Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in catharanthus roseus

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor alpha-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously. Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.

Evidence for the formation of dinor isoprostanes E1 from alpha-linolenic acid in plants.

The free radical oxidation of arachidonic acid is known to generate complex metabolites, termed isoprostanes, that share structural features of prostaglandins and exert potent receptor-mediated biological activities. In the present study, we show that alpha-linolenic acid can undergo a similar oxidation process, resulting in a series of isomeric dinor isoprostanes E1. E-ring dinor isoprostane formation from linolenate was found to be catalyzed by soybean lipoxygenase. The main enzymatic products were 13- and 9-hydroperoxylinolenate but in addition, two dinor isoprostane E1 regioisomers were formed with a yield of 0.31%. Identification and quantification of two dinor isoprostane E1 regioisomers in plant cell cultures was achieved by a negative chemical ionization gas chromatography-mass spectrometry method using [18O]dinor isoprostanes E1 as internal standards. Endogenous levels of these compounds were determined in four taxonomically distant plant species and found to be in the range of 4.5 to 60.9 ng/g of dry weight. Thus analogous pathways in animals and plants exist, each leading to a family of prostaglandin-like compounds derived from polyunsaturated fatty acids. It remains to be shown whether the dinor isoprostanes exert biological activities in plants as has been demonstrated for their C20 congeners in mammals.

Induction of 12-oxo-phytodienoic acid in wounded plants and elicited plant cell cultures.

Jasmonic acid (JA) is rapidly biosynthesized from alpha-linolenic acid in plants upon contact with pathogens or wounding, and triggers gene activation, leading to the synthesis of defensive secondary metabolites and proteins. Despite the recent finding that its precursor, 12-oxo-phytodienoic acid (PDA), is a more powerful inducer of gene activation, interest has focused so far almost exclusively on JA. A validated negative chemical ionization-gas chromatography-mass spectrometry method has been developed that allows the simultaneous quantification of endogenous 12-oxo-PDA and JA in plant tissues. In six out of eight plant species tested maximal levels of 12-oxo-PDA exceeded peak levels of JA by approximately 3- to 5-fold after elicitation with a yeast cell wall preparation or when plants were wounded. These experiments support the hypothesis that 12-oxo-PDA acts as the predominant jasmonate signal in most plants, whereas JA remains an active metabolite of its precursor. Furthermore, JA but not 12-oxo-PDA was shown to be secreted into the medium from cultured plant cells, suggesting that JA may also act as an intercellular signal.