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Chen et al. BMC Plant Biology (2019) 19:390 https://doi.org/10.1186/s12870-019-1999-0 RESEARCH ARTICLE Open Access A novel R2R3-MYB from grape hyacinth, MaMybA, which is different from MaAN2, confers intense and magenta anthocyanin pigmentation in tobacco Kaili Chen1,2,3, Lingjuan Du1,2,3, Hongli Liu1,2,3 and Yali Liu1,2,3* Abstract Background: The primary pigments in flowers are anthocyanins, the biosynthesis of which is mainly regulated by R2R3-MYBs. Muscari armeniacum is an ornamental garden plant with deep cobalt blue flowers containing delphinidin-based anthocyanins. An anthocyanin-related R2R3-MYB MaAN2 has previously been identified in M. armeniacum flowers; here, we also characterized a novel R2R3-MYB MaMybA, to determine its function and highlight similarities and differences between MaMybA and MaAN2. Results: In this study, a novel anthocyanin-related R2R3-MYB gene was isolated from M. armeniacum flowers and functionally identified. A sequence alignment showed that MaMybA contained motifs typically conserved with MaAN2 and its orthologs. However, the shared identity of the entire amino acid sequence between MaMybA and MaAN2 was 43.5%. Phylogenetic analysis showed that they were both clustered into the AN2 subgroup of the R2R3-MYB family, but not in the same branch. We also identified a IIIf bHLH protein, MabHLH1, in M. armeniacum flowers. A bimolecular fluorescence complementation assay showed that MabHLH1 interacted with MaMybA or MaAN2 in vivo; a dual luciferase assay indicated that MaMybA alone or in interaction with MabHLH1 could regulate the expression of MaDFR and AtDFR, but MaAN2 required MabHLH1 to do so. When overexpressing MaMybA in Nicotiana tabacum ‘NC89’, the leaves, petals, anthers, and calyx of transgenic tobacco showed intense and magenta anthocyanin pigments, whereas those of OE-MaAN2 plants had lighter pigmentation. However, the ovary wall and seed skin of OE-MaMybA tobacco were barely pigmented, while those of OE-MaAN2 tobacco were reddish-purple. Moreover, overexpressing MaMybA in tobacco obviously improved anthocyanin pigmentation, compared to the OEMaAN2 and control plants, by largely upregulating anthocyanin biosynthetic and endogenous bHLH genes. Notably, the increased transcription of NtF3′5′H in OE-MaMybA tobacco might lead to additional accumulation of delphinidin 3-rutinoside, which was barely detected in OE-MaAN2 and control plants. We concluded that the high concentration of anthocyanin and the newly produced Dp3R caused the darker color of OE-MaMybA compared to OE-MaAN2 tobacco. Conclusion: The newly identified R2R3-MYB transcription factor MaMybA functions in anthocyanin biosynthesis, but has some differences from MaAN2; MaMybA could also be useful in modifying flower color in ornamental plants. Keywords: Anthocyanin biosynthesis, Flower color, Monocot plants, R2R3-MYB, Transcription regulation, Transgenic tobacco * Correspondence: lyl6151@126.com 1 College of Landscape Architecture and Arts, Northwest A&F University, Yangling 712100, Shaanxi, People’s Republic of China 2 Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Yangling 712100, Shaanxi, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. BMC Plant Biology (2019) 19:390 Background A desirable floral color is a typical trait of ornamental plants [1]; it is mainly attributable to anthocyanins, carotenoids, and betalains [2]. Anthocyanins are the primary pigments in flowers, fruit, and other plant tissues, imparting a wide range of colors, from red to violet/blue [3]. They also function in flower pollination, enhancing the tolerance of plants to biological and abiotic stress. Moreover, they are important in the food industry, where they are used as safe and natural colorants [2]. The anthocyanin biosynthetic pathway, in which anthocyanin biosynthetic genes and their regulators are essential to producing anthocyanins, has been the subject of extensive and thorough study [4]. In recent years, genetic engineering has proven to be a promising approach to flower color modification [5]; in ornamental plants, the main approach has been molecular breeding through genetic regulation of anthocyanin synthesis [6]. Although there are many examples of successful flower modification engineering in various ornamental species, either by overexpressing or silencing of diverse anthocyanin biosynthetic genes, such as in gerbera [7], rose [4], carnation [8], and chrysanthemum [9, 10], regulatory proteins may also offer considerable potential in controlling anthocyanin biosynthesis. Anthocyanin biosynthetic regulators include R2R3MYB transcription factors (TFs), their partners the basic helix-loop-helix (bHLH) proteins, and WD-repeat (WDR) proteins; together, these form the MYB-bHLHWDR (MBW) complex, in which R2R3-MYBs, which regulate anthocyanin biosynthesis, are the key TFs determining the intensity and patterning of pigmentation in plants [8]. There have been numerous studies of R2R3MYBs in both dicot and monocot ornamental plants. In dicot ornamental plants, for example, AmRosea1, AmRosea2, and Amvenosa in Antirrhinum majus have been shown to control anthocyanin accumulation in different flower parts [11]. In Lisianthus, overexpression of AmRosea1 leads to an increased level of anthocyanins and a changed color in the petals and sepals of transgenic plants [12]. Additionally, expression of both AmRosea1 and AmDelila (which encode a bHLH protein related to anthocyanin pigmentation in snapdragon flowers) has been shown to induce a high concentration of delphinidin- (Dp-) based anthocyanins in the peel and flesh of transgenic tomato fruits [13]. GMYB10, an anthocyanin-promoting R2R3-MYB in Gerbera hybrida, was overexpressed in the Gerbera cultivar ‘Terra Regina’ (pelargonidin-based flowers), obviously enhancing the anthocyanin level, and inducing novel cyanidin (Cy) biosynthesis [7, 14]. In monocot flowers, Lilium LhMYB12Lat [15], Anthurium andraeanum AaMYB2 [16], and Muscari armeniacum MaAN2 [17] have been shown to promote anthocyanin accumulation in tobacco plants. In Page 2 of 15 the model plant Arabidopsis thaliana, PAP1 and PAP2 increased anthocyanin accumulation when overexpressing as transgenes in Arabidopsis, tobacco, and Rosa hybrida [18–20]. Therefore, identifying these anthocyanin regulators is meaningful in molecular breeding aimed at modifying flower color in ornamental plants, and will even be useful in the commercial production of anthocyanins for use as colorings in the food industry. Grape hyacinth (M. armeniacum) is a species of perennial bulbous monocotyledonous plants that blossom in mid-spring, and is often used as a garden ornamental. Its flowers are a naturally deep cobalt blue color, containing Dp-based anthocyanins [21, 22], and it provides suitable material for the study of blue flower coloration in monocots [23]. In our previous study, we only screened one anthocyanin-related R2R2-MYB unigene by functional annotation in the transcriptome of M. armeniacum flowers [23], and then named MaAN2, which was identified that it could induce anthocyanin accumulation in tobacco, and interacted with a bHLH protein, AtTT8, to regulate the expression of anthocyanin late biosynthetic genes (LBGs; i.e., MaDFR and MaANS) [17] . In the present study, we excavated a novel anthocyanin-related R2R3-MYB TF named MaMybA, using a typical anthocyanin-regulator AtPAP1 from the M. armeniacum flowers transcriptome [23], by local BLASTP (BLAST protein database). We found that MaMybA differed from MaAN2, and we further verified the function of MaMybA in anthocyanin biosynthesis by heterologous expression in tobacco. The study aimed to determine the regulatory mechanism that governs coloration in M. armeniacum flowers, identify a valuable anthocyanin-promoting regulatory gene for use in the modification of flower colors in ornamental plants, and determine similarities and differences between MaMybA and MaAN2 in anthocyanin synthesis. Results Sequence alignment and phylogenetic analysis of R2R3-MYB MaMybA isolated from M. armeniacum One R2R3-MYB unigene was screened with local BLASTP, using anthocyanin-related R2R3-MYB TF AtPAP1 from an M. armeniacum flower transcriptome [23]. Its full-length cDNA sequence was obtained by RACE-PCR. The 711 bp open reading frame (ORF) of MaMybA encoded 237 amino acids. The cDNA sequence was submitted to the NCBI GenBank (accession number MF663728). Sequence alignment showed that the highly conserved MYB domain (i.e., the R2R3 repeats) is present in the Nterminal of MaMybA, MaAN2, and other anthocyaninrelated R2R3-MYBs (Fig. 1a). In addition, a conserved motif [D/E]LX2[R/K]X3LX6LX3R, which is necessary to the interaction of the MYB domain with the R-like Chen et al. BMC Plant Biology (2019) 19:390 Page 3 of 15 Fig. 1 Multiple alignment, phylogenetic analysis, and transcription expression of MaMybA. a The sequence alignment of partial amino acids deduced from MaMybA, MaAN2, and other R2R3-MYBs. R2R3 repeats are indicated above the alignment. Three conserved amino acids [arginine (R), valine (V), and alanine (A)] in the R2R3 repeats of anthocyanin-promoting MYBs in dicots are marked using a red solid circle. The motif [D/E]LX2[K/R]X3LX6LX3R in the R3 repeat is necessary for interactions with a bHLH (basic Helix-Loop-Helix) protein, and is indicated by dark solid triangles. The typical motif [K/R]P[Q/R]P[Q/R] in the AN2 subgroup and KAX[K/R]C[S/T] in the C1 subgroup are shown by a black box and a blue box, respectively. b Phylogenetic analysis of entire amino acid sequences deduced from MaMybA, MaAN2, and other anthocyanin-related R2R3MYBs. The phylogenetic tree was constructed by the maximum-likelihood method using MEGA 6.0 software. The numbers next to the nodes indicate bootstrap values from 1000 replicates. Human cMYB (M15024) was used as a rooting outlier. R2R3-MYB protein sequences of different plant species were retrieved from the GenBank database, and their GenBank accession numbers are listed in brackets. c Expression profile of MaMybA in different tissues of Muscari armeniacum; MaActin was the reference gene. Error bars indicate the standard deviations (SD) of average results bHLH proteins [24], was present in the R3 repeat of MaMybA, MaAN2, and other anthocyanin-related R2R3-MYBs (Fig. 1a). When considering the entire amino acid sequence, MaMybA showed only 43.5% shared identity with MaAN2, 47.3% shared identity with EgVIR (an R2R3-MYB controlling seed color in Elaeis guineensis [25], and 43.07% shared identity with AcMYB1 (an R2R3-MYB regulating the anthocyanin accumulation of bulbs in Allium cepa) [26] (Fig. 1a). Moreover, MaMybA and MaAN2 shared three conserved amino acids [Arg (R), Val (V), and Ala (A)] in the R2R3 repeats, and a highly conserved motif [K/R]P[Q/ R]P[Q/R] in the C-terminal with MYBs in the AN2 subgroup from eudicots and some monocots (Fig. 1a). A phylogenetic analysis indicated that MaMybA, along with MaAN2, AtPAP1, LhMYB6, and AcMYB1, was clustered into the AN2 subgroup, and was most closely associated with AcMYB1 (Fig. 1b). However, MYBs from the monocot families Poaceae and Orchidaceae were grouped into the C1 subgroup (Fig. 1b). Additionally, the relative expression of MaMybA in the roots, bulbs, leaves, and five different floral developmental stages (S1~S5) showed that MaMybA was predominantly expressed in pigmented flowers. The mRNA level of MaMybA was very low in the roots, bulbs, and flowers of stage S1, which was much lower than in the leaves. The MaMybA transcript in flowers gradually increased, peaking at stage S3, and then Chen et al. BMC Plant Biology (2019) 19:390 slightly decreased from stages S3 to S4, before showing a fall by stage S5 (Fig. 1c). MaMybA was localized in the cell nucleus and had transcriptional activation ability In order to detect the subcellular localization of MaMybA, the plasmids 35S:GFP (Green fluorescent protein) and MaMybA-GFP were separately cotransformed with AtHY5-(ELONGATED HYPOCOTYL5; AT5G11260) mCherry into Arabidopsis mesophyll protoplasts. The GFP fluorescence of MaMybA was visualized to overlap with the mCherry fluorescence of AtHY5, which is a known nuclear protein [27], indicating that MaMybA was localized in the nuclei of A. thaliana protoplasts (Fig. 2). We performed a transactivation assay in yeast in order to assess the transcriptional activation ability of MaMybA. As shown in Additional file 1: Figure S1, the yeasts only transformed with the positive control (pGBKT7-53 + pGADT7-T), and the pGBKT7/MaMybA plasmid was able to grow in SD/-Trp media to which were added 40 μg mL− 1 X-α-gal and 200 ng mL− 1 aureobasidin A (AbA), and exhibited blue plaques. However, the negative control was not able to grow in SD/-Trp media plus 200 ng mL− 1 AbA or 40 μg mL− 1 X-α-gal and 200 ng mL− 1 AbA. The results showed that MaMybA has transcriptional activation ability. MabHLH1 interacted with MaMybA or MaAN2 in vivo R2R3-MYBs have been reported to interact with the IIIf bHLH protein that regulates anthocyanin biosynthesis [28–30]. We screened one anthocyanin-related bHLH unigene from the transcriptome of M. armeniacum with local BLASTP, using the R-like IIIf bHLH protein AtTT8. A 2001 bp full-length cDNA was obtained from M. armeniacum flowers using a reverse transcriptionpolymerase chain reaction (RT-PCR), and named MabHLH1 (MF663729). MabHLH1 contained a highly conserved MYB interaction region in the N-terminal, and a bHLH DNA binding domain in the C-terminal (Additional file 2: Figure S2a). The phylogenetic analysis showed that MabHLH1 was grouped with LhbHLH1 Fig. 2 Subcellular localization of MaMybA. The transient coexpression of MaMybA-GFP with AtHY5-mCherry in Arabidopsis thaliana mesophyll protoplasts showed that MaMybA and AtHY5 were co-located in the nucleus. Bars: 10 μm Page 4 of 15 and ZmLC into the LC/JAF13/DEL clade of the IIIf subgroup of the flavonoid-related bHLH proteins (Additional file 2: Figure S2b), and was also constitutively expressed in different tissues of M. armeniacum (Additional file 2: Fig. S2c). Subcellular localization and transcriptional activation ability assays showed that MabHLH1 was localized in the nucleus and had transactivation ability, thus meaning it was a TF (Additional file 3: Figure S3). A bimolecular fluorescence complementation (BiFC) assay was then conducted to verify the interaction between MabHLH1 and MaMybA or MaAN2 in vivo (Fig. 3a). Co-expressing either YC/ MaMybA with YN/MabHLH1 or YC/MabHLH1 with YN/MaAN2 in Nicotiana benthamiana leaves revealed yellow fluorescent protein (YFP) fluorescence in the epidermal cell nuclei (Fig. 3a). Thus, MabHLH1 was shown to be an interacting protein from the anthocyanin-related R2R3-MYBs in M. armeniacum, which was able to interact with MaMybA or MaAN2 in vivo. MaMybA activated the anthocyanin biosynthetic gene promoters In order to investigate whether MaMybA or MabHLH1 is able to control expression of the anthocyanin biosynthetic genes, a dual-luciferase assay was performed on N. benthamiana leaves (Fig. 3b). The promoters of the anthocyanin early biosynthetic gene (EBG) MaCHS (M. armeniacum; KY781171) and the key LBGs, AtDFR (A. thaliana; AT5G42800) and MaDFR (M. armeniacum; KY781169), were fused to LUC (firefly luciferase). MaMybA, MaAN2, and MabHLH1 were driven by 35S promoters. Each or both of the TFs were co-infiltrated with each promoter. The results showed that single or both of the TFs could not increase the promoter activity of MaCHS. However, MaMybA alone could activate the MaDFR and AtDFR promoters, while neither MaAN2 nor MabHLH1 were able to activate both. When co-expressing MaMybA with MabHLH1, the promoter activities of MaDFR and AtDFR were significantly promoted, by 1.6 and 1.1 times higher than when infiltrated with MaMybA alone, respectively (Fig. 3b). When co-infiltrating MaAN2 with MabHLH1, the promoter activities of MaDFR and AtDFR were also significantly promoted, 4.0 and 22.2 times higher, when compared with MaAN2 infiltrated alone, respectively (Fig. 3b). Therefore, the results indicated that, MaMybA alone or in interaction with MabHLH1, could control the transcription of MaDFR, but MaAN2 required a direct interaction with MabHLH1 to do so. Heterologous expression of MaMybA in tobacco strongly promoted anthocyanin accumulation In order to characterize the function of MaMybA, MaMybA driven by a 35S promoter was transformed Chen et al. BMC Plant Biology (2019) 19:390 Page 5 of 15 Fig. 3 Bimolecular fluorescence complementation (BiFC) assay and dual luciferase assay. a BiFC assay was performed to confirm the interaction between MabHLH1 and MaMybA or MaAN2 in Nicotiana benthamiana leaves. Bars: 25 μm. b Dual luciferase assay of MabHLH1 with or without MaMybA or MaAN2: effects on the transcriptional regulation of MaCHS, MaDFR, and AtDFR promoters, respectively, in N. benthamiana leaves. The results show promoter activity expressed as the ratio of the MaCHS, MaDFR, and AtDFR promoters, LUC to 35S: REN. Error bars indicate the standard deviations (SD) of average results into tobacco (N. tabacum ‘NC89’) leaf discs by an Agrobacterium-mediated transformation. Three T1 overexpressing MaMybA (OE-MaMybA) transgenic lines, designated as OE#1, OE#2, and OE#3, were generated (Fig. 4a, b). Additionally, the T1 overexpressing MaAN2 tobacco (the most pigmentation line (i.e., line 2) among three OE-MaAN2 transgenic plants in respect to Chen et al. [17]) was used as the positive control (OEMaAN2), and the empty vector transgenic plant was used as the negative control (control). The results showed that MaMybA strongly increased anthocyanin production and conferred an intense red-purple color in tobacco, especially in the leaf, calyx, petal, and anther (Fig. 4a-c), while the ovary wall and seed skin were barely anthocyanin pigmentation (Fig. 4c, d). However, OE-MaAN2 tobacco presented a much lighter magenta color in the leaf, calyx, and anther than did OEMaMybA tobacco; however, the ovary wall and seed skin were both pigmented with a reddish color, and the corolla was deep pink (Fig. 4a-d). Floral color is related to the internal or surface tissue morphology of floral organs, and to the type and content of anthocyanin pigments in the floral cells; pigment, however, is the key determinant [1]. We first determined the content and type of anthocyanin in tobaccos using high-performance liquid chromatography (HPLC). The anthocyanin concentration in the leaves and corollas of three OE-MaMybA tobaccos was obviously higher than in the OE-MaAN2 and control tobaccos (Fig. 4e, f). Specifically, the anthocyanin level in three lines of OE- Chen et al. BMC Plant Biology (2019) 19:390 Page 6 of 15 Fig. 4 Phenotypic observation and total anthocyanin content of the leaves and corollas of the control, OE (overexpressing)-MaAN2, and three OE-MaMybA line (OE#1, OE#2, and OE#3) tobaccos. Phenotype of total plant (a), phenotype of flower (b), calyx, anthers, filament, and ovary of a flower (c), and seeds (d). A quantitative determination of the anthocyanin content of the leaves (e) and corollas (f) in the control, OE-MaAN2, and three OE-MaMybA line tobaccos. The anthocyanin extracts from the leaves and corollas were analyzed using high-performance liquid chromatography and were each monitored at 530 nm. A standard curve of cyanidin 3-rutinoside content was used as a control to calculate the anthocyanin content in the tobacco. Error bars indicate standard deviations (SD) in the average anthocyanin content MaMybA leaves was 1.25- to 2-fold higher than that of OE-MaAN2 leaves. The level of anthocyanin in three lines of OE-MaMybA corollas was 1.8- to 4.3-fold higher than that of OE-MaAN2 corollas, and was 6.9- to 16.7fold higher than that of the control corollas (Fig. 4e, f). Anthocyanins were virtually undetectable in the control tobacco leaves (Fig. 4e, Fig. 5a, Additional file 4: Figure S4). Next, we detected two main anthocyanins (peaks 1 and 2) in the leaves and corollas of three OE-MaMybA lines (OE#3 as the representative in Fig. 5a; Additional file 4: Figure S4), while primarily a single anthocyanin (peak 2) was detected in the corollas of the control and three OEMaAN2 lines mentioned in Chen et al. [17] (line 2 as the representative in Fig. 5a, Additional file 4: Figure S4). Previous studies have shown the primary anthocyanin in tobacco to be cyanidin 3-rutinoside (Cy3R) [31]; the retention times of the delphinidin 3-rutinoside (Dp3R) and Cy3R standards were very close to Chen et al. BMC Plant Biology (2019) 19:390 Page 7 of 15 Fig. 5 Anthocyanin composition analysis of the leaves and corollas of the control, OE-MaAN2, and OE-MaMybA tobaccos. High-performance liquid chromatography analysis of anthocyanin extract (a), and its corresponding hydrolysate (b) in the leaves and corollas of the control, OEMaAN2, and OE-MaMybA tobaccos. According to the chromatographic peak in the sequence, the standards are delphinidin 3-rutinoside, cyanidin 3-rutinoside, and pelargonidin 3-rutinoside (from left to right) in diagram a; and delphinidin, cyanidin, petunidin, pelargonidin, and malvidin (from left to right) in diagram b. The mass spectrum and structure patterns of delphinidin 3-rutinoside (c) peaks 1 and 2 (Fig. 5a), respectively; and Dp (peak a) and Cy (peak b) were detected by hydrolysis (Fig. 5b). Therefore, we made a preliminary speculation that peaks 1 and 2 represented Dp3R and Cy3R, respectively. Moreover, the two anthocyanins were confirmed by ultra-performance liquid chromatography (UPLC)time-of-flight (TOF)-tandem mass spectrometry (MS/ MS) analysis. In detail, peak 1 showed typical molecular ions at a mass-to-charge ration (m/z) of 611.1601 [M]+ (C27H31O16), which was consistent with the molecular weight of Dp3R. In addition, two product ions, one at m/z 465.1018 and one at m/z 303, indicated that one molecule of glucose and one of rhamnose were released from one molecule of Dp (Fig. 5c). Additionally, the metabolite was also used to search the SciFinder and Reaxys databases. Peaks 1 and 2 were therefore identified as representing Dp3R and Cy3R (Additional file 5: Figure S5), respectively. Additionally, microscopic observations indicated that no anthocyanin was found in any of these cell types in the control tobacco (Additional file 6: Figure S6ad). Anthocyanin accumulated primarily in the palisade parenchyma, abaxial epidermal cells, and trichomes in OE-MaAN2 and OE-MaMybA tobacco leaves (Additional file 6: Figure S6e-l). Anthocyanin was also scattered in the adaxial epidermal cells, spongy parenchyma, and hypodermal parenchyma cells (Additional file 6: Fig. S6 g, h, k, l). Moreover, there were no morphological changes in the epidermal cells of leaves and corollas between the control, OE-MaAN2, and OE-MaMybA plants, according to scanning electron microscopy (Additional file 7: Figure S7). Chen et al. BMC Plant Biology (2019) 19:390 Overexpression of MaMybA in tobacco upregulated the expression levels of anthocyanin pathway genes We first performed a quantitative real-time polymerase chain reaction (qRT-PCR) assay to test MaMybA transcripts in the transgenic tobaccos, and found that MaMybA was expressed in the leaves and corollas of three transgenic lines (OE#1, OE#2, and OE#3), but not in the negative control plants (i.e., control, Fig. 6a, d). In addition, MaAN2 was also expressed in the positive control tobacco (i.e., OE-MaAN2, Fig. 6a, d). Next, we conducted a qRTPCR assay of anthocyanin biosynthetic and TF genes in the leaves and corollas of the control, OE-MaAN2, and OEMaMybA tobacco. The results showed that all the anthocyanin structural genes, including NtCHS, NtCHI, NtF3H, NtF3′H, NtF3′5′H, NtDFR, NtANS, and NtUFGT, were upregulated in the leaves and corollas of transgenic plants, especially in OE#2 and OE#3 (Fig. 6b, e). The two endogenous bHLH TF genes NtAn1a and NtAn1b, which regulate flavonoid synthesis in tobacco [32], were also upregulated in the leaves and corollas of the OE-MaMybA plants (Fig. 6c, f). However, the transcripts of NtAN2 were barely detected in the leaves of all genotypes of tobaccos, and those in the corollas of OE-MaAN2 and OE-MaMybA plants were lower than those in the control (Fig. 6c, f). Discussion Anthocyanins are the primary pigments in flowers. Anthocyanin biosynthesis is regulated by R2R3-MYB TFs, or the Page 8 of 15 MBW complex [33]. R2R3-MYB TFs involved in flower coloration have been found in various ornamental plants, including Gerbera hybrida, Lilium, and Anthurium [14–16]. In grape hyacinth, previous study has shown that an anthocyanin-related R2R3-MYB, MaAN2, induced the obvious increment of cyanidin biosynthesis in tobacco and relied on AtTT8 or MabHLH1 to activate the LBG expression [17] (Fig. 3b). In the present study, we isolated a novel R2R3-MYB, MaMybA, which differed from MaAN2. Heterologous expression of MaMybA conferred intense and dark anthocyanin pigmentation in tobacco, and produced a new anthocyanin, Dp3R. The study determined some precise differences between MaMybA and MaAN2 functioning in anthocyanin biosynthesis, and further revealed the regulatory mechanism responsible for coloration in M. armeniacum flowers. MaMybA is an R2R3-MYB TF in the AN2 subgroup of the R2R3-MYB family Anthocyanin-promoting R2R3-MYB TFs have specific structures and typical motifs. In M. armeniacum, we characterized an R2R3-MYB MaMybA, similar to MaAN2, which contained three conserved amino acids (Arg, Val, and Ala) in the highly conserved R2R3 domain of N-terminal region and a typical motif [K/R]P[Q/ R]P[Q/R] in the C-terminal region. These conserved amino acids and typical motif have also been reported as Fig. 6 Relative expression of anthocyanin biosynthetic genes and endogenous transcription factor (TF) genes in the leaves and corollas of the control, OE-MaAN2, and three independent OE-MaMybA transgenic plants (OE#1, OE#2, and OE#3). The expression profiles of MaMybA in the leaves (a) and corollas (d) of the control, OE#1, OE#2, and OE#3; the expression profile of MaAN2 in the leaves (a) and corollas (d) of the OEMaAN2 tobacco; the expression levels of anthocyanin biosynthetic genes (NtCHS, NtCHI, NtF3H, NtF3′H, NtF3′5′H, NtDFR, NtANS, and NtUFGT) in the leaves (b) and corollas (e) of different tobacco genotypes; and the relative expression of three endogenous TF genes (NtAN2, NtAn1a and NtAn1b) in the leaves (c) and corollas (f) of different tobacco genotypes. NtTubA1 was used as the internal control. Error bars indicate the standard deviations (SD) of average results Chen et al. BMC Plant Biology (2019) 19:390 present in anthocyanin-promoting R2R3-MYBs of the AN2 subgroup in dicots [7, 11, 20] and a few monocots [15–17, 34] (Fig. 1a, b). Notably, MaMybA and MaAN2 in monocot plants were not found to contain the motif KAX[K/R]C[S/T] in the C1 subgroup of R2R3MYBs from the Poaceae and Orchidaceae plant families (Fig. 1a, b). Additionally, the motif [D/E]LX2[R/ K]X3LX6LX3R in the R3 repeat of MaMybA and MaAN2 (Fig. 1a) indicated that MaMybA and MaAN2 could interact with bHLH TFs. MaMybA was identified as a TF because it was localized in the cell nucleus, and had transcriptional activation ability (Fig. 2, Additional file 1: Figure S1). To our knowledge, all anthocyanin-related dicot R2R3-MYB TFs have been grouped into the AN2 subgroup of the R2R3-MYB family. In monocots, only those MYBs in Poaceae and Orchidaceae are clustered into the C1 subgroup [30, 35, 36]; the other monocot MYBs are placed in the AN2 subgroup [16, 17, 25, 26, 34]. Therefore, more anthocyanin-related R2R3-MYBs should be excavated in order to determine the regularity of clustering between dicots and monocots. Interestingly, phylogenetic analysis indicated that the evolutionary relationship of MaMybA was closer than that of MaAN2 to AcMYB1. MaMybA and MaAN2 shared an identity of only 43.5% in their whole amino acid sequences. The relative expression of MaMybA in different tissues indicated that it was predominantly expressed in pigmented flowers (Fig. 1c). Its expression was coordinated with, although slightly earlier than that of MaAN2, and also with pigment accumulation during flower development [17]. The large family of R2R3-MYB is the major family of MYB proteins. R2R3-MYBs all have the highly conserved R2R3 domain at the N-termini, which decide the regulatory specificity and offer the MYB nuclear localization signals, while the C-terminal regions often possess transactivation or DNA-repression motifs [37, 38]. In Arabidopsis, the R2R3-MYBs are divided into 22 subgroups according to the recurring amino acid motifs in the C-termini [39]. Specifically, the R2R3-MYBs of AN2 subgroup (i.e., subgroup 6) which consist of the conserved motif ([K/R]P[Q/R]P[Q/R]) in the C-terminal regions function in inducing anthocyanin biosynthesis [34]; the R2R3-MYBs of subgroup 4 contained the conserved EAR motif (LxLxL) in the C-termini function in reducing anthocyanin biosynthesis [40]. Previous study also showed that R2R3-MYBs which contain highly conserved motifs and exon lengths have similar functions between Arabidopsis and Vitis, although low sequence homology in the C-terminal regions of all R2R3-MYB proteins. Thus, despite occurrence of diversification, the highly conserved regions could maintain the specific functionality, while variations in Page 9 of 15 C-terminal regions could facilitate the gaining of new or cooperative functions [37]. In planta, there are numerous examples where two or more R2R3-MYB genes, functioning in anthocyanin biosynthesis, are found within a single plant species, such as AtPAP1 and AtPAP2 in Arabidopsis [20]. However, some R2R3-MYB anthocyanin regulators found in the same plant show diversity through functioning in anthocyanin pigmentation in different tissues or organs; for example, Petunia AN2 is responsible for flower limb pigmentation, while AN4 mainly regulates anther anthocyanin accumulation [41]; in Solanum lycopersicum, two different R2R3 MYB, SlANT1 and SlAN2, both could induce anthocyanin biosynthesis, but only the latter one functions in anthocyanin synthesis in vegetative tissues under high light or low temperature conditions [42]. In grapevine, four anthocyanin-related R2R3 MYBs have different amino acid lengths of C-termini. Particularly, VvMYBA2 has two C-terminal repeats, while VvMYBA1 has one and VvMYBA3 lacks this motif. Besides, MYBA1 and MYBA2 function fruit coloration, but MYBA3 loses the function of accumulating anthocyanin. These differences could cause novel or cooperative functions [43]. In this study, each of MaMybA and MaAN2 only contained one conserved motif (KPQPQ) in the Cterminal region. Furthermore, premature termination of the C-terminal domain did not occur in MaMybA or MaAN2 (the amino acid lengths of MaMybA and MaAN2 were 237 and 240, respectively), and the two MYBs could perform complete functions in anthocyanin biosynthesis in tobacco. Thus, MaAN2 might be the paralog of MaMybA by gene duplication in the process of evolution, and the variation in C-terminal indicated that they might possess redundant, overlapping or cooperative functions in grape hyacinth. So, based on this, the experimental evidence on functionality of the variable C-terminal between MaMybA and MaAN2 is a future direction of our work. MaMybA alone or in interaction with MabHLH1 could activate anthocyanin biosynthetic gene promoters Anthocyanin biosynthesis is regulated by MYB-bHLH complexes in almost all plant species [33]. In the present study, the resulting MabHLH1 contained an MYB interaction region on the N-terminal and a bHLH DNA binding domain at the C-terminal region, together with other IIIf bHLH proteins involved in flavonoid biosynthesis [28, 30, 44] (Additional file 2: Figure S2a). The phylogenetic analysis indicated that MabHLH1 was grouped into the LC/JAF13/DEL clade of the IIIf bHLH TF family [26, 45] (Additional file 2: Figure S2b). These findings indicated that MabHLH1 might play a role in anthocyanin biosynthesis in M. armeniacum. However, MabHLH1 was constitutively expressed in different Chen et al. BMC Plant Biology (2019) 19:390 tissues, and was not concomitant with the pigmentation of flower development (Additional file 2: Figure S2c), which has also been found in apples [46], grapes [47], and Phalaenopsis [36]. MabHLH1 interacted with either MaMybA or MaAN2 in vivo; this was confirmed by the BiFC assay (Fig. 3a). In the dual luciferase assay, we found that the promoter activity of MaCHS (EBG) was induced by none of all TFs (each or both of MYB-bHLH complexes) in this assay, which was consistent with the result that the activity of MrCHS promoter was not response to MrMYB1, or MrbHLH1, or MrMYB1-MrbHLH1 in Myrica rubra [48]. MaMybA alone was able to regulate the expression of MaDFR (LBG), in contrast to MaAN2, which relied on MabHLH1 to control MaDFR expression (Fig. 3b). In maize, ZmP alone can activate flavonoid biosynthetic genes, but ZmC1 require a direct interaction with bHLH protein R/B to do so [49]; in Phalaenopsis, PeMYB11 is dependent on PebHLH1, while PeMYB2 and PeMYB12 are independent of PebHLH1 in regulating PeDFR expression [36]. The regulation pattern of ZmP, PeMYB2, and PeMYB12 is consistent with that of MaMybA. However, in Arabidopsis, AtPAP1 interacts with AtTT8 or AtGL3 to regulate AtDFR expression [50, 51]. PhAN2 also requires bHLH cofactor PhAN1 or PhJAF13 to enhance promoter activity in Petunia PhDFR [44]; and the MdMYB10MdbHLH3 and MdMYB10-MdbHLH33 complexes can each activate the expression of MdDFR in apples [28]. These regulation patterns are highly consistent with that of MaAN2. Phenotypic differences between OE-MaMybA and OE-MaAN2 tobacco can be ascribed to the high concentration of anthocyanin and the newly produced Dp3R in OE-MaMybA tobacco The ectopic expression of a gene in a model plant quickly facilitates its functional identification, and visible anthocyanin pigmentation could directly demonstrate its function in anthocyanin biosynthesis [52]. Many studies show that overexpressing anthocyanin-related R2R3MYB genes could promote anthocyanin production in tobacco by genes such as AtPAP1 [18, 20], and AaMYB2 [16]. In this study, overexpressing MaMybA in tobacco induced a high level of anthocyanin production in the leaf, petal, anther, and calyx, which all presented with mulberry pigmentation (Fig. 4a-c), while the ovary wall and seed skin were barely pigmented (Fig. 4c, d). However, OE-MaAN2 tobacco presented a magenta leaf, calyx, anther, and a deep pink corolla, which was much lighter than in OE-MaMybA tobacco (Fig. 4a-c); the ovary wall and seed skin of OE-MaAN2 tobacco showed a reddish color, which were different from that of OEMaMybA (Fig. 4c, d). A microscopic observation of OE- Page 10 of 15 MaMybA tobacco leaves indicated that anthocyanin was primarily present in the palisade parenchyma, trichomes, and lower epidermis, with some pigment scattered in the upper epidermis and spongy mesophyll; and the pigmentation was much deeper in comparison with OE-MaAN2 transgenic plants (Additional file 6: Figure S6). In OEAtPAP1 tobacco, the pigmentation cells were primarily found in the trichomes and lower epidermis [18]. Thus, different R2R3-MYBs influence anthocyanin accumulation in different tissues or cells to various extents, and the distribution of anthocyanin might affect plant color. Previous studies have demonstrated that the primary anthocyanin in the tobacco corolla is Cy3R [31]. In the present study, a newly produced anthocyanin, Dp3R, was found in the leaves and corollas of OE-MaMybA tobacco, in addition to Cy3R, the primary anthocyanin (Fig. 5a); this differed from OE-MaAN2 tobacco, in which the primary anthocyanin in the leaves and corollas was found to be Cy3R (Fig. 5a). Additionally, the anthocyanin levels of the leaves and corollas in three OEMaMybA transgenic lines were all greater than 2.0 mg g− 1 fresh weight (FW), while those in OE-MaAN2 plants were both lower 2.0 mg/L FW (Fig. 4e, f). Previous study has shown that two anthocyanin regulatory genes (Arabidopsis R2R3 MYB gene mPAP1 and Zea mays bHLH-type gene B-Peru) overexpressing in N. tabacum L. to yield an enhanced anthocyanin content, and also produced a new type pigment Dp3R in the flower limb of transgenic tobacco, which presented a dark red color [53]. The hydrolysate of anthocyanin extracts from the leaves and corollas of OE-MaMybA tobacco also showed that Cy was the primary anthocyanidin, followed by Dp (Fig. 5b); this was similar to the results of anthocyanin hydrolysis from OE-AtPAP1 tobacco [18]. Previous studies have shown that the reddish-purple flower of grape hyacinth (M. comosum ‘Plumosum’) contains two main anthocyanidins (Dp and Cy) that contribute to its color [22]. Therefore, we concluded that the red Cy-based derivatives, together with the blue Dp-based derivatives and the high anthocyanin concentration, contributed to the dark reddish-purple color in OE-MaMybA tobacco. Moreover, when overexpressing different R2R3-MYB genes in model plants, the resultant plant phenotypes were distinct in anthocyanin localization, type and content, possibly because different R2R3-MYB TFs regulate different target genes to induce different anthocyanins in different tissues. R2R3-MYB proteins of MBW complexes play core roles in anthocyanin accumulation, and the MBW complexes could probably induce the entire anthocyanin biosynthesis pathway [8]. In the present study, expression levels of almost all the anthocyanin biosynthetic genes (NtCHS, NtCHI, NtF3H, NtF3’H, NNtDFR, NtANS, and NtUFGT) and endogenous bHLH TF genes (NtAn1a