Plant materials
Samples of Camellia sinensis cv. Shucazao (Variety Approval number: CHN20022008), were obtained from the experimental tea garden of Anhui Agricultural University in Hefei, China (north latitude 31.86, east longitude 117.27, altitude 20 m above mean sea level). Leaves were collected at five different stages (bud, 1st leaf, 2nd leaf, and 3rd leaf, older leaf), stem and root, snap frozen in liquid nitrogen and stored at −80°C.
Healthy tissue culture seedlings were used for light and sucrose induction experiments. Seedlings were cultured in normal light–dark cycle (light/dark: 14 h/10 h) in N6 medium containing 3% sucrose, and subcultured every 20 days by transferring about 5 g (fresh weight) to fresh medium. Six separate culture flasks were selected from the light and sucrose treatments. For light treatment, plates were exposed to 50 ± 5 μmolm−2 s−1 light (Cool white, 55 W, Philips, Netherlands) for 7 days, and culture flasks covered with aluminum foil were used as full darkness controls. For sucrose treatment, the seedlings were subcultured in the previously described medium or the previously described medium containing additional 90 mM/L sucrose for 7 days. Total RNA was isolated from leaves for quantitative real time polymerase chain reaction (qRT-PCR) in three independent experiments. The morphology of tea seedlings were captured with a Cannon 600D camera (Cannon, Japan).
The yeast strain (Saccharomyces cerevisiae cv. WAT11) and the tobacco variety (Nicotiana tabacum cv. G28), were kindly provided by Conagen Inc (Bedford, MA, USA) and University of Science and Technology of China (Hefei, Anhui, China), respectively.
End-to-end PCR
The CsF3′5′H gene from the NCBI database was subjected to standard end-to-end PCR reactions, with the primers designed according to the cDNA sequence (synthesized by Invitrogen, Shanghai, China; Additional file 2: Table S1). The cDNA strands for end-to-end PCR were synthesized with Phusion® High-Fidelity DNA Polymerase (New England Biolabs, USA). PCR products were gel purified using the MiniBEST Agarose Gel Extraction Kit (Takara, DaLian, China), ligated into a pMD18-T vector, and transformed into E. coli DH5α competent cells for sequencing. The results were assembled using DNAMAN 7 software (Lynnon, Canada). Briefly, end-to-end PCR was performed under the following conditions: 98°C for 30 s, 30 cycles at 98°C for 30 s, 58°C for 10 s, 72°C for 40 s, and a final extension at 72°C for 10 min.
Validation of expression by qRT-PCR
Total RNA was isolated from Camellia sinensis organs with RNAiso Plus (Takara, DaLian, China) and RNAiso-mate for Plant Tissue (Takara, DaLian, China), according to the manufacturers’ instructions.
All primers were blasted against the NCBI database to guarantee specificity. Values were normalized against the expression levels of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in tea plant [21] and actin in tobacco [38]. The first strand cDNA samples for qRT-PCR were synthesized with the PrimeScript® RT reagent Kit (Takara, DaLian, China). The PCR mixture contained cDNA template (approximately 0.01 μg/μL), 10 μL SYBR Green PCR Master Mix (Takara), and 200 nmolL−1 of each gene-specific primer in a final volume of 20 μL. Real-time PCR was performed using a CFX96™ optical reaction module (Bio-Rad, USA) as follows: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s (58°C for 30 s for root) in 96-well optical reaction plates. The amplification specificity was verified by melting curve analysis (55–95°C). Data were expressed as mean value of three replicates, normalized against the expression levels of GAPDH or actin. The relative expression was derived by the 2-ΔΔCt method. △CT = CT,target -CT,internal standard, −△△CT = −(△CT, target -△CT, control), where CT,target and CT,internal standard are cycle threshold (CT) values for targets and housekeeping genes, respectively.
Transformation of tobacco plants with CsF3′5′H transgenes
The Gateway® Cloning System was used to construct the vectors provided by Prof Xiang [39] of the University of Science and Technology of China. CsF3′5′H PCR products were obtained by end-to-end PCR and ligated into pMD18-T vectors. The CsF3′5′H - pMD18-T plasmids were amplified in E. coli strain DH5α and used as PCR templates. The PCR primer pairs for linking the attB adaptors are listed in Additional file 2: Table S1. PCR products were purified, transferred to pMD18-T and confirmed by sequencing. The correct plasmid was cloned into the entry vector pDONR207 by Gateway® BP Clonase® Enzyme mix according to the manufacturer’s instructions (Invitrogen, USA). The resulting entry pDONR207- clones were selected on gentamycin plates and validated by restriction enzyme digestion. Entry vectors were then transferred into the Gateway plant transformation destination vector pCB2004 using Gateway® LR Clonase™ (Invitrogen, USA). Recombinant colonies pCB2004-CsF3′5′H and control pCB2004 vectors were selected on kanamycin plates and validated by restriction enzyme digestion, followed by transformation into EHA105 by electroporation at 2500 V for about 5.5 ms.
A single colony containing each target construct was confirmed by PCR and used for genetic transformation of tobacco. EHA105-pCB2004-CsF3′5′H and EHA105-empty pCB2004 were inoculated in liquid LB medium containing 50 mg/L kanamycin and 50 mg/L spectinomycin. Cells were allowed to grow in the dark at 28°C, for 20–22 h at 200 rpm to OD600 = 0.6, then pelleted by centrifugation (6000 rpm, 10 min) followed by two washing steps with liquid MS medium containing 100 μmol/L acetosyringone (Sigma, R40456). The leaf disc approach was used for tobacco transformation, with 25 mg/L phosphinothricin selection [40].
Construction of the yeast strain Saccharomyces cerevisiae‘WAT11’ vector for CsF3′5′H expression
PCR products of VvF3′5′H, FS, FSII, FSIII were obtained by end-to-end PCR, gel purified, and ligated into pENTR™/TEV/D-TOPO vectors using Top cloning (pENTR™/TEV/D-TOPO® Cloning Kits, Invitrogen, USA). Then, the entry vectors pENTR-VvF3′5′H, pENTR-CzyF3′5′H-1, pENTR-CzyF3′5′H-2, and pENTR-CzyF3′5′H-3 were cloned into the destination vector pYES-dest52 using Gateway® LR Clonase™ enzyme (Invitrogen, USA). The resulting pYES-dest52-VvF3′5′H, pYES-dest52-FSI, pYES-dest52-FSII, and pYES-dest52-FSIII were transformed into Saccharomyces cerevisiae WAT11 with Frozon-EZ yeast Transformation II™ (Zymo Research, USA).
Yeast cells were propagated at 28°C for 12 h in 10 ml SD-U liquid medium containing 20 g/l glucose, by inoculation of a single colony from a SGlu plate. The thalli collected were transferred into 10 ml SD-U medium containing 20 g/l galactose, and grown at 28°C for 5 h.
For substrate specificity experiments, N, E, DHK, DHQ, K, and Q were separately added into the yeast culture to a final concentration of 5 μM, and incubated at 28°C for 10 h. Reactions were terminated by sonication for 15 min and addition of ethyl acetate. Products from each reaction were extracted three times with 10 ml ethyl acetate, evaporated and re-dissolved in 150 μl methanol for HPLC analysis at 280–370 nm.
Microsome preparation
Protein synthesis was indiced in the yeast culture by the addition of galactose and the microsomal yeast fraction was prepared with MgCl2 as described by Olsen et al. [11]. Protein quantities were estimated according to the Bradford method. The microsome was dissolved in 1.0 to 1.5 ml TEG (30% glycerol in 50 mM Tris–HCl with 1 mM EDTA) on ice. All buffers/solutions and centrifuge were pre-cooled to 4°C.
Enzyme extraction from Camellia sinensis
About 2 g of tea leaves were homogenized under liquid nitrogen, and total protein was extracted with 0.1 molL−1 phosphate-buffered saline (PBS, pH 7.4) containing an equivalent amount of PVPP, then centrifuged at 15000 g for 10 min at 4°C. The supernatants were used to assess F3′5′H activity. Protein concentrations of enzyme extract were determined by spectrometric analysis using Coomassie Brilliant Blue G-250.
Enzyme assays
All enzyme assays were carried out in phosphate buffer. In the multi-enzyme incorporative reaction system, the F3′5′H assay solution was incubated at 28°C for 30 min (for microsomes) or 1 h (for crude enzyme extract) in 100 mM phosphate buffer (pH 7.0) containing 1 mM NADPH, 1–300 μM substrates. Enzyme reactions were terminated by adding ethyl acetate. Products from each reaction were extracted three times with an equal volume ethyl acetate, evaporated and re-dissolved in 500 μl methanol for HPLC analysis at 280–370 nm.
Flavonoid pigment preparation and analyses
Anthocyanin aglycones were extracted with 1.6 ml methanol containing 20% water from about 500 mg frozen tobacco flowers. After centrifugation at 6,000 g at 4°C for 5 min, supernatants were extracted three times by equal volume of ethyl acetate, and the extracts were added to 1/3 volume of 4 M HCl aqueous solution for acid-hydrolysis by heat treatment at 90°C for 1 h. Hydrolysates were tested by HPLC at 530 nm.
HPLC and MS analyses
Mass spectra were acquired using the electrospray ionization in the negative ionization modes at fragmentation voltages of 175 V over the range of m/z 100 to 2000 on the UPLC-QQQ-MS/MS (Waters 2478, Waters Instruments) with drying gas flow of 12 L min−1, a drying gas temperature of 350°C, a nebulizer pressure of 35 psi, and capillary voltages of 3500 V.
The HPLC consisted of a quaternary pump with a vacuum degasser, thermostatted column compartment, autosampler and diode array detector (DAD). A Phenomenex Synergi 4u Fusion-RP80 column (5 μm, 250*4.6 mm) was used at a flow rate of 1.0 mL min−1. The column oven temperature was set at 25°C. The mobile phase consisted of 1% acetic acid in water (A) and 100% acetonitrile (B). The gradient increased linearly from 0 to 10% B (v/v) at 5 min, to 15% B at 15 min, 40% B at 20 min, 60% B at 22 min, and maintained at 10% B to 25 min. The DAD was set at 280 and 340 nm for real-time monitoring of the peak intensities. Ultraviolet (UV) spectra were recorded continuously from 200 to 600 nm for plant component identification.
Among the standards used, N, E, P, DHK, DHQ, and DHM were quantified at 280 nm, whereas K, Q, and myricetin (M) were quantified at 365 nm. All products, except P, were identified and quantified by mass spectrums (MS) and peak area compared with standards. Since standard samples of 5, 7, 3′, 4′, 5′-pentahydroxyflavanone were unavailable, P was identified with LC-MS, and its relative concentration was quantified using E as the molar equivalent. All samples were run in triplicate for both quantitation and multivariate statistical analysis.
Bioinformatics and statistical analyses
The phylogenetic tree was constructed using protein sequences from several plant F3′5′H, F3′H, and Cinnamic acid 4-hydroxylase (C4H) enzymes retrieved from the NCBI database by ClustalW of MEGA5 (accession numbers are given in the phylogenetic tree, Figure 2). The phylogenetic tree was constructed according to the neighbor-joining method. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The evolutionary distances were computed using the p-distance method. Evolutionary analyses were conducted in MEGA5 (web page: http://www.megasoftware.net/).
Data were presented as the mean ± SD of three independent measurements. The statistical significance of differences between groups was determined with Student’s t-test using SPSS software (SPSS, Chicago, IL, USA). P < 0.05 was considered statistically significant.
Supporting data
The data set(s) supporting the results of this article is (are) included within the article (and its additional file(s)). The cDNA and protein sequences from several plant F3′5′H, F3′H and C4H enzymes retrieved from the NCBI web page (http://www.ncbi.nlm.nih.gov/).