Identification And Map-based Gene Cloning Of A Chlorophyll Biosynthesis Deficiency Mutant Cbd1 In Rice (Oryza Satival.)

High photosynthetic efficiency is one of the determinants of rice yield.Chlorophyll is a major pigment in botanic photosynthesis and its content associates with not only the leaf color but also the photosynthetic efficiency.It is of great significance to dig genes involved in Chlorophyll biosynthesis by mutants with altered leaf color and reveal their roles in photosynthesis,shedding light on the mechanism of high photosynthetic efficiency and paving the way for super rice breeding.In this study,we characterized a Chlorophyll biosynthesis deficiency mutant(named as cbdl),whose Chlorophyll(Chls)and Carotenoids(Cars)content were decreased to two-thirds of the wild type and displays light-green leaves during the whole life period.With the treatment of gradient temperature,the Chls content in wild type and cbdl reduced simultaneously,which means that cbdl is not sensitive to temperature.By transmission electron microscopy(TEM)analysis,we found that thylakoid lamellar structures kept intact in cbd1,similar to that in wild type.Likely,several agromonic traits between wild type and cbdl,such as plant height,tiller number,panicle length,length and width of flag leaves and grain weights were also identical.Interestingly,the number of grain per panicle was increased in cbdl than in wild type.Genetic analysis showed that a recessive nuclear gene was responsible for the light-green phenotype in cbdl.By map-based gene cloning stragety,the causal gene was restricted within a 57kb-interval on chromosome 10,flanked by marker D5 and RM25669,wherein 10 putative genes were located.After candidate gene cloning and sequence alignment,a 433bp mPingA miniature inverted-repeat transposable element insertion in the promoter region of OsHemA(about 1.1 kb upstream of the start codon)was identified,leading to a reduced expression of OsHemA gene and its protein.OsHemA encodes Glutamyl-tRNA reductase(GluTR),which initiates the Chlorophyll and Heme biosynthesis and catalyzes Glutamyl-tRNA to glutamate-1-semialdehyde.The light-green leaf color in OsHemA gene overexpression transgenic lines was recovered,and also Chls and Cars contents were elevated to the nonnal level compared to wild type,which indicates that down-regulation of OsHemA gene is responsible for the decline of pigments.HemA gene was first identified in Bacterial and HemA-like proteins widely exist in archaea,cyanobacteria and higher plants.In contrast to multiple homologs in other species,only one unique counterpart exists in rice.We found OsHemA transcripts are expressing in all tested tissues but abundant mainly in green tissues.A similar transcription pattern with a lower transcription level was observed in cbdl mutant.Consistently,genes involved in the pathway of Chlorophyll biosynthesis were consequently down-regulated in the mutant,which confirmed that GluTR is rate-limiting in Chlorophyll biosynthesis.GluTR contains conserved N-terminal and C-terminal domains;the former participates in combining t-RNA and NADP,while the latter promotes the dimer formation.Through transcient transformation in rice protoplasts,HemA-GFP fusion protein mainly locates at chloroplasts,whereas GFP-HemA protein pervades in both cytoplasm and chloroplasts.In summary,OsHemA gene is unique in rice and its encoding protein performs in chloroplasts,initiating the biosynlthesis of chloroplasts.