Phenotypic Characterization And Gene Cloning Of A Glume Lesion Mimic Glmm1 Mutant In Rice

Rice lesion mimic mutants spontaneously form necrotic spots in the absence of various adversity stresses,which can even cause plant death in severe cases.The production of rice lesion mimic mutants is often associated with ROS（Reactive Oxygen Species）outbreaks,uncontrolled PCD（Programmed Cell Death）,metabolic disorders,and overexpression of defense-related genes,making them ideal materials for studying the mechanisms underlying the occurrence of PCD and defense responses in rice.It is important to reveal the coordination mechanism of rice lesion mimic related genes and defense reactions in rice for breeding new disease-resistant varieties of rice.In this study,agronomic trait analysis,diaminobenzidine（DAB）staining,chlorophyll content determination,scanning electron microscopy（SEM）analysis,and silicon content determination were performed on wild-type material LR005 and glume lesion mutant glmm1（glume lesion mimic 1）,and the F₂population constructed by glmm1 and japonica rice material L422 was used as the genetic mapping population,and the glmm1 gene was localized by map cloning and BSA-seq strategy,and construct genetic complementation vectors and perform genetic transformation validation,the main findings are as follows:1.Neither glumes nor leaves of wild type and glmm1 showed lesion mimic phenotypes at the beginning stage of heading.Ten days after heading,brown spots gradually began to appear on the glumes and middle of the leaves of the mutant,and the leaf tips began to dry out.To maturity,the brown spots on the leaves continue to increase,the leaf tip dries more seriously,and the glumes are covered with brown spots,until completely brown.Investigation of agronomic traits showed that plant height,spike length,number of grains per spike,yield,and thousand-grain weight were significantly lower in glmm1.It is shown that the glmm1 mutant not only produces lesion mimic but also causes deterioration of agronomic traits.2.Using 1%DAB solution to stain the glumes and flag leaf of the wild type and glmm1at the heading stage,the results showed that the glumes and flag leaf of glmm1 were stained brown,while the staining of the wild type was not obvious.this indicates that glmm1 accumulated a large amount of H₂O₂on the glumes and leaves,suggesting that the production of glmm1 lesion mimic may be due to excessive accumulation of H₂O₂.3.The use of chlorophyll meter（SPAD-502PLUS）to determine the content of sword chlorophyll content of wild and glmm1,showing that there is no significant difference in chlorophyll content,indicating that the formation of glmm1 lesion mimic has nothing to do with chlorophyll content.4.Scanning electron microscope of the glumes and leaves of wild type and glmm1showed that the phyllosilicate and siliceous cells of the wild type glumes and leaves were intact with normal structure and smooth surface before and after the production of glmm1lesion mimic,while the phyllosilicate and siliceous cells of the glumes and leaves of glmm1 were damaged with the rough and wrinkled surface,indicating that the gene mutation of glmm1 caused the destruction of its glume phyllosilicate and siliceous cells of leaves.5.Genetic analysis showed that glmm1 was controlled by a single recessive gene.We mapped 501 individual strains of F₂population constructed by glmm1 and L422 were mapped to the 68kb between the Y3 and Y5 markers of chromosome 2,which contained10 ORFs.Sequencing revealed that only LOC＿Os02g51110 gene was mutated,causing the coding amino acid position 238 to change from Ile to Thr.The results of the Mut Map analysis were consistent with those of the map clone.Transgenic complementation confirmed that the LOC＿Os02g51110 gene was the target gene.6.The LOC＿Os02g51110 gene encodes the silicon transporter protein,which affects silicon absorption in roots.The silicon accumulation in glmm1 was reduced by the determination of the total silicon content in the glume and leaves,which further proved that glmm1’s lesion mimic phenotype was caused by mutation of LOC＿Os02g51110 gene.