NtbHLH1, a JAF13-like bHLH, interacts with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus

Background Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus (Narcissus tazetta L. var. chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear. Results In this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6-transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6. Conclusions Our results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-021-03050-1.

The IIIf subfamily bHLHs can be further divided into two clades: JAF13 clade and AN1 clade. In Solanaceous plants, AN1 is directly involved in the activation of the biosynthetic genes, whereas JAF13 is involved in the regulation of AN1 transcription [26]. In maize, overexpression of ZmLC can promote the expression of ZmDFR and ZmANS, key genes of the flavonoid biosynthesis pathway, and promote the accumulation of anthocyanins [20]. Therefore, in different species, and in particular across the dicot and monocot division, the function of bHLHs may differ.
R2R3-MYB proteins are the largest class of transcription factors in plants and are involved in a wide range of regulatory functions including regulation of various secondary metabolites [10]. R2R3-MYB transcription factors have highly conserved DNA-MYB domains, which consist of a series of highly conserved amino acid sequences and spacer sequences [23].
Zea mays ZmC1 was the first identified R2R3-MYB transcription factor shown to regulate anthocyanin biosynthesis [29]. Most MYBs found in plants that can promote flavonoid metabolism are R2R3-MYBs. For example, PyMYB10 regulates anthocyanin biosynthesis in pears [13],DkMYB4 in persimmons promotes the accumulation of proanthocyanidins by directly binding promoters of some structural genes of the proanthocyanidin pathway [1,2]. VvMYBPA1 activates VvLAR and VvANR promoters to up-regulate proanthocyanidin biosynthesis in grape peel [7].
The MYB-bHLH-WD40 (MBW) complex has a crucial role in the regulation of flavonoid biosynthesis. In heterologous systems, the monocot members of this complex can activate anthocyanin expression in dicots; petunia transformed with ZmC1 has activated expression of PhCHSJ and PhDFRA [32]. Tobacco transformed with ZmLC can activate the expression of tobacco CHS and DFR [28]. Freesia FhGL3L and FhTT8L can interact with AtPAP1 and AtTT2, thereby regulate the flavonoid biosynthesis pathway [22].
Chinese narcissus (Narcissus tazetta L. var. chinensis) which belongs to the monocot Amaryllidaceae family is a perennial bulbous plant and shows high ornamental value [39]. Flower colors of Chinese narcissus are only yellow or white. The composition of flavonoid compounds in petals and corona was flavonols, but no anthocyanins are found. In addition, proanthocyanidins are found accumulate mainly in basal plates, but not detected in flowers [36]. Our recent research found that NtMYB2, NtMYB3 and NtMYB5 [38] are transcriptional repressors of flavonoid biosynthesis in Chinese narcissus. However, activation of flavonoid biosynthesis in Chinese narcissus is still not clear [4,5].
In the current investigation, a JAF13-like bHLH homolog, NtbHLH1 and a R2R3-MYB homolog, NtMYB6 were isolated from Chinese narcissus and functionally characterized. NtbHLH1 and NtMYB6 were ectopically expressed in transgenic tobacco, increasing the expression level of flavonoid and proanthocyanidin biosynthesis genes. Dual luciferase assays showed that co-expression of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese narcissus DFR gene (NtDFR). Yeast two-hybrid (Y2H) analysis confirmed the interaction of NtbHLH1 and NtMYB6. Our results suggest that Ntb-HLH1 plays a role in flavonoid biosynthesis and interacts with NtMYB6 to directly promote proanthocyanidin biosynthesis in Chinese narcissus.

Cloning and Sequence Analysis of NtbHLH1
Previously, an RNA-Seq-based transcriptome database from flowers and basal plates of Chinese Narcissus was generated [36]. By blast-match to sequences in this database, one potential flavonoid-related bHLH gene was identified and designated as NtbHLH1. The 3´ end sequence of NtbHLH1 was obtained by 3´ RACE and then the full length of open reading frame (ORF) was cloned by RT-PCR (reverse transcription PCR) (Supplemental Fig. 1) (GenBank number QDS02912.1). NtbHLH1 has an ORF of 1,896 bp, encoding a polypeptide of 632 amino acids. The predicted NtbHLH1 protein contains two domains, N-terminal bHLH-MYC_N domain and C-terminal HLH domain that are conserved in flavonoidrelated bHLH-type TFs (Fig. 1A). These two domains have been predicted to be important for the bHLH protein to perform transcriptional regulatory function [37].

Expression pattern of NtbHLH1 in Chinese narcissus
The expression pattern of NtbHLH1 in different tissues of Chinese narcissus was analyzed by qRT-PCR (Quantitative real-time PCR) (Fig. 2). The expression level of NtbHLH1 in basal plates was the highest, as compared to petals, and corona. This tissue has been shown to accumulate proanthocyanidin in previous research [36]. This result suggests that NtbHLH1 has the potential to play a role in the biosynthesis of flavonoids in basal plates.

Over-expression of NtbHLH1 in Tobacco
Transgenic tobacco over-expressing NtbHLH1 displayed an increase in red appearance in the petals as compared to controls (Fig. 3A). All flowers of transgenic lines had higher anthocyanin content than wild type tobacco. Floral anthocyanin content of three transgenic lines was between 1.7 to 3.24 fold higher than control tobacco petals (Fig. 3B). Total flavonoid content of the flowers of the three transgenic lines increased significantly compared to the control (Fig. 3C).
To check the effects of ectopic over-expression of NtbHLH1 on endogenous tobacco genes encoding the tobacco flavonoid biosynthesis pathway, qRT-PCR analysis on the flowers was performed. The results indicated that over-expression of NtbHLH1 affected the transcript levels of tobacco flavonoid biosynthesis genes. Expression of the tobacco bHLH AN1 and the structural genes CHS, FLS, LAR, F3H, DFR, ANR were up-regulated (Fig. 4). The transcript level of early pathway gene CHS was 1.3 to 2.3 times higher and late pathway gene LAR was 4.5 to 13.5 times higher in the transgenic lines than the control. These data indicate that NtbHLH1 promotes not only anthocyanin biosynthesis in the petals but also flavonoid biosynthesis in transgenic tobacco plants.

Cloning and sequence analysis of NtMYB6
Because NtbHLH1 is mainly expressed in basal plates of Narcissus, we analysed the RNA-Seq database [36] for potential transcriptional partners for this bHLH. A R2R3-MYB was identified which also showed higher expression levels in basal plates. This R2R3-MYB gene was cloned (Supplemental Fig. 3) and named NtMYB6. Sequence analysis showed that NtMYB6 has a full length ORF of 768 bp encoding a polypeptide of 256 amino acids (Gen-Bank number KY645961). Protein sequence alignment between NtMYB6 and other R2R3-MYBs showed that NtMYB6 contained the conserved R2 and R3 DNA binding domains at the N-terminus (Fig. 5A).
Phylogenetic analysis showed that NtMYB6 was placed in a clade of R2R3-MYB proteins that include DkMYB4, VvMYBPA1 and AtTT2, which have been characterized as regulatory activators of proanthocyanidin biosynthesis related gene expression, suggesting that NtMYB6 may act as a positive regulator of Narcissus proanthocyanidin biosynthesis (Fig. 5B).
The expression of NtMYB6 gene in Chinese narcissus was examined by real-time qRT-PCR. The results confirmed that NtMYB6 expression was higher in basal plates. Expression levels of NtMYB6 in perianth and corona were significantly lower (Fig. 6).

Over-Expression of NtMYB6 in tobacco
Compared to the wild type tobacco, the flowers of tobacco expressing the NtMYB6 gene displayed a decrease in color (Fig. 7A). Anthocyanin content decreased and proanthocyanidin contents increased significantly in transgenic flowers (Fig. 7B, C). QRT-PCR analysis showed that expression levels of tobacco F3'H and LAR were significantly up-regulated, while the expression levels of other flavonoid pathway genes was either unaffected or down-regulated (Fig. 8).

Fig. 2
Expression of NtbHLH1 in different tissues of Chinese narcissus. P indicates the petals. C denotes for corona. B represents for basal plates. The bars indicate the standard error of three biological replicates. Letter represents a significant difference at the level of P < 0.05 using SPSS statistical analysis Dual luciferase assay A dual luciferase system in Agrobacterium tumefaciensinfiltrated Nicotiana benthamiana leaves was used to investigate the function of NtbHLH1 and NtMYB6. Ntb-HLH1 and NtMYB6 were independently infiltrated or coinfiltrated into tobacco leaves, along with the promoter of Narcissus DFR or LAR fused to luciferase. It was found that NtbHLH1 alone did not activate the NtDFR promoter, however, NtMYB6 alone can activate the NtDFR promoter (Fig. 9). Co-infiltration of both NtbHLH1 and NtMYB6 showed a significant increase in NtDFR promoter activity, resulting in approximately 14-fold increase in the promoter activity of NtDFR compared to the control and eightfold increase compared to NtMYB6 alone. NtbHLH1 and NtMYB6 alone or co-infiltration showed little effect on NtLAR promoter (Fig. 9). These results indicate that NtMYB6 mediated activation of NtDFR promoter is enhanced by the bHLH TF NtbHLH1.

Protein-Protein Interaction between NtbHLH1 and NtMYB6
Yeast two-hybrid (Y2H) was performed to investigate the interaction between NtbHLH1 and NtMYB6. Firstly, the auto-activation was tested for NtbHLH1. Auto-activation test results showed that pGBKT7-NtbHLH1 was not able to grow in SD/-Trp/-Ade and SD/-Trp/-His medium, and did not turn blue in SD/-Trp /X-ɑ-Gal medium. This indicates that NtbHLH1 does not have auto-activation activity  (Fig. 10A). pGADT7 and pGBKT7 empty vectors were used as negative controls. Yeast cells transformed with BD-NtbHLH1 and AD-NtMYB6 showed growth on both SD/-Trp/-Leu and SD/-Trp/-Leu/-Ade/-His media, and their color turned to blue when grown on SD/-Trp/-Leu/-His/-Ade /X-ɑ-Gal medium. By contrast, the negative controls could grow on SD/-Trp/-Leu medium, but not on SD/-Trp/-Leu/-Ade/-His medium (Fig. 10B). These results confirm the protein-protein interaction between NtbHLH1 and NtMYB6.

NtbHLLH1 is a bHLH Transcription Factor That Regulates Flavonoid Biosynthesis
In this study, we isolated a bHLH transcription factor NtbHLH1 from Chinese Narcissus. NtbHLH1 has a conserved bHLH-MYC_N terminal structure and a HLH domain, and belongs to the JAF13 clade of IIIf subfamily which includes PhJAF13 (Petunia hybrida), ZmLC (maize), and AtGL3 (Arabidopsis). In dicotyledons, JAF13-like bHLH interacts with R2R3-MYB protein promoting the expression of a AN1-like bHLH, and then AN1 interacts with an R2R3-MYB to activate the expression of biosynthetic genes in the flavonoid pathway [3]. In Arabidopsis, JAF13-like AtGL3 is expressed at an early stage of anthocyanin synthesis, and an MBW complex PAP1-GL3-TTG1 was first formed. This MBW complex activates the expression of AtTT8, and then another MBW complex which includes AtTT8 activates the expression of structural genes related to flavonoid biosynthesis [6].
Ectopic expression of NtbHLH1 in tobacco demonstrates that it functions as a positive regulator of flavonoid biosynthesis. The expression of NtbHLH1 increased anthocyanin and total flavonoid contents of transgenic tobacco petals by activating the expression of tobacco genes encoding anthocyanin-related enzymes and the bHLH AN1, suggesting NtbHLH1 may regulate both AN1 and structural genes directly. In monocotyledons, JAF13-like bHLHs can interact with MYBs to directly promote the expression of structural genes in the flavonoid biosynthesis pathway. For example, Freesia hybrida FhGL3 and FhTT8 can activate transcription of AtDFR with AtAPA1 and the activation capacity of FhGL3 with AtPAP1 is higher than FhTT8 with AtPAP1 [20]. Our results suggest that NtbHLH1 may have a function similar to JAF13-like bHLHs in monocotyledons.

NtMYB6 is an activator of proanthocyanidin biosynthesis
Phylogenetic analysis shows that NtMYB6 is highly similar to the R2R3-MYB transcription factors DkMYB4 and VvMYBPA1 which have been shown to activate proanthocyanidin biosynthesis. The ectopic overexpression of VvMYBPA1 in Arabidopsis induced elevated biosynthesis of proanthocyanidins in roots, hypocotyls, and apical meristems. Transient expression assays revealed the ability of VvMYBPA1 to activate the proanthocyanidin-specific branch point genes VvANR and VvLAR1 [7]. Ectopic expression of DkMYB4 induced proanthocyanidin accumulation in the kiwifruit callus and directly activates DkANR transcription [1,2]. Ectopic expression of NtMYB6 in tobacco activated the expression of LAR gene and induced PA accumulation in transgenic flowers suggesting NtMYB6 possibly has similar regulatory functions in proanthocyanidin biosynthesis.

NtbHLH1 interacts with NtMYB6 to activate NtDFR expression and promote proanthocyanidin biosynthesis
NtbHLH1, a flavonoid-related bHLH regulator, was found to have higher expression levels in basal plates compared to petals and corona of Chinese Narcissus. In the same tissue, the expression level of NtMYB6 was also elevated. Previous studies have shown that proanthocyanidins were accumulated mainly in basal plates of Chinese Narcissus [36]. Therefore the expression profiles of NtbHLH1 and NtMYB6 were positively associated with the pattern of proanthocyanidin accumulation. Yeast two-hybrid experiment confirmed the interaction of NtbHLH1 with NtMYB6. The accumulation of proanthocyanidin is likely because the expression of NtDFR and NtLAR was significantly up-regulated in basal plates [36]. Dual luciferase reporter assays showed that combined with NtbHLH1, NtMYB6 activated the expression of NtDFR more significantly, but in this heterologous system this combination did not activate the expression of NtLAR. However, ectopic expression of NtMYB6 in tobacco can activate the expression of tobacco LAR gene. Other factors may be involved in how NtbHLH1 interacts with NtMYB6 to promote activation of the Narcissus LAR gene. One of possible reasons why NtDFR has low expression level in petals and corona is the low expression levels of Ntb-HLH1 and NtMYB6.

Conclusions
We have demonstrated that NtbHLH1 functions as a positive regulator of flavonoid biosynthesis and NtMYB6 positively regulates the proanthocyanidin biosynthesis. NtbHLH1 is likely to form a MBW complex with NtMYB6 to regulate the expression of NtDFR directly in Chinese Narcissus.

Plant materials
Chinese narcissus cultivar 'Jin Zhan Yin Tai' was used in this experiment. Petals, corona, and basal plates were collected for RNA extraction. Tobacco (Nicotiana tabacum) was used in stable transformation. Chinese narcissus cultivar 'Jin Zhan Yin Tai' and seeds of tobacco were obtained from Institute of Genetics and Breeding in Horticultural Plants, Fujian Agriculture and Forestry University.

Gene cloning and sequence analysis
Total RNA was extracted from basal plates using Up Plus RNA Kt (TransGen Biotech, Beijing, China).  Table 1. After getting the 3´ end, the fulllength ORF was amplified by RT-PCR from cDNA using gene-specifc forward and reverse primers (Supplemental Table 1). The total 25 µL PCR mixture (Yesen, Shanghai, China) contained 12.5 µL 2 × Canace ® PCR buffer, 1 µL cDNA, 1.25 µL each primer and 8.75 µL double distilled water. The PCR reaction was carried out as follows: preheating at 98 ℃for 3 min, 34 cycles at 98 ℃ for 30 s, the annealing temperature was 60 ~ 65 ℃ for 1 min and 72 ℃ for 30 s, then an extension at 72 ℃ for 5 min.
The amino acid sequence of NtbHLH1 and NtMYB6 were aligned by using DNAMAN6.0 software with default parameters (Lynnon Corporation, San Ramon, CA, USA). The whole protein sequence of flavonoidrelated bHLHs belonging to the subgroup IIIf transcriptional factors and flavnoid-related R2R3-MYBs were used to develop a phylogenetic tree using MEGA7 software with default parameters. Bootstrap was 1000.

Real time qPCR analysis
The expression of NtbHLH1 and NtMYB6 in different tissues of Chinese narcissus was analyzed by real time qRT-PCR using the Light cycler ® 480 real time PCR (Roche Diagnostics, Indianapolis, IN, USA) and SYBR Premix Ex Taq (Takara, Beijing, China) according to the manufacturer's instructions. The conditions of qRT-PCR were as follows: one cycle of denaturation (95 C, 30 s), then 40  Table 1). The comparative Ct method was carried out to estimate the gene expression level. Three technical and biological replications for each sample were performed.

Vector construction and tobacco transformation
Plant expression vector was constructed using the In-Fusion HD Cloing Kit (Takara, Beijing, China). The forward primer was added with an EcoRI restriction site and reverse primer with a HindIII restriction site (Supplemental Table 1). The sequence was then cloned into expression vector pSAK277. The constructed expression vector pSAK277-NtbHLH1 and pSAK277-NtMYB6 were transferred into Agrobacterium GV3101 by freeze-thaw method.
Stable transformation of tobacco used the the leaf disc method [18]. Transformed tobacco shoots were screened on MS basal medium adding 100 mg·L −1 Kan and then were confirmed by RT-PCR. The transgenic tobacco plants were then transferred to a mixture of soil for their adaptation under normal condition.

Total anthocyanin extraction and quantification
The samples were collected from the center of the fresh flowers of transgenic tobacco. Samples were powdered in liquid nitrogen and transferred into a clean tube containing 10 ml of methanol (1% HCl) and kept overnight at room temperature. The extracts were homogenized and centrifuged at 13,000 rpm (10 min, 4 °C) and the upper aqueous phase was subjected to spectrophotometric quantification at 530, 620 and 650 nm using a Spectrophotometer. The relative anthocyanin content was determined using the following formula: OD = (A530 − A620 ) − 0.1(A650 − A620). The total anthocyanin concentration was determined using a molar extinction coefficient of 46,200 mol·cm −1 [40].

Yeast two hybrid assay
For Y2H experiments, pGADT7 (harboring the GAL4 activation domain (AD)) and pGBKT7 (harboring the GAL4 DNA-binding domain (BD)) were uesd. The ORF fragments of NtMYB6 and NtbHLH1 were inserted into pGADT7 and pGBKT7, seperately, generating AD-NtMYB6 and BD-NtbHLH1 construct. The BD-NtbHLH1 was transformed alone or together with AD-NtMYB6 into the yeast strain Y2HGold using the PEG/LiAc method. The autoactivation activities of Ntb-HLH1 were tested. The co-transformed colonies were selected on SD medium lacking leucine and tryptophan (SD/-Leu/-Trp), and screened for growth on quadruple dropout SD medium lacking adenine, histidine, leucine and tryptophan (SD/-Ade/-His/-Leu/-Trp). To further confirm the positive interactions, X-α-Gal was used for the assay of β-galactosidase activity.

Dual luciferase assay
The promoter sequences of Chinese narcissus LAR gene (NtLAR) [31] and DFR gene (NtDFR) [19] were cloned and inserted into multiple cloning site of vector pGreenII 0800-LUC separately as the reporter cassette. pSAK277-NtbHLH1 and pSAK277-NtMYB6 driven by the cauliflower mosaic virus (CaMV) 35S promoter were used as the effector cassettes. PSAK277 with β-glucuronidase (GUS) gene was used as the negative control. Dual luciferase assay was conducted in Nicotiana benthamiana leaves according to the previous report [4,11]. The confluent bacteria was resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES, 200 µM Acetosyringone) and incubated at room temperature without shaking for 3 h before infiltration. Firefly luciferase (LUC) values relative to the Renilla luciferase (REN) control were measured using the machine Multifunctional Microplate Reader (Infinite M200 PRO, TECAN, Austria).