Abstract
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.