text: This invention relates to the field of compositions and methods for inhibiting the enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase in rose thereby prolonging the shelf-life of cut flowers as well as reducing leaf yellowing and petal abscission during shipping and storage.
  A variety of factors cause wilting and natural abscission in flowers, particularly after a cutting of the plant or when flowers have been removed from the plant. Such factors include increased oxygen levels, wounding, chemical stress, and the plant's own production of ethylene. Of these factors, the plant's production of ethylene, has been shown to play a key role in natural senescence, the degenerative process which generally leads to controlled cell death in plants, but also in the degradation of flowers after they have been cut.
  Ethylene, in all higher plants, is important to plant growth and development from seed germination, seedling growth to flowering and senescence (Abeles, F. B. et al. (1992), In: Ethylene in Plant Biology. Eds. Abeles, F. B. et al., Academic Press, New York, pp 285-291 and 1-13; Yang, S. F. et al. (1984), Annu. Rev Plant Physiol:35, 155-189). Ethylene production in plants can also be associated with trauma induced by mechanical wounding, chemicals, stress (such as produced by temperature and water amount variations), and by disease. Hormones can also stimulate ethylene production. Such ethylene, also sometimes called "stress ethylene", can be an important factor in storage effectiveness for plants. Moreover, exposure of plant tissue to a small amount of ethylene often may be associated with increased production of ethylene by other adjacent plants. This autocatalytic effect may be often associated with losses in marketability of plant material during storage and transportation (Abeles et al., supra; Yang et al., supra).
  The ethylene biosynthetic pathway in plants was established by Adams and Yang (Adams D. O., et al., (1979) Proc. Nat'l Acad Sci USA 76: 170-174)). The first step involves the formation of S-adenosyl-L-methionine (AdoMet) from methionine by S-adenosyl-L-methionine synthetase. AdoMet is then converted into 1-aminocyclopropane-1-carboxylate (ACC), the direct precursor of ethylene in higher plants. This conversion is catalyzed by ACC synthase (S-adenosyl-L-methionine methyl thioadenosine-lyase, EC4.4.1.14), the rate limiting step in the ethylene biosynthetic pathway. (See also Kionka C., et al., (1984) Planta 162:226-235; Amrhein N. et al., (1981) Naturwissenschaften 68: 619-620; Hoffman N. E., et al., (1982) Biochem Biophys Res Commun 104:765-770).
  Knowledge of the biosynthetic pathway for ethylene formation has been fundamental in developing strategies for inhibiting ethylene production in plants. One approach has been to use chemical inhibitors to inhibit the synthesis or activity of ethylene, two of the most common being aminoethoxyvinylglycine and aminooxyacetic acid (Rando, R. R., 1974, Science, 185, 320-324 and in Ethylene in Plant Biology, (Abeles, F. B., et al., eds. Academic Press, p. 285)). However, chemical methods find limited use because such methods are expensive and the beneficial effect they provide is generally only short-lived.
  A second approach has been to over express ACC deaminase, an enzyme which metabolizes ACC, thereby eliminating an intermediate in the biosynthesis of ethylene (Klee, et al., (1991) Cell 3: 1187-1193) (See also Theologis, A., et al. (1993), Cellular and Molecular Aspects of the Plant Hormone Ethylene, p. 19-23). Because ACC deaminase is a bacterial enzyme, it is heterologous, and thus, external to the plant. Thus, it is unlikely that this approach will yield a modification that will be stable from generation to generation.
  Yet another approach involves attempts to genetically inhibit the production of the enzymes involved in the biosynthesis of ethylene or to inhibit the biosynthesis of the enzymes directly. This approach has the advantage of not only altering the way the plant itself functions irrespective of external factors but also of presenting a system which reproduces itself, that is, the altered plant's progeny will have the same altered properties for generations to come.
  Initial efforts to better understand the enzymes which catalyze the reactions in the biosynthesis of ethylene have involved the identification and characterization of the genes encoding for AdoMet synthetase, ACC synthase, and ACC oxidase (See also Kende H., 1993, Annu Rev Plant Physiol Mol Biol 44:283-307). Some of the genes encoding for ACC synthase have been identified for a number of plants. For instance, ACC synthase sequences have been identified for zucchini (Sato T., et al., (1989) Proc. Natl Acad Sci USA 86:6621-6625), winter squash (Nakajima, N., et al., (1990) Plant Cell Physiol 31:1021-1029), tomato (Van Der Straeten, D., et al., (1990) Proc Natl Acad Sci USA 87:4859-4863); (Rottmann, W. H., et al., (1991) J Mol Biol 222:937-961), apple (Dong, J. G., et al., (1991) Planta 185:38-45), mung bean (Botella, J. R., et al., (1992a) Plant Mol Biol 20:425-436; Botella, J. R., et al., (1993) Gene 123: 249-253; Botella, J. R., et al., (1992b) Plant Mol Biol 18: 793-797); Kim, W. T., et al., (1992) Plant Physiol 98:465-471), carnation (Park, K. Y., et al., (1992) Plant Mol. Biol., 18, 377-386), Arabidopsis thaliana (Liang, X., et al., (1992) Proc Natl Acad Sci USA 89:11046-11050; Van Der Staeten, D., et al., (1992) Proc Natl Acad Sci USA 89:9969-9973), tobacco (Bailey, B. A., et al., (1992) Plant Physiol 100: 1615-1616), rice (Zarembinski, T. I., et al., (1993) Mol Biol Cell 4: 363-373), mustard (Wen, C. M., et al., (1993) Plant Physiol 103:1019-1020), orchid (O'Neill, S. D., et al., (1993) Plant Cell 5: 419-432), broccoli (Pogson, B. J., et al., (1995) Plant Physiol 108:651-657), and potato (Schlagnhaufer, C. D., et al.. (1995) Plant Mol. Biol. 28:93-103).
  That ACC synthase is involved in the ethylene pathway is confirmed by the fact that increased levels of ACC synthase mRNA correlate with an increased activity of ACC synthase in plants during fruit ripening and flower senescence. Similar correlation is also observed in response to exogenous signals caused either by wounding or due to treatment with hormones such as auxin, cytokinin and ethylene. Interestingly, the expression of different classes of ACC synthase occurs from a variety of signals in many plants, e.g. four different ACC synthase genes have been shown to be differentially expressed in tomato fruit, cell cultures, and hypocotyls during ripening, wounding, and auxin treatment (Olson, D. C., et al (1991) Proc. Natl. Acad. Sci. USA 88:5340-5344; and Yip, W. K., (1992) Proc. Natl. Acad. Sci. USA 89:2475-2479). Differential expression of two ACC synthase genes has also been observed in winter squash during wounding or by auxin (Nakajima, et al. (1990) Plant Cell Physiol, 31; 1021-29 and (1991) Plant Cell Physiol, 32; 1153-63). Similar differential regulation of expression ACC synthase genes takes place in carnation flowers by wounding or during senescence (Park, K. Y., et al., (1992) Plant Mol. Biol., 18, 377-386). The evolution of ACC synthase genes into a multigene family that responds differentially during plant development or in response to stimuli external to the plant (Rottmann, W. H., et al., (1991) J Mol Biol 222:937-961) may be a reflection of the importance of ethylene in plants. (See also Slater, A., et al., (1985) Plant Mol Biol 5:137-147). (Smith, C. J. S., et al., (1986) Planta 168; 94-100 and Smith, C. J. S., et al. (1988) Nature 334;724-26). (Hamilton, A. J., et al., (1990) Nature 346:284-286; Kock, M., et al., (1991) Plant Mol Biol 17:141-142).
  The discovery of the foregoing and of other properties has lead to an understanding that it may be desirable to attempt to genetically alter the production of ethylene in plants. This approach, however, may be considered in some ways delicate. Elimination of ethylene is not a desired result as in many instances it will kill the plant. Modulation of ethylene--at the appropriate times--is the critical goal, not elimination of it entirely. This has been attempted at least two points in the pathway: the production of ACC by ACC synthase, and the oxidation of ACC by a different enzyme, ACC oxidase. Because the ACC synthase approach can permit stable modulation and not only total elimination of ethylene, it is a preferred technique. To date, however, successful reduction of the production of ethylene through an alteration at the ACC synthase step in the pathway has only been accomplished in one plant, tomato (Oeller, et al. (1991) Science 254:437-39). In spite of the seemingly simple conceptual nature of this goal, the actual accomplishment of an alteration of the ethylene biosynthetic pathway through the ACC synthase technique has remained elusive. This is particularly true for the rose plant, perhaps due to the fact that the identification of full length genes can be difficult for plants. As discussed later, this may, in part, be due to the fact that isolation of full length or high quality RNA has been deemed "notoriously difficult" for plants. (John, M. E., Nucleic Acids Research 20:2381, 1992, and Logemann, J. et al, Anal Biochem 163, 16-20, 1987).
  Efforts by others highlight some of the difficulty involved. Recently, Arteca's laboratory (Wang, T. W. et al., (1995) Plant Physiol. 109:627-636) studied two cDNA molecules encoding ACC synthase from a white flower variety of a flowering geranium plant (Pelargonium x hortorum cv Snow Mass Leaves). As their publication explained (perhaps after the fact), these researchers tried to identify and characterize two clones, GAC-1 and GAC-2. In spite of their efforts, they were only able to completely identify one of those cDNA gene sequences, GAC-1. Their study examined the expression of these ACC synthase genes in different plant parts of the geranium and in response to stress induced by osmotic changes (sorbitol) or metal ions (CuCl.sub.2). It also evaluated the effects of ethylene on auxin 2,4-D induction in geranium leaves. The study indicated that GAC-1 expression was induced only by stress, whereas expression of GAC-2 appeared to be developmentally regulated. Furthermore, these authors speculated about possible future "transfer of antisense GAC-1, GAC-2 . . . into Pelargonium tissues through the Agrobacterium transformation or particle bombardment." This confirms a desire in the art for an ACC synthase approach to altering ethylene production in such plants. In spite of this desire, however, the isolation and identification of some, if not all, the ACC synthase gene sequences--for geranium remained elusive. In similar fashion, rose as well has remained elusive.
  Although several plant ACC synthase genes have been identified and sequenced, the current invention describes ACC synthase gene sequences which were previously unknown and which are not believed to have been easily discoverable. As mentioned, one factor which may have militated against an expectation of successfully cloning a plant gene is the particular difficulty in obtaining high-quality and full-length RNA from plants. Indeed, this process has been characterized as "notoriously difficult" by at least more than one practitioner of the art (John, M. E., Nucleic Acids Res. 20:2381, 1992 and Logemann, J., et al, Anal Biochem 163, 16-20, 1987)). While this proved to be true for the present inventor, these difficulties were overcome by assessing a new approach to the RNA isolation process. The current inventor, after finding traditional RNA isolation methods to be ineffective, was forced to develop a non-traditional approach described herein. Basically, even though those of ordinary skill in the art had long desired to identify some gene to manipulate to alter the production of ethylene in some plants, in this case, they failed to realize that the problem lay in the need for a better isolation process. Even though the implementing technology for this process had long been available, those in the art apparently failed to realize how to use that technology to achieve the results now described. To some extent they simply may not have defined the problem, preventing the achievement the goals sought. Their efforts may properly be characterized as having taught away from the direction taken by the present inventor and, thus, the results achieved here should be considered unexpected.
  Difficulties in isolating full-length mRNA in the specific case of geranium and rose are also further reflected by the fact that one of the sequences encoding ACC synthase in a geranium isolated by the current inventor (clone pPHSacc49), though it may bear some similarity to portions of the clone termed GAC-2 by Wang et al., supra, (which, in any case, may have been discovered after the making of the present invention) is actually considerably longer than GAC-2. This highlights the difficulty in successfully isolating a full-length mRNA molecule using standard RNA isolation procedures in certain plant materials including roses. However, the high quality RNA (as defined below) isolated by the current inventor is evidenced by the fact that full length cDNA clones were obtained in a different plant, and all of them could be successfully expressed in an in vitro expression system. In each case, full length ACC synthase (enzyme) protein is synthesized in vitro. In contrast, even later publications by Arteca's group do not describe the actual in vitro expression of any of the isolated DNA clones. In fact the cDNA for the GAC-2 gene was never isolated. Rather, only a partial sequence was merely deduced from the sequence of genomic clones.
  This is significant because it highlights the difficulty in isolating and thereby identifying full length ACC synthase genes. Those of ordinary skill in the art had faced the same challenge. Derivation of DNA encoding ACC synthase from a genomic clone rarely is successful, and therefore, simply would not necessarily provide a reasonable expectation of success to one of ordinary skill. Only by utilizing a new and different approach did the present invention successfully identify not only one but several full length ACC synthase gene sequences from the geranium plant. The same technique applies to the identification of the ACC synthase gene sequence from the rose plant. Basically, it was this high quality library containing full length cDNA clones which allowed the present inventor to successfully achieve direct cloning of ACC synthase cDNA. The prior art did not discover these sequences because the genes did not exist in the available libraries. It was this new approach which overcame the problems faced, but not solved, by others and resulted in the extraordinary successes described herein. The extraordinary success of the present invention--a nearly one hundred fold increase in positive identifications is a consequence of the new technique for RNA isolation and cDNA identification, and not the result of analogous knowledge gained from the efforts of others. Mere comparison to other genes in the same or different plants did not and could not have yielded the successes described here. The existence of the cDNAs of interest in the library was the governing factor. Thus, even with a viable identification process, successful identification of the rose ACC synthase gene, let alone the actual alteration of the plants themselves by means of this knowledge, would not have been likely.
  Additionally, it should be understood that knowledge of the full length sequence of a gene from other plants simply does not necessarily lead one to the sequences of the homologous genes in the rose plants. First, as mentioned earlier, the genes encoding ACC synthase have evolved into a multigene system in some cases. There appears to be no single gene, but rather a family of genes in most cases. Thus, knowledge of one gene in one plant species is not certain to lead to one (or several) homologous or analogous genes in another plant species. Second, because known ACC synthase genes are typically so diverse in their nucleotide sequences, knowledge of one would not lead a person of ordinary skill in the art to an expectation of success in isolating the ACC synthase gene from rose.
  Antisense technology is a well known approach to create a plant that produces less of a selected protein. Through this technology, a plant is altered by introducing a foreign DNA sequence that encodes an mRNA product complementary to part or all of the plant's "sense" mRNA encoding the protein. The presence of antisense RNA inhibits RNA function within a cell (and whole organism). Antisense RNA can bind in a highly specific manner to its complementary sense RNA resulting in blockade in processing and/or translation of the sense mRNA. Antisense RNA may also disrupt interactions between sense mRNA and sequence-specific RNA binding proteins. Antisense technology may be employed to inhibit the synthesis of an enzyme involved in ethylene biosynthesis. The genes identified by the current inventor and disclosed herein have been used for the conception and implementation of antisense sequences specific for ACC synthase mRNA. Introduction of DNA encoding such antisense RNA sequences into a rose plant is highly probable to result in a plant which stably produces less ethylene.
  The incorporation of antisense RNA in plants as a means to inhibit the synthesis of enzymes has been described by various investigators. Rothstein, et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84: 8439, found that antisense RNA inhibited nopaline synthase (nos) in tobacco. Smith, C. J. S., et al. (1988) Nature 334: 724, reported that antisense RNA inhibited polygalacturonase in tomato. Others have used antisense RNA to inhibit the synthesis of enzymes involved in ethylene formation. Oeller, P. W., et al., (1991) Science 254: 437-439, expressed RNA antisense to ACC synthase in tomato plants. Others have expressed antisense RNA to a different ethylene forming enzyme (EFE), ACC oxidase, in carnation and tomato (Michael, M. Z., et al., 1993, In: Pech, J. C., et al., eds., Cellular and Molecular Aspects of the Plant Hormone Ethylene (Kluwer Academic Publishers, pp. 298-302); Hamilton, A. J., et al. (1990) Nature 346: 284-287; Gray, et al. (1993), in Pech, J. C., et al., supra, pp. 82-89; Murray, A. J., et al. (1993) in Pech, J. C., et a., supra,, pp. 327-328). The above work with antisense RNA may also be applicable to efforts to stably incorporate the sequences identified by the current inventor and their antisense sequences into a rose plant. Similarly, the success in expressing antisense RNA for ACC synthase in tomato plants may also be applicable (Oeller, et al., supra). It is noteworthy, and perhaps surprising, that neither of the foregoing disclosures have led to the long sought goal of stably altering ethylene production in rose plants. Hence, no altered rose plants expressing reduced levels of ethylene has been described. The incorporation of ACC synthase antisense DNA into a rose plant has remained elusive because the complete ACC gene sequences were not available prior to the present invention. The discoveries disclosed herein enable the production of an appropriately altered rose plant which will express ACC synthase antisense sequences and stably produce reduced levels of ethylene.