The role of heterotrimeric G proteins in gibberellin (GA) induction of a-amylase gene expression was examined in wild oat aleurone protoplasts. Mas7, a cationic amphiphilic tetradecapeptide that stimulates GDP/GTP exchange by heterotrimeric G proteins, specifically induced alpha-amylase gene expression and enzyme secretion in a very similar manner to GA1. In addition, Mas7 stimulated expression of an alpha-Amy2/54:GUS promoter and reporter construct in transformed protoplasts. Both Mas7 and GA1 induction of alpha-amylase mRNA were insensitive to pertussis toxin. Hydrolysis-resistant nucleotides were introduced into aleurone protoplasts during transfection with reporter gene constructs. GDP-beta-S, which inhibits GDP/GTP exchange by heterotrimeric G proteins, completely prevented GA1 induction of alpha-Amy2/54:GUS expression, whereas GTP-gamma-S, which activates heterotrimeric G proteins, stimulated expression very slightly. Novel cDNA sequences from Galpha and Gbeta subunits were cloned from wild oat aleurone cells. By using RNA gel blot analysis, we found that the transcripts were expressed at a low level. Heterotrimeric G proteins have been implicated in several events during plant growth and development, and these data suggest that they may be involved in GA regulation of alpha-amylase gene expression in aleurone.