Transcriptome Analysis Of Alfalfa In Response To Saline-alkali Stress And Functional Characterization Of MsNAC47

Alfalfa(Medicago sativa L.)is a potentially high-yielding economically valuable crop plant cultivated worldwide.Alfalfa perform highly resistant to various abiotic stresses,including freezing,salinity and saline-alkali stress.In this study,saline-alkaline stress tolerance mechanism was uncovered based on transcriptome analysis of alfalfa in response to saline-alkaline stress.Comparative transcriptome analysis of salt and saline-alkali stress in alfalfa was presented for candidate genes screen.Furthermore,MsNAC47 gene was selected as candidate gene to reveal the role of calcium metabolism regulated by NAC47 in plant salt-alkaline tolerance.1.Transcriptome analysis of alfalfa in response to saline-alkali stressDe novo transcriptome analysis was performed to elucidate the mechanism underlying plant saline-alkaline tolerance.Whole alfalfa seedlings treated with 100m M mixed saline-alkaline(Na₂CO₃:Na HCO₃=1:2)for 0 day(control),1 day(short-term treatment,1d),and 7 days(long-term treatment,7d)were used for transcriptome sequencing.A total of 4,137 differentially expressed genes(DEG)were obtained,2,286and 2,233 DEGs were found in the short-term and long-term treatment,respectively.109(1 d)and 96(7 d)DEGs were annotated as transcription factors,including AP2,WRKY,MYB and NAC gene families.DEGs were enriched in 14 GO pathway,reactive oxygen scavenging related genes were significantly induced,flavonoid biosynthesis and modification pathway were up-regulated,ion transport,ferritin,organic acid synthesis and calcium metabolic pathway genes presented higher expression.Moreover,expression of DEG in reserve carbohydrate synthesis and light pathway were inhibited.Compared transcriptome analysis between salt and saline-alkali stress showed that,8 genes were screened involving carbon assimilation,organic acid synthesis and other metabolic pathways that presented differential expression pattern under such stresses.Among these genes,MsNAC47 gene presented down-regulated under salt stress and up-regulated under saline-alkali stress,indicating that the metabolic pathway regulated by MsNAC47 gene had deversity tolerance mechanism under salt and saline-alkali stress.2.Isolation and characterization of alfalfa MsNAC47 geneThe full-length c DNA sequence of MsNAC47 gene was 1023 bp,and encoding a protein of 340 amino acids.MsNAC47 showed highly homologous with At NAC47 in Arabidopsis and presented 94%similar to Mt NAC47 in Medicago truncatula.MsNAC47gene was expressed in all tissue and the highest expression was found in flower,followed by fruit.The expression pattern analysis showed that MsNAC47 exhibited different expression patterns in roots,but similar expression patterns in leaves under saline-alkali stress.Arabidopsis homologous gene mutant nac47 was used for characterization MsNAC47 gene function.Root length assay under salt and saline-alkali indicated that absence of NAC47 gene decrease plant saline-alkali stress tolerance and may provide positive influence in salt stress tolerance.Phenotype assay under salt and saline-alkali showed that nac47 presented serious wilting and chlorosis with Col-0 when suffered 60m M Na HCO3 treated for 7 days,accompanied by higher MDA accumulation and chlorophyll degradation.However,higher salt tolerance was observed in nac47 under300m M Na Cl treatment with lower oxidative damage and chlorosis.Revertant by overexpression of MsNAC47 gene in nac47 exhibited similar phenotype with Col-0 and showed resumed tolerance to saline-alkali stress.Measurement of ion content showed that nac47 presented higher Na⁺/K⁺under saline-alkali stress,but lower of that under salt stress.However,the accumulation of Ca²⁺in nac47 was significantly lower than that in wild type under both stresses.The deletion of NAC47 gene may reduce Ca²⁺transport pathway of mutant plants,which resulted weaken tolerance to saline-alkali stresses.3.MsNAC47 regulates calcium dependent saline-alkali specific response pathwayFurther study on ion flux and Ca²⁺imaging assay showed that the deletion of NAC47 gene in Arabidopsis altered the Ca²⁺signal trait in response to stress,and reduces the efficiency of sodium transport.The strength of Ca²⁺signal and the recovery time of resting Ca²⁺concentration in the nac47 were lower than that in the wild type,and indicated that NAC47 was participated in Ca²⁺signaling pathway.Combined with transcriptome analysis and expression analysis of calcium and sodium transporters,Ms CAX3,Ms NCL1 and Ms NHX7 genes showed co-expression pattern with MsNAC47 gene under saline-alkali stress.The promoter regions of the co-expressed NCL,CAX and NHX family genes were analyzed,and the potential MsNAC47 recognition sites in the upstream promoter region were obtained.Y1H assay showed that the expression of Ms CAX3 and Ms NCL1 was regulated by MsNAC47 gene.MsNAC47 may regulate the calcium transport under salt and saline-alkali stress by regulating Ms CAX3 and Ms NCL1.