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 beta-glucuronidase (GUS) reporter gene. GUS activities were obtained upon coinjection of either Box II/-90GUS or Unit I/-46GUS with oat phytochrome A (phyA) and GTP gamma S, 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.
Wu
Y
Y
Laboratory of Plant Molecular Biology, Rockefeller University, New York, NY 10021-6399, USA.
Hiratsuka
K
K
Neuhaus
G
G
Chua
N H
NH
D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology*; D006152:Cyclic GMP / Q000494:pharmacology*; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008027:Light; D018551:Solanum lycopersicum / Q000235:genetics; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000235:genetics; D010834:Phytochrome / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D014675:Vegetables / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects
D010834:Phytochrome; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000217:Acyltransferases; C020141:flavanone synthetase; D012273:Ribulose-Bisphosphate Carboxylase; D006152:Cyclic GMP; D002118:Calcium