In barley leaves a group of genes is expressed in response to treatment with jasmonates and abscisic acid (ABA) [21]. One of these genes coding for a jasmonate-induced protein of 23 kDa (JIP-23) was analyzed to find out the link between ABA and jasmonates by recording its expression upon modulating independently, the endogenous level of both of them. By use of inhibitors of JA synthesis and ABA degradation, and the ABA-deficient mutant Az34, as well as of cultivar-specific differences, it was shown that endogenous jasmonate increases are necessary and sufficient for expression of this gene. The endogenous rise of ABA did not induce synthesis of JIP-23, whereas exogenous ABA did not act via jasmonates. Different signalling pathways are suggested and discussed.

In tomato plants wounding leads to up-regulation of various plant defense genes via jasmonates (Ryan, 1992; Bergey et al., 1996). Using this model system of jasmonic acid (JA) signalling, we analyzed activity of octadecanoids to express JA-responsive genes. Leaf treatments were performed with naturally occurring octadecanoids and their molecular mimics such as coronatine or indanone conjugates. JA responses were recorded in terms of up- or down-regulation of various genes by analyzing transcript accumulation, and at least partially in vitro translation products and polypeptide pattern of leaf extracts. The data suggest: (i) 12-Oxo-phytodienoic acid and other intermediates of the octadecanoid pathway has to be ß-oxidized to give a JA response, (ii) Octadecanoids which can not be ß-oxidized are inactive, (iii) JA, its methyl ester (JM), and its amino acid conjugates are most active signals in tomato leaves leading to up regulation of mainly wound-inducible genes and down-regulation of mainly genes, (iv) Some compounds carrying a JA/JM- or JA amino acid conjugate-like structure induce/repress only a subset of genes suggesting diversity of JA signalling.

Leaves of barley (Hordeum vulgare L. cv. Salome) treated with jasmonic acid (JA), its methyl ester (JM), or its amino acid conjugates exhibit up-regulation of specific genes and down-regulation of house-keeping genes, This transcriptional regulation exhibits several specificities, (i) The (-)-enantiomers are more active, and conjugates are mainly active if they carry an L-amino acid moiety, (ii) The various JA-responsive genes respond differentially to enantiomeric and chiralic forms, (iii) Both JA and its amino acid conjugates exhibiting no or negligible interconversion induce/repress genes. (C) 1997 Federation of European Biochemical Societies.

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, Mla12, ormlo5. 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.

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the target of the herbicide glyphosate [N-(phosphonomethyl)glycine], exists in two molecular forms in Euglena gracilis. One form has previously been characterized as a monofunctional 59 kDa protein. The other form constitutes a single domain of the multifunctional 165 kDa arom protein. The two enzyme forms are inversely regulated at the protein and mRNA levels during light-induced chloroplast development, as demonstrated by the determination of their enzyme activities after non-denaturing polyacrylamide gel electrophoresis and Northern hybridization analysis with a Saccharomyces cerevisiae ARO1 gene probe. The arom protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination. In contrast, the monofunctional EPSP synthase and its mRNA are induced by light, the increase in mRNA abundance preceding accumulation of the protein. The two enzymes are localized in different subcellular compartments, as demonstrated by comparing total protein patterns with those of isolated organelles. Glyphosate-adapted wild-type cells and glyphosate-tolerant cells of a plastid-free mutant of E. gracilis, W10BSmL, were used for organelle isolation and protein extraction, as these cell lines overproduce EPSP synthase and the arom protein, respectively. Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartmentalization of the monofunctional EPSP synthase. These conclusions are further supported by the observation that EPSP synthase precursor, produced by in vitro translation of the hybrid-selected mRNA, was efficiently taken up and processed to mature size by isolated chloroplasts from photoautotrophic wild-type E. gracilis cells, while the in vitro-synthesized arom protein was not sequestered by isolated Euglena plastids.

Cells of the plastid-free mutant line of Euglena gracilis var. bacillaris, W10BSmL, can be adapted to glyphosate [N-(phosphonomethyl)glycine] by gradually increasing the concentration of the herbicide in the culture medium. The molecular basis of glyphosate tolerance is the selective ca. ten-fold overproduction of the multifunctional arom protein catalyzing steps 2-6 in the pre-chorismate pathway. Determination of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (E.C.2.5.1.19), shikimate:NADP+ oxidoreductase (E.C.1.1.1.25) and shikimate kinase (E.C.2.7.1.71) activities after non-denaturing gel electrophoresis, in combination with two-dimensional separations, revealed an increase in all three enzyme activities associated with overproduction of a 165 kDa protein in cells adapted to 6 mM glyphosate. Further evidence for an involvement of the multifunctional arom protein in aromatic amino acid synthesis in the plastid-free W10BSmL cells was obtained by Northern hybridization with ARO1-, aroA-, aroL- and aroE-specific Saccharomyces cerevisiae gene probes encoding the entire arom protein or parts of the EPSP synthase, shikimate:NADP+ oxidoreductase and shikimate kinase domains, respectively. Overproduction in adapted relative to control cells of a 5.3 kb transcript that cross-hybridized with all of the different probes could be demonstrated. The elevated content of the arom transcript correlated with a selective amplification of two out of five genomic sequences that hybridized with the S. cerevisiae ARO1 gene probe in Southern blots. One of the amplified genomic fragments is assumed to encode the previously identified monofunctional 59 kDa EPSP synthase, which is thought to be an organellar protein, that accumulates to a certain extent in its enzymatically active precursor form of 64.5 kDa in the plastid-free W10BSmL cells.