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.

Figure 1.

Comparison of subunit composition of the plastid RNA polymerase from mustard after 2D BN-PAGE and glycerol gradient centrifugation. A, Purification schemes and resulting proteins. B, PEP subunit composition obtained by 2D BN-PAGE (left, large gel, 7%–17%) and SDS-PAGE after glycerol gradient centrifugation (right, mini gel, 5%–15%). Two representative gels are shown. Total protein (150 μg) was separated and fixed, and proteins were stained with silver. Running directions of the first and second dimensions are indicated by arrows. Sizes of marker proteins separated in parallel on the same gel are given in the margins. Single subunits within the PEP complexes that gave significant hits in the databases are indicated by consecutive numbering. Corresponding proteins within the two preparations are connected by lines. Asterisks mark proteins not reproducibly found in the complexes. For identity and detailed data of mass spectrometry, see Table I and Supplemental Table S1. MALDI, Matrix-assisted laser-desorption ionization. [See online article for color version of this figure.]

Figure 2.

Analysis of the novel β′-s subunit. A, Detected peptides of the two β′-subunits. Peptides identified by mass spectrometry are given within the RpoC₁ amino acid sequence. Gray background, peptides identified in both subunits. Overlaps between neighboring peptides are indicated in dark gray. Gray background underlined, peptides of β′-s. Black background with white letters, peptide solely identified in the β′-l subunit. Positions of the spliced intron (triangle), the editing site (underlined S), and the proposed alternative splice acceptor site (arrow) are indicated. B, Results of RT-PCR analysis and corresponding gene models indicating potential splice variants of rpoC₁. Left panel, ethidium bromide-stained RT-PCR products using Arabidopsis and Sinapis cDNA and rpoC₁-specific primers. The top band represents a product generated from the genomic DNA, as depicted in the right panel. The bottom band represents a product generated from the spliced RNA. Right panel, gene model and splice variants. The coding sequence is given as the thick black bar and the intron as the thin black bar. A potential alternatively spliced area is given as the hatched bar.

Figure 3.

Identification and analysis of homozygous pap3/ptac10, pap6/fln1, and pap7/ptac14 T-DNA insertion mutants. A, Schematic presentation of the corresponding genes showing the positions of the T-DNA insertions as confirmed by PCR and subsequent sequencing. B, RT-PCR amplification of the pap3/ptac10, pap6/fln1, and pap7/ptac14 genes using gene-specific primers given in A. Lines homozygous for pap3/ptac10, pap6/fln1, and pap7/ptac14 T-DNA insertion fail to express the wild-type (WT) allele. Asterisks indicate bands derived by genomic DNA. C, Wild-type and homozygous pap3/ptac10, pap6/fln1, and pap7/ptac14 plants germinated on petri dishes with medium. Seeds of the T-DNA insertion line were surface sterilized and placed on sterile agar plates containing Murashige and Skoog medium supplemented with 2% Suc.

Figure 4.

Checkpoint model describing the reconfiguration of the plastid RNA polymerase complex as an essential step in plastid development. The scheme depicts the structural assembly of the plastid-encoded RNA polymerase, starting with the expression of the rpo genes by the NEP enzyme, resulting in the formation of the basic PEP-B enzyme. Interaction with σ-factors is assumed. Upon initiation of photomorphogenesis, it is modified first by posttranslational changes of rpo subunits (via unknown modifying enzymes) and second by the addition of PAPs, generating the structurally more complex PEP-A. White arrows indicate the flow of events required for PEP-A buildup. Thin black arrows indicate the action or involvement of nucleus-encoded proteins delivered in a fixed sequence that follows a distinct developmental program in the nucleus. Thick black arrows indicate transcription activity. Dotted lines indicate the possible impact of a PAP gene knockout in the nucleus on PEP-A. The lacking subunit is indicated by a cross, its inhibitory feedback by dotted lines. Numbers refer to discussed possibilities causing the observed phenotypes of PAP knockout mutants. For further details, see text.