Map-Based Cloning And Functional Analysis Of Two Novel Leaf-Color Genes YGL8 And BSL1 In Rice(Oryza Sativa L.)

The chlorophyll biosynthesis pathway is complex and comprises a series of conserved enzymatic reactions,which plays a vital role in photosynthesis and other cellular process responsible for plant growth and development.Cell death is a fine regulated process,which is also essential for both development and defense response in plants.However,their molecular mechanisms remain unclear.In this research,we identified two rice leaf-color mutants,yellow-green leaf 8(ygl8)and brown spotted leaf 1(bsll).Compared with the wild-type plant,we analyzed the phenotype and physiology of the mutants.Then we cloned the YGL8 and BSL1 genes by map-based cloning method.Functional analysis of these genes provided the basis on understanding the regulation pathway of chlorophyll biosynthesis,chloroplast development and cell death.The main results of YGL8 gene are summarized as follows:1.The ygl8 mutant was isolated from tissue cultures of Japonica variety Kita-ake,which exhibited obvious yellow-green leaves from the seedling stage.Compared to its wild-type plants,the mutant leaves contained less Ch1 content and higher Ch1 alb ratio.Additionally,it was a temperature-insensitive leaf color mutant.Transmission electron microscopy analysis revealed that chloroplast development was impaired and delayed in the ygl8 mutant.As the development of leaves,the chlorotic plants turned green accompanied by restorations in Ch1 content and chloroplast ultrastructure.However,the agronomic traits of the ygl8 mutant were significantly different from those of the wild-type plants.2.Genetic analysis of the ygl8 locus indicated that the yellow-green leaf phenotype was controlled by a single recessive nuclear gene.And then the ygl8 locus was mapped to a 24.6-kb interval on the short arm of chromosome 1.Within the target region,five ORFs had been predicted.Sequence alignments showed that a single nucleotide substitution from A-to-G was present in the third exon of ORF2(LOC_Os01g17170)from the ygl8 mutant,which caused a conserved amino acid residue change(Asn182Ser).Furthermore,complementation and RNA interference tests confirmed that ORF2 was the YGL8 gene responsible for the mutant phenotype.YGL8 encoded a catalytic subunit of MgPME cyclase in rice.By the analysis of the Ch1 precursor,the ygl8 mutant accumulated the cyclization reaction substrate,but the product content of Pchlide was significantly decreased.In addition,YGL8 was constitutively expressed in various tissues,with more abundance in young photosynthetic tissues.Its functional defect could affect the expression of some nuclear genes associated with Ch1 biosynthesis.3.Based on amino acid sequence alignment,YGL8 shares high identity with its orthologs in other species.Phylogenetic analysis showed that YGL8 belongs to a typical monocots group.Subcellular localization demonstrated that YGL8 was localized in chloroplast,and N-terminal 40 amino acid residues are enough for its localization.Using yeast two-hybrid assays.we found that YGL8 interacted with OsLCAA.Furthermore,the interactions between YGL8 and OsLCAA,YGL8 and OsFLU1,YGL8 and OsPORB,OsLCAA?OsPORB were confirmed by BiFC assays.However,the ygl8 mutantion did not affect their interactions.These results revealed that the FLU-YGL8-LCAA-POR complex might be present in chloroplast.The main results of BSL1 gene are summarized as follows:1.Two bsl1 mutants were screened from the 60Co-? ray irradiated population of indica variety 9311.Compared with the wild-type plants,the bsll-1 mutant showed a dwarf phenotype with retarded growth,and small brown lesions were dispersed over the surface of mature leaves.By the analysis of tissue cross section,the internode cells of the bsl1-1 mutant were reduced in size and number.The mesophyll cells were chlorosis and accumulated a large amount of anthocyanins.Moreover,the levels of ROS were increased in the internal and surrounding regions of the lesion.Based on the observations of cell ultrastructure,the bsl1-1 mutant showed a PCD phenotype,including cytoplasmic shrinkage and vacuolization,organelle swelling and disruption.2.Genetic analysis showed the bsl1 lesion phenotype was recessive and controlled by a single locus.Then the bsll locus was fine mapped to a 21-kb region on the long arm of chromosome 12,which contained four predicted ORFs.Sequence alignments showed that the bsll-1 and bsll-2 mutant had a single base substitution on the tenth and second exon of ORFI,respectively,which resulting in an amino acid change.Genetic complementation and overexpression tests confirmed that ORF1 was the BSL1 gene responsible for the mutant phenotype.BSL1 encoded an ATP-grasp domain containing protein.Expression analysis showed that YGL8 was constitutively expressed in various tissues,with higher expression levels in leaves and panicles.And its expression was up-regulated in the lesion leaves of the bsll-1 mutant.Furthermore,some genes associated with ROS and cell death were induced to express in the bsl1-1 mutant especially the gene CATA and OsAtg4.Therefore,BSL1 acts as a new member regulating the cell death signaling pathway.