Molecular biological study on bule light signaling system in stomatal guard cells.

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Molecular mechanisms in blue light response of stomata were investigated regarding protein phosphorylation and dephosphorylation in guard cell protoplasts from Vicia faba. Blue light-dependent proton pumping has been shown to be inhibited by myosin light chain kinase (MLCK) inhibitor ML-7, suggesting that MLCk or MLCK-like protein may be involved in this stomatal response. We found that the antibody against MLCK from chicken gizzard reacted with guard cells proteins with molecular masses of 32 and 17kD.cDNA library of Vicia guard cells was immunoscreened using this polyclonal antibody. Two cDNA clones, VFPK1 and VFPK2, encoding putative protein kinases, were isolated from the library, and their nucleotide sequences were determined. The deduced amino acid sequences of VFPK1 and VFPK2 contain 271 and 219 amino acid residues, respectively, and are 88% identical at overlapping amino acid sequence level. The amino acid sequence revealed that they conserve invariant amino acid residues of kinase domain, and exhibited slight similarity to known protein kinases (<17%). Fusion protein of VFPK1 with glutathione-S-transferase was expressed in E.coli and the protein had protein kinase activity. These results suggest that VFPK1 cDNA encodes new protein kinase. However, the function and role in stomatal responses should be elucidated.Furthermore, we found protein phosphorylation of 80 kD protein in guard cells in response to blue light. The blue light-dependent protein phosphorylation was inhibited by MLCK inhibitor, supporting the notion that MLCK-like protein might be involved in blue light response of stomatal guard cells.