GhCTSF1, a short PPR protein with a conserved role in chloroplast development and photosynthesis, participates in intron splicing of rpoC1 and ycf3-2 transcripts in cotton.

Cotton is one of the most important textile fibers worldwide. As crucial agronomic traits, leaves play an essential role in the growth, disease resistance, fiber quality, and yield of cotton plants. Pentatricopeptide repeat (PPR) proteins are a large family of nuclear-encoded proteins involved in organellar or nuclear RNA metabolism. Using a virus-induced gene silencing assay, we found that cotton plants displayed variegated yellow leaf phenotypes with decreased chlorophyll content when expression of the PPR gene GhCTSF1 was silenced. GhCTSF1 encodes a chloroplast-localized protein that contains only two PPR motifs. Disruption of GhCTSF1 substantially reduces the splicing efficiency of rpoC1 intron 1 and ycf3 intron 2. Loss of function of the GhCTSF1 ortholog EMB1417 causes splicing defects in rpoC1 and ycf3-2, leading to impaired chloroplast structure and decreased photosynthetic rates in Arabidopsis. We also found that GhCTSF1 interacts with two splicing factors, GhCRS2 and GhWTF1. Defects in GhCRS2 and GhWTF1 severely affect intron splicing of rpoC1 and ycf3-2 in cotton, leading to defects in chloroplast development and a reduction in photosynthesis. Our results suggest that GhCTSF1 is specifically required for splicing rpoC1 and ycf3-2 in cooperation with GhCRS2 and GhWTF1.

GhYGL1d, a pentatricopeptide repeat protein, is required for chloroplast development in cotton.

BACKGROUND: The pentatricopeptide repeat (PPR) gene family, which contains multiple 35-amino acid repeats, constitutes one of the largest gene families in plants. PPR proteins function in organelles to target specific transcripts and are involved in plant development and growth. However, the function of PPR proteins in cotton is still unknown. RESULTS: In this study, we characterized a PPR gene YELLOW-GREEN LEAF (GhYGL1d) that is required for cotton plastid development. The GhYGL1d gene has a DYW domain in C-terminal and is highly express in leaves, localized to the chloroplast fractions. GhYGL1d share high amino acid-sequence homology with AtECB2. In atecb2 mutant, overexpression of GhYGL1d rescued the seedling lethal phenotype and restored the editing of accD and ndhF transcripts. Silencing of GhYGL1d led to the reduction of chlorophyll and phenotypically yellow-green leaves in cotton. Compared with wild type, GhYGL1d-silenced cotton showed significant deformations of thylakoid structures. Furthermore, the transcription levels of plastid-encoded polymerase (PEP) and nuclear-encoded polymerase (NEP) dependent genes were decreased in GhYGL1d-silenced cotton. CONCLUSIONS: Our data indicate that GhYGL1d not only contributes to the editing of accD and ndhF genes, but also affects the expression of NEP- and PEP-dependent genes to regulate the development of thylakoids, and therefore regulates leaf variegation in cotton.

Two pivotal RNA editing sites in the mitochondrial atp1mRNA are required for ATP synthase to produce sufficient ATP for cotton fiber cell elongation.

RNA editing is a post-transcriptional maturation process affecting organelle transcripts in land plants. However, the molecular functions and physiological roles of RNA editing are still poorly understood. Using high-throughput sequencing, we identified 692 RNA editing sites in the Gossypium hirsutum mitochondrial genome. A total of 422 editing sites were found in the coding regions and all the edits are cytidine (C) to uridine (U) conversions. Comparative analysis showed that two editing sites in Ghatp1, C1292 and C1415, had a prominent difference in editing efficiency between fiber and ovule. Biochemical and genetic analyses revealed that the two vital editing sites were important for the interaction between the α and ß subunits of ATP synthase, which resulted in ATP accumulation and promoted cell growth in yeast. Ectopic expression of C1292, C1415, or doubly edited Ghatp1 in Arabidopsis caused a significant increase in the number of trichomes in leaves and root length. Our results indicate that editing at C1292 and C1415 sites in Ghatp1 is crucial for ATP synthase to produce sufficient ATP for cotton fiber cell elongation. This work extends our understanding of RNA editing in atp1 and ATP synthesis, and provides insights into the function of mitochondrial edited Atp1 protein in higher plants.