Research On Overwintering Adaptability Evaluation And Cold Resistance Mechanism Of Alfalfa

The alfalfa planting areas in China were mainly in the northern regions.In traditional alfalfa planting areas.Low temperature stress affects the growth,development and productivity of alfalfa.The basic research on the cold-resistant mechanism of alfalfa varieties will be very important for the selection of cold-resistant varieties and the guidance of production practice.The cold resistance of alfalfa had determined by environmental factors and its own genetic factors.Cold adaptation in autumn and overwintering resistance in winter were the main cold resistance processes of alfalfa.In recent years,the research on cold resistance of alfalfa had mainly focused on the response and regulation of its physiological and molecular mechanisms to chilling stress above the freezing point.Therefore,this research was based on the natural overwintering process of alfalfa.The relationship between morphological characteristics indexes and overwintering rate was analyzed of different alfalfa varieties.Compared the changes of major cold resistance physiological indicators in their roots and crowns during the process of overwintering.Controlled experiments were conducted indoors to study the changes of relevant physiological processes in alfalfa during cold adaptation process.RNA-seq and LC-MS non-targeted metabolomics technology approaches were used to study the changes of cold-resistant genes and key metabolites in cold-adapted alfalfa at the antifreeze stage from the transcription and metabolism levels,discovering related cold resistance genes and the core regulatory network.Accordingly,the cold resistance mechanism of alfalfa was systematically studied from the morphological characteristics,physiological and biochemical processes,and the level of transcription and metabolism.The main experimental results were as follows:（1）The overwintering rate of alfalfa was correlated with its total root length,root surface area,average taproot diameter,root volume,root tips,root forks,crown depth in soil,aboveground biomass per plant,and plant height.The overwintering rate was positively correlated with the content of proline in roots and crowns during soil thawing and the content of soluble sugar in crowns at the beginning of overwintering.The principal component analysis showed that the root characteristic indexes（root surface area,root forks,total root length and root tips）were the main indicators affecting the overwintering rate of alfalfa.The changes of the main cold-resistant physiological indicators in roots and crowns during different periods of overwintering were as follows:The activity of superoxide dismutase in the roots of most varieties was higher at the end of cold acclimation and soil thaw,its average activity at soil thaw was 1.38 times higher than at the beginning of soil freezing,and the trend in the crowns was opposite to that in the roots;the activity of peroxidase in roots began to decrease at the end of cold adaptation,and its average root activity at the end of cold adaptation was 45068.32U·min^-1·g^-1 FW,but maintained high activity in crowns until the beginning of soil freezing;the activity of catalase in the roots and crowns were similar,and its activity reached to the top at the end of cold adaptation,the average catalase activities of crowns and roots at the end of cold adaptation were 673.46 and 409.77 U·min^-1·g^-1 FW respectively;The content of proline in roots and crowns were the lowest at the initial stage of cold adaptation,and reached the highest when the soil was frozen,the average roots and crowns proline content was 8.76 and 5.15 times higher at the time of soil freezing than at the beginning of cold acclimation respectively;the malondialdehyde content of roots and crowns continued to decrease during the cold adaptation stage with an average decrease of 70.86%and 51.75%respectively,during the subsequent overwintering process,its content remained stable within a certain range.‘WL319HQ’,‘Alfaqueen’,‘Sun-god’and‘AC Caribou’alfalfa varieties with developed root system and strong expansibility had better overwintering ability in the natural overwintering process.（2）During the cold adaptation process,the osmotic pressure,ascorbic acid,glutathione reduced,and glutathione oxidized contents of alfalfa continued to increase.By elevating the osmotic pressure of cytosol,alfalfa could increase the concentration of cytosol and enhance the osmoregulatory ability of tissue cells.With the increase of the contents of three antioxidant substances,alfalfa was capable of improving its antioxidant capacity.The relative conductivity all showed an increasing trend followed by a decreasing trend,indicating that the cell membrane of alfalfa was damaged in the early stage of cold adaptation,resulting in ion leakage.However,with the continuation of the adaptation process,the cold resistance of alfalfa was initially formed,which reduced the amount of ion leakage.The content of superoxide anion and hydrogen peroxide continued to increase,but the increasing trend slowed down in the late cold adaptation period（7-14 d）compared to the early period（0-7 d）.Cold adaptation caused decreases in chlorophyll a,b content,net photosynthetic rate,transpiration rate,non-photochemical quenching coefficient,actual quantum yield of photosystemⅡ,photosystemⅡmaximum photochemical quantum yield,and electron transfer rate.（3）In the HH,a cold-resistant variety,5825 differentially expressed genes were identified,while in the WL,a non-cold-resistant variety,5903 differentially expressed genes were identified.In the HH,the number of both up-and down-regulated differentially expressed genes was more than that of WL at the early stage of freezing stress（0.5h and 3h）,especially the number of up-regulated differentially expressed genes was relatively high,as well as the early response of HH to freezing stress was more rapid.The results of differentially expressed gene enrichment analysis showed that the KEGG pathways that were significantly enriched by the two varieties of differentially expressed genes together were Ca²⁺signaling,plant hormone signaling,and plant MAPK signaling,and the number of differentially expressed genes enriched in these pathways was higher.In addition,differentially expressed genes related to flavonoid biosynthesis,starch and sugar metabolism,photosynthesis had also involved in the response of alfalfa to freezing stress.The main functions of differentially expressed genes were reflected in biological processes such as signal transduction,transcriptional regulation,antioxidant synthesis and promotion the decomposition of macromolecular carbohydrates into small molecular oligosaccharides.The reason for differential cold tolerance between two alfalfa species could be the difference in expression patterns of differentially expressed genes in the biological processes of linoleic acid metabolism,phenylpropanoid biosynthesis,diterpenoid biosynthesis,glutathione metabolism and Isoflavone biosynthesis.（4）Using metabolomics analysis,a total of 62 key differential metabolites were identified,which were organic acids and derivatives（21）,phenylpropanoids and polyketides（10）,carbohydrates and carbohydrates and carbohydrate conjugates（9）,lipids and lipid-like molecules（6）,and nucleosides,nucleotides and analogues（6）.Analyzed by differential metabolite KEGG enrichment analysis,the differential metabolites of cold-resistant varieties were significantly enriched to 8 metabolic pathways,while that of non-cold-resistant varieties were significantly enriched to 13pathways.Through network analysis of differential metabolite metabolic pathways,alfalfa regulated amino acid anabolism（glutamic,aspartic）,central carbohydrate metabolism,metabolism of lipid compounds（tocotrienol,ω-3 polyunsaturated fatty acid）,phenylpropanoid anabolism（hydroxycinnamate,flavonoid）,metabolism of nucleotides and organic heterocyclic compounds,thereby regulating physiological functions such as antioxidant levels,osmotic regulation,citric acid cycle,chlorophyll synthesis,synthesis of second messenger signaling molecules and hormone regulation in alfalfa under freezing stress.Additionally,the major reason for the difference in cold resistance between the two alfalfa varieties might be the differential metabolite expression abundance of oxidized glutathione,flavonoid compounds,uroporphyrinogen III,γ-aminobutyric acid,ε-tocopherol,γ-tocotrienol and dihydrozeatin under freezing stress.In this research,the key morphological indicators of alfalfa affecting overwintering in the process of cold resistance were preliminarily identified.The key genes and metabolites of alfalfa responding to freezing stress were analyzed and screened,and a core metabolic regulatory network was constructed.This research provides a reference for further research on the cold resistance mechanism of alfalfa.