Molecular Cloning and Characterization of a Guanylyl Cyclase, PnGC-1, Involved in Light Signaling in Pharbitis nil

Guanylyl cyclases (GCs) are enzymes involved in the biosynthesis of cyclic guanosine monophosphate (cGMP). Here we report the cloning and characterization of a new guanylyl cyclase, designated PnGC-1, from Pharbitis nil. This gene encodes a protein of 286 amino acids, with a calculated molecular mass of 32 kDa. The predicted amino acid sequence contains all typical features and shows high identity with known plant GCs. The GST-PnGC-1 was catalytically active in E. coli cells and the purified, recombinant PnGC-1 was able to convert GTP to cGMP in the presence of Mn2+. Moreover, the enzyme activity was strongly inhibited by a specific sGC inhibitor, NS2028, whereas in the case of nitric oxide, an animal sGC stimulator, no positive effect was observed. Besides the identification of the PnGC-1 as a guanylyl cyclase, it was shown that a transcript of PnGC-1 was present in every tested organ of the light- or dark-grown plants; however, the highest expression level was found in dark-treated plants. The PnGC-1 mRNA level in the cotyledons exhibited diurnal oscillations under short-day conditions (8/16-h photoperiod). Meanwhile, monitoring of transcript levels in cotyledons exposed to a special photoperiodic regime (24 h light of low intensity then 24 h long night with or without far-red light before the night) revealed that a stabile phytochrome is involved in this process. These data unequivocally identify the product of the PnGC-1 gene as a guanylyl cyclase and emphasize the potential that soluble GC can be an element of light signal transduction.

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