PTAC7 Regulates Chloroplast Gene Expression In Arabidopsis Thaliana

Chloroplast is a semiautonomous organelle, which contains the DNA genetic material. Chloroplast gene expression in higher plant cells involves at least two types of RNA polymerases: a plastid-encoded bacterial-type RNA polymerase(PEP), and a nucleusencoded phage-type RNA polymerase(NEP). PEP is the main RNA polymerase of chloroplast, is composed of four subunit, α, β, β’, β’’, and numerous associated proteins. Its regulatory mechanism is very complex, as well as the functions of most components remain unclear.pTAC7 is one of PEP complex. Two knockout mutants of pTAC7, ptac7-1 and ptac7-2, were previously identified in our lab, and the two null pTAC7 mutants displayed the blockage of thylakoid formation, thus were seedling lethal. The transcriptional levels of all investigated PEP-dependent chloroplast genes were obviously decreased compared with those of wild type.Thus, pTAC7 is important for chloroplast gene expression.In this discussion, the genomics of pTAC7 fused MYC and GFP tag can successfully complement the ptac7-2 mutant, respectively. These results indicated that the two fusion proteins are functional. The pTAC7: GFP fluorescence was present in chloroplast, which is merged with chlorophyll, through a laser confocal microscope to observation, demonstrating pTAC7 protein lacated to chloroplast. Inact chloroplasts, thylakoid and strom from the pTAC7:MYC and pTAC7:GFP transgenic plants and the protein were subjected to western blotting analysis. The results showed that pTAC7 was localized in chloroplast and mainly associated with thylakoid. Immunoprecipitation and mass spectrometry in pTAC7: MYC plants, a total of 120 chloroplast-localization were indentified. Including the the core of PEP, rpoA,rpoB,rpoC1,rpoC2 and other PAPs components, pTAC2, pTAC3, pTAC10, pTAC6, pTAC14, Trxz, FLN1, FLN2, FSD2. Thylakoid membrane proteins from pTAC7:MYC transgenic plants were fractionated by BN-PAGE in the first dimension and by SDS-PAGE in the second dimension. It showed that pTAC7 located in the complex of 669 kD, which merged with rpoB. These results indicated that pTAC7 was another PAP proteins, which was closely to the core subunits. In addition, besides to PEP complex components, we also fond other proteins in chloroplast, seven ribosomal proteins, two molecular chaperones and two PPR proteins participated in procession of plastid transcription. These proteins may be as the candidate proteins interacting with pTAC7. This paper provides a theoretical basis for further study of pTAC7.