Identification of promoter elements involved in the cytosolic Ca2+-mediated photoregulation of maize cab-m1 expression

Changes in cytoplasmic Ca2+ levels are involved in the regulation of several plant genes. However, to our knowledge, no regions of genes or specific cis elements have been shown to be involved in the regulation of plant gene expression by cytosolic Ca2+ signaling. The maize (Zea mays) gene cab-m1, which encodes a light-harvesting chlorophyll a/b-binding apoprotein, is positively photoregulated in mesophyll cells (MC) but not in bundle-sheath cells (BSC). This gene is highly preferentially expressed in maize MC versus BSC. In situ transient expression assays have revealed that exposure of tissues to ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), which chelates Ca2+, blocks the photostimulation of cab-m1 full promoter (-1026 to +14) activity in MC of leaf segments of dark-grown maize seedlings. EGTA has no effect on expression in BSC. These results suggest that light-induced elevation of the cytosolic Ca2+ concentration in MC is required for the enhancement of cab-m1 expression in MC. Deletion of the sequence from -1026 to -360 completely abolished Ca2+ responsiveness of cab-m1 expression in MC. On the other hand, a 54-bp fragment in the 5' flanking region (-953 to -899 relative to the translation start site) conferred Ca2+ responsiveness on a -359 core promoter: reporter gene, suggesting that Ca2+ signaling is mediated via specific sequences in this short fragment. Furthermore, possible involvement of Ca2+-calmodulin in the signal transduction chain for regulating cab-m1 expression was suggested by the results of inhibitor experiments.