Calcium and cGMP target distinct phytochrome-responsive elements

Previous work using microinjection into single cells of the tomato aurea mutant demonstrated that phytochrome A-dependent activation of rbcS and chs genes was mediated by calcium and cGMP, respectively. This work sought to identify promoter cis-elements that respond to these two small molecules. Box II and Unit I, derived from rbcS-3A and chs promoters, respectively, were previously shown to function as light-responsive cis-elements. Eleven copies of Box II and four copies of Unit I were linked 5′ to the ?90 and ?46 35 S promoters, respectively, and, both constructs were fused to the β-glucuronidase (GUS) reporter gene. GUS activities were obtained upon co-injection of either Box II/-90GUS or Unit I/-46GUS with oat phytochrome A (phyA) and GTPγS, an activator of heterotrimeric G proteins. The activation of Box II/-90GUS by phyA was insensitive to the cGMP antagonist, Rp-cGMPS, although anthocyanin accumulation, but not chloroplast development, was totally blocked in the injected cells. Consistent with this result, calcium, but not cGMP, induced Box II/-90GUS activity. In contrast to Box II/-90GUS, phyA-dependent activation of Unit I/-46GUS activity was blocked by Rp-cGMPS. Moreover, cGMP, not calcium, induced Unit I/-46GUS activity. Control experiments showed that ?90 GUS and ?46 GUS were inactive in the presence of calcium and cGMP, respectively. These results provide evidence that Box II and Unit I are targets of the calcium and cGMP pathways, respectively. Interestingly, calcium activation of Box II/-90GUS was repressed by a high concentration of cGMP and cGMP induction of Unit I/-46GUS was blocked by a high concentration of calcium/CaM. Thus, these two small cis-elements can also serve as targets of the reciprocal control mechanisms that operate to regulate the activities of the two phyA signaling branches.