| China is a country with more than 1,000 million acres saline-alkali land.Soil salinization could inhibit plant growth by reducing the absorption of water and nutrient,and therefore affect crop yield.Alfalfa is moderately salt-resistant.In addition,its strong nitrogen-fixing ability could be helpful to improve soil quality.As a high-quality legume forage,the demand for alfalfa has increased rapidly during the development of animal husbandry in China,which caused a huge gap between alfalfa domestic supply and demand,and required import from other countries.Therefore,developing alfalfa industry and breeding new cultivars of salt-resistant alfalfa could not only make full use of the large area of salinealkali land,but also be the fundamental way to increase alfalfa yield domestically and to reduce alfalfa supply shortage.In our lab,an alfalfa cDNA library was constructed by SSH(Suppression subtractive hybridization)to acquire gene expression profile influenced by salt stress.A salt-inducible EST(express sequence tag)was found to be homologous to HD-Zip transcription factor,which indicated its possible function in salt stress response.To identify its specific function on salt stress response,and to provide theoretical support for salt-resistant alfalfa breeding,research methods from molecular biology,transcriptome,and physiological and biochemical analysis are applied in current study.The results are listed as below.We cloned this gene from alfalfa,and named it as MsHB7.Sequence analysis show that MsHB7 belongs to HD-Zip transcription factor.Transactivation assay show that it has transactivation activity.The expression profile analyses show that MsHB7 is inducible under abiotic stresses,including salt,drought and ABA treatment.Alfalfa overexpressing MsHB7 show increased salt sensitivity comparing to wild type.In addition,expressions of ABA responsive genes,including MsABI1 and MsSnRK2.6,and antioxidant enzyme coding genes,including MsCatalase4 and MspxdC,are reduced in MsHB7overexpressing alfalfa comparing to wild type after salt treatment.We systematically identified 15 HD-Zip I genes from the Medicago truncatula and 11 HD-Zip I genes from Medicago genome database.Transcription profiles of MtHD-Zip I under salt,drought and ABA treatment are constructed and show that HD-Zip I genes are either up-regulated or downregulated under theses stresses.In the meanwhile,by sequence BLAST and phylogenetic analysis,we confirmed the close evolutionary relationship between MsHB7 and MtHB7.Therefore,MsHB7overexpressing Medicago truncatula lines and two mthb7 mutants screened from Medicago truncatula Tnt1 mutant database are used for further study.MsHB7-overexpressing Medicago truncatula lines are more sensitive to salt treatment while mthb7 mutants are more resistant to it compared with wild type.The transcriptomic analysis of MsHB7overexpressing Medicago truncatula under salt stress is conducted,and 485 DEGs(Differentially expressed gens)are found "before salt treatment" versus "after salt treatment",suggesting that MsHB7 could play a negative role in alfalfa orMedicago truncatula salt stress regulation through repressing ABA signaling transduction or ROS(Reactive oxygen species)scavenging.POD(Peroxidase)and GST(Glutathione S-transferase)enzyme activity assays show that MsHB7-overexpressing Medicago truncatula,mthb7 mutants and wild type have similar enzyme activities under normal growth condition;however,under salt stress treatment,enzyme activities in MsHB7-overexpressing Medicago truncatula are the lowest,while mthb7 mutants have the highest enzyme activities.Promoter sequences analysis,qRT-PCR analysis and dual luciferase reporter assay show that MsHB7 could repress salt stress induced ABA signaling transduction through directly up-regulating the expression of PP2C genes,which are negative regulators in ABA signaling pathway.In addition,MsHB7 could also improve endogenous ABA levels by directly up-regulating the expression of NCED1,a gene coding for ABA biosynthesis ratelimiting enzyme.In summary,MsHB7 could play a negative role in salt stress regulation through repressing ABA signaling transduction and ROS scavenging.Study of MsHB7 salt responsive mechanism could provide theoretical supports for salt-resistant alfalfa germplasm creation and new cultivar breeding. |
