Transcriptome Analysis Reveals The Mechanism Of Nitric Oxide On Regulating Drought Stress Response In Medicago Sativa L.

Alfalfa?Medicago sativa L.?is one of the most widely cultivated perennial forage legumes in the world,and play an important role in grassland-livestock for its abundant nutritional value.Drought stress as one of the main abiotic stress factors,which severely restricts the seed germination and seedling growth of alfalfa.Therefore,it is vital importance to investigate the effect of drought stress on alfalfa and drought-adaptation mechanisms,as well as finding ways to improve alfalfa drought resistance to overcome the constraints of alfalfa cultivation in arid regions.Nitric oxide?NO?is a simple gaseous signaling molecule that can easily diffuse through the cell membrane,and regulated myriad biological processes,such as plant development,growth and stress response,etc.So far,the research-related to NO mitigation stress predominantly focused on salinity stress,the species was also primarily centered on Arabidopsis thaliana,Zaa mays L.,Solanum lycopersicum,Triticum aestivum L.and Avena sativa L.However,there are few reports about NO application on forages,especially alfalfa.Furthermore,researches about NO regulated alfalfa stress-resistance were mostly at physiological and biochemical levels,the molecular mechanism of NO on alfalfa drought tolerance is still unclear,and there is need further exploration.In this study,drought stress was simulated by polyethylene glycol 6000?PEG-6000?,sodium nitroprusside?SNP?as an exogenous NO donor,and Carboxy-PTIO?cPTIO?as an endogenous NO scavenger.Different drought-tolerance types of alfalfa‘Algonquin',‘Sanditi',and‘Gloden empress'were as experimental material.Then,morphological,physiological and biochemical analysis,and transcriptome sequencing?RNA-Seq?technology were used to reveal the mechanism of NO on regulation alfalfa drought stress resistance by study the differential responses of variety drought-resistance alfalfa,germination and growth morphology,antioxidant system,carbon assimilation and carbon metabolism,and transcriptome changes of alfalfa in germination and seedling stage.The main results as follows:?1?Exogenous NO donor SNP pretreated different varieties of alfalfa under drought stress significantly decreased MDA concentration,increased proline,soluble sugar and soluble protein content,and also increased the activity of superoxide dismutase?SOD?,peroxidase?POD?,catalase?CAT?,ascorbate peroxidase?APX?.Subordinate function method was used to analysis eight resistance-related indicators,the results show that the sensitivity of three alfalfa varieties to exogenous NO in order from strong to weak was:Sanditi>Golden empress>Algonquin.Thus,we select Sanditi as experimental materials to investigate the regulation mechanism of NO on alfalfa drought resistance.?2?Exogenous SNP soaking seeds inducing the germination rate,germination potential,germination index,vitality index,dry weight and water content of the 7 days bud were increased,root length was decreased under drought stress;however,cPTIO soaking seeds inhibited seed germination,decreased bud dry weight and root length.Spraying SNP or cPTIO on alfalfa seedling leaves has no significant effects on the stem length and root length,while the number of branches was significantly increased under SNP application,the number of lateral roots was significantly decrease under cPTIO application.Furthermore,under drought stress condition,exogenous supplementation of SNP reduced the accumulation of O₂??,H₂O₂ and MDA,and increased·OH scavenging rate in germination period bud and seedling leaves,the activity of APX,glutathione peroxidase?GPX?and glutathione reductase?GR?,as well as the content of ascorbic?AsA?and reduced glutathione?GR?were increased.All of these physiological change result in oxidative damage caused by drought stress was alleviated.Conversely,supplementation of cPTIO lead to reactive oxygen levels significantly increased,antioxidant enzymes activity and antioxidant compound content were decreased,thus further accelerated drought stress damage.?3?Exogenous supplementation of SNP improved the activity of fructose-1,6-bisphosphatase?TBA?,glyceraldehyde 3-phosphate dehydrogenase?GAPDH?,ribulose-bisphosphate carboxylase?Rubisco?participate in Calvin cycle,and also increased the activity of pyruvate dehydrogenase?PDH?,citrate synthase?CS?,2-oxoglutarate dehydrogenase??-KGDH?,succinate dehydrogenase?SDH?and malate dehydrogenase?NADPH-MDH?involved in Citrate cycle.On the contrary,supplementation of cPTIO cause a down-regulation of the expression of key enzyme genes involved in calvin cycle and citrate cycle under drought stress,and the enzyme activity was decreased.This suggested that exogenous cPTIO application could suppress the speed of carbon fixation in Calvin cycle and carbon metabolism.?4?In the alfalfa germination period,RNA sequencing-based transcriptomics analysis was performed to compare the global transcriptional profiles in the bud under different treatments.There were 3524 different expressed genes?DEGs?between PEG treatment and control?FDR<0.01,FC?2?,According to GO and KEGG pathway enrichment analyses,DEGs were significantly enriched in isoflavonoid,flavonoid,phenylpropanoid,monoterpenoid,sesquiterpenes and triterpenes biosynthesis;photosynthesis,photosynthesis-antenna proteins,starch and sucrose metabolism,glutathione metabolism and plant hormone signal transduction pathway.There are 2208 DEGs were found between PEG+NO and PEG treatment?FDR<0.01,FC?2?,and the DEGs were significantly involved in phenylpropanoid biosynthesis,nitrogen metabolism,inositol phosphate metabolism,as well as starch and sucrose metabolism.There are3461 DEGs were found between PEG-NO and PEG treatment?FDR<0.0001,FC?2?,endogenous NO-mediated DEGs mainly involved in citrate cycle,pyruvate metabolism,glyoxylate and dicarboxylate metabolism,arginine biosynthesis and metabolism,and carbon fixation in photosynthetic organisms pathway,in addition,mostly DEGs were down-regulated.?5?In the seedling period,RNA sequencing-based transcriptomics analysis was performed to compare the global transcriptional profiles in the seedling leaf under different treatments.There were 947 DEGs between PEG and CK treatment were observed?FDR<0.01,FC?2?,most of these DEGs were involved in carotenoid and flavonoid biosynthesis.A total of 852 DEGs were identified between PEG+NO and PEG treatment?FDR<0.01,FC?2?,and GO and KEGG analyses revealed that most DEGs involved in biosynthesis of secondary metabolites?such as phenylpropanoid,stilbenoid,diarylheptanoid and gingerol,flavonoid,carotenoid?,and almost all genes were down-regulated.Number of DEGs identified between PEG-NO and PEG treatment were 2891?FDR<0.01,FC?4?,most of genes participated in carbon fixation in photosynthetic organisms,carbon metabolism,citrate cycle,oxidative phosphorylation and alanine,aspartate and glutamate metabolism.