The Mechanism Of GhNAC1 Regulating Salt Tolerance In Cotton

The area of saline land in China is approximately 100 million hectares,leading to negative effects on the growth and yield of crops due to soil salinization.Cotton,an important cash crop in China,is known for its strong salt tolerance and is often grown as a"pioneer crop"in saline and alkaline areas.However,the salt tolerance of cotton is limited,and its yield is also affected by salt stress.Genetic engineering is,therefore,an important approach to develop new salt-tolerant cotton germplasm and explore and develop saline and alkaline land.NAC,a transcription factor in plants,plays a crucial role in regulating drought resistance and salt tolerance.Overexpression of genes such as Arabidopsis At NAC019/055/072,rice ONAC022,and tobacco Sl NAC35 have been found to improve plant stress tolerance in model plants like Arabidopsis and rice.Although cotton has a large number of NAC genes,there are few reports on the mechanism of NAC genes regulating salt tolerance in cotton plants,and the specific regulatory mechanism is still not clear.In our laboratory,during the early stages of the cotton salt stress transcriptome,we discovered that several NAC transcription factor genes in cotton were induced by salt stress,with GhNAC1being highly induced.Therefore,we cloned the GhNAC1 gene,constructed a GhNAC1 gene silencing vector,and infected cotton seedlings.We found that under Na Cl treatment,the salt tolerance of the GhNAC1 silenced cotton seedlings decreased.To further study the mechanism by which GhNAC1 regulates cotton salt tolerance,our laboratory constructed a GhNAC1 gene overexpression vector and transformed cotton,obtaining transgenic cotton with excess expression of the GhNAC1 gene.Based on the preliminary work,this paper screened homozygous transgenic cotton seedlings and obtained two stable transgenic homozygous cotton seedlings,L2-3 and L3-7,with excess expression of the GhNAC1 gene.Using these homozygous cotton seedlings,we conducted in-depth research and revealed the mechanism by which GhNAC1 regulates cotton salt tolerance.The main experimental results are as follows:1.Cotton seedlings were treated with Na Cl,PEG,ABA and H₂O₂respectively.After 6 hours of treatment,the expression of GhNAC1 gene in cotton leaves and roots was measured.It was found that Na Cl,PEG,ABA and H₂O₂could significantly increase the expression of GhNAC1gene in leaves and roots,indicating that GhNAC1 gene was induced by drought and salt stress,and ABA and H₂O₂signals might participate in the induced expression of GhNAC1 under drought and salt stress.2.GhNAC1 gene can induce the expression of ABA synthesis gene Gh NCED5,improve the ABA content in plants,and enhance the salt tolerance of transgenic cotton seedlings.Under normal conditions,the ABA content between the homozygous transgenic GhNAC1 transgenic lines L2-3 and L3-7 and wild-type cotton was not significantly different.The salt stress treatment significantly increased the Gh NCED5 gene expression and ABA content of transgenic lines L2-3and L3-7 and wild-type cotton;however,the ABA content and Gh NCED5 expression of ABA synthesis genes of transgenic lines L2-3 and L3-7 were significantly higher than WT,indicating that GhNAC1 gene could increase ABA content by promoting Gh NCED5 expression under salt stress.3.GhNAC1 gene can induce the expression of Gh SOS1,Gh NHX1 and Gh NHX6,thus reducing the content of Na⁺and ionic toxicity.Salt stress treatment significantly increased the gene expression of Gh SOS1,Gh NHX1 and Gh NHX6 in transgenic lines L2-3 and L3-7 and wild-type cotton,increased the Na⁺content of cotton and decreased the K⁺content.However,the expression of Gh SOS1,Gh NHX1,and Gh NHX6 genes in L2-3 and L3-7 of two transgenic cotton under 200 m M was significantly higher than WT control;compared with the WT,the Na⁺content in transgenic lines L2-3 and L3-7 decreased by 29.76%and 23.28%,respectively,and K⁺content increased by 29.87%and 30.26%,respectively.These results indicated that overexpression of GhNAC1 gene could improve Gh SOS1,Gh NHX1 and Gh NHX6 gene expression under salt stress,thus reducing the Na⁺content to reduce ion toxicity.4.GhNAC1 gene can improve the antioxidant capacity of plants and reduce the content of H₂O₂and MDA.Salt stress significantly increased the antioxidant enzyme SOD activity,H₂O₂content and MDA content of WT and transgenic lines L2-3 and L3-7 cotton seedlings.However,compared with WT,the content of SOD in transgenic plants L2-3 and L3-7 under salt stress increased by 71.86%and 77.30%respectively,and the content of H₂O₂decreased by 15.00%and15.95%respectively.These results showed that overexpression of GhNAC1 gene could increase the activity of antioxidant enzyme SOD and reduce the content of ROS in cotton,thus reducing the stress of ROS.5.In order to further explain the molecular mechanism of GhNAC1 gene improving salt tolerance of cotton.We selected WT,VIGS-GhNAC1 gene silenced cotton seedlings and GhNAC1 gene overexpressed cotton seedlings for transcriptome analysis.30 differentially expressed genes related to salt stress,and 15 stress-related WRKY,NAC,GATA stress transcription factor genes were screened.These genes may play a role in the downstream of GhNAC1 gene and regulate the salt tolerance of cotton.The specific role of these genes will be further studied in the future.Overall,this study demonstrated that salt and drought stress can induce the expression of the GhNAC1 gene in cotton,and overexpression of the GhNAC1 gene can improve salt tolerance in cotton.This is primarily due to the fact that GhNAC1 gene overexpression can enhance the expression of Gh SOS1,Gh NHX1 and Gh NHX6,reducing Na⁺concentration in cotton leaves and mitigating ion toxicity in the plant.Additionally,GhNAC1 gene overexpression can also increase ABA content,improve stress tolerance in cotton,and enhance reactive oxygen scavenging ability,reducing H₂O₂content and ROS in cotton.Overall,this study elucidated the mechanism by which the GhNAC1 gene improves salt tolerance in cotton,identified new GhNAC1 gene cotton germplasm with enhanced salt tolerance,and provided new salt-tolerant germplasm materials for the cultivation of new varieties of salt-tolerant cotton.