Vacuolar H⁺-ATPase Is Required for Efficient Vacuolar Phosphate Storage and Systemic Pi Homeostasis in Arabidopsis

The vacuolar H⁺-ATPase (V-ATPase) plays a crucial role in facilitating nutrient ions storage in vacuoles, whereas its direct impact on vacuolar phosphate (Pi) accumulation has not been fully elucidated. Previous research revealed that the absence of VPT1 and VPT3, two major vacuolar Pi influx transporters, significantly affected vacuolar Pi storage. This study shows that disrupting V-ATPase function could mimic the vpt1 vpt3 mutant phenotypes. The vha-a2 a3 mutant, lacking V-ATPase activity, had lower vacuolar Pi levels, higher cytoplasmic Pi and increased resistance to As(V) toxicity under sufficient Pi conditions. Complementation assays in Pi transport-deficient yeast confirmed that high pH suppressed VPT1 activity, while overexpressing VPT1 couldn't overload Pi in vacuoles of vha-a2 a3 mutants. These data illustrate the reliance of VPT1's activity on V-ATPase-generated proton gradients. Furthermore, we find V-ATPase activity correlates positively with Pi availability, and varying across developmental stages. During flowering, V-ATPase activity decreases to enhance Pi allocation in xylem sap for long-distance transport when external Pi is replete, akin to the vpt1 vpt3 mutant. Thus, V-ATPase could cooperate with VPT proteins to regulate Pi homeostasis at both subcellular and systemic levels.