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.

WHIRLY (WHY) family proteins, a small family of single-stranded DNA (ssDNA) binding proteins, are widely found in plants and have multiple functions to regulate plant growth and development. However, WHY in rice has received less attention. In this study, we continued our previous study on OsTRX z that is important for chloroplast development. OsTRX z was discovered to interact with OsWHY1, which was confirmed using yeast two-hybrid, pull-down, and BiFC assays. Subsequently, the oswhy1 mutants were obtained by CRISPR/Cas9, which exhibited an albino phenotype and died after the three-leaf stage. Consistent with this albino phenotype, low amounts of Chl a, Chl b, and Car were detected in the oswhy1-1 mutant. Moreover, the oswhy1-1 mutant had chloroplasts with disrupted architecture and no stacked grana and thylakoid membranes. Subcellular localization showed that the OsWHY1-GFP fusion protein was targeted to the chloroplast. What’s more, OsWHY1 was found to be preferentially expressed in young leaves and was involved in chloroplast RNA editing and splicing. Mutation of OsWHY1 significantly affected the expression of chloroplast and ribosome development-related and chlorophyll synthesis-related genes. In conclusion, OsWHY1 contributes to early chloroplast development and normal seedling survival in rice. These results will further elucidate the molecular mechanism of chloroplast development and expand our understanding of WHY1 functions.

Improving a plant’s level of tolerance to oxidative stress can frequently also enhance its tolerance to several other abiotic stresses. Here, a screen of a japonica type rice T-DNA insertion mutant library identified a highly oxidative stress-sensitive mutant. The line exhibited premature leaf senescence, starting at the three-leaf stage, and the symptoms were particularly severe from the five-leaf stage onwards. The leaves progressively lost chlorophyll, suffered protein degradation and were compromised with respect to their photosynthetic activity; their leaf mesophyll and bulliform cells became shrunken, and several senescence-associated genes (SAGs), senescence-associated transcription factor genes (SATFs) and autophagy-related genes (ATGs) were progressively up-regulated. The product of the gene inactivated by the mutation, identified via positional cloning, was putatively a ubiquitin-conjugating enzyme. The gene was denoted here as RLS1 (reactive oxygen species-sensitive leaf senescence1). The phenotype of plants in which RLS1 was knocked down using RNA interference was comparable to that of the rls1 mutant. A comparative analysis of the knock-out line and the wild type leaves showed that the former accumulated more hydrogen peroxide and more malondialdehyde, expressed a heightened level of superoxide dismutase activity and a decreased level of catalase activity, and exhibited an altered transcriptional profile with respect to several SAGs, SATFs and ATGs, and that these effects were magnified when the plants were exposed to oxidative stress. The product of RLS1 is presumed to be a critical component of the rice oxidative stress response and is involved in ROS (reactive oxygen species)-mediated leaf senescence.

Thioredoxin z (TRX z) plays a significant role in chloroplast development by regulating the transcription of chloroplast genes. In this study, we identified a pentatricopeptide repeat (PPR) protein, rice albino seedling-lethal (RAS), that interacts with OsTRX z. This interaction was initially discovered by using a yeast two-hybrid (Y2H) screening technique and was further validated through Y2H and bimolecular fluorescence complementation (BiFC) experiments. RAS contains 16 PPR motifs and features a small MutS-related (SMR) domain at its C-terminus. CRISPR/Cas9-generated ras mutants exhibited an albino seedling-lethal phenotype characterized by abnormal chloroplast structures and a significantly reduced chlorophyll content. RAS localizes to the chloroplast and is predominantly expressed in young leaves. Mutations in RAS affect RNA editing at the rpl2, rps14, and ndhA sites, as well as RNA splicing at the rpl2, atpF, and ndhA transcripts within the chloroplast. Furthermore, the expression levels of genes associated with chloroplast formation are altered in the ras mutant. Both OsTRX z and RAS were found to interact with chloroplast signal recognition particle (cpSRP) proteins, indicating that their proper localization within the chloroplast may be dependent on the SRP pathway. Collectively, our findings highlight the critical role of RAS in chloroplast development, as it is involved in RNA processing and the regulation of chloroplast gene expression.

High light and high temperature (HLHT) stress induces the production of damaging reactive oxygen species (ROS) in many plants. Recently, we described a HLHT-sensitive rice (Oryza sativa) mutant, local lesions (ls1), that exhibits local lesions under HLHT, due to DNA damage and excess ROS accumulation. Here, we determined that an HLHT treatment induced the local lesion phenotype in ls1 within 6 h. Corroborating this result, we found that transient HLHT treatment influenced the expression of many genes in the ls1 mutant, while affecting the growth and development of young leaves.