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A defined range of guard cell calcium oscillation parameters encodes stomatal movements

Nature volume 411, pages 1053–1057 (2001)Cite this article

Abstract

Oscillations in cytosolic calcium concentration ([Ca2+]cyt) are central regulators of signal transduction cascades1, although the roles of individual [Ca2+]cyt oscillation parameters in regulating downstream physiological responses remain largely unknown. In plants, guard cells integrate environmental and endogenous signals to regulate the aperture of stomatal pores2 and [Ca2+]cyt oscillations are a fundamental component of stomatal closure3,4. Here we systematically vary [Ca2+]cyt oscillation parameters in Arabidopsis guard cells using a ‘calcium clamp’3,5,6,7 and show that [Ca2+]cyt controls stomatal closure by two mechanisms. Short-term ‘calcium-reactive’ closure occurred rapidly when [Ca2+]cyt was elevated, whereas the degree of long-term steady-state closure was ‘calcium programmed’ by [Ca2+]cyt oscillations within a defined range of frequency, transient number, duration and amplitude. Furthermore, in guard cells of the gca2 mutant8, [Ca2+]cyt oscillations induced by abscisic acid and extracellular calcium had increased frequencies and reduced transient duration, and steady-state stomatal closure was abolished. Experimentally imposing [Ca2+]cyt oscillations with parameters that elicited closure in the wild type restored long-term closure in gca2 stomata. These data show that a defined window of guard cell [Ca2+]cyt oscillation parameters programs changes in steady-state stomatal aperture.

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Figure 1: Increasing the number of [Ca2+]cyt transients decreases steady-state stomatal aperture.
Figure 2: The period and duration of [Ca2+]cyt transients alters steady-state stomatal aperture.
Figure 3: Preceding [Ca2+]cyt oscillation kinetics program steady-state stomatal aperture.
Figure 4: Altered [Ca2+]cyt oscillation kinetics and stomatal closure in the gca2 mutant.
Figure 5: Altered [Ca2+]cyt oscillation kinetics in gca2 prevent long-term stomatal closure.

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Acknowledgements

We thank J. Harper for discussions and N. Spitzer and R. Tsien for comments on the manuscript. This research was supported by NIH, Department of Energy and National Science Foundation grants to J.I.S. and a DFG grant and Fonds der Chemischen Industrie to E.G.

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Authors and Affiliations

  1. Division of Biology and Center for Molecular Genetics, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, 92093-0116, California, USA

    Gethyn J. Allen, Sarah P. Chu, Carrie L. Harrington, Yat Y. Tang & Julian I. Schroeder

  2. ZMBP-Pflanzenphysiologie, Universität Tübingen, Auf der Morgenstelle 1, Tübingen, 72076, Germany

    Karin Schumacher

  3. Technische Universität, München Lehrstuhl fur Botanik, Freising–Weihenstephan, D-85350, Germany

    Thomas Hoffmann & Erwin Grill

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Correspondence to Julian I. Schroeder.

Supplementary information

Supplementary data Figure 1

(GIF 20.7 KB)

Hyperpolarization-activated "calcium-clamp" and [Ca2+]cyt oscillations in Arabidopsis guard cells expressing yellow cameleon 2.1.

(a) Stomata were opened in high KCl (100 mM) (depolarizing) buffer for 2.5h in the light. Guard cells subsequently exhibited a stable resting [Ca2+]cyt level in the 100 mM KCl buffer. (b) Stomatal aperture was stable after the 2.5 h opening period (t=0) and for a subsequent 3 h period (t=3) when cells were maintained in the 100 mM KCl buffer. (c) Repetitive exchanges to low KCl (0.1 mM) hyperpolarizing buffer induced a series of [Ca2+]cyt transients composing a [Ca2+]cyt oscillation (upper panel). Oscillations were abolished if extracellular calcium was removed with 10 mM EGTA (lower panel). (d) Steady-state stomatal closure was induced when five hyperpolarizations of 5 min duration and a 10 min period (5 x 5) were applied in the presence of extracellular calcium, but was reduced in the absence of extracellular calcium (5 x 5 + EGTA). Apertures were measured 3 h following the onset of oscillations imposed as (i) and (ii) in (c). Apertures are mean ± SEM of 160 stomates from n=4 replicates.

Supplementary data Figure 2

(GIF 16 KB)

Steady-state stomatal closing is enhanced when [Ca2+]cyt elevations oscillate.

(a) Three 5 min [Ca2+]cyt transients imposed in guard cells (upper panel) induced a total integrated [Ca2+]cyt increase of 3332 ± 422 nM· min (see shaded area, n=22 guard cells). A 15 min [Ca2+]cyt increase induced by stepwise decreases from 100 to 10, 1 and 0.1 mM KCl at the indicated points in the presence of 10 mM Ca2+ (lower panel) induced a total integrated [Ca2+]cyt increase of 3678 ± 97 nM· min (see shaded area, n=30 guard cells). (d) Steady-state stomatal closure was greater (P< 0.001) following three 5 min [Ca2+]cyt transients (left bar, i) than one 15 min [Ca2+]cyt elevation (right bar, ii) even though the total [Ca2+]cyt increases were similar (P> 0.56).

Supplementary data Figure 3

(GIF 13 KB)

Stomatal aperture changes measured during [Ca2+]cyt oscillations with 1 min duration and 10 min periods in wild type.

Oscillations composed of three transients of 1 min duration at a 10 min period were imposed in wild type guard cells. These oscillations induced 26.5 ±8.6 % calcium reactive closure, but despite being at the optimal 10 min period to elicit closure only resulted in a 2.4 ±5.3% decrease in steady-state aperture, compared to » 20% for 2 min duration transients (Fig 2c,d in main text). These data suggest that a minimum transient duration greater than 1 min is required to elicit significant calcium programmed aperture changes.

Supplementary data Figure 4

(GIF 21 KB)

Reducing the amplitude of [Ca2+]cyt oscillations in guard cells reduces stomatal aperture.

(a) Three 5 min [Ca2+]cyt transients imposed in guard cells in the absence (upper panel) or presence (middle panel) of 50 m M of the Ca2+ channel blocker La3+ (n=16 cells). Transients were also imposed by 5 min exchanges between the high (100 mM) KCl buffer and a buffer containing 5 mM KCl, 50 m M Ca2+ (n=16 cells) (lower panel). Limiting calcium influx with 50 m M La3+ (a, ii) or by reducing the hyperpolarization step and extracellular calcium (a, iii) reduced the amplitude of [Ca2+]cyt transients by » 70 %. (b) Steady state stomatal closure was only 2 ± 7 % when the amplitude of [Ca2+]cyt transients was reduced with La3+ and 6 ± 6 % when the amplitude was reduced by 5 mM KCl and 50 µM Ca2+ buffers. These data together suggest that the amplitude of [Ca2+]cyt transients composing oscillations needs to reach a critical level before closure is elicited.

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Allen, G., Chu, S., Harrington, C. et al. A defined range of guard cell calcium oscillation parameters encodes stomatal movements. Nature 411, 1053–1057 (2001). https://doi.org/10.1038/35082575

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