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We have previously shown that the expression of SmCP which encodes Solanum melongena cysteine proteinase is ethylene-inducible and is under circadian control. To understand the regulation of SmCP, a 1.34-kb SmCP 5'-flanking region and its deletion derivatives were analyzed for cis-elements using GUS and luc fusions and by in vitro binding assays. Analysis of transgenic tobacco transformed with SmCP promoter-GUS constructs confirmed that the promoter region -415/+54 containing Ethylene Responsive Element ERE(-355/-348) conferred threefold ethylene-induction of GUS expression, while -827/+54 which also contains ERE(-683/-676), produced fivefold induction. Using gel mobility shift assays, we demonstrated that each ERE binds nuclear proteins from both ethephon-treated and untreated 5-week-old seedlings, suggesting that different transcriptions factors bind each ERE under varying physiological conditions. Binding was also observed in extracts from senescent, but not young, fruits. The variation in binding at the EREs in fruits and seedlings imply that organ-specific factors may participate in binding. Analysis of transgenic tobacco expressing various SmCP promoter-luc constructs containing wild-type or mutant Evening Elements (EEs) confirmed that both conserved EEs at -795/-787 and -785/-777 are important in circadian control. We confirmed the binding of total nuclear proteins to EEs in gel mobility shift assays and in DNase I footprinting. Our results suggest that multiple proteins bind the EEs which are conserved in plants other than Arabidopsis and that functional EEs and EREs are present in the 5'-flanking region of a gene encoding cysteine proteinase.

We have previously shown that SmCP, the gene encoding Solanum melongena cysteine proteinase, is expressed during developmental events associated with programmed cell death (PCD) suggesting its involvement in protein degradation during these events (Xu and Chye, Plant Journal 17 (1999) 321–327). Here, we investigated the regulation of SmCP expression and showed that it is ethylene-inducible and is under circadian control. This circadian rhythm is entrained by light/dark (LD) cycling with peak expression in the late light period, as opposed to that in early light for rbcS, suggesting that protein degradation and photosynthesis are temporally separated by circadian control. Northern blot analysis shows that the pattern of ethylene induction of SmCP is consistent with our previous observation of its significantly increased expression at leaf senescence and fruit ripening when endogenous ethylene is abundant. To further understand SmCP regulation, we have cloned the SmCP promoter and identified a G-box (CACGTG) at −85/−80 by DNase I footprinting analysis of the −221/+17 region. Its specific interaction with nuclear proteins in S. melongena leaves and fruits was confirmed by competitive electrophoretic mobility shift assays using oligonucleotides containing the G-box and mutant derivatives. G-box binding activity was stronger in senescent than young fruits. In circadian-regulated leaves, stronger binding activity coincided with peak circadian expression of SmCP. This correlation between binding activity and expression suggests that G-box binding factors enhance SmCP transcription and that the G-box likely plays a role in circadian regulation of genes affected by LD cycling.

We have previously shown that SmCP, the gene encoding Solanum melongena cysteine proteinase, is expressed during developmental events associated with programmed cell death (PCD) suggesting its involvement in protein degradation during these events (Xu and Chye, Plant Journal 17 (1999) 321-327). Here, we investigated the regulation of SmCP expression and showed that it is ethylene-inducible and is under circadian control. This circadian rhythm is entrained by light/dark (LD) cycling with peak expression in the late light period, as opposed to that in early light for rbcS, suggesting that protein degradation and photosynthesis are temporally separated by circadian control. Northern blot analysis shows that the pattern of ethylene induction of SmCP is consistent with our previous observation of its significantly increased expression at leaf senescence and fruit ripening when endogenous ethylene is abundant. To further understand SmCP regulation, we have cloned the SmCP promoter and identified a G-box (CACGTG) at -85/-80 by DNase I footprinting analysis of the -221/+17 region. Its specific interaction with nuclear proteins in S. melongena leaves and fruits was confirmed by competitive electrophoretic mobility shift assays using oligonucleotides containing the G-box and mutant derivatives. G-box binding activity was stronger in senescent than young fruits. In circadian-regulated leaves, stronger binding activity coincided with peak circadian expression of SmCP. This correlation between binding activity and expression suggests that G-box binding factors enhance SmCP transcription and that the G-box likely plays a role in circadian regulation of genes affected by LD cycling.