Regulatory Mechanism Of MYB And BHLH Transcription Factors On Anthocyanin Biosynthesis In Potato Tuber

Potato(Solanum tuberosum L.)is one of the world’s most important food crops,and is recognized as a source of health-promoting antioxidants for the human diet.It has been found that pigmented potato cultivars are a rich source of anthocyanins,in particular acylated derivatives.As the largest group of plant flavonoids,anthocyanins are the main pigments responsible for the red-blue colour of potatoes.Besides attracting pollinators and aiding in seed dispersal in flowers and fruits,anthocyanins have key roles in protection against UV radiation and cold temperatures and response to drought stress.Moreover,anthocyanins have potential health benefits in humans,such as protection against some cancers and neuronal and cardiovascular diseases.Therefore,a high anthocyanin potato has potential as a new cultivar with enhanced health benefits.It’s been reported that anthocyanin biosynthesis is regulated at the level of transcription by transcriptional factors of the MYB,basic helix-loop-helix(b HLH)and WD40 classes via a various array of studies on different plant species,among these MYB TFs,R2R3 MYBs constitute the largest TF gene family in plants.In this thesis,comparative transcriptome analysis of white and purple cultivars was carried out using high-throughput RNA sequencing in order to discover the differentially expressed genes between two datasets of RNA-Seq libraries(White skin vs Purple skin and White flesh vs Purple flesh),especially new transcription factors which are regulating anthocyanin biosynthesis,the function of anthocyanin-related transcription factors and regulatory mechanism of anthocyanin biosynthesis in potato were further investigated.To identify genes encoding anthocyanin biosynthetic steps and regulatory transcription factors involving in anthocyanin biosynthesis in potato tuber,we performed the next-generation sequencing technology by using Illumina Hi Seq 2000 in paired-end(PE)mode on purple skin and purple flesh of purple cultivar and white skin and white flesh of white cultivar.Using a comparison of the RNA-sequence datasets,we identified 10,499 genes(4,387 up-regulated while 6,112 down-regulated)between WS and PS libraries and 8,157 genes(3,685 up-regulated while 4,472 down-regulated)between WF and PF libraries were found to be differentially expressed.We analyzed differentially expressed genes related to anthocyanin biosynthesis between the cultivars and discovered new versions of the pathway genes and potential transcription factors.These included 48 biosynthetic genes,26 MYB and 27 b HLH TFs in PS vs.WS library and 45 biosynthetic genes,21 MYB and 17 b HLH TFs in PF vs.WF library.We further validated 11 differentially expressed genes that are likely to be involved in anthocyanin biosynthesis or the transcriptional regulation of the pathway by using q PCR with gene-specific primers and the results showed that there was a good correlation between RNA-seq data and q PCR data.In addition,putative SNPs were identified and validated in UFGT,AN1 and b HLH1.We used the transcriptome results to study the large UFGT gene family which plays a role in anthocyanin biosynthesis in the potato tuber.The present transcriptome analysis provides valuable information regarding anthocyanin biosynthesis in potato,including differential responses of gene family members and regulatory transcription factor candidates.We further investigated function of candidate MYB and b HLH TFs from cultivated potatoes.We characterized three variants of R2R3-MYB St AN1 from four differentially pigmented genotypes and found amino acid variants in the C-terminus of the MYB St AN1,termed R0,R1,and R3,due to the presence of a repeated 10-amino acid motif.These variant MYBs showed some expression in both white and pigmented tubers.We found several new alleles or gene family members of R2R3 MYBs,St MYBA1 and St MYB113,which were also expressed in white potato tubers.From functional analysis in tobacco,we showed that the presence of a C-terminal 10-amino acid motif is optimal for activating anthocyanin accumulation.Engineering a motif back into a MYB lacking this sequence enhanced its activating ability.Versions of St MYBA1 and St MYB113 can also activate anthocyanin accumulation in tobacco leaves.In heterologous system,anthocyanin accumulation was induced by overexpressing three variants of St AN1 and St MYBA1 in all tissues of transgenic tobacco lines and the elevated expression levels of endogenous b HLH partners are essential for anthocyanin production in plant tissues,and we further found that light is required for its function on anthocyanin accumulation in over-expressing St MYBA1 system.We also isolated five family members of potato Stb HLH1,and one St JAF13,from potato to test their ability to interact with MYB variants.The results showed that two alleles of Stb HLH1 from white skin and red skin are non-functional,while three other Stb HLH1 s have different co-regulating abilities,and need to be activated by St JAF13,Stb HLH1 s and St JAF13 showed similar expression profiles,with higher levels detected in genotypes with pigmented skin and flesh.Combined with expression analysis in potato tuber,results suggest that three AN1 alleles(St AN1-R0,St AN1-R1,and St AN1-R3)have functionally important roles in anthocyanin accumulation,Stb HLH1 and St JAF13 are key co-regulators of anthocyanin biosynthesis,while the transcripts of MYB variants St AN1,St MYBA1,and St MYB113 are well expressed,even in the absence of pigmentation.The majority of transcripts of St AN1 are truncated in unpigmented tissues according to RNA-Seq results.In white potato tubers,failure to activate expression of Stb HLH1 and St JAF13 may be due to differential processing of the St AN1 transcripts and non-functional alleles of Stb HLH1.