Positional Cloning And Function Analysis Of Spotted LEAF 42 (SPL42) In Rice

In this study,a novel mimic mutant spotted leaf 42（spl42）was obtained from the Japonica rice cultivar（cv.）Ningjing 6 afer ethyl methanesulfonate mutagenesis.We carried out the phenotypic characterization and functional analysis of spl42 mutant.The results and analysis can provide evidences for the spotted leaf mutant occurrence and disease resistance mechanism.The specific research results were inciuded as following:1.The phenotype of the mutant spl42:the spotted leaf appeared at the beginning of the old leaves from the three-leaf and four-leaf stage under field conditions and gradually spread from the middle of the leaves to the whole leaves.There were tiny and reddish brown spots on the leaves.Two weeks after transplanting,the old leaves were covered with spots,while the new leaves had fewer and smaller spots.At the heading stage,the flag leaves of the plant heading had spots and no spots were found in the leaf sheaths and glumes.Under the field conditions,the agronomic traits were measured and the mutant spl42 showed a significant decrease in plant height,length of the panicle,length of the flag leaf,seed setting rate compared with the wild type,while the width of flag leaf of the spl42mutant was decreased compared with the wild type.There was no difference in the number of effective tillers,grain length,grain width,and 1000-grain weight.In the seedling stage,the contents of chlorophyll were significantly lower in the spot leaves for the mutant spl42compared with the wild type.spl42 is exhibited more serious phenotype at high temperature.There were significant differences in the parameters of photosynthetic indexes between the mutant spl42 and wild type.Transmission electron microscopy（TEM）analysis showed that the chloroplasts were normal and the thylakoid structure were intact and arranged tightly in wild-type leaves.There were two types of chloroplasts in the spl42 mutant leaves:one kind of chloroplast developed normally but the chloroplast became larger than the wild type.The body arrangement becomes relatively loose.Another kind of chloroplast is broken and degraded,and the degradation of the thylakoid in the chloroplast of spl42 damaged the photosynthesis ability of the plant.q RT-PCR analysis indicated that the expression of senescence-related genes were significantly up-regulated while chlorophyll synthesis and chloroplast development related genes were significantly down-regulated in the spl42.2.Physiology and biochemistry of spl42 mutant:there were obvious brown deposits in the spl42 spot leaves compared with the wild type after DAB（3,3’-diaminobenzidine）staining.The higher H₂O₂ content and the lower activity of CAT were dected in the spot leaves for spl42 compared with the wild type.The results indicated that there was H₂O₂deposition in spl42 leaves.There were obvious blue deposits in the spl42 spot leaves compared with the wild type after NBT（Nitrotetrazolium blue chloride）staining.The activities of superoxide dismutase（SOD）and peroxidase（POD）of mutant spl42 were significantly higher than the wild type.q RT-PCR analysis indicated that the expression of CATB,AOX1a and AOX1b for the reactive oxygen scavenging system gene were significantly up-regulated in the spl42.In addition,the significantly higher content of malondialdehyde（MDA）in the spot leaves for spl42 was found compared with the wild type,and the expression of death-related genes is disorder.These results indicate that there had PCD in spl42 leaves.3.The disease resistance of spl42 mutant:the results of the rice blast inoculation experiment showed that spl42 was more resistant compared the wild type when inoculated with RO1-1.The resistance-related genes of JA signaling genes including AOS2,LOX,PAD4,JAMyb,PBZ1 and WRKY45 genes in spl42 were expressed significantly higher than the wild type,but there was significantly lower in the expression of JAZ8,CHS1 and WRKY85 than the wild type.The resistance-related genes of SA signaling genes including PR1a,PR1b,PR3,PR4,PR10,NPR1,PAL2,PAL3,PAL4,PAL5 and PAL7 genes in spl42were expressed significantly higher than the wild type.These results indicate that the SPL42 may affect the expression of JA and SA signaling genes,thereby regulating the disease resistance of plants.4.Genetic analysis indicated that the spl42 mutation was controlled by a single recessive nuclear gene.The SPL42 locus was mapped to a 123 kb interval between the markers2-3.6-1 and2-3.7-3 on the long arm of chromosome 2,which contains 14 open reading frames（ORF）.Genome DNA sequencing confirmed that ORF9（Os02g0168800）was mutated and considered to be a candidate gene for SPL42.A C→T single base mutation occurred on the spl42 mutant,resulting in the replacement of alanine（Ala）by valine（Val）.Os02g0168800 encodes a porphobilinogen deaminase,which is a key enzyme in the chlorophyll synthesis pathway.The complementation,CRISPR knockout verified that Os02g0168800 is the SPL42 gene.Tissue expression experiments showed that SPL42 was constitutively expressed in various tissues,with the highest expression in mature leaves.5.The subcellular localization of protoplasts and tobacco showed that SPL42 was localized in chloroplasts.The mutation of SPL42 resulted in decreased enzyme activity and increased enzyme precursor content.RNA editing site sequencing experiment,yeast double hybrid and tobacco double molecule fluorescence experiments showed that SPL42 interacts with Os MORF8-1 and Os MORF8-2 to affect RNA editing.6.RNA-Seq results showed that most genes related to chlorophyll metabolism,carotenoid biosynthesis and photosynthesis were down-regulated,and most genes related to ROS-related flavonoids and disease resistance processes were up-regulated in spl42 mutant.In summary,the spotted lesf phenotype is controlled by a recessive nuclear gene encoding a porphobilinogen deaminase.spl42 mutant showed significant changes in physiological and biochemical parameters,which in turn promotes resistance-related genes.The study of SPL42 gene can further analyze the rice molecular mechanism of disease resistance.