2.4 External CA activity
The CAext activity was measured as in Fernández et al. (2018) with small modifications, using commercial CA (Sigma, C4396) as a positive control and to check activity linearity (Figure S1). A 50 mL plastic tube was placed inside a container filled with ice that maintained the temperature at 0-4°C. Approximately 60 mg FW leaf was placed in the tube containing 10 mL of buffer (pH 8.5): 50 mM Tris, 2 mM DTT, 15 mM ascorbic acid, 5 mM Na2-EDTA and 0.3% w/v polyvinylpyrrolidone (PVP). Temperature and pH were simultaneously measured using a pH meter. The reaction was started by rapidly introducing 5 mL of ice-cold CO2 saturated water and pH was recorded over time. The relative enzyme activity (REA) was determined using the equation below:
REA= (Tb/Ts)-1 (1)
where Tb and Ts are the times in seconds required for the pH to drop from pH 8.3 to 7.9 in the non-catalyzed (without sample) and catalyzed reactions, respectively. The REA was expressed on a fresh weight basis. In the leaves grown at LC and HC, external CA activity was measured in the presence of 0.1 mM and 0.2 mM AZ as well as 0.3 mM DIDS.