The cyclic derivative of 13(S)-hydroperoxolinolenic acid, 12-oxophytodienoic acid, serves as a signal transducer in higher plants, mediating mechanotransductory processes and plant defenses against a variety of pathogens, and also serves as a precursor for the biosynthesis of jasmonic acid, a mediator of plant herbivore defense. Biosynthesis of 12-oxophytodienoic acid from ␣-linolenic acid occurs in plastids, mainly in chloroplasts, and is thought to start with free linolenic acid liberated from membrane lipids by lipase action. In Arabidopsis thaliana, the glycerolipid fraction contains esterified 12-oxophytodienoic acid, which can be released enzymatically by sn1-specific, but not by sn2-specific, lipases. The 12-oxophytodienoyl glycerolipid fraction was isolated, purified, and characterized. Enzymatic, mass spectrometric, and NMR spectroscopic data allowed us to establish the structure of the novel oxylipin as sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride. The novel class of lipids is localized in plastids. Purified monogalactosyl diglyceride was not converted to the sn1-(12-oxophytodienoyl) derivative by the combined action of (soybean) lipoxygenase and (A. thaliana) allene oxide synthase, an enzyme ensemble that converts free ␣-linolenic acid to free 12-oxophytodienoic acid. When leaves were wounded, a significant and transient increase in the level of (12-oxophytodienoyl)-monogalactosyl diglyceride was observed. In A. thaliana, the major fraction of 12-oxophytodienoic acid occurs esterified at the sn1 position of the plastid-specific glycerolipid, monogalactosyl diglyceride.

SummaryRecent evidence suggests that oxidized lipid-derived molecules play signi®cant roles in inducible plant defence responses against microbial pathogens, either by directly deterring parasite multiplication, or as signals involved in the induction of sets of defence genes. The synthesis of these oxylipins was hypothesized to be initiated by the phospholipase A 2 -mediated release of unsaturated fatty acids from membrane lipids. Here, we demonstrate that, in tobacco leaves reacting hypersensitively to tobacco mosaic virus, a strong increase in soluble phospholipase A 2 (PLA 2 ) activity occurs at the onset of necrotic lesion appearance. This rapid PLA 2 activation occurred before the accumulation of 12-oxophytodienoic and jasmonic acids, two fatty acid-derived defence signals. Three PLA 2 isoforms were separated and the most active enzyme was partially puri®ed, its N-terminal sequence displaying similarity with patatin, the major storage protein in potato tubers. Three related tobacco patatin-like cDNAs, called NtPat1, NtPat2 and NtPat3, were cloned, with NtPat2 encoding the PLA 2 isolated from infected leaves. RT±PCR experiments showed a rapid transcriptional activation of the three NtPat genes in virus-infected leaves, preceding the increase in PLA 2 activity. Recombinant NtPat1 and NtPat3 enzymes were active in an assay using labelled bacterial membranes, and also displayed high bona ®de PLA 2 activity on phosphatidylcholine substrate. These results point to a possible new role of patatin-like phospholipases in inducible plant defence responses. The induction kinetics together with the enzymatic activity data indicate that the NtPat proteins may provide precursors for oxylipin synthesis during the hypersensitive response to pathogens.

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We investigated the effect of UV radiation on early signaling events in the response of young tomato plants (Lycopersicon esculentum) to wounding. Ultraviolet‐C (<280 nm) and UVB/UVA (280–390 nm) radiation both induced 48 kDa myelin basic protein kinase activity in leaves. The activation was associated with phosphorylation of tyrosine residues on the kinase, which is indicative of protein kinases of the mitogen‐activated protein kinase family. Ultraviolet‐C irradiation resulted in a strong proteinase inhibitor synthesis, as reported previously (Conconi et al., Nature 383, 826–829, 1996). Under the conditions used, UVB/UVA radiation did not induce proteinase inhibitor synthesis but resulted in a strong potentiation of systemic proteinase inhibitor synthesis in response to wounding. The UVB/UVA‐irradiated plants that were subsequently wounded accumulated 2.5–4‐fold higher levels of proteinase inhibitor I when compared to wounded non‐irradiated plants. The potentiating effect was most prominent in the systemic unwounded leaf of a wounded plant. Levels of 12‐oxo‐phytodienoic acid and jasmonic acid that have been well documented to increase in response to wounding were not detected in response to UVB/UVA irradiation alone. The effect of UVB/UVA radiation in potentiating plant defense signaling should be further considered as a factor that may influence the ecological balance between plants and their predators.