Dissection Of The Molecular Mechanisms Underlying Differential Anthocyanin Accumulation In Radish(Raphanus Sativus L.)

Radish is one of the most important anthocyanin rich root vegetables consumed in various parts of the world.Some genotypes of radish have colored flesh and/or skin due to pigmentation brought by accumulation of anthocyanin.Anthocyanins are a big group of naturally occurring water-soluble pigments that belong to larger group of ubiquitous secondary metabolites referred to as flavonoids,synthesized from the phenylpropanoid pathway.They exhibit potential human health benefits,which include antihypertensive,inhibition of cell proliferation,anti-inflammatory,antimutagenic,antioxidant and antimicrobial properties.Radishes have been found to differ in pigmentation across different genotypes,developmental stages and tissues.However,the molecular mechanisms underlying the differential accumulation in different tissues and at different growth stages in radish remained vague.The characteristic of differential anthocyanin accumulation in the plant makes it an invaluable system for studying regulatory mechanisms of anthocyanin,providing new insights into radish culture.The objective of this study was therefore to analyze the phenotypic,spatial and temporal expression regimes of key structural genes that encode the enzymes of the anthocyanin biosynthetic pathway.Transport mechanisms in the downstream pathway could also contribute to differential anthocyanin accumulation and therefore MATE(Multidrug and Extrusion Compound)transporter genes associated with flavonoid transport in radish were identified,isolated and analyzed.Differentially expressed MATE transcripts were also identified from DEG(differentially expressed genes)experiments.In addition,microscopic and grafting experiments were also done to provide information on differential anthocyanin accumulation and anthocyanin transport,respectively.Lastly a genome-wide analysis of the myeloblastosis(MYB)transcription factors were identified and characterized,and the transcripts of those implicated in regulation of anthocyanin biosynthesis profiled in different tissues from different stages of growth,across four color variant radishes.From the total transcript sequences obtained through illumina sequencing,102 assembled transcripts,and 20 candidate genes were identified to be involved in anthocyanin biosynthesis Fifteen genomic sequences were isolated and sequenced from radish taproot.The length of these sequences was between 900 bp to 1,579 bp,and the unigene coverage to all of the corresponding cloned sequences was more than 93%.Gene structure analysis revealed that RsF3’H is intronless and anthocyanin biosynthesis genes(ABGs)bear asymmetrical exons,except RsSAM.Anthocyanin accumulation showed a gradual increase in the leaf of the red radish and the taproot of colored cultivars during development,with a rapid increase at 30 DAS and the highest content at maturity.Spatial-temporal transcriptional analysis of 14 genes revealed detectable expressions of twelve ABGs in various tissues at different growth levels.The investigation of anthocyanin accumulation and gene expression in four color variant radish cultivars,at different stages of development,indicated that total anthocyanin correlated with transcript levels of ABGs,particularly RsUFGT,RsF3H,RsANS,RsCHS3 and RsF3 ’H1.Our results suggest that these candidate genes play key roles in phenotypic and spatial-temporal anthocyanin accumulation in radish through coordinated regulation and the major control point in anthocyanin biosynthesis in radish is RsUFGT.The present findings lend invaluable insights into anthocyanin biosynthesis and may facilitate genetic manipulation for enhanced anthocyanin content in radish.The recent sequencing of the radish genome allowed the identification of the genes which encode proteins with high similarity to the MATE family.Additionally,from differential expression data,1110 DETs(Differentially expressed transcripts)were found to be associated with transport activity,from which fifty-one unigenes(4.5%)were identified as sequences encoding proteins with a significant similarity to MATE genes.Of these,21 and 30 were up-regulated at the cortex splitting and taproot thickening stages respectively.A homology based search between functionally validated MATE proteins from other species and those from radish was done to identify the MATE genes involved in flavonoid transport.Nine MATE genes from radish were found to have a high similarity with flavonoid MATEs and subsequently,their cDNA was isolated.The nine genes were named RsMATE1-RsMATE9.Their proteins were found to comprise of 479-519 residues,with theoretical isoelectric points ranging from 5.18-8.60,with molecular masses of 52-56kDa.The predicted putative transmembrane segments of the RsMATEs were 12,all lacking an N-terminal signal peptide.All RsMATE proteins shared 35.4%-87.2%identity,among which RsMATE3,RsMATE7,RsMATE8 and RsMATE9 were highly similar to the MATE-type flavonoid transporters.RsMATE1 and RsATE4 may be involved in the transport of toxic cations and detoxification of xenobiotics.RT-qPCR revealed that transcript levels of these genes were both temporally and spatially regulated.Microscopic analysis and grafting experiments provided evidence that anthocyanins are differentially translocated in plant tissues and more so,that long distance transport is involved in the sequestration of anthocyanin.The present study also conducted a genome-wide analysis of MYB genes in radish,denoted as RsMYBs.The study included identification of putative MYB members,analysis of their phylogenetic relationships,gene structures,GO annotations,and transcript profiling.Totally,187 RsMYBs were identified and were further found to cluster into 31 subfamilies.The R2R3 encoding MYB genes maintained a conserved gene structure,having three exons and two introns,against six exons and five introns in genes encoding R1R2R3 repeat proteins.MYB gene sequence analysis led to the identification of conserved and over-represented cis-motifs.Motif analysis also revealed that gene function could be associated with the number and organization of conserved domains.Subsequently,transcript profiles of 14 RsMYB genes associated with regulation of anthocyanin revealed that the MYBs respond to tissue and developmental cues.Three RsMYBs were identified as possible anthocyanin repressors while eleven are possible enhancers.Globally,this research would provide vital information on radish genes that could be used as elite candidate genes for anthocyanin metabolic engineering studies in future.