Report

Alteration of Stimulus-Specific Guard Cell Calcium Oscillations and Stomatal Closing in Arabidopsis det3 Mutant

See allHide authors and affiliations

Science  29 Sep 2000:
Vol. 289, Issue 5488, pp. 2338-2342
DOI: 10.1126/science.289.5488.2338

Abstract

Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutantdet3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.

Cytosolic calcium ([Ca2+]cyt) oscillations are an integral component of cell signaling, and the frequency, amplitude, and spatial localization of oscillations control the efficiency and specificity of cellular responses in animals (1–3). In plant cells [Ca2+]cyt oscillations are induced by multiple stimuli (4–9); however, it remains unknown whether oscillations are required to elicit physiological responses in plants. Here we show that the Arabidopsis det3mutant abolishes guard cell [Ca2+]cytoscillations and stomatal closure in response to oxidative stress and extracellular calcium ([Ca2+]ext), but not to abscisic acid (ABA) and cold. Restoring [Ca2+]ext-induced [Ca2+]cyt oscillations in det3guard cells rescued stomatal closure, suggesting that [Ca2+]cyt oscillations are essential for stomatal closure.

Stomatal closure follows increases in guard cell [Ca2+]cyt (10), and endomembrane calcium transport contributes to the [Ca2+]cyt signal (7,11–13). Genetic impairment of endomembrane calcium transport could therefore provide a direct approach for dissecting [Ca2+]cyt signals. Thede-etiolated 3 (det3) Arabidopsismutant has reduced endomembrane energization owing to a 60% reduction in expression of the C-subunit of the V-type H+–adenosine triphosphatase (V-ATPase) (14).det3 was identified by its failure to repress light-specific development in the dark; it has a dwarf phenotype, but normal stomatal development (14). Figure 1 shows DET3 promoter–driven DET3–green fluorescent protein (GFP) fluorescence inArabidopsis guard cells (Fig. 1A), protoplasts (Fig. 1C), and isolated vacuoles (Fig. 1D) and indicates that DET3 is expressed in guard cells and is localized on multiple membranes including both endoplasmic reticulum (ER) and vacuolar membranes. We hypothesized that endomembrane de-energization in det3 guard cells could affect [Ca2+]cyt signaling because Ca2+ sequestration into intracellular stores is H+ gradient–dependent in many organisms (15–19).

Figure 1

Localization in Arabidopsisguard cells of a COOH-terminal DET3-GFP fusion expressed under the control of the DET3 promoter in plasmid pPZP221 (14). (A) GFP fluorescence is excluded from the vacuoles (vc), chloroplasts (ch), and nucleus (nu) and is concentrated in the ER. Bar, 5 μm. (B) Autofluorescence in nontransformed cell. Bar, 5 μm. (C) In guard cell protoplasts, GFP fluorescence is excluded from vacuoles (vc) and the nucleus (nu) and is concentrated in the ER. Bar, 4 μm. (D) In ruptured guard cell protoplasts fluorescence was associated with released vacuolar membranes (vc) and the collapsed nucleus (nu), but not the plasma membrane (pm). Bar, 4 μm.

Wild-type (WT) Arabidopsis stably expressing yellow cameleon 2.1 (YC2.1) under the control of the constitutive 35S promoter was used to measure [Ca2+]cyt signals in stomatal guard cells (20–22). Oscillations in [Ca2+]cyt were rapidly induced by 1 or 10 mM extracellular calcium in all 84 WT guard cells tested (Fig. 2, A and B). [Ca2+]cyt oscillations were smaller and of higher frequency for 1 mM [Ca2+]ext (average [Ca2+]cyt at peak ≈ 160 nM; period = 161 ± 20 s) than for 10 mM [Ca2+]ext([Ca2+]cyt ≈ 1020 nM; period = 396 ± 23 s) [see also (4, 8)]. Oscillations ceased after 45 to 60 min (Fig. 2B) and preceded maximal stomatal closure, which was measured after 3 hours. In det3guard cells, increasing [Ca2+]ext from 0 to 1 or 10 mM caused immediate [Ca2+]cyt increases which, unlike in the WT, subsequently failed to oscillate but consisted of small, rapid spikes superimposed on prolonged [Ca2+]cyt increases (n = 55 of 56 cells) (Fig. 2, C and D) ([Ca2+]cyt ≈ 350 nM for 1 mM [Ca2+]ext and ≈ 1250 nM for 10 mM [Ca2+]ext). Additionally, the total integrated [Ca2+]cyt increase over an ≈30-min period was higher in det3 [5910 ± 785 nM·min (n = 6)] than in WT [3010 ± 263 nM·min (n = 6)] at 10 mM [Ca2+]ext.

Figure 2

[Ca2+]ext-induced guard cell [Ca2+]cyt oscillations and stomatal responses in WT and det3. (A) [Ca2+]cyt oscillations in a WT guard cell induced by 1 mM [Ca2+]ext (n= 32 from 16 stomates). Oscillations had a mean peak 535/480 nm emission ratio of 2.47 ± 0.4. (B) [Ca2+]cyt oscillations in a WT guard cell induced by 10 mM [Ca2+]ext(n = 52 from 26 stomates). Oscillations had a mean peak 535/480 nm ratio of 3.21 ± 0.16. The trace is from the right-hand cell of the stomate in the lower panel. Images indicate the 535/480 nm ratio at points *1 to *4. (C) Increase of det3guard cell [Ca2+]cytinduced by 1 mM [Ca2+]ext(n = 21 cells from 11 stomates). Increases had a mean peak 535/480 nm ratio of 2.83 ± 0.31. (D) Increase ofdet3 guard cell [Ca2+]cytinduced by 10 mM extracellular calcium (n = 34 from 17 stomates). Increases had a mean peak 535/480 nm ratio of 3.26 ± 0.39. The trace is from the right-hand cell of the stomate in the lower panel. Images indicate the 535/480 nm ratio at points #1 to #4. (E) Increasing [Ca2+]ext caused stomatal closure in WT (○) but not det3 (•). Data are the mean ± SEM of 120 stomata from four replicates for each [Ca2+]ext.

The absence of [Ca2+]ext-induced [Ca2+]cyt oscillations in det3guard cells was consistent with a marked effect on [Ca2+]ext-induced stomatal closure. Addition of [Ca2+]ext to preopened WT stomata caused closure, whereas in det3 stomatal closure was abolished at all [Ca2+]ext concentrations tested (Fig. 2E).

To further investigate the role of [Ca2+]cytoscillations and det3 in guard cell signaling, we measured [Ca2+]cyt and stomatal movements in response to other stimuli. In WT guard cells ABA caused repetitive [Ca2+]cyt transients (Fig. 3A), or more prolonged oscillations (Fig. 3B), with a magnitude of ∼500 nM and a period of 468 ± 41 s (n = 40) for transients and 333 ± 35 s (n = 6) for oscillations (Fig. 3, A and B). Indet3, ABA caused [Ca2+]cyttransients or oscillations with near-identical magnitudes (Fig. 3, C and D) and periods (476 ± 32 s, n = 26 for transients and 328 ± 36 s, n = 4 for oscillations; P > 0.15 for WT versus det3periods for both transients and oscillations). ABA also induced stomatal closure to the same extent in WT and det3 (Fig. 3E).

Figure 3

ABA-induced guard cell [Ca2+]cyt oscillations and stomatal responses in WT and det3. (A) Repetitive, transient [Ca2+]cyt increases in a WT guard cell induced by 10 μM ABA (n = 40 from 28 stomates). Transients had a mean peak 535/480 nm ratio of 3.07 ± 0.29. (B) Oscillations of WT guard cell [Ca2+]cyt induced by 10 μM ABA (n = 6 from three stomates). Oscillations had a mean peak 535/480 nm ratio of 2.91 ± 0.31. (C) Repetitive, transient increases in det3 guard cell [Ca2+]cyt induced by 10 μM ABA (n = 26 from 15 stomates). Transients had a mean peak 535/480 nm ratio of 3.09 ± 0.22. (D) Oscillations ofdet3 guard cell [Ca2+]cyt induced by 10 μM ABA (n = 4 from two stomates). Oscilla- tions had a mean peak 535/480 nm ratio of 2.98 ± 0.31. Transients occurred in both guard cells of all responsive stomata (ABA-induced [Ca2+]cytincreases were observed in 81% of cells, n = 76 from 96 stomates). (E) ABA induced stomatal closure in WT (○) and det3 (•). Data are the mean ± SEM of 120 stomata from four replicates.

Hydrogen peroxide (H2O2) also induced [Ca2+]cyt increases in WT guard cells (23, 24), consisting of one or two separate transients (Fig. 4A) with peak magnitudes of ∼700 nM (n = 24). In det3guard cells H2O2 caused larger (≈1450 nM), more prolonged [Ca2+]cyt increases (Fig. 4B,n = 22 cells), and H2O2-induced stomatal closure was abolished (Fig. 4C). H2O2-induced [Ca2+]cyt signaling in guard cells requires activation of a plasma membrane calcium influx current (I Ca) that shows enhanced activity at negative membrane potentials (24). Electrophysiological measurement (22) of I Ca in guard cell protoplasts showed identical I Ca activation by H2O2 in WT and det3 (Fig. 4, D and E).

Figure 4

Guard cell [Ca2+]cytoscillations and stomatal responses to H2O2 and cold in WT and det3.(A) Repetitive, transient increases in WT guard cell [Ca2+]cyt induced by 100 μM H2O2 (n = 24 from 13 stomates). Transients had a mean peak 535/480 nm ratio of 3.11 ± 0.31 and a period, when two increases occurred, of 366 ± 31 s. (B) Increase of det3 guard cell [Ca2+]cyt induced by 100 μM H2O2 (n = 22 from 13 stomates). Increases had a mean peak 535/480 nm ratio of 3.31 ± 0.46. (C) One hundred micromolar H2O2induced stomatal closure in WT (left bars) but not det3(right bars). Data are the mean ± SEM of 180 stomata from three replicates. Hyperpolarization activated calcium-permeable currents in (D) WT (n = 9) and (E)det3 (n = 6) guard cell protoplasts induced by H2O2. (F) Oscillations of WT guard cell [Ca2+]cyt induced by cold (n = 20 from 10 stomates). Oscillations had a mean peak 535/480 nm ratio of 2.66 ± 0.31. (G) Oscillations ofdet3 guard cell [Ca2+]cyt induced by cold (n = 18 from nine stomates). Oscillations had a mean peak 535/480 nm ratio of 2.61 ± 0.29. (H) Cold induced stomatal closure in WT (left) and det3 (right). Data are the mean ± SEM of 180 stomata from three replicates.

Cold increases plant cell [Ca2+]cyt(25). In WT guard cells cold elicited small, repetitive [Ca2+]cyt transients (amplitude ≈ 125 nM [Ca2+]cyt; frequency, 154 ± 11 s; n = 20 cells) (Fig. 4F). Cold-induced transients of similar amplitude and frequency (amplitude ≈ 125 nM [Ca2+]cyt; frequency, 162 ± 21 s;n = 18 cells) were measured in det3 guard cells (P < 0.01) (Fig. 4G). Cold elicited stomatal closure to the same extent in det3 and WT (P< 0.05) (Fig. 4H).

Calcium contents (predominantly vacuolar and ER) measured by elemental x-ray microanalysis (26) in intact guard cells of open stomates were indistinguishable between WT anddet3 (calcium as weight percent of total K+, Na+, and Ca2+ = 34.3 ± 1.2% for WT and 34.7 ± 0.7% for det3, n = 90 each). These data suggest that the residual 40% V-ATPase activity is sufficient to allow long-term intracellular calcium accumulation indet3. Therefore, disruption of [Ca2+]ext- and oxidative stress–induced [Ca2+]cyt oscillations in det3guard cells (Figs. 2, C and D, and 4B) does not appear to be due to depletion of intracellular calcium stores.

The correlation in det3 between stimuli for which guard cell [Ca2+]cyt oscillations are disrupted and stomatal closure is abolished strongly suggests that [Ca2+]cyt oscillations are necessary for stomatal closure. To critically test this hypothesis, we experimentally imposed [Ca2+]cyt oscillations in guard cells using hyperpolarization-mediated calcium influx (9,11). Exchanging the bathing solution every 300 s between high-KCl (depolarizing) and low-KCl (hyperpolarizing) buffers (22), and adding [Ca2+]extconcomitantly with the hyperpolarizing buffer, imposed repetitive [Ca2+]cyt increases in WT guard cells (Fig. 5A, n = 20 cells) and resulted in stomatal closure (Fig. 5B, left panel, “Post Osc”). Removing external calcium with 10 mM EGTA prevented oscillations (n = 8) and inhibited stomatal closure when external K+ was repetitively exchanged (27). One 300-s hyperpolarization resulted in a single calcium transient, indicating that spontaneous oscillations were not initiated (Fig. 5C,n = 12 cells). Continuous transfer into the hyperpolarizing buffer (0.1 mM K+) (Fig. 5D) induced oscillations with a higher frequency than oscillations induced by 10 mM [Ca2+]ext at 5 mM K+[period = 178 ± 31 s, n = 32 compared with 396 ± 23 (Fig. 2B)] and resulted in stomatal closure (Fig. 5B, left panel, “Hyp”). However, imposing hyperpolarizations by decreasing external K+ stepwise from 100 mM to 10, 1, and 0.1 mM, concomitant with the addition of 10 mM [Ca2+]ext, produced prolonged [Ca2+]cyt increases in WT guard cells (Fig. 5E) similar to [Ca2+]ext-induced [Ca2+]cyt increases in det3(compare Figs. 2, C and D, and 5E). Notably, prolonged [Ca2+]cyt increases in WT also failed to elicit stomatal closure (Fig. 5B, left panel, “Step”).

Figure 5

Repetitive hyperpolarizations impose [Ca2+]cyt oscillations in guard cells. (A) [Ca2+]cyt oscillations in a WT guard cell resulting from exchanges between depolarizing (100 mM KCl, zero added Ca2+) (▪) and hyperpolarizing buffers (0.1 mM KCl, 10 mM Ca2+) (□) (n = 20 from 10 stomates). (B) (Left) Inducing [Ca2+]cytoscillations close pre-opened [Control (Pre-osc)] stomata in WT (Post Osc). Oscillation-induced closure is elicited by continuous hyperpolarization (Hyp) but inhibited by prolonged [Ca2+]cyt increase induced by stepwise hyperpolarization (Step). (Right) Oscillation-induced closure indet3. Data are the mean ± SEM of 160 stomata from four replicates. (C) One exchange from depolarizing to hyperpolarizing buffer (n = 12 from six stomates). (D) Continuous transfer to hyperpolarization buffer (n = 32 from 16 stomates). (E) A stepwise hyperpolarization induces prolonged [Ca2+]cyt increases (n = 20 from 10 stomates). (F) Depolarizing to hyperpolarizing buffer exchanges as in (A) in det3 guard cells (n = 18 from 11 stomates).

In det3 guard cells, repetitive hyperpolarizations and the concomitant addition of 10 mM [Ca2+]ext also induced [Ca2+]cyt oscillations (Fig. 5F,n = 18 of 22 cells), which contrasted with the prolonged [Ca2+]cyt increases induced by [Ca2+]ext at constant external K+(Fig. 2, C and D). Remarkably, imposing [Ca2+]cytoscillations in det3 guard cells elicited [Ca2+]ext-induced stomatal closure (Fig. 5B, right panel). Therefore, by restoring oscillations in det3guard cells, the impairment of [Ca2+]ext-induced stomatal closure was rescued.

The [Ca2+]cyt oscillations elicited by ABA, cold, [Ca2+]ext, and oxidative stress had different amplitudes and frequencies (Figs. 2, A and B; 3, A and B; and 4, A and F), and all induced stomatal closure. Oscillations in [Ca2+]cyt result from the interaction of three processes: extracellular calcium influx, intracellular calcium release, and sequestration into intracellular stores or across the plasma membrane (28, 29). In guard cells the stimuli [Ca2+]ext (4) and oxidative stress (24) activate calcium influx from the extracellular space. In det3 guard cells these stimuli caused an initial [Ca2+]cyt increase (Figs. 2, C and D, and 4B), suggesting that this initial calcium influx is unaffected, as also indicated by identical I Caactivation in WT and det3 (Fig. 4, D and E). However, indet3 these stimuli caused prolonged [Ca2+]cyt increases, suggesting that disruption of [Ca2+]cyt oscillations is due to impaired endomembrane Ca2+ uptake. Disruption of endomembrane Ca2+ uptake in det3 could occur by a number of possible mechanisms. Ca2+/H+antiporter activity, which is involved in Ca2+ homeostasis in plant cells (16), may be reduced. Alternatively, a proposed direct facilitation of Ca2+-ATPase activity by V- ATPases (17, 19) might be disrupted indet3. Additionally, endomembrane lumen or cytosolic pH changes in det3 may have effects on Ca2+transporters.

Disruption of [Ca2+]cyt oscillations in response to [Ca2+]ext and oxidative stress but not ABA and cold suggests that different mechanisms generate oscillations for these stimuli. These mechanisms may involve different Ca2+ transporters located at separate intracellular calcium stores, as is found in animal cells (18, 19), although certain components can be shared among stimuli (9, 24,25).

In det3 guard cells, the perfect correlation between those stimuli for which [Ca2+]cyt oscillations were disrupted and stomatal closure was abolished strongly suggests that calcium oscillations are required for stomatal closure. Imposing oscillations rescued [Ca2+]ext-induced stomatal closure in det3, supporting previous hypotheses that calcium oscillations are required for physiological responses in guard cells (30). Prolonged [Ca2+]cyt increases in det3 (Fig. 2, C and D) or WT (Fig. 5, B and E) failed to elicit stomatal closure, suggesting, as in animal cells (1–3), that disruption of oscillations has a negative effect on some physiological responses. Therefore, regulation of the mechanism(s) in guard cells necessary to mediate stomatal closure is probably encoded by a pattern of periodic [Ca2+]cyt increases. Overall, these findings strongly suggest that in Arabidopsis guard cells, [Ca2+]cyt oscillations are essential to elicit stomatal closure.

  • * To whom correspondence should be addressed. E-mail: gallen{at}biomail.ucsd.edu, julian{at}biomail.ucsd.edu

  • These authors contributed equally to this work.

  • Present address: ZMBP-Pflanzenphysiologie, Universitaet Tuebingen, Auf der Morgenstelle 1, 72076 Tuebingen, Germany.

REFERENCES AND NOTES

View Abstract

Stay Connected to Science

Navigate This Article