Research On Transcriptional Regulation Of Anthocyanin In Red-and White-fleshed Strawberry

Strawberry(Fragaria X ananassa Duch.),a perennial herbaceous plant belonging to the Rosaceae,is widely-consumed economically important fruit,because of its enriched nutrients(such as vitamin C,vitamin A,anthocyanins etc.),unique flavor,sweet,and bright color.Anthocyanin is not only an important antioxidant conferring health profit for people,but also responsible for the color formation of strawberry fruit.It is also the core index of judging the appearance quality and commodity value of strawberry fruit.Therefore,increasing the content of anthocyanin in fruits is of great guiding significance and practical value for improving fruit quality and promoting the breeding of high quality germplasm resources of fruit trees.The presence of different colors of strawberry fruits in nature,especially those in light colors such as yellow and white,provides us a good opportunity for unravelling the mechanism of anthocyanin accumulation.At present,strawberry with phenotypes in different colors has been successfully applied to investigate the regulation mechanism of anthocyanin biosynthesis.However most of them are limited to diploid strawberry such as woodland strawberry(Fragaria vesca)or Fragaria pentaphylla,and most of them are focus on the known regulatory factors related to anthocyanin biosynthesis and regulation.In this study,two cultivated octoploids strawberries with identical genetic background were used,one is ’Benihoppe’ with red-flesh,and the other is ’Xiaobai’ with white-flesh.The key regulators involved in the anthocyanin biosynthesis were investigated and finally a network demonstrating the regulation fo anthocyanin in strawberry was constructed.The main research contents and results are as below:1.The content of two main anthocyanin components(Pelargonidin 3-glucoside,Pg3G and Cyanidin 3-glucoside,Cy3G)in the skin(outer red layer)and flesh of 5 developmental stages of’Benihoppe’ and ’Xiaobai’ were determined by HPLC(High-performance liquid chromatography).The results showed that only Pg3G was accumulated in the flesh of ’Benihoppe’,and the content of Pg3G increased gradually with the fruit development and ripening,while there was no anthocyanin accumulated in the flesh of ’Xiaobai’.Both Pg3G and Cy3G were accumulated in the skin of ’Benihoppe’ and ’Xiaobai’,the content of Pg3G was about 2-3 times higher than that of Cy3G,and the content of total anthocyanin in the skin of ’Benihoppe’ was higher than that in the skin of ’Xiaobai’.2.The full length of FaANS(Anthocyanin synthase)cDNA was cloned using homologous cloning method,the expression of FaANS in ’Benihoppe’ and ’Xiaobai’ fruit during fruit ripening was subsequently detected,and finally FaANS was transiently overexpressed in ’Xiaobai’ fruit using agrobacterium-mediated method.As results,the expression level of FaANS in ’Xiaobai’fruits was much lower than that in ’Benihoppe’.And it was found that there was no significant phenotypic change in the FaANS-overexpressed fruit,indicating there was no anthocyanin accumulation observed3.RNA sequencing(RNAseq)of ’Benihoppe’ and’Xiaobai’ flesh at white stage and full red stage was carried out.The expression patterns of anthocyanin biosynthetic genes and regulatory factors were analyzed.The results showed that,compared with ’Benihoppe’,the expression of almost all the key genes(CHS,CHI,F3H,F3 ’H,DFR,ANS and UFGT)involved in anthocyanin biosynthesis pathway were inhibited in ’Xiaobai’,while the expression level of F3 ’H was not significantly different between the two cultivars because of its very low expression in ’Xiaobai’and ’Benihoppe’.Transcription factor MYB1R was up-regulated and a WD40 TF AN11 was significantly down-regulated in ’Xiaobai’4.HPLC was performed to determine the ascorbic acid(AsA)content during development and ripening,the expression of genes involved in AsA metabolic pathways was analyzed based on the transcriptome data.The results revealed that anthocyanin and AsA were potentially related,’Benihoppe’ with high anthocyanin content had high levels of AsA,which was mainly affected by the regulation of D-galacturonate pathway.In ’Xiaobai’,the expression of PME,PG and GalUR in this pathway significantly decreased compared with that in ’Benihoppe’.Moreover,the expression of sucrose synthase involved in sucrose synthesis was inhibited in ’Xiaobai’,while the expression levels of genes involved in auxin,GAs and ethylene signals(such as A UX/IAA,GA20ox,ERF)were significantly enhanced in ’Xiaobai’ comparing with that in ’Benihoppe’5.Based on RNAseq data,anthocyanin related IncRNAs was screened and identified using bioinformatic method.As results,50,601 transcripts were screened as predicted IncRNAs,19 and 12 of which were down-regulated/up-regulated in white stage of white-fleshed ’Xiaobai’respectively,49 and 20 of them were down-regulated or up-regulated in full red stage of ’Xiaobai’comparing with ’Benihoppe’.Moreover,6 and 4 IncRNAs were down-regulated or up-regulated in both full red stage and white stage respectively.Prediction of the precursors and target genes of known microRNAs in strawberry showed us 130 lncRNAs might be used as precursors of 80 microRNAs,of which only one IncRNA was differentially expressed in ’Benihoppe’ and ’Xiaobai’;392 microRNAs could target 60 differentially expressed IncRNAs,suggesting that IncRNAs might participate in anthocyanin mainly through being targets of microRNAs.A regulatory network of anthocyanin biosynthesis involving IncRNAs was finally constructed6.FaMYB10 was overexpressed in ’Benihoppe’ and ’Xiaobai’ using agrobacterium-mediated method,the anthocyanin content in overexpressed fruits was measured by HPLC.The results showed us,overexpression of FaMYB10 successfully turned white flesh of ’Xiaobai’ into red,the accumulation of Cy3G was restored in the flesh of both ’Benihoppe’ and ’Xiaobai’,and the content of Cy3G in the skin was higher than that in the flesh.The expression levels of anthocyanin-related structural genes and regulators were detected by qPCR.As results,the expression levels of key genes involved in anthocyanin biosynthesis including CHS,CHI,F3’H,DFR,ANS and UFGT were significantly up-regulated comparing with the control.Especially,F3 ’H expressed at a rare level in the control fruit,while expressed at a high level in the FaMYB10-overexpressed’Benihoppe’ and ’Xiaobai’ fruit,indicating that its expression was regulated by FaMYB10,and the low expression of F3 H was probably the key reason of Cy3G blocking in ’Benihoppe’ and’Xiaobai’.