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Black carrots are potent sources of anthocyanin for the natural food color industry as their anthocyanins contain very high percentages of acylated anthocyanins which are much more stable than non-acylated anthocyanins. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway activated by a triad of R2R3-MYB, bHLH and WD40 transcription factors (TFs). Recent studies in black carrots have elucidated major anthocyanin related structural genes and also regulatory TFs. However, the active TFs responsible for anthocyanin production in black carrots differ between cultivars. We have previously shown by RNAseq that DcMYB113 (LOC108213488), a R2R3-MYB TF, was up-regulated in colored as compared to non-colored tissues of the black carrots 'Superblack' and 'CH05544' and that this upregulation was positively correlated with anthocyanin content. However, this gene showed no upregulation in the black carrot 'Nightbird' also included in that study. In the present study, we present a novel R2R3-MYB DcMYB113_NB (LOC108212072) and a complementary bHLH DcEGL1_NB (LOC108210744) isolated from the RNA of 'Nightbird'. Their functionality as anthocyanin regulators was confirmed by their simultaneous expression under the control of a constitutive promoter in the background of the orange carrot 'Danvers 126'. Transformants showed activation of the structural anthocyanin genes and accumulation of anthocyanins across leaves, stems and taproots. Interestingly, the anthocyanin profile of the transformants showed increases of 20 to 30% in acylated anthocyanins as compared to 'Nightbird' resulting in transformants with almost 100% acylated anthocyanins.

Key Message: The simultaneous expression of AmRosea1 and AmDelila transcription factors from snapdragon can activate the anthocyanin pathway in orange carrots, leading to the synthesis and accumulation of anthocyanins in the taproots. Anthocyanins are phenolic compounds produced in various parts of plants. They are used as natural food dyes and are reported as beneficial antioxidants for humans. Black carrot is an important source for anthocyanins; however, the reason for the lack of anthocyanin production in the orange carrot is unknown. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway that has previously been reported to be activated by a triad of R2R3-MYB, basic helix-loop helix (bHLH) and WD40 transcription factors (TFs). In the current study, orange carrots were turned purple by simultaneous expression of R2R3-MYB and bHLH TFs, i.e. AmRosea1 and AmDelila from snapdragon (Antirrhinum majus). Simultaneous transgenic expression of the TFs under a constitutive promoter in the orange carrot cultivar 'Danvers 126' lead to consistent upregulation of anthocyanin-related biosynthetic genes and significant accumulation of anthocyanins in leaves, stems and taproots. Highest overall content of soluble anthocyanins in the taproot among the transformants amounted to 44.38 mg g -1 dry weight. The anthocyanin profile of the transformants were significantly different from the profile in the reference black carrot 'Deep Purple'. The main anthocyanins present in the transformed taproots were cyanidin 3-xylosyl(sinapoylglucosyl)galactoside, whereas the main anthocyanin present in Deep Purple was cyanidin 3-xylosyl(feruloylglucosyl)galactoside. This study confirms the presence of the necessary biosynthetic genes in orange carrots for production of anthocyanins and demonstrates the absence of suitable R2R3-MYB and bHLH TFs for stimulating anthocyanin biosynthesis in the orange carrot.

Background: Anthocyanins are water-soluble colored flavonoids present in multiple organs of various plant species including flowers, fruits, leaves, stems and roots. DNA-binding R2R3-MYB transcription factors, basic helix-loop-helix (bHLH) transcription factors, and WD40 repeat proteins are known to form MYB-bHLH-WD repeat (MBW) complexes, which activates the transcription of structural genes in the anthocyanin pathway. Although black cultivars of carrots (Daucus carota L.) can accumulate large quantities of anthocyanin in their storage roots, the regulatory genes responsible for their biosynthesis are not well characterized. The current study aimed to analyze global transcription profiles based on RNA sequencing (RNA-Seq), and mine MYB, bHLH and WD40 genes that may function as positive or negative regulators in the carrot anthocyanin biosynthesis pathways., Results: RNA was isolated from differently colored calli, as well as tissue samples from taproots of various black carrot cultivars across the course of development, and gene expression levels of colored and non-colored tissue and callus samples were compared. The expression of 32 MYB, bHLH and WD40 genes were significantly correlated with anthocyanin content in black carrot taproot. Of those, 11 genes were consistently up- or downregulated in a purple color-specific manner across various calli and cultivar comparisons. The expression of 10 out of these 11 genes was validated using real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)., Conclusions: The results of this study provide insights into regulatory genes that may be responsible for carrot anthocyanin biosynthesis, and suggest that future focus on them may help improve our overall understanding of the anthocyanin synthesis pathway.

Background: Black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) constitute a valuable source of anthocyanins, which are used as natural red, blue and purple food colourants. Anthocyanins and phenolic compounds are specialised metabolites, accumulation of which often requires elicitors, which act as molecular signals in plant stress responses. In the present study, ethephon, an ethylene-generating compound was explored as enhancer of anthocyanin and phenolic contents during growth of 'Deep Purple' black carrots. The effects of ethephon on several parameters were investigated, and the expression of biosynthetic anthocyanin genes was studied during growth and anthocyanin accumulation., Results: Roots of ethephon-treated carrot plants exhibited an increase in anthocyanin content of approximately 25%, with values ranging from 2.25 to 3.10 mg g -1 fresh weight, compared with values ranging from 1.50 to 1.90 mg g -1 fresh weight in untreated roots. The most rapid accumulation rate for anthocyanins, phenolic compounds, soluble solids and dry matter was observed between 10 and 13 weeks after sowing in both untreated and ethephon-treated carrots. The differences in anthocyanin contents between untreated and treated carrots increased for several weeks after the ethephon treatment was terminated. Five cyanidin-based anthocyanin forms were identified, with variable relative abundance values detected during root growth. Overall, the expression of the anthocyanin biosynthetic genes analysed (PAL1, PAL3, F3H1, DFR1, LDOX2) increased in response to ethephon treatment, as did the expression of the MYB1 transcription factor, which is associated with activation of the phenylpropanoid pathway under stress conditions. In addition, a correlation was proposed between ethylene and sugar contents and the induction of anthocyanin synthesis., Conclusions: This study presents a novel method for enhancing anthocyanin content in black carrots. This finding is of economic importance as increased pigment concentration per unit of biomass implies improved profitability parameters in food colour production. We provide new insight into the accumulation patterns of the different cyanidin-based anthocyanins and phenolic compounds during root growth. Moreover, we show that enhanced anthocyanin content in ethephon-treated carrots is accompanied by increased expression of anthocyanin biosynthetic genes.