Albino Leaf 2 is involved in the splicing of chloroplast group I and II introns in rice.

Chloroplasts play an essential role in plant growth and development through manipulating photosynthesis and the production of hormones and metabolites. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, identification of novel components is still lacking. We isolated a rice (Oryza sativa) mutant, termed albino leaf 2 (al2), using genetic screening. Phenotypic analysis revealed that the al2 mutation caused obvious albino leaves at the early developmental stage, eventually leading to al2 seedling death. Electron microscopy investigations indicated that the chloroplast structure was disrupted in the al2 mutants at an early developmental stage and subsequently resulted in the breakdown of the entire chloroplast. Molecular cloning illustrated that AL2 encodes a chloroplast group IIA intron splicing facilitator (CRS1) in rice, which was confirmed by a genetic complementation experiment. Moreover, our results demonstrated that AL2 was constitutively expressed in various tissues, including green and non-green tissues. Interestingly, we found that the expression levels of a subset of chloroplast genes that contain group IIA and IIB introns were significantly reduced in the al2 mutant compared to that in the wild type, suggesting that AL2 is a functional CRS1 in rice. Differing from the orthologous CRS1 in maize and Arabidopsis that only regulates splicing of the chloroplast group II intron, our results demonstrated that the AL2 gene is also likely to be involved in the splicing of the chloroplast group I intron. They also showed that disruption of AL2 results in the altered expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded polymerases and nuclear-encoded chloroplast genes. Taken together, these findings shed new light on the function of nuclear-encoded chloroplast group I and II intron splicing factors in rice.

Exon skipping in IspE Gene is associated with abnormal chloroplast development in rice albino leaf 4 mutant.

The formation of leaf color largely depends on the components of pigment accumulation in plastids, which are involved in chloroplast development and division. Here, we isolated and characterized the rice albino leaf 4 (al4) mutant, which exhibited an albino phenotype and eventually died at the three-leaf stage. The chloroplasts in al4 mutant were severely damaged and unable to form intact thylakoid structure. Further analysis revealed that the candidate gene encodes 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), which participates in the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis. We further demonstrated that the mutation at the exon-intron junction site cause alternative splicing factors fail to distinguish the origin of the GT-AG intron, leading to exon skipping and producing a truncated OsIspE in the al4 mutant. Notably, disruption of OsIspE led to the reduced expression of chloroplast-associated genes, including chloroplast biosynthetic and translation related genes and photosynthetic associated nuclear genes (PhANGs). In summary, these findings reveal that OsIspE plays a crucial role in chloroplast biogenesis and provides novel insights into the function of CMK during chloroplast development in rice.

Gene mapping and transcriptome profiling of a practical photo-thermo-sensitive rice male sterile line with seedling-specific green-revertible albino leaf.

Abnormal environment weather can cause rice photoperiod-thermo-sensitive genic male sterile (PTGMS) lines fertile or partially fertile and thus cause the mixture of true hybrids with selfing seeds. Seedling-specific green-revertible albino leaf color mutant can be used to distinguish the real hybrids. Besides, it can also be used as an ideal material to research the development of chloroplast and biosynthesis of chlorophyll. The phenotype of leaf color mutants includes light green, yellowing, albino, green-revertible albino. Gene mutations affecting the synthesis and degradation of photosynthetic pigments, lycopene and heme, the differentiation and development of chloroplast, gibberellins (GAs) biosynthesis, can change the leaf color. We have created a PTGMS line with seedling-specific green-revertible albino leaf named W01S. The leaf phenotype, pollen sterility and fertility, agronomic traits, heredity, gene mapping and RNA-Seq of the differentially expressed genes between albino and green-revertible leaves were investigated. The results showed that W01S is a practical PTGMS line as Pei'ai 64S. The mutation of candidate gene Os03g0594100 (ent-isokaurene C2-hydroxylase-like) in W01S can be related to the biosynthesis of GAs, indole acetic acids, ethylene.