In vitro analysis of light-induced transcription in the wheat psbD/C gene cluster using plastid extracts from dark-grown and short-term-illuminated seedlings

Abstract

We describe a plastid in vitro transcription system that reflects characteristic features of the light-regulated transcription observed in vivo. Multiple transcripts of the wheat (Triticum aestivum) psbD/ C gene cluster comprise six distinct 5′ ends including four transcription initiation sites designated as D/C-1 through D/C-4. Transcripts from one particular site, D/C-3, were found to be conspicuously enhanced in abundance after 4 h of illumination in vivo. The plastid extract prepared from 5-d-old dark-grown wheat seedlings was capable of transcribing from the D/C-2 and D/C-4 sites in vitro but had almost no transcription activity from the light-responsive D/C-3 site (the D/C-1 site was not examined). The plastid extract from 4-h-illuminated seedlings initiated transcription from the light-responsive site (D/C-3). Transcription from the D/C-2 and D/C-4 sites was not enhanced by using the extract from 4-h-illuminated seedlings, indicative of specific activation of the light-responsive promoter on the D/C-3 site by the extract from 4-h-illuminated seedlings. The plastid extract from 4-h-illuminated seedlings was divided into two fractions on a heparin-Sepharose column, into which the light-induced component(s) responsible for activation of the D/C-3 promoter and RNA polymerase were separated. The fraction containing the component(s) activating the D/ C-3 promoter induced the transcription activity from the D/C-3 site in the plastid extract from dark-grown seedlings. It is concluded that the plastid extract from 4-h-illuminated seedlings contains some light-regulatory component(s) that activate specifically the light-responsive promoter.