Gene Cloning And Functional Analysis Of Spotted And Rolled Gene SPRL1 In Rice

Rice spotted leaf mutant,also known as lesion mimic mutant,is an important leaf color mutant resulting from spontaneously localized cell death lesions in the absence of pathogens.Such mutants are ideal material’s for studying reactive oxygen species(ROS)accumulation and deciphering the signal pathways of programmed cell death(PCD)in the plants.Previously we screened a spotted and rolled leaf(sprl1)mutant in indica rice restorer line Lehui188(188R)using ethyl methanesulfonate(EMS)-treatment.In this study,we carried out phenotype analysis on sprl1 mutant,as well as gene cloning and functional analysis of sprl1 were performed,and the results are as follows:(1)The sprl1 mutant displayed reddish-brown lesions on leaves throughout the whole growth period.Before the three-leaf stage,sprl1 also possessed extremely inwardly rolled leaves.Photosynthetic pigments measurement showed that the contents of these pigments in sprl1 were severely reduced than those of the wild type.Cytological observation found,the rolling leaf phenotype of sprl1 was resulted from reducing the number and size of bulliform cells in leaf blades.Transmission electron microscopy analysis showed that the chloroplasts were tiny and abnormal,the grana stacks were thinner and the starch granules were increased in sprl1.At maturity,plant height,the number of productive panicles per plant,spikelet number of main panicle,panicle length,and grain yield per plant of sprl1 were significantly decreased compared with those of the wild type.(2)Physiological and biochemical analysis showed that the leaves of sprl1 exhibited deep blue and brownish respectively under nitro blue tetrazolium(NBT)and 3,3’-diaminobenzidin(DAB)staining.These results indicated that ROS were highly accumulated in sprl1 leaves.Physiological indexes showed that the activity of SOD(superoxide dismutase)and POD(peroxidase)of sprl1 were increased significantly.Moreover,trypan blue(TB)staining showed that cell death occurred in sprl1 leaves.(3)The genetic analysis suggested that the spotted and rolled leaf phenotype of sprl1 is controlled by a single recessive nuclear gene.Os PPO1(LOC＿Os01g18320)was identified as the candidate gene of sprl1 by whole-genome resequencing and Mut Map analysis.Sequence comparison showed that sprl1 carries a single nucleotide C-to-T substitution at position 516 in sprl1 mutant,causing an amino acid change from Arg-143 to Trp in the encoded protein.(4)HPLC analysis resulted that sprl1 accumulated excess Proto(gen)Ⅸ,suggesting that the mutant indeed has a defective PPO1.(5)Complementary transgenic lines of sprl1 showed leaves phenotype and main major agronomic traits of sprl1 were almost restored to the wild type level.This result confirmed that the single nucleotide mutation in Os PPO1 is responsible for the spotted and rolled leaf phenotype of sprl1.(6)Temperature-and light-sensitivity experiments found that the sprl1 displayed more necrotic lesions under high light conditions than under low light conditions,and no obvious necrotic lesion was observed under dim light conditions.Besides,sprl1 mutant showed severe necrotic lesions under 22°C conditions,but only a few necrotic lesions were observed under30°C conditions.Therefore,the spotted leaf phenotype of sprl1 mutant is sensitive to light intensity and temperature.(7)Subcellular localization analysis suggested that Os PPO1 protein is localized in the chloroplast.Expression pattern analysis with q RT-PCR indicated that Os PPO1 was expressed in all the tested tissues and mainly expressed in green tissues.We examined the expression levels of 12 genes which involved in tetrapyrrole biosynthesis,4 photosynthesis-related genes and 7 genes which related to active oxygen scavenging.The result showed that all the genes related to tetrapyrrole synthesis and photosynthesis were significantly decreased,while the gene involved in ROS accumulation were dramatically increased in the sprl1 mutant.In addition,we also detected transcript levels of leaf rolling-related genes at the two-leaf stage and five-leaf stage respectively.The result showed these genes were all remarkably downregulated at the two-leaf stage,and those genes were returned to the wild type levels at the five-leaf stage.(8)Overexpression of Os PPO1 in japonica cultivar Zhonghua 11 displayed normal leaf phenotype and was not significant different from control group.Importantly,overexpression of Os PPO1 confer a diphenyl ether herbicides resistance in rice under natural conditions in paddy field,but had no distinct influence on plant growth and grain yield.Therefore,the Os PPO1 could have potential values of application in Diphenyl ether-herbicide resistance.