Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells

myo-Inositol hexakisphosphate (InsP(6)) is the most abundant inositol phosphate in cells, yet it remains the most enigmatic of this class of signaling molecule. InsP(6) plays a role in the processes by which the drought stress hormone abscisic acid (ABA) induces stomatal closure, conserving water and ensuring plant survival. Previous work has shown that InsP(6) levels in guard cells are elevated in response to ABA, and InsP(6) inactivates the plasma membrane inward K+ conductance (I-K,I-in) in a cytosolic calcium-dependent manner. The use of laser-scanning confocal microscopy in dye-loaded patch-clamped guard cell protoplasts shows that release of InsP(6) from a caged precursor mobilizes calcium. Measurement of calcium (barium) currents I-Ca in patch-clamped protoplasts in whole cell mode shows that InsP(6) has no effect on the calcium-permeable channels in the plasma membrane activated by ABA. The lnsP(6)-mediated inhibition Of I-K,in can also be observed in the absence of external calcium. Thus the InsP(6)-induced increase in cytoplasmic calcium does not result from calcium influx but must arise from InsP(6)-triggered release of calcium from endomembrane stores. Measurements of vacuolar currents in patch-clamped isolated vacuoles in whole-vacuole mode showed that InsP(6) activates both the fast and slow conductances of the guard cell vacuole. These data define InsP(6) as an endomembrane-acting calcium-release signal in guard cells; the vacuole may contribute to InsP(6)-triggered Ca2+ release, but other endomembranes may also be involved.