Physiological And Molecular Mechanisms Of Exogenous Melatonin Alleviating Imidacloprid Stress In Cucumber (Cucumis Sativus L.)seedlings

Plant diseases and insect pests occur frequently in cucumber（Cucumis sativus L.）plants under high temperature and high humidity.In the process of prevention and control,large amounts of pesticides are applied,which seriously affect crop growth,pollute the environment,and even concentrate in the food chain,posing a threat to the ecological environment and human health.Melatonin（Mel）,as a new plant regulatory substance,plays an important regulatory role in response to adversity stress.At present,there are few studies on the degradation and metabolism of plant pesticides by Mel.Therefore,this study analyzed the effects of exogenous MEL on photosynthesis,As A-GSH cycle,nitrogen metabolism,nutrient absorption and transcriptomics of cucumber seedlings under IMD stress,and explored the physiological and molecular regulatory mechanisms of exogenous Mel on cucumber seedlings under IMD stress.This study provided a theoretical basis for reducing IMD damage in protected cucumber cultivation.The main results are as follows:1.2.75mm-IMD significantly inhibited the net photosynthetic rate（Pn）and chlorophyll content（Chl）of cucumber plants,and affected the normal growth of cucumber seedlings.Exogenous root application of 50 mΜMel significantly increased stomatal opening,decreased MDA content and increased chlorophyll content of cucumber seedlings,effectively alleviated photosynthetic and membrane damage caused by IMD stress,and increased Mel content in roots and leaves of cucumber seedlings,which accelerated the degradation of IMD.2.Exogenous Mel significantly increased the maximum photochemical quantum yi eld（Fv/Fm）,photochemical quenching coefficient（QL）and photosystem II actual phot onic quantum yield（ΦPSII）of cucumber seedlings under IMD stress.At the same tim e,exogenous Mel treatment helped to repair the damage of chloroplast structure cause d by IMD stress,significantly reduced the number of osmiophilic particles,and mainta ined the relative integrity of thylakoid structure.In addition,the activities of 1,5-ribul ose bisphosphate carboxylase/oxygenase（Rubisco）and fructose 1,6-bisphosphatase（FB P）in leaves were significantly increased by 2.4%and 38.9%by exogenous Mel,whic h alleviated the photosynthetic damage caused by IMD stress.By decreasing the biosy nthesis of fructose,sucrose and soluble protein,the normal carbon metabolism and os motic regulation of cucumber seedlings were maintained.3.Mel treatment significantly inhibited the accumulation of H₂O₂ and O₂^·-in the leaves of cucumber seedlings under IMD stress,the activity of glutathione reductase（GR）in mesophyll cells was improved,the reduction of oxidized glutathione（GSSG）and the regeneration of reduced glutathione（GSH）were catalysed;meanwhile,Mel also enhanced the ability of dehydroascorbic acid reductase（DHAR）to accept electrons generated by GSH,reduced DHA to ascorbic acid（As A）,improved the free radical scavenging efficiency of As A—GSH cycle system,enhanced the ROS scavenging ability of cucumber seedlings,and further alleviated the oxidative damage caused by IMD stress.In addition,Mel significantly induced the activity of GST and the expression of GST1,GST2 and GST3,which promoted the degradation and metabolism of IMD and maintained the steady state of oxidation and reduction in plants.4.Exogenous Mel significantly improved the activities of nitrate reductase（NR）,nitrite reductase（Ni R）,glutamine synthase（GS）and glutamate synthase（GOGAT）in cucumber seedling leaves during nitrogen assimilation under IMD stress,and maintained the normal uptake of nutrients by plants.At the same time,exogenous Mel promoted the absorption of a large number of elements（N,P,K）,medium elements（Ca,Mg）and trace elements（Zn,Fe,Mn）in the roots,stems and leaves of cucumber seedlings under IMD stress,and the promotion effect was most significant in the roots.5.Transcriptome sequencing showed that a total of 3042 differentially expressed genes（DEGs）were identified in treated cucumber leaves.Compared with IMD-Mel+IMD,523differentially expressed genes were up-regulated and 118 genes were down-regulated.Gene Ontology（GO）analysis showed that most DEGs were annotated to transition metal ion binding,membrane assembly,and secondary metabolic processes.Kyoto Encylopeida of Genes and Genomes（KEGG）enrichment is mainly involved in glutathione metabolism,MAPK signaling pathway--plant,phenylalanine metabolism,plant hormone transduction and plant-pathogen interaction.Further analysis of DEGS in GO and KEGG terms found that exogenous Mel may promote the degradation of IMD in cucumber seedlings by regulating the expression of Lactie,Phenylalanine ammonia lyase,Respiratory explosion oxidase homologous protein,Cytochrome P450 gene,WRKY transcription factor,Mitogen-activated protein kinase,Ethylene response factor,b HLH transcription factor and MYC2 transcription factor.In conclusion,exogenous Mel can improve the tolerance of cucumber seedlings to IMD by maintaining the stability of photosynthetic system,regulating redox homeostat,promoting nutrient absorption and inducing the expression of stress resistance genes.This study provided a theoretical basis for reducing pesticide damage in protected cucumber cultivation.