Characterization of the barley chloroplast transcription units containing psaA-psaB and psbD-psbC.

Four plastid genes, psaA, psaB, psbD and psbC, were localized on the barley plastid genome. PsaA was adjacent to psaB in one transcription unit and psbD was adjacent to psbC in a second transcription unit. The transcription units containing psaA-psaB and psbD-psbC are separated by approximately 25 kbp on the barley plastid genome and are transcribed convergently. Transcripts hybridizing to each transcription unit were characterized by northern blot analysis, S1 protection experiments and primer extension analysis. Two 5.3 kb transcripts hybridize to psaA-psaB. The two transcripts have a common 5' end but differ at their 3' ends by about 26 nucleotides. The transcription unit which contains psbD-psbC also includes trnS(UGA), trnG(GCC), and an open reading frame which codes for a 62 amino acid protein. Six large transcripts ranging from 5.7 kb to 1.7 kb hybridize to the psbD-psbC transcription unit as well as several RNAs of tRNA size. The large transcripts arise from three 5' ends and two clusters of 3' ends. The 3' ends map near trnG(GCC) and trnS(UGA) and could be generated by RNA processing or termination of transcription. Two of the six transcripts hybridize to psbC but not psbD suggesting that translation of psbD and psbC could occur on separate RNAs

The role of RpoT;3 in chloroplast development and gene expression

The real-time PCR assay method was used to quantify the RNA abundance of twenty-eight plastid genes in a range of tissues and developmental stages of Arabidopsis thaliana. Three groups of co-regulated genes were identified. Three trn genes (Cluster I) showed differential expression in siliques. Genes encoding components of the plastid transcription and translation apparatus, the energetic apparatus as well as two genes encoding components of the plastid protease and acetyl-CoA carboxylase, showed maximum transcript accumulation at the 2-day stage (Cluster IIA and IIB). Finally, the genes encoding components of the photosynthetic apparatus of Cluster III reached maximum transcript abundance in later stages of chloroplast development. This coordinated expression of plastid genes reflects the presence of regulatory mechanisms that modulate plastid gene expression in different plant tissues and developmental stages. We identified an Arabidopsis mutant, rpoZ191, in which a T-DNA is inserted in the RpoT;3 gene encoding the plastid-targeted phage-type NEP enzyme. The mutant displays a general reduction in growth, as well as a delay in greening and in chloroplast development. Real-time PCR analysis of plastid RNA accumulation showed that the RpoT;3 mutation caused a significant decrease in plastid transcript accumulation at the 2-day stage and a smaller inhibition at the 5-day stage. No major effects of the RpoT;3 mutation on the accumulation of plastid transcripts was observed in mature seeds and 5-day roots. Additionally, plastid transcript accumulation in mutant siliques was not significantly different from the wild-type, except for trnfM-CAU and trnW-CCA, which showed enhanced transcript levels. Taken together, these data indicate that the RpoT;3 NEP enzyme plays an important role in the overall transcription of plastid genes during the early phases of chloroplast and leaf differentiation. Furthermore, a functional RpoT;3 is required for the activation of selected nucleus-encoded plastid-localized proteins. However, the enhanced activity of RpoT;3 during the early stages of chloroplast differentiation is not due to an increase in RpoT;3 mRNA abundance. We suggest that post-transcriptional mechanisms (e.g. phosphorylation, specificity factors) activate the transcription of plastid RpoT;3 - transcribed genes during the early stages of plastid development