Albino Leaf 2 Is Involved In The Splicing Of Chloroplast Group ? And ? Introns In Rice (Oryza Sativa L.)

In plants, chloroplasts play an essential role in plant growth and development through manipulating the photosynthesis and the production of hormones and metabolites.Chloroplast morphology and chlorophyll content are of great importance to the photosynthetic efficiency and plant growth and development. In general, defects of chloroplast development result invisualized leafcolor variation in plant, whichare potential materials for the studying of chloroplast development and photosynthesis.Numerous genes or regulators involved in chloroplast differentiation and development have been isolated and characterized. However, identification of novel components is still lacking and the molecular mechanism underlying chloroplast development still needs to be further determined.Our previous studies showed that dezons of rice mutants with various leaf color, termed albino leaf mutants(als), were isolated from T-DNA insertion lines(Zhonghua 11 background)by genetic screening. Among them, the al1 mutant has been well-characterized. Here, we are particularly interesting in the investigation of al2 mutant. Our results are summaried as following:1. Phenotypic analysis demonstrated that an apparent albino phenotype was observed in the young buds ofal2 mutant as compared to that in wild type at the early developmental stage. Subsequently, this phenotype was more obvious and spread around the entire leaf at the third-leaf stage, eventually led to al2 seedling lethality. Consistantly, chlorophyll contents were significantly reduced in al2 compared to that in wild type.2.Transmission electron microscopy investigations of the chloroplast structuresindicated that the formation of thylakoids and chloroplastwas severely affected in al2 mutants at the early developmental stage, eventually resulted in the breakdown of the entire chloroplast.3. Our previous studies suggested that the albino leaf phenotype of al2 mutant might be caused by the insertion of T-DNA into the ninth exon of Loc_Os09g19850. Here, our results demonstrated that the transcript level of Loc_Os09g19850 determined by semi-quantitative PCR was completely eliminated in al2 as compared to that in wild type, implying that Loc_Os09g19850 was the candidate AL2(Albino Leaf 2) gene. Further analysis showed thatnine exons and eight introns are present in Loc_Os09g19850, which encodes a chloroplast group IIA intron splicing facilitator(CRS1) in rice.4. Genetic complementation assay confirmed that Loc_Os09g19850wasthe AL2 gene in rice. Five independent complementation lines were obtained, and the albino leaf phenotype of al2 was fully rescued by the Loc_Os09g19850 transgene, as well as the chlorophyll changes and the transcript level of Loc_Os09g19850. In addition, six independent AL2-knockdown lines were obtained, and the al2-like phenotypeswere observed in these transgenic rice, indicating that AL2 positively regulate chloroplast development5. Expression pattern analysesindicated that AL2 was expressed from germination untill the mature stage. In addition to the young leaves, AL2 was also expressed in the non-green tissues at the seedling stage, such as root hair. The most abundance of AL2 were detected in leaf at the mature stage, while GUS staining indicated that AL2 was also expressed in panicle, spikelet and culm, as well as leaf sheath. Cross section illustrated that AL2 was expressed in the endodermis of culm. In consistence, q RT-PCR results indicated that the AL2 gene was expressed in the root, culm, leaf, young panicle and mature panicle, while the highest expression of AL2 was detected in leaf. In addition,subcellular localization showed that the AL2 protein was localized in the chloroplast.6. Previous reports haveimplicatedthat CRS1 is involed in the splicingof chloroplast group IIA atp Fintronin Arabidopsis and maize. Our results showed that the expression level of atp F was significantly reduced in al2 compared to that in wild type, suggesting that the rice AL2 may be a functional CRS1. Moreover, determination of expression levels of chloroplast genes containing group IIB introns, including ndh A, ndh B, pet Dandycf3, as well astrn L containing the chloroplast group I introns by q RT-PCR in al2 suggested that AL2 is also likely involved in the splicing of both chloroplast group I and II introns.7. The expressions of chloroplastassociated genes,includingchlorophyll biosynthetic genes(CBGs)(Os HAP3 A, Os HAP3 B, Os HAP3 C, Os PPR1, YGL1, Cab1 R, Cab, Hem A and Cao), nuclear-encoded chloroplast genes(NECGs)(psb O, psa D, psa E, psb P, lhcb2 and rbc S), and two plastid-encoded polymerase(PEP)(psa A and psb A), were significantly reducedin al2 as compared to that in wild type, whereas expression levels ofother two PEP genes, includingpsa B and rps14, and nucleus-encoded polymerase(NEP), includingatp A, pet A, rpo B and rps2, were not significantly changed. Taken together, we propose that AL2 coordinates the expression of a subset of chloroplast associated genes to regulate the chloroplast development in rice.In summary, we identified a chloroplast development associated gene, AL2. The albino leaf phenotype of al2 mutant was caused by the disruption of AL2 function. Molecular cloning and functional analyses indicated that the Loc_Os09g19850 is the AL2 target gene, which encodes a CRS1 in rice. The q RT-PCR analyese suggested that AL2 is likely involved in the splicing of both chloroplast group I and II introns, and AL2 coordinates the expression of a subset of chloroplast associated genes to regulate the chloroplast development in rice.Taken together, our findings shed new lights on the molecular mechanism of CRS1 in regulating the development of chloroplast.