs. 7(A) and 7(B)). By comparison, the transformed bacteria co-expressing ACS5 and EOLl, or ACS 5 and EOL2, were unable to grow on minimal media without the addition of ACC (FIG. 7(A) and 7(B)). This growth inhibition demonstrated the inhibition of ACS5 enzymatic activity by the EOL proteins. Unfortunately, the effect of ETOl expression on the growth of growth of bacterially expressed ACS5 could not determined because ETOl expression was toxic for the bacteria (data not shown).

Example 7 - Genetic Requirement ofthe ACS5 Gene for the etol Phenotype

To further demonstrate the direct interaction of ETOl with ACS activity inplanta, a double mutant was prepared and analyzed between etol-4 and eto2-2 (originally referred to as cin5-l; Vogel et al, 1998), a recessive loss-of-function mutant allele of the ACS5 gene. Crosses were performed following Guzman and Ecker, 1990. Progeny ofthe FI were genotyped using a dCAPS marker for the etol-4 allele, and a PCR marker for the T-DNA insertion of eto2-2 mutant. The phenotype ofthe progeny ofthe double mutant plants was analyzed 3 days after germination entirely in the dark on Murashige and Skoog media in the presence of air, or 10 ppm ethylene (C₂H ). FIG. 8 photographically compares the resulting phenotypes, showing wild-type plants (Col-0) in column 1, etol-4 plants in column 2, eto2-2 plants in column 3, and etol-4 eto2-2 double mutant plants in column 4. In contrast with the dominant eto2-l allele that overproduces 20-fold of ethylene, the eto2-2 allele did not accumulate significantly higher amount of ethylene. Instead, it had a defect in the cytokinin-induced triple response phenotype.

In the double mutant, the etol phenotype in etiolated seedlings appeared suppressed, both when grown in the air and in ethylene (FIG. 8, column 4). The seedlings restored a long hypocotyl, without exaggerated hook and long root. The genotype ofthe etol-4 eto2-2 double mutant was verified by PCR. When combined with the preceding examples, this result confirmed that ETO 1 negatively regulated the activity of ACS5 in etiolated seedlings.

Each and every patent, patent application and publication that is cited in the foregoing specification is herein incorporated by reference in its entirety.

While the foregoing specification has been described with regard to certain preferred embodiments, and many details have been set forth for the purpose 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 ofthe details described herein can be varied considerably without departing from the spirit and scope ofthe invention. Such modifications, equivalent variations and additional embodiments are also intended to fall within the scope ofthe appended claims.

Claims

What is claimed is:

1. An isolated nucleic acid encoding a plant negative regulator of ethylene biosynthesis, wherein the encoded product comprises at least one BTB/POZ (Broad- Complex, tramtrack, and brie a brac.l poxvirus and zinc finger) domain in its N-terminus, and at least one tetratricopeptide repeat (TPR) motifs in its C-terminus.

2. The nucleic acid according to claim 1 , comprising ETHYLENE-OVERPROD UCER1 (ETOl), and active fragments thereof.

3. The nucleic acid according to claim 2, comprising SEQ ID NO:2.

4. The nucleic acid according to claims 2 or 3, further comprising mutants, derivatives, paralogs, orthologs or homologs of ETOl, which encode an expression product having ETOl -activity in a plant cell, and wherein the mutant, derivative, paralog, ortholog or homolog is at least 40% homologous to ETOl or SEQ ID NO:2.

5. The nucleic acid according to claim 1, comprising a member ofthe ETOl gene family, which is an ETOl-Like (EOL) gene, and active fragments thereof.

6. The nucleic acid according to claim 5, wherein the EOL gene is EOLl or EOL2.

I. The nucleic acid according to claim 6, comprising SEQ ID NO:3 or SEQ ID NO:4.

8. The nucleic acid according to claims 5-7, further comprising mutants, derivatives, paralog, orthologs or homologs of an EOL gene, which encode an expression product having EOL-activity in a plant cell, and wherein the mutant, derivative, paralog, ortholog or homolog is at least 40% homologous to EOLl, EOL2, SEQ ID NO:3, or SEQ ID NO:4.

9. A purified preparation of a polypeptide encoded by the nucleic acid of any of claims 1-8, and active fragments thereof.

10. The polypeptide according to claim 9, comprising ETOl.

I I . The polypeptide according to claim 9, comprising SEQ ID NO: 19.

12. The polypeptide according to claim 9-11, further comprising mutants, derivatives, paralogs, orthologs, homologs, and analogs of ETOl, having ETOl -activity in a plant cell, wherein the mutant, derivative, paralog, ortholog, homolog or analog is at least 40% homologous to ETOl or SEQ ID NO: 19.

13. The polypeptide according to claims 9-12, comprising a member ofthe ETOl family, which is an ETOl-Like (EOL) gene, and active fragments thereof.

14. The polypeptide according to claim 13, wherein the EOL gene is EOLl or EOL2.

15. The polypeptide according to claim 14, comprising SEQ ID NO:20 or SEQ ID NO:21.

16. A recombinant cell comprising the isolated nucleic acid of any of claims 1-9.

17. A vector comprising the isolated nucleic acid of any of claims 1-9.

18. An antibody specific for a plant ETOl or EOL polypeptide, paralog, ortholog, homolog, analog, derivative or fragment thereof, wherein the polypeptide has ETOl or EOL activity in a plant cell.

19. An isolated nucleic acid sequence comprising a sequence complementary to all or part ofthe nucleic acid sequence of one of claims 1-9.

20. The nucleic acid according to claim 19 having antisense activity at a level sufficient to regulate, control, or modulate the ethylene biosynthesis activity of a plant, plant cell, organ, flower or tissue comprising same.

21. A plant, plant cell, organ, flower, tissue, seed, or progeny comprising nucleic acid according to any of claims 1-8 or 16-20.

22. A transgenic plant, the cells, organs, flowers, tissues, seeds or progeny of which comprise the nucleic acid according to any of claims 1-8 or 16-20.

23. A transgenic plant, the cells, organs, flowers, tissues, seeds or progeny of which comprise the polypeptide according to any of claims 9-15.

24. An isolated nucleic acid of one of claims 1-8 or 16-20, further comprising a promoter sequence operable in a plant cell.

25. The isolated nucleic acid of claims 1-8, 16-20 or 24, further comprising a reporter gene operably fused thereto, or a fragment thereof having reporter activity.

26. A transgenic plant, the cells, organs, flowers, tissues, seed, or progeny of which comprise a transgene comprising an isolated nucleic acid of claim 24.

27. A method for manipulating in a plant the nucleic acid according to any of claims 1- 9, 16-20 or 24 permit the regulation, control or modulation ofthe ethylene response in a plant or plant cell, organ, flower, tissue, seed or progeny comprising same.

28. The method according to claim 27, wherein said regulation, control or modulation initiates or enhances the germination, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit ripening, insect, herbicide or pathogen resistance, abscission, or response to stress, injury or pathogens in the plant or plant cell, organ, flower, tissue, seed or progeny comprising same.

29. The method according to claim 27, wherein said regulation, control or modulation inhibits or prevents the germination, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit ripening, insect, herbicide or pathogen resistance, abscission, or response to stress, injury or pathogens in the plant or plant cell, organ, flower, tissue, seed or progeny comprising same.

30. A method of identifying a compound capable of affecting ethylene biosynthesis in a plant or plant cell comprising: providing a cell comprising an isolated nucleic acid encoding a polypeptide according to claims 9-15, having a reporter sequence operably linked thereto; adding to the cell a compound being tested; and measuring the level of reporter gene activity in the cell, wherein a higher or lower level of reporter gene activity in the cell compared with the level of reporter gene activity in a second cell to which the compound being tested was not added is an indicator that the compound being tested is capable of affecting the ethylene biosynthesis of a plant.

31. A method for generating a modified plant with modified ethylene biosynthesis activity as compared to that of comparable wild type plant comprising introducing into the cells ofthe modified plant an isolated nucleic acid encoding ETOl or a gene in the EOL family, wherein the nucleic acid ofthe ETOl or EOL gene regulates ethylene biosynthesis ofthe modified plant.

32. The method for generating a modified plant of claim 31, wherein ethylene biosynthesis is enhanced or activated in the modified plant.

33. The method for generating a modified plant of claim 31, wherein ethylene biosynthesis is reduced or blocked in the modified plant.

34. A method for manipulating ethylene biosynthesis in a plant cell comprising: operably fusing an ETOl or EOT gene, or an operable portion thereof to a plant promoter sequence in the plant cell to form a chimeric DNA, and generating a transgenic plant, the cells of which comprise said chimeric DNA, whereupon controlled activation ofthe plant promoter, manipulates expression of ETOl or EOT, which operates as a regulator of ethylene biosynthesis in the plant cell.