P from five cucumber seedlings was regarded as a biological replicate, and each and every remedy incorporated 3 replicates. The relative gene expression was calculated making use of the 2- Ct approach [58]. Each primer of CsRBOHs and CsBZRs is presented in Table S1. 2.six. Chlorophyll Fluorescence-Induced Kinetic Curve (OJIP) Measurement The determination with the fast chlorophyll (chl) fluorescence-induced kinetics curve and the calculation of relevant fluorescence parameters had been carried out in line with the solutions of Turan et al. [47] and Di et al. [59]. Just after CS-ACC, cucumber seedling leaves have been placed in darkness having a smaller clip for a minimum of 30 min and then the continuous excitation fluorescence was measured using a Plant Efficiency Analyzer (Handy PEA, Hansatech, UK) [60]. Plants had been measured with 3000 ol -2 -1 pulsed red light, plus the fluorescence signal was recorded from 10 to 1 s at an initial recording speed of 100,000 times per second. The OJIP curve was analyzed using the JIP test as previously described by Turan et al. [47] and Masoj ek et al. [61]. two.7. Measurement of the Electrolytic Leakage; Contents of Proline, H2 O2 , and Chlorophyll; and NADPH Oxidase Activity Measurement on the electrolytic leakage was carried out as outlined by Fang et al. [62]. An FE30 conductivity meter (Mettler Toledo International Co., Ltd., Zurich, Switzerland) was made use of to measure the distilled water (EC3) and conductivity of 0.3 g of cucumber seedling leaves placed in 30 mL of distilled water before boiling (EC1) and right after boiling (EC2), respectively. The following calculation formula was employed: Electrical leakage =(EC1 – EC3) 100 (EC2 – EC3)(2)A Comin kit (Suzhou Comin Biotechnology Co., Ltd., Suzhou, China) was utilized to establish the proline and H2 O2 content material. In total, 0.1 g of fresh cucumber seedling leaves were collected along with the extract was added. Then, the samples were ground and centrifugedAntioxidants 2022, 11,6 ofto get the crude enzyme extract for evaluation in accordance with the directions with the kit. Measurement from the proline content was carried out employing the sulfosalicylic acid assay at OD520 nm [63]. The H2 O2 content material was determined by the TiCl4 precipitation assay at OD415 nm [64].Histone deacetylase 1/HDAC1 Protein custom synthesis The NADPH oxidase activity was determined using an ELISA kit (Jiangsu MEIMIAN Co.Annexin A2/ANXA2, Human , Ltd.PMID:24456950 , Yancheng, China) in accordance with the manufacturer’s directions. Detection on the chlorophyll content was conducted as described by Arnon [65] with minor modifications. The contents of chlorophyll a (Ca), chlorophyll b (Cb), and carotenoid (Cc) were calculated as follows: Ca = 13.95 OD665 – six.88 OD649 Cb = 24.96 OD649 – 7.32 OD665 Cc = two.eight. Statistical Analysis SPSS 18.0 (SPSS, Inc., Chicago, IL, USA) was used to analyze the significance of difference in Turkey HSD (p 0.05) and Student’s t test (p 0.05). The data have been repeated 3 occasions. three. Final results 3.1. Cold Anxiety Induced the Acquisition of Cold Strain Memory in Cucumber Seedlings and Enhanced the Acquired Cold Tolerance A tester cold tension (1 C) was applied for 24 h to decide the cold tolerance of the treatment options. The cold tolerance was significantly enhanced following a 1.five h recovery immediately after induction at ten C (Figure 1A). In contrast, the cold tolerance was considerably decreased following a 48 h recovery period just after induction at 10 C (Figure 1A). Nevertheless, the cucumber seedlings showed stronger cold tolerance following a 48 h recovery soon after cold anxiety acclimation (CS-ACC) that integrated the following regime: 1 h at 10 C, 1.five.
