Molecular analysis of plant G proteins

Our lab previously proposed that blue-light signaling in pea is G protein mediated based on biochemical and immunological. To test and strengthen this hypothesis with respect to the blue-light stimulated transcription of the nuclear encoded Lhcb genes, we have employed the G protein activating peptide mastoparan (mas). In response to treatment of seven day old seedlings of pea and Arabidopsis with mastoparan from Polistes jadawagae, the endogenous steady state levels of Lhcb transcripts increase over the levels of mock treated seedlings. Even more striking is the finding that following mas treatment, GUS transcript levels increase in transgenic seedlings harboring the pea blue-light activated promoter PsLhcb1*4 fused to the GUS reporter gene ( PsLhcb1*4::GUS), but not in those carrying the blue-light unresponsive construct PsLhcb1*3::GUS. Based on the results described here it seems likely that the expression of Lhcb genes, including those that respond specifically to blue light, results from the stimulation of a G protein dependent pathway.; Toward identifying the G protein(s) that mediate the blue-light response of the Lhcb genes, we report here the cloning and characterization of two G protein alpha-subunits from pea: PGA1 and PGA2. Based on DNA gel blot analysis, PGA1 and PGA2 are the only Galpha homologous sequences in pea. RT-PCR analysis reveals that PGA1 and PGA2 transcripts are present in a variety of adult pea tissues. However, PGA2 mRNA is consistently detected at a lower level than PGA1 and demonstrates some degree of tissue specificity relative to PGA1. In the apical bud of pea seedlings, PGA1 and PGA2 transcripts decrease in response to 24 hrs of white light following growth for 6 days in darkness. We have used the G protein mediated, yeast mating pathway to analyze the function of PGA1 and PGA2 in vivo. Only PGA1 downregulates the mating pathway, but through a mechanism that is independent of Gbetagamma sequestration. Unexpectedly, both PGA1 and PGA2 promote growth through a mating pathway independent mechanism.