Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose

Abstract

Abscisic acid (ABA) is a plant hormone involved in the response of plants to reduced water availability. Reduction of guard cell turgor by ABA diminishes the aperture of the stomatal pore and thereby contributes to the ability of the plant to conserve water during periods of drought. Previous work has demonstrated that cytosolic Ca2+ is involved in the signal transduction pathway that mediates the reduction in guard cell turgor elicited by ABA. Here we report that ABA uses a Ca2+-mobilization pathway that involves cyclic adenosine 5'-diphosphoribose (cADPR). Microinjection of cADPR into guard cells caused reductions in turgor that were preceded by increases in the concentration of free Ca2+ in the cytosol. Patch clamp measurements of isolated guard cell vacuoles revealed the presence of a cADPR-elicited Ca2+-selective current that was inhibited at cytosolic Ca2+ >/= 600 nM. Furthermore, microinjection of the cADPR antagonist 8-NH2-cADPR caused a reduction in the rate of turgor loss in response to ABA in 54% of cells tested, and nicotinamide, an antagonist of cADPR production, elicited a dose-dependent block of ABA-induced stomatal closure. Our data provide definitive evidence for a physiological role for cADPR and illustrate one mechanism of stimulus-specific Ca2+ mobilization in higher plants. Taken together with other recent data [Wu, Y., Kuzma, J., Marechal, E., Graeff, R., Lee, H. C., Foster, R. & Chua, N.-H. (1997) Science 278, 2126-2130], these results establish cADPR as a key player in ABA signal transduction pathways in plants.

Figure 1

Microinjection of cADPR into guard cell cytosol elevates [Ca²⁺]_cyt. (A) Typical results for the change in [Ca²⁺]_cyt after microinjection of cells, by using pipettes filled with ADPR or water, into the cytosol of fura-2-loaded guard cells of open stomata of C. communis (n = 5 and 2 cells respectively). Data shown are from a cell injected with ADPR. A transient increase in [Ca²⁺]_cyt was observed only during the 10-s current pulse. (B) Sustained increase in [Ca²⁺]_cyt following microinjection of cADPR into the cytosol of fura-2-loaded guard cells of open stomata of C. communis (n = 5 cells). [Ca²⁺]_cyt remained elevated for at least 10 min after the 10-s current pulse. (C) Oscillations in [Ca²⁺]_cyt (amplitude, 200 nM; period, 3.75 min) occasionally observed following microinjection of cADPR into the cytosol of fura-2-loaded guard cells of open stomata of C. communis (n = 2 cells). For A–C bar = 5 min. (D) ADPR (data shown) or water injected into the cytosol caused no change in guard cell turgor (right-hand guard cell, arrowed). (E) In contrast, cADPR injected into the cytosol caused a reduction in guard cell turgor (right-hand guard cell, arrowed). (Bars in D and E = 5 μm.)

Figure 2

Guard cell whole vacuole currents elicited by cADPR. (A) Whole vacuole currents from Vicia faba guard cell vacuole in the absence of cADPR in symmetric 200 mM KCl and other conditions as shown. (B and C) Currents from the same vacuole respectively in the presence of 100 nM cADPR on the cytosolic (bath) side of the membrane and then 20 min after onset of bath perfusion with cADPR-free solution. (D) I–V relationship for whole vacuole instantaneous currents before (○) and after (•) addition of 100 nM cADPR, conditions as in A and B. (E) Difference relationship for cADPR-induced currents, with conditions as in D (■) or with the addition of 20 mM CaCl₂ (a_Ca = 4.9 mM) to the pipette solution (□). The I–V difference relationship for controls carried out with 100 nM noncyclic ADPR is also shown (▵). In D and E all data are replicates from 7 vacuoles. (F) Instantaneous currents induced by 100 nM cADPR (as in E, ■) at +100 mV (○) and −100 mV (•) over a range of [Ca²⁺]_cyt concentrations. Each point is the mean ± SEM of n = 7 or 8.

Figure 3

Effect of inhibitors of the cADPR signaling pathway on ABA-induced stomatal closure. (A) Representative plot showing half stomatal aperture measurements after challenge of a guard cell with 1 μM ABA, where 8-NH₂-cADPR had previously been loaded into the cell cytosol by pressure microinjection (○). Also shown are similar measurements for the uninjected cell from the same stoma (•). (A Inset) Half stomatal aperture measurements of a cell previously loaded with ADPR and subsequently challenged with 1 μM ABA (○) and the uninjected cell from the same stoma (•). (B) Stomatal aperture measurements from isolated epidermal strips incubated under conditions that promote stomatal opening (50 mM KCl/10 mM Mes continuously perfused with CO₂-free air under constant illumination) for 2 h and subsequently incubated for a further 2 h in the same buffer containing a range of nicotinamide concentrations in the absence (▵) or presence (□) of 1 μM ABA for the final 1 h of the experiment. (C) Stomatal aperture measurements from isolated epidermal strips incubated for 3 h under conditions promoting stomatal opening in the presence or absence of inhibitor or ABA. Bar a, buffer only; bar b, 50 mM nicotinamide; bar c, 1 μM ABA; and bar d, 1 μM ABA and 50 mM nicotinamide. For B and C the data are the means ± the SEMs. A total of 120 and 160 stomatal aperture measurements for each data point were obtained for B and C, respectively.