pTAC10, an S1-domain-containing component of the transcriptionally active chromosome complex, is essential for plastid gene expression in Arabidopsis thaliana and is phosphorylated by chloroplast-targeted casein kinase II

摘要

In higher plant chloroplasts, the plastid-encoded RNA polymerase (PEP) consists of four catalytic subunits and numerous nuclear-encoded accessory proteins, including pTAC10, an S1-domain-containing protein. In this study, pTAC10 knockout lines were characterized. Two ptac10 mutants had an albino phenotype and severely impaired chloroplast development. The pTAC10 genomic sequence fused to a four-tandem MYC tag driven by its own promoter functionally complemented the ptac10-1 mutant phenotype. pTAC10 was present in both the chloroplast stroma and thylakoids. Two-dimensional blue native polyacrylamide gel electrophoresis (BN-PAGE), and immunoblotting assays showed that pTAC10:MYC co-migrates with one of the PEP core subunits, RpoB. A comprehensive investigation of the plastid gene expression profiles by quantitative RT-PCR revealed that, compared with wild-type plants, the abundance of PEP-dependent plastid transcripts is severely decreased in the ptac10-1 mutant, while the amount of plastid transcripts exclusively transcribed by NEP either barely changes or even increases. RNA blot analysis confirmed that PEP-dependent chloroplast transcripts, including psaB, psbA and rbcL, substantially decrease in the ptac10-1 mutant. Immunoblotting showed reduced accumulation of most chloroplast proteins in the ptac10 mutants. These data indicate the essential role of pTAC10 in plastid gene expression and plastid development. pTAC10 interacts with chloroplast-targeted casein kinase 2 (cpCK2) in vitro and in vivo and can be phosphorylated by Arabidopsis cpCK2 in vitro at sites Ser95, Ser396 and Ser434. RNA-EMSA assays showed that pTAC10 is able to bind to the psbA, atpE and accD transcripts, suggesting a non-specific RNA-binding activity of pTAC10. The RNA affinity of pTAC10 was enhanced by phosphorylation and decreased by the amino acid substitution Ser434-Ala of pTAC10. These data show that pTAC10 is essential for plastid gene expression in Arabidopsis and that it can be phosphorylated by cpCK2.