Physiological Mechanism And Molecular Regulation Network Analysis Of Drought Tolerance In Peanut Seedling Stage

The seedling stage is the key period of vegetative growth and morphogenesis of peanut,and drought stress will cause serious interference to the yield and quality in the later period.In the face of increasingly severe drought conditions,it is the most economical and effective method to investigate the physiological and molecular mechanism of peanut response to drought stress at seedling stage,explore its biological potential of efficient use of water,and cultivate peanut varieties with strong drought tolerance without increasing irrigation input of water.In this study,drought-tolerant variety NH5 and drought-sensitive variety FH18 were used as experimental materials to explore the morphology,physiological response,transcriptional regulation and non-coding RNA of peanuts at seedling stage in the field shed and indoor light-temperature culture room.The main research results are as follows:1.Drought stress restricted the vegetative growth of peanut at seedling stage,and the growth almost stopped after 20 days of drought treatment.The main stem height of NH5 and FH18decreased by 52.01%and 56.04%compared with the control,respectively.The activities of the key photosynthetic enzymes FBA and Rubisco were significantly increased under drought stress,and the peak activities were 0.74 to 4.51 times higher than that of the control.Meanwhile,the net photosynthetic rate,stomatal conductance and intercellular CO₂ content were significantly decreased,and stomatal limit value was increased.Chlorophyll fluorescence parameters showed thatΦPSⅡof NH5 and FH18 decreased by 51.20%and 76.02%,q P decreased by 38.33%and 60.94%,and NPQ increased significantly at 20 days under drought stress.The chloroplast structure of NH5 was clear and complete at 20 days,and the thylakoid stack was significantly thickened after rehydration,but the chloroplast lamellar structure of FH18 was seriously damaged and did not recover at 5 days after rehydration.The fruit number per plant,full fruit number,100 fruit weight and 100 kernel weight of NH5 and FH18 decreased,and the average yield of the two years decreased by 31.94%and 55.92%,respectively.In addition,after drought stress at seedling stage,grain protein content increased while oil content decreased,and amino acid contents such as proline and threonine increased by 2.50%-50.01%.2.Under drought stress at seedling stage,primary reaction of photosynthesis of peanut was significantly affected.The PSI terminal receptor pool of the two cultivars became smaller,the Q_A in FH18 was over-reduced and the oxygen-releasing complex was damaged after 24h drought stress.The energy absorbed,captured and dissipated per unit reaction center of NH5increased by 6.9%～15.4%,and the characters of FH18 increased by 9.6%～33.3%,indicating the decrease of active reaction centers.The quantum yield of the reaction center decreased significantly,and the PIabs and PItotal decreased by 29.12%-39.11%and 46.72%-49.01%,respectively.ROS accumulation in NH5 and FH18 resulted in a significant increase in TBARs content.The activity of NH5 MDHAR and GR increased by 2.81 times and 6.28 times respectively,which was conducive to the increase of AsA content and the stability of AsA/DHA ratio.3.Comparative transcription analysis of FH18 and NH5 under drought stress identified5511 and 2360 sustainably differentially expressed genes,respectively.Enrichment analysis showed that the pathways of response to stimulation,antioxidant activity,plant hormone signal transduction and glutathione metabolism were significantly enriched in the two cultivars,reflecting the similarities in drought stress response mechanism.Through KEGG enrichment of differentially expressed genes at each time point,it was found that multiple pathways related to photosynthesis were significantly enriched,indicating that drought stress seriously affected the photosynthetic performance of peanuts.In addition,855 and 554 differentially expressed transcription factors were identified in FH18 and NH5,respectively.There were a large number of bHLH,MYB,HB-HD-ZIP,C2H2,and NAC families,which indicated their important roles in drought tolerance regulation of peanut.4.Non-coding RNA identification and competitive endogenous regulatory network analysis showed that 428～442 miRNAswere identified in each peanut library,and the length was concentrated in 21 nt and 24 nt.Novel miR＿73 and novel miR＿416 have been identified as key miRNAsin regulating drought tolerance of peanut through interaction networks with target genes.They improved drought tolerance by regulating the biosynthesis of keratin,suberin and wax,stomatal movement,water transport and cell wall synthesis.In addition,a total of 1021lncRNAswere differentially expressed,and 673 pairs of ceRNAswere identified in NH5,which were involved in ABC protein transport and sulfur metabolism under drought stress.Six lncRNAswere obtained from the center of ceRNAscompetitive regulation network.Their target genes regulated peanut response to drought stress by promoting plant growth and regulating the expression of drought-tolerant genes.5.The physiological characters and transcriptional regulation of peanut were analyzed by weighted coexpression network,and the genes were divided into 16 modules by hierarchical clustering tree.Through the correlation between genes and physiological traits and the comparison of gene expression patterns between varieties,three key gene modules were obtained which were significantly related to physiological traits.A large number of genes were concentrated in pathways related to transmembrane transport and photosynthesis in the module,and 21 hub nodes were identified in the competitive endogenous regulatory interaction network constructed in the module.These hub genes encode protein kinase,E3 ubiquitin ligase,potassium transporter,pentapeptide repeating protein and aspartic protease,which regulate peanut to improve drought stress tolerance.