| Low temperature in early spring is the key factor limiting the yield and quality of peanut in northeast China,which mainly occurs in the two stages of germination and seedling.In agricultural production,the selection and breeding of cold-tolerant varieties is the most direct and effective means to solve the problem of chilling injury.Therefore,it is very important for peanut production in northeast China to screen cold-tolerant germplasms and reveal the mechanism of peanut cold tolerance at germination and seedling stages.In this study,68 peanut varieties(lines)mainly planted in northeast China were used as experimental materials,and their cold tolerance was evaluated at germination and seedling stages in the climatic chamber and field,respectively.Then,using cold-tolerant and cold-sensitive varieties,the cold tolerance mechanism of peanut was studied at physiological,biochemical,and molecular biological levels through morphological and physiological indexes,high-throughput transcriptome sequencing and lipidome detection,and the main findings were as follows:1.A comprehensive evaluation system of peanut cold tolerance was constructed by evaluating the cold tolerance of 68 peanut germplasm in germination,seedling and field.At germination stage,the measured results of seed vigor of 68 peanut germplasm after treatments at 10℃,8℃,6℃ and 4℃ for 7 days indicated that the cold treatment at 6 ℃ for 7 days can be used as the suitable condition for cold tolerance evaluation of large-scale peanut germplasm.And a total of 18 cold-tolerant peanut germplasm,38 intermediate peanut germplasm and 12 cold-sensitive peanut germplasm were screened.At seedling stage,leaf area,shoot fresh weight and cold tolerance grade were most closely related to peanut cold tolerance and can be used as evaluation indicators for cold tolerance.In the field,cold tolerance of various peanut germplasm was evaluated by emergence rate,emerging ability and yield component factors.Finally,the most cold-tolerant peanut variety NH5 and the most cold-sensitive peanut variety FH18 at both germination and seedling stages,suitable for planting in northeast China,were identified.2.Cold stress can significantly inhibit the growth and development of peanut seedlings,in which the degree of inhibition in the aboveground part was significantly greater than that in the underground part.Cold stress can also increase the membrane permeability and electrolyte leakage rate in peanut leaves,while NH5 can maintain cell osmotic balance by increasing soluble protein and free proline content.Under cold stress,the content of MDA,O2·-,and H2O2 in NH5 and FH18 increased,but the growth level in NH5 was less than that in FH18.It was because that the activities of SOD,POD,CAT,and APX in NH5 were always at higher levels,which inhibited the accumulation of reactive oxygen species.And the increased activities of CAT and APX may be the main reason for keeping intracellular H2O2 at a low level all the time.Under cold stress,the stomatal limiting factor was the main reason for the decrease of photosynthetic rate.The chlorophyll fluorescence parameters Fv/Fm,ΦPSII,q P and ETR also showed significant declines,while NH5 could inhibit the decrease of photosynthetic activity and electron transfer rate in PSⅡ during the early stage of cold stress,thereby maintaining the photochemical efficiency.Under cold stress,the leaf cell structure of FH18 was significantly damaged,mainly reflected in the destroyed membrane system and chloroplast structure,the increased number of mitochondria and the accumulation of toxic substances.Conversely,NH5 can keep the integrity of cell structure under cold stress,thus maintaining the stability of membrane system and normal photosynthesis.3.Based on the comparative transcriptome analysis of cold-tolerant variety NH5 and cold-sensitive variety FH18 under cold stress,a total of 759 peanut cold-tolerance genes were identified.Functional enrichment analysis of differentially expressed genes(DEGs)showed that peanut cold responsive genes were significantly enriched in plant hormone signal transduction and plant pathogen interaction pathways,while peanut cold-tolerance genes were significantly enriched in membrane lipid metabolism and fatty acid metabolism pathways.A total of 59 lipid-related genes regulating peanut cold tolerance were identified,and mainly involved in the synthesis of stored lipid TAG,membrane lipid metabolism and fatty acid metabolism,and most of them were significantly up-regulated.In addition,445 peanut cold-tolerance transcription factors(TFs)belonging to 69 families also were identified,of which b HLH,MYB,NAC,ERF,WRKY and C2H2 families had the most TF members and 76.62% were up-regulated.Functional enrichment results showed that cold-tolerance TFs were most significantly enriched in plant hormone signal transduction pathway.Among them,arahy.108W4S(MGP),arahy.FK7XM9(SAP11),arahy.2MPX2Y/arahy.8LB9ZD(DREB2C)can regulate peanut cold tolerance through ABA signal transduction pathway,while arahy.J8HG2W(ICE1),arahy.0RH9QK(MYC2)and arahy.2KRW4X(MYC4)can enhance peanut cold tolerance through JA signal transduction pathway.4.Based on comparative lipidomic analysis,a total of 20 lipid components and 168 lipid molecular species were detected from peanut leaves.Under cold stress,MGDG,PC,PG,PE and LPG in peanut leaves were significantly reduced,and the decline was greater in FH18.Instead,PA,PI,PS,LPC and LPE all increased,and the increase of PA in FH18 was significantly higher than that of NH5.In addition,DGDG,SQDG,DAG and TAG in NH5 also increased significantly.The calculated results of DBI showed that,under cold stress,the membrane lipid unsaturation of NH5 increased significantly,while that of FH18 decreased significantly.The decrease of C36:6-MGDG content was the main reason for the lower membrane lipid unsaturation,while the substantial increase of C36:6-DGDG content in NH5 was the main reason for the higher membrane lipid unsaturation.Moreover,a total of 22 fatty acid components were detected from peanut leaves by GC-MS.Of which,C16:0,C16:1,C18:0,C18:1,C18:2,C18:3 and C20:4 were the main fatty acids in peanut leaves.Under cold stress,the total fatty acid content,unsaturated fatty acid content and fatty acid unsaturated index increased,while the saturated fatty acid content decreased significantly,and the changed levels in the cold-tolerant variety were higher.5.Based on the integrated analysis of transcriptome and lipidome,a lipid metabolism regulation network of peanut cold tolerance was constructed.Under cold stress,the phospholipid synthesis pathway in endoplasmic reticulum,and the galactolipid synthesis pathway and the α-linolenic acid metabolism pathway in chloroplast were activated,besides,most of the genes that catalyze these reactions were up-regulated.The upregulation of PAP1 and CDS1/2 under cold stress can inhibit the damage of membrane lipid peroxidation caused by excessive accumulation of PA.The upregulation of MGD,DGD1 and SQD2 caused the increase of DGDG and SQDG content,which was crucial to the maintenance of chloroplast structural integrity and normal photosynthesis.α-Linolenic acid metabolism and fatty acid β-oxidation may improve peanut cold tolerance by regulating the JA signal transduction pathway. |
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