Effect Of CmWRKY44 And CmWRKY49 On Regulating β-Carotene Accumulation In Oriental Melon

Oriental melon（Cucumis melo L.）was one of main cultivated vegetables in China,and it was popular with people due to their flavor and nutrients.There are many varieties of oriental melon with diverse flesh colors,among which orange-flesh oriental melon mainly accumulates carotenoids.Carotenoids as major nutrients have many bioactive functions including antioxidant activity,delay aging and improve immunity.Human do not synthesize carotenoid,and they take the carotenoid through daily diet.Currently,the carotenoid metabolism pathway has been clearly elucidated.In many horticultural crops,transcription factors affect carotenoid content by regulating the expression of key genes in the carotenoid metabolism pathway,thereby elevating fruit quality.However,the regulatory mechanism of carotenoid metabolism in melon is still unclear.Therefore,two cultivars with different flesh color,‘HDB’（orange flesh）and‘YMR’（white flesh）were used as experiment materials in this study.Combing the targeted carotenoid metabolomic analysis and the expression of key genes in carotenoid metabolism pathway,which showed thatβ-carotene is a major carotenoid component and CmPSY1 is a key gene controlling carotenoid biosynthesis in orange flesh-color oriental melon.The transcription factors CmWRKY44 and CmWRKY49 was selected and identified through bioinformatics analysis,yeast one hybridization assay,luciferase report assay and Agrobacterium tumefaciens-mediated transient transformation assay,and they could regulate CmPSY1-mediatedβ-carotene accumulation in oriental melon fruit.Subsequently,the interaction factors CmERFⅣ-4 and CmMAPK7 were screened out by yeast related assay,GUS assay and dual luciferase complementary imaging assay.Finally,the oriental melon fruit（25 days after anthesis）was treated with exogenous ethephon(200 mg·L^-1).The expressions of CmWRKYs and their interaction factors were analyzed andβ-carotene content was measured,indicating that ethylene influence the expression of CmPSY1 by inducing the transcript of CmWRKY44 and CmWRKY49,thereby affectingβ-carotene accumulation in oriental melon.The main findings were follows:1.Targeted carotenoid metabolomic analysis for two cultivars at mature stage.Twelve carotenoid components were detected in‘HDB’,and the main carotenoid component wasβ-carotene(74μg·g^-1FW),accounting for 54%of the total carotenoid content.However,only 5 carotene components were detected in‘YMR’,and theβ-carotene content is 1.1μg·g^-1 FW.Theβ-carotene content in‘HDB’was approximately 70 times higher than that in‘YMR’,which is the main pigment resulted in difference in flesh color between the two cultivars.Theβ-carotene content was sharply increased at 25 days after anthesis.The expression profile of carotenoid metabolism related genes showed that the trend of their expression was firstly upregulated and then downregulated,and reach the peak at 25 days after anthesis.Additionally,the expression of CmPSY1 is the higher up-regulation than that of other genes during fruit ripening,and is also a rate-limiting enzyme for carotenoid synthesis.Therefore,it is regarded as a key gene affecting carotenoid biosynthesis in oriental melon.2.Transcriptome sequencing was performed at different developmental stages（5,15,25,30,and 35 d after anthesis）of two cultivars oriental melon,and statistical and enriched analysis were performed for the differentially expressed genes.The results showed that the highest number of differentially expressed genes（3353）was at 25 days after anthesis,and the highest number of differentially expressed transcription factors（184）was also at 25 days after anthesis,indicating that significant difference in physiological activity in this stage.At the same time,GO and KEGG enrichment analysis showed that these differentially expressed genes were mainly enriched in metabolic processes including photosynthesis related pathways,carotenoid metabolism related pathways,and transcriptional regulatory activities.In addition,the weighted co-expression network analysis（WGCNA）of key carotenoid metabolism genes was conducted to screen out three highly related modules,and the classification and expression analysis of transcription factors in the three modules were conducted.It was found that most transcription factors were members of the WRKY,MADS,ERF/AP2,and MYB gene families.It was speculated that these transcription factors might play an important role in regulating carotenoid metabolism in oriental melon.3.The cis-element prediction of the CmPSY1 promoter was performed through bioinformatics analysis,and it was found that the promoter contains light responsive elements,hormone responsive elements,stress responsive elements,and transcription factor binding sites.Transcription factors CmWRKY44 and CmWRKY49 that may regulate the expression of CmPSY1 were screened through combing the transcriptome and yeast one hybrid assay.Moreover,GUS,luciferase report assay and Agrobacterium-mediated instantaneous transformation assay further demonstrated that CmWRKY44 and CmWRKY49 could directly bind to the promoter of CmPSY1 and activate its expression,which affectβ-carotene accumulation in oriental melon.4.Combined with bioinformatics analysis,CmERFⅣ-4 and CmMAPK7 as interaction factors of CmWRKY44 and CmWRKY49 were selected through yeast one hybrid and yeast two hybrid experiments,respectively.The DNA-protein interaction assay further demonstrated that CmERFⅣ-4 can bind to the promoter of CmWRKY44 and promote its transcription.Meanwhile,the fluorescence complementary imaging assay further proves that CmWRKY49 could interact with CmMAPK7.Additionally,the oriental melon fruit（25 days after anthesis）was sprayed with exogenous ethephon(200 mg·L^-1),which proves that ethylene could induce the expression of CmERFⅣ-4 and CmWRKY44,which promote the expression of CmPSY1 through a cascade reaction,thereby promotingβ-carotene accumulation in oriental melon.Ethylene could also induce the expression of CmMAPK7 and CmWRKY49,which interact with each other to induce the expression of CmPSY1,thus affectingβ-carotene accumulation in oriental melon.In summary,β-carotene is a major carotenoid component and CmPSY1 is a key gene regulatingβ-carotene biosynthesis in orange flesh-color oriental melon.CmWRKY44 and CmWRKY49 could active the expression of CmPSY1 by directly binding to its promoter.Additionally,the expression of CmWRKY44 and CmWRKY49 and their interaction factors were induced by ethylene.