Chloroplast transcription at different light intensities. Glutathione-mediated phosphorylation of the major RNA polymerase involved in redox-regulated organellar gene expression

Previous, studies using purified RNA polymerase from mustard (Sinapis alba) chloroplasts showed control of transcription by an associated protein kina se. This kinase was found to respond to reversible thiol/disulfide formatio n mediated by glutathione (GSH), although at concentrations exceeding those thought to exist in vivo. In the present study, several lines of evidence are presented to substantiate the functioning of this regulation mechanism, also in vivo: (a) Studies on the polymerase-associated transcription kinas e revealed that at appropriate ATP levels, GSH concentrations similar to th ose in vivo are sufficient to modulate the kinase activity; (b) GSH measure ments from isolated mustard chloroplasts showed considerable differences in response to light intensity; (c) this was reflected by run-on transcriptio n rates in isolated chloroplasts that were generally higher if organelles w ere prepared from seedlings incubated under high-light as compared with gro wth-light conditions; (d) the notion of a general transcriptional switch wa s strengthened by in vitro experiments showing that the kinase not only aff ects the transcription of a photosynthetic gene (psbA) but also that of a n onphotosynthetic gene (trnQ); and (e) the polymerase-kinase complex reveale d specific differences in the phosphorylation state of polypeptides dependi ng on the light intensity to which the seedlings had been exposed prior to chloroplast isolation. Taken together, these data are consistent with GSH a nd phosphorylation-dependent regulation of chloroplast transcription in viv o.