Mp96 readers

Samples were ground in liquid nitrogen to a fine powder using a TissueLyser II (Qiagen). Ascorbate content was assayed using a protocol that takes advantage of the specificity of ascorbate oxidase, adapted from Bergmeyer (1987) , using 40 and 100 mg FW for leaves and fruits, respectively, extracted in 400 µL 0.1 M HCl at 4 °C. For the determination of total ascorbate, 20 µL of extract was first incubated 10 min at 25 °C in 0.15 M HEPES/KOH pH 7.5 and 0.75 mM DTT. N-Ethyl maleimide was added to reach 0.035% w/v. After 10 min, 1 unit.mL⁻¹ of ascorbate oxidase was added. After 20 min, phenazine ethosulfate and thiazolyl blue mix were added for final concentrations of respectively 0.3 and 0.6 mM. The thiazolyl blue mix was prepared as follows: 10 mM thiazolyl blue, 0.2 M Na₂HPO₄, 0.2 M citric acid, 2 mM EDTA, and 0.3% v/v Triton X100 at pH 3.5. All steps were performed in a polystyrene microplate at room temperature. Measurements were performed at 570 nm in MP96 readers (SAFAS, Monaco). For reduced ascorbate, the same protocol was used, except steps involving DTT and N-ethylmaleimide were omitted.