Figure 3.

VAMP711 negatively regulates PM H⁺-ATPase activity. A, PM H⁺-ATPase activity was measured in Col-0, vamp711-6, vamp711-7, and OE-VAMP711-15/27. Plasma membrane vesicles of Col-0, vamp711-6, vamp711-7, OE-VAMP711-15/27 were isolated from 4-week-old soil-grown plants treated with 200 mm of NaHCO₃ for 3 d. Error bars represent sd; Student’s t test was used to assess statistical significance (P ≤ 0.05). Significant differences were represented by different lowercase letters. The experiment was repeated three times independently. B, Comparison of PM H⁺-ATPase activity from A. C, Net H⁺ fluxes in different plant materials. Five-day-old seedlings of Col-0, vamp711-6, vamp711-7, and OE-VAMP711-15/27 were treated with a buffer (0.5 mm KCl, 0.1 mm CaCl₂, 75 mm NaCl, and 0.03 mm HEPES, pH 7.8) for 30 min, and then the H⁺ fluxes in the root tips was detected by the noninvasive microtest technique. Error bars represent sd (seedling number ≥10); Student’s t test was used to assess statistical significance (P ≤ 0.05). Experiment was repeated three times independently. D, Calculated net H⁺ fluxes from C. Error bars represent SD; Student’s t test was used to assess statistical significance (P ≤ 0.05). Significant differences were represented by different lowercase letters. E, VAMP711 inhibits PM H⁺-ATPase activity in vitro. Fifty nanograms of plasma membrane vesicles from Col-0 was incubated with 500 ng of VAMP711-LS protein for 15 min at room temperature. Error bars represent SD; Student’s t test was used to assess statistical significance (P ≤ 0.05). Significant differences were represented by different lowercase letters. The experiment is described in the “Materials and Methods” and repeated three times independently. F, Comparison of PM H⁺-ATPase activity from E. G, VAMP711 inhibits the PM H⁺-ATPase activity of AHA2 in the RS72 yeast strain. VAMP711 and an empty vector were transferred into the RS72 yeast strain containing AHA2. The endogenous H⁺-ATPase gene in RS72 was induced by Gal (gal). When AHA2 and VAMP711 were transferred into the RS72 yeast strain, the growth of the yeast depended on AHA2 activity on Glc (glu) medium. Photographs were taken after 3 to 5 d of growth on the indicated medium. Panels show yeast serial half dilutions. Experimental details are provided in the “Materials and Methods.” The experiment was repeated at least three times independently.