We have recently proposed that one way that plant growth-promoting rhizobacteria (PGPR) stimulate plant growth is through the activity of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which causes a lowering of plant ethylene levels resulting in longer roots. As part of an effort to understand the role of this enzyme in PGPR, the genes for ACC deaminase from two PGPR, Enterobacter cloacae CAL2 and UW4, have been isolated. These genes are highly homologous to the ACC deaminase genes from Pseudomonas strains 6G5 and F17 and similar to the ACC deaminase gene from Pseudomonas sp. strain ACP. The region downstream (i.e., at the 3'-terminal end) of the strain UW4 ACC deaminase gene has a potential hairpin-like transcription termination site. The regions upstream of the strains UW4 and CAL2 ACC deaminase genes contain putative ribosome-binding sites; however, the promoter sequences have not yet been identified. Southern hybridization experiments suggest that there is a single copy of the ACC deaminase gene in Enterobacter cloacae strains UW4 and CAL2 and that there may be several different types of ACC deaminase genes in different microbes. The cloned ACC deaminase gene can be expressed in Escherichia coli enabling this bacterium to grow on ACC as a sole source of nitrogen and confers upon both Escherichia coli and Pseudomonas spp. strains that are transformed with this gene the ability to promote the elongation of the roots of canola seedlings.

The seed oils of higher plants contain many different fatty acids that determine the value of a particular oil for human nutrition or as a source of industrial chemicals (Murphy, 1994; Ohlrogge, 1994). For this reason, there is considerable interest in bringing about useful changes in the fatty acid composition of oilseed crops. One of the most promising areas of research in this regard relates to protein engineering of acyl-ACP desaturases (Cahoon et al., 1997b).The acyl-ACP desaturases are a family of closely related, soluble enzymes that catalyze insertion of the first double bond in a saturated fatty acyl chain (Cahoon et al., 1997a). The most widely occurring member of the family is the ⌬9-18:0-ACP desaturase, which is responsible for 18:1 synthesis in plants (Nagai and Bloch, 1968; Shanklin and Somerville, 1991; Thompson et al., 1991). In addition, several other acyl-ACP desaturases have been described: the ⌬4-16:0-ACP desaturase of coriander seed (Cahoon et al., 1992), the ⌬6-16:0-ACP desaturase from blacked-eyed Susan (Thunbergia alata) seed (Cahoon et al., 1994a), and the ⌬9-14:0-ACP desaturase of geranium trichomes (Schultz et al., 1996). The characterization of these variant enzymes and the availability of a crystal structure for the castor Ricinus communis L. ⌬9-18:0-ACP desaturase (Lindqvist et al., 1996) raises the possibility that protein engineering can be used to manipulate the double-bond position and substrate specificities of these enzymes. Unusual monounsaturated fatty acids produced by redesigned desaturases may be useful for generating seed oils with altered physical properties and new commercial applications.Based on previous studies (Cahoon et al., 1992(Cahoon et al., , 1994a Cahoon and Ohlrogge, 1994b; Schultz et al., 1996), seeds and other plant tissues that accumulate large amounts of unusual monounsaturated fatty acids represent potential sources of variant forms of acyl-ACP desaturases. The seed oils of several plant species are rich sources of 16:1⌬9 and its elongation product, 18:1⌬11 (Chisholm and Hopkins, 1965). These unusual fatty acids can account for 25 to 80% of the seed oils of such species. A likely route of synthesis of 16:1⌬9 and 18:1⌬11 would initially involve the ⌬9 desaturation of 16:0-ACP by an acyl-ACP desaturase with enhanced specificity for this substrate relative to known ⌬9-18:0-ACP desaturases. In this regard, a diverged acyl-ACP desaturase was recently identified in milkweed seed (Cahoon et al., 1997a)

Summary Ethylene evolved during compatible or susceptible disease interactions may hasten and/or worsen disease symptom development; if so, the prevention of disease-response ethylene should reduce disease symptoms. We have examined the effects of reduced ethylene synthesis on Verticillium wilt (causal organism, Verticillium dahliae) of tomato by transforming tomato with ACC deaminase, which cleaves ACC, the immediate biosynthetic precursor of ethylene in plants. Three promoters were used to express ACC deaminase in the plant: (i) CaMV 35S (constitutive expression); (ii) rolD (limits expression specifically to the site of Verticillium infection, i.e. the roots); and (iii) prb-1b (limits expression to certain environmental cues, e.g. disease infection). Significant reductions in the symptoms of Verticillium wilt were obtained for rolD- and prb-1b-, but not for 35S-transformants. The pathogen was detected in stem sections of plants with reduced symptoms, suggesting that reduced ethylene synthesis results in increased disease tolerance. The effective control of formerly recalcitrant diseases such as Verticillium wilt may thus be obtained by preventing disease-related ethylene production via the tissue-specific expression of ACC deaminase.

A mutant of Arabidopsis contained increased levels of 18:3 fatty acids and correspondingly decreased levels of 18:2. The fatty acid phenotype was strongly expressed in root and seed tissues and this observation, together with other data, suggested that the mutation leads to increased activity of the endoplasmic reticulum 18:2 desaturase encoded by the FAD3 gene. Cel-blot analysis of RNA from wild-type and mutant plants established that FAD3 transcript levels were increased 80% in the mutant relative to the wild type. Cenetic analysis demonstrated a linkage between the new mutation and the fad3 locus. Linkage of the mutation to fad3 raises the possibility that the lesion is an alteration to the promoter or another regulatory region of the FAD3 gene, which results in increased transcription.A characteristic feature of the membranes in plant cells is their very high level of fatty acid unsaturation compared with membranes of other eukaryotes. Whereas glycerolipids from animal tissues typically contain an average of 2.0 to 2.5 double bonds/ molecule in the hydrocarbon chains (Harwood et al., 1986;Marsh, 1990), the number of double bonds/molecule in glycerolipids from higher plants is 3 to 3.4 from nonphotosynthetic tissues and 4.3 to 5.0 for chloroplasts (Harwood, 1980). The chloroplast membranes of plant cells are composed entirely of the uncharged galactolipids MGD and DGD, which make up about 75% of the total chloroplast thylakoid lipid content (Harwood, 1980). Polyunsaturated trienoic fatty acids (18:3 and 16:3) constitute up to 80% of the fatty acids in this organelle. This high level of membrane unsaturation is achieved by the synthesis of polyunsaturated a-linolenate (A9, 12, 15, and 18:3) and hexadecatrienoic acid (A7, 10, 13, and 16:3). The insertion of double bonds at the 12 and 15 positions in a-linolenate occurs in plants but not in other higher eukaryotes; therefore, 18:2 and 18:3 are two essential fatty acids in human nutrition.In plant cells lipid metabolism is initiated by the synthesis of 16:O-ACP and 18:O-ACP by the reactions of a type I1 fatty acid synthase (Ohlrogge and Browse, 1995). The 18: O-ACP is quickly and efficiently desaturated to 18:l-ACP

SummaryThe constitutive expression of the pea ABR17 (abscisic acid-responsive 17) cDNA, which is a member of the group 10 family of pathogenesis-related proteins (PR 10), in Arabidopsis thaliana is reported. The presence of ABR17 transcripts and the protein in the three transgenic lines is demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) and two-dimensional electrophoresis followed by tandem mass spectrometry, respectively. Three independently derived transgenic lines containing ABR17 germinated better in the presence of salt, cold temperature or both. Furthermore, the transgenic plants also exhibited enhanced tolerance to freezing temperature, suggesting the potential utility of the ABR17 gene to engineer multiple stress tolerance. In order to obtain insights into the mechanism underlying ABR17 -mediated stress tolerance, we have compared the proteome of a transgenic line with that of its wild-type counterpart. Several proteins were observed to be significantly altered in the transgenic line, including some with a role(s) in photosynthesis, stress tolerance and the regulation of gene expression. Our findings are discussed within the context of available genes to engineer multiple stress tolerance as well as the biological activities of the ABR17 protein.