Map-based Cloning And Function Analysis Of Two Lesion Mimic And Early Senescence Gene LMES3 And LMES4 In Rice

Leaf senescence of plants is a complex biological process,usually occurring at the last stage of leaf development,which is regulated by many environmental signals gene networks.Lesion mimic and early senescence mutants is a kind of ideal material to study the mechanism of plant defense response,which can spontaneously form necrotic spots similar to allergic reactions,and most can show resistance to one or more pathogens.Although,numerous studies have illustrated some mechanisms of plant leaf senescence and the defense response,but the molecular regulatory network still need to be further enriched and analyzed,which will lay the foundation for molecular design breeding and breeding new varieties of anti-premature senescence and high yield.In this study,two stably inherited lesion mimic and early senescence mutants were obtained by EMS mutagenesis of the rice variety Yundao 32(Y32)seeds.They were named as:lmes3(lesion mimic and early senescence 3)and lmes4(lesion mimic and early senescence 4).After phenotypic identification,gene mapping and cloning,resistance identification and functional analysis,the main results are as follows:1.lmes3 mutant exhibited normal phenotype in seedling stage,but root development was blocked.The rust phenotype appeared in the leaves during early tillering stage,and the number of spots increased gradually according to the growth of plants.At booting stage,the leaf tip begins to yellow and a premature senescence phenotype appears.During the heading stage,the yellowing started from leaf tip and expanded to bottom along the edge.In the mature stage,the leaf senescence was accelerated and the spots were spread and connected.Then the leaves became yellow gradually,and finally the whole plant exhibited yellow-brown and withered.The premature senescence phenotype was induced by low temperature treatment at the seedling and tillering stage in lmes3.The lmes4 mutant was also showed normal phenotype at the seedling stage and lesion mimic phenotype of leaves similar to the hypersensitive response(HR)was occurrenced at the early tillerling stage.At the same time,the leaf cholorosis was observed in the mesophyll,but the vein remained green.At the heading stage,the yellow of leaf tip aggravated in lower leave and upper functional leaf began to yellow accompanied with water-soaked spots.In the mature stage,the leaf tip presented yellow completely,and the yellowing extended from the leaf tip to the middle until the entire leaf blade over 1/3 completely withered.After 20 days of maturity,the leaves of lmes4 showed premature senescence with the leaf tip completely withered and only parts of the leaf blade remained green.Compared with the wild-type,the heading date of the lmes3 was 3 days later,and the heading date of the lmes4 was 2 days later.The plant height,seed setting rate and 1000-grain weight were all significantly reduced,but the panicle length,effective panicle number and grain number per spike were not significantly different in lmes3 and lmes4.2.Genetic analysis showed that the phenotypes of lmes3 and lmes4 mutants were controlled by single recessive nuclear genes.The LMES3 and LMES4 genes were cloned by map-based cloning.After preliminary mapping and fine mapping,the LMES3 gene was finally located in the interval of 55 kb between P27 and P33 on chromosome 5,and the LMES4 gene was located in the region of about 48 kb between M25 and M48 on chromosome 1.Through the annotation query on the RGAP(Rice Genome Annotation Project)website,the localization region of LMES3 contained 8 ORFs,and a single base mutation of G to A in the second ORF was found by sequencing,resulting in a replacement of glutamic acid(Glu)to lysine(Lys).The further transgenic complementation and overexpression experiments confirmed that the candidate gene is LMES3.The mapping region of LMES4 gene contained 7 ORFs and a single base substitution from G to A was found in the 6th ORF,causing the encoded amino acid to be replaced by tryptophan(Try)to terminate the codon which led to early termination of translation and the loss of protein function.3.LMES3 and LMES4 are two new genes involved in leaf senescence that have not been reported.Expression pattern analysis showed that LMES3 gene was expressed in all test tissues and highest in stems.Subcellular localization analysis showed that LMES3 protein was mainly located in the cell membrane.4.The decrease of chlorophyll content is an important physiological index of leaf senescence.The results showed that the leaf chlorophyll content of the lmes3 and lmes4 mutants were significantly lower than that of the wild type.The aging-induced SR((ST Y GREEN)gene plays an important role in the regulation of chlorophyll degradation and metabolism.qRT-PCR(quantitative real-time PCR)analysis showed that SGR was significantly up-regulated in leaves of lmes3 and lmes4 mutants.The decrease of chlorophyll content indicates that the lmes3 and lmes4 mutants have already entered the process of leaf premature aging at the physiological level.In the dark induction,the leaves of lmes3 and lmes4 mutants were all faster and more violent than those of the wild type,which showed that the leaf senescence were significantly accelerated in lmes3 and lmes4.5.The ultrastructure of mesophyll cells showed that the thylakoids in the chloroplasts of the lmes3 and lmes4 mutants were disordered,the slices were irregular,the number of osmiophilic particles increased,the volume enlarged,the color deepened,and the number of chloroplasts decreased.These results showed that the mutation of LMES3 and LMES4 genes affected the development of the structure of chloroplasts,which led to the decrease of chlorophyll content in the mutant and the premature senescence of the leaves.Scanning electron microscopy revealed that stomatal reduction within the leaf area of the lmes4 mutant and silicified claws arranged around the stomata,suggesting that its water transpiration was weakened,which may lead to weakening of the water absorption process due to transpirational pull.So,it may be some of the mineral salts(inorganic salts)to plant leaves by the water absorption and flow,and the normal physiological activities of leaves were blocked,resulting in premature senescence of leaves.6.Histochemical staining showed that reactive oxygen species(ROS)-superoxide anion(O2-)and hydrogen peroxide(H2O2)accumulated in leaves of lmes3 and lmes4 mutants,and malondialdehyde(MDA)content increased in leaves,which demonstrated the premature senescence of leaves had occurred in lmes3 and lmes4.In order to explore the metabolic processes of ROS in lmes3 and lmes4 mutants,the antioxidant enzymes peroxidase(POD),superoxide dismutase(SOD)and catalase(catalase)were tested.In lmes3 and lmes4 leaves,POD and SOD activity increased significantly,while catalase(CAT)activity decreased significantly,which presented the lmes3 and lmes4 mutants may be actively responding to the accumulation of O2-and H2O2,whereas the decrease of CAT activity in leaves of lmes3 and lmes4 mutants may not be sufficient to clear these extra H2O2,resulting in large accumulation of H2O2.Therefore,the accumulated ROS may play an important role in leaf senescence and defense responses in lmes3 and lmes4.7.qRT-PCR analysis showed that senescence related genes Osl2 and Osl85,senescence-associated transcription factors OsWRKY23 and OsNAC2 were all up-regulated,nuclear encoded photo synthetic system related genes rbcS,lhcA and lhcB were all down-regulated in leaves of lmes3 and lmes4.These results confirmed that the leaves of lmes3 and lmes4 mutants showed a premature aging phenotype at molecular level.8.After inoculation with Xanthomonas oryzae Zhe173(Z173),the wild-type Y32 plants showed a typical bacterial leaf blight response,whereas the mutant plants significantly increased resistance to bacterial blight.qRT-PCR analysis showed that the expression of PRla,PBZ1,and OsWRKY45 defense genes were all significantly up-regulated in lmes3 and lmes4 leaves.These results indicated that mutations in the LMES3 and LMES4 activated defense responses,resulting in increase of plant disease resistance.These results exhibited that the gene mutations of LMES3 and LMES4 lead to premature senescence and defense response.ROS(O2-and H2O2)may be involved in these two biological responses.It also shows that the LMES3 is a multifunctional protein that plays an important role in the premature aging of leaves,defense responses and abiotic stress pathways,and its biochemical mechanism mechanism remains to be further studied.