Nucleoid-Enriched Proteomes in Developing Plastids and Chloroplasts from Maize Leaves: A New Conceptual Framework for Nucleoid Functions      

Abstract

Plastids contain multiple copies of the plastid chromosome, folded together with proteins and RNA into nucleoids. The degree to which components of the plastid gene expression and protein biogenesis machineries are nucleoid associated, and the factors involved in plastid DNA organization, repair, and replication, are poorly understood. To provide a conceptual framework for nucleoid function, we characterized the proteomes of highly enriched nucleoid fractions of proplastids and mature chloroplasts isolated from the maize (Zea mays) leaf base and tip, respectively, using mass spectrometry. Quantitative comparisons with proteomes of unfractionated proplastids and chloroplasts facilitated the determination of nucleoid-enriched proteins. This nucleoid-enriched proteome included proteins involved in DNA replication, organization, and repair as well as transcription, mRNA processing, splicing, and editing. Many proteins of unknown function, including pentatricopeptide repeat (PPR), tetratricopeptide repeat (TPR), DnaJ, and mitochondrial transcription factor (mTERF) domain proteins, were identified. Strikingly, 70S ribosome and ribosome assembly factors were strongly overrepresented in nucleoid fractions, but protein chaperones were not. Our analysis strongly suggests that mRNA processing, splicing, and editing, as well as ribosome assembly, take place in association with the nucleoid, suggesting that these processes occur cotranscriptionally. The plastid developmental state did not dramatically change the nucleoid-enriched proteome but did quantitatively shift the predominating function from RNA metabolism in undeveloped plastids to translation and homeostasis in chloroplasts. This study extends the known maize plastid proteome by hundreds of proteins, including more than 40 PPR and mTERF domain proteins, and provides a resource for targeted studies on plastid gene expression. Details of protein identification and annotation are provided in the Plant Proteome Database.

Effects of Reduced Chloroplast Gene Copy Number on Chloroplast Gene Expression in Maize

“…The strong visible phenotype of w2 mutants contrasts with those findings, and shows that w2 function is essential for chloroplast biogenesis. This is consistent with proteome studies, which have detected W2 but not its paralog in various chloroplast subfractions (Majeran et al, 2012).…”

“…The division of labor between organelle DNA polymerases differs in Arabidopsis, where a pair of paralogs function redundantly to support DNA replication in both organelles (Parent et al, 2011). W2 has been detected in proteome analyses of purified chloroplasts (Majeran et al, 2012), but the W2 paralog has not been detected in either chloroplasts or mitochondria. Our results are consistent with the possibility that W2 is the only source of DNA polymerase in chloroplasts, as the residual chloroplast DNA signal in the most severely affected samples (approximately 1% of normal in the apical section of w2-mum2 leaves) might arise from small revertant sectors.…”

New insights into plastid nucleoid structure and functionality

“…The TAC fraction is prepared from plastid membranes by release of the nucleoids with detergents and by subsequent purification involving sepharose gel filtration and other steps (Krause and Krupinska 2000;Pfalz et al 2006). Recent proteomic analyses of these (Pfalz et al 2006;Melonek et al 2012) and other nucleoid-enriched fractions (Phinney and Thelen 2005;Majeran et al 2012) have revealed that several of the newly identified DNA binding proteins were not inherited from the prokaryotic ancestors. Plastid proteins homologous to the abundant prokaryotic HU protein, on the other hand, were not detected in higher plants which stands in contrast to their identification in some algal plastids (Karcher et al 2009), in the apicoplasts of apicomplexans (Sato et al 2003) as well as in some dinoflagellates (Chan et al 2006).…”

“…Accordingly, core subunits of the plastid-encoded RNA polymerase, PEP, have been identified in most of the proteomic studies of plastid nucleoid fractions of dicots (i.e. 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).…”

“…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.…”