Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells

Abstract

(RS)-2-cis, 4-trans-abscisic acid (ABA), a naturally occurring plant stress hormone, elicited rapid agonist-specific changes in myo-inositol hexakisphosphate (InsP(6)) measured in intact guard cells of Solanum tuberosum (n = 5); these changes were not reproduced by (RS)-2-trans, 4-trans-abscisic acid, an inactive stereoisomer of ABA (n = 4). The electrophysiological effects of InsP(6) were assessed on both S. tuberosum (n = 14) and Vicia faba (n = 6) guard cell protoplasts. In both species, submicromolar concentrations of InsP(6), delivered through the patch electrode, mimicked the inhibitory effects of ABA and internal calcium (Ca(i)(2+)) on the inward rectifying K(+) current, I(K,in), in a dose-dependent manner. Steady state block of I(K,in) by InsP(6) was reached much more quickly in Vicia (3 min at approximately 1 microM) than Solanum (20-30 min). The effects of InsP(6) on I(K,in) were specific to the myo-inositol isomer and were not elicited by other conformers of InsP(6) (e.g., scyllo- or neo-). Chelation of Ca(2+) by inclusion of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or EGTA in the patch pipette together with InsP(6) prevented the inhibition of I(K,in), suggesting that the effect is Ca(2+) dependent. InsP(6) was approximately 100-fold more potent than Ins(1,4,5)P(3) in modulating I(K,in). Thus ABA increases InsP(6) in guard cells, and InsP(6) is a potent Ca(2+)-dependent inhibitor of I(K,in). Taken together, these results suggest that InsP(6) may play a major role in the physiological response of guard cells to ABA.

Figure 1

Activation of inositol phosphate metabolism in guard cells. Inositol phosphates in ³H-labeled guard cells (○), mixed with internal ¹⁴C (no symbol) standards (A) and ³²P (no symbol) standards (B). The traces shown in A and B were from HPLC runs performed on different extracts of ³H-labeled guard cells. The bar graph represents the distribution of label in different inositol phosphates, as a percentage of the total label in InsP₁ to InsP₇, from control (transparent bars) or (RS)-2-cis, 4-trans-abscisic acid-treated (hashed bars) guard cells (C).

Figure 2

InsP₆ and increased Ca_i²⁺ inactivate I_K,in but not I_K,out in Solanum GCPs. Current traces of I_K,in and I_K,out (Left) and corresponding I/V curves (Right) were measured at the indicated times shown above each set of current traces (referring to the time after achieving whole-protoplast configuration). Control experiment carried out with ≈0.1 μM free Ca²⁺ in the patch pipette (A). Typical experiments with pipette solutions containing ≈1.3 μM free Ca²⁺ (B) and those containing 20 μM InsP₆ (with ≈0.1 μM free Ca²⁺) (C).

Figure 3

Stereospecific inactivation of I_K,in by InsP₆ conformers is relieved by buffering Ca_i²⁺ in Solanum GCPs. Current traces of I_K,in and corresponding I/V curve obtained with pipette solutions containing 1 μM myo-InsP₆ (A), 1 μM neo-InsP₆ (B), and 1 μM myo-InsP₆ together with 20 mM BAPTA (C).

Figure 4

At the submicromolar range, InsP₆ is more potent in the inhibition of I_K,in than Ins (1,4,5)P₃ in both Solanum (A and B) and Vicia GCPs (C–F). In A–F, I/V scans were taken at different times, as indicated in the graph; the amount of inositol polyphosphate used is also indicated. A, C, and E are experiments carried out with InsP₆ in the patch pipette. B, D, and F are experiments carried out with Ins (1,4,5)P₃ in the patch pipette. Note the remarkable lack of effect of Ins (1,4,5)P₃ compared with InsP₆-induced large inhibition of I_K,in at either concentration. This figure also highlights the speed at which GCPs from Vicia respond to InsP₆ [at −200 mV, 90% block in only 9 min (C)] when compared with GCPs from Solanum (at −200 mV, 60% block in 25 min; A).