Analysis of barley chloroplast psbD light-responsive promoter elements in transplastomic tobacco

The plastid gene psbD encodes D2, a photosystem II reaction center chloroph yll-binding protein. psbD is transcribed from a conserved chloroplast promo ter 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 t o the uidA reporter gene and introduced into the tobacco (Nicotiana tabacum L.) plastid genome through homologous recombination. Primer extension anal ysis of transcripts from the psbD-LRP-uidA construct showed that the barley psbD-LRP was activated in tobacco by blue or white light. Transcription fr om this construct was also regulated by circadian cycling indicating that t he 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, mutatio n 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) di d not alter promoter activation by blue light, or responsiveness to circadi an cycling. These in vivo studies confirm the importance of the psbD-LRP -1 0 promoter element and AAG-box in light regulation and demonstrate that the se elements are sufficient to mediate circadian cycling of the barley psbD promoter.