Physiological And Molecular Mechanisms In The Difference Of Submergence Tolerance Between Wild And Cultivated Soybeans

Waterlogging stress is one of the main factors restricting agricultural production.Most crops have weak tolerance to waterlogging stress,which will cause crop growth disruption and yield decline.The main reason is that the low oxygen and weak light environment caused by waterlogging stress hinders the normal respiration and photosynthesis.The massive accumulation of CO₂leads to changes in cytoplasmic p H,the restriction of ATP and carbohydrate synthesis,and the large accumulation of ROS,which ultimately leads to the serious impact on the normal physiological and biochemical processes of plants.Therefore,it is very important to further study the physiological and molecular mechanisms of crop flooding response and explore ways or means to improve flood tolerance to ensure crop yield and promote sustainable agricultural development.In this study,wild soybean（Glycine soja Seib.et Zucc.）PI342618B（P18B）and cultivated soybean[Glycine max（L.）Merr.]Kefeng 1（KF1）were used as experimental materials.We characterized the fundamental responses difference to complete submergence in leaves of two kinds of soybean materials at the physiological and transcriptomic levels.Two members of the glutathione S-transferase gene family,Gs GSTU24 and Gs GSTU42,one member of the cyclic nucleotide gated channel gene family GSCNGC20.1 were selected as submerging-resistant candidate genes by transcriptome sequencing to study the molecular mechanism of submergence tolerance.The main results are as follows:Under the complete submergence,the physiological responses of P18B and KF1soybean seedlings were compared in terms of growth phenotype,photosynthetic characteristics,respiratory metabolism and ROS steady-state level.The results showed that the leaves of P18B and KF1 seedlings appeared withered and yellow after flooding treatment,and the number of dead cells increased significantly,while P18B suffered relatively light damage;the chlorophyll content and Fv/Fm value of the two soybean leaves decreased,and the decrease of KF1 was greater.The content of starch and soluble carbohydrates in leaves gradually decreased with the prolongation of flooding treatment time,while the activities of key enzymes involved in glycolysis,PDC and ADH,showed an upward trend.After 3 or 5 days of submergence,the above indicators in P18B leaves were significantly higher than KF1,and relatively more ATP was generated;from the analysis of the steady-state level of ROS,the activity of CAT in P18B was relatively high and maintained Relatively low MDA and H₂O₂content and O₂.^-production rate.It can be seen that the wild soybean P18B material has obvious advantages over the cultivated soybean KF1 in terms of photosynthetic capacity,anaerobic respiration metabolism and ROS homeostasis maintenance under waterlogging stress.The purpose of this study was to analyze and compare the differences between the leaves of wild soybean P18B and cultivated soybean KF1 in response to flooding stress from the perspective of transcriptomics.We used Illumina Hi Seq?2000 for transcriptomic sequencing of two soybean materials under waterlogging stress.It was found that 56.9%of the 11202 DEGs of P18B were up-regulated,while only 44.4%of the 9905 DEGs of KF1were up-regulated.Therefore,wild soybean P18B had more up-regulated DEGs involved in the response to flooding stress than cultivated soybean KF1.KEGG pathway enrichment analysis revealed that carbohydrate metabolism,energy metabolism,amino acid metabolism,vitamin metabolism,terpenoids and poly ketones metabolism were significantly inhibited in P18B and KF1 leaves under waterlogging stress.Plant hormone signaling pathway,isoflavone biosynthesis and metabolism of some amino acids were significantly improved.However,in environmental adaptation,signal transduction,gene transcription,protein translation and other processes,P18b material showed obvious advantages,that is,the number of up-regulated genes in these processes is more than the number of down-regulated genes.KF1 is the opposite.In conclusion,responses of wild soybean P18B to environmental adaptation,signal transduction and glutathione metabolism were significantly higher than those of cultivated soybean KF1 under flooding stress,which may be the reason for the different flooding tolerance of the two materials.The plant-specific tau-class of glutathione S-transferase（GSTU）has high stress-inducing expression characteristics that can play an important protective role in plant tolerance to different environmental stresses.To explore the mechanisms underlying submergence and reoxygenation tolerance in soybean plants,the transcriptomic analysis of leaves under submergence was firstly performed using the highly submergence-tolerant G.soja accession（P18B）and the submergence-sensitive G.max cultivar（KF1）,and two potential submergence-responsive GSTU genes,Gs GSTU24 and Gs GSTU42 were identified.Their physiological functions in tolerance to submergence and subsequent de-submergence were compared in Gs GSTU24-and Gs GSTU42-overexpressing hairy root composite soybean plants or transgenic Arabidopsis plants.The results showed that,overexpression of Gs GSTU24 or Gs GSTU42 could alleviate submergence and subsequent de-submergence stress on soybean hairy root composite plants or transgenic A.thaliana seedlings by enhancing antioxidant ability for leaf ROS detoxification or homeostasis reestablishment,and by improving the chlorophyll content and quantum efficiency of chlorophyll fluorescence（Fv/Fm）for maintenance of leaf photosynthetic capacity.Our results further indicate that,the genes Gs GSTU42 or Gs GSTU24 from high submergence-tolerant G.soja P18B could be considered as the vital submergence tolerance determinants for utilization in molecular breeding designs for flooding-tolerant crop improvement in the future.Through transcriptomics sequencing analysis,CNGC20.1 gene was obtained as a potential submergence-tolerant response gene.Analysis of its protein properties showed that GsCNGC20.1 has 6 transmembrane structures,and the C-terminal has a highly conserved cyclic nucleotide binding domain;this protein is a membrane-localized protein and interacts with GsCa ML and GsCa M4.The submergence tolerance function of GsCNGC20.1 was further analyzed by overexpression of soybean hairy roots and transgenic Arabidopsis.The results showed that:GsCNGC20.1 overexpression soybean rooting combination plants or Arabidopsis seedlings suffered from flooding stress are all reduced,which is mainly through increasing the leaf Ca²⁺content of the plant under submergence stress and the PDCs and ADHs in the anaerobic respiration metabolism pathway.The expression level in turn increases the activity of PDC and ADH to maintain a relatively high ATP level.In conclusion,wild soybean strain P18B has obvious advantages over cultivated soybean variety KF1 in maintaining ROS homeostasis and anaerobic respiration metabolism.Gs GSTU24/42 enhances the resistance to flooding and reoxygenation stress by participating in ROS homeostasis reconstruction.GsCNGC20.1 can improve the tolerance to waterlogging stress by regulating Ca²⁺signal transduction and further enhancing anaerobic metabolism.