Cloning And Functional Study Of A White And Lesionmimic Leaf 1 In Rice(Oryza Sativa L.)

Riboflavin is the precursor of FMN and FAD that serve as indispensable cofactor to maintain normal metabolism,which plays pivotal roles in mitochondrial electron transport chain,tricarboxylic acid cycle,fatty acidβ-oxidation,amino acid catabolism,redox homeostasis,chromatin remodeling,DNA repair,apoptosis and secondary metabolites biosynthesis.Rice（Oryza sativa）is a staple crop and model plant,and it is of great biological significance and practical value to explore the molecular mechanism of riboflavin effect on rice growth and development.Our previous study isolated a rice mutant white and lesion-mimic 1（wll1）and cloned WLL1（LOC＿Os04g42000）,which encoded the rate-limited enzyme―lumazine synthase of riboflavin biosynthesis pathway.The G-to-A mutation at position 211 in LOC＿Os04g42000 led to Gly to Arg at position 71.Compared with the wild type,the mutation of WLL1resulted in irregular white and lesion-mimic phenotypes in leaves.At present,there were few reports on riboflavin-defective mutants in rice.The wll1 provided an ideal material for exploring riboflavin functions in the regulation of chloroplast development in rice.In this paper,the molecular mechanism of WLL1regulating chloroplast development was analyzed in rice by means of genetics,cell biology,biochemistry and molecular biology and transcriptomics.The main findings are as follows:（1）Compared with the wild type,the chloroplasts were abnormally developed,severely degraded and harbored more osmophilic particles in wll1.The mesophyll cells in the white-spot parts were shrunken,the number of mesophyll layers was significantly reduced with the occurrence of cell death.（2）The wll1 mutant phenotype was completely rescued when the wild type genomic DNA of LOC＿Os04g42000 was introduced.At the same time,the LOC＿Os04g42000 knockout lines by CRISPR-Cas9 showed similar phenotypes with the wll1 mutant.Compared with the wild type,the contents of riboflavin,FAD and FMN were significantly reduced in wll1;1 m M FAD treatment could rescue the phenotype of wll1.The above tests demonstrated that LOC＿Os04g42000 was responsible for the phenotypes of wll1.Therefore,LOC＿Os03g50860 was the target gene of WLL1.（3）The GUS staining and RT-q PCR results showed that WLL1 was mainly expressed in roots,followed by panicles and leaves,and lower in stems and sheaths.Subcellular localization analysis indicated that WLL1 protein was localized in the chloroplast.（4）Over-accumulated ROS were detected in wll1.Trypan blue staining and TUNEL assay showed that the mesophyll cells of wll1 showed obvious DNA damage and cell necrosis,accompanied by expression up-regulation of antioxidant genes.These results indicated that WLL1 mutation led to imbalance of ROS in rice,and over-accumulation of ROS led to DNA damage,organelles degradation,and even cell death in the severe cases.（5）Transcriptome analysis suggested that mutation of WLL1 could affect cytokinin metabolism.As well known,the cytokinin plays vital roles in chlorophyll biosynthesis and chloroplast development,and the disorder of cytokinin metabolism affects chlorophyll biosynthesis and chloroplast development.In addition,GO and KEGG enrichment analysis of DEGs showed that the antioxidant metabolism pathways were significantly enriched,and the expressions of glutathione transferases,heat shock proteins and CYP450s in the oxidative stress pathway were significantly up-regulated.These data also proved that WLL1 mutation resulted in oxidative stress in wll1.（6）The contents of t ZR,i P and i PR were significantly increased in wll1,suggesting that WLL1mutation could affect endogenous cytokinins homeostasis.We speculated that the riboflavin defect led to decreased activities of FAD-dependent flavinenzyme CKXs,which were responsible for degradation of CK.In addition,cytokinin-response and TCS-GUS staining assays confirmed that CK signaling was significantly enhanced in wll1,which could accelerate the synthesis of ALA.Further studies showed that the ALA content was significantly increased in wll1,accompanied with the expression up-regulation of HEMA1 encoding a rate-limiting enzyme of ALA synthesis.ALA was a photosensitizer,and the uncontrolled flow of ALA triggered a large amount of ROS production,which might be one of the main reasons for ROS accumulation in wll1.（7）WLL1 mutation induced the metabolic disorders of tetrapyrrole.The contents of chlorophyll intermediates Proto IX,Mg-proto IX and Pchlide were significantly decreased in wll1.We speculated that the impairment of riboflavin biosynthesis affected the activity of FDA-dependent flavoenzyme PPOX in tetrapyrrole pathway,resulting in the block of protoporphyrogen IX to protoporphyrin IX.The photooxidation of protoporphyrogen IX led to the production of ROS,which might be another main reason for the accumulation of ROS in wll1.ROS accumulation eventually resulted in DNA damage,organelle degradation and cell death.（8）Y2H and Bi FC assays demonstrated that WLL1 and RS formed a multimeric complex to catalyze the riboflavin synthesis.The rs mutant created by CRISPR/CAS9 showed a similar phenotype to wll1.In addition,we predicted the multimeric structure of WLL1 by homology modeling.The results showed that five monomers of WLL1 could assemble into a pentamer,and twelve pentamers further assembled into an icosahedron.Mutated subunit interactions were predicted using PISA,a tool for protein crystal surface accessibility and interfacial interactions.After the mutation of Gly⁷¹to Arg⁷¹,Arg⁷¹ could form hydrogen bonds with Ser¹¹⁸ of adjacent monomers within the same pentamer,thereby enhancing the interaction.However,the closest interatomic distance between the side chain of Arg⁷¹ and Arg¹⁰⁶ of the adjacent monomer in the adjacent pentamer was only 2.62 angstroms,which might generate electrostatic repulsion and eventually affect the icosahedron stability.In addition,WLL1 protein was expressed in E.coli BL21（DE3）,and an about 40 nm spherical structure was observed under transmission electron microscope.