Hormonal Regulation of α-Amylase Gene Transcription in Wild Oat (Avena fatua L.) Aleurone Protoplasts

Abstract

The time of appearance and relative amounts of .alpha.-amylase mRNA in wild oat (Avena fatua L.) aleurone protoplasts incubated with 1 micromolar gibberellin A4 (GA4) were closely correlated with the amounts of .alpha.-amylase enzyme secreted by the protoplasts. In the absence of GA4, or when protoplasts were incubated with 25 micromolar abscisic acid (ABA) together with 1 micromolar GA4 no .alpha.-amylase mRNA was detected and only very low levels of .alpha.-amylase were secreted. Nuclei were isolated in high yields (65-71%) from aleurone protoplasts and in an in vitro transcription system displayed characteristics of a faithful DNA-dependent RNA synthesizing system. The time course of incorporation of [3H]-UTP suggested that the RNA synthesized was mainly run off transcription and therefore that the transcripts produced in vitro were those being synthesized in the protoplasts at the times when the nuclei were isolated. By hybridizing in vitro synthesized [32P]RNA to barley .alpha.-amylase cDNA and control filters we have estimated that 90 .+-. 10 ppm of the transcripts synthesized by nuclei isolated from GA4 treated protoplasts can be attributed to .alpha.-amylase sequences and that statistically insignificant amounts of these transcripts are obtained from control and GA4 plus ABA treatments. The results suggest that GA4 and ABA influence the transcription of .alpha.-amylase genes in aleurone protoplasts of wild oat.