Regulatory Function Of Exogenous Melatonin On Anthocyanin Biosynthesis In Red Pear Fruit

Anthocyanins are important components in the peel of red pears and contribute to the appearance of the fruit.Research into anthocyanins is important for improving pear quality,health benefits,and commercial value.Melatonin is produced in all plant species and has been identified as an important plant growth regulator.In a previous field experiment,we found that melatonin treatment significantly affected pear fruit color development.However,the molecular mechanism remains unclear.This study aims to elucidate the mechanism and pathway of the influnence of melatonin on anthocyanin biosynthesis and fruit coloration in‘Nanhong’pear（Pyrus ussuriensis）,and to provide a theoretical basis for regulated method of anthocyanin biosynthesis in pear fruit.The representative results are as follow:1.The applications were performed during the pre-color-change period by spraying 200μM of melatonin on bagged pear fruits.We found that treatment with melatonin had a significant effect on color development.The concentrations of anthocyanins and flavonols were enhanced by melatonin treatment,whereas hydroxycinnamate and flavanol concentrations were reduced.Quantitative real-time polymerase chain reaction analyses indicated that the transcription levels for most anthocyanin biosynthetic genes and anthocyanin-related transcription factors were induced by melatonin.Melatonin application also stimulated the expression of melatonin biosynthesis-related genes and consequently caused an increase in endogenous melatonin concentration.These results provide insights into melatonin-induced fruit coloration and will facilitate application of exogenous melatonin in agriculture.2.Transcriptome analysis revealed PuERF27,a melatonin-induced ethylene response factor（ERF）,as a candidate gene.Transient overexpression of PuERF27 is essential for enhancing anthocyanin accumulation and activating anthocyanin biosynthesis genes,whereas PuERF27 silencing repressed anthocyanin accumulation.Yeast one-hybrid and GUS assays indicated that PuERF27 could bind directly to,and positively regulate,the PuMYB10 and PuGSTF12 promoters.Furthermore,PuERF27 together with PuMYB10 enhanced the activation of PuGSTF12 promoter.These results indicate that melatonin-induced PuERF27expression may directly promote the expression of structural genes,or indirectly through PuMYB10,thereby leading to the biosynthesis and transport of anthocyanins.3.We also found that application of exogenous melatonin increased endogenous jasmonic acid（JA）content in pear fruits.The effect of melatonin on anthocyanin biosynthesis was abolished by inhibitors of JA.After melatonin treatment,the expression of PuOPR2 was significantly up-regulated.Yeast one-hybrid and GUS assays indicated that PuERF27 bound to PuOPR2 promoter,and positively regulate its expression.Transient overexpression of PuOPR2is essential for enhancing anthocyanin and JA accumulation and activating anthocyanin biosynthesis genes,whereas PuOPR2 silencing repressed anthocyanin accumulation.4.The signaling cross-talk between melatonin and NADPH oxidase（RBOH）-mediated ROS during anthocyanin biosynthesis were investigated.We found that application of exogenous melatonin not only induced anthocyanin biosynthesis,but also increased endogenous H₂O₂ and O₂^·-content in pear fruits.The effect of melatonin on anthocyanin biosynthesis was abolished by inhibitors of RBOH.We also observed that genes encoding RBOH（PuRBOHF）,which was activated by PuERF27,were ubiquitously and highly expressed after melatonin treatment.Transient PuRBOHF overexpression significantly enhanced anthocyanin accumulation and activated transcription of anthocyanin biosynthesis genes,whereas PuRBOHF silencing repressed melatonin-promoted anthocyanin accumulation and H₂O₂ production.Moreover,RBOH-derived H₂O₂ induced PuMYB10 transcription,and PuRBOHF enhanced the PuMYB10-induced activation of the PuUFGT promoter.PuMYB10,in turn,activated PuRBOHF transcription,revealing a positive feedback loop.These results provide molecular evidence supporting the essential roles of PuRBOHF-dependent H₂O₂ in melatonin-induced anthocyanin accumulation in pears.Taken together,exogenous melatonin treatment may regulate anthocyanin accumulation of pear fruit through three ways:melatonin-activated PuERF27 may directly promote the expression of PuGSTF12,or indirectly through PuMYB10,thereby leading to the biosynthesis and transport of anthocyanins.Subsequently,PuERF27 also promotes the expression of PuOPR2,increasing endogenous JA content,thereby modulating anthocyanin accumulation.PuRBOHF are also induced by PuERF27,thereby increasing apoplastic H₂O₂ production;this apoplastic H₂O₂ then diffuses,or is transported,into the cytosol,inducing PuMYB10transcription and promoting anthocyanin accumulation.