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.

‘Whirly’ proteins comprise a plant-specific protein family whose members have been described as DNA-binding proteins that influence nuclear transcription and telomere maintenance, and that associate with nucleoids in chloroplasts and mitochondria. We identified the maize WHY1 ortholog among proteins that coimmunoprecipitate with CRS1, which promotes the splicing of the chloroplast atpF group II intron. ZmWHY1 localizes to the chloroplast stroma and to the thylakoid membrane, to which it is tethered by DNA. Genome-wide coimmunoprecipitation assays showed that ZmWHY1 in chloroplast extract is associated with DNA from throughout the plastid genome and with a subset of plastid RNAs that includes atpF transcripts. Furthermore, ZmWHY1 binds both RNA and DNA in vitro. A severe ZmWhy1 mutant allele conditions albino seedlings lacking plastid ribosomes; these exhibit the altered plastid RNA profile characteristic of ribosome-less plastids. Hypomorphic ZmWhy1 mutants exhibit reduced atpF intron splicing and a reduced content of plastid ribosomes; aberrant 23S rRNA metabolism in these mutants suggests that a defect in the biogenesis of the large ribosomal subunit underlies the ribosome deficiency. However, these mutants contain near normal levels of chloroplast DNA and RNAs, suggesting that ZmWHY1 is not directly required for either DNA replication or for global plastid transcription.

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Comparative genomics has provided evidence for numerous conserved protein domains whose functions remain unknown. We identified a protein harboring ''domain of unknown function 860'' (DUF860) as a component of group II intron ribonucleoprotein particles in maize chloroplasts. This protein, assigned the name WTF1 (''what's this factor?''), coimmunoprecipitates from chloroplast extract with group II intron RNAs, is required for the splicing of the introns with which it associates, and promotes splicing in the context of a heterodimer with the RNase III-domain protein RNC1. Both WTF1 and its resident DUF860 bind RNA in vitro, demonstrating that DUF860 is a previously unrecognized RNA-binding domain. DUF860 is found only in plants, where it is represented in a protein family comprising 14 orthologous groups in angiosperms. Most members of the DUF860 family are predicted to localize to chloroplasts or mitochondria, suggesting that proteins with this domain have multiple roles in RNA metabolism in both organelles. These findings add to emerging evidence that the coevolution of nuclear and organellar genomes spurred the evolution of diverse noncanonical RNA-binding motifs that perform organelle-specific functions.DUF860 ͉ mitochondria ͉ plastid T he evolution of mitochondria and chloroplasts from bacterial endosymbionts was accompanied by large scale transfer of genes to the nucleus (1). Accordingly, many organellar proteins are encoded by nuclear genes of bacterial ancestry that retain their ancestral function. However, during the long coevolution of mitochondria and chloroplasts with their host cell, both organelles acquired features that are not typical of their bacterial ancestors. The origin of the genes that confer such traits is only beginning to be elucidated.The complex RNA metabolism characteristic of plant mitochondria and chloroplasts provides striking examples of acquired, nonprocaryotic traits. For example, both organellar genomes are rich in introns, RNAs in both organelles are modified by RNA editing, and posttranscriptional events have the predominant role in determining gene product abundance (2, 3-5). Many proteins that participate in such processes were not derived from the endosymbiont, but rather emerged in the context of nuclear-organellar coevolution. For example, the pentatricopeptide repeat (PPR) protein family is found only in eucaryotes, where it has been implicated in RNA-related processes in mitochondria and chloroplasts (6). Current data argue that PPR proteins generally function as RNA-interaction platforms, but they appear to be derived from the tetratricopeptide repeat (TPR) motif, a more ancient motif that binds protein ligands.Here, we present evidence that a previously uncharacterized protein family defined by ''domain of unknown function 860'' (DUF860) fits this general paradigm. We show that the DUF860 protein WTF1 (''what's this factor?'') is required for the splicing of group II introns in chloroplasts, that it associates in vivo with its genetically-defined RNA ligands, and tha...