Functional characterization of a liverworts bHLH transcription factor involved in the regulation of bisbibenzyls and flavonoids biosynthesis
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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.
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.
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.
KeywordsbHLH transcription factor Liverworts Bisbibenzyls Flavonoids P. appendiculatum
4coumarate: coenzyme A ligase
Cinnamic acid 4-hydroxylation
Double bond reductase
Phenylalanine ammonia lyase
In plants, the structural genes responsible for flavonoids biosynthesis are regulated largely at the transcriptional level, controlled by two classes of transcription factors (TF) (MYB and bHLH) along with the WD40 proteins. The bHLH TFs are essential for the activity of the R2R3 MYB partner, and also provide enhanced activity on promoters containing a cis-regulatory element conserved in several flavonoids and anthocyanins biosynthetic genes . The bHLH family has been divided into 26 sub-groups , which regulate a wide range of cellular processes, including the fate of epidermal cells, the hormonal response, metal homeostasis, photomorphogenesis and floral organ development . The flavonoids biosynthesis related bHLHs have been assigned to a subgroup denoted IIIf. One of the first identified members of the subgroup is the maize R gene, which encodes a regulator of anthocyanins accumulation in the grain . The delila (del) gene encoding bHLH factor regulates the pattern of red anthocyanin pigmentation in Antirrhinum majus plants . The Arabidopsis thaliana bHLH proteins AtTT8, AtGL3 and AtEGL3 are all involved in the synthesis of various flavonoids [17, 18, 19]. ANTHOCYANIN1 (AN1) of petunia is a bHLH transcription factor that is required for the synthesis of anthocyanin pigments . In rice, the products of Rc and Rd are bHLH transcription factors which control proanthocyanidins synthesis in the grain pericarp, while the tobacco genes NtAn1a and NtAn1b encode enhancers of anthocyanin accumulations in the flower . In Dahlia variabilis, DvIVS (a member of the An1 subgroup of bHLH transcription factors) is involved in the regulation of anthocyanins synthesis in the ray florets . In morning glory, IpIVS regulates both proanthocyanidins accumulation in seeds and anthocyanins biosynthesis in flowers . The two grapevine bHLH proteins VvMYC1 and VvMYCA1 are required for the production of, respectively, anthocyanins and proanthocyanidins [24, 25].
The regulation of the flavonoids synthesis by bHLH TFs has been extensively studied in vascular plants, but as yet, little is known concerning their participation in the regulation of bisbibenzyls and flavonoids synthesis in the liverworts. Liverworts represent a division of bryophyte species and are among the earliest diverging lineages of land plants dated to the early Ordovician period (about 488 million to 444 million years ago). Characterization of the bHLH genes involved in the regulation of flavonoids biosynthesis from liverworts will shed light on the elucidation of the origin and evolution of bHLH transcript factors. In our previous investigation, PabHLH has been isolated and functional characterized as a positive regulator of bisbibenzyls biosynthesis from liverworts P. appendiculatum . Here, we characterized another bHLH (PabHLH1) gene from P. appendiculatum by over-expressing in P. appendiculatum and heterologously expressing in A. thaliana. PabHLH1 was positively regulated the bisbibenzyls and flavonoids biosynthesis in P. appendiculatum and stimulated the accumulation of the flavonols and anthocyanins in Arabidopsis.
P. appendiculatum thallus (A little bit samples were originally collected from Sichuan province, China and then cultured and propagated in our green house, and authenticated by Professor Xuesen Wen. A voucher specimen with No. 20091010–01 has been deposited in the School of Pharmaceutical Sciences, Shandong University. No specific permits were required for collecting this sample for research purpose.) was raised in green house delivering a constant temperature of 25 °C and a 12 h photoperiod in Shandong University. Two-month-old thallus were harvested and snap-frozen in liquid nitrogen and stored at − 80 °C. Axenic thallus and callus were raised, respectively, on half strength Murashige and Skoog (1962) (MS) medium  supplemented with 0.5 mg L− 1 6-benzyladenine. The cultures were exposed to a 12 h photoperiod and a day/night temperature regime of 22 °C/20 °C. A. thaliana ecotype Col-0 (purchased from Arabidopsis Biological Resource Center) plants were raised in a growth chamber under a 16 h photoperiod at a constant temperature of 22 °C. The Nicotiana benthamiana (Donated by Professor Fengning Xiang from School of life sciences, Shandong University) plants required for transient expression experiments were soil-grown for 5–6 weeks under a 12 h photoperiod and a day/night temperature regime of 24 °C/22 °C.
Nucleic acid extraction and gene isolation
Total RNA was extracted from P. appendiculatum thallus or A. thaliana seedlings using, respectively, a CTAB-based method  and the RNAiso Plus reagent (TaKaRa, Kusatsu, Shiga, Japan). cDNA was synthesized from preparations of total RNA using PrimeScript™ RT Master Mix (Takara, Otsu, Japan), according to the manufacturer’s protocol. A PabHLH1 fragment lacking the full coding region was identified from P. appendiculatum transcriptome sequencing database (SRP073827). The 3′-RACE method was then applied to recover the missing 3′ sequence: the template for this reaction was 3′-Ready cDNA and the primer pair was PabHLH1-NGP (sequences given in Additional file 1: Table S1), along with the UPM primer provided in the SMART RACE cDNA Amplification kit (Clontech, USA). Once the complete open reading frame (ORF) sequence of PabHLH1 had been acquired, it was amplified from P. appendiculatum thallus cDNA using the primer pair PabHLH1-F/R (sequences given in Additional file 1: Table S1); the amplicon was then inserted into pMD19-T and sequenced for validation purposes.
The predicted PabHLH1 polypeptide sequence was aligned with those of its plant homologs A. thaliana TT8 (CAC14865), Ipomoea purpurea IpIVS (BAD18982) and Vitis vinifera MYC1 (EU447172) using DNAMAN v5.2.2 software (Lynnon Biosoft, Quebec, Canada). A phylogenetic analysis was conducted based on the maximum likelihood method implemented in MEGA v4.0 software , based on 1000 bootstrap replicates.
Stress treatment of P. appendiculatum thallus
One hundred two month old P. appendiculatum thallus were irradiated with a UV-B 311 nm narrow band lamp (Philips, PL-S 9 W/01/2P, Poland) (60 mJ/cm2) or 1 mM salicylic acid (SA) for 10 min, and then harvested after 0, 6, 12, 24, 36, 48 and 60 h. The material was snap-frozen in liquid nitrogen and stored at − 80 °C until processed for qRT-PCR assays as described above.
Quantitative real time PCR (qRT-PCR) analysis
The qRT-PCR approach was used to characterize the transcriptional behavior of PabHLH1 in three different tissues. The expression of PabHLH1 along with that of a number of phenylpropanoid pathway genes, flavonoid structural genes and the bibenzyl synthesis genes were analyzed using qRT-PCR in both wild type and transgenic P. appendiculatum and A. thaliana. The required cDNA template was derived from RNA extracts as described above, and the qRT-PCRs were performed using an Eppendorf Mastercycler ep realplex RealTime PCR System (Eppendorf, Germany). The relevant primers are listed in Additional file 2: Table S2. Each 10 μL reaction comprised 2 μL SYBR, 1 μL of the template cDNA, 0.5 μL of each primer pair (10 μM) and 6.5 μL RNase-free dH2O. The reference gene for the P. appendiculatum samples encodes an elongation factor, as amplified by the primer pair P. appendiculatum elongation F/R . All samples were evaluated in three independent experiments.
Subcellular localization and transcriptional activity assay
The full length ORF (lacking the stop codon) was PCR-amplified using primers including an attB recombination site (sequences given in Additional file 1: Table S1), and the amplicon was subjected to a BP clonase reaction in order to insert it into the vector pDONR207 (Invitrogen, Carlsbad, CA, USA). Recombined plasmids were processed in a Gateway LR clonase reaction (Invitrogen, Carlsbad, CA, USA) with pGWB5 vector to create a PabHLH1-GFP fusion driven by the CaMV 35S promoter . The construct was transferred into Agrobacterium tumefaciens strain EHA105 using the freeze/thaw method and from thence into N. benthamiana epidermal leaf cells or onion epidermal cells  for its transient expression. The onion epidermal cells were incubated with 0.1 mg ml− 1 DAPI for 10 min for staining the nucleus. The GFP and DAPI fluorescence was monitored via confocal laser scanning microscopy (LSM 700, Carl Zeiss, Inc., New York, USA) or fluorescence microscope (Olympus IX71; Olympus Co., Tokyo, Japan). Four different segments of PabHLH1 were generated by Nde I-BamH I or Nco I-BamH I restriction to produce gene fragments corresponding to residues 1–261, 262–460, 461–702 and 1–460 of the full polypeptide sequence; these were ligated separately to the pGBKT7 plasmid (Clontech, USA). The full-length sequence of PabHLH1 lacks the available restriction endonuclease sites, so a single nucleotide substitution (a957c) was created using the Stratagene QuickChange site-directed mutagenesis method. The necessary mutant primers designed by primer X software (www.bioinformatics.org/primerx) (Table S1). The resulting PCR product was gel-purified and digested with Dpn I (Thermo Scientific, USA) at 37 °C for 4 h, and an aliquot was transformed into E.coli DH5α. After sequenced, mutated gene (PabHLH1-a957c) was inserted into the pGBKT7 (Clontech, USA) vector after digested with Nde I and Nco I (the primers used in this study are listed in Table S1). Each construct was then transformed into yeast strain AH109 using the PEG/LiAC method . Yeast transformants were cultured on a synthetic drop-out medium lacking tryptophan (SD-Trp). After their PCR-based validation, transformed colonies were tested for the ability to growth on triple selection SD medium lacking tryptophan, histidine and adenine (SD-Trp-His-Ade).
The PabHLH1 coding sequence harbored within pDONR207 was placed in pGWB5 vector under the control of the CaMV 35S promoter by the Gateway cloning technique , then introduced into A. tumefaciens EHA105. A 1 mL aliquot of an overnight A. tumefaciens culture was inoculated into 100 mL yeast extract peptone medium containing 50 mg/L kanamycin, and held at 30 °C with shaking until the OD600 had reached 1.5–2.0. The cells were harvested by centrifugation (4000 g, 5 min), then re-suspended in 50 mL liquid half strength MS medium (pH 5.2) containing 100 μM acetosyringone (Sigma-Aldrich, St. Louis, MO, USA). About fifty P. appendiculatum thallus were chopped into small pieces and incorporated into the A. tumefaciens suspension. The mixture was held for 1 h with shaking, then cultured for 72 h in the dark at 22 °C on solid half strength MS medium (pH 5.6–5.8) containing 100 μM acetosyringone. The thallus pieces were rinsed three times in sterile deionized water, then transferred to a half strength MS medium containing 25 mg/L hygromycin and 200 mg/L cefotaxime. After 2–3 weeks, surviving thallus were sub-cultured on half strength MS medium containing 50 mg/L hygromycin and 50 mg/L cefotaxime, with the medium being refreshed at two weekly intervals. A. thaliana ecotype Col-0 was transformed with A. tumefaciens GV3101 harboring the transgene using the flower dip method . Regenerated plants were raised at 22 °C under a 16 h photoperiod, and putative T1 transformants were PCR-validated using the primer pair PabHLH1-vector-F/−R (sequences given in Additional file 1: Table S1).
Compositional analysis of P. appendiculatum thallus
Transgenic and non-transgenic P. appendiculatum thallus, grown on half strength MS medium for about 20 days, was freeze-dried. A 25 mg aliquot was suspended in 500 μL methanol and ultrasonicated for 1 h to extract the bibenzyls fraction (baicalein was added as the internal standard). After centrifugation, a 20 μL volume of supernatant was separated via a reverse-phase Luna 5u C18(2) 100A column (4.6 × 250 mm, Phenomenex, CA, USA) using Agilent 1260 series system (Agilent, CA, USA). The HPLC parameters were: solvent A: 0.1% (v/v) aqueous formic acid, solvent B: acetonitrile; the solvents were provided at a flow rate of 0.8 mL/min. The initial solvent mix was 60% A/40% B, followed by 45 min of 33% A/67% B, then 5 min of 10% A/90% B, and finally 5 min of 100% B. The detection wavelength was 280 nm. The major bibenzyls, lunularic acid, riccardin C and riccardin D, were identified on the basis of corresponding standard samples and quantified according to the standard curve of the corresponding compound. For flavonoids analysis, 10 mg freeze-dried thallus was suspended in 800 μl 80% methanol and ultrasonicated for 1 h (myricetin was added as the internal standard). Flavonoids content was determined as aglycones by preparing acid-hydrolyzed extracts. An aliquot of 400 μL of the supernatant was acid-hydrolyzed by adding 120 μL of 3NHCl, incubated at 90 °C for 1 h, and then mixed with 200 μL of methanol. An Agilent 1260 series HPLC system equipped with a Luna 5u C18(2) 100A column (4.6 × 250 mm, Phenomenex, CA, USA) was used for chromatographic analysis. The HPLC conditions were as follows: the mobile phase consisted of solvent A (acetonitrile) and solvent B (1% acetic acid in water, v/v). The gradient elution program was: 0–10 min, 30% solvent A; 10–30 min, 30–45% solvent A; 30–35 min, 45–100% solvent A; 35–45 min, 100–30% solvent A. The detection wavelength was set at 330 nm. The flavonoid was quantified as the relative content in wild type and transgenic P. appendiculatum thallus according to the peak area. All samples were evaluated in three independent experiments.
Flavonoids and lignin determination in transgenic A. thaliana
Ten day old transgenic and wild type (WT) A. thaliana seedlings were freeze-dried and ground to fine powder. For the determination of flavonoids composition, a 15 mg aliquot of the powder was extracted by sonication (1 h) in 800 μL methanol:water (80:20, v/v) . Following centrifugation (16,000 g, 4 °C, 15 min), the supernatant was subjected to reverse phase HPLC using an Luna 5u C18(2) 100A column (4.6 × 250 mm, Phenomenex, CA, USA). The mobile phase was 5–85% acetonitrile and 95–15% water (0.1% v/v formic acid) provided at a flow rate of 0.8 mL/min over 40 min. The detection wavelength was 364 nm. Peaks were identified as specific kaempferol derivatives using MS and UV spectral analysis . Chrysin was used as the internal standard to normalize the peaks and calculate the relative contribution of the various flavonols. Seedlings were visually screened for anthocyanin accumulation after 5 days of culture on anthocyanin gene induction media half strength MS (pH 5.8) consisting of sucrose (3%, w/v) and agar (0.8%, w/v). For the total anthocyanins analysis of transgenic and wild type lines, 100 mg fresh plant material was suspended in 400 μL methanol with 1% HCl (v/v) and ultrasonicated for 1 h. After centrifugation (15,000 g, 10 min), the supernatant was transferred to a fresh tube and the total anthocyanin was determined by measuring the OD at A530 and A657 by using a spectrophotometer (Shimadzu, Kyoto, Japan). The quantity of anthocyanin was determined by calculating absorbance A = (A530–0.25 × A657). The concentration of anthocyanin pigment in the original sample was calculated using the following formula: anthocyanin pigment (mg/L) = (A × MW × DF × 1000)/(ε × 1), where MW is the molecular weight of cyandindin-3-glucoside (449.2), DF is the dilution factor, and ε is the molar absorptivity of cyanindin-3-glucoside (26,900) . The pattern of lignin distribution present in five- to seven-leaf stage plants was obtained by staining stem sections following the previous procedure . The lignin content of the stem tissue was quantified by spectrophotometric (280 nm) analysis of an acetyl bromide extract. The samples were ground into powder in liquid nitrogen and extracted, in turn, with 70% ethanol, chloroform/methanol (1:1 v/v) and acetone for cell wall preparation. According to the acetyl bromide method , 6 mg dried samples were digested with 1 mL 25% acetyl bromide in 70 °C for 30 min, and sequentially added 5 mL acetic acid after cooled in an ice bath for 10 min. Then 300 μL supernatants were mixed by 400 μL of 1.5 M NaOH and 300 μL of 0.5 M hydroxylamine hydrochloride, followed 1.5 mL acetic acid to dilute. The extinction coefficient for A. thaliana lignin was 23.35 g− 1 L cm− 1 at 280 nm. All samples were evaluated in three independent experiments.
The PabHLH1 sequence
Expression of PabHLH1 in different tissues and its response to stress
The sub-cellular localization and transactivation ability of PabHLH1
PabHLH1 over-expression increased the bibenzyls and flavonoids content of the P. appendiculatum thallus
The accumulation of flavonoids and lignin in transgenic A. thaliana
The bHLHs represent one of the largest families of plant TFs, which regulate a wide range of plant developmental and physiological processes , including the biosynthesis of flavonoids. As yet, only one bHLH sequence, a positive regulator of bisbibenzyl biosynthesis, has been isolated from liverworts . In the present investigation, a novel bHLH transcription factor, PabHLH1, was obtained by searching the P. appendiculatum transcriptomic database. Its phylogenetic relationship with bHLHs known to regulate flavonoids synthesis suggested that the gene belongs to the bHLH IIIf sub-group . Analysis of its sequence revealed it has retained both the bHLH region (involved in homo- and heterodimerization) and the basic DNA-binding region . In addition it has retained the His/Lys9, Glu13, and Arg17 (HER motif) residues, which confer binding to the G-box [38, 39]. Besides the bHLH domain, PabHLH1 also harbors N and C terminal domains representing the sites of interaction with myb-type TFs [19, 42]. PabHLH1 displayed both transactivational activity and was deposited in both the nucleus and cytoplasm. Basic helix-loop-helix transcript factors are expected to be localized to the nucleus. However, some bHLH proteins involved in the regulation of flavonoids biosynthesis are also cytoplasm associated .
The bibenzyls are a distinctive class of compounds produced by liverworts and possess a wide spectrum of biological and pharmacological activity. Like the flavonoids and lignin, they are products of the phenylpropanoid pathway, as shown by the fate of radioactively labeled precursors . The synthesis of bibenzyls and flavonoids in liverworts may involve a common set of structural genes and transcriptional regulators. Here, the over-expression of PabHLH1 in P. appendiculatum thallus had the effect of boosting the accumulation of bibenzyls and of up-regulating genes encoding PAL, C4H, 4CL and STCS1 (Fig. 7). The content of bibenzyls and the abundance of PAL, C4H and 4CL transcript was correlated with the transcript abundance of PabHLH1. These results indicated that in P. appendiculatum, PabHLH1 influences bibenzyl synthesis via its up-regulation of structural genes acting early in the phenylpropanoid pathway. The increase in flavonoid content induced by PabHLH1 over-expression was accompanied by a significant up-regulation of the genes encoding the flavonoid synthesis enzymes CHI and CHS.
The constitutive expression of PabHLH1 in A. thaliana enhanced the accumulation of both flavonoids and anthocyanin, but had no effect on the lignin content. At the gene level, the transgenic plants experienced an up-regulation of PAL, 4CL, CHS, CHI, F3H, DFR and FLS, leaving the transcription of the lignin synthesis-associated genes CCR and CAD unaffected. DFR, the first committed enzyme of anthocyanin synthesis, was particularly strongly up-regulated (by > 2 fold). The products of both Petunia hybrida AN1 and AtTT8 control the expression of the flavonoid synthesis pathway structural genes CHS, CHI, F3H, FLS1 and DFR [17, 20], while the morning glory TF IpIVS regulates all these genes except for F3H . DvIVS is a transcription factor that activates anthocyanin biosynthesis genes including DvCHS1, DvF3H, DvDFR, and DvANS in Dahlia variabilis . PabHLH1 appears able to influence the expression of PAL, 4CL, CHS, CHI, F3H, DFR and FLS. These results suggested that the bHLHs transcript factors in IIIf subgroup regulate the flavonoids and anthocyanin biosynthesis through controlling the expression of several structure genes simultaneously.
A number of secondary metabolites, including the bibenzyls and flavonoids, function as abiotic stress protectants, with their synthesis commonly being triggered by environmental stresses. In some cases, the stress signal is transduced by a phytohormone, so that exogenous treatment with SA, for example, can mimic the stress response. As regulators of secondary metabolism synthesis, the bHLH TFs are inducible by stresses such as salinity and extreme temperature, as well as by treatment with phytohormone [25, 43]. Here, the transcription of PabHLH1 in P. appendiculatum thallus responded positively to both SA treatment and UV irradiation, as is also the case for a number of genes encoding key enzymes involved in phenylpropanoid synthesis [10, 11, 44]. The transcription of C4H and 4CL1, which encode enzymes acting in the first two steps of phenylpropanoid synthesis are known to respond positively to exogenous SA treatment [11, 45]. Meanwhile CHS, encoding the “gatekeeper” of flavonoid synthesis, is markedly induced by this treatment . The indication is that PabHLH1 acts to up-regulate a series of genes which contribute to defense against abiotic stress.
A bHLH TF was isolated and characterized from the liverwort P. appendiculatum. When over-expressed, PabHLH1 was able to regulate bisbibenzyl biosynthesis, while when constitutively expressed in A. thaliana, it regulated flavonoid and anthocyanin biosynthesis; in both cases, the gene acted by influencing the transcription of relevant structural genes. The functional identification of liverwort transcription factors is of significance for tracing the evolution of secondary metabolite synthesis, while potentially also suggesting strategies aimed at enhancing the production of valuable molecules in plants.
AXC and HXL conceived the research plan and designed the experiments; YZ, YYZ, HL, XSZ, RN, PYW and SG performed the experiments; AXC and HXL supervised the experiments and analyzed data; YYZ and AXC wrote the paper. All authors read and approved the final manuscript.
This study was supported by the National Natural Science Foundation of China (No. 31370330 and 31770330) and Science & Technology Development Plan Project of Shandong (No. 2016GSF121032). The funders had no role in the experimental design, data collection and analysis or writing the manuscript.
Ethics approval and consent to participate
Plagiochasma appendiculatum sample collection was complied with all relevant institutional, national and international guidelines. In China, Plagiochasma appendiculatum is not a vulnerable species, so no specific permits are required to collect this plant for research purpose.
Consent for publication
The authors declare no conflict of interest in the present investigation.
- 4.Asakawa Y. Chemical constituents of the bryophytes. Berline: Progress in the chemistry of organic natural products. Springer Vienna; 1995. p. 1–562.Google Scholar
- 15.Ludwig SR, Habera LF, Dellaporta SL, Wessler SR. Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci. 1989;86(18):7092–6.PubMedCrossRefGoogle Scholar
- 18.Payne C, Zhang F. Lloyd, A.M. GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1. Genetics 2000; 156(3): 1349–1362.Google Scholar
- 34.Rispail N, Morris P, Webb KJ. Phenolic Compounds: extraction and analysis. Lotus japonicus Handbook. Springer Netherlands 2005; 349–354.Google Scholar
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