Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells

Abstract

myo-Inositol hexakisphosphate (InsP6) is the most abundant inositol phosphate in cells, yet it remains the most enigmatic of this class of signaling molecule. InsP6 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 InsP6 levels in guard cells are elevated in response to ABA, and InsP6 inactivates the plasma membrane inward K ؉ conductance (IK,in) in a cytosolic calciumdependent 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 ICa in patch-clamped protoplasts in whole cell mode shows that InsP6 has no effect on the calcium-permeable channels in the plasma membrane activated by ABA. The InsP6-mediated inhibition of IK,in can also be observed in the absence of external calcium. Thus the InsP6-induced increase in cytoplasmic calcium does not result from calcium influx but must arise from InsP6-triggered release of calcium from endomembrane stores. Measurements of vacuolar currents in patch-clamped isolated vacuoles in whole-vacuole mode showed that InsP6 activates both the fast and slow conductances of the guard cell vacuole. These data define InsP6 as an endomembrane-acting calciumrelease signal in guard cells; the vacuole may contribute to InsP6-triggered Ca 2؉ release, but other endomembranes may also be involved.

Death Don't Have No Mercy and Neither Does Calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and Innate Immunity

“…In each experiment, a minimum of three epidermal peels was used as treatment replicates (as was also done for the experiments shown in Figure 3). block I Ca (Lemtiri-Chlieh et al, 2003). A critical objective of the studies included in this report is to link Ca 2þ conductance through plasma membrane ion channels to (downstream) generation of NO in a signal cascade.…”

“…Application of cAMP to the bath solution activates an inward Ca 2þ (as is convention, Ba 2þ is used as the charge carrier in these patch clamp recordings; Gelli and Blumwald, 1997;Peng et al, 2005) current in wild-type Arabidopsis guard cell protoplasts. We have characterized this ligand-gated inward Ca 2þ channel current in guard cell (for characterization of the guard cell Ca 2þ current, see Lemtiri-Chlieh et al, 2003) and mesophyll cell protoplasts prepared from leaves of wild-type Arabidopsis plants previously (Lemtiri-Chlieh and Berkowitz, 2004). In this prior work, the cAMPactivated current was completely blocked by 50 mM Gd 3þ , and reversal potential (E rev ) of currents recorded in the patch configuration was near the Nernst equilibrium potential of Ba 2þ (E Ba ) and far away from E K and E Cl , evidence consistent with our assertion that the ligand activates a Ca 2þ -conducting channel.…”

“…Guard cell protoplasts were isolated from leaves of 5-to 6-week-old Arabidopsis or 3-week-old V. faba plants exactly as described previously (Lemtiri-Chlieh et al, 2003;Lemtiri-Chlieh and Berkowitz, 2004). After enzymatic digestion of abaxial epidermal peels of leaf tissue and centrifugation purification, guard cells were kept on ice in 1 to 2 mL of medium containing 0.42 M mannitol, 10 mM MES, 200 mM CaCl 2 , 2.5 mM KOH (pH 5.5 and osmolality at 466 mOsm/kg) and used for patch clamp recordings for several hours after isolation.…”

IP3 receptors: the search for structure

“…Closely related sequences (typically , 70% amino acid identity) [6] ( Figure 1a) are expressed in all animals, from starfish to human, and in almost all cell types [5]. Despite many reports of IP 3 -stimulated Ca 2þ release in plants [7], the genomes of fungi and plants lack similar sequences; recent work suggests that IP 6 , rather than IP 3 , might be the physiological regulator of Ca 2þ release in plants [8]. Ryanodine receptors (RyRs), another family of intracellular Ca 2þ channels [9], are the closest relatives of IP 3 Rs, sharing many structural and functional characteristics with them.…”

Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase from Maize: Molecular and Biochemical Characterization

“…During this process, phytic acid appears to act as an endomembrane calcium-releasing signal (Lemtiri-Chlieh et al, 2003). However, the biological significance of phytic acid accumulation in plant seeds remains unclear.…”