| Rice（Oryza sativa L.）is one of the most important crops in the world.During the development,rice suffers various biotic or abiotic stresses,such as temperature,humidity,light,and various pathogens.These kinds of biotic and abiotic stresses usually cause ROS burst,PCD,metabolic pathway disorder,callose accumulation,and increased expression of genes related to defense response in rice,thus resulting in the occurrence of rice lesion mimic phenotype.The rice lesion mimic mutants（LMM）usually spontaneously form necrotic spots of different sizes and colors on its leaves,leaf sheaths,stems or seeds.Most of the rice lesion mimic mutants have enhanced resistance to pathogens.Therefore,rice lesion mimic mutants are excellent materials for studying the mechanism of programmed cell death and defense stress response in plants.The study of genes that cause rice lesion mimic phenotypes is of great significance for understanding the disease resistance mechanism in rice.The new lesion mimic mutant lml11 in this study was identified from japonica rice variety ZH11 by EMS mutagenize.We have carried out the study of lml11 mutant in different aspects including phenotypic analysis,physiological and biochemical analysis,seed filling,rice quality,seed germination analysis,disease resistance analysis,map-based cloning and functional analysis,bioinformatics and transcriptome sequencing analysis.Through these studies and analyses,we understand the mechanism of the lml11 lesion mimic mutant occurrence and disease resistance,which provides some theoretical references for further research on rice lesion mimic mutants.The main results are as follows:1.The phenotypic characteristics of the lml11 mutants:the lml11 mutant began to appear reddish-brown necrotic spots from the three-leaf stage.During the tillering stage,the necrotic spots on the leaves of the lml11 mutant were more obvious and denser.During the heading stage,the leaves of the whole plant of the lml11 mutant were rapidly covered with necrotic spots around two or three weeks.The necrotic spots led to the rapid death of the lml11 mutant leaves.The investigation of agronomic traits showed that some main agronomic traits of the lml11 mutant,such as the plant height,tiller number,panicle length,number of primary branch,number of secondary branch,number of grains per panicle,and seed setting rate decreased significantly.2.The physiological and biochemical characteristics of the lml11 mutant:the contents of photosynthetic pigments（such as chlorophyll a,chlorophyll b and carotenoids）of the lml11 mutant leaves were significantly lower than those in the wild-type leaves.The detection of photosynthesis-related indicators showed that the net photosynthetic rate,stomatal conductance,transpiration rate and intercellular CO2concentration of the lml11 mutant leaves were significantly reduced compared with the wild type.DAB staining showed that the leaves of the lml11 mutant exhibited brown sedimentation.NBT staining showed that the leaves of the lml11 mutant exhibited blue sedimentation.In the H2DCFDA probe staining,the leaf stomata were dyed green fluorescence in the lml11 mutant.The results of physiological indexes showed that the contents of H2O2 and MDA of the lml11 mutant increased significantly,and the activity of CAT enzyme of the lml11 mutant decreased significantly.In conclusion,these results indicated that a large amount of ROS was accumulated in the leaves of the lml11 mutant.Both TUNEL detection and comet experiment showed that the leaves of the lml11 mutant contained a large amount of programmed cell death.The shading experiment and 6-BA treatment proved that the dark conditions lead to the rapid degradation of chloroplasts and the rapid cell death in the leaves of the lml11 mutant,indicating that the lesion mimic phenotype of the lml11 mutant may be dark-induced.3.Grain filling rate and rice quality of the lml11 mutant:compared with the wild type,some of the seeds of the lml11 mutant were almost non-filling,and the other part of the seeds could not be fully filled.The lml11 mutant exhibited a markedly slower grain filling rate that led to decreased significantly grain fresh weight and dry weight at the same period.In addition to the length,the width,thickness and 1000-grain weight of the lml11 mutant seeds were significantly lower than the wild type.And the brown rice of the lml11 mutant have further significant decrease in length,width,thickness and 1000-grain weight than the wild type.The grain quality is affected by the filling and 1000-grain weight.The seeds of the lml11 mutant showed more chalkiness,and opaque.Compared with the wild type,the cross-section of the endosperm of the lml11mutant showed columnar protrusions.The starch granules of the lml11 mutant were spherical or irregular in shape with different sizes,and they were loosely arranged with obvious gaps.The total starch content,amylose content and soluble sugar content in the lml11 mutant were significantly lower than those of the wild type seeds.The protein contents and gelatinization temperature in the lml11 mutant were higher than those in the wild type.4.Seed germination analysis of the lml11 mutant:the mutation of LML11 gene causes the seeds fail to germinate by the normal way of soaking.By using 1μM,5μM and 10μM concentration GA solution treatment,the germination rate of the lml11mutant seeds was significantly increased.After GA treatment for 7 days,the germination rate of the lml11 mutant seeds was even increased to 55%by 1μM and 5μM GA.However,when the GA concentration reached 10μM,the root length and bud length of the lml11 mutant and wild type germinated seeds were inhibited in varying degrees.5.Gene mutation causes the lesion mimic phenotype of the lml11 mutant:the lesion mimic phenotype of the lml11 mutant is controlled by a pair of single recessive genes.By map-based cloning method,we located LML11 to a range of 61.3 kb between the molecular marker M5 and M6.Sequencing revealed that the 277th base G on the CDS of LOC＿Os11g40590 was mutated to base A,which caused the transformation of the 93rd amino acid from valine to isoleucine in the amino acid sequence.The whole genome sequence of the LML11 gene was introduced into the mutant,and the phenotype of the mutant was restored.When the LML11 gene was knocked out in the wild type,the transgenic plants also showed a lesion mimic phenotype.Therefore,LOC＿Os11g40590 is LML11 gene.Tissue expression experiments showed that LML11 was expressed in all tissues.GUS transgenic staining also showed that the LML11 gene was expressed in the embryo of germinating seeds.The expression analysis of the LML11 gene in grain filling showed that its expression reached the highest level around 9 days after filling.The subcellular localization results indicated that LML11 localized in the cytoplasm and the nucleus.6.Bioinformatics analysis of the LML11 gene:the CDS of the LML11 gene has2793 bases and encodes 930 amino acids.The N-terminal of LML11 protein sequence contains a glycine-rich sequence,indicating that the LML11 protein may belong to GRDP（a glycine-rich protein）.Multiple sequence analysis showed that the homologous sequence of LML11 protein had a conserved and unknown functional region in several monocotyledons,dicotyledons and bryophytes,indicating that the functional region was very conserved throughout evolution.We found that the mutant site of the LML11 gene in the mutant was just located in this conserved unknown functional region.Therefore,we speculated that the occurrence of the lesion mimic phenotype of the lml11 mutant was related to the loss of function of the conserved unknown region.7.The disease resistance of the lml11 mutant:compared to the wild type,the lml11 mutant displayed enhanced resistance to the two Xoo races PXO99A and PXOzhe173.However,the resistance of the three overexpression lines to the PXO99A and PXOzhe173 was not significantly different from that of the wild type.Compared to the wild type and three overexpression lines,the expression levels of resistance related genes PR1a,PR1b,PBZI,NAC and PAL in the lml11 mutant were significantly increased.8.Transcriptome sequencing analysis of the lml11 mutant:transcriptome data showed that the transcriptome sequencing quality of the wild type and lml11 was qualified and reproducible.The DEGs analysis of the lml11 mutant and wild-type showed that the expressions of 2149 genes were decreased,and 3380 genes were increased in the lml11 mutant.The expressions of most of the genes involved in plant immune response of the lml11 mutant were significantly higher than that of wild type.The GO enrichment results of the lml11 mutant and wild type showed that the biological processes related to ROS accumulation,including oxygen binding（GO:0019825）,metabolic process（GO:0009607）,secondary metabolic process（GO:0019748）,response to stress（GO:0006950）and kinase activity（GO:0003824）,were significantly enriched.The enrichment results of KEGG pathway showed that the photosynthesis and carbon fixation in photosynthetic organisms were enriched,indicating that the occurrence of the lesion mimic phenotype of the lml11 mutant affected the photosynthesis of leaves. |
