Class I ß-1,3-glucanase in the endosperm of tobacco during germination

Rupture of the seed coat and rupture of the endosperm are separate events in the germination of Nicotiana tabacum L. cv Havana 425 seeds. Treatment with 10-5 M abscisic acid (ABA) did not appreciably affect seed-coat rupture but greatly delayed subsequent endosperm rupture by more than 100 h and resulted in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue. Therefore, ABA appears to act primarily by delaying endosperm rupture and radicle emergence. Measurements of [beta]-1,3-glucanase activity, antigen content, and mRNA accumulation together with reporter gene experiments showed that induction of class I [beta]-1,3-glucanase genes begins just prior to the onset of endosperm rupture but after the completion of seed-coat rupture. This induction was localized exclusively in the micropylar region of the endosperm, where the radicle will penetrate. ABA treatment markedly inhibited the rate of [beta]-1,3-glucanase accumulation but did not delay the onset of induction. Independent of the ABA concentration used, onset of endosperm rupture was correlated with the same [beta]-1,3-glucanase content/seed. These results suggest that ABA-sensitive class I [beta]-1,3-glucanases promote radicle penetration of the endosperm, which is a key limiting step in tobacco seed germination

Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling.

Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I beta-1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5'-deletion analysis of the class I beta-1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I beta-1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I beta-1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I beta-1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these datastrongly suggest that ethylene is signalling ozone-induced expression of class I beta-l,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an additional ethylene independent signalling pathway for ozone-induced expression of genes involved in phytoalexin biosynthesis