Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development      

Abstract

The major RNA polymerase activity in mature chloroplasts is a multisubunit, Escherichia coli-like protein complex called PEP (for plastid-encoded RNA polymerase). Its subunit structure has been extensively investigated by biochemical means. Beside the "prokaryotic" subunits encoded by the plastome-located RNA polymerase genes, a number of additional nucleus-encoded subunits of eukaryotic origin have been identified in the PEP complex. These subunits appear to provide additional functions and regulation modes necessary to adapt transcription to the varying functional situations in chloroplasts. However, despite the enormous progress in genomic data and mass spectrometry techniques, it is still under debate which of these subunits belong to the core complex of PEP and which ones represent rather transient or peripheral components. Here, we present a catalog of true PEP subunits that is based on comparative analyses from biochemical purifications, protein mass spectrometry, and phenotypic analyses. We regard reproducibly identified protein subunits of the basic PEP complex as essential when the corresponding knockout mutants reveal an albino or pale-green phenotype. Our study provides a clearly defined subunit catalog of the basic PEP complex, generating the basis for a better understanding of chloroplast transcription regulation. In addition, the data support a model that links PEP complex assembly and chloroplast buildup during early seedling development in vascular plants.

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

“…Arsova et al demonstrated that Trx-z binds to two fructokinase-like proteins (FLNs) in a thiol-dependent manner, which is essential for plastid-encoded RNA polymerase (PEP)-dependent gene expression and proper chloroplast development (42). In accordance with this report, Trx-z and FLNs were identified as PEP complex components by proteomic studies (44,45). All these results let us hypothesize that the NADPH/NTRC/Trx-z redox cascade acts as a redoxbased switch of PEP-dependent gene expression in chloroplasts, although Trx-z likely receives reducing power from other redoxmediator proteins ( Fig.…”

A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression

“…However, its function in the chloroplast still remains unknown. Both pTAC12 and pTAC14 are shown to be essential components of the PEP complex (Steiner et al, 2011). In this work, our results demonstrated that pTAC14 is directly associated with pTAC12 in the chloroplast.…”

New insights into plastid nucleoid structure and functionality

“…Interestingly, only one sigma factor (Sigma 2) could be identified in the nucleoid preparations performed with maize seedlings (Majeran et al 2012) while no sigma factors were detected in preparations of the soluble RNApolymerase fraction (Suzuki et al 2004;Schröter et al 2010) including fractions enriched in PEP (Steiner et al 2011). The reason for this failure might be that the proteins escaped detection due to very low abundance.…”

“…Arabidopsis, mustard, spinach, pea) (Phinney and Thelen 2005;Pfalz et al 2006;Melonek et al 2012) as well as monocots (i.e., maize) (Majeran et al 2012). A preparation, where only the core PEP and very tightly associated subunits were preserved, contained an additional ten proteins that were termed PEP-associated proteins (for PAPs) (Steiner et al 2011). These PAPs seem to be closely associated accessory subunits of the PEP core.…”

“…Considering the tight association of nucleoids and thylakoids, a direct perception of redox changes in the photosynthetic apparatus by redox sensitive proteins in the nucleoids as also reported for a soluble RNA polymerase preparation from chloroplasts (Schröter et al 2010) must be expected. Sato and Ohta 2001) Unknown (Terasawa and Sato 2009) MFP1 At3g16000 Anchorage to thylakoid membranes (Jeong et al 2003) Chromatin attachment to the nuclear envelope (Meier et al 1996) Whirly1 At1g14410 SsDNA and RNA binding; recombination (Marechal and Brisson 2010;Melonek et al 2010;Pfalz et al 2006;Prikryl et al 2008) Transcription factor and telomere homeostasis (Desveaux et al 2000;Yoo et al 2007) SWIB-4 At3g03590 Unknown (Melonek et al 2012) Component of SWI/SNF chromatin remodeling complexes (Melonek et al 2012) pTAC12/ HEMERA/ PAP5 At2g34640 Component of the plastid transcriptional apparatus, subunit of plastid-encoded RNA-polymerase complex (Gao et al 2011;Pfalz et al 2006;Steiner et al 2011) Phytochrome signaling (Chen et al 2010) SIB1 ? 2 a At3g56710…”