Regulatory Mechanism Of Melatonin On Zanthoxylum Bungeanum Under Chilling Stress

Zanthoxylum bungeanum Maxim.,a plant of the Rutaceae family,is an important economic tree species with both medicinal and edible purposes.Z.bungeanum has low cold resistance and is easily affected by chilling stress,which often causes leaf wilting,flower buds,branches to freeze and even tree trunks to die.Low temperature has become an important limiting factor restricting the development of prickly ash industry.Therefore,taking effective measures to improve the cold resistance of Z.bungeanum is an important way to ensure the normal growth of Z.bungeanum and maintain high and stable yield and good quality of Z.bungeanum.As a new type of plant regulator,melatonin plays an important role in the response of plants to biotic and abiotic stresses.At present,there is no report on melatonin-mediated stress resistance of Z.bungeanum.Therefore,this study used Z.bungeanum cultivar “Hancheng Dahongpao” seedlings as the test material,and explored the effects of foliar spraying of melatonin on the physiology and gene expression of Z.bungeanum seedlings under chilling stress at a low temperature of 4℃,in order to improve the cold resistance of Z.bungeanum and provide a theoretical basis for enhancing its growth adaptability.The main findings are as follows:1.Under chilling stress for 24 h and 48 h,the maximum photochemical efficiency(Fv/Fm)and electron transfer rate(ETR)of photosystem II(PSII)treated with melatonin were significantly higher than those of single low temperature treatment,and the ETR was increased by 36.28% and 41.44%,respectively.Compared with low temperature treatment alone,melatonin treatment significantly reduced the contents of malondialdehyde(MDA)and reactive oxygen species(ROS)in leaves,indicating that melatonin treatment can alleviate the oxidative damage caused by chilling stress.2.After 48 h of stress,melatonin treatment increased peroxidase(POD)activity,which was significantly increased by 18.19% compared with low temperature treatment alone,superoxide dismutase(SOD)activity was significantly decreased by 12.27%,catalase(CAT)activity was not significantly different,indicating that melatonin enhanced the antioxidant capacity of Z.bungeanum leaves under low temperature stress.The contents of soluble sugar and sedum heptulose-1,7-bisphosphatase(SBPase)in the leaves of Z.bungeanum treated with melatonin were significantly increased by 18.53% and 59.14%,respectively.It indicated that melatonin could regulate the resistance of Z.bungeanum to low temperature by promoting the accumulation of soluble sugar,and increase the content of SBPase to improve the carbon assimilation ability.3.The results of transcriptome sequencing showed that compared with the low temperature treatment alone,melatonin pretreatment had 1630(up-regulated 678,down-regulated 952)and 2176(up-regulated 907,down-regulated 1269)genes at 6 h and 24 h,respectively.Gene Ontology(GO)enrichment analysis revealed that melatonin significantly up-regulated differentially expressed genes under early stress were mainly enriched in photosynthesis-related items,and under late stress mainly regulated metabolic processes,ligase,oxidoreductase and hydrolase activities.Pathway analysis of the Kyoto Encyclopedia of Genes and Genomes(KEGG)database showed that the up-regulated differential genes were significantly enriched in photosynthesis,carbon fixation in photosynthetic organisms,and pentose phosphate pathways under stress for 6 h.The significantly annotated pathways up-regulated in differential genes under stress for 24 h were starch and sucrose metabolism,lysine Acid biosynthesis and glycolysis/gluconeogenesis.4.Further analysis of photosynthesis and sugar metabolism pathways found that melatonin treatment up-regulated 21 and 7 photosynthesis-related genes at 6 and 24 h of stress,mainly involving the Psb gene family related to photosystem II.The genes involved in the carbon fixation pathway were up-regulated by 11 and 12,including the important regulators of the Calvin cycle,fructose bisphosphate aldolase,fructose-1,6-bisphosphatase,phosphoribulose kinase and ribulose-phosphate 3-isomerase,chloroplast transketolase,and sedum heptulose-1,7-bisphosphatase related to the synthesis of sedum heptulose.In starch and sucrose metabolism pathways,melatonin up-regulated 32 genes,including sucrose synthase related to sucrose decomposition and synthesis,α-amylase,β-glucosidase and glycogen phosphorylase genes related to starch hydrolysis.These genes facilitate the accumulation of sugar in the leaves of Z.bungeanum.