Cloning of a Ca(2+)-ATPase gene and the role of cytosolic Ca2+ in the gibberellin-dependent signaling pathway in aleurone cells.
The ultimate goal of this investigation was to identify intermediary steps in the gibberellin (GA)-dependent signaling pathway in rice aleurone cells. By using a differential display approach, a number of putative GA-responsive genes were isolated. One of them, a GA-responsive Ca(2+)-ATPase gene, was identified and partially characterized. A genomic clone and a cDNA clone were isolated and sequenced. The deduced amino acid sequence showed that this protein resembles an endoplasmic reticulum membrane Ca(2+)-ATPase. In a transient assay in rice aleurone cells, expression of the introduced Ca(2+)-ATPase cDNA bypassed the GA requirement for stimulating the expression of a major target gene, the alpha-amylase c gene (Osamy-c). This result suggests that GA-dependent expression of this Ca(2+)-ATPase gene (OsCa-atpase) plays an important role in the GA-dependent signal-transduction pathway. To investigate the possible involvement of other proteins and genes that may affect the intracellular Ca2+ level, compounds which can block different putative steps in the signal-transduction pathway were introduced into rice aleurone cells, and then the level of the OsCa-atpase transcript or the Osamy-c transcript was monitored. In the presence of GA, the rice Ca(2+)-ATPase and the Ca2+ channels appeared to co-regulate the local concentration of cytosolic Ca2+. The release of Ca2+ from the internal stores to the cytoplasm was presumably initiated by inositol-1,4,5-triphosphate which reached a peak level within 25 min after GA induction. As a second messenger, Ca2+ binds to calmodulin (CaM), and the Ca2+/CaM complex regulates the cytosolic Ca2+ by affecting expression of the OsCa-atpase. Finally, a working model is proposed for the GA-dependent signaling pathway in aleurone cells.
Chen
X
X
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
Chang
M
M
Wang
B
B
Wu
B
B
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary; D004721:Endoplasmic Reticulum / Q000201:enzymology; D017930:Genes, Reporter; D005875:Gibberellins / Q000378:metabolism*; D008156:Luciferases / Q000096:biosynthesis; D018551:Solanum lycopersicum / Q000201:enzymology; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology / Q000502:physiology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics
D018076:DNA, Complementary; D005875:Gibberellins; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D000516:alpha-Amylases; D000252:Calcium-Transporting ATPases; D002118:Calcium