Morpho-physiological,transcriptomic And Genetic Analyses Of Soybean Responses To Flooding Stress

Flooding stress at the germination and vegetative stage pose a potential threat to the production and yield stability of soybeans.Plants evolved different morphological,anatomical,physiological,and biochemical adaptive features to overcome the drastic effects of flooding stress.However,the potential adaptive mechanisms for flooding tolerance are not well known.Therefore,further studies are needed to decipher the genetic basis and molecular mechanisms of flooding tolerance in soybean.In the present study,we evaluated soybean germplasms to optimize the time point and morphological indicators at the germination stage for seed flooding tolerance.For the identification of potential genetic loci,possible candidate genes,and biochemical pathways involved in seed flooding tolerance mechanism,we also performed genome-wide association study（GWAS）and transcriptome analysis in soybean at the germination stage.Furthermore,to understand the adaptive mechanisms associated with waterlogging tolerance at the seedling stage,we investigated morphological,physiological,biochemical,and gene expressional responses of two contrasting soybean lines under waterlogging stress.We also validated the function of GmPIP1;7（Glyma.14G061500）in response to multiple abiotic stress tolerance through overexpression and CRISPR/Cas9mediated genome editing approaches.1.Detection of Major Genetic Loci and Candidate Genes Controlling Seed Flooding Tolerance in SoybeanTo understand the genetic basis of seed flooding tolerance in soybean at the germination stage,we performed a genome-wide association study（GWAS）using 34,718 single nucleotide polymorphisms（SNPs）in a panel of 243 soybean accessions.Two major SNP markers,Gm₀8₁1971416.and Gm₀8₄6239716,were identified associating with seed-flooding tolerance related traits,viz.,electrical conductivity（EC）and germination rate（GR）across all environments at a significance level of-log10（p）≥3.5.Gm₀5₁000479 and Gm₀1₅3535790 SNP markers were also identified for shoot length（ShL）and root length（RL）traits,respectively.These four significant SNP markers distributed on chromosome 01,05,and 08 were considered as genetic loci for mining potential candidate genes.Within the interval of 500kb up and down streams of the four SNPs,eight candidate genes that have their function directly or indirectly related to stress defense mechanisms were identified based on gene annotation information and available literature.Also,three hub genes were identified from protein-protein interaction（PPI）network analysis for possibly regulating the seed-flooding tolerance in soybean.The findings of the present study will be useful for marker-assisted selection（MAS）in soybean breeding programs as well as gene cloning to explore the mechanisms of seed-flooding tolerance.2.Identification of Genes Associated with Seed flooding Tolerance in Wild Soybean through RNA-Seq Based Transcriptomic AnalysisTo explore the molecular mechanisms underlying seed-flooding tolerance,we investigated the transcriptome profile in root tissues of two contrasting soybean genotypes viz.,PI342618B（Seed flooding tolerant/SFT-tolerant）,and NN86-4（seed flooding sensitive/SFS-sensitive）to seed-flooding stress using RNA-Seq approach.A total of 1563 and 1958 differentially expressed genes（DEGs）were identified in SFT-tolerant and SFS-sensitive,respectively.Both Gene ontology（GO）enrichment and MapMan pathway analyses revealed that response to seed-flooding stress was mostly enriched in the DEGs that are involved in the cell wall,antioxidant activity,catalytic,and transcription factor activities as well as protein metabolism and signaling.Based on gene annotation,GO enrichment,and PPI network analysis,a total of 807 DEGs were screened from the above terms related to the stress,including response to stress,defense response,and response to stimulus for candidate gene prediction analysis.Out of them,51 genes revealing significant opposite gene expression patterns between tolerant and sensitive genotypes along with GmERFVII1,GmERFVII2&GmERFVII3,and MAPK1 were predicted as the possible candidate genes for further analysis.Using quantitative real-time RT-PCR and sequencing analysis,five of the randomly selected ten genes viz.,Glyma.01G231200,Glyma.08G083300,Glyma.06G045400,Glyma.05G215900.and Glyma.15G015100 revealed both significant opposite expression pattern and nucleotide differences,respectively,between SFT-tolerant and SFS-sensitive.These candidate genes can be used for functional studies to develop seedflooding tolerant soybean.3.Comparative Morpho-Physiological,Biochemical and Transcriptional Responses in Two Contrasting Soybean Lines Under Waterlogging StressTo understand the adaptive mechanisms of waterlogging tolerance at the seedling stage,we conducted a comprehensive comparative study of two soybean introgression lines,contrasting with waterlogging stress tolerance based on their morphological,physiological,biochemical,and gene expressional responses.Our observations showed that divergent stress tolerance mechanisms exist between the two introgression lines at morphological,physiological,and biochemical levels with waterlogging tolerant（WT）being comparatively more tolerant than waterlogging sensitive（WS）owing to its maintenance of better growth performance.Higher photosynthesis efficiency,better antioxidant potential,cellular membrane integrity,increased accumulation of proline contents under waterlogging stress identified WT to be the most waterlogging tolerant line.In contrast,increased malondialdehyde level,higher electrolyte leakage,reduced proline contents,and lower antioxidant potential,as well as lower photosynthetic rates in WS,marked it to be sensitive to the waterlogging stress.In addition,transcriptional expression analysis indicated that WT exhibited significantly increased transcripts of several categories of waterlogging-responsive genes in roots under waterlogged conditions compared to WS roots.Taken together,these findings suggest that tolerance to waterlogging is closely associated with the maintenance of high activity of antioxidant enzymes,enhanced photosynthetic performance,and increased expression of stress defensive genes.This study will be useful for the future development of flood-tolerant varieties with improved tolerance to waterlogging.4.Overexpression of GmPIP1;7 enhances abiotic stress tolerance in soybeanPlasma membrane intrinsic proteins（PIPs）are transmembrane channel proteins are involved in the transport of water and small uncharged solutes.These PIPs play an important role in plant abiotic stress responses.However,the mechanisms underlying the role of GmPIP1;7,a soybean plasma intrinsic protein-encoding gene during abiotic stress response remain unclear.In the present study,we generated overexpressing GmPIP1;7 OE1,OE2,and OE3 lines and knockout mutant lines M1,M2,and M3 to elucidate the role of GmPIP1;7 in waterlogging,drought and Cd stress tolerance in soybean.The results revealed that the overexpressing transgenic soybean plants constitutively displayed better abiotic stress survival characteristics.The gene expression of GmPIP1;7 was highly induced by waterlogging,drought,and cadmium（Cd）stress in the roots of overexpressing soybean plants,which exhibited the greatest root activity and better growth performance with fully expanded green leaves compared to wild type（WT）.Furthermore,overexpressing GMPIP1;7 soybean lines showed higher chlorophyll contents and maintained higher photosynthesis efficiency compared to wild type,whereas the opposite phenomena have been observed in the GmPIP1;7 knockout mutants.In addition,peroxidase（POD）,superoxide dismutase（SOD）,and catalase（CAT）activities and proline contents were significantly higher in the GMPIP1;7 overexpression lines compared to WT plants under waterlogging,drought,and Cd stress.These results suggested that GmPIP1;7 enhanced waterlogging,drought,and Cd stress tolerance by increasing photosynthesis e□ciency and improving the antioxidant defense system in soybean.The results of this study will help further research and a better understanding of the molecular mechanisms of waterlogging,drought,and Cd stress tolerance in soybean.