Research On The Regulatory Mechanism Of Anthocyanin Accumulation In Wild Citrus

Citrus is one of the most important fruit crops worldwide,consist of diverse species which can accumulate anthocyanins.Anthocyanins are secondary plant metabolites that belong to the family of compounds known as flavonoids,plays key roles in many aspects like the formation of fruit quality,stress resistance and human health benefits.Recently,the regulation mechanism of anthocyanins in Citrus have only been reported in blood orange（Citrus sinensis）,but few research on other Citrus species which can accumulate anthocyanins have been reported.Pummelo（Citrus maxima）is one of the three basic species of Citrus,all cultivated pummelos do not synthesize anthocyanins,and the peel is mainly yellow or orange yellow.A wild germplasm,called‘purple pummelo’in this study,was discovered in a mountainous area in the Enshi region of Hubei Province,China,by our group.Purple pummelo accumulates anthocyanins in the fruit peel and present a purple color different from cultivated pummelo.The mechanism responsible for purple fruit in purple pummelo is unknown.Chinese box orange（Atalantia buxifolia）,a primitive citrus,can accumulate anthocyanins both in its fruits and its leaves,and the anthocyanins component accumulated in peel and leaves are quite different.It suggested that the regulation mechanism of anthocyanins in the peel and leaves of Chinese box orange is different.Therefore,in the present study,the difference of anthocyanin accumulation phenotypes of purple pummel and normal pummelo have been analyzed,and the transcriptional regulation mechanism of anthocyanin metabolism have been parsed.In addition,we further explored the regulation mechanism of anthocyanins in Chinese box orange,a primitive citrus,based on the research of purple pummelo.The main results are as follows:1.Identification of anthocyanins component and profiling anthocyanin biosynthesis regulatory gene in purple pummelo.Firstly,high-performance liquid chromatography（HPLC）analysis revealed that the predominant anthocyanin components in the peel of purple pummelo were cyanidin-3-glucoside and cyanidin-3-（6-malonylglucoside）.Gene expression profiling in the fruit peel was compared between the purple pummelo and normal pummelo.A total of 1,667 genes were found to be differentially expressed,including 955 up-regulated and 712 down-regulated genes,among them,genes involved in the anthocyanin pathway were up-regulated.qRT-PCR analysis confirmed that seven of the eight structural genes of the anthocyanin pathway（CgCHS1,CgF3H,CgF3’H,CgDFR,CgANS,CgUFGT1 and CgUFGT2）,were highly correlated with the anthocyanin accumulation pattern during fruit development stages.A similar tendency was observed for CgGST,encoding a glutathione-S-transferase potentially involved in the vacuolar accumulation of anthocyanins.In addition,because of the similar expression pattern,CgRuby1 and CgbHLH1 were selected as the candidate critical regulatory gene of purple pummelo.We found a candidate MYB transcription factor at 6.7 kb upstream of CgRuby1gene.The expression pattern at transcription level and protein level of this gene was opposite to that of the CgRuby1 gene during fruit development in purple pummelo by qRT-PCR and western-blot,and the R2R3 domain of this gene was incomplete,we hereafter refer to this gene as CgRuby2^Short.2.Regulation model of anthocyanin synthesis in purple pummelo was proposed.The function of CgRuby1 and CgRuby2^Short was verified by its ectopic expression under the control of the 35S promoter in Arabidopsis thaliana and the yellow strawberry（Fragaria vesca）,CgRuby1 is an activator and CgRuby2^Short is a repressor.In dual luciferase assay,transcriptional activation activity of CgRuby1 was drastically increased when and CgbHLH1 was added,while CgRuby2^Short inhibited the transcriptional activation activity of CgRuby1-CgbHLH1 complex.The analysis of promoter elements and promoter activity indicated that the up-regulation of CgRuby1 in the fruit peel of purple pummelo was due to the intact CAAT-box and TATA-box cis-elements in the promoter,in contrast,the cis-elements in normal pummelo were mutated.The Y1H assays and EMSA assays confirmed that CgRuby1 can bind to the promoters of CgF3’H and CgDFR,while CgRuby2^Short can not.To test the interaction between CgRuby1 and CgRuby2^Short with the putative bHLH partner CgbHLH1,yeast two-hybrid（Y2H）,bimolecular fluorescence complementation（BiFC）and pull-down assays were performed,the results showed that CgRuby1 and CgRuby2^Short can both interact with CgbHLH1.Competitive binding assays indicated that CgRuby2^Short competes with CgRuby1 via binding to the same bHLH partner.Therefore,we infer that CgRuby1 is the key activator of anthocyanin accumulation in purple pummelo,CgRuby2^Short retains the ability to interact with the same partner,CgbHLH1,as CgRuby1,thus acting as a passive competitor in the regulatory complex.3.The influence of environmental factors on anthocyanin regulation in purple pummelo.In bagging experiment,we found that the accumulation of anthocyanin in pummelo was affected by light.The qRT-PCR results confirmed that the key structural gene of anthocyanin biosynthesis and the expression of CgRuby1 were also affected by light.Promoter element and activity analysis indicated that CgRuby1 promoter was a light-induced promoter,which was obviously affected by light,while low temperature had no effect on the activity of CgRuby1 promoter.This results indicated that the regulation mechanism of anthocyanin biosynthesis in purple pummelo was different from that of blood oranges which induced by low temperature.4.Profiling and identification of anthocyanin biosynthesis regulatory gene in Chinese box orange.High levels of anthocyanins were detected in mature fruit and the main compounds were identified as cyanidin and delphinidin derivatives by LC-MS/MS.Comparing the AbRuby2 protein sequence with CgRuby2^Short,we found that the R2R3domain of AbRuby2 was intact,and we named it AbRuby2^Full.Further studies indicated that AbRuby2^Full is a novel activator of anthocyanin biosynthesis in Citrus,it can induce anthocyanin accumulation in strawberry and Arabidopsis,it can also bind to the promoters of anthocyanin structure genes and induce activation activity.The promoter of AbRuby2 contained a mutator-like miniature inverted repeat transposable element（MITE）,we analyzed the methylation levels of cytosine in promoter of AbRuby2 in green and pigmented leaves of Chinese box orange via bisulfite sequencing（BSP）-PCR,the methylation level showed the greatest difference between the green and pigmented leaves.These results indicate that the alleviation of DNA methylation in the promoter of AbRuby2^Full may affect the expression of AbRuby2^Full and ultimately lead to anthocyanin biosynthesis in the pigmented leaves of Chinese box orange.