Isolation And Functional Characterization Of Genes Regulating Anthocyanin Biosynthesis In The Coleoptile And Grain Of Wheat

Anthocyanin,as an antioxidant substance,is able to clear free radicals in cells and plays an important role in preventing cardiovascular diseases and promoting human health.Purple-grained wheat,with plenty of anthocyanin accumulated in its pericarp,is thought to have high nutritional value and considered as a precious germplasm resource.In addition,the accumulation of anthocyanins in different tissues of wheat can be used as an effective morphological marker in wheat breeding.However,the specific regulatory genes and the regulatory network is still unclear.Therefore,it is of great significance to study the molecular mechanism of anthocyanin synthesis in wheat.In the present study,the Inclusive composite interval mapping（ICIM）method was used to detect the QTLs of red coleoptile trait and the comparative genomics tools were used to screened out the candidate genes responsible for red coleoptile.Moreover,the candidate MYB and bHLH transcription factors（TFs）that involved in anthocyanin regulation in wheat pericarp were identified based on transcriptome analysis.The genetic basis and molecular mechanism of these TFs regulating anthocyanin biosynthesis in purple pericarp were studied.In general,this study provides a theoretical basis for revealing the regulatory network of anthocyanin synthesis in wheat coleoptile and pericarp.The main results are as follows:（1）A recombinant inbred line（RIL）population from the cross of common wheat（Triticum aestivum L.）Ning7840×Clark were used as plant material.The ICIM mapping method was used to detect the QTLs of red coleoptile（Rc）.A single QTL was mapped between Xsnp7205 and Xsnp5258 in chromosome 7A.By applying comparative genomics analyses,collinear genomic regions of the Rc locus on wheat chromosome 7A were identified in brachypodium distachyon chromosome 1,sorghum（Sorghum bicolor L.）chromosome 10and rice（Oryza sativa L.）chromosome 6,respectively.In the collinear region,we found two homologous MYB genes,Sb10g06800 of sorghum and Os06g10350 of rice,which were participated to regulate the anthocyanin synthesis.The corresponding homologous gene in wheat was Traes₇AS_A84ABE6EF（TaC1）.Sequence alignment and phylogenetic analysis showed that TaC1 has similar characteristics of MYB TF that regulating anthocyanin biosynthesis.（2）Two allelic variants,TaC1a and TaC1b,were cloned from the RIL progenies with red and white coleoptiles,respectively.Compared with the sequence of TaC1a,the major variation of TaC1b was a 1-bp deletion at 714 bp site.The 1-bp nucleotide deletion causes a frame-shift that result in alerted amino acid sequence of TaC1b.The transient expression mediated by particle bombardment showed that TaC1a was able to regulate anthocyanin synthesis in wheat calluses,while mutated TaC1b loses this function.The mutated site was developed to a Cleaved Amplified Polymorphic Sequence（CAPS）marker and used to detect the genotypes of TaC1 in different materials.The result indicated that TaC1 is a candidate gene regulating anthocyanin synthesis in coleoptiles.（3）Transcriptome sequencing was performed on three pericarp samples of Heixiaomai 76:pericarps of 10 days after pollination（DAP）（H-10d,white pericarp）,pericarps of 17 DAP（H-17d,purple pericarp）,and pericarps of 10 DAP with directly exposed to sunlight（H-10p,purple pericarp）by removing the covered glumes.The obtained raw data was de novo assembled into 113,684 Unigenes.The differentially expressed gene（DEG）analysis was performed between genes expressed in purple and white pericarps.The result showed that5,750 genes were differentially expressed between H-10d vs.H-10p,and 4,216 genes were differentially expressed between H-10d vs.H-17d.Among them,1,833 DEGs were overlapped between H-10d vs.H-10p and H-10d vs.H-17d.（4）According to the information of gene annotation and result of phylogenetic analysis,we found 199 anthocyanin-related biosynthesis genes:three MYB genes,one bHLH gene,192anthocyanin structural genes,and three light response HY5 genes.Among them,some genes,such as CHI,F3H,DFR,and ANS have already been reported in previous studies.In the present study,we found some anthocyanin transporter genes（GST and MATE）which have not been studied before.Moreover,the gene expression profiles were analyzed in three samples.Most of the genes（90%）were thought to be activated by light due to their up-regulated expression in H-10p sample,which directly exposed to sunlight.The remaining genes were thought to be expressed along with the seed development as they were significantly expressed in H-17d.It is speculated that these genes may be activated by internal hormones produced during seed development.（5）qRT-PCR was conducted to investigate expressions of MYB and bHLH genes in five tissues and four pericarp development stages of white-grained variety A14 and purple-grained variety Heixiaomai 76.The result showed that TaPpm1 and TaPpb1 co-regulate the anthocyanin accumulation in purple pericarp.Four TaPpm1 allelic variants（TaPpm1a/b/c/d）with different coding region,were detected in different colored varieties.TaPpb1 has two allelic variants（TaPpb1a and TaPpb1b）with different promoter sequences.There are six imperfect 261 bp repeat units in the promoter region of TaPpb1a,while TaPpb1b has only one261 bp unit in its promoter.（6）The variations in allelic variants of TaPpm1 and TaPpb1 were developed into specific markers and used to detect the genotypes of TaPpm1 and TaPpb1 in 34 wheat varieties with different grain colors.Four purple varieties possess TaPpm1a and TaPpb1a,while the allelic variants TaPpm1b/c/d and TaPpb1b were distributed in non-purple varieties.In F₂ population obtained from the cross between A14 and Heixiaomai 76,plants with purple pericarp have both TaPpm1a and TaPpb1a.The dual luciferase assay showed that the combination of TaPpm1a and TaPpb1 activate the promoter of ANS.TF alone or the combination of TaPpm1b and TaPpb1 failed to activate the promoter of ANS.These results indicated that TaPpm1 and TaPpb1 co-regulate the anthocyanin synthesis in purple pericarp.（7）In the yeast-two hybrid assay,we demonstrated that only TaPpm1a strongly interacts with TaPpb1,while the other three variants TaPpm1b/c/d with mutated protein sequences had weak or no interaction with TaPpb1.Particle bombardment-mediated transient expression showed that TaPpm1a can regulates anthocyanin synthesis in wheat callus,while TaPpm1b/c/d lost this function.We speculated that the mutated sequences of TaPpm1b/c/d affect their interaction with TaPpb1,which further preventing the formation of TaPpm1-TaPpb1 complex and hindering the production of anthocyanin synthesis.（8）The dual luciferase reporter assay showed that the promoter of TaPpb1a initiate the significant higher expression of fused luciferase gene（LUC）than the promoter of TaPpb1b did.As the insert sequence in promoter of TaPpb1a can enhancing the expression of fused genes,we speculated that the variation in the promoter region of TaPpb1 directly affects its expression level.TaPpb1b,with only one 261 bp unit in its promoter,have barely expression and then encoded insufficient amount of TaPpb1 protein,resulting in a decreased TaPpm1-TaPpb1 complex.The less amount of TaPpm1-TaPpb1 complex might regulate less accumulation of anthocyanin in wheat pericarp.