Functional characterization of a liverworts bHLH transcription factor involved in the regulation of bisbibenzyls and flavonoids biosynthesis

Abstract

Background: The basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, play important roles in the regulation of many secondary metabolites including flavonoids. Their involvement in flavonoids synthesis is well established in vascular plants, but not as yet in the bryophytes. In liverworts, both bisbibenzyls and flavonoids are derived through the phenylpropanoids pathway and share several upstream enzymes.

Results: In this study, we cloned and characterized the function of PabHLH1, a bHLH family protein encoded by the liverworts species Plagiochasma appendiculatum. PabHLH1 is phylogenetically related to the IIIf subfamily bHLHs involved in flavonoids biosynthesis. A transient expression experiment showed that PabHLH1 is deposited in the nucleus and cytoplasm, while the yeast one hybrid assay showed that it has transactivational activity. When PabHLH1 was overexpressed in P. appendiculatum thallus, a positive correlation was established between the content of bibenzyls and flavonoids and the transcriptional abundance of corresponding genes involved in the biosynthesis pathway of these compounds. The heterologous expression of PabHLH1 in Arabidopsis thaliana resulted in the activation of flavonoids and anthocyanins synthesis, involving the up-regulation of structural genes acting both early and late in the flavonoids synthesis pathway. The transcription level of PabHLH1 in P. appendiculatum thallus responded positively to stress induced by either exposure to UV radiation or treatment with salicylic acid.

Conclusion: PabHLH1 was involved in the regulation of the biosynthesis of flavonoids as well as bibenzyls in liverworts and stimulated the accumulation of the flavonols and anthocyanins in Arabidopsis.

Keywords: Bisbibenzyls; Flavonoids; Liverworts; P. appendiculatum; bHLH transcription factor.

Fig. 1

The Schema of plant flavonoids biosynthesis. PAL Phenylalanine ammonia lyase, C4H Cinnamic acid 4-hydroxylation, 4CL: 4-coumarate: coenzyme A ligase, CHS: chalcone synthase, CHI: chalcone isomerase, F3H: flavanone 3β-hydroxylase, FLS: flavonol synthase, DFR: dihydroflavonol reductase, ANS: anthocyanidin synthase. DBR: double bond reductase, STCS: stilbenecarboxylate synthase

Fig. 2

Sequence alignment of PabHLH1 and its closest homologs Arabidopsis thaliana TT8 (CAC14865), Ipomoea purpurea Ivory Seed (BAD18982) and Vitis vinifera MYC1 (EU447172). Residues composing the HER motif are shown with red stars. The bHLH domains and the acidic domain are labeled

Fig. 3

Phylogenetic tree of PabHLH1 and other plant bHLH TFs domains. The tree was constructed by the MEGA v4.0 program using neighbor-joining method. TFs sequences used are Vitis vinifera MYC1 (EU447172), Malus domestica bHLH (ADL36597), Theobroma cacao bHLH (XP_007050639.1), Dahlia pinnata bHLH (BAM84239), Petunia hybrida AN1 (AAG25927), Solanum tuberosum bHLH (AGC31677.1), Gentiana triflora bHLH1 (BAH03387), Ipomoea purpurea Ivory Seed (BAD18982), Lotus japonicus TT8 (BAH28881), Zea mays IN1 (AAB03841), Oryza sativa Rc (BAF42668), Arabidopsis thaliana TT8 (CAC14865), Plagiochasma appendiculatum bHLH (MF983804); Plagiochasma appendiculatum bHLH1 (AXO67713); Arabidopsis thaliana MYC1 (NP_001329692.1), Malus domestica bHLH33 (ABB84474), Citus sinensis MYC2 (ABR68793), Vitis vinifera MYCA1 (EF193002); Arabidopsis thaliana GL3 (NP_680372), Arabidopsis thaliana EGL3 (NP_176552.1), Zea mays B (CAA40544), Zea mays Lc (NP_001105339), Petunia hybrid JAF13 (AAC39455), Perilla frutescens RP (BAA75513), Antirrhinum majus Delila (AAA32663)

Fig. 4

The relative bisbibenzyls content and the expression level of PabHLH1 in P. appendiculatum. a Three different tissues of P. appendiculatum, 1, 2: axenic callus and thallus cultured on MS medium; 3: thallus growing in greenhouse. b Relative bisbibenzyls content in three tissues referred to in (a). c PabHLH1 transcript abundance by qRT-PCR in the three tissue types referred to in (a). Data represent mean ± S.D. with three biological repeats. *p < 0.05, ** p < 0.01 according to Student’s t-test

Fig. 5

The transcriptional response of PabHLH1 to stress. Thallus was treated with SA (a), or exposed to UV radiation (b). The reference sequence used for the qRT-PCR was a gene encoding an elongation factor. The results were shown as the mean of three replicate reactions with standard deviations, *p < 0.05, ** p < 0.01 according to Student’s t-test

Fig. 6

Subcellular localization and Transactivation of PabHLH1. The expression cassettes of PabHLH driven by 35S promoter, was transiently expressed in the tobacco leaves (a) and onion epidermal cells (b). The GFP fluorescence is shown in green and the DAPI fluorescence is shown in blue. c Four truncated fragments and the full-length protein were each introduced into pGBKT7, and transformed yeasts selected on both SD-Trp and SD-Trp-His-Ade. Construct containing PabHLH1 residues 1–460 and the full length protein was functional and supported survival on the multiple amino acid deficient medium. The black filled bar indicates the PabHLH1 domain

Fig. 7

PabHLH1 transcription and biochemical analysis in transgenic P. appendiculatum thallus. a The transcription of PabHLH1 in P. appendiculatum wild-type (WT) and the transgenic overexpression PabHLH1 gene thallus (OE). b The contents of flavonoids in WT and OE thallus. c Representative HPLC profiles of methanolic bibenzyls extracted from OE thallus (solid line) and WT thallus (red dashed line). The major bibenzyls identified were: LA = lunularic acid, RC = riccardin C, RD = riccardin D. The internal standard was baicalein. d The bibenzyls contents quantified according to the HPLC analysis of WT and OE P. appendiculatum thallus using corresponding sdandard curve of LA, RC and RD. e The transcript abundance of genes encoding enzymes involved in bibenzyl and flavonoid synthesis in WT and OE thallus. Data represent mean ± S.D. with three biological repeats. *p < 0.05, ** p < 0.01 according to Student’s t-test

Fig. 8

The heterologous expression of PabHLH1 in A. thaliana (a) RT-PCR analysis of expression levels of PabHLH1 in the WT and PabHLH1-OE transgenic Arabidopsis seedlings. WT, wild-type A. thaliana; OE: transgenic A. thaliana lines carrying PabHLH1. b Representative HPLC profiles of methanolic extracts of transgenic Arabidopsis seedlings line (blue solid line) and WT seedlings (red dashed line). The major flavonoid or sinapic acid derivatives are: K1, Kaempferol 3-O-[6″-O-(rhamnosyl) glucoside] 7-O-rhamnoside; K2, kaempferol 3-O-glucoside 7-O-rhamnoside; K3, kaempferol 3-O-rhamnoside 7-O-rhamnoside; K4, kaempferol 3-O-rhamnoside 7-O-glucoside; SM, sinapoyl malate; SG, sinapoyl glucose. The internal standard chrysin are labeled. c The contents of kaempferol derivatives in methanolic extracts of Arabidopsis seedlings by HPLC analysis. d The contents of anthocyanin in methanolic extracts of Arabidopsis seedlings. e Seedlings of 5 days after germination of wild type and transgenic lines OE1, OE2 and OE3, showing the more anthocyanin accumulation in the hypocotyl in transgenic lines indicated with arrow. f qRT-PCR analysis of the genes encoding key enzymes in lines OE1, OE2 and OE3. Data represent mean ± S.D. with three biological repeats. *p < 0.05, ** p < 0.01 according to Student’s t-test.