Plastid ribosomal proteins (PRPs) play important roles in the translation of key proteins involved in chloroplast development and photosynthesis. PRPs have been widely studied in many plant species; however, few studies have investigated their roles in rice. In the present study, we used ethyl methane sulfonate mutagenesis and obtained a novel rice mutant called white green leaf 2 (wgl2). The wgl2 mutants exhibited an albino phenotype from germination through the three-leaf stage, and then gradually transitioned to green through the later developmental stages. Consistent with this albino phenotype, wgl2 mutants had abnormal chloroplasts and lower levels of photosynthetic pigments. Map-based cloning and DNA sequencing analyses of wgl2 revealed a single-nucleotide substitution (G to T) in the first exon of LOC_Os03g55930, which resulted in a substitution of glycine 92 to valine (G92 V). WGL2 encodes a conserved ribosomal protein, which localizes to the chloroplast. Complementation and targeted deletion experiments confirmed that the point mutation in WGL2 is responsible for the wgl2 mutant phenotype. WGL2 is preferentially expressed in the leaf, and mutating WGL2 led to obvious changes in the expression of genes related to chlorophyll biosynthesis, photosynthesis, chloroplast development, and ribosome development compared with wild-type. WGL2 encodes a conserved ribosomal protein, which localizes to the chloroplast. WGL2 is essential for early chloroplast development in rice. These results facilitate research that will further uncover the molecular mechanism of chloroplast development.

Background: The large family of pentatricopeptide repeat (PPR) proteins is widely distributed among land plants. Such proteins play vital roles in intron splicing, RNA editing, RNA processing, RNA stability and RNA translation. However, only a small number of PPR genes have been identified in rice.Results: In this study, we raised a mutant from tissue-culture-derived plants ofOryza sativa subsp. japonica‘Zhonghua 11’, which exhibited a lethal chlorosis phenotype from germination to the third-leaf stage. The mutant was designatedseedling-lethal chlorosis 1(slc1). Theslc1mutant leaves showed extremely low contents of photosynthetic pigments and abnormal chloroplast development, and were severely defective in photosynthesis. Map-based cloning ofOsSLC1revealed that a single base (G) deletion was detected in the first exon ofOs06g0710800in theslc1mutant, which caused a premature stop codon. Knockout and complementation experiments further confirmed thatOsSLC1is responsible for the seedling-lethal chlorosis phenotype in theslc1mutant.OsSLC1was preferentially expressed in green leaves, and encoded a chloroplast-localized PPR protein harboring 12 PPR motifs. Loss-of-function ofOsSLC1affected the intron splicing of multiple group II introns, and especially precluded the intron splicing ofrps16, and resulted in significant increase in the transcript levels of 3 chloroplast ribosomal RNAs and 16 chloroplast development-related and photosynthesis-related genes, and in significant reduction in the transcript levels of 1 chloroplast ribosomal RNAs and 2 chloroplast development-related and photosynthesis-related genes.Conclusion: We characterized a novel chloroplast-localized PPR protein, OsSLC1, which plays a vital role in the intron splicing of multiple group II introns, especially therps16intron, and is essential for early chloroplast development and seedling survival in rice.

The chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are involved in the splicing of chloroplast gene introns. Numerous CRM domain proteins have been reported to play key roles in chloroplast development in several plant species. However, the functions of CRM domain proteins in chloroplast development in rice remain poorly understood. In the study, we generated oscaf1 albino mutants, which eventually died at the seedling stage, through the editing of OsCAF1 with two CRM domains using CRISPR/Cas9 technology. The mesophyll cells in oscaf1 mutant had decreased chloroplast numbers and damaged chloroplast structures. OsCAF1 was located in the chloroplast, and transcripts revealed high levels in green tissues. In addition, the OsCAF1 promoted the splicing of group IIA and group IIB introns, unlike orthologous proteins of AtCAF1 and ZmCAF1, which only affected the splicing of subgroup IIB introns. We also observed that the C-terminal of OsCAF1 interacts with OsCRS2, and OsCAF1–OsCRS2 complex may participate in the splicing of group IIA and group IIB introns in rice chloroplasts. OsCAF1 regulates chloroplast development by influencing the splicing of group II introns.

Gametophytic male sterility (GMS) plays an important role in the study of pollen development and seed propagation of recessive nuclear male sterile lines insensitive to the environmental conditions in hybrid rice breeding. Since the inherent phenotypic and genetic characteristics of GMS, it is very difficult to find and identify the GMS mutants. However, due to the abundance of gene transcription data, a large number of pollen-specific genes have been found, and most of them may be associated with GMS. To promote the study of these genes in pollen development and heterosis utilization, in this study, an easy and efficient method of creating and identifying GMS was established using RNAi and OsMYB76R as a reporter. First, the OsC1/OsMYB76 gene involved in anthocyanin synthesis was modified, and we have validated that the modified OsMYB76R is workable as the same as the pre-modified OsMYB76 gene. Then, the ascorbic acid oxidase gene OsPTD1 was downregulated using RNAi, driven by its own promoter that resulted in abnormal pollen tube growth. Finally, the RNAi elements were linked with OsMYB76R and transformed into an osmyb76 mutant, and the distortion of purple color segregation was found in T₁ and F₁ generations. This indicates that the OsPTD1 GMS was prepared successfully. Compared to current methods, there are several advantages to this method. First, time is saved in material preparation, as one generation less needs to be compared than in the conventional method, and mutation screening can be avoided. In addition, for identification, the cost is lower; PCR, electrophoresis, and other processes are not needed; and no expensive chemicals or instruments are required. Finally, the results are more accurate, with much lower background effects, and no damage to the plant. The result is an easy, efficient, low-cost, and accurate method of preparing and identifying GMS genes.