The flip side of the Arabidopsis type I proton-pumping pyrophosphatase (AVP1): Using a transmembrane H⁺ gradient to...

Energy partitioning and plant growth are mediated in part by a type I H⁺-pumping pyrophosphatase (H⁺-PPase). A canonical role for this transporter has been demonstrated at the tonoplast where it serves a job-sharing role with V-ATPase in vacuolar acidification. Here, we investigated whether the plant H⁺-PPase from Arabidopsis also functions in "reverse mode" to synthesize PP_i using the transmembrane H⁺ gradient. Using patch-clamp recordings on Arabidopsis vacuoles, we observed inward currents upon P_i application on the cytosolic side. These currents were strongly reduced in vacuoles from two independent H⁺-PPase mutant lines (vhp1-1 and fugu5-1) lacking the classical PP_i-induced outward currents related to H⁺ pumping, whereas they were significantly larger in vacuoles with engineered heightened expression of the H⁺-PPase. Current amplitudes related to reverse-mode H⁺ transport depended on the membrane potential, cytosolic P_i concentration, and magnitude of the pH gradient across the tonoplast. Of note, experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing the Arabidopsis H⁺-PPase (AVP1) demonstrated P_i-dependent PP_i synthase activity in the presence of a pH gradient. Our work establishes that a plant H⁺-PPase can operate as a PP_i synthase beyond its canonical role in vacuolar acidification and cytosolic PP_i scavenging. We propose that the PP_i synthase activity of H⁺-PPase contributes to a cascade of events that energize plant growth.