AU5222699A - Copper transporter in ethylene signaling pathway - Google Patents

Description

WO 00/04760 PCT/US99/16591 5 COPPER TRANSPORTER IN ETHYLENE SIGNALING PATHWAY Reference To Related Applications This application claims priority to US Provisional Application 60/093,698, filed July 22, 1998. 10 Government Support This work was supported in part by grants from the National Science Foundation, grant number MCB-95-07166, the United States Department of Energy and the National Institutes of Health, grant 15 number DE-FG02-93ER20104. The government may have certain rights in this invention. BACKGROUND OF THE INVENTION The simple gaseous hormone ethylene (C 2

H

4 ) is involved in a 20 variety of plant growth and developmental processes, including germination, cell elongation, flower and leaf senescence, sex determination and fruit ripening (Abeles et al., (1992) In Ethylene in Plant Biology, 2 nd Ed., New York, NY: Academic Press. A number of biological stresses are known to induce ethylene 25 production in plants, including wounding, abscission, bacterial, WO 00/04760 PCT/US99/16591 5 viral or fungal-infection, and treatment with elicitors, such as glycopeptide elicitor preparations from fungal pathogen cells. In the case of abscission, a particular layer of cells in a zone located between the base of the leaf stalk and the stem (the abscission zone) responds to a complex combination of ethylene 10 and other endogenous plant growth regulators by a process that is, to date, not fully understood. However, the effect of abscission is the controlled loss of part of the plant, typically localized; the result of which is visible as a dead leaf or flower, or as softening or "ripening" of fruit, ultimately 15 leaving the plant wounded at the point of separation. Recent studies have shown that ethylene is also required in the determination of cell fate in the root epidermis (Tanimoto et al., Plant J. 8:943-948 (1995)), the systematic activation of a defense gene upon pathogen attack (Penninckx et al., Plant Cell 20 8:2309-2323 (1996)), the wound response in tomato (O'Donnell et al., Science 274:1914-1917 (1996)), and the formation of symbiotic nitrogen-fixing nodules (Penmetsa et al., Science 275:527-530 (1997)). Thus, regulated ethylene production and the perception of ethylene by receptors in required tissues of plants 25 followed by signal transduction is necessary to establish, control and maintain the complicated developmental systems found in plants, including defensive responses to plant pathogens, chlorosis, senescence and abscission.. The synthetic pathway of ethylene has been well 30 characterized (Kende, Plant Physiol., 91:1-4 (1989)). The conversion of ACC to ethylene is catalyzed by ethylene forming enzyme (Spanu et al., EMBO J 10:2007 (1991) ) . In a closed circular ethylene synthetic pathway, S-adenosyl-1 methionine (SAM) is produced from methionine. Then, in a rate-limiting 35 step, SAM is converted to 1 aminocyclopropane-1-carboxylic acid (ACC) by an ACC synthase. In a final step, ethylene is produced from ACC by an ACC oxidase. The pathway, therefore, utilizes multiple ACC synthases and ACC oxidases. 2 WO 00/04760 PCT/US99/16591 5 Scientists are just beginning to understand the ethylene signal transduction pathway at the molecular level. To address the ethylene signaling mechanisms, a molecular/genetic approach has been applied using the ethylene-evoked triple response phenotype of Arabidopsis thaliana seedlings. In Arabidopsis, the 10 "triple response" typically involves inhibition of root and stem elongation, radial swelling of the stem and absence of normal geotropic response (diageotropism). Etiolated morphology of a plant can be dramatically altered by stress conditions which induce ethylene production, so that, for example, the ethylene 15 induced triple response provides a seedling with the additional strength required to penetrate compacted soils. Based upon the triple response, a dozen Arabidopsis mutants have been isolated into two classes (Ecker, Science 268:667-675 (1995); Johnson & Ecker, Annu. Rev. Genetics 32:227-254 (1998): 20 US Pat. Nos. 5,367,065; 5,444,166; 5,602,322 and 5,650,553, each of which is herein incorporated by reference). One class of mutants, the ein (ethylene insensitive) mutants, show reduced or are completely insensitive to exogenous ethylene. This group includes etrl (Bleecker et al., Science 241:1086-1089 (1988), 25 etr2 (Sakai et al., Proc. Natl. Acad. Sci. USA 95:5812-5817 (1998)), ein2 (Guzman & Ecker, Plant Cell 2:513-523 (1990)), ein3 (Rothenberg & Ecker, Sem. Dev. Biol. Plant Dev. Genet. 4:3-13 (1993)), ein5/ainl [Van der Straeten et al., Plant Physiol. 102:401-408 (1993)), eti (Harpham et al., Ann. Bot. 68:55-62 30 (1991), ein4 and ein6 (Roman et al., Genetics 139:1393-1409 (1995)). The other class of mutants, the constitutive hormone response mutants) display constitutive ethylene response phenotypes in the absence of exogenously applied hormones. Using antagonists of ethylene biosynthesis and activity, this class was 35 further divided into hormone overproducing mutants (etol, eto2 and eto3) (Guzman and Ecker, 1990, supra; Kieber et al., Cell 72:427-441 (1993)) and into constitutive signaling mutants (ctrl; constitutive triple response) (Kieber et al., 1993, supra.). The 3 WO 00/04760 PCT/US99/16591 5 latter mutants display "ethylene" phenotypes even in the presence of inhibitors of ethylene biosynthesis or receptor binding. Genetic epistasis studies indicate that ETRl and EIN4 act upstream of CTRl, while EIN2, EIN3, EIN5, EIN6 and EIN7 act downstream of CTR1 (Roman et al., 1995, supra; Sakai et al., 10 1998, supra; Hua et al., Plant Cell 10:1321-1332 (1998) ). The CTR1 gene encodes a Raf-like serine/threonine protein kinase (Kieber et al., 1993, supra) . Raf is known as a component of a MAP kinase cascade in mammalian cells, indicating that a MAP kinase cascade is involved in the ethylene signaling pathway. 15 Given that a loss-of-function mutation in this gene confers an ethylene constitutive response phenotype, CTRl is presumed to be a negative regulator of the ethylene response pathway. The EIN3 gene and its related genes (EILl, 2 and 3) have been cloned and characterized (Chao et al., Cell 89:1133-1144 (1997)). EIN3 20 protein is presumed to be a new class of transcriptional regulator as evidenced by its sequence specific DNA binding activity in vitro (Solano et al., Genes Dev. 12:3703-3714 (1998)). The mechanism of ethylene perception was first addressed by 25 biochemical approaches (Bleecker et al., (1997) In Biology and Biotechnology of the Plant Hormone Ethylene, AK Kanellis, ed: Kluweer Acad. Publ, pp. 63-70; Hua & Meyerowitz, Cell 94:261-271 (1998)). Ethylene binding assays using whole plant tissue or organs with [1 4 C] ethylene, revealed saturable two class binding 30 sites with different dissociation rates, fast dissociation (half life, <30 minutes) and a slow dissociation (half life >6 hours) in many plant species (reviewed by Sisler, (1990) In The Plant Hormone Ethylene, K. Mattoo & JC Suttle, eds. (Boca Raton, FL: CRC Press), pp. 81-100 (1990). Subsequent competitive binding 35 assays using ethylene antagonists supported the physiological relevance of binding activities; however, attempts to purify the ethylene receptors proved unsuccessful. 4 WO 00/04760 PCT/US99/16591 5 Interestingly, etrl (ethylene receptor) plants display a reduction in ethylene binding activity (Bleecker et al., 1988, supra), indicating that ETR1 encodes a receptor for ethylene. Mutations in the gene can cause a complete loss of the ethylene response. Consequently, it can be concluded that the ETRl gene 10 encodes a receptor for ethylene. The ETR1 gene, isolated by map based chromosomal walking, encodes a membrane-bound histidine kinase (Chang et al., Science 262:539-544 (1993); Chang et al., Proc. Natl. Acad. Sci. USA 92):4129-4133 (1995)). The kinase is similar to an emerging family of eukaryotic histidine kinases, 15 including the osmosensor of budding yeast (Wurgler-Murphy et al., Trends Biochem. Sci. 22:172-176 (1997)), and to sensor molecules in the two component regulatory systems in prokaryotes. Expression of this protein in budding yeast cells was found to confer ethylene binding capability with similar binding 20 characteristics to those found in plant materials, further confirming that the etrl gene encodes an ethylene receptor (Schaller et al., Science 270:1809-1811 (1995)). Moreover, ETR1 protein expressed and partially purified from E. coli has been shown to possess histidine kinase activity (Gamble et al, Proc. 25 Natl. Acad. Sci. USA 95.: 7825-7829 (1998)). Arabidopsis has at least four other ETRl-like genes, ERS1, ETR2, EIN4 and ERS2 (Hua et al., 1995, supra; Hua et al., 1998, supra; Sakai et al., 1998, supra) . The ein4 and etr2 mutants display a similar ethylene insensitive phenotype as that of etrl. 30 Transgenic Arabidopsis plants with an artificial mutant ERS or ERS2 gene, which has the same mutation as etrl-1, also display an ethylene insensitive phenotype. These results indicate that the ETR1-related proteins are involved in the ethylene signaling pathway. Since all identified receptor mutants are dominant, it 35 has been unclear until recently that receptor molecules are positive or negative regulators of ethylene responses. Recent studies on the loss-of-function mutations of ethylene receptor genes have revealed that ethylene receptors negatively regulate 5 WO 00/04760 PCT/US99/16591 5 the ethylene response pathway (Hua et al., 1998, supra). "Never ripe," an ethylene insensitive mutant tomato species, has been identified as bearing a mutation in an ERS-like gene, indicating that the ethylene signaling pathway, at least at the perception of this hormone, is conserved in the plant kingdom. 10 Copper is, of course, an essential metal for aerobic organisms. It serves as a co-factor for enzymes such as cytochrome c oxidase, copper-zinc superoxide dismutase, and lysyl oxidase, and is also required for neurotransmitter synthesis and the maturation of connective tissues in animals (for review Uauy 15 et al., Am. J. Clin. Nutr. 67(suppl)e:952S-959S (1998)). However, paradoxically, copper is highly toxic. The intracellular concentration of copper is tightly regulated and copper is typically sequestered in non-reactive forms. Nevertheless, the basic mechanisms of copper transport and 20 metabolism seem to be highly conserved in evolution (Askwith et al., Trends Biochem. Sci. 23 :135-138 (1998)). In yeast, for example, a plasma membrane-localized copper transporter imports copper ions into the cytoplasm, where they are immediately bound by copper trafficking proteins. Several copper distinct 25 trafficking proteins are known including, Atxlp, Lys7p and Coxl7, which differ in their intracellular targets. Thus, it appears that without a sufficient supply of copper, the receptors are unable to assume the proper protein conformation. By analogy with yeast, reduced copper transport by RAN1 30 localization into a post-Golgi compartment appears to present an abnormal amount of copper to co-localized ethylene receptor apoproteins (Thompson et al., J. Am. Chem. Soc. 105.:3522-3527 (1983)). This may cause altered (or relaxed) ligand specificity allowing the recognition of both ethylene and TCO to function as 35 agonists. From recent studies (Rodriguez et al., Science 283:396-398 (1999)), it appears that a dimerized ethylene receptor requires at least two molecules of copper to assume a correct structure (and ligand specificity). 6 WO 00/04760 PCTIUS99/16591 5 The fact that mutations in copper trafficking genes cause physiological disorders in many eukaryotes, supports the concept that the proper regulation of copper metabolism is crucial. In addition to the Menkes/Wilson disease genes (yeast CCC2/Arabidopsis RANl), human homologues of the yeast cytosolic 10 copper metabolism genes have been uncovered, although not intended to be limiting, including human Ctrl gene (a homologue of yeast CTRl, (Zhou et al., Proc. Natl. Acad. Sci. USA 94:7481 7486 (1997)), human Hahl gene (a homologue of yeast ATX1, (Klomp et al., J. Biol. Chem. 272:9221-9226 (1997)), human Ccsl gene (a 15 homologue to LYS7, (Culotta et al., J. Biol. Chem. 272:23469 23472 (1997)) and human Coxl7 gene (a homologue of yeast COX17, (Amaravadi et al., Hum. Genetics 99:329-333 (1997)). Because ethylene is an olefin, it has been speculated that ethylene is perceived by a plant receptor(s) with transition 20 metals, such as a copper (Burg et al., Plant Physiol. 42:144-152 (1967). Several observations supported this idea. For example, when silver ions (Ag(I)), which are biochemically similar to copper ions (Cu(I)), are added to the media of plant cells in vitro, the ethylene response is inhibited. Moreover, the 25 addition of either copper or silver ions increases the ethylene binding activity of membrane extracts from yeast cells expressing the ETRl protein (Bleecker, 1997, supra; Bleecker et al., Science 283:996-999 (1999)). No known metal binding motifs, however, had been reported prior to the present invention in the predicted 30 amino acid sequence of ETRl or in any of its related proteins, leaving the issue unsolved until the development of the present invention. The present invention, therefore, provides the novel isolated nucleic acid, its expression product, and methods for 35 controlling, modulating or regulating the delivery of essential transition metal ions, particularly copper ions, to the ethylene receptors during the secretion pathway. By thus improving understanding of the ethylene signal pathway, it is now possible 7 WO 00/04760 PCT/US99/16591 5 to develop methods for improving the tolerance of plants to pathogens, as well as for developing 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 10 possible to significantly improve the quality, quantity and longevity of plant food products, such as fruits and vegetables, flowers and flowering ornamentals, and other non-food plant products, such as commercially valuable crops, e.g., cotton or flax, or ornamental green plants, thereby providing more and 15 better products for market in both developed and underdeveloped countries. SUMMARY OF THE INVENTION The present invention is directed to an isolated nucleic 20 acid encoding a plant, plant cell, tissue, flower, organ or other plant part having copper transporter activity, and mutants, derivatives, homologues and fragments thereof encoding a plant, plant cell, tissue, flower, organ or other plant part having copper transporter activity. Also included are the ranl gene, 25 and the genomic nucleic acid sequences and cDNA comprising SEQ ID NO:l and 2. In addition, embodiments of the invention also relate to a plant, plant cell, organ, flower, tissue, seed, or progeny comprising the nucleic acid encoding a plant copper transporter. 30 The invention is also directed to a purified preparation of a polypeptide comprising a plant copper transporter in a plant, plant cell, tissue, flower, organ or other plant part having copper transporter activity, and analogs, derivatives and fragments thereof having such activity. Also included in the 35 invention is RAN1 and the polypeptide having amino acid sequence SEQ ID NO:3, further including an embodiment in which the copper transporter activity is ATP-dependent. 8 WO 00/04760 PCT/US99/16591 5 Further included in embodiments of the invention is a recombinant cell comprising a plant copper transporter, a vector comprising the isolated nucleic acid encoding a plant copper transporter, and an antibody specific for a plant RAN1 polypeptide, analog, derivative or fragment thereof having copper 10 transporter activity. Also included in the invention is an isolated nucleic acid sequence comprising a sequence which is complementary in an antisense orientation to all or part of the nucleic acid encoding a plant copper transporter sequence, and to mutants, derivatives, 15 homologues or fragments thereof encoding a plant, plant cell, tissue, flower, organ or other plant part having copper transporter activity. An embodiment further includes such complementary nucleic acid having antisense activity at a level sufficient to regulate, control, or modulate the copper 20 transporting activity of a plant, plant cell, organ, flower or tissue. In addition, the invention also relates to a plant, plant cell, organ, flower, tissue, seed, or progeny comprising a nucleic acid in an antisense orientation to the nucleic acid encoding a plant copper transporter. 25 The invention is further directed to a transgenic plant, the cells, organs, flowers, tissues, seeds or progeny of which comprise the nucleic acid encoding a plant copper transporter, or a recombinant nucleic acid comprising the nucleic acid encoding a plant copper transporter. Also included in the 30 invention is a transgenic plant, the cells, organs, flowers, tissues, seeds or progeny of which comprise a polypeptide having plant copper transporter activity. In addition, the invention is directed to any of the preceding isolated nucleic acids encoding a plant copper 35 transporter, further comprising a plant RAN1 promoter sequence, or a fragment thereof having promoter activity. Also included in the invention is a vector comprising any of the preceding isolated nucleic acids encoding a plant copper transporter. 9 WO 00/04760 PCT/US99/16591 5 Moreover, an additional embodiment includes any of the preceding isolated nucleic acids encoding a plant copper transporter, further comprising a reporter gene operably fused thereto, or a fragment thereof having reporter activity. Also the invention is directed to a transgenic plant, the 10 cells, organs, flowers, tissues, seed, or progeny of which comprise a transgene comprising an isolated nucleic acid comprising a RAN1 promoter sequence. The invention is further directed to a method for manipulating in a plant a nucleic acid encoding a plant copper 15 transporter, wherein the method permits the regulation, control or modulation of germination, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit ripening, insect, herbicide or pathogen resistance, abscission, or response to stress in the plant. In one embodiment the method is directed 20 to the initiation or enhancement of the germination, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit ripening, insect, herbicide or pathogen resistance, abscission, or response to stress of the plant, in another embodiment the method is directed to the inhibition or 25 prevention of such responses in the plant, plant cell, organ, flower or tissue. In addition, the invention is directed to a method of identifying a compound capable of affecting the transport of copper in the ethylene signaling system in a plant comprising: 30 providing a cell comprising an isolated nucleic acid encoding a plant RAN1 sequence, 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 35 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 expression of a plant ranl gene. 10 WO 00/04760 PCT/US99/16591 5 Moreover, the invention is directed, in one embodiment, to a method for generating a modified plant with enhanced copper transporting activity as compared to that of comparable wild type plant comprising introducing into the cells of the modified plant an isolated nucleic acid encoding RAN1, wherein said ranl nucleic 10 acid is capable of transporting copper within the cells of the modified plant. Another embodiment is directed to a method for generating a plant with diminished or inhibited copper transporting activity as compared to that of a comparable wild type plant comprising binding or inhibiting the copper 15 transporting molecules within the cells of the modified plant by introducing into said cells an isolated nucleic acid encoding a complementary nucleic acid to all or a portion of ranl, wherein said rani nucleic acid would otherwise be capable of transporting copper within the cells of the modified plant. An additional 20 embodiment provides a method for generating a plant with diminished or inhibited copper transporting activity comprising binding or inhibiting the copper transporting molecules within the cells of the modified plant by introducing into said cells an antibody to all or a portion of RAN1, wherein said RAN1 25 polypeptide would otherwise be capable of transporting copper within the cells of the modified plant. Further, the invention is directed to a method for manipulating the expression of RAN1 in a plant cell comprising operably fusing the nucleic acid ranl or an operable portion 30 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, where upon controlled activation of the plant promoter, manipulates expression of RAN1. The invention is also directed to the nucleic acid 35 encoding and the polypeptide expression products of mutant alleles ranl-1 and ranl-2. Additional objects, advantages and novel features of the invention will be set forth in part in the description, examples 11 WO 00/04760 PCT/US99/16591 5 and figures which follow, and in part will become apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE FIGURES 10 Fig. 1 is a model representing the function of RAN1 in the ethylene signal transduction pathway. Filled and unfilled shapes indicate the active and inactive states of signaling pathway components, respectively. Figs. 2A and 2B illustrate the phenotype of the ranl 15 mutants. Fig. 2A shows the seedling phenotype of wild type plants (Col), ranl-1, ranl-2, and ethylene over-producing mutant (etol-5) and constitutive triple response mutant (ctrl-1) in air, transcyclooctene (TCO) or ethylene (C 2

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4 ) . Fig. 2B shows the rosette plant phenotype, in air and in TCO. 20 Figs. 3A and 3B illustrate that ethylene inducible genes are up-regulated in the ranl mutants by TCO treatments. In Fig. 3A, northern blots are shown of total RNA prepared from seedlings treated with hydrocarbon-free air, ethylene or TCO. Arabidopsis a-tublin 3 (TUA3) was used as a control. The same blot was used 25 in three hybridizations. In Fig. 3B, an analysis is shown of the expression of the basic chitinase gene, CHIB, in TCO-treated adult plants. Northern blot analysis was done using total RNA prepared from rosette leaves exposed to hydrocarbon-free air or TCO for three (3) days. 30 Fig. 4 illustrates the fine mapping of the RAN1 gene to -160 kb region at the bottom of chromosome 5 by the identification of three (3) each recombinant chromosomes using the simple sequence length polymorphism (SSLP) markers CIC2AT2L and CIC4ET2R. The assembled BAC contig covering this region is 35 shown. Additionally, new markers developed from the sequence of T19K24 permitted localization of the RANl gene on a single BAC (bacterial artificial chromosome). The predicted gene organization of the BAC insert is drawn according to the 12 WO 00/04760 PCT/US99/16591 5 annotation of T19K24 (GenBank AC002342) . Gray or black boxes represent predicted genes. The intron and exon organization of the RANl gene is shown at the bottom. Arrowheads indicate the locations of the ranl-1 and ranl-2 mutations. Fig. 5 illustrates the genomic nucleotide sequence of the 10 RANl gene, SEQ ID NO:l; GenBank accession #AF091112. Beneath the nucleotide sequence is the predicted amino acid sequence, SEQ ID NO:3. The upper case and lower case characters indicate exon and intron sequences, respectively. The exon-intron boundaries were determined by comparison with the sequence of cDNA, SEQ ID NO:2, 15 GenBank acession # AF082565, which extends from nucleotide -275 through nucleotide 4201. The numbers for the nucleotides are shown on the left, with the A from the first ATG codon assigned the number 1. The amino acid numbers are also indicated. Asterisks indicate in-frame stop codons. 20 Fig. 6A shows a Fet3p oxidase assay in which oxidase activity of the membrane fraction from various yeast cells shown in panel A was measured using an in-gel assay system. The lower panel shows the result of a western blot of membrane fraction samples used in the oxidase assay using anti-HA-tag antibody. 25 Fig. 6B shows a high affinity iron uptake assay in which the iron uptake activity of wild-type or ccc2-disrupted yeast cells expressing various proteins were measured. This included yeast wild-type CCC2, RAN1:HA-tag, ranl-l:HAtag, or ranl-2:HAtag. Averages from three (3) independent experiments are shown. Error 30 bars indicate standard deviations. Fig. 7 illustrates that copper partially suppresses the ranl phenotype. The seeds of various strains were germinated on MS medium absent added copper ions (- CuSO 4 ) or on the same medium containing 12.5 pM CuSO 4 (+ CuSO 4 ) . Both were exposed to 35 hydrocarbon-free air or TCO. After three (3) days of incubation, the seedlings were photographed. The applied concentration of CuSO 4 had no effect on the triple response of etol-5 or ctrl-1 13 WO 00/04760 PCT/US99/16591 5 mutants grown in hydrocarbon-free air or on the growth-promoting activity of TCO treatment of etol-5 seedlings. Figs. 8A - 8C illustrate the constitutive ethylene response phenotypes in 35S::RAN1 transgenic plants. Fig. 8A shows the phenotype of 35S::RAN1 transgenic plants, specifically three-week 10 old transgenic plants. Approximately one-half of the transgenic plants displayed a ctrl-like phenotype (class 1), while the other half displayed normal morphology (class 2) . The photographs were taken at the same magnification. Fig. 8B shows the expression of RANl (upper panel) and the ethylene-inducible basic-chitinase 15 (CHIB) gene (lower panel) in transgenic plants. Northern blots were performed using 20 pg of total RNA per lane from adult plants. In the class 1 transgenic plants, RNA was prepared from five (5) independent Ti-generation plants and loaded in one lane. Fig. 8C shows the ethylene response phenotypes in the transgenic 20 seedlings. Representative dark-grown T2-generation seedlings are shown from ranl-2 and Li (35S::RANl) transgenic plants treated with TCO for three days. Also shown are representative dark grown T2-generation seedlings from 35S::RAN1-containing plants (L6 and L3) and seedlings from the ctrl-1 mutant treated with 25 hydrocarbon-free air for three days. DETAILED DESCRIPTION OF THE INVENTION The invention is based upon the identification, pursuant to a novel screen for antagonist responsive mutants, of an early 30 acting, negative regulator in the ethylene gas signaling pathway in Arabidopsis. As a result of the present invention, new insights into the mechanisms involved in the ethylene signaling pathway are evident. To better understand the mechanisms underlying ethylene perception, the present inventors have 35 isolated mutants in which ethylene recognition is changed. In doing so, mutants have been identified and characterized in the present invention that show an altered ligand specificity of their ethylene receptors. 14 WO 00/04760 PCT/US99/16591 5 An ethylene antagonist, trans-cyclooctene (TCO), activates the ethylene signaling pathway in the class of mutants of this invention, as opposed to inhibiting or blocking the pathway. The mutants are encoded by alleles of ranl (responsive to antagonist 1) . Map based cloning of the RAAT1 gene by the inventors revealed 10 ranl to be a homologue of human Menkes-Wilson disease genes and of the yeast CCC2 gene. The RAN1 protein has been found to advantageously provide copper transporting activity to plant cells. Coincidentally, recent studies of the human Menkes protein, Wilson protein and yeast Ccc2p suggest that those 15 proteins may also have a copper transporter function and deliver copper ions into the secretion pathway. In accordance with the present invention, genetic, molecular and biochemical studies of RAN1 provide the first in vivo evidence of a requirement of copper ions for ethylene 20 perception/signal transmission in plants. Moreover, functional studies of RANI demonstrate that the mechanisms controlling copper trafficking in yeast and plant cells are conserved. Discovery of the requirement of RAN1/copper for the assembly of functional hormone receptors facilitate an understanding of the 25 mechanism of ethylene perception in plants and offers a useful paradigm for copper-dependent signaling processes in other organisms. To identify mutations in novel components of the ethylene gas signal transduction pathway, a screen was initiated for 30 Arabidopsis thaliana mutants that exhibited 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 35 elongation, 2) radial swelling of the stem and 3) exaggeration of the apical hook. A class of constitutive mutants, ctr, display a constitutive triple response in the presence of ethylene biosynthetic inhibitors, and is most likely affected at, 15 WO 00/04760 PCTIUS99/16591 5 or downstream of the receptor. Based on the results of genetic experiments, over-expression of the normal or truncated versions of the regulatory gene ran1 in transgenic plants was identified by the inventors and characterized as an early acting gene in the ethylene response pathway, displaying an ethylene triple response 10 phenotype in response to potent receptor antagonists. It should be appreciated that the present invention is not limited by the proposed models and mechanisms described herein. Thus, it should be understood that models such as the one shown in Fig. 1, for example, present a working model showing the role 15 of RAN1 to deliver copper ions to create functional ethylene receptors, and the like, in the ethylene signal transduction pathway, all of which facilitates understanding of the invention. RAN1 is shown as localized on the membrane of post-Golgi compartments. RAN1 receives copper ions from copper chaperons 20 such as Atxl-like proteins and transports them into a post-Golgi compartment, delivering the metal to membrane-targeted ethylene receptors. After the incorporation of copper ion, the receptors are localized on the plasma membrane, where they functionally coordinate ethylene. 25 Ethylene is an olefin, therefore its receptors are presumed to require coordination of a transition metal for hormone binding activity. In a preferred embodiment of the present invention, the transition metal is a silver ion (Ag (I)) . In a particularly preferred embodiment the transition metal is a copper ion (Cu 30 (I)). Either these or other transition metals are incorporated into the ethylene receptors. Once there, they may inhibit ethylene binding or proper folding of a functional receptor, resulting in constitutively active receptor proteins. Reduction of copper transport causes the Ran~ phenotype. 35 Thus, it appears that without a sufficient supply of copper, the receptors are unable to assume the proper protein conformation. In wild type plants, ethylene binding to its receptor inactivates the activity of ethylene receptors (presumably 16 WO 00/04760 PCT/US99/16591 5 causing a reduction in the histidine kinase activity), and consequently causing induction of the ethylene response through activation (de-repression) of the signaling pathway. In the absence of copper delivery by RAN1 in co-suppressed transgenic plants (class 1), the ethylene receptors are nonfunctional 10 (absent of kinase activity). Consequently, even in the absence of ethylene, the hormone response pathway is constitutively activated. Loss-of-function ethylene receptor mutants have been shown to function as negative regulators of the signaling pathway and 15 show significant functional overlap. Moreover, binding of ethylene to the receptor(s) presumably inhibits biochemical activity. More severe alterations in the expression of RAN1 causes partial or complete activation of ethylene responsive genes and phenotypes. Furthermore, the phenotypes observed in 20 RANl co-suppressed plants were indistinguishable from those described for the quadruple ethylene receptor knockout mutants. The resulting plants were severely stunted, and fully infertile. (Hua & Meyerowitz, 1998, supra), confirming that copper is required by more than one ethylene receptor for functionality. 25 RAN1 activity appears to be required for the functionality of at least four or more of the ethylene receptors. Thus, loss of expression of this single critical regulator of receptor function results in de-repression of the entire ethylene response pathway. Copper is required for assembly of the receptor 30 complex, and in its absence, these proteins appear to be turned over more rapidly (i.e., none of the ethylene receptors may be functional). By "plant" as used herein, is meant any plant and any part of such plant, wild type, treated, genetically manipulated or 35 recombinant, including transgenic plants. The term broadly refers to any and all parts of the plant, including plant cell, tissue, flower, leaf, stem, root, organ, and the like, and also 17 WO 00/04760 PCT/US99/16591 5 including seeds, progeny and the like, whether such part is specifically named or not. By "copper transporter activity" or "copper transporting activity" as used herein, is meant a protein or polypeptide which transports copper or other transition metals to the post-Golgi 10 region, after which the metal is delivered to membrane-targeted ethylene receptors in the ethylene signaling pathway. Although reference is made by example to "copper" in the delivery system, the term is meant to exemplify any transition metal involved in the secretory pathway. 15 As used herein "biologically active" refers to copper transporting activity, meaning a polypeptide or fragment thereof which is capable of transporting a copper ion in the ethylene signal system as shown in the model of the present invention. At the molecular, cellular and whole plant level, and in 20 seedling and adult plants, air-grown ran1 mutants appear identical to ethylene-treated wild-type plants. The dominant nature of ranl suggests that the ethylene-response pathway is normally under positive regulation and loss of function of the RAN1 repressing activity by treatment with TCO results in 25 activation of the constitutive triple response phenotype. The gene corresponding to RAN1 has been cloned as set forth below and the sequence of cDNA clone is described. Physiological, biochemical and genetic evidence indicate that the ranl gene product is required for transduction of the ethylene 30 signal in both etiolated seedling and adult plants. The putative RAN1 polypeptide acts as a positive regulator in the ethylene signal transduction chain, while exposure to the ethylene receptor antagonist trans-cyclooctene (TCO) prevents this morphological transformation. Instead of acting as a competitive 35 inhibitor of ethylene binding in ranl seedlings, TCO treatment causes activation of the triple response, mimicking the effects of ethylene. 18 WO 00/04760 PCT/US99/16591 5 A reduced delivery of copper ions to the ethylene receptors appears to produce a state of suboptimal copper : apoprotein stoichiometry. Such an altered protein conformation results in reduced ligand specificity, thereby allowing TCO, as well as ethylene, to act as agonists. Analogous to multi-receptor 10 knockout mutants, severe reduction of RAN1 function results in copper-depletion of all ethylene receptors, causing de-repression of the signaling pathway and activation of all known ethylene response phenotypes. Several ranl alleles have been identified, as exemplified 15 by ranl-1 and ranl-2. Such mutants are recessive and modulate ethylene perception. Sequence analysis of the exemplified ranl mutant alleles revealed that both mutations cause amino acid substitutions in residues found within conserved protein motifs (metal-binding and ATPase domains), suggesting reduced function. 20 Expression of the mutant ranl proteins in the yeast ccc2 mutant was insufficient to restore full Fet3p activity, supporting the understanding that ranl alleles are partial loss-of-function mutations. Nevertheless, in view of the descriptions provided, it is understood that other alleles and variations would be 25 available to one of ordinary skill in the art. Therefore, additional mutants are also enabled by the present invention that have insertions, deletions, alterations or substitutions within the same conserved protein motif, so long as copper transporting activity is expressed or regulated. 30 Additional evidence for the in vivo requirement of copper for ethylene perception/signaling is provided by studies of the ranl mutants (see the Examples) . Based on several lines of evidence, the ranl mutants are hypomorphs, effecting a reduction in copper-transporting activity. The ranl-1 and ranl-2 alleles 35 contain a miss-sense mutation in the phosphate and metal binding motifs, respectively. Thus, the conclusion that a defect in a copper transporter causes altered ligand specificity of ethylene receptors is supported by the findings that the defect in the 19 WO 00/04760 PCT/US99/16591 5 ranl-1 and ranl-2 mutants allows response to trans- but not cis cyclooctene (CCO) or 1-methylcyclopropene (MCP), potent competitive inhibitors of ethylene-receptor binding. In sum, the invention should be construed to include nucleic acid comprising ranl, or any mutant, derivative, 10 homologue or fragment thereof, which encodes a copper transporter of the ethylene signaling pathway. In accordance with the present invention, nucleic acid sequences include, but are not limited to DNA, including and not limited to cDNA and genomic DNA; RNA, including and not limited to mRNA and tRNA, and may 15 include chiral or mixed molecules. Preferred nucleic acid sequences include, for example, those set forth in SEQUENCE ID NOS: 1 and 2, as well as modifications in those nucleic acid sequences, including alterations, insertions, deletions, mutations, homologues and 20 fragments thereof encoding a regulatory protein in the ethylene response pathway affecting copper transporter activity in the ethylene response pathway. A "fragment" of a nucleic acid is included within the present invention if it encodes substantially the same expression 25 product as the isolated nucleic acid, or if it encodes a peptide having copper transporter capability. The invention should also be construed to include peptides, polypeptides or proteins comprising RAN1, or any mutant, derivative, varient, analogs, homologue or fragment thereof, 30 having copper transporter activity in the ethylene signaling pathway. The terms "protein," "peptide," "polypeptide," and "protein sequences" are used interchangeably within the scope of the present invention, and include, but are not limited to the sequence set forth in SEQUENCE ID NO: 3, the amino acid sequences 35 corresponding to nucleic acid SEQUENCE ID NOS: 1 and 2, as well as those sequences representing mutations, derivatives, analogs or homologues or fragments thereof having copper transporter activity in the ethylene response pathway. 20 WO 00/04760 PCTIUS99/16591 5 The invention also provides for analogs of proteins, peptides or polypeptides encoded by the gene of interest, preferably ranl. "Analogs" can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or 10 by both. "Homologues" are chromosomal DNA carrying the same genetic loci; when carried on a diploid cell there is a copy of the homologue from each parent. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the peptide, 15 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 20 arginine; or phenylalanine and tyrosine. Modifications (which do not normally affect the primary sequence) include in vivo or in vitro chemical derivatization of the peptide, e.g., acetylation or carbonation. Also included are modification of glycosylation, e.g., modifications made to the 25 glycosylation pattern of a polypeptide during its synthesis and processing, or further processing steps. Also included are sequences in which amino acid residues are phosphorylated, e.g., phosphotyrosine, phosphoserine or phosphothreonine. Also included in the invention are polypeptides which have 30 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 35 acids, e.g., D-amino acids or non-naturally occurring synthetic molecules. However, the polypeptides of the present invention are not intended to be limited to products of any specific exemplary process defined herein. 21 WO 00/04760 PCT/US99/16591 5 "Derivative" is intended to include both functional and chemical derivatives, including fragments, segments, variants or analogs of a molecule. A molecule is a "chemical derivative" of another, if it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's 10 solubility, absorption, biological half life, and the like, or they may decrease toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980) . Procedures for 15 coupling such moieties to a molecule are well known in the art. Included within the meaning of the term "derivative" as used in the present invention are "alterations," "insertions," and "deletions" of nucleotides or peptides, polypeptides or the like. A "variant" or "allelic or species variant" of a protein 20 refers to a molecule substantially similar in structure and biological activity to the protein. Thus, if two molecules possess a common activity and may substitute for each other, it is intended that they are "variants," even if the composition or secondary, tertiary, or quaternary structure of one of the 25 molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical. A "fragment" of a polypeptide is included within the present invention if it retains substantially the same activity as the purified peptide, or if it has copper transporting 30 activity. Such fragment of a peptide is also meant to define a fragment of an antibody. In accordance with the invention, the RANl and ranl nucleic acid sequences employed in the invention may be exogenous sequences. Exogenous or heterologous, as used herein, denotes 35 a nucleic acid sequence which is not obtained from and would not normally form a part of the genetic makeup of the plant, cell, organ, flower or tissue to be transformed, in its untransformed state. Plants comprising exogenous nucleic acid sequences of 22 WO 00/04760 PCT/US99/16591 5 RAN1 or ranl mutations are encoded by, but not limited to, the nucleic acid sequences of SEQUENCE ID NOS: 1 and 2, including alterations, insertions, deletions, mutations, homologues and fragments thereof. Transformed plant cells, tissues and the like, comprising 10 nucleic acid sequences of RAN1 or ranl mutations, such as, but not limited to, the nucleic acid sequences of SEQUENCE ID NOS: 1 and 2, are within the scope of the invention. Transformed cells of the invention may be prepared by employing standard transformation techniques and procedures as set forth in Sambrook 15 et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). By the term "nucleic acid encoding" the plant cell and the like having copper transporter activity, as used herein is meant a gene encoding a polypeptide capable of transporting copper as 20 described above. The term is meant to encompass DNA, RNA, and the like. As described in the following Examples, ranl genes encode proteins having specific domains located therein, for example, terminal extensions, transmembrane spans, TM1 and TM2, nucleotide 25 binding folds, a putative regulatory domain, and the C-terminus. A mutant, derivative, homologue or fragment of the subject gene 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 of the present 30 invention. It will be appreciated that the definition of such a nucleic acid encompasses those genes having at least about 40% homology, in any of the described domains contained therein. In addition, when the term "homology" is used herein to refer to the domains of these proteins, it should be construed 35 to be applied to homology at both the nucleic acid and the amino acid levels. Significant homology between similar domains in such nucleic acids or their protein products is considered to be at least about 40%, preferably, the homology between domains is 23 WO 00/04760 PCT/US99/16591 5 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 domains is about 99%, or the protein products thereof. 10 According to the present invention, preferably, the isolated nucleic acid encoding the biologically active copper transporter polypeptide or fragment thereof of the ethylene signal system is full length or of sufficient length to encode a regulated or active copper transporter. In one embodiment the 15 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. In another embodiment, preferably, the purified 20 preparation of the isolated polypeptide having copper transporter polypeptide activity in the ethylene signal system is at least about 60 amino acids in length. More preferably, it 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 25 preferably, at least 700 amino acids in length. In an additional embodiment the polypeptide encodes the full length RAN1 protein or a regulated version thereof. The invention further includes a vector comprising a gene encoding RAN1. DNA molecules composed of a protein gene or a 30 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 35 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 24 WO 00/04760 PCT/US99/16591 5 hybrid gene may be introduced into the viral genome by techniques well known in the art. Thus, the present invention encompasses the expression of the desired proteins in either prokaryotic or eukaryotic cells, or viruses which replicate in prokaryotic or eukaryotic cells. 10 Preferably, the proteins of the present invention are cloned and expressed in a virus. Viral hosts for expression of the proteins of the present invention include viral particles which replicate in prokaryotic host or viral particles which infect and replicate in eukaryotic hosts. Procedures for 15 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 20 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. 25 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 30 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, 35 or the virus or its genetic sequence is introduced into a susceptible host, the cells are grown in media and screened for appropriate activities. Expression of the sequence results in the production of the protein of the present invention. 25 WO 00/04760 PCT/US99/16591 5 Procedures for generating a plant cell, tissue, flower, organ or a fragment thereof, are well known in the art, and are described, for example, in Sambrook et al., supra. Suitable cells include, but are not limited to, cells from yeast, bacteria, mammal, baculovirus-infected insect, and plants, with 10 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 modulated copper transporting capability. Suitable vector and plant combinations will be readily 15 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-mediated leaf disc transformation methods described 20 by Horsch et al., 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 of the desired protein in eukaryotic hosts 25 requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, but are not limited to, the SV40 early promoter (Benoist et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene 30 promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971 6975 (1982)) and the exemplified pYES3 PGK1 promoter. As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic 35 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). 26 WO 00/04760 PCTIUS99/16591 5 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 10 molecules are incapable of autonomous replication, the expression of the desired protein may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome. For expression of the desired protein in 15 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 20 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, 25 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 30 synthesis of mRNA. 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. 35 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 27 WO 00/04760 PCT/US99/16591 5 importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is 10 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 15 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 initiates or enhances the above responses; whereas in another preferred embodiment the method inhibits or prevents the 20 above responses. Thus, methods of the present invention define embodiments in which the copper transporting activity is prevented or inhibited. By "prevention" is meant the cessation of copper transport activity in the ethylene signal pathway. By 25 "inhibition" is meant a statistically significant reduction in the amount of copper transport activity or RAN1 protein detected as compared with plants, plant cells, organs, flowers or tissues grown without the inhibitor or disclosed method of inhibition. Preferably, the inhibitor reduces copper transport by at least 30 20 %, more preferably by at least 50%, even more preferably by 80% or greater, and also preferably, in a dose-dependent manner. Once inhibitors satisfying these requirements are identified, the utilization of assay procedures to identify the manner in which the copper transport is inhibited are particularly useful. 35 Similarly, methods of the present invention are defined in which the copper transporting activity is initiated, stimulated or enhanced if there is a statistically significant increase in the amount of copper transport activity or RAN1 protein detected 28 WO 00/04760 PCT/US99/16591 5 as compared with plants, plant cells, organs, flowers or tissues grown without the enhancer or disclosed method of enhancement. Preferably, the enhancer increases copper transport 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. 10 Once enhancers satisfying these requirements are identified, the utilization of assay procedures to identify the manner in which the copper transport 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 15 all of ranl or of a plant copper transporter gene. The antisense nucleic acid should be of sufficient length as to inhibit expression of the target gene of interest. The actual length of the nucleic acid may vary, depending on the target gene, and the region targeted within the gene. Typically, such a preparation 20 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 25 to, and essentially identical to the non-coding DNA strand of the ranl gene, but which does not encode RAN1. "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 30 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 50%) of the corresponding positions in each of the molecules are occupied by nucleotides which normally base-pair with each other (e.g., A:T 35 and G:C nucleotide pairs.). In yet another aspect of the invention, antibodies are provided which are directed against the copper transporter polypeptide of the ethylene signaling system, preferably RAN1, 29 WO 00/04760 PCTIUS99/16591 5 which 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 RAN1 polypeptide, including functional equivalents of the 10 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. 15 The invention further includes a transgenic plant comprising an isolated DNA encoded plant copper transporter or active fragment thereof, capable of transporting copper in the ethylene signaling system. For instance provided in at least one example if the current invention, a transgenic Arabidopsis plant 20 comprising a yeast cccL transgene rescued by the addition of the ranl, which when expressed confers upon the plant the ability recognize the presence of ethylene, an ability that had been deleted from the original yeast gene. By "transgenic plant" as used herein, is meant a plant, 25 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 30 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 35 antisense DNA encoding the copper transporter of the ethylene signaling pathway, preferably RAN1, may be accomplished by transforming the plant with a plasmid, liposome, or other vector encoding the desired DNA sequence. Such vectors would, as 30 WO 00/04760 PCTIUS99/16591 5 described above, include, but are not limited to the disarmed Agrobacterium tumor-inducing (Ti) plasmids containing a sense or antisense strand placed under the control of a strong constitutive promoter, such as 35CaMV 35S or under an inducible promoter. Methods of generating such constructs, plant 10 transformation and plant regeneration methods are well known in the art once the sequence of the gene of interest is known, for example as described in Ausubel et al., 1993, Current Protocols in Molecular Biology, Greene & Wiley, New York). In accordance with the present invention, plants included 15 within its scope include both higher and lower plants of the Plant Kingdom. Mature plants, including rosette stage plants, and seedlings are included in the scope of the invention. A mature plant, therefore, includes a plant at any stage in development beyond the seedling. A seedling is a very young, 20 immature plant in the early stages of development. Transgenic plants are also included within the scope of the present invention, having a phenotype characterized by the RAN1 gene or ranl mutations. Preferred plants of the present invention, which are 25 affected by the copper transporter in the ethylene signal system 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, 30 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 of the genera Daucus (particularly the species carota, carrot) and Apium (particularly the species graveolens dulce, celery) and the 35 like; the Family Solanacea, particularly of the genus Lycopersicon, particularly the species esculentum (tomato) and the genus Solanum, particularly the species tuberosum (potato) and melongena (eggplant), and the like, and the genus Capsicum, 31 WO 00/04760 PCT/US99/16591 5 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 of the genus Brassica, particularly the species campestris (turnip), oleracea cv Tastie (cabbage), 10 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 more preferred are the leafy 15 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 of the flower on the stem (delayed abscission) is of 20 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 of the leaf stem on the plant (delayed abscission) is of particular relevance. Delayed flowering in 25 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 30 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, Clavibacter michiganense (formerly Coynebacterium michiganense), 35 Pseudomonas solanacearum and Erwinia stewartii, and more particularly, Xanthomonas campestris (specifically pathovars campestris and vesicatoria), Pseudomonas syringae (specifically pathovars tomato, maculicola). 32 WO 00/04760 PCT/US99/16591 5 In addition to bacterial infections, other examples of plant viral and fungal pathogens within the scope of the 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 10 parasitica, Rhizoctonia solani, Botrytis cinerea, Phoma lingam (Leptosphaeria maculans), and Albugo candida. In Arabidopsis, several expressed sequence tags with significant similarity to the functional equivalent of yeast Atxlp have been identified (Himelblau et al., Plant Physiol. 15 117:1227-1234 (1998)), indicating that other steps in intracellular trafficking copper ions may also be conserved in the plant kingdom. Although mutations have not been identified in any of these genes, one or more of these proteins may also be expected play a role in the creation of functional ethylene 20 receptors and would be known to one of ordinary skill in the art in view of the findings of the present invention. Through continued isolation of mutants using the Ran/Ctr screen of the present invention (in particular from segregating families to enable recovery of lethal mutations), it will be possible, in 25 accordance with the present invention, to identify additional genes required for assembly of functional ethylene receptors. The present invention is further described in the following examples. These examples are not to be construed as limiting the scope of the appended claims. 30 EXAMPLES Example 1: Screen for Mutants Responsive to Potent EthyleneReceptor Antagonist. 35 Arabidopsis thaliana ecotype Columbia (Col-0) was used as the parental strain for mutant isolation. EMS (ethyl methane sulfonate) mutagenesis of seeds was performed as described by Guzman and Ecker, 1990, supra. Plant growth in hydrocarbon-free 33 WO 00/04760 PCT/US99/16591 5 air, ethylene and TCO were carried out as described by Kieber et al., 1993, supra. To identify novel components required for ethylene perception/signaling, mutants were screened that displayed hormone responsive phenotypes upon exposure to an antagonist of 10 ethylene action. Trans-cyclooctene (TCO) was chosen as the ethylene antagonist for the screen because this cyclic olefin acts as a potent competitive inhibitor of ethylene binding to its receptor(s) in vitro and in vivo (Sisler, 1990 supra; Schaller et al., 1995, supra). 15 The screening of 30,000 M2-mutagenized seed populations, which were selected from twenty (20) independent lots of seeds that had been mutagenized with EMS, yielded two independent responsive-to-antagonistl (ranl) mutants that displayed a characteristic "ethylene" triple response phenotype in response 20 to treatment with TCO. Complementation tests revealed that these two mutants (ranl-l and ranl-2) were allelic (see Table 1). 34 WO 00/04760 PCT/US99/16591 5 Table 1- Genetic analysis of ran1 mutants Treatment Segregation Rati Cross ng ranl-1 x wild type F1 TCO all wild type F2 TCO ran-a; 44, wild type; 152 1:3 0.68 ranl-2 x wild type Fl TCO all wild type F2 TCO ran-; 28, wild type; 135 1:3 5.32 ranl-1 x ranl-2 F1 TCO all ran~ F2 TCO all ran~ ranl-1 x etrl-1 Fl TCO ran-; 8, wild type; 109 1:15 0.067 F2 ACC ein-b; 151, wild type; 62 3:1 1.88 ranl-2 x etrl-1 F1 TCO ran~; 16, wild type; 137 1:15 4.62 F2 ACC ein; 195, wild type; 60 3:1 0.29 ranl-2 x etrl-1 F1 TCO ran-; 15, wild type 228 1:15 0.0025 F2 ACC ein~; 143, wild type; 58 3:1 1.60 ranl-2 x ein2-12 Fl TCO ran; 25, wild type; 222 1:15 6.31 F2 ACC ein ; 143, wild type; 58 3:1 1.60 a: Ran~ indicates plants that display ethylene response phenotypes when treated with TCO. 10 b: Ein indicates plants that show absence of ethylene response phenotypes. Crosses were performed as described by Guzman and Ecker, 1990, supra. F1 progeny from crosses between wild-type plants 15 and ranl-1 or ranl-2 did not display the triple response phenotype in the presence of TCO, while F2 progeny from self fertilized Fl plants showed a segregation ratio of 1 ran : 3 wild-type plants (see Table 1). This indicated that ranl is a single locus recessive mutant. 20 35 WO 00/04760 PCTIUS99/16591 5 Example 2: Ethylene Response Pathway Is Activated in ran1 Mutants by an Ethylene Antagonist. As previously shown, TCO treatment causes reversion of the triple response phenotype of etol, a mutant that overproduces 10 ethylene, but not the phenotype of cntl, a mutant in which the ethylene signaling pathway downstream of the receptors is constitutively active. Although the ranl mutant seedlings exhibit the triple response-like phenotype in the presence of TCO, the same response is not seen when the seedlings are grown 15 in hydrocarbon-free air (Fig. 2A). Effect of Mutation in Presence of Ethylene Agonist on Seedlings 20 In ranl-1 and ranl-2, exposure to TCO produced effects on seedling growth similar to those evoked by treatment of wild type seedlings with ethylene (Fig. 2A). Measurements of the hypocotyl and root length confirmed these observations. In the presence of TCO, the length of the hypocotyl in ranl-1 and ranl-2 25 seedlings was 3.8 ± 0.7 mm and 4.3 ± 0.5 mm, respectively. In ethylene-treated wild-type seedlings, the length of the hypocotyl was 4.2 ± 0.4 mm. Similarly, TCO-treatment caused inhibition of root growth in ranl seedlings; although the length in ranl mutants (ranl-l; 1.9 ± 0.6 mm, ranl-2; 2.4 0.4 mm) was slightly 30 longer than of wild type in ethylene (1.2 0.2 mm). The degree of curvature of the hypocotyl hook was also measured in wild-type and mutant seedlings. In ethylene, the angle of the hypocotyl hook in wild-type seedlings (250 ± 49.2 ) was indistinguishable from that observed in TCO-treated ranl 35 mutants (ranl-1; 250 ± 23.7 , ranl-2; 253 ± 23.8 ), further demonstrating that TCO activates the ethylene signaling pathway in ranl. 36 WO 00/04760 PCTIUS99/16591 5 In contrast to its effect on the ranl mutants, TCO treatment of wild-type seedlings caused an increase in hypocotyl and root growth, compared with growth in hydrocarbon-free air (Fig. 2A). These results are consistent with previous findings that inhibition of basal level ethylene biosynthesis/perception 10 allows maximal elongation of seedlings (Guzman & Ecker, 1990, supra). Other compounds that inhibit ethylene action, such as silver ion or 1-methylcyclopropane (MCP) (Sisler et al., Plant Growth Regul. 18:79-86 (1996)), failed to induce the triple 15 response in the ranl mutants. As in wild-type plants, these compounds prevented responsiveness to ethylene in ranl mutants, demonstrating that the ranl phenotype was specific to TCO. Induction of the seedling triple response in ranl mutants was stereo-specific. Trans- but not cis-cyclooctene was effective 20 in evoking this morphological transformation. Furthermore, treatment of ranl seedlings with TCO in the presence of AVG (aminoethoxyvinylglycine), a potent inhibitor of ethylene biosynthesis in Arabidopsis seedlings (Guzman & Ecker, 1990, supra), had no effect on the ranl mutant phenotype (Fig. 2A), 25 further demonstrating that the seedling phenotype typifies the response to TCO ethylene antagonist, and that it was not due to an increase in ethylene biosynthesis. Induction of the triple response was examined in ranl plants to different doses of ethylene. However, there appeared 30 to be no significant difference between mutant and wild-type plants, suggesting that the ranl mutations do not alter normal perception of ethylene. When grown in hydrocarbon-free air, the phenotype of ranl-2 seedlings was identical to the wild-type phenotype (Fig. 2A). However, the root length of ranl-l was 35 slightly shorter than that observed in wild-type seedlings. Although these lines were extensively backcrossed, it was possible that the short root phenotype of ranl-1 was caused by a mutation near the RANl locus. However, since ranl-2 did not 37 WO 00/04760 PCT/US99/16591 5 display this phenotype, it was concluded that this effect was allele specific. No other significant growth phenotypes were observed in ranl-l or ranl-2 seedlings or adult plants. Effect of Mutation in Presence of Ethylene 10 Agonist on Adult Plants For analysis of adult plants, seedlings were moved from agar plates containing MS medium (Mirashugi and Skoog medium) to soil, or they were germinated directly on soil, and young rosette 15 plants (after the second true.leaves have emerged) were exposed to hydrocarbon-free air or TCO in 5-liter vessels for twelve (12) days. The vessels were flushed daily with hydrocarbon-free air and supplied with fresh TCO to reduce the accumulation of C02, an inhibitor of ethylene action. 20 To examine the effect of TCO on vegetative growth, young rosette plants were exposed to TCO. The size of the leaves in mutants of ranl-1 and ranl-2 was found to be significantly reduced, as compared to wild type (compare Fig. 2A with Fig. 2B). Leaf shape and petiole length observed in TCO-treated ranl 25 mutants phenocopied the morphology of ctrl-1 and were characteristic of ethylene effects on plant growth (Chao et al., 1997, supra). The size of the leaves of the wild-type plants was also slightly reduced by treatment with TCO (Fig. 2B). However, this 30 effect was assumed to be caused by the toxicity evoked by treatment with a high level of TCO since a similar size reduction was also observed in the constitutive ethylene signaling mutant ctrl-1 plants. Measurement under a microscope of epidermal cells of leaves from wild-type and ranl plants indicated that the 35 overall reduction in the size of TCO treated ranl leaves was due to a decrease in cell elongation. Consequently, the ranl mutations and RANl gene expression products were shown to affect 38 WO 00/04760 PCT/US99/16591 5 not only growth of seedlings, but also growth in adult plants, and it was concluded that in rani mutants the ethylene receptors have altered or relaxed ligand specificity. Effect of Ethylene Agonist on Ethylene-Inducible Genes 10 To determine the effect of an ethylene antagonist on ranl plants at the molecular level, several well-known ethylene inducible genes were treated with TCO and examined. E1305 (Chao et al., 1997, supra) and GST2 (Itzhaki et al., Plant Mol. Biol. 22:43-58 (1993)) expression were examined by northern blot 15 analysis using total RNA from seedlings treated with ethylene or TCO (Fig. 3A). As expected in wild-type plants, the steady state mRNA levels for these two hormone response genes increased upon treatment with ethylene, and decreased to below basal levels by TCO treatment. Exposure to TCO significantly reduced E1305 and 20 GST2 expression in etol-5, whereas their expression was unaffected in the ctrl-l mutant (Fig. 3A), confirming that TCO acts as an inhibitor of ethylene action. In ranl mutants, the steady state levels of E1305 and GST2 mRNAs were not inhibited by TCO, as observed in wild-type 25 seedlings. In fact, consistent with the agonist-like effects of TCO on seedling morphology in ranl plants, TCO caused a slight, but reproducible, increase in the steady state mRNA levels of the two genes (Fig. 3A). 30 Effect of Mutation on Agonist Response in Late Stage Development The effect of the ranl mutation on TCO responsiveness in later stages of development was also examined. Basic-chitinase 35 (CHIB) gene expression is known to be up-regulated by ethylene in adult Arabidopsis plants (Samac et al., Plant Physiol. 93:907 914 (1990)) and dependent upon an intact ethylene signaling pathway (Chen & Bleecker, Plant Physiol. 108:597-607 (1995) ) . Northern blot analysis showed that the CHIB gene expression was 39 WO 00/04760 PCTIUS99/16591 5 induced in ranl mutants by exposure of rosette plants to TCO (Fig. 3B). Taken together, these results demonstrated that in ranl seedlings and adult plants, TCO mimics the action of ethylene in both the morphological and molecular aspects of the response. 10 Expression of the ethylene inducible genes was further examined in rosette plants. The basic chitinase gene (CHIB) and one of the defensin genes (PDF1.2) are known to be induced by ethylene treatment [Samac et al., 1990, supra; Penninckx et al., 1996, supra] . However, northern blot analysis showed that the 15 expression of those genes were induced by one (1) day of TCO treatment in ranl rosette plants (Fig. 3B), further confirming that TCO activates the ethylene signaling pathway in ranl mutants in both seedlings and adults plants. 20 Example 3: RAN1 Acts Early in the Ethylene Gas Signaling Pathway. To determine where RAN1 acts in the ethylene signaling pathway, epistasis analyses were performed using ranl and the 25 ethylene insensitive mutants, etrl and ein2. In the presence of TCO, F2 progenies derived from self-fertilized etrl-1/+, ranl-1/+ or ein2-12/+, ranl-l/+ Fl plants displayed a phenotypic segregation ratio of 1 Ran to 15 wild-type plants. On the other hand, the F2 progeny segregated ein : wild type in ethylene, 30 demonstrating a ratio of 3 Ein~ to 1 wild-type plants. Given that etrl-1 is dominant (Bleecker et al., 1988, supra) and ein2 12 is semi-dominant (J.M. Alonzo and J.R. Ecker, unpublished results), it became apparent that etrl-l and ein2-12 masked the ran1 mutant phenotype. 35 Using a PCR-based allele assay for ranl-1, homozygous ranl mutants were identified among the F2 progenies derived from crosses of ranl-1 to etrl-1 and ein2-12. The phenotypes of both ran1-1, etrl-1 and ran1-1 ein2-12 double mutants were Ein , Ran+, 40 WO 00/04760 PCT/US99/16591 5 confirming that etrl and ein2 are epistatic to ranl. Since ETR1 encodes an ethylene receptor, these genetic studies demonstrated that RAN1 function is required very early in the hormone signaling pathway. Furthermore, etrl-1 and ein2-12 were clearly ethylene 10 insensitive, further supporting the conclusion that TCO treatment activates the ethylene signaling pathway in ranl mutants. After determining the mutation site of ranl-1, it was possible to confirm these results at the molecular level by finding several ranl-1 homozygotes in ein seedlings in F2 progeny from both ranl 15 1 x etr1-1 and ranl-1 x ein2-12 crosses. Example 4: RAN1 Gene Encodes a Copper Transporting P-type ATPase. 20 The ranl-1 mutant gene was mapped with visible markers by crossing it with the W13 and W100 lines (from the Arabidopsis Biological Resource Center at Ohio State University) and examining segregation of the ran] and ttg, yi, tt3, markers. SSLP markers, AthOl09 and ngal29, were used. (Bell & Ecker, 25 Genomics 19:137-144 (1994)). Newly synthesized SSLPs, (simple sequence length polymorphisms) smMLN1 and smMCL19, a cleaved amplified polymorphic sequence (CAPS) CIC4E12R and a derived cleaved-amplified polymorphic sequence (dCAPS) CIC2A12L were used for mapping ranl-l. 30 DNA samples for PCR (polymerase chain reaction) based marker analysis were isolated with CTAB (cetrimethyl amonimumbromide) methods. Briefly, 3-4 young leaves from individual F2 progeny were frozen in liquid nitrogen and ground with vigorous shaking in a Capmix (ESPE, America, Inc., USA) with 35 glass beads. 500 liters of the CTAB solution (100 mM Tris-HCl pH 8.0, 20 mM EDTA pH8.0, 1.4 M NaCl, 3% w/v CTAB) were added, then heated at 65 C for 30 min. After phenol/chloroform extraction, nucleic acids were precipitated by adding equal volume of iso-propanol and resuspended in 100 pl of TE buffer (10 41 WO 00/04760 PCT/US99/16591 5 mM Tris-HCl, 1mM EDTA, pH 8.0). RNase was added to a concentration of 10 g/l, the sample was incubated for 30 min at 37 C, followed by phenol/chloroform extraction and ethanol precipitation. Precipitated nucleic acids were resuspended in 50 pl of TE buffer. PCR reactions (10 mM Tris-HCl pH9.0, 50 mM 10 KCl, 2.5 mM MgCl 2 , 0.2 mM dNTPs, 5 pM each primers, 0.1 % Triton X-100, 1 pl of nucleic acid sample, Taq polymerase, total volume 15 pl, were passed through 35 cycles of 30 seconds at 94 C, 30 seconds at 56 C, 30 seconds at 72 C. To determine the nucleotide sequence of the RAN gene in 15 wild-type plants and ranl mutants, synthetic oligonucleotide primers were made (20-25bp, >50% GC) that would enable sequencing of the entire gene by primer walking. Genomic DNA was prepared from the leaves of wild-type plants, ranl-1 and ranl-2 mutants. Using synthetic primers, four overlapping DNA fragments were 20 amplified by PCR and sequenced after purification through agarose gel electrophoresis. To avoid errors due to PCR, more than four independent PCR samples were mixed and batch sequenced. Synthetic oligonucleotides (5'-3' direction) used for genetic marker production were as follows: 25 smMLN1-a; 5'-GTGGGTTGTTTCCGGCTAAG-3' (SEQ ID NO:4) smMLN1-b; 5'-GCCAGTCACCAGAACCAGC-3' (SEQ ID NO:5) smMCP19-a; 5'-TACCATAGTGTCCTTCAACGG-3' (SEQ ID NO:6) smMCP19-b; 5'-TGGACCTGTAATCGGAGACG-3' (SEQ ID NO:7) T19K24-la; 5'-CTTGGTGATCGAACCACGAAGGGAC-3' (SEQ ID NO:8) 30 T19K24-lb; 5'-CAAAGGACTCAATCTCAACCTACGC-3' (SEQ ID NO:9) T19K24-3a; 5'-TTTCAGTTATACGTGGTTAATTTCGC-3' (SEQ ID NO:10) T19K24-3b; 5'-GTCATGAGATTATGAGGTGCCGAC-3' (SEQ ID NO:11) T19K24-4a; 5'-GCTATTCATCAGTCATCATCCCC-3' (SEQ ID NO:12) T19K24-4b; 5'-GCGATAATTCTAGCATCCGATGC-3' (SEQ ID NO:13) 35 CAPS CIC4El2R-a; 5'-AATGAAACACGCTCATGTGCTCACC-3' (SEQ ID NO:14) CAPS CIC4E12R-b; 5'-TCTCGCAGGAGTTTCCCTTTGAGCC-3' (SEQ ID NO:15) dCAPS CIC2Al2L-a; 5'-AGGACGTGATTGCTTGTGTAGGAGG-3' (SEQ ID NO:16) 42 WO 00/04760 PCT/US99/16591 5 dCAPS CIC2A12L-b (mut); 5'-CAGTTTCTGGTTCAAGGAATAACTT-3' (SEQ ID NO:17) The amplified DNA produced using the CAPS primers was subjected to MboII digestion; whereas the amplified DNA produced using the dCAPS primers was subjected to Xmnl digestion. 10 For epistasis analyses, ranl mutants and etrl-l or ein2-12 were crossed and progeny of individual F1 plants were collected. The F2 seeds were germinated in the presence of ACC (1 aminocyclopropane-1-carboxylic acid) or TCO, then the Ein seedlings in the presence of ACC and Ran~ seedlings in TCO were 15 counted. The ranl-l alleles were distinguished from wild type by digestion of PCR fragments with BsrI; the ranl-1 mutation eliminates a BsrI restriction site. The ranl-l, etrl-l and ranl 1, ein2-12 double mutants were confirmed at molecular level by identifying ranl-1 homozygotes in Ein~ seedlings of F2 progeny 20 from both ranl-l x etrl-l and ran1-1 x ein2-12 crosses using the ranl-l polymorphism. Chromosomal and Fine Mapping of ranl The ranl-1 mutation mapped close to the tt3 gene on 25 chromosome 5, then fine mapping was done using SSLP markers. Based on the analysis of 2018 recombinant chromosomes derived from a cross between ranl-1 and the Landsberg strain (Ler), the position of ranl was narrowed to a region near the CRAl locus (Fig. 4) . Ler was from the Arabidopsis Biological Resource 30 Center at Ohio State University. Development of additional PCR-based genetic markers (smMLN1 and smMCP19) and their use in assembly of a BAC contig, permitted the localization of ranl to an interval 1.8 cM from smMLN1 and 2.6 cM from smMCP19 (Fig. 4) . To further delineate the location 35 of ranl, a CAPS marker, CIC2A12 (Konieczny et al., Plant J. 4:403-410 (1993)) was developed from the right-end of YAC (yeast artificial chromosome) CIC4E12 and a dCAPS sequence, CIC2A12 (Michaels et al., Plant J. 14:381-385 (1998); Neff et al., Plant 43 WO 00/04760 PCTIUS99/16591 5 J. 14:387-392 (1998)) was produced from the left-end of YAC CIC2A12. Using the two genetic markers, several additional BAC clones were identified and a BAC contig spanning about 160 kb was assembled that contained the RAN1 gene (Fig. 4). Among the BACs, one clone unassigned chromosomal location 10 (T19K24; GenBank accession AC002342) was fully sequenced, and a number of gene models were predicted. By identifying recombinant events using two additional SSLP markers (T19K24-1 and T19K24-4) derived from sequences near the BAC ends, the location of RAN1 on the BAC clone was confirmed (Fig. 4). By selecting from among 15 the numerous predicted open reading frames in the RANlregion of BAC T19K24, a predicted protein with similarity to copper transporting P-type ATPases was also identified. Because it has been proposed that the binding of ethylene to its receptor requires the presence of a transition metal, this gene was 20 selected as the candidate for RANl. Genomic Sequencing of ranl Genomic DNA from the region was sequenced in ranl-l and ranl-2 and the parental strain (Col). Single base changes were 25 identified in both ranl alleles (see below) confirming that this gene was, in fact, RANM (Fig. 5). The genomic sequence of RAN1 was recorded at GenBank with accession #AF091112 and is reproduced herein as SEQ ID NO:1. 30 cDNA Sequencing of ranl and Putative Amino Acid Sequence Using the PCR fragments mentioned above, a cDNA library was screened and several cDNAs were isolated. One cDNA (pNH633), containing the full RAN1 coding region, was subjected to sequence analysis. Sequencing from the 5' and 3' ends of a second RAN1 35 cDNA was also performed to confirm the position of initiation and stop codons. Total RNA extraction and northern analysis were performed as described (Kieber et al., 1993, supra). 44 WO 00/04760 PCT/US99/16591 5 Several cDNA clones were isolated from the ethylene-treated etiolated seedling library. Determination of the nucleotide sequence of the longest cDNA and comparison with the genomic sequence revealed that the candidate RAN1 gene contained nine exons and eight introns (Fig. 5), distinct from the computer 10 predicted gene model. The cDNA sequence of RANi was recorded at GenBank with accession #AF082565 and is reproduced herein as SEQ ID NO:2. The amino acid sequence of RAN1 was predicted from the expressed sequences (exons) of the RANl cDNA to be a polypeptide of 1001 amino acid residues, which is reproduced herein as SEQ 15 ID NO:3. Comparing Amino Acid Sequence of RAN1 with Other Known Copper Transporters 20 The amino acid sequence of RAN1 was aligned with other copper transporting P-type ATPases from human (ascession # Menkes Q04656; Wilson P35670), C. elegans (ascession # D83665) and budding yeast (ascession # P38995). These proteins are localized to a post-Golgi compartment where they function to 25 transport copper ions into the secretory pathway, delivering copper to secreted or membrane bound proteins that require this metal for functionality (Petris et al., EMBO J. 15:6084-6095 (1996); Yamaguchi et al., Proc. Natl. Acad. Sci. USA 93:14030 14035 (1996); Yuan et al., J. Biol. Chem. 272:25787-25793 30 (1997)). Significant portions of the polypeptides were found to have identity and conservative changes, to the extent that putative metal binding motifs and predicted functional domains were determined, particularly to the human Menkes disease gene 35 product, ATP7A (Chelly et al., Nature Genetics 3:14-19 (1993); Mercer et al., Nature Genetics 3:20-25 (1993); Vulpe et al., Nature Genetics 3:7-13 (1993)), the human Wilson disease gene product, ATP7B (Bull et al., Nature Genetics 5:327-337 (1993); Chelly & Monaco, Nature Genetics 5:317-318 (1993); Petrukhin et 45 WO 00/04760 PCT/US99/16591 5 al., Nature Genetics 5:338-343 (1993).), a copper transporter of Caenorhabditis elegans (Sambongi et al., J. of Biochem. (Tokyo) 121:1169-1175 (1997)) and yeast Ccc2p (Fu et al., Yeast 11:283 292 (1995); Yuan et al., Proc. Natl. Acad. Sci. USA 92:2632-2636 (1995)). These proteins share several structural features, 10 including 1) amino-terminal metal-binding motifs, 2) a phosphatase domain, 3) a transduction domain, 4) a phosphorylation domain, and 5) an ATP binding domain. The RAN1 protein possesses all of these features in regions of high similarity between RAN1 and other copper transporters. 15 Interestingly, RAN1 and Ccc2p have only two putative metal binding motifs in their N-terminal regions, whereas Menkes and Wilson proteins have six, and a predicted nematode gene has three motifs. Like these proteins, the candidate RAN1 protein contains eight putative membrane-spanning regions. 20 In the ranl-1 mutant allele, a C to T transition was found at nucleotide 1880 (corresponding to genomic sequence with the A from the first ATG codon assigned the number 1). This base change causes an amino acid change from Thr 497 to Ile. Threonine 497 is located in the phosphatase domain, and this 25 residue is conserved in all copper transporters (Fig. 5A). This suggested that the amino acid change found in ranl-1 reduces RAN1 function. In ranl-2, a G to A base change was found at nucleotide 637, changing Gly 173 to Glu. Glycine 173 is not highly 30 conserved among copper transporters, but the corresponding residues in the metal binding repeats of copper transporters are not charged amino acid residues (Fig. 5B). Recent analysis of the three dimensional structure of the fourth metal binding repeat of the Menkes protein indicated that the residue 35 corresponding to Gly 173 of RANI contributes to the hydrophobic core that is required for the correct positioning of the metal binding loop (Gitschier et al., Nature Structural Biology 5:47-54 (1998)). Thus, the conversion from Gly to Glu causes a dramatic 46 WO 00/04760 PCT/US99/16591 5 change in charge and hydrophobicity, conceivably disturbing the structure of the metal binding domain, which is necessary for copper transporter function (Payne et al., J. Biol. Chem. 273:3765-3770 (1998)). Neither of the two mutations, however, affected the 10 expression of the RANi gene, at least at the transcriptional level, since RANl mRNA was detected in both mutant strains to much the same extent as it was detected in wild type plants. Example 5: Expression of RAN1 cDNA rescues the Fet3p 15 deficient phenotype of the yeast ccc2 disruption mutant. Because of its extensive similarity to known copper transporters, RAN1 was predicted to have copper transporting 20 activity. To confirm this hypothesis, yeast complementation analyses were performed using a budding yeast ccc2 disruption (ccc2A) mutant. In budding yeast, Ccc2p delivers copper ions from the lumen to the cytosol of the secretion pathway (Yuan et al., 1995, 25 supra). The copper binding domain of the multi-copper oxidase, Fet3p, is loaded with copper, which is required for Fet3p oxidase activity. Thus, the oxidase activity of Fet3p provides the basis for an in vivo assay for copper transporter activity. In the absence of Ccc2p, Fet3p has no oxidase activity. However, the 30 ccc2~ defect can be rescued by homologous copper transporters from other species (Hung et al., J. Biol. Chem. 272:21461-21466 (1997); Payne et al., 1998, supra; Sambongi et al., 1997, supra). Consequently, to confirm that RAN1 was able to suppress the Fet3p oxidase defect of ccc2A, recombinant plasmids were 35 constructed in which RANl cDNA was under the control of the constitutive PGK1 promoter. The following were used for the yeast complementation analyses: 2908 (strain 7) [MAT( a), his3-200, leu2, trpl-101, ura3-52, ade5] and ccc2(n) (strain 8) [MAT(a), his3-200, trpl 47 WO 00/04760 PCT/US99/16591 5 101, ura3-52, ade5,ccc2::LEU2]. For construction of plasmids containing the ranl-1 mutation, two primers were designed: ranl-la: 5' ATGAATCCATGGTGATTGGTGAATCAGTTCC-3' (SEQ ID NO:18); and ranl-lb: 5'-CAGTCACCATGGATTCATTAACGTAACTTG-3' (SEQ ID 10 NO:19). The underlined T corresponds to the ranl-1 mutation. Italic characters indicate an introduced NcoI restriction site. Using these two primers and two primers corresponding to nucleotides 2632-2608 (#20) and nucleotides 934-958 (#7), two 15 fragments were amplified by PCR using the cloned RAN1 cDNA as template. The ranl-la-#20 PCR fragment was digested with NcoI and BspEI, while ranl-lb-#7 PCR fragment was digested with NcoI and StuI. The cDNA region from StuI to BspEI sites was exchanged with ranl-la-#20 and ranl-lb-#7 PCR fragments. 20 For construction of plasmids containing the ranl-2 mutation, two primers were designed. ranl-2a: 5'-AGCTTTGTCGACAT CATTAGAAGAAGTTGAG-3' (SEQ ID NO: 20); and ranl-2b: 5' -AATGATGTCGACAAAGCTACCACTGCTC-3'. (SEQ ID NO: 21). 25 The underlined A corresponds to the ranl-2 mutation. Italic characters indicate an introduced SailI restriction site. Using these two primers and another primer corresponding to nucleotides 1439-1416 (#18) and the T7 primer for the sequence of pBluescript, two fragments were amplified by PCR using the 30 cloned RANl cDNA as template. The ranl-2a-#18 PCR fragment was digested with SalI and StuI, while the ranl-lb-T7 PCR fragment was digested with SalI and ClaI. The ClaI to StuI region was exchanged with ranl-2a-#18 and ranl-lb-T7 PCR fragments. Wild type and mutant RAN1 cDNAs were introduced into pYES3 (PGK1 35 promoter). Transformation of yeast with various plasmids was performed using the lithium acetate method of Ito et al., J. Bacteriol. 153:163-168 (1983). 48 WO 00/04760 PCT/US99/16591 5 Yeast cells were grown in YPD (yeast, peptone and dextrose) medium at 30 0 C to a density of 0.3 OD600. The cells were harvested by centrifugation and washed with ice-cold extraction buffer (25.mM Tris pH 7.5, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM leupeptin, 10 mM antipain) . Yeast cells were 10 homogenized in the extraction buffer by mixing with glass beads. After removal of large particles, homogenates were centrifuged (20,000 x g) at 4 0 C for 20 min. Pellets were washed in extraction buffer, resuspended in the same buffer containing 1% Triton X-100, and centrifuged again to precipitate undissolved 15 materials. Protein samples (25 mg) were mixed with the same volume of SDS page buffer (100 mM sodium phosphate, pH 7.2; 0.5% SDS; 10% (w/v) glycerol) and separated by SDS/PAGE without prior heating. After electrophoresis, the gel was equilibrated with 0.005% 20 Triton X-100 in 10% (w/v) glycerol, and then soaked in 3 mM p phenylenediamine dihydrochloride (Sigma), 100 mM sodium acetate, pH 5.8, 1 mM NaN 3 . The gel was air-dried in the dark at room temperature. To detect RAN1 protein in yeast, a triple repeat of the HA-tag sequence was introduced into RAN1, ran1-1 and ranl 25 2 cDNAs, just before the stop codon. Western blot analysis was performed using monoclonal anti-HA antibody. Transformants of the ccc2A strain, in which the plasmid DNA containing RANl cDNA, were found to have detectable Fet3p oxidase activity, although the activity was somewhat less than that of 30 the wild type. By comparison, the same strain transformed with vector plasmid DNA alone, did not have such activity (Fig. 6A). In addition, copper transporter activity could be determined by monitoring the iron uptake of yeast cells since Fet3p has a pivotal role in high-affinity iron uptake in budding 35 yeast (Stearman et al, Science 271:1552-1557 (1996)). The iron uptake assay was performed as described by Dancis et al., J. Biol. Chem. 269:25660-25667 (1994) . Cells from the transformed yeast strains were inoculated into 100 pl of YPD in the wells of 49 WO 00/04760 PCT/US99/16591 5 a microtiter plate and grown at 300C. After 14 hrs of incubation, the cells were diluted 1:5 into fresh YPD medium in a volume of 50 pl. After incubation for an additional 2 hrs, the 55Fe radionuclide (Amersham 37-50 mCi/mg iron) was added in 50 p1 of citrate buffer. The uptake was allowed to proceed for 2 hours 10 at which point the cell number was determined by measuring the turbidity (OD720). The cells were then collected on glass fiber filters and washed with water. The filters were counted in a Beckman scintillation counter and the cell-associated radioactivity was calculated. For preparation of the 15 radionuclide, 55Fe was prepared by reduction with sodium ascorbate and diluted to a concentration of 2 pM in citrate buffer (50mM sodium citrate pH 6.5, glucose 5%) . The iron preparation was then added to the cells in microtiter wells. Without Ccc2 function, iron uptake of yeast cells was found 20 to be reduced. In the ccc2A cells expressing RANl cDNA, high affinity iron uptake was restored relative to the negative control cells, which carried only the empty vector, (Fig. 6B), further demonstrating that RAN1 has copper transporter activity. To confirm that the ranl mutant gene products have reduced 25 activity, the ranl-1 mutation and the ranl-2 mutation were respectively introduced into wild type RAN1 cDNA. Then, ccc2A was transformed with the plasmid containing those mutant cDNAs. Both the ranl-1 and ranl-2 defects were found to impair copper transporting activity, although notably the ccc2A transformants 30 harboring the ran1 mutant cDNAs had a significantly reduced Fet3p oxidase activity and high affinity iron uptake activity when compared to RAN. While transformants containing ranl-2 cDNA also showed reduced activity, the level of Fet3p oxidase activity and iron uptake activity was higher than that observed in 35 transformants expressing ranl-1. Western blot analysis revealed that RAN1, ranl-2 and ranl-2 proteins accumulated to the same level in the yeast. 50 WO 00/04760 PCTIUS99/16591 5 One explanation for the somewhat greater activity of the ranl-2 may be that in RAN1, which contains only a single functional metal binding motif, there is sufficient copper transporting activity in yeast, but not in plant cells. As shown previously, expression of a Wilson's protein containing only a 10 single binding motif was sufficient for the functional complementation of yeast ccc2 deletion mutant (Iida et al., FEBS Letts 428:281-285 (1998)). Example 6: Presence of Copper Ions Suppresses the Ethylene 15 Phenotype of ran1 Plants. The addition of copper ions to the media suppresses the defect in Fet3p activity of the ccc2 disrupted mutant (Fu et al., 1995, supra; Yuan et al., 1995, supra). Since RAN1 encodes a 20 Ccc2p homologue, it was important to determine whether or not the addition of copper ions to the plant growth media could suppress the ranl phenotype, analogous to the effect on the yeast ccc2 mutants. Consequently, various concentrations of CuSO 4 (from 0.1 pM to 100 pM) were added to the growth media to evaluate the 25 effect on the germination and growth of seedlings of Arabidopsis. At concentrations of 1 pM and below, the CuSO 4 did not have an apparent effect. By contrast, however, concentrations of 50-100 pM CuSO 4 significantly inhibited germination. Interestingly, an intermediate CuSO 4 concentration of 10-15 30 pM, which did not have any obvious effects on germination and seedling growth, partially suppressed the ranl phenotype of both ranl-1 and ranl-2 (Fig. 7). Specifically, the copper supplemented growth media prevented the ranl seedlings from responding to TCO, as evidenced by the lack of the triple 35 response phenotype. However, CuSO 4 at this concentration did not interfere with TCO-mediated inhibition of the triple response phenotype of etol-5 or ctrl-1 (Fig. 7). Thus, it is not likely that CuSO 4 , as a compound per se, affects the triple response phenotype or the downstream ethylene signaling pathways. 51 WO 00/04760 PCT/US99/16591 5 Fet3p is also involved in high affinity iron uptake. The human Fet3p homologue, ceruloplasmin, has been implicated in the iron metabolism in human cells, and its activity is dependent on the proper functioning of the Menkes/Wilson proteins. Thus, to evaluate the possibility that the ranl phenotype could also have 10 been caused by a deficiency in high affinity iron uptake in Arabidopsis, iron was added to the plant media to determine whether or not the ranl phenotype was suppressed in TCO. Several concentrations of ferric ammonium sulfate (from 30 pM to 1 mM) and of FeEDTA (from 1 pM to 1 mM) were added to the seedling 15 growth media, but no effect could be detected on the rani phenotype in the presence of TCO. Therefore, it was concluded that although the phenotype of the ranl mutants is caused by a defect in copper metabolism, the phenotype is not related to a similar defect in iron metabolism. 20 Example 7: Constitutive Activation of Ethylene Responses in RAN1 Transgenic Plants To further explore the function of RAN1, the effect of 25 ectopic gene expression on seedling development/hormone responsiveness was examined. To transform the plants the RAN cDNA was inserted between the CaMV 35S promoter and the nopaline synthase (NOS) terminator sequence of pROK2 vector plasmid (Baulcombe et al., Nature 321:446-449 (1986)) yielding the 30 plasmid pNH634. Then pNH634, containing the full length RANl cDNA, was introduced into Agrobacterium C58 strain and the resulting strains were used to transform ranl-l, ranl-2 and wild type plants by the vacuum infiltration method, according to Bechtold et al., CR Acad. Science (Paris) 316:1194-1199 (1993)). 35 Interestingly, independent of their different genetic backgrounds, two distinct phenotypic classes of transformants were observed. Approximately one-half of all Ti-generation transgenic lines displayed a constitutive ethylene response phenotype (class 1, Ctr~), while the other half of transformants 52 WO 00/04760 PCT/US99/16591 5 did not show an obvious growth phenotype (class 2, Ctr*) (Fig. 8A). When treated with TCO, class 2 transformants were no longer able to respond to the antagonist, indicating that the introduced 35S:RAN1 transgene had complemented the ranl mutation. Northern blot analysis revealed that class 2 transgenic plants expressed 10 a high level of RAN1 mRNA (Fig. 8B, upper panel). In contrast, the level of RAN1 mRNA found in plants displaying a strong constitutive ethylene response (Ctr~) phenotype was much reduced (Fig. 8B, upper panel), an effect likely due to co-suppression of the endogenous RAN1 gene. 15 The severe Ctr- phenotype observed in the RANl transgenic plants suggested that activation of the ethylene-signaling pathway was occurring. To examine this hypothesis, the expression of the ethylene-responsive basic-chitinase (CHIB) gene was examined in the 35S::RAN1 transformed plants (classes 1 and 20 2). Northern blot analysis of mRNA from plants that displayed a Ctr- phenotype (class 1) revealed a significantly elevated level of expression of basic-chitinase compared to expression in untransformed plants (Fig. 7B, lower panel) . By comparison, phenotypically wild-type plants (class 2) showed only basal level 25 expression of basic-chitinase mRNA. These results confirm the association between reduced RAN1 expression, the Ctr~ phenotype and activation of ethylene responsive gene expression in the class 1 CaMV35S:RAN1 transgenic lines. Because it was not possible to carry out further genetic 30 characterization of the class 1 plants due to complete infertility in all lines, subsequent generations of class 2 plants segregating for the 35S::RAN1 transgene were studied. T2 generation plants from twenty-four (24) independent transformants were permitted to self-fertilize, and the progeny were examined 35 for ethylene phenotypes. Seedlings from two 35S::RANli lines (L3 and L6) when grown in hydrocarbon-free air, displayed a constitutive triple response phenotype identical to ctrl mutants (Fig. 8C). 53 WO 00/04760 PCT/US99/16591 5 In addition, treatment of L3 and L6 seedlings with ethylene action inhibitors, silver ion or MCP (1-methylcyclopropane), had no effect on the triple response phenotype, indicating that activation of the ethylene response in these lines was not due to overproduction of the hormone. Moreover, after several weeks 10 of growth, the resulting plants developed an adult morphology identical to that observed in RANi co-suppressed plants (class 1) . These studies revealed that a reduction of RANl expression results in constitutive activation of ethylene morphology in both seedlings and adult plants. 15 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 20 that certain of the details described herein can be varied considerably without departing from the basic principles of the invention. Such modifications and additional embodiments are also intended to fall within the scope of the appended claims. 54

Claims (31)

1. An isolated nucleic acid encoding a plant, plant cell, tissue, flower or organ having copper transporter 10 activity, and mutants, derivatives, homologues and fragments thereof encoding a plant, plant cell, tissue, flower or organ having copper transporter activity.

2. The nucleic acid according to claim 1, comprising ranl. 15

3. The nucleic acid according to claims 1 or 2, comprising SEQ ID NO:1.

4. The nucleic acid according to claims 1 or 2, comprising 20 SEQ ID NO:2.

5. A purified preparation of a polypeptide encoded by the nucleic acid of one of claims 1-4, comprising a plant, plant cell, tissue, flower or organ having copper 25 transporter activity, and analogs, homologues, derivatives, varients and fragments thereof having copper transporter activity.

6. The polypeptide according to claim 5, comprising RAN1. 30

7. The polypeptide according to claim 6, comprising SEQ ID NO:3.

8. The polypeptide according to claim 6, wherein RAN1 copper 35 transporter activity is ATP-dependent.

9. A recombinant cell comprising the isolated nucleic acid of one of claims 1-4. 55 WO 00/04760 PCT/US99/16591 5

10. A vector comprising the isolated nucleic acid of one of claims 1-4.

11. An antibody specific for a plant RAN1 polypeptide, and 10 homologues, analogs, derivatives or fragments thereof having copper transporter activity.

12. An isolated nucleic acid sequence comprising a sequence complementary to all or part of the nucleic acid sequence 15 of one of claims 1-4, and to mutants, derivatives, homologues or fragments thereof encoding a plant, plant cell, tissue, flower or organ having copper transporter activity. 20

13. The nucleic acid according to claim 12 having antisense activity at a level sufficient to regulate, control, or modulate the copper transporting activity of a plant, plant cell, organ, flower or tissue. 25

14. A plant, plant cell, organ, flower, tissue, seed, or progeny comprising nucleic acid according to one of claims 1-4 or 12-13.

15. A transgenic plant, the cells, organs, flowers, tissues, 30 seeds or progeny of which comprise the nucleic acid according to one of claims 1-4 or 12-13.

16. A transgenic plant, the cells, organs, flowers, tissues, seeds or progeny of which comprise the recombinant 35 nucleic acid according to claim 9. 56 WO 00/04760 PCT/US99/16591 5

17. A transgenic plant, the cells, organs, flowers, tissues, seeds or progeny of which comprise the polypeptide according to one of claims 5-8.

18. An isolated nucleic acid of one of claims 1-4 or 12-13, 10 further comprising a plant RAN1 promoter sequence, or a fragment thereof having promoter activity.

19. A vector comprising the isolated nucleic acid of one of claims 1-4 or 12-13 or 18. 15

20. The isolated nucleic acid of claim 18, further comprising a reporter gene operably fused thereto, or a fragment thereof having reporter activity. 20

21. A transgenic plant, the cells, organs, flowers, tissues, seed, or progeny of which comprise a transgene comprising an isolated nucleic acid comprising a RAN1 promoter sequence. 25

22. A method for manipulating in a plant the nucleic acid according to one of claims 1-4 or 12-13 to permit the regulation, control or modulation of germination, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit ripening, insect, herbicide or 30 pathogen resistance, abscission, or response to stress in said plant.

23. The method according to claim 22 wherein said regulation, control or modulation initiates or enhances the 35 germination, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit ripening, insect, herbicide or pathogen resistance, abscission, or response to stress in said plant. 57 WO 00/04760 PCT/US99/16591 5

24. The method according to claim 22, wherein said regulation, control or modulation inhibits or prevents the germination, cell elongation, sex determination, flower or leaf senescence, flower maturation, fruit 10 ripening, insect, herbicide or pathogen resistance, abscission, or response to stress in said plant.

25. A method of identifying a compound capable of affecting the transport of copper in the ethylene signaling system 15 in a plant comprising: providing a cell comprising an isolated nucleic acid encoding a plant RAN1 sequence, having a reporter sequence operably linked thereto; adding to the cell a compound being tested; and 20 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 25 compound being tested is capable of affecting the expression of a plant ranl gene.

26. A method for generating a modified plant with enhanced copper transporting activity as compared to that of 30 comparable wild type plant comprising introducing into the cells of the modified plant an isolated nucleic acid encoding RAN1, wherein said ranl nucleic acid is capable of transporting copper within the cells of the modified plant. 58 WO 00/04760 PCTIUS99/16591 5

27. A method for generating a plant with diminished or inhibited copper transporting activity as compared to that of a comparable wild type plant comprising binding or inhibiting the copper transporting molecules within 0 the cells of the modified plant by introducing into said cells an isolated nucleic acid encoding a complementary nucleic acid to all or a portion of ranl, wherein said ranl nucleic acid would otherwise be capable of transporting copper within the cells of the modified 5 plant.

28. A method for generating a plant with diminished or inhibited copper transporting activity as compared to that of a comparable wild type plant comprising binding 0 or inhibiting the copper transporting molecules within the cells of the modified plant by introducing into said cells an antibody to all or a portion of RAN1, wherein said RAN1 polypeptide would otherwise be capable of transporting copper within the cells of the modified 5 plant.

29. A method for manipulating the expression of RAN1 in a plant cell comprising: operably fusing the nucleic acid ranl or an operable 0 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, where upon controlled activation of the plant promoter, manipulates expression 5 of RAN1. 59 WO 00/04760 PCT/US99/16591 5

30. A mutant allele ranl-1 capable of expressing RANI, wherein ranl-1 is characterized by a C to T transition at nucleotide 1880 of ranl cDNA, effecting a change in amino acid sequence from RAN1 at nucleotide Thr497 to Ile. 10

31. A mutant allele ranl-2 capable of expressing RAN1, wherein ranl-2 is characterized by a G to A transition at nucleotide 637 of ranl cDNA, effecting a change in amino acid sequence from RAN1 at nucleotide Gly173 to Glu. 15 60