Plant materials and growth conditions
Nicotiana benthamiana Domin plants were grown in a climate chamber under fluorescent light (16-h light/8-h dark) at 25°C. A Eucalyptus camaldulensis Dehnh. clone (Myrtaceae; seed lot 19708; Australian Tree Seed Centre, CSIRO) was cultured hydroponically in a growth chamber as previously described (Tahara et al. 2018). Leaf samples were collected, immediately frozen in liquid nitrogen, and stored at − 80°C until analyzed.
Taxonomic re-classification of HT distribution
Families and genera with HT-containing species determined on the basis of the Cronquist system of classification (Engelhardt et al. 2016; Okuda et al. 2000; Table S1, Fig. 2) were revised according to the Angiosperm Phylogeny Group classification (APG IV 2016).
RNA extraction
Total RNA was extracted from E. camaldulensis and N. benthamiana leaves using the hexadecyltrimethylammonium bromide (CTAB) method and purified using the SV Total RNA Isolation System (Promega) as previously described (Tahara et al. 2018).
Plasmid construction
First-strand cDNA was synthesized from the total RNA extracted from E. camaldulensis leaves and then used as the template to amplify target cDNA sequences by PCR with specific primer sets (Table S2). The recombinant plasmids for the transient expression in N. benthamiana leaves were created as follows. For the biosynthesis of gallic acid, the intein-UBQ sequence for polycistronic expression (Zhang et al. 2019) was sandwiched by the open reading frames of EcDQD/SDH2 (Accession No. LC487989) and EcDQD/SDH3 (LC487990) and subsequently inserted into p35SHSPstarG4_L4R1, which is the same as p35SHSPG (Oshima et al. 2011) except for the mutated HindIII site in the HSP terminator and the different Gateway att site (attL4–attR1) (Fig. 3a). The same method was used to prepare the construct for UGT84A25 (LC189069) and UGT84A26 (LC189071), which are responsible for the subsequent reaction, but the construct was inserted into p35SHSPG, which contains the Gateway attL1–attL2 site (Oshima et al. 2011; Fig. 3b). Finally, the inserted constructs in these two plasmids and the corresponding sequence in the empty vector were inserted into the pGWB501 vector (Nakagawa et al. 2009), which has the Gateway attR4–attR2 site, via a multisite Gateway reaction to prepare the recombinant plasmids for the transient expression of SDH, UGT, and SDH-UGT.
Transient heterologous gene expression in N. benthamiana
The recombinant plasmids were individually introduced into Agrobacterium tumefaciens strain GV3101 cells harboring the transformation helper plasmid pSoup via electroporation (Hellens et al. 2000).
Overnight cultures were harvested by centrifugation and the pellets were resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES pH 5.6, and 100 µM acetosyringone). The OD600 values of the suspensions were adjusted to 0.5, after which the suspensions were incubated for 2 h at room temperature prior to the infiltration of N. benthamiana using a needleless syringe. The infiltrated plants were cultivated in the climate chamber. At specific time-points, leaves were collected and immediately frozen in liquid nitrogen.
Quantitative real-time PCR (RT-qPCR) analysis
PrimeScript RT Master Mix (Takara) was used to synthesize cDNA from the extracted total RNA. The RT-qPCR analysis of EcDQD/SDH2, EcDQD/SDH3, UGT84A25, and UGT84A26 transcript levels was performed using TB Green Premix Ex Taq II (Takara) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The gene-specific primers are listed in Table S2. The target sequences were amplified using the E. camaldulensis cDNA as the template and then cloned into a pBlueScript II SK (+) vector. The plasmid solution for each gene was serially diluted (10-fold from 108 molecules µL− 1 to 101 molecules µL− 1) and used for the standard curve assay. The PCR efficiency for each primer pair was determined using relative standard curves and the following equation as described by Bustin et al. (2009): PCR efficiency = 10− 1/slope−1. Each gene-specific primer pair amplified a single product of the correct size with high efficiency (> 90%). The N. benthamiana gene encoding elongation factor 1-α (NbEF-1α) was selected for the normalization of transcript levels, which are presented herein as the copy number per picogram of total RNA.
Identification and quantification of HTs and related compounds
Leaf samples (1 g) were ground to a powder in liquid nitrogen using a cell disruptor (Multi-Beads Shocker, Yasui Kikai) for the subsequent extraction with 1 mL ice-cold 70% (v/v) aqueous acetone. The samples were centrifuged at 4°C and the supernatant (50 µL) was diluted with 450 µL 0.1% (v/v) formic acid in water. Extracts were analyzed using an ultra-performance liquid chromatography (UPLC) system (ACQUITY UPLC H-Class, Waters) coupled with a quadrupole time-of-flight mass spectrometry (Q-TOF-MS) system (Xevo G2-XS QTof, Waters). The UPLC conditions were as follows: column, ACQUITY UPLC HSS T3 Column (particle size 1.8 µm, 2.1 mm × 100 mm; Waters); column temperature, 40°C; solvent A, 0.1% (v/v) formic acid in water; solvent B, acetonitrile; flow rate, 0.5 mL min− 1. The gradient was 0.1–25% eluent B (at 0–10 min after injection), 25–99% (10–10.1 min), 99–0.1% (12–12.1 min), and 0.1% (12.1–15 min). The Q-TOF-MS conditions were as follows: ionization mode, electrospray ionization; acquisition mode, MSE or MS/MS negative-sensitivity mode; acquisition range, 40–1,200; capillary voltage, 1.0 kV; cone voltage, 30 V; source temperature, 120°C; desolvation temperature, 500°C; cone gas flow, 50 L h− 1; desolvation gas flow, 1,000 L h− 1; collision energy, 6 eV (low energy) or 10–45 eV (high energy). Compounds in the extracts were identified by comparing their retention times and product ion spectra with those of authentic standard compounds (β-glucogallin, BOC Sciences; 3-glucogallic acid and 4-glucogallic acid, Synthose). The measured mass of the compounds was consistent with their elemental composition (Table S3). Each compound was quantified according to a standard curve plotted using the peak areas in the extracted ion chromatograms of the deprotonated molecule m/z values.
Extraction of crude protein from plants and UGT activity assay
Frozen fresh leaves (0.5 g) were mixed with 2 mL ice-cold extraction buffer (100 mM phosphate buffer pH 7.5 and 150 mM NaCl) supplemented with 0.5 g PVPP and then homogenized in a mortar. The homogenized samples were centrifuged. The supernatant was used for the overnight precipitation with ammonium sulfate (80% saturation) at 4°C. The precipitate was suspended in 1 mL storage buffer [100 mM MES pH 5.5, 100 mM NaCl, and 10% (v/v) glycerol]. After a centrifugation at 10,000 × g for 5 min, the supernatant was desalted using the Amicon Ultra-0.5 Centrifugal Filter Device (Merck Millipore). The protein concentrations of desalted extract were estimated using the Qubit Protein Assay Kit (Thermo Fisher Scientific) prior to the UGT activity assay.
The UGT activity assay was performed using a 100 µL reaction mixture containing 100 mM MES (pH 5.5), 4 mM UDP-glucose, 2 mM gallic acid, 3 mM 2-mercaptoethanol, and 50 µg desalted crude protein. The reaction mixture was incubated at 30°C for 3 h. All reactions were terminated by adding an equal volume of methanol and then samples were centrifuged. The supernatant was transferred to a new tube for the UPLC-Q-TOF-MS analysis.
Statistical analysis
The metabolite concentrations and gene expression levels are presented herein as the mean ± SE for at least three replicates. Data were analyzed using Student’s t-test or the Tukey–Kramer test with BellCurve for Excel version 4.04 (Social Survey Research Information).