Influence of mitochondrial genome rearrangement on cucumber leaf carbon and nitrogen metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO2 assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH4+ assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu

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

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 th

Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria

RNA editing is converting hundreds of cytosines into uridines during organelle gene expression of land plants. The pentatricopeptide repeat (PPR) proteins are at the core of this posttranscriptional RNA modification. Even if a PPR protein defines the editing site, a DYW domain of the same or another PPR protein is believed to catalyze the deamination. To give insight into the organelle RNA editosome, we performed tandem affinity purification of the plastidial CHLOROPLAST BIOGENESIS 19 (CLB19) PPR editing factor. Two PPR proteins, dually targeted to mitochondria and chloroplasts, were identified as potential partners of CLB19. These two proteins, a P-type PPR and a member of a small PPR-DYW subfamily, were shown to interact in yeast. Insertional mutations resulted in embryo lethality that could be rescued by embryo-specific complementation. A transcriptome analysis of these complemented plants showed major editing defects in both organelles with a very high PPR type specificity, indicating that the two proteins are core members of E+-type PPR editosomes

RNA editing in mitochondrial trans-introns is required for splicing

In plant mitochondria, gene expression of translatable mRNAs is a complex process with two critical steps, RNA editing and splicing. We studied the role of RNA editing on non-coding regions of the mat-r-nad1e-nad5c transcript from wheat mitochondria. This RNA contains two trans-introns, 3'-nad1-I4 and 3'-nad5-I2, involved in different trans-splicing events, ensuring the association of nad1d-nad1e and nad5b-nad5c exons from nad1 and nad5 mRNAs respectively. The C-to-U editing changes studied here affect homologous positions on 3'-nad1-I4 and 3'-nad5-I2. It is proposed that these base changes are necessary to place an Adenosine residue in a bulging conformation characteristic of domain VI (D6) from group II introns. In this work, we investigated the role of RNA editing events on 3'-nad1-I4 and 3'-nad5-I2 in the trans-splicing process using in vivo and in organello approaches. When the branched intermediates formed during the splicing process were analyzed, the C residues from D6 intron domains from 3'-nad1-I4 and 3'-nad5-I2 were found changed to U, suggesting that RNA editing of these residues could be mandatory for splicing. This assumption was tested by expressing recombinant mat-r-nad1e transgenes introduced into mitochondria by electroporation. Mutation of the editing target residue dramatically affected trans-splicing. Interestingly, the exon joining efficiency was not recovered by compensatory mutations, suggesting that the role of RNA editing is not confined to the restoration of the secondary structure of domain D6 of the intron. Our results strongly support the hypothesis that RNA editing in trans-introns precedes maturation, and is required for the splicing reaction. In addition, this is the first report using an in organello approach to study the trans-splicing process, opening the way to future studies of this peculiar mechanism

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications

DOI/ 10.1101/gr.2544504International audienc