The Molecular Basis Of Salicylic Acid Regulating Alfalfa Response To Low Temperature

Alfalfa（Medicago sativa L.）is one of the most important cultivated perennial forage legume species in the world based on its high biomass yield,rich nutritional value,good palatability,high capacity for nitrogen fixation,and strong ecological adaptability.However,under extreme environmental conditions,low temperature stress is a major factor that substantially attenuates alfalfa growth,development,productivity,and distribution.Moreover,unusual abrupt temperature changes lead to serious economic losses in the winter and later spring frost events.Therefore,clarifying the mechanisms responsible for low temperature tolerance during periods of abrupt low temperature stress is important for the breeding of novel alfalfa varieties.Low temperature induced mounts of hormone changes in plants,including Salicylic acid（SA）.SA is a natural phenolic phytohormone and is a vital hormone that participates in numerous plant physiological processes,including seed germination,vegetative growth,nodulation in legumes,and stomatal closure.Furthermore,SA plays a critical role in the resistance to biotic and abiotic stress,such as low temperature stress.Thus,exogenous SA has been considered as one of the most effective methods to improve low temperature tolerance.Whereas,there were no any studies on the molecular basis of salicylic acid regulating alfalfa response to low temperature.Thus,this study compares the physiological and molecular responses in different type of cold resistant alfalfa varieties（cold-resistant Gannong No.3and cold-sensitive WL326GZ）to reveal the molecular basis in low temperature stress.In addition,we compares the difference of seed gemination,survival,physiological,hormones changes and molecular mechanism in WL326GZ between exogenous SA and control.The main research results and findings are as follows:（1）This study performed survival rates,physiological indicators,and transcriptome profiling of the leaves from two genotypes of alfalfa,cold-resistant‘Gannong NO.3’and cold-sensitive‘WL326GZ’exposure to-10°C at 0,0.5,1 and 2 hours.The results showed that the‘Gannong No.3’had a higher survival rate and antioxidant enzyme activities（SOD,POD,CAT,and APX）compared to‘WL326GZ’in freezing stress.The RNA-seq reveals there were total of 121,366 genes were identified,and 7,245 differentially expressed genes（DEGs）between the control and treated leaves.In particular,the DEGs in‘Gannong NO.3’were mainly enriched in the metabolic pathways and biosynthesis of secondary metabolites,and most of the DEGs in‘WL326GZ’were enriched in the metabolic pathways,the biosynthesis of secondary metabolites,and plant-pathogen interactions.Moreover,the weighted gene co-expression network analysis（WGCNA）showed that ATP-binding cassette（ABC）C subfamily genes and PR1（the marker gene in SA signaling pathway）were strongly impacted by low temperature stress,indicating that ABCC8 and ABCC3 are critical to develop the low temperature tolerance.Moreover,our data revealed that numerous Ca²⁺signal transduction and CBF/DREB1 pathway-related genes were severely impacted by the low temperature resistance,which is believed to alleviate the damage caused by low temperature stress.（2）Under low temperature stress at-5℃for 16 and 24 hours,this study analysis the germination rate,germination potential,vigor index,and germination index of‘WL326GZ’pretreatment with different concentration of SA.The results showed that when the SA concentration was between 300-500μM,it significantly improved the relative germination rate,relative germination potential,relative vigor index,and relative germination index of alfalfa seeds after freezing stress at-5℃for 16 hours.Whereas in 24 hours,the SA concentration at 500μM significantly improved the relative germination rate,germination potential,vigor index,and germination index.（3）The leaf samples of alfalfa seedlings pretreatment with 0μM SA（CK）and 200μM（Treatment）,which were exposed to low temperature stress（-10°C）for 0,0.5,1,and 2h and allowed to recover at normal temperature in a growth chamber for 2 days,which were detected the changes of the phenotypical,physiological,hormonic and RNA-seq transcriptome in‘WL326GZ’.The results showed that the survival rate of exogenous SA to alfalfa was higher7.66%than that of the control.And exogenous SA could significantly increase the chlorophyll content and antioxidant enzyme activities（SOD,POD,CAT,and APX）in the alfalfa leaves,but there were no significantly changes in the content of malondialdehyde（MDA）.Furthermore,exogenous SA increased the content of endogenous SA,MEJA,IAM,TAM,IAA-GLU,IAA,CTZ,Zeatin,JA-ILE,IAA-ASP,KT,GA1,and GA7,while it decreased the content of OPDA,JA,IAN,TZR,i PR,and GA4 in low temperature stress.The transcriptome analysis reveals exogenous SA could improve the accumulation of free SA in alfalfa leaves primarily through the phenylalanine ammonia-lyase pathway.Moreover,the mitogen-activated protein kinase（MAPK）signaling pathway-plant play a critical role in SA alleviating low temperature stress.In addition,the weighted gene co-expression network analysis（WGCNA）found that MPK3,MPK9,WRKY22（downstream target gene of MPK3）,and TGACG-binding factor 1（TGA1）are candidate hub genes involved in low temperature stress defense through the SA signaling pathway.Combine the results of survival rate,physiological changes and RNA-seq showed that SA could induce MPK3 to regulate WRKY22 to participate in low temperature stress for the expression of genes in SA signaling pathway（NPR1-dependent pathway and NPR1-independent pathway）.Which resulted in enhancing the production of antioxidant enzymes such as SOD,POD,and APX,to increases the low temperature stress tolerance of alfalfa plants.（4）the results of combination survival rate,physiological changes,hormonic changes and RNA-seq reveals exogenous SA improve the endogenous SA and GA content,decrease content of JA and OPDA in alfalfa leaves due to the increased of GA content leading to degradant of DELLA proteins,which in turn accelerates the activities of antioxidant to clear ROS and increased the content of SA.In addition,the degradation of DELLA proteins leads to a decrease in the expression of PDF1 gene in the JA signaling pathway.And OPDA,as a precursor of JA,is synthesized into JA via the OPR3 gene.The expression of OPR3 were decreased to the content of OPDA reduction.Furthermore,the expression of MPK4 in SA treatment is lower than in control,which improved the expression of EDS1 and PAD4,leading to increase of SA content through analysis the suppression synthesis of SA in MAPK pathway.And with the increased of SA content,the higher expression of WRKY70 in SA treatment than in control to positive regulate the SA signaling pathway and negative regulate the JA signaling pathway.Whereas in the expression of MYC2 as a hub gene in WGCNA,which opposite with WRKY70.Therefore,the function of MYC2 is opposite with WRKY70.In a word,SA synergistic GA and antagonistic JA signaling pathway to increase the survival rate in exogenous SA of alfalfa under low temperature stress.This study preliminarily explored the effect of exogenous SA on improving the survival rate,antioxidant enzyme activity,endogenous hormone content changes,and molecular basis of alfalfa in low temperature stress.And identified the key pathways and hub genes of SA in enhancing the low temperature stress of alfalfa.However,these candidate hub genes,the molecular mechanisms of SA improved the low temperature stress of alfalfa,and the patterns of endogenous hormone changes remain to be further studied.