Folin phenol reagent

Alkaline protease activity was measured by Folin-Ciocalteu method described previously^{59 (link)} with slight modification. The suitably diluted enzyme (50.0 μl) was added to 150.0 μl 50.0 mM NaOH-glycine buffer (pH 10.5) containing 2% (w/v) casein and the reaction mixture was incubated at 60 °C for 10 min. The reaction was terminated by adding 200.0 μl 0.4 M trichloroacetic acid (TCA). A blank control was conducted by adding TCA before the enzyme. After centrifuging at 13,000 × g for 10 min, 50.0 μl supernatant was pipetted into another tube with 250.0 μl 0.4 M Na₂CO₃ and 50.0 μl Folin-Phenol reagent (Shanghai Sangon, Shanghai, China). The mixture was then incubated at 40 °C for 20 min and the absorbance at 680 nm was measured. Tyrosine was used as the standard. One unit (U) of protease activity was defined as the amount of enzyme liberating 1.0 μg of tyrosine per minute at 60 °C under the standard assay conditions. All experiments were repeated three times.

Total proteins were extracted from leaves with extraction buffer (10 mM Hepes-KOH, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% [v/v] Triton X-100, 56 mM dithiothreitol, and protease inhibitor cocktail). Protein concentrations were determined using Folin-Phenol Reagent (Sangon Biotech) according to the manufacturer’s protocol. The proteins were separated by 15% SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore). Anti-HHL1 has been described previously (17 (link)), and anti-FLAG was purchased from Sigma. Antibodies against photosynthetic proteins were purchased from Agrisera (Cat.#:D1:AS05084;D2:AS06146;CP43: AS06110;CP47: AS05920;LHCA1: AS01005; ATPB: AS05085;PSBP: AS0614), and the signals were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

On the day of harvest, peach fruits of uniform commercial maturity (fruit hanging time of approximately 75 days) with no disease or mechanical wounding were selected by experienced farmers and transported to the laboratory. In total, 87 peach fruits were obtained. Sulfuric acid was purchased from Wuxi Jiahong Chemical Trading Co. Ltd. Ultrapure water was produced using a Milli‐Q system. Folin‐phenol reagent was purchased from Sangon Biotech Co., Ltd. All other chemicals and reagents used were of the highest analytical grade commercially available.