Genetic Analysis And Fine Mapping Of Rice‐like Lesions Lml Mutant

Lesion mimic mutants spontaneously exhibit necrotic spot phenotype without the pathogen infection and adversity stress.At the site of lesion mimic,there are reactive oxygen species accumulated and programmed cell death,some lesion mimic usually have a enhanced resistance and senescence phenotype.Therefore,the acquisition of rice lesion mimic mutants can further analyze the mechanism of cell death and resistance.In this study,lesion mimic leaf(lml)mutant was induced with Japonica rice Wuyunjing 7 by ethyl methane sulfonate(EMS).This mutant showed a small brown lesion at the 3-leaf stage,and its formation was induced by light and high temperature.The area and number of lesions became larger with the plants developed,and the leaves showed senescence at the heading.The gene was maped in the chromosome 12 approximately 220 kb region of the M18 and M23 markers,genomic sequence analysis revealed that the LOC_Os12g16720 carries a single nucleotide transition(G-C),resulting the changing of amino acid coding,the arginine becoming threonine.The gene was verified by genomic complementation analysis and it was found that the mutant phenotype was restored,which was consistent with the wild type,indicating that LOC_Os12g16720 is the target gene of LML.Analysis of the expression pattern of the LML gene indicated that it was expressed in roots,stems,leaves,leaf sheaths and ears,and its protein was localized in the endoplasmic reticulum.Histochemical analysis and physiological analysis showed that the activity of antioxidant enzymes decreased and the accumulation of hydrogen peroxide occurred during the formation of lesions;trypan blue staining and aniline blue staining showedthat cell membrane damage and callose accumulation in mutant cells;paraffin sectioning and TUNEL assay analysis showed programmed cell death in mutant leaves.In addition,down-regulation of photosynthesis-related gene expression and up-regulation of senescence gene in mutant,and the decreasing of chlorophyll content,chloroplast degradation,indicate the senescence occure in mutant leaves.Analysis of drought treatment of wild-type and mutant,the wild type was found to have high drought tolerance.After analyzing its drought tolerance mechanism,the expression of the self-gene and drought-tolerant gene in the wild type was significantly up-regulated after drought,indicating that the LML gene may be a drought-responsive gene.In addition,in further analysis,it was found that the stomata of the wild type and the mutant remained closed after the drought treatment,and the waxy layer accumulation around the wild type stomata remained intact,the wax layer in the mutant was severely damaged,and the wax layer plays an important role in preventing water loss.In the determination of the relative water content of the cells,it was found that the water in the mutant was significantly lower than that in the wild type,suggesting that the LML gene is involved in regulating the rice drought resistance mechanism.