| Alfalfa(Medicago sativa L.)is the most widely cultivated and applied forage in the world.It has precious nutritional value and high yield.It is known as the "king of forage" and is mainly distributed in northwest,northeast and northern China.With the development of animal husbandry in southern China,the demand for high-quality green fodder is also increasing,and alfalfa,as a high-quality legume forage,is gradually promoted from northern to southern.Alfalfa is susceptible to summer high-temperature in the growing season,leading to a decline in yield and quality.Therefore,it is of great scientific significance to study the physiological and molecular mechanisms of alfalfa in response to high-temperature stress.Here,we first studied the effect of hightemperature stress on the phenotypes and physiological indicators of different varieties of alfalfa,and selected high-temperature-resistant and high-temperature-sensitive varieties of alfalfa.Second,the molecular mechanism of alfalfa's response to hightemperature stress was analyzed using RNA-Sequencing.Finally,the role of exogenous salicylic acid(SA)in mitigating alfalfa's high-temperature stress was investigated by applying SA.The research results are as follows: 1.Effect of heat stress on alfalfa growth and physiological traits To investigate the effect of heat stress,four-week-old seedlings of 15 alfalfa cultivars were exposed to 38/35 °C day/night temperature for 7 days and various growth and physiological parameters were measured.Results showed that heat stress significantly reduced the biomass,relative water content(RWC),chlorophyll content,and increased the electrolyte leakage(EL),malondialdehyde(MDA),soluble sugar(SS)and soluble protein(SP)contents of most alfalfa cultivars.Based on membership function value,Bara310SC(0.86)and Magna995(0.80)were heat-tolerant,while Gibraltar(0.24)and WL712(0.24)were heat sensitive.The above four cultivars were selected for futher validation by chlorophyll a fluorescence analysis.Heat stress significantly affected the photosynthetic activity of heat-sensitive cultivars.The overall results indicate that Bara310 SC and WL712 are heat-tolerant and heat-sensitive cultivars,respectively.2.Transcriptome analysis of alfalfa under heat stress To explore the molecular mechanisms of alfalfa's response to high-temperature stress,the 4-week-old seedlings the heat tolerant alfalfa(Bara310SC)were subjected to hightemperature treatment at 38 °C for 4 h,and the leaves before and after high-temperature treatment were used for RNA-sequencing.The results showed that a total of 177278 unigenes were obtained from the six alfalfa c DNA libraries.Compared with the control,the expression of 4649 genes in the leaves treated with high-temperature changed significantly.Among them,the expression of 2406 genes was significantly induced by high-temperature,while the expression of 2243 genes was significantly inhibited by high-temperature.The q RT-PCR analysis of 10 randomly selected differentially expressed genes verified the accuracy of the RNA-seq results.Subsequently,GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)analyses of differential genes in response to high-temperature stress were performed.GO analysis showed that these differential genes were mainly concentrated in 'protein metabolic process','oxidoreductase activity' and 'cytosolic part'.KEGG analysis found that 'ribosome','oxidative phosphorylation','aminoacyl-t RNA biosynthesis' and 'citrate(TCA)cycle' are the main pathways in response to high-temperature stress.3.Effect of SA in protecting alfalfa from heat stress damages To investigate the role of exogenous SA in protecting alfalfa from heat-induced damage,four-week-old alfalfa(cv.WL712)seedlings were treated with 0.25 m M or 0.5 m M SA for five days and then with heat stress(38/35 °C day/night)for 3 days,and various growth and physiological traits were measured.The results showed that exogenous SA pretreatment could improve leaf morphology,plant height,biomass,chlorophyll content,and photosynthetic efficiency of alfalfa under heat stress.Meanwhile,SA could alleviate heat-induced membrane damage by reducing electrolyte leakage(EL)and malondialdehyde(MDA)content,and regulate the activities of antioxidant enzymes including catalase(CAT),superoxide dismutase(SOD),and peroxidase(POD).Overall,exogenous SA improved alfalfa heat tolerance with more prominent effect at lower concentration(0.25 m M).In conclusion,the study provided basic information for understanding of the growth,physiology and transcriptome responses of alfalfa to heat stress.In addition,this study also revealed fundamental insights into the SA-mediated physiological adaptation of alfalfa plants to heat stress,and exogenous SA can be utilized to alleviate heat stressinduced damages in alfalfa.Further functional studies will be conducted to investigate the potential role of heat-responsive candidate genes or transcription factors in alfalfa thermotolerance improvement. |
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