Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings expressing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca2+]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H2O2. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca2+]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.
Magnaporthe grisea special races 98-186-1G1 and 97-23-2D1 induce incompatible and compatible reactions respectively with rice Xiushui? The elicitor from the cell wall of M. grisea race 98-186-1G1, termed IE, strongly in-duced the HR response in suspension cultures of rice Xiushui? including increased PAL activity, transcription of pal , pr1, chi, cell death and the generation of NO. The elici-tor prepared from the cell wall of M. grisea race 97-23-2D1, named CE, was much less efficient at inducing such effects. The NOS enzyme inhibitors L-NAA and PBITU suppressed the production of NO induced by IE in Xiushui?rice. The increased PAL activity and transcription of pr1, pal, chi genes induced by IE were blocked by L-NAA, PBITU or CPTIO pretreatment. Direct treatment of rice cultures with the NO donor (SNP) also induced the transcription of pr1, pal and chi genes. These data implicated that NO acted as a signal mediating the HR induced by IE in rice and showed that NO, in combination with H2O2, is necessary for induc-tion of cell death by IE in rice suspension cells.
Chitosan (CHN) specially induced the activities of 39 kD and 42 kD protein kinases in ginseng cells, which could be suppressed by an inhibitor of mitogen-activated protein kinase (MAPK) pathway, PD98059. The immunoprecipitation (IP) using MAPK antibody or kinase assay in vitro also showed that CHN-induced 42 kD and 39 kD protein kinases belonged to the MAPK family. PD98059 suppressed CHN-induced transcriptions of ginseng squalene synthase and ginseng squalene epoxidase genes (gss and gse), CHN-induced accumulation of b-Amyrin synthase (b-AS) and synthesis of saponin. These results showed that CHN-induced activities of MAPKs were necessary for the CHN-induced saponin synthesis. EGTA and LaCl3 suppressed CHN-induced 39 kD and 42 kD MAPK activities. Ruthenium red (RR) could suppress CHN-induced 39 kD activity. All of them suppressed CHN-induced saponin synthesis. These results indicated that CHN-induced increment of cytosolic calcium was necessary for CHN-induced saponin synthesis. PD98059 also suppressed CHN-induced oxidative burst (in-cluding the increment of activity of plasma membrane NADPH oxidase and production of H2O2), but diphenylene iodonium (DPI), dimethylthiourea (DMTU) and 2,5-dihydroxycinnamic acid methyl ester (DHC) could not suppress CHN-induced MAPK activities, which indicated that MAPK was possibly function upstream of CHN-induced oxidative burst.
