| Quality-related characteristics of pepper（Capsicum spp.）are prone to change during postharvest storage,such as discoloration,nutritional quality and loss of fruit hardness,which will result in the shrinkable shelf life and reduced value of edible and commodity.The contents variation of chlorophyll and carotenoid in the fruit is an important reason for the color change of pepper after harvest.As a new biological preservative,melatonin has been widely used in a variety of vegetables,but it has rarely been reported in pepper.In order to delay color change of postharvest pepper fruits and prolong their shelf time,the 75 pepper germplasm resources were collected and evaluated in this paper.Two fresh pepper fruits（S01 and S59）were treated with exogenous melatonin and its effects on quality of pepper fruits were analyzed through monitoring quality variation during storage period and carotenoid metabolism pathway.Afterwards,transcriptome sequencing technology was applied to excavate the key transcription factor Ca RAP2-12,which was proved to adjust the carotenoid synthesis by melatonin.Furthermore,it was preliminarily analyzed that the molecular mechanism of melatonin affecting on carotenoid accumulation in pepper fruit by regulating Ca RAP2-12.The main results were made as follows:（1）Analysis of fruit quality evaluation of 75 pepper germplasm resources.The fruit morphology and secondary metabolite content of 75 chili germplasm resources belonging to four species of pepper（C.annuum,C.chinense,C.frutescens,and C.bacaccatum）were investigated.The results showed that had a greater variation in terms of fruit weight,size,total phenols,total soluble sugars,total soluble solids,titratable acidity,total soluble proteins,carotenoids,anthocyanins,and capsaicin content.In addition,in terms of the two most important capsaicinoid components in pepper fruit,capsaicin and dihydrocapsaicin,C.chinense had the highest content,while some germplasm resources of C.annuum did not detect these two capsaicinoids.S23 and S24（C.chinense）had the highest capsaicin content,S67（C.annuum）had higherβ-carotene content,and S68（C.annuum）had higher total fatty acids and ascorbic acid.Eighteen types of fatty acids were detected in pepper fruit,of which linoleic acid（polyunsaturated fatty acid）was the main one.Correlation analysis showed a positive correlation between the content of capsaicinoids and fatty acids.The color of pepper fruit was related to the content and proportion of chlorophyll,carotenoids,and anthocyanins.（2）The effect of exogenous melatonin on pepper fruit quality.The postharvest fruits of S01and S59 were treated with different concentrations of melatonin（0,25,50,100,200,400μmol/L）at 20℃to analyze their effects on fruit color-related quality（CIELAB color space L*a*b*value,chlorophyll,carotenoid）and hardness.The results revealed that exogenous melatonin could delay the decrease of L*value of S59 fruits and increase a*value of S01 fruits during storage.Several genes related to chlorophyll degradation have been reported in the literature,namely Ca CLH,Ca PPH,and Ca PAO.It was found that the relative expression level of the three genes was significantly up-regulate and thus contributed to chlorophyll degradation after S01 fruits subjected exogenous melatonin.Moreover,the same rule was applied to the expression levels of carotenoid biosynthesis-related genes Ca PSY3,Ca PDS,Ca LCYB1 and Ca CRTZ1,which induced the accumulation of carotenoid and the optimum concentration was 50μmol/L for S01 fruits.When S59fruits were treated with exogenous melatonin,the hardness was maintained due to the lower expression of cell wall structure-related genes Ca PG（polygalacturonase）and Ca EG（endo-β-1,4-glucanase）.According to the result,100μmol/L melatonin was optimal for S59 hardness.Interesting,100μmol/L exogenous melatonin could delay the senescence of S59 green pepper fruits,which was owe to the upregulated expression of Ca CAT and Ca POD genes,the enhanced activities of antioxidant enzyme CAT and POD,the rasied ratios of GSH/GSSG and As A/DHA,but the down concentrations of O2-,H2O2 and MDA concentrations in intracellular.（3）Transcriptome analysis of pepper fruits treated with exogenous melatonin.In order to further elucidate the regulation mechanism of melatonin treatment on carotenoid synthesis of S01fruits,transcriptional sequencing was conducted on S01 pepper fruits treated with different melatonin concentrations（0,25,50,100μmol/L）at different storage stages（0,3,6,9,12d）.The results showed that 550～5003 differentially expressed genes were detected between the melatonin treatment group and the control group at different storage stages.Gene Ontology（GO）enrichment analysis revealed that these differentially expressed genes were mainly pooled in cellular metabolism,catalysis,transport and other functions in the same storage.Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis furtherly made these genes clear,which were significantly concentrated in some metabolic pathways,such as secondary metabolites and carotenoid biosynthesis,etc.,indicating that melatonin treatment has a significant effect on carotenoid biosynthesis of pepper fruit.Moreover,Ca PSY3 and Ca CRTZ1 were highly expressed at each storage period after melatonin treatment,suggesting that melatonin may regulate the synthesis of carotenoids in pepper by controlling the high expression of the two genes.The mainly differentially expressed transcription factors were found between the melatonin treatment group and the CK group,including AP2/ERF,b HLH,C2H2,MYB,NAC and WRKY transcription factor families,etc.Total 59 differentially expressed AP2/ERFs were identified in the transcriptome of pepper fruit.Among them,there was a similar variation trend between the carotenoid content of pepper fruit and the expression levels of Ca RAP2-12（Ca3g15066）,Ca2g13177,Ca6g25362,Ca5g23514,Ca3g15489,Ca4g20436,Ca4g19760,Ca1g03278 and Ca1g00644.Hence,these AP2/ERFs may be involved in the regulation of carotenoid synthesis in pepper fruit in response to melatonin treatment.（4）Molecular Mechanism of transcription factor Ca RAP2-12 regulating carotenoid biosynthesis of pepper.In this study,118 AP2/ERF gene family members were identified in the reference genome of Zhangshugang pepper,which belonged to three family members:ERF（85）,DREB（31）and AP2（2）.Prediction of subcellular localization showed that most of the AP2/ERF protein family genes were localized in the nucleus,and a few members were localized in the cytoplasm,chloroplast and mitochondria.The 118 AP2/ERF family members mainly exist on chromosome 1 of pepper.The ORF frame of Ca RAP2-12 gene（Ca3g15066）was 1164 bp,encoding387 amino acid residues,which was a hydrophilic stable nuclear protein and belonged to the AP2/ERF family members.Five cis-elements related to hormone response were found in the upstream 2000bp of Ca RAP2-12 gene in pepper,hinting that there were cis-elements interacting with melatonin in the promoter of Ca RAP2-12 gene.The expression of Ca RAP2-12 was significantly increased after fruits treated with melatonin,indicating that Ca RAP2-12 gene played an important role in the melatonin signal response of pepper.When Ca RAP2-12 of pepper seedlings was silenced based on VIGS technology,the expression of Ca PSY3 and Ca LCYB2 were significantly downregulated,which leading to the total carotenoid contents in leaves was significantly decreased.However,carotenoid biosynthetic structural genes were all significantly down-regulated except Ca CRTZ1 after Ca RAP2-12 in pepper fruit was silenced.If Ca RAP2-12 of pepper fruits was overexpressed transiently,the expression of a series of genes were significantly up-regulated,including Ca PSY2,Ca PSY3,Ca PDS,Ca ZDS,Ca Crt ISO1,Ca Crt ISO2,Ca LYCB1,Ca LYCB2,Ca CRTZ1 and Ca CRTZ2.By Dual Luciferase Reporter assay and Yeast One-Hybrid assay,it was verified that transcription factor Ca RAP2-12 can bind to the promoter of Ca PSY3,thereby positively regulating the expression of a key gene Ca PSY3 related to carotenoid synthesis. |
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