A MYB transcription factor, DcMYB6, is involved in regulating anthocyanin biosynthesis in purple carrot taproots

Carrots are widely grown and enjoyed around the world. Purple carrots accumulate rich anthocyanins in the taproots, while orange, yellow, and red carrots accumulate rich carotenoids in the taproots. Our previous studies indicated that variation in the activity of regulatory genes may be responsible for variations in anthocyanin production among various carrot cultivars. In this study, an R2R3-type MYB gene, designated as DcMYB6, was isolated from a purple carrot cultivar. In a phylogenetic analysis, DcMYB6 was grouped into an anthocyanin biosynthesis-related MYB clade. Sequence analyses revealed that DcMYB6 contained the conserved bHLH-interaction motif and two atypical motifs of anthocyanin regulators. The expression pattern of DcMYB6 was correlated with anthocyanin production. DcMYB6 transcripts were detected at high levels in three purple carrot cultivars but at much lower levels in six non-purple carrot cultivars. Overexpression of DcMYB6 in Arabidopsis led to enhanced anthocyanin accumulation in both vegetative and reproductive tissues and upregulated transcript levels of all seven tested anthocyanin-related structural genes. Together, these results show that DcMYB6 is involved in regulating anthocyanin biosynthesis in purple carrots. Our results provide new insights into the regulation of anthocyanin synthesis in purple carrot cultivars.

In this study, a gene encoding an R2R3-type MYB, designated as DcMYB6, was isolated from a purple carrot cultivar. The correlation between its expression with anthocyanin production in purple and non-purple carrots was analyzed. The function of DcMYB6 was also analyzed by overexpression in Arabidopsis plants. These results will further our understanding of how anthocyanin synthesis is regulated in carrots.

Results
Sequence analysis of DcMYB6. The amplification products of the genomic DNA sequence and the open reading frame (ORF) sequence of DcMYB6 from the carrot cultivar 'Deep purple' are shown in Supplementary Fig. S1. The genomic DNA sequence of DcMYB6 was 1,801 bp long while the ORF sequence of DcMYB6 was 903 bp long, encoding a polypeptide of 300 amino acids ( Supplementary Fig. S1A). Alignment analysis of genomic DNA and ORF sequences revealed that the DcMYB6 gene consisted of two introns and three exons ( Supplementary Fig. S1B).
Next, we conducted an alignment analysis of the deduced amino acid sequence of DcMYB6 with those of other MYB TFs related to anthocyanin biosynthesis. Like other MYB TFs, DcMYB6 contained the highly conserved R2R3 domain at the N-terminus (Fig. 2). DcMYB6 showed high sequence homology with other MYB TFs within the R2R3 domain, sharing the highest identity (85%) with LeANT1 and the lowest identity (80%) with AmVENOSA. However, all the MYB TFs showed little homology in the C-terminus sequence to the R2R3 domain. When whole sequences were compared, DcMYB6 shared the highest identity (44%) with PhAn2 and the lowest identity (32%) with AmROSEA1.
The alignment showed that the [D/E]Lx2[R/K]x3Lx6Lx3 R motif, also known as the bHLH motif 25 , which is required for the interaction with bHLH proteins, was present in the R3 domain of all the analyzed MYB TFs (Fig. 2). The conserved ANDV motif that has been identified in MYB TFs in the anthocyanin pathway in the Rosaceae 26 was also present in all of the analyzed MYB TFs and was modified to [A/G]NDV. Besides these motifs, the MYB TFs contained the motif KPRPR[S/T]F defined by Stracke et al. 27  Quantitative real-time PCR analysis of DcMYB6 in purple and non-purple carrot taproots. At the 90-day-old stage, purple carrot cultivars had accumulated rich anthocyanins whereas anthocyanins were barely detectable in, or absent from non-purple carrot cultivars 16 . Using specific primer pairs, qRT-PCR analyses were performed to quantify the transcript levels of DcMYB6 in purple and non-purple carrots at this stage. The transcript levels of DcMYB6 in the taproots of three purple carrot cultivars ('Deep purple' , 'Purple 68' , and 'Tianzi2hao') were approximately 10-229-fold higher than those in the taproots of six non-purple carrot cultivars ('Kuroda' , 'Sanhongliucun' , 'Junchuanhong' , 'Bejo1719' , 'Qitouhuang' , and 'Baiyu'). Among the three purple carrot cultivars, 'Tianzi2hao' had the highest transcript level of DcMYB6 and 'purple 68' had the lowest. Among the six non-purple carrot cultivars, 'Baiyu' had the lowest transcript level of DcMYB6 and 'Sanhongliucun' had the highest (Fig. 3).

Subcellular localization of DcMYB6 protein.
To investigate the subcellular localization of DcMYB6, the DcMYB6 coding sequence was fused in-frame to the 5′ terminus of the gene encoding GFP, and the construct was transiently expressed in onion cells. In onion cells expressing GFP alone, fluorescence was localized in the cytoplasm and nucleus (Fig. 4 up). Onion cells expressing the DcMYB6-GFP fusion protein showed a strong signal in the nucleus (Fig. 4 down).

Overexpression of DcMYB6 in transgenic Arabidopsis induced anthocyanin production. The
DcMYB6 gene driven by the CaMV 35 S promoter was overexpressed in Arabidopsis plants to test its function. Arabidopsis seedlings of three homozygous CaMV 35 S:DcMYB6 transgenic lines (DcMYB6-1, DcMYB6-2, and DcMYB6-3) and one control transgenic line, which were selected on MS agar plates containing hygromycin, showed β -glucuronidase (GUS) activity (Fig. 5A). A PCR product of approximately 900 bp corresponding to the DcMYB6 coding sequence was detected in all three CaMV 35 S:DcMYB6 transgenic Arabidopsis lines analyzed, whereas no such PCR product was amplified from control transgenic plants (Fig. 5B).

Up-regulation of anthocyanin biosynthetic genes in transgenic Arabidopsis overexpressing
DcMYB6. Among the three CaMV 35 S:DcMYB6 transgenic Arabidopsis lines, DcMYB6-1 plants showed the lowest DcMYB6 transcript levels and DcMYB6-3 plants showed the highest (Fig. 7). As expected, DcMYB6 transcripts were undetectable in the control Arabidopsis plants. The results of the qRT-PCR analyses also determined which endogenous anthocyanin pathway structural genes were up-regulated in the transgenic Arabidopsis plants overexpressing DcMYB6. Compared with the control line, the transgenic Arabidopsis plants overexpressing DcMYB6 showed significantly increased transcript levels of AtCHS (chalcone synthase), AtCHI (chalcone isomerase), AtF3H (flavanone 3-hydroxylase), AtF3′H (flavonoid 3′ -hydroxylase), AtDFR (dihydroflavonol 4reductase), AtLDOX (leucoanthocyanidin dioxygenase), and AtUGT78D2 (Fig. 7). Among the three transgenic Arabidopsis lines overexpressing DcMYB6, DcMYB6-1 plants showed the lowest transcript levels of these structural genes and DcMYB6-3 plants showed the highest. Transcripts of these structural genes were undetectable or barely detectable in the control Arabidopsis plants.

Discussion
Anthocyanins are water-soluble pigments responsible for purple colors in carrots. In plants, TFs such as MYB, bHLH, and WD40 upregulate the expression of structural genes in the anthocyanin biosynthesis pathway. Two previous studies showed that the expression levels of all anthocyanin pathway structural genes were significantly lower in non-purple carrot cultivars than in purple carrot cultivars, which possibly resulted from the inactivation of regulator genes 16,28 . In other plant species, many R2R3-MYB TFs are known to control anthocyanin biosynthesis by regulating structural genes in the anthocyanin pathway 4,16,17,22,25 . However, little is known about the R2R3-MYB TFs involved in regulating the anthocyanin pathway in carrot. A previous study reported that DcMYB3 and DcMYB5 might upregulate the activity of the DcPAL3 promoter 29 . In the present study, a gene encoding R2R3-MYB, namely DcMYB6, was isolated from 'Deep purple' , a purple carrot cultivar.    The presence of these motifs suggested that DcMYB6 may be involved in regulating anthocyanin biosynthesis. In several other plant species, the expression of many R2R3-MYB genes in the anthocyanin pathway is strong correlated with anthocyanin accumulation. For example, MdMYB10 was found to be highly expressed highly in red-fleshed apple and in the colored skin of white-fleshed apple, but was virtually undetectable in the white cortex of white-fleshed apple 19 . In the present study, DcMYB6 transcript levels corresponded well with anthocyanin pigmentation; there were much higher transcript levels in all three 90-day-old purple carrot taproots than in 90-day-old taproots of the six non-purple carrot cultivars. Therefore, DcMYB6 is probably involved in regulating anthocyanin biosynthesis in purple carrot taproots.
The reason why DcMYB6 transcript levels were much lower in non-purple carrots than in purple carrots is still unknown. In peach (Prunus persica), the heterodimer of BL and PpNAC1 was shown to activate transcription of the anthocyanin-related MYB, PpMYB10.1 30 . In European pear (Pyrus communis), methylation of the PcMYB10 promoter reduced PcMYB10 expression levels and resulted in a peel color change from red to green 31 . Insertions and deletions in the promoter region have been shown to affect the expression levels of anthocyanin-related MYBs in some species, such as apple (Malus × domestica) and grapevine (V. vinifera) 20,22 . In future work, we will attempt to establish the reason for the different transcript level of DcMYB6 in purple and non-purple carrots.
In several studies, overexpression of anthocyanin-related MYB TFs in heterologous plant species led to enhanced anthocyanin accumulation 32,33 . In this study, transgenic Arabidopsis plants overexpressing DcMYB6 exhibited a clearly darker color and accumulated higher levels of anthocyanins in both vegetative and reproductive tissues, compared with those in control Arabidopsis plants. Furthermore, qRT-PCR analyses of the three transgenic Arabidopsis lines with different transcript levels of DcMYB6 and different total anthocyanin levels showed that higher transcript levels of DcMYB6 led to greater anthocyanin accumulation. Also, the transcript levels of all seven tested anthocyanin-related structural genes were much higher in transgenic Arabidopsis plants overexpressing DcMYB6 than in control Arabidopsis plants. Together, these results indicate that DcMYB6 could enhance anthocyanin accumulation in Arabidopsis by upregulating anthocyanin-related structural genes, and suggest that DcMYB6 regulates anthocyanin biosynthesis in purple carrots.
In conclusion, an R2R3-MYB TF, DcMYB6, was isolated from a purple carrot cultivar and was found to be involved in regulating the anthocyanin biosynthetic pathway. The results of this study provide important information on the pigmentation of purple carrots. Other TFs such as bHLH and WD40 that form complexes with MYB proteins and together regulate anthocyanin biosynthesis have not yet been identified in carrots. In future work, we will test whether overexpression of DcMYB6 in non-purple carrot cultivars leads to anthocyanin accumulation.

Methods
Plant materials. Three purple carrot cultivars ('Deep purple' , 'Purple 68' , and 'Tianzi2hao'), three orange carrot cultivars ('Kuroda' , 'Sanhongliucun' , and 'Junchuanhong'), and three yellow carrot cultivars ('Bejo1719' , 'Qitouhuang' , and 'Baiyu'), which are widely cultivated in China, were chosen for this work. Seeds were grown in a controlled artificial climatic chamber under the same conditions as previously described 16 . Arabidopsis thaliana ecotype Columbia was grown under the same conditions. RNA and DNA extraction from carrots and cDNA preparation. Total RNA was extracted from taproots of 90-day-old carrot plants using an RNAsimple Total RNA Kit (Tiagen, Beijing, China). First-strand cDNA was synthesized using the PrimeScript ™ RT reagent kit with gDNA Eraser (Perfect Real Time; Takara, Dalian, China). cDNA was diluted 20-fold for gene cloning and qRT-PCR analyses. Genomic DNA was isolated from young leaves with a DNAsecure plant kit (Tiangen).
Isolation of genomic DNA and cDNA sequence of DcMYB6. AtPAP1 (AAG42001) was BLASTed against our CarrotDB: a genomic and transcriptomic database for carrot 34 and the high-quality carrot genome which spans 421.5 Mb and accounts for ~90% of the estimated genome size (473 Mb) 35 . Two transcript contigs showing high sequence identity with AtPAP1 and with higher FPKM values in purple carrots than in non-purple carrots were identified in the transcriptomic database of CarrotDB. After assembling these two transcript contigs, an ORF of 903 bp was identified and predicted to be a MYB TF using Pfam (http://pfam.xfam.org/). This MYB TF was designated as DcMYB6 in this study. Two genomic sequence scaffolds (scaffold 016995 and scaffold 029424) that matched the ORF sequence were identified in the genomic database of CarrotDB. However, no genomic sequence matching the ORF sequence was found in the high-quality carrot genome.
The ORF sequence of DcMYB6 was amplified from cDNA produced from 90-day-old 'Deep purple' carrot taproots using PrimeSTAR HS DNA polymerase (Takara, Otsu, Japan) with the forward primer (5′ -CGCGCGGATCTTCCAGAGATTATGCATCCAAAGGCTTTGAAGAAT-3′ ) and reverse primer (5′ -CACGCCTGCCGTTCGACGATTTTAACTATAATCCAAGTTAAGAAGGTCCC-3′ ). The ORF sequence was then cloned into the pMD19-T simple vector (Takara, Otsu, Japan) using the ClonExpress II One Step Cloning Kit (Vazyme Biotech Co. Ltd., Nanjing, China) before sequencing (Genscript, Nanjing, China). The same pairs of primers were also used to amplify the genomic clone of DcMYB6 from genomic DNA extracted from carrot leaves. The full-length ORF and DNA sequences of DcMYB6 have been deposited in the GenBank database under the accession numbers KY020445 and KY020446, respectively. Subcellular localization analysis. The protein-coding region of DcMYB6 was amplified with the forward primer (5′ -CACCATCACCATCACGCCATGATGATCAAGAGCACTGGTAATCC-3′ ) and the reverse primer (5′ -CACTAGTACGTCGACCATGGCACTATAGTCCTGGTTGAGAAGATCCC-3′ ), and was subcloned into the pA7-GFP vector at the Nco I site to create the CaMV 35 S:DcMYB6-GFP fusion construct. This construct and the pA7-GFP empty vector (as control) were both bombarded into onion epidermal cells using a Biolistic PDS-1000 instrument (Bio-Rad, Hercules, CA, USA). After incubation at 25 °C for at least 16 h in the dark, samples were observed under a confocal laser scanning microscope.

Overexpression vector construct preparation and Arabidopsis transformation.
The coding sequence of DcMYB6 was amplified with the for ward primer (5′ -T T TACA AT TACCATGGGATCCATGCATCCAAAGGCT T TGAAGAAT-3′ ) and the reverse primer(5′ -ACCGATGATACGAACGAGCTCTTAACTATAATCCAAGTTAAGAAGGTCCC-3′ ), and then subcloned into the binary vector pCAMBIA-1301 under the control of the CaMV 35 S promoter and the pea rbcSE9 terminator to create the CaMV 35 S:DcMYB6 construct. This construct was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and then transformed into Arabidopsis using the floral-dip method 36 . Transgenic Arabidopsis plants carrying the DcMYB6 gene were identified by selection on half-strength Murashige and Skoog (MS) agar plates containing 35 mg/L hygromycin, assaying for GUS activity, and detecting the presence of the transgene by reverse transcription PCR with the forward primer (5′ -ATGCATCCAAAGGCTTTGAAGAAT-3′ ) and the reverse primer (5′ -AAGCACAACAAATGGTACAAG-3′ ), which were designed according to the sequence of DcMYB6 and the pea rbcSE9 terminator, respectively. Three transgenic Arabidopsis lines (DcMYB6-1, DcMYB6-2, and DcMYB6-3) with black leaves were used for further experiments. Arabidopsis plants transformed with the pCAMBIA-1301 empty vector served as controls.
Determination of total anthocyanin content. Total anthocyanins were extracted from 40-day-old transgenic Arabidopsis plants (T3) as described previously 37 . Total anthocyanin quantities are presented in mg cyanidin 3-O-glycoside equivalents per 100 g fw (mg/100 g fw). Three biological replicates were analyzed for each sample.
Quantitative real-time PCR expression analysis. The mRNA levels of the DcMYB6 gene in 90-day-old carrot taproots and in 40-day-old transgenic Arabidopsis plants were determined by qRT-PCR with the forward primer (5′ -GCCATAGGGCACAAGCACTCT-3′ ) and the reverse primer (5′ -GATCCCAATTTCCGCAAACAA-3′ ). Total RNA was extracted from 40-day-old transgenic Arabidopsis plants and used to synthesize cDNA using the method described above. To determine the transcript levels of anthocyanin pathway structural genes in transgenic Arabidopsis, qRT-PCR assays were performed with the primers listed in Supplementary Table S2. The DcActin1 gene was used as an internal standard in carrot with the same primers as described previously 16,38 , while the AtActin2 gene was used as an internal standard for normalization in Arabidopsis and was amplified using the primers listed in Supplementary Table S2. Experiments were conducted using three biological replicates for each sample. The relative gene transcript level was calculated with the 2 −ΔΔCT method 39 . To compare DcMYB6 expression patterns among purple and non-purple carrots at the 90-day-old stage, the Δ Δ C T was calculated by subtracting Δ C T of 'Kuroda' from Δ C T of all carrot cultivars. To compare the transcript levels of DcMYB6 and anthocyanin pathway structural genes among transgenic Arabidopsis plants, the Δ Δ C T was calculated by subtracting Δ C T of the AtF3H (flavanone 3-hydroxylase) gene in DcMYB6-1 Arabidopsis plants from the Δ C T of all tested genes in transgenic Arabidopsis plants.