*4.2. Pigments Content Measurement*

The contents of total chlorophyll and carotenoids were determined by spectrophotometric analyses. About 0.1 g leaves (accurately record the weight) were cut into pieces and rinsed with distilled water. Add 1 mL extracting solution (95% ethanol) and 50 mg calcium carbonate powder in mortar, the mixtures were grinded fully in low light conditions and transferred to 10 mL glass test tube. The mortar was rinsed with the extracting solution, all the washing solution was transferred into the glass tube and replenished to 10 mL with the extracting solution. Keep the glass tube in the dark until the tissue is completely whitened. Add 200 μL leach liquor in a 96-well plate, the automatic microplate reader (Sunrise, TECAN, Switzerland) was set to zero by extracting solution, then the absorbance values of 663 nm, 645 nm and 470 nm were determined and recorded as A663, A645 and A470, respectively. The calculation formula is as follows: total chlorophyll content (mg/g) = 0.02 × (20.21 × A6458.02 × A663) × N/M, total carotenoid concentration (mg/g) = 0.02 × [(1000A470–3.27Ca–104Cb)/229] × N/M (N represents dilution multiple, M represents sample fresh quantity).

The content of total anthocyanins was determined by colorimetry method. The sample was dried in the oven (37 ◦C) to a constant weight and crushed. After passing through a 40-mesh sieve, about 0.1 g sample was weighed (accurately recording the weight). Add 1 mL extracting solution (95% ethanol: 1.5 mol/L HCL = 85:15) in samples and the mixtures were extracted at 4 ◦C for 24 h. Then the mixtures were centrifuged at 8000× *g* for 10 min at room temperature, and the supernatant was taken to be tested. The automatic microplate reader (Sunrise, Switzerland) was preheated more than 30min, and reagent A (PH 1.0 buffer, 0.2mol/L KCL : 0.2mol HCL = 25:67) and reagent B (PH 4.5 buffer, 1 mol NaAC : 1 mol HCL: H2O = 100:60:90) were preheated more than 10 min. Add 180 μL reagent A to 20 μL supernatant and let stand for 15 min, then the absorbance values at 530 nm and 700 nm were determined and were recorded as A1 and A2, respectively. Add 180 μL reagent B to 20 μL supernatant and let stand for 15 min, then the absorbance values at 530 nm and 700 nm were determined and were recorded as A3 and A4, respectively. The calculation formula is as follows: total anthocyanin concentration (μg/g) = 33.4 × [(A1 − A2) − (A3 − A4)] × N/M (N represents dilution multiple; M represents sample dry weight, g).

The content of total flavonoids was determined by colorimetry method. The sample was dried in the oven (37 ◦C) to a constant weight and crushed. After passing through a 40-mesh sieve, about 0.1 g sample was weighed (accurately recording the weight). Add 1 mL extracting solution (60% ethanol), the mixtures A were extracted by ultrasonic method (ultrasonic power: 300 W, crushing time: 5 s, interval time: 8 s) at 60 ◦C for 30 min. Then, the mixtures were 12,000 rpm for 10 min at room temperature, and the supernatant A was replenished to 1 mL by extracting solution and taken to be test. The automatic microplate reader (Sunrise, Switzerland) was preheated more than 30 min, set to 470 nm and zero by distilled water. Reagent A (5% nitrous acid), reagent B (10% aluminum nitrate), reagent C (4% sodium hydroxide solution) and standard (10 mg/mL tannic acid standard solution) were prepared. The supernatant was treated according to the instruction manual of the kit for the

determination of total flavonoids in plants (Figure S6). The mixtures B was mixed well and placed in a water bath at 37 ◦C for 45 min. Then the mixtures B was centrifuged at 10,000× *g* for 10 min at room temperature, and 200 uL supernatant B was used to determine absorbance values at A470 in a 96-well plate. The calculation formula is as follows: the flavonoids concentration (mg/g) = C × N × V/M (C represents the content of flavonoids observed in the standard curve, mg/mL; N represents dilution multiple; V represents the total volume of extracting solution, ml; M represents the dry weight of sample, g).

Flavonoids-targeted metabolomics analysis was performed by liquid chromatography-mass spectrometry (LC-MS) at Metware Biotechnology Co.,Ltd (Wuhan, China) as described by [37], with small modifications. Metabolomics analysis includes two parts: metabolomics experiment and data analysis, based on the metabolite data obtained from experimental design, sample collection and processing, metabolite extraction and metabolite detection and analysis. It can carry out the identification of metabolites and the quality control analysis of sample data, and screen out some differential metabolites, so as to predict and analyze the related functions of the metabolites of the samples.
