Isolation And Characterization Of Anthocynin-specific Pathway Genes And Transcription Factors In Tobacco

Anthocyanins are one of the most important group metabolites produced in plants. Theroles of anthocyanins are their ability not only to attract pollinators and seed dispersers butalso to protect plants from insect attack, to attenuate UV-B radiation and to protectchloroplasts from the adverse effects of excess light. Recent studies suggest that theconsumption of anthocyanins is able to lower the risk of cardiovascular disease, diabetes,arthritis and cancer due to their anti-oxidant and anti-inflammatory activities. The synthesis ofanthocyanin is derived from a branch of the flavonoid biosynthetic pathway. Studies onanthocyanin biosynthesis pathway genes and regulatory factors should be possible not only toengineer improved plant resistance by generating or manipulating anthocyanins but also topromote metabolic engineering of health-protecting flavonoids in important plants, and thusto help human beings to obtain food with increased antioxidant capacity. Tobacco is amember of one of the most agriculturally important plant families (Solanaceae) and has beenused as a model in plant biotechnology for decades due to its ease of transformation andrelatively short generation time. Moreover, the fast growing tobacco cell lines such as BrightYellow-2(BY-2) and Xanthi (used in the present study) are an excellent system for studyinggene expression and secondary metabolism. However, to the best of our knowledge, thefunctional characterization of anthocyanin biosynthesis pathway genes and regulatory factorshas been reported rarely to date. Herein we report the molecular cloning and functionalanalyses of the key regulator, basic helix-loop-helix (bHLH) transcription factors (TFs), andthe key enzymes, Dihydroflavonol-4-reductase (DFRs), of anthocyanin biosynthesis pathwayin tobacco flowers. The main conclusions obtained from this project are as follows:1. Two bHLH TFs, NtAn1a and NtAn1b, have been isolated from tobacco (Nicotianatabacum) flowers. Sequences alignment demonstrated that NtAn1a and NtAn1b share highsequence homology with other known flavonoid-related bHLH TFs. Phylogenetic analysisdivided NtAn1a and NtAn1b into the clade close to PhAn1from petunia, TT8fromArabidopsis and VvMYC1from grape.2. PCRs by gene-specific primers using cDNA and genomic DNA clarified thatNtAn1a and NtAn1b are originated from two ancestors of tobacco, N. sylvestris and N. tomentosiformis, respectively. Analysis of the genomic sequences revealed that both NtAn1aand NtAn1b contain eight introns and nine exons.3. Quantitative PCR (qPCR) revealed that NtAn1a and NtAn1b are predominantlyexpressed in flowers. A1.1kb NtAn1a promoter fragmen was isolated using a PCR-basedgenome walking method. The GUS expression profile driven by the NtAn1a promoter isessentially identical to that of NtAn1genes, indicating this floweral tissue-specific expressionis controlled by the NtAn1promoter4. Yeast one-hybrid and transient protoplast assays suggested that both NtAn1a andNtAn1b are transcriptional activators. Ectopic expression of the two genes in tobacco lead toincreased anthocyanin accumulation in flowers and the elevated anthocyanin content isassociated with the increase in NtAn1a or NtAn1b transcripts. In transgenic tobaccoexpressing NtAn1a or NtAn1b, both subsets of early (Chalcone synthase, CHS; Chalconeisomerase, CHI; Flavanone3-hydroxylase, F3H) and late (Dihydroflavonol4-reductase,DFR；Anthocyanidin synthase, ANS) flavonoid pathway genes were up-regulated.5. Yeast two-hybrid assays showed that NtAn1a and NtAn1b have strongprotein–protein interactions with R2R3-MYB TF, NtAn2. Transient tobacco protoplast assaysimplied that NtAn1a, NtAn1b and NtAn2cloud not activate the promoters of two keyanthocyanin pathway genes, dihydroflavonol reductase (DFR) and chalcone synthase (CHS)individually. However, in combination (NtAn1a+NtAn2or NtAn1b+NtAn2), they activatethe two promoters. The promoter activation is severely repressed by the dominant repressiveforms either NtAn1a or NtAn2, created by fusing the SRDX repressor domain to the TFs. thisresults pointed out that NtAn1and NtAn2act in concert to regulate the anthocyanin pathwayin tobacco flowers.6. RT–PCR manifested that over expression of NtAn2induces expression of bothNtAn1a and NtAn1b in tobacco leaves, while over expression of NtAn1genes has no effecton NtAn2expression. These results indicate that NtAn1a and NtAn1b are regulated by NtAn2,a floral tissue specific MYB TF; however, the NtAn1proteins do not regulate NtAn2.7. Yeast two-hybrid assays illustrated that the maize R2R3MYB TF, C1, stronglyinteracts with bHLH TF Lc, but it does not interact with either NtAn1a or NtAn1b. This resultprovides an explanation to why C1fails to induce anthocyanin when expressed alone intobacco.8. Two dihydroflavonol4-reductase (Dfr) cDNAs, NtDfr1and NtDfr2, were isolatedfrom tobacco flowers. Sequences alignment revealed that NtDfr1and NtDfr2share highsequence homology with other known Dfr genes isolated from other species. Phylogeneticanalysis divided NtDfr1and NtDfr2into the clade close to the ones from petunia and potato. 9. Transcript analysis uncovered that both Dfr genes express only in flowers and areunder strict developmental control; highly expressed in young flowers and the expressiongoes down significantly in mature flowers.10. RT-PCR on flower cDNA of N. sylvestris and N. tomentosiformis were performedusing allele-specific primers. Sequence analysis of the PCR products revealed that NtDfr1originated from N. tomentosiformis, whereas NtDfr2derived from N. sylvestris.11. Over-expression of NtDfr1or NtDfr2in tobacco results in pigment accumulation inflowers, while RNAi-mediated suppression of NtDfr in tobacco resulted in white to pale pinkflowers. The levels of NtDfr1or NtDfr2mRNA in flowers from transgenic tobacco lines weresignificantly higher than control. However, the NtDfr trascript levels in transgenic line weresignificantly reduced compared to the control flowers. This suggests that the Dfr genesderived from both parents are functional in tobacco.12. Tobacco protoplast transient assay demonstrated that NtDfr is a direct target of thebHLH TF, NtAn1and R2R3-MYB, NtAn2. NtDfr expression is regulated by a complex ofNtAn1and NtAn2.