Phosphatidylinositol (PtdIns) is a structural phospholipid that can be phosphorylated into various lipid signaling molecules,
designated polyphosphoinositides (PPIs). The reversible phosphorylation of PPIs on the 3, 4, or 5 position of inositol is
performed by a set of organelle-specific kinases and phosphatases, and the characteristic head groups make these molecules
ideal for regulating biological processes in time and space. In yeast and mammals, PtdIns3P and PtdIns(3,5)P2 play crucial
roles in trafficking toward the lytic compartments, whereas the role in plants is not yet fully understood. Here we identified
the role of a land plant-specific subgroup of PPI phosphatases, the suppressor of actin 2 (SAC2) to SAC5, during vacuolar
trafficking and morphogenesis in Arabidopsis thaliana. SAC2-SAC5 localize to the tonoplast along with PtdIns3P, the presumable
product of their activity. In SAC gain- and loss-of-function mutants, the levels of PtdIns monophosphates and bisphosphates
were changed, with opposite effects on the morphology of storage and lytic vacuoles, and the trafficking toward the vacuoles
was defective. Moreover, multiple sac knockout mutants had an increased number of smaller storage and lytic vacuoles, whereas
extralarge vacuoles were observed in the overexpression lines, correlating with various growth and developmental defects.
The fragmented vacuolar phenotype of sac mutants could be mimicked by treating wild-type seedlings with PtdIns(3,5)P2, corroborating
that this PPI is important for vacuole morphology. Taken together, these results provide evidence that PPIs, together with
their metabolic enzymes SAC2-SAC5, are crucial for vacuolar trafficking and for vacuolar morphology and function in plants.