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

Abstract

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.

Keywords: BHLH; Chinese Narcissus; Flavonoid; MYB; Proanthocyanidin.

Fig. 1

(A) Amino acid sequence comparison between NtbHLH1 and six flavonoid-related bHLHs. Two conserved domains (bHLH-MYC_N domain and HLH domain) among known bHLH TFs regulating anthocyanin flavonoid biosynthesis are underlined. (B) A neighbor-joining phylogenetic tree of IIIf subfamily plant bHLHs. Numbers next to the nodes are bootstrap values from 1,000 replications. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances that were used to infer the phylogenetic tree. The following deduced amino acid sequences were retrieved from GenBank databases: Arabidopsis thaliana AtTT8 (NP_192720.2), AtEGL3 (NP_001332705.1), AtGL3 (NP_001185302.1); Asparagus officinalis EGL1-like (XP_020260845.1); Elaeis guineensis GLABRA 3 (XP_010918535.1); Ananas comosus EGL1-like (XP_020084606.1); Chinese narcissus NtbHLH1 (QDS02912.1). Zea mays ZmLC (P13526.1), ZmB-Peru (CAA40544.1), ZmIN1 (AAB03841.1); Lilium hybrid division I LhbHLH1(BAE20057.1), LhbHLH2(BAE20058.1); Chinese narcissus NtbHLH1 (QDS02912.1); Malus domestica MdbHLH33 (ABB84474.1), MdbHLH3 (ADL36597.1); Fragaria vesca subsp. vesca FvEGL1 (XP_004308377.1); Vitis vinifera VvMYC1 (NP_001268182.1), VvMYCA1 (NP_001267954.1); Arabidopsis thaliana AtMYC (NP_191957.2), Lotus japonicus LjGL3 (BAJ10680.1), LjTT8 (BAH28881.1); Perilla frutescens PfMYC-RP (BAA75513.1), PfF3G1 (BAC56998.1); Antirrhinum majus AmDEL (AAA32663.1); Ipomoea nil InDEL (XP_019171149.1), InIVS (XP_019197480.1); Petunia x hybrida phJAF13 (AAC39455.1), PhAN1 (AAG25927.1); Solanum tuberosum StGL3-like (NP_001275132.1), StAN1 (ALA13578.1); Nicotiana tabacum NtAN1a (NP_001312042.1), NtAN1b (NP_001312452.1), NtJAF13a (NP_001311775.1), NtJAF13b (AHY00341.1); Fragaria x ananassa FabHLH3 (AFL02463.1); Nicotiana sylvestris NsAN1 (NP_001289495.1); Nicotiana tomentosiformis NtAN1 (NP_001289454.1)

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

Fig. 3

Anthocyanin contents of transgenic tobacco plants carrying NtbHLH1 gene. (A) Flower color comparison between wild type (WT) and transgenic lines (1–3, 1–4 and 1–7). (B) Anthocyanin contents. (C) Total flavonoid contents. The bars represent the standard error of thrice biological replication. Small letters showed a significant difference at the level of p < 0.05 by using SPSS statistical analysis

Fig. 4

Expression analysis of flavonoid biosynthesis pathway genes in flowers of NtbHLH1 transgenic tobacco. 1–3, 1–4 and 1–7 means three transgenic lines. The bars indicate the standard error of three biological replicates. Star represents the significant difference from wild type at the level of p < 0.05 using SPSS test

Fig. 5

(A) Comparison of amino acid sequences between NtMYB6 and nine R2R3- MYBs. Three conserved domains (R2 domain, R3 domain and [D/E]Lx₂[R/K]x₃Lx₆LX₃R) are underlined. (B) A neighbor-joining phylogenetic tree of plant R2R3-MYB sequences. Numbers next to the nodes are bootstrap values from 1,000 replications. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances that were used to infer the phylogenetic tree. The following deduced amino acid sequences were retrieved from GenBank databases: Chinese narcissus NtMYB6 (KY645961.1); Gossypium hirsutum GhMYB4 (XP_016708004.1); Antirrhinum majus AmMYB308 (P81393.1); Arabidopsis thaliana AtMYB4 (NP_195574.1),AtMYB7 (NM_127224.6), AtMYB32 (NP_195225.1), AtMYB6 (NP_192684.1), AtTT2 (NP_198405.1), AtMYB75 (NM_104541.4), AtMYB90 (NM_105310.4); Zea mays ZmMYB42 (HQ858694.1); Vitis vinifera VvMYBC2-L-1 (NP_001268133.1), VvMYBC2-L2 (GQ903730.1), VvMYB4 (NP_001268129.1), VvMYBPA1 (NP_001268160.1), VvMYBPA2 (ACK56131.1), VvMYBBA1 (XP_010664911.1); Fragaria x ananassa FaMYB1 (AF401220.1); Petunia x hybrida PhMYB27(AHX24372.1); Populus tremula x Populus tremuloides PtrMYB182 (AJI76863.1); Diospyros kaki DkMYB2 (BAI49719.1), DkMYB4 (BAI49721.1), Solenostemon scutellarioides SsMYB3 (EF522163.1); Populus tremuloides PtMYB134 (ACR83705.1); Litchi chinensis LcMYB1 (KY302802.1); Malus x domestica MdMYB10a (DQ267898.1); Prunus avium L. Tieton PaMYB10 (KR259845.1), Hibiscus syriacus HsMYB6 (KAE8700978.1); Herrania umbratica HuMYB308 (XP_021280377.1); (BAI49721.1); Elaeis guineensis EgMYB6 (XP_019705406.1)

Fig. 6

Expression of NtMYB6 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

Fig. 7

Transgenic tobacco plants carrying NtMYB6 gene. (A) Flower color of wild type (WT) and transgenic lines (L-1 and L-2). (B) Anthocyanin contents. (C) Proanthocyanidin contents. The bars represent the standard error of thrice biological replication. Small letters showed a significant difference at the level of p < 0.05 by using SPSS statistical analysis

Fig. 8

Expression analysis of flavonoid biosynthesis pathway genes in flowers of NtbMYB6 transgenic tobacco. L-1 and L-2 means the transgenic line. The bars indicate the standard error of three biological replicates. Star represents the significant difference from wild type at the level of p < 0.05 using SPSS test

Fig. 9

Dual luciferase assay of Narcissus DFR or LAR promoter activity. Luc and Ren values were measured 3 days after infiltration. The bars represent the standard error of thrice biological replication. Small letters showed a significant difference at the level of p < 0.05 by using SPSS statistical analysis

Fig. 10

(A) Auto-activation of the bait plasmid. a, b, c and d indicate the different mediums of SD/-Trp, SD/-Trp/-Ade, SD/-Trp/-His and SD/-Trp/X-α-Gal. (B) Yeast two-hybrid analysis of interactions between NtbHLH1 and NtMYB6. a, b indicate the different mediums of SD/-Trp/-Leu, SD/-Trp/-Leu/-Ade/-His/X-α-Gal