MISF2 Encodes an Essential Mitochondrial Splicing Co-factor Required for nad2 mRNA Processing and Embryo Development in Arabidopsis thaliana

Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players of plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo-development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1 and thus respiratory complex I biogenesis are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot-dicot divergence, ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.

CAF Proteins Help SOT1 Regulate the Stability of Chloroplast ndhA Transcripts

Protein-mediated RNA stabilization plays profound roles in chloroplast gene expression. Genetic studies have indicated that chloroplast ndhA transcripts, encoding a key subunit of the NADH dehydrogenase-like complex that mediates photosystem I cyclic electron transport and facilitates chlororespiration, are stabilized by PPR53 and its orthologs, but the underlying mechanisms are unclear. Here, we report that CHLOROPLAST RNA SPLICING 2 (CRS2)-ASSOCIATED FACTOR (CAF) proteins activate SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), an ortholog of PPR53 in Arabidopsis thaliana, enhancing their affinity for the 5′ ends of ndhA transcripts to stabilize these molecules while inhibiting the RNA endonuclease activity of the SOT1 C-terminal SMR domain. In addition, we established that SOT1 improves the splicing efficiency of ndhA by facilitating the association of CAF2 with the ndhA intron, which may be due to the SOT1-mediated stability of the ndhA transcripts. Our findings shed light on the importance of PPR protein interaction partners in moderating RNA metabolism.

Evolutionary pattern of the presence and absence genes in Fragaria species

Presence and absence polymorphisms (PAPs) exist extensively and have been investigated in different organisms. However, PAPs have rarely been detected between strawberry species at the genome level. This study identified the presence and absence genes (P/A genes) between wild strawberry species (Fragaria vesca) and octoploid cultivated species (F. × ananassa) under a relatively strict criterion. In total, 333 P/A genes present in the wild strawberry but absent in the cultivated strawberry were detected. Of the P/A genes, 91.89% (306/333) were single genes, and only 8.11% were confirmed as multi-genes. The majority of the identified P/A genes in Fragaria were generated by tandem duplications. The P/A genes were unevenly distributed on the seven chromosomes of woodland strawberry, and they clustered preferentially near the telomeric regions of the chromosomes. The P/A genes tended to encode proteins with domains closely associated with responses to varying ecological factors, such as PPR, Protein kinases (PKs), NB-ARC, F-box and EF-hand domains. This indicated that the P/A genes were associated with coping with biotic and abiotic stresses to improve the adaptability of plants to changing environments.

GmPGL2, Encoding a Pentatricopeptide Repeat Protein, Is Essential for Chloroplast RNA Editing and Biogenesis in Soybean

Chloroplast biogenesis and development are highly complex processes requiring interactions between plastids and nuclear genomic products. Pentatricopeptide repeat (PPR) proteins play an essential role in the development of chloroplasts; however, it remains unclear how RNA editing factors influence soybean development. In this study, a Glycine max pale green leaf 2 mutant (Gmpgl2) was identified with decreased chlorophyll contents. Genetic mapping revealed that a single-nucleotide deletion at position 1949 bp in the Glyma.05g132700 gene in the Gmpgl2 mutant, resulting in a truncated GmPGL2 protein. The nuclear-encoded GmPGL2 is a PLS-type PPR protein that localizes to the chloroplasts. The C-to-U editing efficiencies of rps16, rps18, ndhB, ndhD, ndhE, and ndhF were reduced in the Gmpgl2 mutant. RNA electrophoresis mobility shift assay (REMSA) analysis further revealed that GmPGL2 binds to the immediate upstream sequences at RNA editing sites of rps16 and ndhB in vitro, respectively. In addition, GmPGL2 was found to interact with GmMORF8, GmMORF9, and GmORRM6. These results suggest that GmPGL2 participates in C-to-U RNA editing via the formation of a complex RNA editosome in soybean chloroplasts.

Comprehensive Transcriptome Analyses Reveal Candidate Genes for Variation in Seed Size/Weight During Peanut (Arachis hypogaea L.) Domestication

Seed size/weight, a key domestication trait, is also an important selection target during peanut breeding. However, the mechanisms that regulate peanut seed development are unknown. We re-sequenced 12 RNA samples from developing seeds of two cultivated peanut accessions (Lines 8106 and 8107) and wild Arachis monticola at 15, 30, 45, and 60 days past flowering (DPF). Transcriptome analyses showed that ∼36,000 gene loci were expressed in each of the 12 RNA samples, with nearly half exhibiting moderate (2 ≤ FPKM < 10) expression levels. Of these genes, 12.2% (4,523) were specifically expressed during seed development, mainly at 15 DPF. Also, ∼12,000 genes showed significant differential expression at 30, 45, and/or 60 DPF within each of the three peanut accessions, accounting for 31.8–34.1% of the total expressed genes. Using a method that combined comprehensive transcriptome analysis and previously mapped QTLs, we identified several candidate genes that encode transcription factor TGA7, topless-related protein 2, IAA-amino acid hydrolase ILR1-like 5, and putative pentatricopeptide repeat-containing (PPR) protein. Based on sequence variations identified in these genes, SNP markers were developed and used to genotype both 30 peanut landraces and a genetic segregated population, implying that EVM0025654 encoding a PPR protein may be associated with the increased seed size/weight of the cultivated accessions in comparison with the allotetraploid wild peanut. Our results provide additional knowledge for the identification and functional research into candidate genes responsible for the seed size/weight phenotype in peanut.

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.