Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice

Abstract

The chloroplast is an important organelle that performs photosynthesis as well as biosynthesis and storage of many metabolites. Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in protein synthesis. However, the relationship between chloroplast development and aaRSs still remains unclear. In this study, we isolated a rice albino 1 (ra1) mutant through methane sulfonate (EMS) mutagenesis of rice japonica cultivar Ningjing 4 (Oryza sativa L.), which displayed albinic leaves in seedling stage due to abnormal chloroplast development. Compared with wild type (WT), ra1 showed significantly decreased levels of chlorophylls (Chl) and carotenoids (Car) in 2-week-old seedlings, which also showed obvious plastidic structural defects including abnormal thylakoid membrane structures and more osmiophilic particles. These defects caused albino phenotypes in seedlings. Map-based cloning revealed that RA1 gene encodes a glycyl-tRNA synthetase (GlyRS), which was confirmed by genetic complementation and knockout by Crispr/Cas9 technology. Sequence analysis showed that a single base mutation (T to A) occurred in the sixth exon of RA1 and resulted in a change from Isoleucine (Ile) to Lysine (Lys). Real-time PCR analyses showed that RA1 expression levels were constitutive in most tissues, but most abundant in the leaves and stems. By transient expression in Nicotiana benthamiana, we found that RA1 protein was localized in the chloroplast. Expression levels of chlorophyll biosynthesis and plastid development related genes were disordered in the ra1 mutant. RNA analysis revealed biogenesis of chloroplast rRNAs was abnormal in ra1. Meanwhile, western blotting showed that synthesis of proteins associated with plastid development was significantly repressed. These results suggest that RA1 is involved in early chloroplast development and establishment of the plastidic ribosome system in rice.