Protein phosphorylation, Ca2+ channel, ABA and stomata

 

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

by Köhler B., Blatt M. R. (2002)

Barbara Köhler, Michael R. Blatt

in Plant J. 32,185–194. – DOI: 10.1046/j.1365-313X.2002.01414.x – 

Wiley Online Library | PubMed

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313X.2002.01414.x/full

Summary

Protein phosphorylation and cytosolic-free [Ca²⁺] ([Ca²⁺]_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 Ca²⁺ channel at the plasma membrane of stomatal guard cells, promoting Ca²⁺ 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. Ca²⁺ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K_1/2, 1.3 ± 0.7 mm) but not the non-hydrolyzable analog ATPγS.

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 Ca²⁺ 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 Ca²⁺ current and [Ca²⁺]_i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca²⁺]_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 Ca²⁺ channel or associated proteins, and they are consistent with a role for protein (de-)phosphorylation in ABA signalling mediated directly through Ca²⁺ channel gating that leads to [Ca²⁺]_i increases in the guard cells.

 

ABA, Ca2+ and stomatal guard cells

Photo credit: NCBI

P_o is suppressed by micromolar [Ca²⁺]_i. Means ± SE of P_o from mean open times of 100-s recordings at −120 mV (n = 3). Ca²⁺ added on the cytosolic side (inside) during inside-out recordings against a background of 30 mM Ba²⁺ and with 10 mM Ba²⁺ outside. (Insets) Segments of traces at each [Ca²⁺]_i. Data from one patch. Scale: vertical, 1 pA; horizontal, 1 s.

Ca²⁺ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid.

by Hamilton D. W. A.,  Hills A., Kohler B., Blatt M. R. (2000)

in Proc. Natl Acad. Sci. USA, 97, 4967–4972. –

CrossRef | PubMed | CAS | ADS

http://www.ncbi.nlm.nih.gov/pubmed/10781106

Abstract

In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by means of Ca(2+) entry from outside and release from intracellular stores. The mechanism(s) for Ca(2+) flux across the plasma membrane is poorly understood.

Because [Ca(2+)](i) increases are voltage-sensitive, we suspected a Ca(2+) channel at the guard cell plasma membrane that activates on hyperpolarization and is regulated by ABA.

We recorded single-channel currents across the Vicia guard cell plasma membrane using Ba(2+) as a charge-carrying ion. Both cell-attached and excised-patch measurements uncovered single-channel events with a maximum conductance of 12.8 +/- 0.4 pS and a high selectivity for Ba(2+) (and Ca(2+)) over K(+) and Cl(-).

Unlike other Ca(2+) channels characterized to date, these channels rectified strongly toward negative voltages with an open probability (P(o)) that increased with [Ba(2+)] outside and decreased roughly 10-fold when [Ca(2+)](i) was raised from 200 nM to 2 microM. Adding 20 microM ABA increased P(o), initially by 63- to 260-fold; in both cell-attached and excised patches, it shifted the voltage sensitivity for channel activation, and evoked damped oscillations in P(o) with periods near 50 s. A similar, but delayed response was observed in 0.1 microM ABA.

These results identify a Ca(2+)-selective channel that can account for Ca(2+) influx and increases in [Ca(2+)](i) triggered by voltage and ABA, and they imply a close physical coupling at the plasma membrane between ABA perception and Ca(2+) channel control.