The Mechanism Of CgMYB58 Regulating The Biosynthesis Of Lignin And Carotenoid In ‘Huanong Redfleshed’ Pummelo Juice Sacs

Juice sac granulation is a common physiological disorder in citrus fruit,which occurs before and post citrus fruit harvest.While longer storage period always accompanied with more severe granulation.High lignin accumulation is a major feature of citrus fruit granulation.Lignin biosynthetic pathway and its regulation mechanism have been extensively studied in the model plant,while the mechanism of its accumulation and regulation in granulated citrus fruit juice sac has not been fully understood.Therefore,it is crucial to reveal the lignin regulation mechanism,and to explore an effective measure to inhibit the granulation process.In this study,12 pummelo cultivars at maturity stage and 4 pummelo at 6 developmental stages were used.Based on extensive metabolomic and transcriptomic data,a series of transcription factors that were significantly related to lignin content were excavated,and one of them was verified.Results are shown as follows:1.Based on LC-QTOF-MS,GC-MS and HPLC platforms analyzed the primary and secondary metabolites in pummelo juice sacs.A total of 57 citrus metabolites were identified,including 22 carotenes,17 amino acids,6 flavonoids,7 sugars,3 organic acids,and 1 myo-inositol,and the total lignin content.The highest content of carotenoids was accumulated in Huanong Red-fleshed pummelo(HR),such as the upstream carotene and zeaxanthin.However,HR is also the most prone to granulation,which specifically accumulated high lignin content in the juice sacs at in the mature stage.The carotenoids content increased sharply started at 115 days post anthesis(DPA)in HR,while lignin increased sharply at 145 DPA.Although there was a difference of one month,the total content of lignin at the five developmental stages and phytofluene had a correlation coefficient of 0.93.2.Based on transcriptomic data and metabolic data co-expression analysis to find candidate transcription factors.Juice sacs of HR,HB pummelo(HB),and Kao Pan pummelo(KP)at 55,85,115,145,and 205 DPA were collected for RNA-Seq sequencing analysis,with three biological replicates.The transcript levels of the following genes sharply increased from 145 DPA to 205 DPA in HR: Cg PAL1,-2,-3,-4,and-5;Cg C4H;Cg4CL1;Cg C3H;Cg CCo AOMT3 and-4;and Cg LAC4,-5,-7,-11,-13,-15,and-22.Based on lignin distinct accumulation between HR and other pummelos,the total content of lignin corresponding to 45 RNA-Seq samples was used as a bait.In FPKM library of RNA-Seq,the genes with correlation coefficient of lignin content ≥0.75 were retrieved,including 24 transcription factors.Among these transcription factors,CgMYB58 is critical transcription factors governing to lignin accumulation.3.CgMYB58 modulates multiple lignin biosynthetic genes.Overexpression of CgMYB58 significantly increase the lignin content in the mesocarp(albedo)of HR and KP.The stable transformation in citrus calli,lignin content in CgMYB58 overexpression lines(CgMYB58-OE)was significantly higher than wild-type RM(WT-RM).19 lignin biosynthetic gene expression levels significantly up-regualtied in CgMYB58-OE4,CgMYB58-OE11 compared to WT-RM.Dual luciferase transcriptional activity assay confirmed that CgMYB58 significantly activated the promoter activities of Cg PAL1,Cg PAL2,Cg4CL1,and Cg C3 H in tobacco leaves.Moreover,yeast one-hybrid assay indicated that CgMYB58 can directly bind to promoter region of Cg PAL2 and Cg C3 H in HR.Notably,electrophoretic mobility shift assay(EMSA)verified that CgMYB58 protein directly bind to the AC elements in the above genes’ promoter.Taken together,CgMYB58 directly bind to the promoter regions of Cg PAL1,Cg PAL2,Cg4CL1,and Cg C3 H thereby regulating the lignification process HR.4.Treatment of pummelo juice sacs in vitro with the exogenous abscisic acid(ABA)and indole-3-acetic acid(IAA)disturbed the juice sacs granulation.Phytohormone responsive elements were analyzed in the promoter regions of CgMYB58,Cg PAL1,Cg PAL2,Cg4CL1,and Cg C3 H.Exogenous ABA can accelerate the lignin accumulation in KP,HB and HR,with visible granulation in HR,whereas IAA decreased the lignin content thus reduce the granulation of HR.Above two hormones interfere with the biosynthesis and accumulation of lignin in exogenous cultured juice sacs by affecting the expression level of CgMYB58 and its target genes Cg PAL1,Cg PAL2,Cg4CL1,and Cg C3 H.5.CgMYB58 promotes the accumulation of carotenoids by up-regulating the expression of Cg BCH gene.The expression of Cg BCH gene in the transcriptome during pummelo developmental was positively correlated with the expression of CgMYB58.In the CgMYB58 transgenic calli,the expression of Cg BCH was significantly increased.And the EMSA experiment verified that CgMYB58 can directly bind to the AC cisbinding element on the Cg BCH promoter.In conclusion,this study based on extensive metabolomic and transcriptomic data,a series of transcription factors significantly correlated to lignin contents were screened and preliminary verified the mechanism of CgMYB58 in response to IAA and ABA to accelerate the citrus juice sacs granulation by regulating lignin accumulation.Thus,our study explored a practical method to suppress the occurrence of citrus granulation by adding IAA.