Physiological And Molecular Regulation Of Exogenous Melatonin Treatment In Loquat (Eriobotrya Japonica Lindl.) Leaves Under Drought Stress

Loquat（Eriobotrya japonica Lindl.）,a subtropical ever-green fruit tree,belongs to the family Rosaceae.Loquat is deeply loved by people based on its delicious flesh,rich nutrition and high medicinal value.Loquat has a shallow root system with few fibrous roots,and the proportion of fibrous roots to total roots is less than that of general fruit trees,so it required more water.Loquat has a certain tolerance to drought,but it is prone to drought damage during flowering,young fruit and fruit maturity during growth.Moreover,loquat is generally planted on mountain slopes with weak water retention capacity and poor irrigation conditions,resulting in a serious reduction in fruit set and yield of loquat,so seasonal drought is a major factor limiting the yield and fruit quality of loquat.Melatonin（MT）,a small molecule naturally present in plants,is effective as a growth regulator and antioxidant to alleviate abiotic stresses,including drought,but its underlying mechanisms remain largely unclear.In this study,we investigated the regulatory mechanism of melatonin regulation of drought tolerance in loquat based on histological techniques using’Huabai No.1’loquat as experimental material,and the main results are as follows:1.This study analyzed the protective effect and regulatory mechanism of MT on drought stress in grafted loquat seedlings under drought stress.The physiological results showed that MT could effectively alleviate chlorophyll degradation and malondialdehyde（MDA）accumulation in loquat leaves under drought stress.In addition,MT also improved the net photosynthetic efficiency and chlorophyll fluorescence of loquat leaves,thereby increasing the starch content.Under drought conditions,melatonin significantly reduced ABA content,but increased IAA,CTK and BR content.The metabolomic results showed that melatonin could alter different metabolic pathways such as phenylpropane metabolic pathway,galactose metabolism,flavonoid biosynthesis,flavonoid and flavonol biosynthesis to regulate the drought resistance of the plant during different stages of drought.2.The transcriptome results showed that 3600 up-regulated genes and 6005down-regulated genes were identified in the CK13/CK1 group,while 4748 up-regulated genes and 6568 down-regulated genes were identified in the M13/M1 group.KEGG enrichment analysis revealed that melatonin mainly regulates plant-pathogen interactions,plant signaling,starch and sucrose metabolic pathway,and phenylpropane biosynthetic pathways.Specifically,melatonin could induce CNGCs and CAM/CMLs in the Ca²⁺signaling pathway and down-regulate key genes involved in starch and sucrose metabolism such as Su S5 and Su S6.In addition,melatonin could trigger the IAA and CTK signaling transduction pathways by up-regulating the expression of genes such as AUX/IAAs,AHK4 and A-ARR at 1 d,whereas at 13 d of drought,melatonin could upregulate PYL4 and downregulate EBF1 and ERF1B and other related genes.In addition,melatonin also regulated a large number of transcription factors such as WRKYs,NACs,ERFs,and b HLHs.Taken together,these results provide a new perspective to study the regulatory mechanisms of MT in drought stress and the gene function analysis network of MT-mediated signaling pathways.3.From the above results,we screened for two genes specific for melatonin under drought stress:a brassinolide synthesis gene Ej DET2 specific and the WRKY family transcription factor Ej WRKY1.Further isolation and functional characterization of the loquat Ej DET2 and Ej WRKY17 genes revealed that they are both cytosolic-localized genes.Seed germination and root elongation were inhibited under ABA and mannitol treatment conditions.There was no significant difference in seed germination rate between the Ej DET2 overexpression and wild-type lines under ABA treatment,but cotyledon greening rates in transgenic lines were significantly higher than that of the wild type and was tolerant to mannitol.The seed germination rates of the Ej DET2overexpression lines were lower than that of the wild-type under 1μM ABA treatment.However,under 300 m M mannitol treatment,the Ej DET2 overexpression lines had significantly higher seed germination rates.Under ABA treatment,there was no significant difference in germination rate between Ej WRKY17 and the wild type,but there was a higher cotyledon greening rate.In addition,Ej DET2 and Ej WRKY17 had higher root elongation in mannitol-treated medium.Then,Ej DET2 and Ej WRKY17 were overexpressed in Arabidopsis thaliana and drought resistance phenotyping experiments were carried out on transgenic plants and wild-type plants.Compared to the wild type,the accumulation of reactive oxygen species(H₂O₂ and O^2-)and MDA were significantly inhibited in the transgenic plants after drought treatment.The observation of leaf stomata after ABA treatment and the analysis of expression changes of several stress response functional genes revealed that Ej DET2 and Ej WRKY17 could regulate the tolerance to drought through the ABA-dependent signaling pathway.