*3.4. Uncoupled PSI and PSII Electron-Flow Determination*

Electron-flow activities were monitored by an oxygen monitor yellow spring instrument model 5300A using a Clark-type electrode. All reaction mixtures were illuminated with filtered light (5 cm filter of 1% CuSO4 solution) from a projector lamp (GAF 2660) at room temperature. For each reaction, a blank experiment was performed with chloroplasts in the reaction medium. Uncoupled PSII from H2O to DCPIP was measured through the reduction of DCPIP-supported O2 evolutions, monitored polarographically. The reaction medium for assaying PSII activity was composed by the same basal electron-transport medium, but in the presence of 1 μM 2,5-dibromo-3-methyl-6 isopropyl-1,4-*p*-benzoquinone (DBMIB), 100 μM DCPIP, and 300 μM K3[Fe(CN)6] and 6 mM NH4Cl [28].

To determine the uncoupled partial reaction of PSII from water to SiMo, solutions of 200 μM of SiMo and 10 μM of DCMU were added to the solution used for the PSII reactions (3 mL), then chloroplasts (20 μg Chl/mL) were added and illuminated for 1 min [29].

Uncoupled PSI electron transport from the reduced DCPIP with sodium ascorbate to MV was determined in a similar form in a basal noncyclic electron-transport medium. However, the following reagents were added: 10 μM DCMU, 100 μM DCPIP, 50 μM MV, 300 μM sodium ascorbate, and 6 mM NH4Cl [30]. All measurements were performed in triplicate and compared to negative control (solvent, dimethyl sulfoxide (DMSO)).
