Enzyme extraction and assay

For enzyme assay, shoots (0.5 g) were homogenized in 50 mM K-P buffer (pH 7.0) containing 100 mM KCl, 1 mM ascorbate, 5 mM β-mercaptoethanol, and 10% (w/v) glycerol using a pre-chilled mortar and pestle. The homogenates were centrifuged at 11,500×g for 15 min and the supernatant was used to determine protein content and enzyme activity. During extraction, all procedures were performed at 0–4 °C.

To determine ascorbate peroxidase (APX, EC: 1.11.1.11) activity, enzyme extract was added to reaction buffer containing 50 mM K-P buffer (pH 7.0), 0.5 mM AsA, 0.1 mM H₂O₂, 0.1 mM EDTA. The decreased absorbance at 290 nm for 1 min was observed and APX activity was calculated using an extinction coefficient of 2800 M⁻¹ cm⁻¹ (Noctor et al. 2016), and expressed as µmol min⁻¹ mg⁻¹ protein.

According to the method of Noctor et al. (2016), monodehydroascorbate reductase (MDHAR, EC: 1.6.5.4) activity was assayed and calculated using an extinction coefficient of 6200 M⁻¹ cm⁻¹, and expressed as nmol min⁻¹ mg⁻¹ protein. Enzyme extract was added to reaction buffer containing 50 mM Tris–HCl buffer (pH 7.5), 2.5 mM AsA, 0.2 mM NADPH.

Dehydroascorbate reductase (DHAR, EC: 1.8.5.1) activity was determined according to the method of Noctor et al. (2016). Enzyme extract was added to reaction buffer containing 50 mM K-P buffer (pH 7.0), 2.5 mM GSH, 0.1 mM EDTA, 0.1 mM DHA. DHAR activity was calculated using an extinction coefficient of 14,000 M⁻1 cm⁻¹, and expressed as nmol min⁻¹ mg⁻¹ protein.

To determine glutathione reductase (GR, EC: 1.6.4.2) activity, enzyme extract was added to reaction buffer containing 20 mM K-P buffer (pH 7.8), 1 mM EDTA, 0.1 mM GSSG, 1.35 mM NADPH. The decreased absorbance at 340 nm for 1 min was observed and GR activity was calculated using an extinction coefficient of 6200 M⁻¹cm⁻¹ (Noctor et al. 2016), and expressed as nmol min⁻¹ mg⁻¹ protein.

Catalase (CAT, EC: 1.11.1.6) activity was assayed as described by Noctor et al. (2016) and calculated using an extinction coefficient of 40 M⁻1 cm⁻¹, and expressed as µmol min⁻¹ mg⁻¹ protein.