Analysis Of The Expression Profile And Molecular Mechanisms Of Alfalfa (Medicago Sativa L.) In Response To Graphene Treatments

The special structure of nanomaterials allows them to have many unique and excellent properties.With the wide application in industry and agriculture,nanomaterials will inevitably enter the environment through a variety of ways,and may further accumulate in the high nutrient level organisms through the food chain.Numerous studies have shown that nanoparticles have positive effects on plants,such as promoting seed germination,improving growth,enhancing nitrogen metabolism and photosynthesis process,as well as promoting fertilizer nutrient absorption and utilization.While,some studies have found that nanoparticles can cause toxicity to plants.When present in a large quantity,nanoparticles can adversely affect germination,plant root,leaf growth and crop biomass.In latest years,soil salinization has end up more and more serious,causing serious negative effects on agricultural production and environmental protection.Alfalfa（Medicago sativa L.）,as a high-quality perennial forage,is mostly planted in saline-alkali areas despite its developed root system and strong resistance.Its growth,development,yield and quality are also affected by saline-alkali stress.Therefore,it is a great significance to study the interaction among alfalfa,nanoparticles and ecological environment.Our research is mainly divided into two parts.In the first part,Golden Empress with weak resistance was used as the test material,and graphene with appropriate concentration was used as an important substance for alleviating salt and alkali stresses.Transcriptome sequencing was used to screen out differential expression genes that responded to salt and alkali stresses under graphene.Then enrichment analysis of GO and KEGG Pathway was performed on DEGs to explore the possible biological functions,biochemical metabolic pathways and signal transduction pathways.And the molecular mechanism of graphene improving salt and alkali tolerance of alfalfa was studied from transcriptome level.In addition,the full-length sequence and functional annotation of alfalfa transcripts were obtained by single-molecular long-read sequencing（SMRT）technology,which can be used as the reference genome for transcriptome analysis in this study.This is helpful to improve the quality of data analysis,and it can also be used to conduct in-depth studies on salt and alkali stress tolerance from the aspects of transcription factor and transcriptome structure,which could reveal the adaptive mechanism（s）of graphene improving alfalfa resistance.In the second part,using alfalfa cultivars with different resistance include Golden Empress（weak resistance）and Bara 310SC（resistance）as the experimental material in the sand culture,the leaf metabolites under moderate and severe graphene stresses were compared to reveal the changes of differential metabolite contents and metabolic pathways under graphene stress.In addition,combined with 2+3 generation transcriptome sequencing,differential expression genes responding to stress were screened out,and enrichment analysis of GO and KEGG Pathway was performed on the DEGs to explore their possible biological functions,biochemical metabolic pathways and signal transduction pathways.The transcriptome and metabonomics of two alfalfa cultivars were studied to indicate the possible mechanisms of adaptation to graphene stress.The main results are as follows:（1）It was found that the suitable concentration of graphene(5 g kg^-1)could effectively increase the fresh weight and dry weight of seedlings,increase the chlorophyll content and improve antioxidant reductase activity.Due to osmosis,ion interaction and oxidative damage to cell membranes,salt,alkali and severe graphene stresses increased the relative electrolyte leakage（RC）,hydrogen peroxide（H₂O₂）,superoxide anion（O₂·^-）and malondialdehyde（MDA）contents of alfalfa leaves and roots,whereas significantly decreased the contents of chlorophyll a and b and the activities of superoxide dismutase（SOD）,catalase（CAT）and peroxidase（POD）compared with the control.The results also showed that 5 g kg^-1 graphene had positive effects on plants under salt and alkali stresses,such as enhancing photosynthesis and improving antioxidant systems to promote nutrient uptake and increase yield.（2）A total of 19.4 GB of clean reads were generated using SMRT sequencing,including265811 CCS and 219162 FLNC,and 12960 non-redundant transcripts were obtained,of which 12389 transcripts were annotated.RNA-seq analysis revealed that 930,1114 and 880DEGs were identified in graphene-treated（C1 and C2）,graphene-salt-treated（S1,S2 and S4）and graphene-alkali treated（A1,A2 and A4）leaves,respectively.And hundreds of genes were involved in hormone signaling,photosynthesis,respiration and transcriptional regulation.Notably,5 g kg^-1 graphene-treated leaves showed improved expression patterns of specific differential genes for antioxidant defense systems and photosynthesis,and rapidly gave alfalfa higher abiotic tolerance.（3）Based on metabolome and transcriptome characteristics,alfalfa cultivars with different resistance were used to explain the physiological response of leaves to different concentrations of graphene stress.Under moderate and severe graphene stress(10 and 20 g kg^-1),leaf nitrogen metabolism,antioxidant defense system and photosynthesis were significantly affected.A total of 406 metabolites in leaves were identified by untargeted metabolomics analysis（UPLC-ESI-MS/MS）of different resistance alfalfa cultivars.Among them,62（26 up-regulated,36 down-regulated）and 110（49 up-regulated,49up-regulated）were found in Golden Empress under moderate and severe stress,respectively.Whereas 13（8 up-regulated,5 down-regulated）and 58（30 up-regulated,28down-regulated）differential metabolites were identified in Bara 310SC,respectively.Transcriptome data showed that 250003 FLNCS were generated and 65241 non-redundant transcripts were obtained.The leaves of Golden Empress and Bara 310SC obtained 7583and 6163 DEGs under moderate stress,and 9136 and 9254 DEGs under severe stress,respectively,co-expressing 1125 DEGs.Metabolome and transcriptome data indicated that the number of differentially expressed metabolites and differentially expressed genes was positively correlated with the level of graphene stress.（4）Under moderate and severe graphene stresses(10 and 20 g kg^-1),metabolome analysis showed that alfalfa adapted to graphene stress by changing the contents of amino acids and their derivatives,lipids（triglycerides and glycerophospholipids）,secondary metabolites（organic acids,isoflavones and flavonoids）,nucleotides and their derivatives.Transcriptome results indicated that key differential genes were involved in photosynthesis,antioxidant reduction,amino acid metabolism,sucrose and starch metabolism.Transcription proteomics and metabolomics comprehensive analysis revealed that differential genes and metabolites involved in amino acid,nicotinic acid and nicotinamide metabolism pathways in Golden Empress and Bara 310SC under the graphene stress were significant enrichment.This further indicates amino acid metabolic,nicotinic acid and nicotinamide metabolic pathways are the key of alfalfa to adapt to the stress of graphene metabolic pathways.