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).
