Analysis of Barley Chloroplast PsbD Light-responsive Promoter Elements in Transplastomic Tobacco

The plastid gene psbD encodes D2, a photosystem II reaction center chlorophyll-binding protein. psbD is transcribed from a conserved chloroplast promoter that is activated by blue, white, or UV-A light. In this study, various forms of the barley (Hordeum vulgare L.) chloroplast psbD-LRP were fused to the uidA reporter gene and introduced into the tobacco (Nicotiana tabacum L.) plastid genome through homologous recombination. Primer extension analysis of transcripts from the psbD-LRP-uidA construct showed that the barley psbD-LRP was activated in tobacco by blue or white light. Transcription from this construct was also regulated by circadian cycling indicating that the barley psbD-LRP could respond to light modulated regulatory pathways in tobacco. Mutation of the psbD-LRP prokaryotic -10 promoter element reduced transcription to very low levels in all light regimes. In contrast, mutation of a prokaryotic -35 promoter element had no effect on transcription from the psbD-LRP. Deletion or mutation of an upstream activating element, the AAG-box (-36 to -64), also reduced transcription from the construct to very low levels. In contrast, deletion of the upstream PGT-box (-71 to -100) did not alter promoter activation by blue light, or responsiveness to circadian cycling. These in vivo studies confirm the importance of the psbD-LRP -10 promoter element and AAG-box in light regulation and demonstrate that these elements are sufficient to mediate circadian cycling of the barley psbD promoter.