Background

Chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate using NADPH. To be active, this enzyme needs to be reduced by thioredoxins (TRX), ubiquitous thiol-disulfide oxidoreductases (Issakidis et al., 1994; Collin et al., 2003). In C3 plants, NADP-MDH is involved in the export of reducing power from the chloroplast to the cytosol via the malate valve (Scheibe, 2004). TRX-dependent activation of NADP-MDH makes the link between the chloroplast electron transport chain, the redox state of the chloroplast, and the other cell compartments (Scheibe and Dietz, 2012; Heyno et al., 2014). Hence, the redox state of NADP-MDH is considered as a proxy for the plant leaf cellular redox state. NADP-MDH redox state in protein preparations or in plant extracts is deduced from the ratio between initial/extractable (i.e., activity of the enzyme or extract, without pre-treatment) and maximal activity/enzyme capacity (i.e., activity of the enzyme or in the extract, after reductive activation by TRX) (Issakidis et al., 1994; Keryer et al., 2004).

NADP-MDH activity can be easily assayed spectrophotometrically by monitoring oxaloacetate-dependent NADPH oxidation at 340 nm (Jacquot et al., 1995). Here, we developed a plate-adapted protocol to assay NADP-MDH activity of a large number of samples at the same time, associated with a semi-automatized data analysis procedure using a user-friendly R script. Compared with the classic method in a 1 mL spectrophotometer cuvette, the plate format allows increasing the experimental replicates and/or tested samples or conditions, for a gain of time (estimated divided by three), and in data precision and reliability, at a lower cost (divided by five). We implemented this method to measure NADP-MDH activity in Arabidopsis leaf protein extracts and using purified preparations of recombinant sorghum NADP-MDH. Our method is applicable to measure the activity of virtually any plant species.