Anthocyanins

The analysis was previously described by Sokół-Łętowska et al. [51 (link)]. The HPLC-PDA analysis was performed using a Dionex (Germering, Germany) system equipped with the diode array detector model Ultimate 3000, quaternary pump LPG-3400A, autosampler EWPS-3000SI, thermostated column compartment TCC-3000SD, and controlled by Chromeleon v.6.8 software (Thermo Scientific Dionex, Sunnyvale, CA, USA). The Cadenza Imtakt column C5-C18 (75 × 4.6 mm, 5 μm) was used. The mobile phase was composed of solvent C (4.5% aq. formic acid, v/v) and solvent D (100% acetonitrile). The elution system was as follows: 0–1 min 5% D in C, 20 min 25% D in C, 21 min 100% D, 26 min 100% D, 27 min 5% D in C. The flow rate of the mobile phase was 1.0 mL/min and the injection volume was 20 μL. The column was operated at 30 °C. Iridoids were detected at 245 nm, flavan-3-ols at 280 nm, phenolic acids and their derivatives at 320 nm, flavonols, flavanonols, flavones and flavanones at 280 and 360 nm, and anthocyanins at 520 nm.
Loganic acid and its derivatives were expressed as mg of loganic acid equivalents (LAE) per 100 g fresh weight (fw), loganin, sweroside and their derivatives as loganin equivalents (LoE) per 100 g fw, anthocyanins as cyanidin 3-O-glucoside equivalents (CygE) per 100 g fw, derivatives of quercetin and taxifolin as quercetin 3-O-glucoside equivalents (QgE) per 100 g fw, luteolin -O-dihexoside-hexoside as luteolin 7-O-glucoside equivalents (LgE) per 100 g fw, caffeoylquinic acids as mg of 5-O-caffeoylquinic (chlorogenic) acid equivalents (ChAE) per 100 g fw. Solutions of standards (1 mg/ml) were dissolved in 1 mL of methanol. The appropriate amounts of stock solutions were diluted with 50% aqueous methanol (v/v) acidified with 1% HCl in order to obtain standard solutions. Analytical characteristics for determination of phenolic compounds and iridoids are shown in Table S3.

Catechins and caffeine were extracted from the samples according to the method described by Shan et al. [46 ] with minor modifications. Briefly, 0.1 g of freeze-dried tea leaf tissue was ground in liquid nitrogen with a mortar and pestle and extracted with 3 mL 80 % methanol in an ultrasonic sonicator for 10 min at 4 °C. After centrifugation at 6,000 rpm for 10 min, the residues were re-extracted twice as described above. The supernatants were combined and diluted with 80 % methanol to a volume of 10 mL. The obtained supernatants were filtered through a 0.22 μm organic membrane before HPLC analysis.
The catechin and caffeine contents in the extracts were measured using a Waters 2695 HPLC system equipped with a 2489 ultraviolet (UV)-visible detector. A reverse-phase C18 column (Phenomenex 250 mm × 4.6 mm, 5 micron) was used at a flow rate of 1.0 mL/min. The detection wavelength was set to 278 nm, and the column temperature was 25 °C. The mobile phase consisted of 0.17 % (v/v) acetic acid (A) in water, 100 % acetonitrile (B), and the gradient elution was as follows: B 6 % from 0 to 4 min, to 14 % at 16 min, to 15 % at 22 min, to 18 % at 32 min, to 29 % at 37 min, to 45 % at 45 min, to 45 % at 50 min, to 6 % at 51 min and to 6 % at 60 min. Then, 10 μL of the filtrate was injected into the HPLC system for analysis. The filtered sample (10 μL) was injected into the HPLC system for analysis. Samples from each stage of leaf development were analyzed in triplicate.
Amino acids were extracted with hot water [47 , 48 (link)]. Specifically, 0.15 g of freeze-dried tea leaves was ground in liquid nitrogen with a mortar pestle and extracted with 5 mL deionized water for 20 min in a water bath at 100 °C. After centrifugation at 6,000 rpm for 10 min, the residues were re-extracted once as described above. The supernatants were combined and diluted with water to a volume of 10 mL. The supernatants were also filtered through a 0.22 μm membrane before HPLC analysis. Theanine in tea was detected using a Waters 600E series HPLC system equipped with a quaternary pump and a 2489 ultraviolet (UV)-visible detector. A reverse-phase C18 column (Phenomenex 250 mm × 4.6 mm, 5 micron) was used at a flow rate of 1.0 mL/min. The column oven temperature was set to 25 °C. The detection wavelength was set to 199 nm for analysis [49 ]. The mobile phase consisted of 0.05 % (v/v) trichloroacetic acid (A) in water, 50 % acetonitrile (B), and the gradient elution was as follows: B 0 % (v/v) to 100 % at 40 min, to 100 % at 45 min and to 0 % at 60 min [31 ]. Then, 5 μL of the filtrate was injected into the HPLC system for analysis.
Amino acids in tea were detected using a Waters 600E series HPLC system equipped with a quaternary pump, a 2475 fluorescence detector and a 2489 ultraviolet (UV)-visible detector. The Waters AccQ•Tag method [50 (link)] with a Waters AccQ•Tag column (Nova-Pak C18, 4 μm, 150 mm × 3.9 mm) was employed to detect various amino acids according to the protocol of the AccQ•Fluor Reagent Kit [51 , 52 (link)]. To determine the linearity of the chromatographic techniques, calibration plots of standards were constructed based on peak areas (y) using solutions of various concentrations (x). All plots were linear in the examined ranges; the linear ranges for different concentrations of standard compounds are shown in the plots (μg mL⁻¹). The R² value refers to the correlation coefficient of the equation for calculating the content of a compound. The standard compounds C, EC, EGC, ECG, EGCG, GC, theanine and caffeine were purchased from Shanghai Winherb Medical Technology, Ltd., China.
Anthocyanin was extracted as follows: 0.1 g freeze-dry tea leaf tissue was ground in liquid nitrogen and extracted with 5 mL extraction solution (80 % methanol: 1 % hydrochloric acid [HCl]) using an ultrasonic sonicator for 10 min at room temperature. After centrifugation at 6,000 rpm for 10 min, the residues were re-extracted twice as described above. The supernatants were combined and diluted with extraction solution to 10 mL, followed by extraction with trichloromethane. The anthocyanin content was determined by colorimetry at 525 nm [53 (link)].

The RNA-seq data were used to perform co-expression network analysis using R language (v4.2.1). In order to calculate the adjacent order function formed by the gene network and the difference coefficients of different nodes, the TOM similarity algorithm calculates the co-expression correlation matrix to express the gene correlation in the network. The correlation network diagram was drawn by extracting the non-weight coefficients (weight) of anthocyanin-related genes in the matrix. STRING software (https://version-11-5.string-db.org/) was used to reveal a co-expression plot [33 (link), 70 (link)].

The total anthocyanin content was determined in accordance with the method of Xu et al. (2017) (link) using functional leaves (the third to fifth leaves from the main branches) of two-year-old seedlings of QHP, ZSY, and L2025.