Effects Of Phosphorus And Potassium Fertilization On Cold Resistance And Yield Of Alfalfa And Mechanisms

Alfalfa（Medicago sativa L.）is an important leguminous forage with the largest cultivation area in the world.It plays an important role in animal husbandry production and agricultural planting structure adjustment and ecological construction in China.However,the severely cold winter is one of the main limiting factors for the development and growth of the alfalfa industry in the northeast and northwest of our country.In addition to breeding alfalfa varieties with strong cold resistance,taking necessary cultivation measures is an important means to improve the cold resistance of alfalfa and reduce the loss caused by low temperature,fertilization is a one cultivation strategy.But so far,people have focused on the research of fertilization on the production performance of alfalfa,and little is known about the effects on the cold resistance of alfalfa and the impact mechanism.In this study,Zhongcao No.3 was used as the experimental material to research the effects of phosphorus and potassium fertilizer on cold resistance,production performance,and nutrition quality of alfalfa in Siziwang county and Tuozuo county of Inner Mongolia from 2020.And alfalfa clones were used as experimental materials,the root crowns of alfalfa during the cold acclimation period after P fertilization were analyzed by transcriptome and metabolomics,to screen the differential metabolites and differential expressed genes（DEGs）under different P fertilization treatments,and to study the expression patterns of DEGs and possible metabolic pathways enriched by differential metabolites and DEGs.Results will provide a theoretical basis for revealing the mechanism of P fertilization improving the cold resistance of alfalfa.The main conclusions are as follows:（1）Within the suitable range of P and K fertilizer increased the contents of soluble sugar,soluble protein,phosphorus and potassium in the root crown of alfalfa during overwintering;and increased the overwintering rate of alfalfa,among which P fertilizer had a better effect on improving the winter survival rate.（2）Applied suitable P and K fertilizer increased alfalfa forage yield by increasing shoots plant^-1and shoot mass;and increased alfalfa’s CP,reduced the contents of ADF and NDF,improved the forage quality,but limited;and also promoted the absorption of mineral elements such as P,K,Ca and Mg,and improved the nutrient content of alfalfa.However,if excessive fertilizer was applied,the forage quality would be reduced.（3）Within the appropriate range of fertilizer for alfalfa improved the nutrient status of the soil.Application of P fertilizer increased soil organic matter,total nitrogen and available P,and decreased available K;application of K fertilizer increased soil organic matter and available K,but decreased available P;fertilization with P and K increased soil organic matter,available phosphorus,available potassium and soil p H.（4）Based on UPLC-MS/MS technology,the metabolomics analysis was performed of alfalfa during the cold-accumulation period under different P fertilization levels.There were 22 and 10 differential metabolites,13 and 6 up-regulated differential metabolites in P1 and P4 treatments compared with the control before cooling,respectively;and there were 24 and 12 differential metabolites,12 and 6 up-regulated differential metabolites in P1 and P4 treatments compared with the control after cooling,respectively.Low temperature stress produced 21,60 and 13 differential metabolites in alfalfa under three P levels,respectively,among which the up-regulated differential metabolites were 11,25 and 6,respectively.Through KEGG metabolic pathway enrichment analysis,it was found that low temperature and phosphate fertilizer mainly affected the metabolism of amino acids and carbohydrates.Further study found that phosphorus treatment could improve the biosynthesis of lactobiose,L-serine and N-acetyl-L-phenylalanine,which may be the reason that P fertilizer improves the cold resistance of alfalfa.（5）Based on the Illumina Hi-Seq 4000 high-throughput sequencing platform,the alfalfa root crowns during the cold-accumulation period after the application of P fertilizer were subjected to transcriptome sequencing analysis.Before cooling,the DEGs of alfalfa treated with low and high P fertilization were 26 and 27 up-regulated,and 25 and 137 down-regulated compared with the control,respectively.After cooling,the DEGs of alfalfa treated with low and high P fertilization were 23 and 173 up-regulated,and 26 and 51 down-regulated compared with the control,respectively.The number of DEGs under P fertilization treatments after cooling were more than that of control.GO functional annotation results were mainly concentrated in the subcategories such as metabolic process,cellular process,response to stimulus,biological regulation,cell,organelle,membrane,catalytic activity and binding.KEGG metabolic pathways were significantly enriched in the biosynthesis of secondary metabolites,starch and sucrose metabolism,linoleic acid metabolism,amino sugar and nucleotide sugar metabolism,phenylpropanoid biosynthesis,pentose and glucuronate interconversions,cysteine and methionine metabolism,alpha-linolenic acid metabolism,galactose metabolism,so the synthesis and metabolism of sugars and amino acids play an important role in the cold resistance of alfalfa,and P fertilizer can affect the expression of related genes.（6）Combined analysis of physiological indexes and WGCNA results,two related specific modules are obtained.Hub genes were screened by estimating gene connectivity in their corresponding network which might play key roles in the low-temperature response process of alfalfa.Combined analysis of transcriptome and metabolome revealed that P fertilizer could regulate the internal environment of alfalfa under low-temperature stress at the metabolic and transcriptome level.By affecting the changes in the contents of metabolites such as L-serine,isocitrate,lactobiose and N-acetyl-L-phenylalanine,the cold resistance of alfalfa improved.L-serine is mainly regulated by genes of MGL、HMT3、CAS1,BGLU11,PAT,CAS2,ACO1,SAMS2 and RFS1;N-acetyl-L-phenylalanine is mainly regulated by genes of MGL and HMT3;lactobiose is mainly regulated by the gene of RFS6.