Publikationen - Molekulare Signalverarbeitung

Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 + AOC1 and AOC4 + AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles.

In barley leaves 13-lipoxygenases (LOXs) are induced by salicylate and jasmonate. Here, we analyse by metabolic profiling the accumulation of oxylipins upon sorbitol treatment. Although 13-LOX-derived products are formed and specifically directed into the reductase branch of the LOX pathway, accumulation is much later than in the cases of salicylate and jasmonate treatment. In addition, under these conditions only the accumulation of jasmonates as additional products of the LOX pathway has been found.

In barley leaves 13-lipoxygenases are induced by jasmonates. This leads to induction of lipid peroxidation. Here we show by in vitro studies that these processes may further lead to autoxidative formation of (2E)-4-hydroxy-2-hexenal from (3Z)-hexenal.

Onset of acquired resistance of barley (Hordeum vulgare) chemically induced by 2,6-dichloroisonicotinic acid (DCINA) correlated with the accumulation of mRNA homologous to cDNA pHvJ256 which codes for a soluble leaf-thionin with a Mr. of 6 kDa [Wasternacket al., 1994a]. In the present work, we extend this finding by showing that the thionin transcript also accumulated following treatment of barley with the resistance-inducing compounds 3,5-dichlorosalicylic acid (DCSA), salicylic acid (SA), and an extract fromBacillus subtilis. The polypeptide showed antifungal activity against the biotrophic cereal pathogensErysiphe graminis f.sp.hordei andPuccinia graminis f.sp.tritici which may indicate a possible role in the mechanism of acquired resistance in barley. A thionin transcript hybridizing to pHvJ256 accumulated also in response to application of jasmonates, or treatments that elevated endogenous amounts of the plant growth substance, pointing to the possibility that signaling mediating defense responses in barley involves jasmonates. However, a topical spray application of jasmonic acid (JA) or jasmonate methyl ester (JM) did not protect barley leaves against infection byE. graminis. Performing a kinetic analysis by an enzyme immunoassay specific for (−)-JA, (−)-JM, and its amino acid conjugates, accumulation of jasmonates was detected in osmotically stressed barley but not at the onset of chemically induced or genetically based resistance governed by the powdery mildew resistance genesMlg, Mla 12, ormlo 5. Furthermore, the jasmonate-inducible proteins JIP-23 and JIP-60 were strongly induced following JM- but not DCINA-treatment or inoculation withE. graminis. Hence, in barley, no indications were found in favour for the previously proposed model of a lipid-based signaling pathway via jasmonates mediating expression of resistance in plants against pathogens.