The expression of beta-1,3-glucanase (betaGlu) and chitinase (Chn) was investigated in the testa, cotyledons, and embryonic axis of germinating Pisum sativum L. cv. 'Espresso generoso' seeds. High concentrations of betaGlu and Chn activity were found in the embryonic axis. Treatment with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that an early, 4-fold induction of betaGlu activity in the embryonic axis during the first 20 h after the start of imbibition is ethylene-independent. This initial increase was followed by a later 4-fold ethylene-dependent induction in the embryonic axis starting at 50 h, which is after the onset of ethylene evolution and after completion of radicle emergence. The betaGlu activity in cotyledons increased gradually throughout germination and was ethylene-independent. In contrast, the ethylene-independent Chn activity increased slightly after the onset of radical emergence in the embryonic axis and remained at a constant low level in cotyledons. Immunoinactivation assays and immunoblot analyses suggest that early betaGlu activity in the embryonic axis is due to a 54-kDa antigen, whereas late induction is due to a 34.5-kDa antigen, which is likely to be the ethylene-inducible class I betaGlu G2 described for immature pea pods. Increases in Chn in the embryonic axis were correlated with a 26-kDa antigen, whereas amounts of the additional 32- and 20-kDa antigens remained roughly constant. Thus, ethylene-dependent and ethylene-independent pathways regulate betaGlu and Chn during pea seed germination. The pattern of regulation differs from that of leaves and immature pods, and from that described for germinating tobacco seeds. The functional significance of this regulation and its underlying mechanisms are discussed.

Stochastic and nonstochastic post-transcriptional gene silencing (PTGS) in Nicotiana sylvestris plants carrying tobacco class I chitinase (CHN) and beta-1,3-glucanase transgenes differs in incidence, stability, and pattern of expression. Measurements with inhibitors of RNA synthesis (cordycepin, actinomycin D, and alpha-amanitin) showed that both forms of PTGS are associated with increased sequence-specific degradation of transcripts, suggesting that increased RNA turnover may be a general feature of PTGS. The protein synthesis inhibitors cycloheximide and verrucarin A did not inhibit degradation of CHN RNA targeted for PTGS, confirming that PTGS-related RNA degradation does not depend on ongoing protein synthesis. Because verrucarin A, unlike cycloheximide, dissociates mRNA from ribosomes, our results also suggest that ribosome-associated RNA degradation pathways may not be involved in CHN PTGS.

Class I beta-1,3-glucanase (betaGLU I) is transcriptionally induced in the micropylar endosperm just before its rupture prior to the germination (i.e. radicle emergence) of Nicotiana tabacum L. cv. 'Havana 425' seeds. Ethylene is involved in endosperm rupture and high-level betaGLU I expression; but, it does not affect the spatial and temporal pattern of betaGLU I expression. A promoter deletion analysis of the tobacco betaGLU I B gene suggests that (1) the distal - 1452 to - 1193 region, which contains the positively acting ethylene-responsive element (ERE), is required for high-level, ethylene-sensitive expression, (2) the regions - 1452 to - 1193 and -402 to 0 contribute to downregulation by abscisic acid (ABA), and (3) the region -402 to -211 is necessary and sufficient for low-level micropylar-endosperm-specific expression. Transcripts of the ERE-binding proteins (EREBPs) showed a novel pattern of expression during seed germination: light or gibberellin was required for EREBP-3 and EREBP-4 expression; EREBP-4 expression was constitutive and unaffected by ABA or ethylene; EREBP-3 showed transient induction just before endosperm rupture, which was earlier in ethylene-treated seeds and inhibited by ABA. No expression of EREBP- and EREBP-2 was detected. In contrast to betaGLU I, EREBP-3 and EREBP-4 were not expressed specifically in the micropylar endosperm. The results suggest that transcriptional regulation of betaGLU I could depend on: activation of ethylene signalling pathways acting via EREBP-3 with the ERE as the target, and ethylene-independent signalling pathways with targets in the proximal promoter region that are likely to determine spatial and temporal patterns of expression.

Stochastic and nonstochastic post-transcriptional gene silencing (PTGS) in Nicotiana sylvestris plants carrying tobacco class I chitinase (CHN) and beta-1,3-glucanase transgenes differs in incidence, stability, and pattern of expression. Measurements with inhibitors of RNA synthesis (cordycepin, actinomycin D, and alpha-amanitin) showed that both forms of PTGS are associated with increased sequence-specific degradation of transcripts, suggesting that increased RNA turnover may be a general feature of PTGS. The protein synthesis inhibitors cycloheximide and verrucarin A did not inhibit degradation of CHN RNA targeted for PTGS, confirming that PTGS-related RNA degradation does not depend on ongoing protein synthesis. Because verrucarin A, unlike cycloheximide, dissociates mRNA from ribosomes, our results also suggest that ribosome-associated RNA degradation pathways may not be involved in CHN PTGS.