Transcription Factor Regulating Anthocyanin Biosynthesis Research Articles

Transposon insertions regulate genome‐wide allele‐specific expression and underpin flower colour variations in apple (Malus spp.)

SummaryAllele‐specific expression (ASE) can lead to phenotypic diversity and evolution. However, the mechanisms regulating ASE are not well understood, particularly in woody perennial plants. In this study, we investigated ASE genes in the apple cultivar ‘Royal Gala’ (RG). A high quality chromosome‐level genome was assembled using a homozygous tetra‐haploid RG plant, derived from anther cultures. Using RNA‐sequencing (RNA‐seq) data from RG flower and fruit tissues, we identified 2091 ASE genes. Compared with the haploid genome of ‘Golden Delicious’ (GD), a parent of RG, we distinguished the genomic sequences between the two alleles of 817 ASE genes, and further identified allele‐specific presence of a transposable element (TE) in the upstream region of 354 ASE genes. These included MYB110a that encodes a transcription factor regulating anthocyanin biosynthesis. Interestingly, another ASE gene, MYB10 also showed an allele‐specific TE insertion and was identified using genome data of other apple cultivars. The presence of the TE insertion in both MYB genes was positively associated with ASE and anthocyanin accumulation in apple petals through analysis of 231 apple accessions, and thus underpins apple flower colour evolution. Our study demonstrated the importance of TEs in regulating ASE on a genome‐wide scale and presents a novel method for rapid identification of ASE genes and their regulatory elements in plants.

Functional MYB transcription factor gene HtMYB2 is associated with anthocyanin biosynthesis in Helianthus tuberosus L

BackgroundTuber color is an important trait for Helianthus tuberosus L. (Jerusalem artichoke). Usually, purple tubers with high anthocyanin content are more nutritious than white tuber. But, the molecular mechanism underlying it is unknown.ResultsIn the current study, high-throughput RNA-sequencing was used to compare the transcriptomes between plants with tubers with red or white epidermis. Compared with the white-skinned tubers of cultivar QY3, anthocyanin biosynthesis structural genes had greater expression in the red-skinned tubers of cultivar QY1, indicating that the anthocyanin biosynthesis pathway was activated in ‘QY1’; quantitative PCR confirmed this difference in expression. HtMYB2 (Unigene44371_All) was the only MYB transcription factor, homologous to the MYB transcription factor regulating anthocyanin biosynthesis, expressed in the red tuber epidermis of ‘QY1’. The anthocyanin concentration in the root, stem, leaf, flower, and tuber epidermis of ‘QY1’ was higher than in ‘QY3’, especially tuber epidermis. Correspondingly, HtMYB2 had greater expression in these tissues of ‘QY1’ than in ‘QY3’. The expression of HtMYB2 was associated with anthocyanin accumulation in the different tissues. Overexpression of HtMYB2 activated the anthocyanin biosynthesis pathway, accumulating the pigment in leaves of transgenic tobacco, supporting the model that HtMYB2 regulated anthocyanin biosynthesis. Further experiments found that HtMYB2 had the same coding sequence and genomic sequence in ‘QY1’ and ‘QY3’, but that there were several single nucleotide polymorphisms and one insertion–deletion (indel) mutation of 21 nucleotides in the promoter region between the two alleles. The deletion of three nucleotides “AAA” made the promoter of ‘QY1’ predicted to contain one more possible promoter region. A specific primer, based on the indel, could differentiate between cultivars with red or white tuber epidermis. The genetic variation in HtMYB2 was associated with the tuber skin color in a natural population.ConclusionsRNA-seq can successfully isolate the candidate gene (HTMYB2) controlling anthocyanin biosynthesis in purple epidermis of Jerusalem artichoke tuber. HTMYB2 can regulate anthocyanin biosynthesis in plants and is closely related to the formation of purple phenotype in tubers. This study should be useful in understanding the genetic mechanism underlying different tuber skin colors and in breeding new H. tuberosus cultivars with different tuber skin colors.

A bHLH transcription factor TsMYC2 is associated with the blue grain character in triticale (Triticum×Secale).

RNA-Seq was employed to compare the transcriptome differences between the triticale lines and to identify the key gene responsible for the blue aleurone trait. The accumulation of anthocyanins in the aleurone of triticale results in the formation of the blue-grained trait, but the identity of the genes associated with anthocyanin biosynthesis in the aleurone has not yet been reported. In this manuscript, RNA-Seq was employed to compare the transcriptome differences between the triticale lines HM13 (blue aleurone) and HM5 (white aleurone), and to identify the key genes responsible for the blue aleurone trait. There were 32,406 differentially expressed genes between HM13 and HM5. Seventy-three unigenes were homologous to the structural genes related to anthocyanin biosynthesis, and the average transcript level of the structural genes was higher in HM13 than in HM5, so that quantitative differences between the two lines in transcription rates could be the cause of the blue aleurone. The MYB and bHLH transcription factors had two homologous unigenes, but contained only one differentially expressed unigene each. The relative transcript level of bHLH Unigene5672_All (TsMYC2) in HM13 was 42.71 times that in HM5, while the relative transcript level of the MYB transcription factor Unigene12228_All in HM13 was 2.20 times that in HM5. qPCR experiments determined the relative transcript level of TsMYC2 in developing grain, with the expression of TsMYC2 in grain being the highest compared with that in root, stem or leaf tissue. TsMYC2 was homologous to the bHLH transcription factor regulating anthocyanin biosynthesis and contained three entire functional domains: bHLH-MYC_N, HLH and ACT-like, which were important for exercising regulation of anthocyanin biosynthesis as a bHLH transcription factor. Transient expression of ZmC1 and TsMYC2 could induce anthocyanin biosynthesis in white wheat coleoptile cells, demonstrating that TsMYC2 was a functional bHLH transcription factor. These results indicated that TsMYC2 was associated with the blue aleurone trait and could prove to be a valuable gene with which to breed new triticale cultivars with the blue aleurone trait.

PdMYB118, isolated from a red leaf mutant of Populus deltoids, is a new transcription factor regulating anthocyanin biosynthesis in poplar

A new anthocyanin biosynthesis transcription factor PdMYB118, which could be used for the genetic engineering of colorful tree species, was indentified from a red leaf mutant of Populus deltoids. In higher plants, the biosynthesis of anthocyanins is regulated by several classes of transcription factors (TFs), including R2R3-MYB, bHLH and WD-repeat proteins. In this work, we isolated an MYB gene regulating anthocyanin biosynthesis from a red leaf mutant of Populus deltoids, which accumulated more anthocyanins in the leaves and showed higher expression levels of anthocyanin biosynthesis genes than did the wild type. Gene expression analyses of all TFs regulating anthocyanin biosynthesis demonstrated that only a MYB118 homologous gene, PdMYB118, was up-regulated in the mutant compared with the wide type. Subcellular localization analyses in poplar leaf mesophyll protoplasts showed that PdMYB118-YFP fusion protein was specifically located in nucleus. When transiently expressed in poplar leaf protoplasts, PdMYB118 specifically promoted the expression of anthocyanidin biosynthesis genes. Dual-luciferase assays revealed that PdMYB118 can directly activate the promoters of these genes. When overexpressed in Shanxin Yang (P. davidiana × P. bolleana), a hybrid clone commercially grown for landscaping in the northern part of China, transgenic plants overexpressing PdMYB118 produced more anthocyanins in the leaves and turned their color into redness when grown in both greenhouse and field. Consistently, transcripts of some important anthocyanidin biosynthesis genes were significantly increased in the leaves of transgenic plants. All these results indicate that PdMYB118 functions as an essential transcription factor regulating anthocyanin biosynthesis in poplar and could be used for the genetic engineering of colorful tree species.

Frame-shift Mutation Causes the Function Loss of TaMYB-A1 Regulating Anthocyanin Biosynthesis in Triticum aestivum

Red coleoptile is an easily observed trait in Triticum aestivum and can provide some protection against stress. Here, TaMYB-A1 or TuMYB-A1, homologous to TaMYB-D1, which controls red coleoptile formation in the common wheat cultivar ‘Gy115’, was isolated from eight T. aestivum and 34 T. urartu cultivars. The genome sequence of TaMYB-A1 was 867 bp with an intron of 93 bp, which was similar to the MYBs regulating anthocyanin biosynthesis in T. aestivum but different from other MYB transcription factors regulating anthocyanin biosynthesis. TaMYB-A1 had an integrated DNA-binding domain of 102 amino acids and a transcriptional domain of 42 amino acids, which was responsible for regulating anthocyanin biosynthesis. TaMYB-A1 was assigned to the same branch as the MYBs regulating anthocyanin biosynthesis in a phylogenetic tree. A transient expression analysis showed that TaMYB-A1 induced ‘Opata’ coleoptile cells to synthesize anthocyanin with the help of ZmR. A non-functional allele of TaMYB-a1 existed in common wheat cultivars containing rc-a1. One single nucleotide was deleted 715 bp after the start codon in TaMYB-a1 compared with TaMYB-A1. The deletion caused a frame shift mutation, destroyed the DNA transcription activator domain, and resulted in TaMYB-a1 losing its ability to regulate anthocyanin biosynthesis in ‘Opata’ coleoptile cells. Those cultivars with functional TaMYB-A1 or TuMYB-A1 have red coleoptiles. The isolation of TaMYB-A1 should aid in understanding the molecular mechanisms of coleoptile traits in T. aestivum.

Development of a Broad bean wilt virus 2-based expression vector for gentian

Japanese gentian is one of the most important ornamental flowers in Japan. Its most attractive trait is the vivid blue flower, whose color results from polyacylated anthocyanins. Other important traits in gentian breeding include flower shape, flowering time, and overwintering characteristics. To show the molecular mechanism by which these traits are regulated, transgenic gentian plants are often generated by Agrobacterium-mediated transformation. However, this method is not suitable for all gentian cultivars because of the difficulties in regeneration and/or transformation. Also, it takes a long time to obtain transgenic plants and analyze their phenotypes. Therefore, it is important to develop a more efficient method to analyze gene function in gentian. In the present study, we developed a novel viral expression vector for efficient gene functional analysis in gentian using Broad bean wilt virus 2 (BBWV-2) infectious cDNA. First, we expressed two reporter genes, GFP and GUS, in gentian using the BBWV-2 vector. Fluorescence from GFP was observed in various organs including the leaf, flower, root, and overwintering bud. β-Glucuronidase (GUS) activity was detected in the inoculated leaf, but not in upper uninoculated leaves because the GUS insert was unstable in the BBWV-2 vector. Next, we inoculated the BBWV-2 vector carrying GtMYB3, a transcription factor regulating anthocyanin biosynthesis, fused with SRDX, which encodes the EAR transcriptional repression domain. Expression of GtMYB3-SRDX via the BBWV-2 vector partly changed the gentian petal color from blue to white. The expression levels of genes encoding anthocyanin biosynthetic enzymes (flavanone 3-hydroxylase, dihydroflavonol-4-reductase, anthocyanidin synthase, flavonoid 3′,5′-hydroxylase, and anthocyanin 5,3′-aromatic acyltransferase) were markedly lower in the white parts of petals than in the blue parts. These results clearly indicated that GtMYB3-SRDX expressed via the BBWV-2 vector was functional in gentian flowers. This BBWV-2-based gene expression system enabled us to analyze a phenotypic change in gentian flower color within a few months.

A reverse genetics approach identifies novel mutants in light responses and anthocyanin metabolism in petunia

Flower color and plant architecture are important commercially valuable features for ornamental petunias (Petunia x hybrida Vilm.). Photoperception and light signaling are the major environmental factors controlling anthocyanin and chlorophyll biosynthesis and shade-avoidance responses in higher plants. The genetic regulators of these processes were investigated in petunia by in silico analyses and the sequence information was used to devise a reverse genetics approach to probe mutant populations. Petunia orthologs of photoreceptor, light-signaling components and anthocyanin metabolism genes were identified and investigated for functional conservation by phylogenetic and protein motif analyses. The expression profiles of photoreceptor gene families and of transcription factors regulating anthocyanin biosynthesis were obtained by bioinformatic tools. Two mutant populations, generated by an alkalyting agent and by gamma irradiation, were screened using a phenotype-independent, sequence-based method by high-throughput PCR-based assay. The strategy allowed the identification of novel mutant alleles for anthocyanin biosynthesis (CHALCONE SYNTHASE) and regulation (PH4), and for light signaling (CONSTANS) genes.

Identification and characterization of seed-specific transcription factors regulating anthocyanin biosynthesis in black rice

Black rice is rich in anthocyanin and is expected to have more healthful dietary potential than white rice. We assessed expression of anthocyanin in black rice cultivars using a newly designed 135 K Oryza sativa microarray. A total of 12,673 genes exhibited greater than 2.0-fold up- or down-regulation in comparisons between three rice cultivars and three seed developmental stages. The 137 transcription factor genes found to be associated with production of anthocyanin pigment were classified into 10 groups. In addition, 17 unknown and hypothetical genes were identified from comparisons between the rice cultivars. Finally, 15 out of the 17 candidate genes were verified by RT-PCR analysis. Among the genes, nine were up-regulated and six exhibited down-regulation. These genes likely play either a regulatory role in anthocyanin biosynthesis or are related to anthocyanin metabolism during flavonoid biosynthesis. While these genes require further validation, the results here underline the potential use of the new microarray and provide valuable insight into anthocyanin pigment production in rice.

Identification and Characterization of R2R3-MYB and bHLH Transcription Factors Regulating Anthocyanin Biosynthesis in Gentian Flowers

Gentian plants have vivid blue-colored flowers, caused by accumulation of a polyacylated anthocyanin 'gentiodelphin'. We previously performed expression analysis of gentiodelphin biosynthetic genes, and hypothesized that the white-flowered gentian cultivar 'Polarno White' might have resulted from the mutation of certain regulatory factors responsible for anthocyanin biosynthesis in flower petals. In this study, we isolated 26 R2R3-MYB gene fragments including four full-length cDNAs (GtMYB2a, GtMYB2b, GtMYB3 and GtMYB4) and one basic helix-loop-helix (bHLH) gene (GtbHLH1) from blue-flowered gentian by degenerate PCR and rapid amplification of cDNA ends (RACE). Phylogenetic tree analysis showed that GtMYB3 was categorized into a clade involved in anthocyanin biosynthesis including petunia AN2 and Arabidopsis PAP1. On the other hand, GtbHLH1 exhibited high identity with petunia AN1 based on both phylogenetic and genomic structural analyses. Temporal profiles of GtMYB3 and GtbHLH1 transcript levels corresponded well with those of gentiodelphin accumulation and their biosynthetic genes in petals. Yeast two-hybrid analysis showed that GtbHLH1 interacted with GtMYB3. Moreover, transient expression analysis indicated that the co-expression of GtMYB3 and GtbHLH1 could enhance the promoter activities of late anthocyanin biosynthetic genes in tobacco BY2 cells. We also revealed that in cv. 'Polarno White' the GtMYB3 genes were mutated by insertions of transposable elements or uncharacterized sequences, indicating that the white coloration was caused by GtMYB3 mutation. These results strongly suggested that GtMYB3 and GtbHLH1 are involved in the regulation of gentiodelphin biosynthesis in gentian flowers.