Ion [35]. The MDA content at 532 nm was calculated by subtracting the absorbance at 600 nm. 2.5. Leaf Photosynthesis, Chlorophyll Fluorescence Parameters, and Chlorophyll Content The net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration price (Tr), and intercellular CO2 concentration (Ci) of the leaves have been measured by the transportable photosynthetic method (li-6400, Li-COR, Lincoln, NE, USA). Leaf photosynthetic parameters had been determined at ten a.m. following the plants had been treated with diverse concentrations of NaCl and treated with diverse concentrations of calcium chloride for one week. The mature leaves had been dark-adapted for 20 min without the need of isolation, plus the fluorescence kinetic parameters at space temperature have been measured applying a portable modulation chlorophyll fluorescence instrument (PAM-2500 Walz, Effeltrich, Germany). For the chlorophyll content material, 0.03 g of fresh leaves had been extracted within a 10 mL pigment extraction resolution containing absolute ethanol and acetone (1:2, v/v) at 25 C for 12 h within the dark. The absorbance in the supernatant at 470, 645, and 663 nm was then measured working with an ultraviolet spectrophotometer. Chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll content have been calculated in accordance with [36]. two.six. Determination of K+ , Na+ , and Ca2+ To ascertain the K+ , Na+ , and Ca2+ ion concentrations, we meticulously washed fresh root, stem, and leaf samples with deionized water, placed them in an oven at 105 C for 20 min, and after that kept the temperature continuous at 80 C till the samples have been absolutely dried. The dried plant samples had been then grounded in a five mL centrifuge tubes applying a high-throughput plant tissue ball milling instrument (Scientz-192, Xinzhi Biotechnology Co., Ltd., Ningbo, China). A total of 0.three g of each and every sample powder was weighed, and 5 mL of nitric acid and 1 mL of perchloric acid were added for wet digestion. The K+ , Na+ , and Ca2+ contents of plant tissue extracts and typical samples (National Institute of Metrology, Beijing, China) had been determined by inductively coupled plasma optical emission spectrometer (ICP-OES; PerkinElmer, Optima 8300, Waltham, MA, USA). The concentration of K+ , Na+ , and Ca2+ is defined as K+ , Na+ , and Ca2+ content (mg) per unit tissue (g) [37]. 2.7. Extraction and LC S Analysis of Phenolic Compounds two.7.1. Chemicals and Reagents UPLC-grade acetonitrile and methanol had been bought from Fisher Scientific (Pittsburgh, PA, USA). All other reagents have been of analytical purity. Ultrapure water was prepared by a Milli-Q method (Millipore, Bedford, MA, USA) water purification program. The reference compounds necessary for the experiment had been all purchased from ChromaDex Inc. (Santa Ana, CA, USA), like p-hydroxycinnamic acid, p-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, genistein, abscisic acid, petunidin, naringenin, hesperidin, quercetin-3-O-rhamnoside, chlorogenic acid, ferulic acid, myricetin, luteolin, catechin, cinnamic acid, p-coumaric acid, hesperetin, quercetin, caffeic acid, L-phenylalanine, naringin, kaempferol, liquiritigenin, isoliquiritigenin, and vanillic acid. The purities of those requirements had been greater than 98 .Agriculture 2021, 11,five of2.7.two. Preparation of Test Sample Remedy Gleditsia Bentiromide Cancer sinensis plant tissues (root, stem, and leaf) treated with unique therapies (CK, S1, S2, S1 + C1, S1 + C2, S1 + C3) had been grounded after which ultrasonically extracted (100 kHz, 40) for 45 min by adding 10 mL of 70 methanol. Immediately after filtration, the.