Figure 5.

Reduction of tonoplast V-ATPase activity in cbl2 cbl3 double mutant results in alkalinization of vacuolar pH. (A) The cbl2 cbl3 double mutant phenocopies small size and leaf-tip necrosis of the vha2-a2 vha-a3 double mutant. Scale bar = 10 mm. (B) Vacuolar H⁺-ATPase (left panel) and H⁺-PPase (right panel) hydrolytic activity was determined from microsomal membranes of 4-week old plants of wild-type (Col-0), cbl2, cbl3, cbl2 cbl3 and vha-a2 vha-a3. Results are shown as percentage of the Col-0 control activity. Values are mean ± SE of at least three replicate experiments. (C) The images show emission intensities of mesophyll protoplast vacuoles loaded with BCECF at 488 nm (the first column, red) and 458 nm (the second column, green). The ratio images indicate an increased vacuolar pH in cbl2 cbl3 mutant than in wild-type (the third column). Pseudo-color scale on the right indicates intensity of fluorescence in which yellow and red represent minimum and maximum intensity, respectively. Scale bar = 10 μm. (D) Quantification of the luminal pH in wild-type Col-0 and cbl2 cbl3 double mutant vacuoles. Error bars represent SE of 60 measurements from 15 different intact vacuoles. (E) ATP-dependent H⁺ transport into purified tonoplast vesicles from Col-0 and cbl2 cbl3 plants was monitored by the quenching of quinacrine fluorescence. Results are mean ± SE of at least three replicates. Asterisks indicate statistically significant difference between wild-type Col-0 and cbl2 cbl3 mutant (Student's t-test, ^*P < 0.05).