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.
Endogenous elicitor, termed cellulase-degraded cell wall (CDW), was prepared from the cell wall of suspension-cultured ginseng (Panax ginseng C.A. Meyer) cells via cellulase degradation. CDW activated the NADPH oxidase activity of isolated plasma membranes and stimulated in vivo H2O2 generation in ginseng cell suspensions. CDW also increased the activity of phenylalanine ammonia lyase (PAL), expression of a P. ginseng squalene epoxidase (sqe) gene and saponin synthesis. NADPH oxidase inhibitors inhibited both in vitro NADPH oxidase activity and in vivo H2O2 generation. Induction of PAL activity, saponin synthesis and sqe gene expression were all inhibited by such inhibitor treatments and reduced by incubation with catalase and HA scavengers. These data indicate that activation of NADPH oxidase and generation of H2O2 are essential signalling events mediating defence responses induced by the endogenous elicitor(s) present in CDW.
NAD(P)H oxidases were detected in suspension cultured cells of ginseng (Panax ginseng C. A. Meyer). The activities of these enzymes were induced by an elicitor (Cle) extracted from cell walls of Col-letotrichum lagerarium. In addition, Cle induced an oxidative burst and enhanced the synthesis of saponin, activity of phenylalanine ammonialyase (PAL) , accumulation of chalcone synthase (CHS) and the transcription of a hydroxyproline-rich glycoprotein gene ( hrgp ) . Pre-treatments with DPI and quinacrine (two inhibitors of mammalian neutrophil plasma membrane NADPH oxidase) for 30 min prior to Cle addition blocked the NAD(P)H oxidase activity induced by Cle. These inhibitors also inhibited the release of H2C2, the synthesis of saponin, PAL activity and CHS accumulation. Our data revealed homology between plasma membrane NAD(P)H oxidases of mammalian neutrophil cells and ginseng suspension cells. They also indicated that deactivated NAD(P)H oxidases catalysed the release of H2O2 and that H2O2 was functioning as a second messenger stimulating PAL activity, saponin synthesis and hrgp transcription. Elevations of Ca2 + and protein phos-phorylation/dephosphorylation were required for this defense process. We propose that NAD(P)H oxidases mediate the processes of Cle-induced defense responses in ginseng suspensions, and postulate the existence of a signalling cascade including extracellular Cle stimulation, activation of plasma membrane NAD(P)H oxidases, release of H2O2, and the intracellular responses of metabolism and gene transcription in ginseng 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 β-Amyrin synthase (β-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 (including 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. Keywords mitogen-activated protein kinase - chitosan - saponin
