Physiological Response And Transcriptome Analysis Of Cultivated And Wild Soybean Under Salt Stress

Soybean is one of the most important food crops in China and plays an important role in Chinese food structure and diet.As an abiotic stress,salt stress can significantly limit the yield and quality of soybean grown in saline-alkaline soils.Wild soybean(Glycine soja)is a close relative of cultivated soybean(Glycine max)but has higher genetic diversity than cultivated soybean,which makes wild soybean highly adaptable to abiotic stress.It is an important and effective way to improve the saline-alkali resistance of cultivated soybean by using wild soybean germplasm resources.Deeper investigation on the salt tolerance mechanism of wild soybean can provide scientific base for better understanding and utilization of its inborn germplasm resources.In this study,wild and cultivated soybean cultivars were used as materials and the physiological responses of the soybean seedlings of the two materials to salt stress were compared.The relationship between root ion flux and ion channel response to salt stress was further explored by Microelectrode ion flux measurement.Then transcriptome sequencing for the roots of soybean seedlings were conducted to preliminarily identify the genes controlling salt resistance differentially expressed under salt stress.The main results were as follows:(1)Under 100 mmol·L-1 NaCl stress,the apparent damage of wild soybeans was less than that of cultivated soybeans,and the fresh weight,plant height and root length of the two soybeans decreased similarly.There was no significant difference in soluble protein content in roots between cultivated soybean and wild soybean after salt stress.However,the contents of other osmotic substances in leaves and roots increased significantly,and the increments of wild soybean were higher than those of cultivated soybean,especially the content of proline in wild soybean was significantly higher.SOD,POD,CAT activities and MDA contents of cultivated soybean and wild soybean increased significantly after salt stress,and the increment of antioxidant oxidase activities of wild soybean was higher than that of cultivated soybean,especially the POD and CAT activities in wild soybean root were higher than those of cultivated soybean,which also resulted in a small increase of MDA content in wild soybean root.These results indicated that wild soybean was mainly dependent on proline to regulate osmotic potential,and root POD and CAT to resist secondary oxidative damage caused by salt stress.(2)Under salt stress,K+content of cultivated soybean and wild soybean decreased and Na+ accumulated in soybean seedlings.However,the decrease of K+content of wild soybean was relatively low,and the increase of Na+ content was high in leaves but low in roots.Meanwhile,compared with cultivated soybean,wild soybean had lower K+ efflux velocity,higher Na+ efflux velocity and higher H+influx velocity.These indicated that wild soybean could maintain higher K+content in plants by reducing K+efflux,and balance K+/Na+ratio by differentiating Na+efflux velocity in leaf vacuoles and high Na+efflux velocity in roots.(3)Correlation analysis of the flux of different ions in the root system,showed that the flux of K+,Na+and Na+,H+were negatively correlated with each other.However,under the salt shock,wild soybeans may repolarize the plasma membrane through H+efflux to reduce the depolarizing K+ efflux induced by NaCl which resulted in a negative correlation between H+flux and K+flux.After inhibitor treatments,TEA(a K+channel inhibitor)inhibited K+and Na+efflux but enhanced H+absorption in wild soybeans,amiloride(a Na+/H+antiporter inhibitor)inhibited K+,Na+and H+absorption in wild soybeans,vanadate(a PM H+-ATPase inhibitor)inhibited Na+and H+absorption in wild soybeans but enhanced K+efflux.The inhibition of K+channels and Na+/H+antiporters both reduced the efflux of K+and Na+,indicating that K+and Na+were competitive,which was consistent with their negative correlation.H+absorption was enhanced by TEA,while K+efflux was enhanced by vanadate,which was consistent with the negative correlation between H+and K+flux under salt shock,indicating that the combined action of K+channel and proton pump contributed to K+retention.Inhibition of Na+/H+antiporter and proton pump both reduced Na+efflux and H+influx,suggesting that the combined action of Na+/H+reverse transporter and proton pump was conducive to Na+efflux.(4)Transcriptome sequencing technology was used in this study to further explore the molecular mechanism for the salt tolerance and the regulation of ion channel related genes in soybean.A total of 2090 and 3959 differentially expressed genes were identified in cultivated soybean and wild soybean,respectively,with 525 co-expressed genes.GO analysis showed that the differential genes of cultivated soybean and wild soybean were respectively enriched in carbohydrate synthesis and metabolic function and microtubule-based movement.KEGG pathway analysis showed that the differential genes of cultivated soybeans were enriched in phenylpropanoid biosynthesis,glutathione metabolism and other pathways,while those of wild soybeans were enriched in DNA replication and fatty acid metabolism,among which MAPK signaling pathway was more important.The above GO and KEGG results were further confirmed by GSEA enrichment analysis.The up-regulated expression of protein ubiquitination and MAPK signaling pathway of wild soybean were identified.Through WGCNA analysis of the relationship between gene expression and physiological traits,we found that the blue module had a high correlation,and the functional annotation focused on trehalose metabolic process and 2-oxocarboxylic acid metabolism.The expressions of POD and GST shared by genes related to antioxidant enzymes were up-regulated,SOD gene of cultivated soybean was down-regulated,CAT of wild soybean was up-regulated,and the upregulated range of wild soybean gene was higher than that of cultivated soybean,which was consistent with the changes of enzyme activity in tested physiological data.The cation/H+antiporter(CHX20)and protein sodium potassium root defective(NaKR2)of wild soybean with ion channel-related differential gene were downregulated,which may negatively regulate Na+transporters.K+efflux antiporters(KEA2 and KEA3),K+transporters(KT1,KT2,and POT 10),and Stelar K+outward rectifying channel(SKOR)were up-regulated in wild soybean,and these potassium channels actively maintained K+balance in wild soybean.In wild soybean root vacuolar membrane proton pump VP 1 was down-regulated,indicating that the root vacuolar membrane proton pump activity was not high.In conclusion,the osmotic regulation and antioxidant capacity of wild soybean were higher than those of cultivated soybean under salt stress,and the root ion flux was involved in regulating the physiological response of soybean to salt stress.Transcriptome analysis further confirmed that ion flux in soybean roots was related to channel protein expression.K+channel gene was up-regulated to reduce K+efflux,and cation/H+reverse transporters made Na+ efflux to balance K+/Na+ratio.The high coordination of MAPK signaling pathway and other pathways resulted in higher salt tolerance of wild soybean than cultivated soybean.