Protein phosphorylation activates the guard cell Ca²⁺ channel and is a prerequisite for gating by abscisic acid

Abstract

SummaryProtein phosphorylation and cytosolic-free [Ca 2þ ] ([Ca 2þ ] i ) contribute to signalling cascades evoked by the water-stress hormone abscisic acid (ABA) that lead to stomatal closure in higher-plant leaves. ABA activates an inward-rectifying Ca 2þ channel at the plasma membrane of stomatal guard cells, promoting Ca 2þ entry by shifting the voltage-sensitivity of the channels. Because many of these effects could be mediated by kinase/phosphatase action at the membrane, we examined a role for protein (de-)phosphorylation in plasma membrane patches from Vicia guard cells. Ca 2þ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K 1/2 , 1.3 AE 0.7 mM) but not the non-hydrolyzable analog ATPcS. ABA activation of the channel required the presence of ATP and like ABA, the 1/2 A-type protein phosphatase antagonists okadaic acid (OA) and calyculin A (CA) enhanced Ca 2þ channel activity by increasing the open probability and number of active channels. Neither ATP nor the antagonists affected the mean open lifetime of the channel, suggesting an action through changes in closed lifetime distributions. Like ABA, OA and CA shifted the voltage-sensitivities of the Ca 2þ current and [Ca 2þ ] i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca 2þ ] i rise evoked by hyperpolarization and delayed its recovery. These results demonstrate a membrane-delimited interaction between 1/2 A-type protein phosphatase(s) and the Ca 2þ channel or associated proteins, and they are consistent with a role for protein (de-)phosphorylation in ABA signalling mediated directly through Ca 2þ channel gating that leads to [Ca 2þ ] i increases in the guard cells.

Control of Guard Cell Ion Channels by Hydrogen Peroxide and Abscisic Acid Indicates Their Action through Alternate Signaling Pathways

“…In the presence of 500 m H 2 O 2 , the open probability (P o ) increased from 0.0004 Ϯ 0.0002 to 0.03 Ϯ 0.01 and the number of channels rose 6 Ϯ 2-fold (n ϭ 6). Analysis of open and closed lifetime distributions from isolated patches with a single channel in H 2 O 2 indicated open ( o , 0.8 Ϯ 0.03 and 3.5 Ϯ 0.4 ms) and closed ( c , 1.1 Ϯ 0.3, 6 Ϯ 3, and 223 Ϯ 39 ms) lifetimes comparable with those obtained previously in ABA (Hamilton et al, 2001;Kö hler and Blatt, 2002). Like ABA, H 2 O 2 had no measurable effect on single channel amplitude (Hamilton et al, 2000; see also Fig.…”

“…Qualitatively, H 2 O 2 action on the Ca 2ϩ channel was comparable with that of ABA and phosphatase antagonists (Hamilton et al, 2000;Kö hler and Blatt, 2002), arising from increases in open probability and a recruitment of "cryptic" channels (compare with Kö hler and Blatt, 2002). In the presence of 500 m H 2 O 2 , the open probability (P o ) increased from 0.0004 Ϯ 0.0002 to 0.03 Ϯ 0.01 and the number of channels rose 6 Ϯ 2-fold (n ϭ 6).…”

“…1) under the same conditions we used previously to characterize its response to ABA and protein phosphorylation (Hamilton et al, 2000;Kö hler and Blatt, 2002). At a voltage of Ϫ150 mV, activation by H 2 O 2 occurred both in the cell-attached configuration (Fig.…”

Biochemical and Physiological Changes in Rice Plants Due to the Application of Herbicides1

“…The increase in the levels of O 2 -* due to environmental stress lead to the formation of other oxygen reactive species, such as * OH (Halliwell, 2006). Once accumulated in the cells, H 2 O 2 activate the calcium passage channels in the vacuole membrane, increasing its concentration in cytosol (Kohler & Blatt, 2002), which leads to the depolarization of the guard cells, potassium efflux, turgor loss and, as a consequence, it can cause the closing of stomata.…”

Roles of ion channels and transporters in guard cell signal transduction

“…In isolated GC membrane patches, ATP or type 1/2A phosphatase inhibitors prolong Ca 2+ channel activity, suggesting protein phosphorylation as a proximate regulator of these channels [123]. Dominant negative mutations in PP2C-type protein phosphatases abi1 and abi2 disrupt ABA-activation of Ca 2+ channels [17,124].…”