Introduction

Dandelions (Taraxacum spp.) have been used as medicinal herbs and functional foods for several centuries1,2. The increasing demand for dandelion products, such as tea, wine, syrup, and coffee, has promoted the industrialization of dandelion production3. The total phenolic compound concentrations in different tissues of Taraxacum mongolicum ranged from 37.12 to 68.89 mg GAE/g4,5,6,7. However, these levels in Taraxacum antungense have not been tested in previous studies. Polyphenolic compounds, including phenolic acids, flavonoids, and anthocyanins, have many biological activities8,9,10. In Taraxacum antungense Kitag, chlorogenic acid (CGA) and caffeic acid (CA) have antioxidative benefits, being hepatoprotective and having diuretic activities11,12,13; rutin and luteolin are used to treat several diseases, such as Parkinson’s disease, severe acute respiratory syndrome, hepatitis, and cancer14,15,16. However, the concentration of these functional active constituents in T. antungense is lower than that in other medicinal plants, such as Lonicera japonica and Eucommia ulmoides, which restricts dandelion industrialization14. Bioengineering strategies could potentially increase the polyphenolic compounds in Taraxacum; however, a better understanding of the polyphenolic compound biosynthesis pathway is required.

Since the 1990s, the polyphenol biosynthesis pathway has been reported in several medicinal model plants, such as Salvia miltiorrhiza, L. japonica, and Dendranthema morifolium17,18. In the first three steps of polyphenol biosynthesis, the key enzymes are phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), and cinnamate 4-hydroxylase (C4H), which catalyze the synthesis of p-coumaroyl-CoA from phenylalanine19. CA is directly catalytically synthesized by coumarate 3-hydroxylase from p-coumaroyl-CoA, whereas CGA is catalytically synthesized by hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and hydroxycinnamoyl-CoA shiki-mate/quinate hydroxycinnamoyl transferase (HCT)20. However, in T. antungense, only HQTs (TaHQT1/2) have been isolated, identified, and assessed, and TaHQT2 is the last rate-limiting enzyme in chlorogenic acid biosynthesis21. For flavonoids, p-coumaroyl-CoA is a branch and precursor compound. The first two steps in the catalytic synthesis of naringenin from p-coumaroyl-CoA are via chalcone synthase and chalcone isomerase (CHS and CHI, respectively)8. Naringenin is catalyzed by flavonoid synthase and flavonoid-3′-hydroxylase (FNS and F3′H, respectively) in plants to synthesize luteolin8,22. Naringenin is also catalyzed by flavonol-3-dehydrogenase (F3H), flavonol synthetase (FLS), and UDP-glucoronosyl/UDP-glucosyl transferase (UFGT) to synthesize rutin23,24,25. PAL, CHS, CHI, and F3′H increase CGA and luteolin concentrations in L. japonica, whereas in Fagopyrum tataricum, the rutin concentration is increased by upregulation of FLS and UFGT gene expression25,26. Identification of these polyphenol biosynthesis pathway genes will lay the foundation for further genetic engineering research in T. antungense (Fig. 1).

Fig. 1: Biosynthetic pathway of the main polyphenols (caffeic acid, chlorogenic acid, rutin, and luteolin) in Taraxacum antungense.
figure 1

Phenylalanine ammonia-lyase (PAL); cinnamate 4-hydroxylase (C4H); 4-coumarate-CoA ligase (4CL); Coumarate 3-hydroxylase (C3H); 5-O-(4-coumaroyl)-D-quinate 3′-monooxygenase (C3′H); hydroxycinnamoyl-coenzyme (Co)A shikimate/quinate hydroxycinnamoyl transferase (HCT); hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT); caffeoylshikimate esterase (CSE); UDP-glucosyltransferase (UGT84); Chalcone synthase (CHS); flavonoid synthase (FNS); Chalcone isomerase (CHI); and hydroxycinnamoyl D-glucose quinate hydroxycinnamoyl transferase (HCGQT). Three arrows indicate multi-steps & routes. Blue indicates what has been reported in this article, and gray indicates information from other plants

Full size image

Plant polyphenolic compounds are important components acting against biotic and abiotic stresses21,25. Plant transcription factor (TF) family members, such as MYB11/12/111 and WRKY18/40/60, respond to biotic and abiotic stress to regulate the expression of downstream structural genes and ultimately promote the biosynthesis of polyphenols18,19,27. Coexpression analysis of TFs and biosynthesis pathway gene expression levels often showed a highly linear relationship27,28. Thus, TFs may be used as a tool not only to improve a plant’s ability to adapt to the environment but also to increase polyphenolic compound production in plants21.

Basic helix-loop-helix (bHLH) TFs are one of the largest families that regulate the expression of key enzyme genes in plants28. Among these TFs, myelocytomatosis oncogene (MYC) TFs (representative bHLH TFs) often participate in secondary metabolite accumulation: AaMYC2 (Artemisia annua L) regulates artemisinin biosynthesis, NtMYC2 (Nicotiana tabacum) regulates nicotine biosynthesis, and CrMYC2 (Catharanthus roseus) regulates the expression of alkaloid biosynthesis genes that respond to methyl jasmonate (MeJA)29,30. In the medicinal model plant S. miltiorrhiza, SmMYC2a/2b regulates key enzyme genes for phenolic acid biosynthesis31. Upregulation of SmPAL by SmbHLH37 leads to increased phenolic acid accumulation32. Moreover, the MeJA-responsive SmbHLH53 TF regulates enzymatic genes involved in the salvianolic acid B biosynthesis pathway in S. miltiorrhiza33. However, the function of bHLH TFs in T. antungense polyphenol biosynthesis and their regulatory models have been poorly reported34.

TFs regulate gene expression levels by combining with cis-acting elements of functional gene promoters; bHLH TFs specifically bind to E-box28,35. In T. antungense, the TaHQT2 gene promoter was obtained and was found to contain various cis-acting elements, specifically four E-boxes (CANNTG) (Supplementary Fig. S1 and Table S2). E-boxes are widely distributed in the promoter region of key enzymes in polyphenol biosynthesis36. Therefore, it was speculated that bHLH TFs bind to the TaHQT2 promoter to participate in polyphenol biosynthesis in T. antungense.

In this study, a T. antungense bHLH TF, TabHLH1, which was obtained through yeast one-hybrid (Y1H) screening, had high homology to bHLH1 from the Gerbera hybrid cultivar. Polyphenol concentration analysis and quantitative real-time polymerase chain reaction (qRT-PCR) results showed that TabHLH1 increased CGA and luteolin biosynthesis by increasing TaHQT2, Ta4CL, TaCHI, and TaF3′H gene expression levels in T. antungense transgenic lines. Functional analysis of TabHLH1 suggested that it regulates the biosynthesis of CGA and luteolin, enhancing the understanding of the routes of polyphenol biosynthesis and providing a structure for future metabolic engineering of T. antungense.

Results

Isolation and characterization of TabHLH1

To identify bHLH TFs involved in CGA biosynthesis of T. antungense, Y1H assays were applied to screen the T. antungense cDNA library, and the TaHQT2 promoter was used as bait. Approximately 860 bp of the TaHQT2 promoter sequence (proTaHQT2) was cloned after two rounds of amplification, and several elements were identified, including an androgen response element, TATA-box, CAAT-box, CGTCA/TGACG-motif, estrogen-responsive element, long terminal repeat, E-box, P-box, TGA-box, and light-responsive elements (Fig. S1 and Table S2). A Y1H cDNA library of T. antungense was created with a titer of approximately 5 × 107 colony-forming units/mL. PCR results showed that the length of T. antungense cDNA ranged from 200–2000 bp (Fig. S2). A 200 bp DNA fragment containing four E-boxes (proTaHQT2-E-box-1, -2, -3, and -4) that were identified in proTaHQT2 (from –685 to –810) within 860 bp of the ATG start codon was cloned. The isolated gene coding protein was able to bind to the proTaHQT2 CATGTG motif (Fig. 2). The results also showed that pMutant-TaHQT2 interacted with the isolated gene coding protein.

Fig. 2: Y1H assay.
figure 2

pAbAi-E-box grew normally on 300 ng/mL aureobasidin A (AbA); however, pAbAi-pMutant-E-box was inhibited by 300 ng/mL AbA. Therefore, 400 ng/mL was the minimum concentration of AbA used to examine the interaction between TabHLH1 and the E-boxes. We used the pAbAi-E-box and pAbAi-pMutant-E-box to transform Y1H gold yeast and then made new Y1H competent cells. Subsequently, the pGADT7-TabHLH1 recombinant vector was transformed into new Y1H competent cells. The cotransformed pAbAi-E-box + pGADT7-TabHLH1 grew on 400–500 ng/mL AbA; however, pAbAi-pMutant-E-box + pGADT7-TabHLH1 was unable to grow and needed a higher concentration of AbA. The difference between the pAbAi-E-box and pAbAi-pMutant-E-box was that the E-box pAbAi-E-box contained CAAATG, CATTTG, CATGTG, and CAATTG motifs. In the pAbAi-pMutant-E-box, AAAAAA was used instead of the CATGTG motif

Full size image

The isolated protein acquired from the nonredundant protein database (using the Basic Local Alignment Search Tool for protein [BLASTP]) showed high homology to the Gerbera hybrid cultivar bHLH1 (GhbHLH1: 69% identity and 78% positivity) and Gynura bicolor bHLH1 (GbbHLH: 66% identity and 76% positivity) (Fig. 3A). The full-length TabHLH1 cDNA was 1566 bp and encoded a 521-amino acid protein (accession number: MH683054) (Fig. 3B). Multiple sequence alignment identified a conserved bHLH–MYC_N domain and an HLH domain in TabHLH1 (pfam 14215 and pfam 00010) using the Pfam online tool (http://pfam.xfam.org/search/sequence). Based on these results, we deduced that TabHLH1 was a bHLH TF.

Fig. 3: Characterization and subcellular localization of TabHLH1.
figure 3

A Phylogenetic analysis of TabHLH1 and related bHLH TFs. B Sequence alignment of TabHLH1 sequences in T. antungense. TabHLH1 (in this article, QBQ52949.1; Taraxacum antungense bHLH1), GhbHLH1 (CAA07615.1; Gerbera hybrid cultivar bHLH1), GbbHLH1 (BAJ17663.1; Gynura bicolor bHLH1), AabHLH1 (PWA53551.1; Artemisia annua bHLH1), DpbHLH (BAJ33516.1; Dahlia pinnata bHLH), CcbHLH1 (KVH95430.1; Cynara cardunculus var. scolymus bHLH1), CsbHLH1 (AOY10782.1; Camellia sinensis bHLH1), RgbHLH1 (ATZ76571.1; Rubus genevieri bHLH1) and AtbHLH1 (AAB72192.1; Arabidopsis thaliana bHLH1). The conserved domains pfam14215 (bHLH-MYC-N) and pfam00010 (HLH) are indicated by straight lines. The neighbor-joining method in MEGA software (version 8.0) was used to construct the phylogenetic tree. Bootstrap percentage (1000 replicates) values are listed above the branches. The TabHLH1 protein is labeled with a red circle. The black line indicates the bHLH-MYC-N motif; pfam14215. The red line indicates the HLH motif; pfam 00010. C Subcellular localization of the TabHLH1 protein in N. benthamiana leaves. Tobacco leaf cells were transiently transformed with recombinant plasmids containing TabHLH1-YFP or YFP (control group). DAPI staining indicates localization in the nucleus

Full size image

Subcellular localization showed that the yellow fluorescent protein (YFP) (control group; empty vector) was distributed uniformly throughout the cell, whereas TabHLH1-YFP fluorescence (treatment group; TabHLH1-YFP vector) was observed in the nucleus. This was confirmed using the positive control nuclear stain 4′,6-diamidino-2-phenylindole (DAPI) (Fig. 3C).

Biochemical analyses of the main polyphenol compounds in T. antungense and expression profiling of TabHLH1

The total phenolic concentrations of T. antungense in different tissue samples ranged from 32.37–66.23 mg GAE/g, and the highest concentrations of total phenolics were found in flowers, followed by leaves, roots, and stems (Fig. 4A, B). The four phenolic acids (CGA, CA, rutin, luteolin) in different tissues of T. antungense showed significant differences. CGA concentrations were highest in the roots, followed by flowers, stems, and leaves; CA concentrations were highest in the flowers, followed by leaves and flowers; rutin concentrations were highest in roots and flowers, followed by leaves and stems; luteolin concentrations were highest in leaves, followed by flowers and roots, while CA and luteolin were not detected in stems (Fig. 4C).

Fig. 4: Basic biochemical analyses of T. antungense and the TabHLH1 elicitor response.
figure 4

A Different tissues (leaves, roots, flowers, and stem) of wild T. antungense. B Total polyphenol concentrations in different tissues. C CGA, CA, luteolin, and rutin concentrations in different tissues. nd: not detected. D The expression levels of polyphenol biosynthesis pathway enzyme genes (PAL, 4CL, C4H, HCT, HQT1, HQT2, CHS, CHI, FNS, F3′H) and TabHLH1 TF in different tissues of T. antungense. The expression levels of different genes in roots were set to 1. E The effects of different hormones on TabHLH1 gene expression levels. Treatment with 100 μM methyl jasmonate (MeJA), 100 μM salicylic acid (SA), 100 μM abscisic acid (ABA), 100 μM gibberellin (GA3), 100 μM ethylene (ET), or 500 μM NaCl for different time points (0, 1, 3, 6, 9, 12, 16, and 24 h). The expression levels of TabHLH1 at 0 h were set to 1. The TaActin gene was selected as an internal reference in this article

Full size image

To study the correlation between key polyphenol biosynthesis enzyme genes and TabHLH1 TF in different tissues, one-year-old wild flowering T. antungense was used for expression analysis by qRT-PCR. The results showed that HQT1 was highly expressed in roots; CHS, CHI, 4CL, FNS, and F3′H were highly expressed in flowers followed by leaves; HCT, PAL, HQT2, and C4H were highly expressed in leaves followed by flowers. For TabHLH1, the expression level was highest in leaves, followed by flowers, roots, and stems (Fig. 4D).

To study the effects of various biotic and abiotic stresses on the expression level of TabHLH1, NaCl, MeJA, salicylic acid (SA), abscisic acid (ABA), ethephon (ET), and gibberellic acid (GA3) treatments were performed at different time points in T. antungense leaves, and then qRT-PCR was used for expression analysis. Under NaCl treatment (500 ng/mL), TabHLH1 expression increased more than 40-fold in 3 h, indicating that TabHLH1 was most responsive to NaCl treatment. After 3 h of ABA treatment, the expression level of TabHLH1 reached 13-fold and had the same tendency as its expression under NaCl treatment. The expression level of TabHLH1 was also affected by 100 μM MeJA; TabHLH1 expression in leaves increased at 3 h and then slowly declined to approximately 1.3-fold at 24 h. Similar results were obtained with SA, ET, and GA3 hormone treatments (Fig. 4E).

Identification of transgenic plants

To evaluate the regulatory mechanism of TabHLH1 in T. antungense polyphenol biosynthesis, the overexpression vector pRI101-TabHLH1-YFP was constructed and transformed into T. antungense leaves according to Liu et al. (2018)14. Seventeen independent transgenic lines were identified using p35SF as the forward primer (according to the 35S promoter sequence) and TabHLH1R as the reverse primer through genomic PCR (Table S1). Three transgenic lines (TabHLH1-OE2, TabHLH1-OE7, and TabHLH1-OE13) with higher expression levels of OE-TabHLH1 than the control lines were selected for further experiments (Fig. S3 and Fig. 5A). At the same time, three RNAi-TabHLH1 transgenic lines (RNAi-1, RNAi-19, and RNAi-22) containing pCAMBIA1300-35S-TabHLH1 were identified according to Liu et al., 201918. qRT-PCR showed that TabHLH1 expression levels in the OE-TabHLH1 transgenic lines were significantly higher than those in the wild-type (WT) line, while the expression levels of RNAi-TabHLH1 were lower than those in the WT transgenic lines (Fig. 5B).

Fig. 5: Generation of T. antungense transgenic lines.
figure 5

A Agrobacterium tumefaciens-mediated transformation generated transgenic T. antungense; (1) callus induction for treatment with recombinant Agrobacterium GV3101 carrying the TabHLH1-overexpressing construct, (2) transgenic callus, (3) shoot induction, (4) root induction, (5) well-rooted transgenic plant, (6) transplantation of transgenic plants. B Expression level of TabHLH1 in different transgenic lines. C Determination of polyphenol concentrations, including those of chlorogenic acid (CGA), caffeic acid (CA), rutin, and luteolin, by HPLC in T. antungense transgenic lines. D Expression levels of the key enzyme genes in the CGA and luteolin biosynthesis pathways in control and TabHLH1 overexpression transgenic lines

Full size image

Role of TabHLH1 in polyphenol biosynthesis

The concentrations of CGA, CA, rutin, and luteolin in the TabHLH1 transgenic lines were determined by high-performance liquid chromatography (HPLC). CGA concentrations increased in the transgenic lines compared to those in the control lines (0.73 ± 0.054 mg/g dry weight [DW]), with the highest concentration in OE-7 (1.18 ± 0.113 mg/g DW) and the lowest in RNAi-19 (0.34 ± 0.141 mg/g DW). There were no significant differences in CA and rutin concentrations between the transgenic and control lines (Fig. 5C). Luteolin concentrations in OE-13 (7.07 ± 0.687 mg/g DW) increased compared to those in the control lines (4.83 ± 0.345 mg/g DW) and were lowest in RNAi-19 (3.84 ± 0.441 mg/g DW). To identify the CGA and luteolin biosynthetic genes regulated by TabHLH1, the expression levels of key enzyme genes in the CGA and luteolin biosynthesis pathways in T. antungense were first determined. Ta4CL, TaHQT2, TaCHI, and TaF3′H were all upregulated in the TabHLH1-overexpression lines to various degrees. TaHQT2 expression was the most significantly increased among the four upregulated genes (Fig. 5D).

In vivo and in vitro evaluation showed that TabHLH1 increased TaHQT2 and Ta4CL expression

The gene promoters of TaPAL, TaC4H, Ta4CL, TaHCT, and TaHQT1/2 from the CGA biosynthetic pathway all contain bHLH cis-acting elements33. Herein, dual-luciferase (LUC) assays were performed to investigate whether TabHLH1 increased the expression of these genes. Reporter and effector vector construction are shown in Fig. 6A. Fluorescence analysis indicated the intensity of gene expression (Fig. 6A). Of the six examined genes, the ratio of LUC/Renilla (REN) was detected only for Ta4CL and TaHQT2 and was significantly higher than that of the control, with a 6.26- and 10.08-fold increase, respectively (Fig. 6B). Furthermore, TabHLH1 binding sites (bHLH-responsive cis-elements) were detected using Y1H assays and electrophoretic mobility shift assays (EMSAs). TabHLH1 directly combined with the TaHQT2 and Ta4CL promoters through the E-box motif (CATGTG) (Fig. 6C, D). These results indicated that TabHLH1 directly increased TaHQT2 and Ta4CL gene expression, thereby modulating CGA accumulation.

Fig. 6: Dual-LUC and EMSAs proved that TabHLH1 activates the expression of Ta4CL, TaHQT2, TaCHI, and TaF3′H.
figure 6

A Dual‐LUC assays indicating that TabHLH1 increases the expression levels of Ta4CL, TaHQT2, TaCHI, and TaF3′H. B The relative fold change of LUC/REN (Renilla) indicates that the expression levels of Ta4CL, TaHQT2, TaCHI, and TaF3′H can be activated by TabHLH1. C Y1H assay results indicated that TabHLH1 directly binds to Ta4CL and TaHQT2 in yeast cells. The CATGTG motif has not been found in the promoter of TaF3′H. D EMSA results indicate that TabHLH1 directly binds to the E-box elements (CATGTG) from the TaHQT2 and Ta4CL promoters. The cold-probe sequences were the same as the labeled probes but without biotin labeling

Full size image

TabHLH1 increased luteolin biosynthesis

Luteolin is also produced in plants using phenylalanine as a precursor16. Hence, we measured luteolin concentrations in the transgenic lines. Luteolin concentrations were significantly higher in the OE-TabHLH1 transgenic line and lower in the RNAi-TabHLH1 line than in the control groups (Fig. 5C). In addition, two of the luteolin biosynthetic pathway genes, TaCHI and TaF3′H, were strongly upregulated in the OE-TabHLH1 line, while TaCHI and TaF3′H were significantly downregulated in the RNAi-TabHLH1 line compared to that in the control groups (Fig. 5D). Dual-LUC assays showed that TabHLH1 significantly increased TaCHI (harboring the CATGTG motif in their promoter) and TaF3′H (without the CATGTG motif) expression levels. The ratio of LUC/REN increased by 7.94-fold for TaCHI and 6.11-fold for TaF3′H compared to that in the control groups (Fig. 6B). However, Y1H assay results indicated that the E-box (CATGTG motif) in the TaCHI promoter cannot directly interact with TabHLH1 (Fig. 6C). Together, these results indicated that TabHLH1 increased both CGA and luteolin concentrations in T. antungense transgenic lines.

Discussion

Basic information on the polyphenols in T. antungense

In different tissues of T. antungense, the total polyphenol concentrations are highest among flowers, followed by leaves, roots, and stems, showing the same tendency as in T. mongolicum4. Total polyphenols, including chlorogenic, caffeic, quinic, caffeoylquinic, ferulic, cinnamic, caftaric, benzoic, vanillic, protocatechuic, gallic, cumaric acid, chrysoeriol, and vanillin, have been found in Taraxacum species1. Flowers and leaves have higher polyphenol concentrations than roots and stems. The CGA and CA levels in T. mongolicum were generally lower than those in T. antungense, while luteolin levels were significantly higher4. The possible reasons are various factors, including growth environments, sample extraction, and HPLC methods, which together cause significant differences in CGA, CA, rutin, and luteolin concentrations in T. antungense and closely related species4,21,37. The relationship among the four compounds and total polyphenol concentrations should be further studied.

TabHLH1 potentially regulates polyphenols in T. antungense

First, the HQT2 gene promoter was used as bait to screen functional TFs. CGA is widely distributed in many plants, such as T. antungense, L. japonica, Solanum lycopersicum, and Solanum tuberosum20,21,38. In the CGA biosynthesis pathway, key enzyme genes, such as PAL, C4H, 4CL, and HCT/HQT, are widely reported14,20. However, the regulation of CGA biosynthesis targeting the key enzyme HQT has not been previously reported20,21. Gene promoters are the core link between TFs and structural gene expression39,40,41. Through analysis of the TaHQT2 promoter (four CANNTG motifs), it is speculated that TaHQT2 is regulated by bHLH TFs (Fig. S1 and Table S2)33,41. Herein, using proHQT2 as a probe, we first obtained a bHLH TF named TabHLH1 through Y1H assays.

Second, coexpression analysis can be used to screen and preliminarily identify the correlation between transcription factors and key enzyme gene expression34,40. Under ABA treatment, SmbZIP1 and salvianolic acid biosynthesis pathway gene expression levels significantly increased, and coexpression analysis showed a highly linear relationship40. By analyzing TabHLH1 together with polyphenol biosynthesis pathway gene expression levels in T. antungense different tissues, it can be concluded that TabHLH1 potentially regulates the expression of key enzyme genes in polyphenol biosynthesis and thus affects the concentration of polyphenols in T. antungense32,33. Therefore, polyphenol analysis, Y1H assays, and coexpression analysis provide a theoretical basis for screening related TFs for designing molecular breeding strategies to improve T. antungense quality.

TabHLH1 increased CGA biosynthesis in T. antungense

In this study, full-length TabHLH1 was isolated and cloned from T. antungense and showed the highest identity with the Gerbera hybrid cultivar GhbHLH1. Both GbbHLH1 and GhbHLH1 play a vital role in the regulation of anthocyanin and dihydroflavonol accumulation37,42,43. In plants, anthocyanin and CGA have the same precursor and share the first three key enzymes41. In this study, salt stress significantly increased TabHLH1 gene expression levels, with the same tendency as that of GhbHLH1, which is consistent with earlier findings42. However, under ABA treatment, TabHLH1 expression increased more than 10-fold, which has not been reported in GbbHLH142. A possible reason for this finding may be species evolution and functional redundancy in multiple gene families31. TabHLH1 is located in the nucleus, similar to other nucleus-localized bHLH TFs, such as AaMYC2-Like and MdMYC244,45. Therefore, TabHLH1 plays a vital role in hormone and salt stress signal regulation, which ultimately assists plants in their response to a variety of biotic and abiotic stresses in their natural environment14,26. The relationship among hormones, salt stress signal regulation, and TabHLH1 expression should be further studied.

Multiple TFs, such as SmbHLH37 and SmbHLH53, increase polyphenol concentrations through PAL or other key enzymes32,33. By analyzing the CGA concentrations in TabHLH1 transgenic lines (OE-7 and RNAi-19), CGA concentrations in the OE-7 transgenic line increased nearly 63.6% compared to that in the WT, while CGA concentrations in RNAi-19 decreased to 53.2%, suggesting that TabHLH1 significantly increased CGA concentrations in T. antungense. TaHQT2 was identified as the key enzyme that can directly synthesize CGA in previous studies21. In this study, Ta4CL and TaHQT2 gene expression levels were positively correlated with the CGA concentration. Furthermore, dual-LUC and EMSA demonstrated that TabHLH1 bound directly to the TaHQT2 promoter region. These results together demonstrated that TaHQT2 was a target of TabHLH1; thus, TabHLH1 played a positive role in regulating CGA biosynthesis.

p-Coumarin-CoA is a precursor compound for the synthesis of downstream phenolic acids, flavonoids, and anthocyanins7,42. 4CL participates in the front-end enzymatic reaction of polyphenol biosynthesis and directly catalyzes the biosynthesis of p-coumarin-CoA (Fig. 1). In this study, TabHLH1 increased the expression level of Ta4CL, thereby regulating metabolic flow in plants to accumulate polyphenols, which is consistent with CsbHLH1 directly activating the Cs4CL promoter44,46,47. Dual-LUC, Y1H, and EMSA demonstrated that TabHLH1 bound directly to the Ta4CL promoter region. These results suggested that Ta4CL was another target of TabHLH1.

TabHLH1 increased CHI and F3′H gene expression levels in vivo to promote luteolin biosynthesis in T. antungense

Flavonoid biosynthesis regulation has been studied in-depth, particularly in the medicinal plant S. miltiorrhiza19,33,48. Functional genes, including key enzyme genes and numerous TFs, are required for luteolin biosynthesis29,44. In this study, TabHLH1 significantly increased luteolin concentrations (OE-7 increased to 1.46-fold, and RNAi-19 decreased to 0.78-fold) in transgenic lines compared to that in the WT. In addition, concentrations of the key luteolin biosynthesis enzymes TaCHI and TaF3′H were directly influenced by overexpression/RNAi of TabHLH1, suggesting that TabHLH1 may interact with TaCHI and TaF3′H. Dual-LUC results showed that TabHLH1 increased the expression levels of two pathway genes involved in luteolin biosynthesis. However, TabHLH1 binds indirectly to the promoters of TaCHI and TaF3′H (Fig. 6C). The possible reason may be that TabHLH1 interacts with other proteins, such as MYB partners, which can directly interact with TaCHI and TaF3′H. These results indicated that TabHLH1 was positively involved in regulating luteolin biosynthesis and metabolic flow, and the direct target was Ta4CL, while the potential targets were TaCHI and TaF3′H. The MYB-bHLH-WD40 complex often increases the expression level of flavonoid biosynthesis genes to control the production of anthocyanins49. CsbHLH1 (CsMYC1) interacts with CsbHLH42, CsWD40, CsMYB60 and itself to regulate flavonoid biosynthesis in cucumber47. In T. antungense, further studies should focus on proteins that interact with TabHLH1 to regulate polyphenol biosynthesis.

Based on these results, a functional model for the role of TabHLH1 in polyphenol biosynthetic regulation in T. antungense is provided (Fig. 7). In summary, TabHLH1 increased TaHQT2 and Ta4CL expression levels, leading to increased CGA concentrations. This is the first dandelion bHLH protein identified as being involved in CGA pathway regulation. In addition, TabHLH1 promoted the expression of luteolin biosynthesis genes (TaCHI and TaF3′H) to increase the accumulation of luteolin. These studies provide new insights into the role of TabHLH1 in the regulation of polyphenol biosynthesis. In addition, these findings lay the foundation for further exploration of the molecular mechanisms and potential functional genes of secondary metabolite biosynthesis in T. antungense.

Fig. 7: Proposed model of the role of TabHLH1 in regulating polyphenol biosynthesis.
figure 7

Using the HQT2 promoter E-box region as a probe, a bHLH transcription factor (named TabHLH1) was obtained by yeast one-hybrid screening. TabHLH1 significantly promoted the expression levels of key enzyme genes in the polyphenol biosynthesis pathway, including 4CL, HQT, CHI, and F3′H, thus increasing CGA and luteolin accumulation

Full size image

Materials and Methods

Plant materials

T. antungense biomaterials were collected and transplanted in the greenhouse of our laboratory, as previously reported34. N. benthamiana and T. antungense seeds were sown in substrate/vermiculite (3/1)-admixture soil and transplanted in pots for 4–6 weeks for hormone treatment experiments. Plants were maintained at a constant temperature of 25 °C under 16/8 h light/dark cycles for use in transient expression analysis14.

TaHQT2 promoter cloning and Y1H screening

The CTAB method was used to extract dandelion plant DNA, and RNase was used to remove the remaining RNA. Fusion primers and nested integrated PCR were used to obtain the TaHQT2 5′-end promoter47,50. All primers are listed in Table S1. PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to analyze the promoter cis-element sequences.

The TaHQT2 promoter (four E-boxes located from –691 bp to –806 bp, Fig. S1) was constructed in pABAi to create the pAbAi-proTaHQT2 recombinant vector using the BamH I and Hind III restriction sites. BstBI was used to digest recombinant plasmids, linearized pAbAi-proTaHQT2 plasmids were transformed into the yeast strain (Y1H), and then, the resulting strains were tested on SD/–Ura media containing aureobasidin A (AbA) at concentrations ranging from 100–500 ng/mL.

A Y1H cDNA library of T. antungense was constructed using the Matchmaker™ one-hybrid library construction & screening kit PT3529-1 (PR732190, Takara Biomedical Technology Co., Ltd., Beijing, China). Yeast recombinant vector (GAL4-AD-sec) was extracted from the primary library and transformed into Y1H containing pAbAi-proTaHQT2. After rescreening on SD/-Leu-Ura medium with higher AbA concentrations than listed above, positive pGADT7-sequence yeast strains were used for sequencing analysis. The PCR thermocycler program used was as follows: 94 °C for 10 min, 40 cycles of denaturation, annealing, and extension (94 °C for 30 s, 50 °C for 45 s, and 72 °C for 90 s, respectively), and a final extension at 72 °C for 10 min.

Bioinformatics analysis and isolation of TabHLH1

A bHLH TF protein isolated from T. antungense, which was designated TabHLH1, was found to interact with the E-boxes of TaHQT2. The gene sequence was compared to Taraxacum kok-saghyz Rodin (accession number: GWHAAAAM043215)36. The complete coding sequence was obtained using homologous cloning. The target cDNA fragment was connected to the 18 T vector (Takara Biomedical Technology Co., Ltd., Beijing, China) for sequencing analysis. BLAST alignment (http://www.ncbi.nlm.nih.gov/BLAST/) was used to search for orthologs. ClustalX (version 1.81) was used for multiple sequence alignment through the neighbor-joining method using 1000 repetitions. A molecular phylogenetic tree was constructed using the MEGA program (version 8.0)14.

Elicitor treatment and subcellular localization of TabHLH1

Methyl jasmonate (MeJA), salicylic acid (SA), ethephon (ET), gibberellin (GA), and abscisic acid (ABA) purchased from Sigma-Aldrich (Shanghai, China) were directly dissolved in distilled water at a final concentration of 100 mM. NaCl (Aladdin, Shanghai, China) was dissolved at a concentration of 500 mM, and distilled water was used as a control. Two-month-old well-grown T. antungense transgenic and WT plants were chosen for different treatments. The above elicitors were filter-sterilized through a 0.45 μm filter membrane (Pall Corporation, NY, USA) and added to the cultures at a final concentration of 100 μM. Tissues from different parts of the plants were collected after 0, 1, 3, 6, 9, 12, 16, and 24 h of treatment.

To identify the in vivo subcellular location of TabHLH1, the coding sequence of TabHLH1 was fused with a reporter sequence. The complete coding sequence of TabHLH1 (without the TAG stop codon), including the restriction sites Nde I (in the 5′-end) and Sal I (in the 3′-end), was amplified and subcloned into the pMD19-T simple vector (Takara Biomedical Technology Co., Ltd., Beijing, China). The plasmid pRI101-YFP (containing the yellow fluorescent protein gene) was double-digested using the same enzymes to create a recombinant vector termed pRI101-TabHLH1-YFP, and the insert was sequenced using the 35S (forward) and TabHLH1R (reverse) primers (Fig. S2 and Table S1). The fused recombinant expression plasmid was transformed into N. tabacum. pRI101-YFP was used as a control to perform the transient expression assay21,40.

Transformation of T. antungense

The pCAMBIA1300-35S-X (restriction sites: BamH I/Spe I for sense and Kpn I/Sac I for antisense sequences) RNAi expression vector was used for RNAi-TabHLH1 recombinant plasmid creation21. The SPLRNAi gene was used as intron X. The middle region of TabHLH1 (631–839 bp) was used for vector construction (nonconserved region). Both pCAMBIA1300-35S-TabHLH1 and pRI101-TabHLH1-YFP were transformed into Agrobacterium tumefaciens strain GV3101. pCAMBIA1300-35S-X and the pRI101-YFP vector were used as controls. After positive identification, Agrobacterium harboring different recombinant plasmids were used for injection into plants. Following a previously published protocol14, Agrobacterium infection was used for genetic transformation to obtain T. antungense transgenic plants.

Analysis of gene expression profiles

Different tissues or transgenic lines of T. antungense were used for total RNA extraction, followed by cDNA synthesis, which was performed following the abovementioned methods50,51. qRT-PCR was performed using gene-specific primer pairs for PAL, C4H, 4CL, HCT, HQT2, CHS, CHI, and F3′H (Supplemental Table S1) using three technical replicates. Based on the 2−∆∆Ct method, qRT-PCR was performed, and relative expression levels were calculated using β-actin as a reference gene14.

Measurement of polyphenol concentrations by HPLC

HPLC was used to investigate the concentrations of four polyphenols (CGA, CA, rutin, and luteolin) in the T. antungense plant materials from the control groups and transgenic lines (Table S3). For transgenic lines, 3-month-old whole plants (containing roots and leaves) were dried and dehydrated at −20 °C to constant weight, ground into powder, and then used as samples. Samples were ultrasonically extracted for polyphenol compounds and passed through a 0.22 μm filter membrane for HPLC, as previously described14,23. HPLC conditions for polyphenol detection were as described in the previous reports34.

Measurement of total phenolic concentrations

The total phenolic concentrations of T. antungense in different tissues were extracted with Folin-Ciocalteu reagent as previously reported4,5. Then, 500 µL of T. antungense extract was added to 1.5 mL FC reagent (0.2 mg/mL) and mixed. Two milliliters of 7.5% Na2CO3 reagent and 2 mL distilled water were added. Then, the mixture was incubated at 25 °C for 1 h (in the dark). The absorbance of the mixture was recorded at 727 nm, and 60% methanol was used as a control. The total phenolic concentrations of T. antungense samples were calculated according to milligrams of gallic acid equivalents per gram dry weight of the sample (mg GAE/g)4

Dual-LUC assay

The pCAMBIA2300+-TabHLH1 vector acted as an effector and was transferred into A. tumefaciens strain GV3101 (pCAMBIA2300+-vector was used as a control). The promoters of key enzyme genes, including Ta4CL, TaHQT2, TaCHI, and TaF3′H, were cloned separately into the pGreen0800-rec plasmid. The pGreen0800-promoter recombinant vectors, separately with the helper vector pSoup19, were cotransformed into GV3101. The Renilla vector was used as an internal control. Both the reporter and effector strains were mixed in equal proportions (3 mL each), slowly cultivated for 2 h, and injected into the leaves of 2-month-old N. benthamiana. After incubation in the dark for 2–3 days, commercial dual-LUC reaction reagents (Promega Biomedical Technology Co., Ltd., Beijing, China) were used to perform dual-LUC assays on leaf samples18. Three biological replicates were measured for each sample.

Y1H assay

The Y1H assay was performed differently from the Y1H screening and used the pB42AD/pLacZ system, which has been previously described40. The full-length TabHLH1 ORF fragment was amplified, sequenced, and cloned into the effector plasmid (pB42AD). For the reporter plasmid pLacZ, a triple tandem copy of the E-box (NNNNCATGTGNNNN) motif near 4 bp from every promoter (TaHQT2, Ta4CL, TaCHI) was inserted by using EcoR I and Xho I as restriction endonuclease sites. After identification by sequencing, recombinant effector plasmids (pB42AD-TabHLH1) and recombinant reporter plasmids (pLacZ-TaHQT2, pLacZ-Ta4CL, pLacZ-TaCHI) were cotransformed into yeast strain EGY48a. Transformants were cultivated on SD/-Ura/-Trp medium for 2 d and then transferred (by using distilled water-diluted 1000-fold) to SD/-Ura/-Trp medium with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) for another 1–2 d. Empty pB42AD and pLacZ plasmids were used as negative controls and cotransformed into EGY48a strains.

EMSAs

The complete sequence of TabHLH1 was inserted into the BamH I and Sal I sites of the pGEX4T-1 plasmid and then transformed into Escherichia coli (BL21 or DE3 strain). Isopropyl-D-thiogalactoside was used to induce recombinant protein expression overnight (16 h), and the GST-tagged protein purification kit (Transgen Biotech Co., Ltd., Beijing, China) was used to purify recombinant proteins. Biotin-labeled 5′- and 3′-ends of the TaHQT2 promoter were synthesized by Shanghai Sangon Co. (Shanghai, China), and the two biotin-labeled primers were annealed to form double-stranded DNA fragments. The purified recombinant proteins and DNA fragments were incubated in 10× EMSA binding buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China) at 25 °C for 30 min. DNA fragments without biotin labeling were used as an internal control. The DNA-protein complex was electrotransferred to a wet electromembrane and examined following the manufacturer’s instructions by using a chemiluminescent nucleic acid detection module kit (Beyotime Biotechnology Co., Ltd., Shanghai, China)19,27,48,52.

Statistical analyses

Statistical comparisons were performed using SPSS v19.0 software. Error bars represent the SE of three biological replicates. All data are presented as the mean±standard deviation (SD). Statistical significance was assessed using Student’s t test (#: decrease, *: increase, P < 0.05) for all the experiments involved in this article (for the different tissues, transgenic lines, and control groups).