SLO2, a mitochondrial pentatricopeptide repeat protein affecting several RNA editing sites, is required for energy metabolism

Pentatricopeptide repeat (PPR) proteins belong to a family of approximately 450 members in Arabidopsis, of which few have been characterized. We identified loss of function alleles of SLO2, defective in a PPR protein belonging to the E+ subclass of the P-L-S subfamily. slo2 mutants are characterized by retarded leaf emergence, restricted root growth, and late flowering. This phenotype is enhanced in the absence of sucrose, suggesting a defect in energy metabolism. The slo2 growth retardation phenotypes are largely suppressed by supplying sugars or increasing light dosage or the concentration of CO(2) . The SLO2 protein is localized in mitochondria. We identified four RNA editing defects and reduced editing at three sites in slo2 mutants. The resulting amino acid changes occur in four mitochondrial proteins belonging to complex I of the electron transport chain. Both the abundance and activity of complex I are highly reduced in the slo2 mutants, as well as the abundance of complexes III and IV. Moreover, ATP, NAD+, and sugar contents were much lower in the mutants. In contrast, the abundance of alternative oxidase was significantly enhanced. We propose that SLO2 is required for carbon energy balance in Arabidopsis by maintaining the abundance and/or activity of complexes I, III, and IV of the mitochondrial electron transport chain.     

高等植物叶绿体RNA编辑研究进展

RNA编辑普遍存在于陆生植物中,在高等植物叶绿体中以C→U的替换为主,可能是叶绿体产生功能蛋白的重要方式。近年来,使用体外分析、叶绿体转化和紫外交联等技术,使叶绿体RNA编辑机制的研究取得较大进展。本文对这些新的进展进行了概述,并对高等植物叶绿体RNA编辑研究中有待解决的问题进行了展望。     

The pentatricopeptide repeat gene OTP51 with two LAGLIDADG motifs is required for the cis-splicing of plastid ycf3 intron 2 in Arabidopsis thaliana

Summary The Arabidopsis thaliana chloroplast contains 20 group-II introns in its genome, and seven known splicing factors are required for the splicing of overlapping subsets of 19 of them. We describe an additional protein (OTP51) that specifically promotes the splicing of the only group-II intron for which no splicing factor has been described previously. This protein is a pentatricopeptide repeat (PPR) protein containing two LAGLIDADG motifs found in group-I intron maturases in other organisms. Amino acids thought to be important for the homing endonuclease activity of other LAGLIDADG proteins are missing in this protein, but the amino acids described to be important for maturase activity are conserved. OTP51 is absolutely required for the splicing of ycf3 intron 2, and also influences the splicing of several other group-IIa introns. Loss of OTP51 has far-reaching consequences for photosystem-I and photosystem-II assembly, and for the photosynthetic fluorescence characteristics of mutant plants.     

The evolution of chloroplast RNA editing

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.     

OsPPR1, a pentatricopeptide repeat protein of rice is essential for the chloroplast biogenesis

In this paper, we report a novel pentatricopeptide repeat (PPR) protein gene in rice. PPR, a characteristic repeat motif consisted of tandem 35 amino acids, has been found in various biological systems including plant. Sequence analysis revealed that the gene designated OsPPR1 consisted of an open reading frame of 2433 nucleotides encoding 810 amino acids that include 11 PPR motifs. Blast search result indicated that the gene did not align with any of the characterized PPR genes in plant. The OsPPR1 gene was found to contain a putative chloroplast transit peptide in the N-terminal region, suggesting that the gene product targets to the chloroplast. Southern blot hybridization indicated that the OsPPR1 is the member of a gene family within the rice genome. Expression analysis and immunoblot analysis suggested that the OsPPR1 was accumulated mainly in rice leaf. Antisense transgenic strategy was used to suppress the expression of OsPPR1 and the resulted transgenic rice showed the typical phenotypes of chlorophyll-deficient mutants; albinism and lethality. Cytological observation using microscopy revealed that the antisense transgenic plant contained a significant defect in the chloroplast development. Taken together, the results suggest that the OsPPR1 is a nuclear gene of rice, encoding the PPR protein that might play a role in the chloroplast biogenesis. This is the first report on the PPR protein required for the chloroplast biogenesis in rice.     

The RanBP2 zinc finger domains of chloroplast RNA editing factor OZ1 are required for protein–protein interactions and conversion of C to U

In the chloroplast, organelle zinc finger 1 (OZ1) is a RanBP2-type zinc finger (Znf) protein required for many RNA editing events, a process by which specific cytosines are enzymatically converted to uracils as a correction mechanism for missense mutations in the organelle genomes. RNA editing is carried out by a large multi-protein complex called the ‘editosome’ that contains members of the pentatricopeptide repeat (PPR) protein family, the RNA editing factor interacting protein (also known as MORF) family and the organelle RNA-recognition motif (ORRM) family, in addition to OZ1. OZ1 is an 82-kDa protein with distinct domains, including a pair of Znf domains and a unique C-terminal region. To elucidate the functions of these domains, we have generated truncations of OZ1 for use in protein–protein interaction assays that identified the C-terminal region of OZ1, as well as the Znf domains as the primary interactors with PPR proteins, which are factors required for site-specificity and enzymatic editing. Expression of these OZ1 truncations in vivo showed that the Znf domains were required to restore chloroplast RNA editing in oz1 knockout plants. Mutation of key structural residues in the Znf domains showed that they are necessary for editing and required for interaction with ORRM1, a general editing factor with an RNA-binding domain. These functional characterizations of the Znfs and novel C-terminal domain contribute to our understanding of the model for the chloroplast plant editosome.