Morpho-physiological Characters,gene Mapping And Transcriptome Profile Of A Leaf Lesion Mimic Mutant In Soybean

Plants have developed a unique and sophisticate molecular defense mechanisms to protect themselves from pathogen attacks,such as the hypersensitive response(HR),which induces programmed cell death(PCD),and the formation of necrotic lesion mimics,which inhibit pathogen spread in plant host tissues.Some mutants that have developed spontaneous necrotic lesions in the absence of pathogen infection,abiotic stress and mechanical damage,are called lesion mimic mutants(LMM).Localized cell death in LMM resembles that caused by HR,and most of these mutants display increased expression of pathogenesis-related genes,which results in enhanced resistance to many different pathogens and restricting further invasion or spread of pathogens to adjacent cells.To further elucidate the molecular mechanisms of the HR and disease resistance pathways,many LMMs have been identified and characterized in different plants based on differences of the lesion phenotypes.However,only a few LMMs were reported in soybean,and no related gene was cloned until now.In the present study,we isolated a new leaf LMM named spotted leaf-1(spl-1)from the progeny of an elite cultivar NN1138-2 treated with ethyl methane sulfonate(EMS).The objectives of the present study were to investigate the morphological,physiological and biochemical changes of the LMM spl-1 leaves,and to reveal the inheritance and map the target gene of the leaf lesion mimic mutant.We also implicated RNA-sequence(RNA-seq)technology to compare the transcriptome profile between spotted leaf-1(spl-1)mutant and wild-type soybean to explore the molecular mechanisms regulating lesion mimic.1.The necrotic lesions appeared to start from older leaves and then progress to young upper leaves in spl-1 mutant.Reactive oxygen species(ROS)accumulation have been involved in promoting lesion mimic phenotype.Thus,ROS scavenging enzymes,such as superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT),play significant regulatory roles in spotted leaf lesion mimic.In this study,histochemical staining revealed that lesion formation might result from PCD and excessive reactive oxygen species(ROS)accumulation.The chlorophyll content was significantly lower in the mutant leaf relative to the wild-type,and also antioxidant activities viz.,SOD,POD)and CAT,as well as the malondialdehyde(MDA)contents,were detected higher in spl-1 than in the wild-type.MDA indicator of cell membrane damage is generally present in higher levels in mutants,compared to their wild-type.This finding indicated that ROS accumulation and chlorophyll degradation in cells might be responsible for cell death and lesion formation.The DAB and Trypan blue staining assay confirmed that the spl-1 mutant was affected from a hypersensitive reaction and exhibited PCD with a visible phenotype at necrotic sites.2.According to segregation analysis of mutant phenotype in two genetic populations derived from crossing spl-1 mutant with two genotypes viz.,W82 and PI378692,the spotted leaf phenotype of spl-1 is controlled by a single recessive gene named Iml.The lm1 locus governing mutant phenotype of spl-1 was identified in 3.15 Mb genomic region on chromosome 04 through MutMap analysis,which was further verified and fine mapped by simple sequence repeat(SSR)marker-based genetic mapping.Genetic linkage analysis narrowed the genomic region(lm1 locus)between the SSR markers BARCSOYSSR₀4₁429 and BARCSOYSSR₀4₁435,covering the physical distance of?76.23 kb.Eight annotated candidate genes were found within the lm1 region.qRT-PCR expression analysis revealed that among these eight genes,only Glyma.04g242300 a homolog of plantacyanin(PLC)protein family showed highly significant expression levels in wild-type relative to the spl-1 mutant.However,sequencing data showed no nucleotide difference between spl-1 and its wild type within the coding regions of these genes.3.Results showed 588 differentially expressed genes(DEGs)in total in spl-1 vs wild-type plant leaves,including 198 up-regulated and 390 down-regulated DEGs at(log2 ratio?2,q?0.05.A broad range of Gene Ontology(GO)functions of transcripts categories were classified into Molecular Function(1337,49.30%)followed by Biological Process 810(29.87%)and Cellular Component 565(20.83%).Based on KEGG analysis 64 pathways were obtained,biosynthesis of secondary metabolites(40 DEGs)and metabolic pathways(34 DEGs)were the most enriched pathway in lesion mimic phenotype of soybean.GO and annotations suggested that many DEGs were involved in pathogenesisrelated proteins regulating salicylic acid(SA)signaling pathway,LRR proteins responsible for innate immunity,ion channel regulators,heat shock proteins(HSPs),hormone signaling as well as clathrin-associated protein.The ROS scavenging enzymes with chlorophyll degradation and photosynthesis-related differentially-expressed genes were up-regulated and down-regulated,respectively.Furthermore,many DEGs were encoded transcription factors(TFs)and essential regulatory proteins involved in signal transduction.Quantitative Real-Time PCR(qRT-PCR)analysis was performed to validate the RNA-Seq data.In total 12 DEGs were randomly selected to analyze the gene expression that was correlated with lesion mimic response and covering almost all the primary functions in various biological pathways,including biosynthesis of secondary metabolites,metabolism and plant hormone signaling transduction.Finally,from the above results obtained so far,it is possible to put forward a hypothesis for the molecular mechanisms underlying lesion mimic mutant of soybean,which would be helpful and accelerate the elucidation of their molecular mechanisms for lesion formation and enhanced/decreased disease resistance.