Overexpression Of NtGCN2 Enhance Salt Tolerance In Transgenic Tobacco

Salt stress is one of the main abiotic stresses that restrict crop production,which causes to decline crop yield and quality.The main damages caused by salt stress include osmotic stress,ion stress and oxidative stress.In order to survive under salt stress,plant cell usually adapt to balance osmotic,ion,and redox homeostasis.GCN2(General control non-derepressible 2)is a Ser/Thr protein kinase,it exists extensively in plants and plays important roles in the response of plants to biotic or abiotic stress.This.kinase could down-regulate protein biosynthesis by phosphorylation of the alpha subunit of eIF2(Eukaryotic translation initiation factor 2)to maintain the balance between plant growth and stress response.Although salt stress could activate the gene expression of yeast GCN2,it still unclear wether plant GCN2 involved in salt stress response and what is the mechanism.In this study,we used NtGCN2-overexpressed transgenic tobacco plants to explore the function of NtGCN2 in salt tolerance in tobacco.The research results mainly include the following aspects:Firstly,NtGCN2-overexpressed transgenic tobacco plants and K326(50-days-old)were used to analyze the main function of NtGCN2 by RNA-seq techonolgy.The results showed that a total of 454 differentially expressed genes(DEGs)were found in the NtGCN2-overexpressed lines with comparison of K326,of which 311 genes were up-regulated and 143 genes were down-regulated.The results revealed that 22 of the DEGs were annotated as salt tolerance related,antioxidant enzyme,ion transport channel protein-related,and proline synthesis genes and up-regulated in NtGCN2-overexpressed lines.suggesting that GCN2 might be involved in salt stress response.Secondly,the seed germination rate of NtGCN2-overexpressed lines were analyzed under different concentrations of NaCl(0 mM,50 mM,100 mM and 150 mM)treatment.It was showed no significant difference under low concentrations of NaCl(0 mM and 50 mM)treatment,while it showed higher seed germination rate in NtGCN2-overexpressed lines than that of the K326 under high concentrations of NaCl(100 mM and 150 mM)treatment.Among them,the germination rate of NtGCN2-overexpressed lines were showed significantly higher than that of K326(10%)under 150 mM NaCl treatment.Also,the NtGCN2 overexpressed lines was exhibited higher root length than that of the K326.Thirdly,the gene expression of NtGCN2 and salt stress response related genes were determined under the treatment of 100 mM NaCl by qRT-PCR.These results showed that gene expression of NtGCN2 increased firstly and decreased,it reached the peak about 3 h after 100 mM NaCl treatment.Gene expression of salt-tolerance related gene including NtLEA5,NtLEA14,NtLTP4,NtHAK,NtABF2 and NtSOS1 were higher in NtGCN2-overexpressed lines than that of the K326.These results indicated that NtGCN2 might up-regulate the gene expression of salt tolerance-related genes to enchance salt tolerance in transgenic plant.Finally,maximum leaf area,chlorophyll content and net photosynthetic rate were measured in NtGCN2 transgenic tobacco plants under salt stress.These results showed that maximum leaf area,chlorophyll content and net photosynthetic rate were significantly higher in the transgenic tobacco plants under 100 mM NaCl treatment.The determination of the proline(Pro)and malondialdehyde(MDA)content in the leaves of the NtGCN2 transgenic tobacco showed that compared with the K326 control,the content of free proline in the leaves of the NtGCN2-overexpressed lines were increased while the content of MDA were decreased.In.addition,NtGCN2 overexpressed line showed lighter histochemical staining,higher antioxidant enzyme activies and its gene expression.The Na+,K+,and Ca2+ ion concentrations was showed that contents of the Na+/Ca2+ in leaves of the NtGCN2-overexpressed transgenic tobacco were decreased significantly,while the K+ contents were increased.These results indicate that NtGCN2 might respond to salt stress by increasing the maximum leaf area,increasing photosynthesis rate,maintaining cell membrane stability,improving leaf antioxidant capacity and regulating intercellular ion concentration.In this study,we found that NtGCN2 is involved in the salt stress response.It promotes seed germination,increases root length and photosynthetic rate,improves leaf antioxidant capacity,maintains cell membrane stability,and regulates concentration of intercellular ions by regulating the expression of salt tolerance-related genes.This study provides new ideas for the functional research of GCN2 and the breeding of salt-tolerant varieties.