Calcium and cGMP target distinct phytochrome‐responsive elements

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Abstract
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.

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