Lander-type stress bomb (Soil Moisture Gear Corp., Santa Barbara, CA, USA). Fresh and dry weightarea (LA) was of recovery period, in remedy, also as in manage plants. Total leaf of both, WT and flacca genotypes wasareameter (LI-COR, Lincoln, NE, USA), and specific leaf location was carried out by LI-3100 determined upon all three drought episodes and immediately after 3-days of recovery period, inthe equation: SLA = Leafcontrol plants. measurements (LA) performed calculated applying treatment, as well as in area/DW. All Total leaf location have been was performed by LI-3100 areameter per genotype and therapy. and specific leaf area with four distinctive plants (LI-COR, Lincoln, NE, USA), was calculated utilizing the equation: SLA = Leaf area/DW. All measurements have been performed with4.3. Extraction and Evaluation of Abscisic Acid Content 4 distinctive plants per genotype and remedy. Determination of abscisic acid (ABA) content inside the tomato leaves was performed as 4.three. Extraction and Analysis of Abscisic Acid 2020 [51]. ABA concentration was measured utilizing indirect described in Zivanoviet al., Content c Determination of abscisic acid (ABA) content inside the tomato leaves was monoclonal antibody for enzyme-linked immunosorbent assay (ELISA) with MAC 252 performed as described inABA (John Innes Centre, Colney, Norwich, UK).was measured using measured at 405 nm Zivanovi et al., 2020 [51]. ABA concentration Plate contents were indirect enzyme-linked a Scaffold Library MedChemExpress microplate reader (Sunrise, Tecan, Switzerland). by immunosorbent assay (ELISA) with MAC 252 monoclonal antibody for ABA (John Innes Centre, Colney, Norwich, UK). Plate contents were measured at 405 nm 4.four. Determination of Tecan, Switzerland). by a microplate reader (Sunrise, Leaf Proline Content material As a way to decide proline content, frozen leaf samples were homogenized in liquid nitrogen, extracted in three (w/v) sulfosalicylic acid and centrifuged at 14,000g for ten min at 4 C. The obtained supernatant was mixed with acidic ninhydrin and glacial acetic acid (1:1:1, v/v/v) and incubated for 60 min on 100 C. The reaction mixture was placed on ice and extracted with toluene (1:1, v/v). The toluene fraction was made use of for determination of proline by measuring absorbance at 520 nm, with toluene as blank [121]. four.five. Determination of Total Leaf Ascorbate Content and Ascorbate Redox State The frozen leaf tissues have been homogenized in 1.five meta-phosphoric acid with 2 mM EDTA and centrifuged at 14,000g for 8 min at four C. The lowered form of ascorbate was measured as outlined by Morina et al. [122]. Briefly, ascorbate (Asc) concentration was determined as absorbance decreased at 265 nm after adding one WZ8040 Epigenetic Reader Domain particular unit of ascorbatePlants 2021, 10,14 ofoxidase (Sigma-Aldrich, Darmstadt, Germany) inside the reaction mixture consisting of 300 mM potassium phosphate buffer (pH 5.5) and sample. Determination of the total ascorbate content material was performed based on Vidovic et al. [123] with some modifications. In order to identify total Asc, the samples were diluted 8 times and incubated with 2.five U ascorbate oxidase in potassium phosphate buffer (pH four.5) for 1 min to complete Asc oxidation. Immediately after that, reaction mixture was treated with potassium hydroxide to achieve pH eight and instantly derivatized with ortho-phenylenediamine (o-PDA) for ten min in the dark. Reaction was stopped with 85 H3 PO4 and samples obtained were loaded on a reversedphase C18 column (5.0 , 250 4.six mm Luna C18 (2); Phenomenex Ltd., Torrance, CA, USA) utilizing the Shimadzu LC-20AB.