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Eukaryotic-Type Plastid Nucleoid Protein Ptac3 Is Essential for Transcription by the Bacterial-Type Plastid RNA Polymerase

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Eukaryotic-Type Plastid Nucleoid Protein Ptac3 Is Essential for Transcription by the Bacterial-Type Plastid RNA Polymerase Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase Yusuke Yagia,b,c, Yoko Ishizakic, Yoichi Nakahirac, Yuzuru Tozawad, and Takashi Shiinac,1 aFaculty of Agriculture and bInstitute of Advanced Study, Kyushu University, Fukuoka 812-8581, Japan; cGraduate School of Life and Environmental Science, Kyoto Prefectural University, Kyoto 606-8522, Japan; and dCell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan Edited by André T. Jagendorf, Cornell University, Ithaca, NY, and approved April 3, 2012 (received for review November 24, 2011) Plastid transcription is mediated by two distinct types of RNA pol- Thus, two types of RNAP have distinct roles in chloroplast ymerases (RNAPs), bacterial-type RNAP (PEP) and phage-type transcription in higher plants. RNAP (NEP). Recent genomic and proteomic studies revealed that It has been proposed that light-dependent initiation of tran- higher plants have lost most prokaryotic transcription regulators scription by PEP is controlled by light-induced expression of and have acquired eukaryotic-type proteins during plant evolu- nuclear-encoded plastid σ-factors (6). Contrarily, several evi- tion. However, in vivo dynamics of chloroplast RNA polymerases dences suggest that phosphorylated PEP may tightly bind to the and eukaryotic-type plastid nucleoid proteins have not been di- promoter region to arrest transcription in the dark (9–11). Dark- rectly characterized experimentally. Here, we examine the associ- induced phosphorylation of PEP and/or σ-factors by redox-reg- ation of the α-subunit of PEP and eukaryotic-type protein, plastid ulated plastid transcription kinase (PTK) may be a key step in transcriptionally active chromosome 3 (pTAC3) with transcribed light-dependent plastid gene transcription. Thus, molecular regions in vivo by using chloroplast chromatin immunoprecipita- mechanism of light-dependent transcription in chloroplasts still tion (cpChIP) assays. PEP α-subunit preferentially associates with remains controversial. PEP promoters of photosynthesis and rRNA genes, but not with Plastid DNAs are densely packed into protein–DNA com- NEP promoter regions, suggesting selective and accurate recogni- plexes called “plastid nucleoids,” as well as bacterial nucleoids. tion of PEP promoters by PEP. The cpChIP assays further demon- However, higher plants and moss have lost prokaryotic major PLANT BIOLOGY strate that the peak of PEP association occurs at the promoter- nucleoid proteins including Hu during evolution (12, 13), proximal region and declines gradually along the transcribed whereas several eukaryotic-type proteins such as PEND (14), region. pTAC3 is a putative DNA-binding protein that is localized to MFP1 (15), SiR (16), and CND41 (17) have been identified as chloroplast nucleoids and is essential for PEP-dependent transcrip- major components of nucleoids in higher plants. Furthermore, tion. Density gradient and immunoprecipitation analyses of PEP recent proteome analysis identified eukaryotic-type chloroplast revealed that pTAC3 is associated with the PEP complex. Interest- nucleoid proteins including putative DNA/RNA-binding pro- ingly, pTAC3 associates with the PEP complex not only during teins as components of a chloroplast-derived DNA–protein transcription initiation, but also during elongation and termina- complex termed pTAC (plastid transcriptionally active chromo- tion. These results suggest that pTAC3 is an essential component some) (18) and a blue native (BN)-PAGE separated basic PEP of the chloroplast PEP complex. In addition, we demonstrate that complex (19). These findings suggest that chloroplasts have lost light-dependent chloroplast transcription is mediated by light-in- most of prokaryotic nucleoid proteins involved in DNA packag- duced association of the PEP–pTAC3 complex with promoters. This ing, replication, transcription, and translation and acquired study illustrates unique dynamics of PEP and its associated protein eukaryotic-type chloroplast nucleoid proteins during evolution pTAC3 during light-dependent transcription in chloroplasts. (20). Because vascular plants lack prokaryotic transcription reg- ulators such as DNA-binding proteins and transcription elonga- lastids are DNA-containing organelles unique to plant cells tion factors except for σ-factors, chloroplast transcription might Pand are thought to have originated from an ancestral cya- be mediated by a unique hybrid system of prokaryotic-type RNA nobacterial endosymbiont. Whereas cyanobacteria contain over polymerase and eukaryotic-type accessory factors, However, the 3,000 genes, the plastid genome in higher plants consists of small, role of the nonprokryotic nucleoid proteins in plastid transcrip- circular, double-stranded DNA (120–150 kbp) encoding ∼120 tion remains largely unknown. genes for photosynthesis and gene expression machineries (1, 2), Earlier studies on pTAC proteins and PEP-associated proteins in planta indicating massive transfer of chloroplast genes to nuclear ge- (PAPs) have focused mainly on analyses using mutant nome during evolution (3). Vascular plants have evolved a com- and transgenic plants [e.g., pTAC2, pTAC6, and pTAC12 (18); plex transcriptional network that is mediated by two types of PAP3/pTAC10, PAP6/FLN1, and PAP7/pTAC14 (19); ET1 (21); RNA polymerases (RNAPs): cyanobacterium-derived plastid- Trx-z (22)]. However, mutations in plastid transcription-related encoded plastid RNA polymerase (PEP) and nuclear-encoded genes occasionally give rise to drastic pleiotropic phenotypes such as albino or pale green plants due to reduced accumulation phage-type RNA polymerase (NEP). PEP is composed of four of plastid rRNA and tRNAs, which are mainly transcribed by catalytic subunits and a promoter recognition subunit, σ-factor α β β′ β′′ PEP, and impaired translation of chloroplast-encoded essential (4). Genes for PEP core subunits, , , , and were retained proteins. Thus, it is sometimes difficult to characterize the in plastid genomes as rpoA, rpoB, rpoC1, and rpoC2 during plant evolution, but genes for σ-factors involved in transcription ini- tiation, have been transferred to the nuclear genome (5), which Author contributions: Y.Y., Y.N., and T.S. designed research; Y.Y., Y.I., Y.N., and Y.T. allows the nucleus to control PEP transcription initiation in performed research; Y.Y. analyzed data; and Y.Y. and T.S. wrote the paper. response to developmental and environmental cues (recently The authors declare no conflict of interest. reviewed in ref. 6). PEP is responsible for transcription of pho- This article is a PNAS Direct Submission. tosynthesis genes in chloroplast in response to light. On the other 1To whom correspondence should be addressed. E-mail: [email protected]. hand, housekeeping genes encoding PEP core subunits and ri- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. bosomal proteins are transcribed by the phage-type NEP (7, 8). 1073/pnas.1119403109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1119403109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 molecular function of pTAC proteins in chloroplast transcription dependent association of the PEP–pTAC3 complex with PEP using null mutants. Chromatin immunoprecipitation (ChIP) promoters. This study provides unique insight into the role of assays have been widely used to study protein–DNA interactions the eukaryotic-type PEP accessory protein pTAC3 in PEP- and map the localization of proteins to specific DNA sequences dependent transcription in chloroplasts. in the cell nucleus and bacterial nucleoids. However, very little work has been done using ChIP to analyze the association of Results PEP and its accessory proteins with transcribed regions in pTAC3 Is a Land Plant-Specific Protein That Is Localized to Plastid chloroplast genomes. Here, we examine the association of the Nucleoids. To investigate whether pTAC3 is an evolutionally ac- α-subunit of PEP and pTAC3 with transcribed regions in vivo quired component of plastid transcription, we examined its evo- fi by using chloroplast chromatin immunoprecipitation (cpChIP) lutionary history. BLAST and position-speci c iterative (PSI)- assays. A pTAC3 has been identified in various PEP-containing BLAST searches revealed that pTAC3 is conserved among land plants including moss and higher plants, but not in cyanobacteria chloroplast fractions (18, 19, 23, 24). The pTAC3 contains a SAP and algae (Fig. 1B and Fig. S1), suggesting a functional conser- DNA-binding domain (Pfam 02037), which is found in matrix vation in embryophytes. TargetP and PSORT, subcellular locali- attachment region binding protein (25) (Fig. 1A). In this study, zation prediction programs, suggest that AtpTAC3 (At3g04260) cpChIP assays demonstrate that pTAC3 associates with the PEP contains a putative transit peptide of 29 amino acid residues at the complex not only during transcription initiation, but also during N terminus. To examine the subcellular localization of pTAC3, we elongation and termination. In addition, we demonstrate light- expressed GFP-tagged pTAC3 in Arabidopsis. Full-length Atp- TAC3 cDNA was fused to the GFP coding region and transiently expressed under the control of the constitutive CaMV35S pro- A B moter in protoplasts prepared from Arabidopsis mesophyll cells Vv (Fig. 1C). It has been reported that GFP-fused plastid DNA- Pt Gm binding proteins such as PEND localized to plastid nucleoids :100aa Rc Bd (26). As shown
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