Ion transport across the tonoplast and plasma membrane play a central role in stomatal functioning



The plasma membrane and tonoplast of guard cells

by Willmer C. M., Pantoja O. (1992)

  • C. M. Willmer,
  • O. Pantoja,


In: Plant Membranes. Springer, Dordrecht – Plant Membranes : 220-238 –


The forces needed to open stomata are osmotically generated by accumulation of K+ by guard cells and concurrent uptake of external C1 and/or intracellular synthesis of malate to maintain electroneutrality.

Potassium, C1 and malate collect in vacuoles of guard cells during opening and exit during closure.

Environmental factors and some hormones stimulate and modify the ion fluxes thereby influencing stomatal movements. Thus ion transport across the tonoplast and plasma membrane play a central role in stomatal functioning.


Proton pumping ATPase at the plasma membrane and tonoplast of stomata

Screen Shot 2018-09-18 at 20.17.18
Fig. 2. Blue light stimulated H+ -efflux from GCP of C. communis. OCP were isolated according to PANTOJA and WILLMER (1988). OCP (c. 1 X 106) were placed in 1 cm3 medium (250 mol m- 3 mannitol, 0.1 mol m- 3 MES, 10 mol m- 3 KCI, 1 mol m- 3 CaCI2) in the well of a Clark-type O2 electrode (DW 1; Hansatech, Norfolk, UK) and illuminated with continuous red (>600 nm) light (800 [lmol m- 2 S-I). Pulses (30 s) of blue «500 nm) light (150 [lmol m- 2 S-I) were superimposed at the point indicated. H+ -efflux was measured using a micro-combination pH probe

Some properties of proton pumping ATPase at the plasma membrane and tonoplast of guard cells

Fricker M. D., Willmer C. M. (1990)

M. D. FRICKER I), and C. M. WILLMER 2)

I) University of Oxford, Department of Plant Sciences, Oxford, U.K.

2) University of Stirling, Department of Biological and Molecular Sciences, Stirling, U.K.


in Biochem. Physiol. Pflanzen 186: 301–308 – –


The kinetics and magnitude of light-stimulated H+ -efflux at the plasma membrane of guard cells can be directly correlated with stomatal opening in epidermal strips, under conditions where K+ fluxes and osmotic changes have already been well characterised (e.g. MACRoBBIE 1988).

Our data is consistent with the action of a plasma membrane ATPase in generating the H+ -efflux. Purification of GCP has allowed more detailed investigation of the spectral requirements for lightstimulated H+ -efflux and has facilitated electrophysiological studies using patch clamp techniques. GCP also provide a homogeneous cell type as the starting point for biochemical studies on guard cells.

The ATP-hydrolysing activities of both the plasma membrane ATPase and tonoplast ATPase, and the H+ -transport properties of the tonoplast ATPase can be distinguished in crude homogenates of GCP.

Application of electrophoretic separation in conjunction with labelling techniques may allow resolution of the individual polypeptides of these ATPases.

Vanadate inhibits blue light-stimulated swelling of stomatal protoplasts



Vanadate inhibits blue light-stimulated swelling of Vicia guard cell protoplasts

by Amodeo G., Srivastava A., Zeiger E. (1992)

Gabriela AmodeoAlaka SrivastavaEduardo Zeiger

Department of Biology, University of California, Los Angeles, CA 90024
Laboratory of Biomedical and Environmental Sciences, University of California, Los Angeles, CA 90024
2 Permanent address: Department of Agronomy-CERZOS, Laboratory of Plant Physiology, Universidad Nacional del Sur, Bahía Blanca, Argentina.

in Plant Physiol. 100: 1567-1570 – PMCID: PMC1075821 – PMID: 16653159


When supplied under low chloride concentrations, vanadate inhibits the blue light-stimulated swelling of Vicia faba L. guard cell protoplasts in a dose-dependent fashion.

The volume of guard cell protoplasts incubated in 10 mm K-imino-diacetic acid, 0.4 mmannitol, and 1 mm CaCl2 remained essentially constant under 1000 μmol m−2 s−1 red light, but increased an average of 27% after 8 min of the addition of 50 μmol m−2 s−1 blue light to the background red light. At 500 μm, vanadate completely inhibits the response to blue light.

Vanadate also inhibits the swelling of guard cell protoplasts stimulated by the H+-ATPase agonist fusicoccin. The vanadate sensitivity of the blue light-stimulated swelling implicates a proton-pumping ATPase as a component of the sensory transduction of blue light in guard cells.

ATPase and phosphatase activity in stomatal protoplasts



Vanadate sensitive ATPase and phosphatase activity in guard cell protoplasts of Commelina

by Fricker M. D., Willmer C. M. (1987)

Screen Shot 2018-09-18 at 16.16.28


in J. Exp. Bot. 38: 642–648 – –

Phosphatase activity was measured in extracts of guard cell protoplasts of Commelina communis L. using the artificial substrate p-nitrophenylphosphate. A pH optimum of 5.8 to 6.3 was determined.

Ammonium molybdate (Ol mol m−3) and sodium vanadate (1–0 mol m−3) gave almost complete inhibition of phosphatase activity at pH 60. ATPase assays were, therefore, conducted in the presence of 0–2 mol m −3 molybdate and vanadate was used as a specific inhibitor of plasmamembrane ATPase activity.

Vanadate sensitive ATPase activity showed a pH optimum of 6.6 and activity was stimulated by KC1. These properties are characteristic of plasmamembrane proton pumping ATPases in other systems and suggest that proton extrusion in guard cells could be mediated by a similar enzyme.

The maximum ATPase activity is sufficient to account for all the proton flux observed during the stomatal opening response.

Stomata from npq1 lack a specific response to blue light



Stomata from npq1, a zeaxanthin‐less Arabidopsis mutant, lack a specific response to blue light

by Frechilla S., Zhu J., Talbott L. D., Zeiger E. (1999)

Silvia Frechilla, University of California, Los Angeles

Jianxin Zhu, University of California, Los Angeles

Lawrence D. Talbott, University of California, Los Angeles

Eduardo Zeiger, University of California, Los Angeles


in Plant and Cell Physiology 40: 949-954 – DOI | 10.1093/oxfordjournals.pcp.a029627 –

The Arabidopsis mutant npql, which cannot accumulate zeaxanthin because of a defective violaxanthin deepoxidase, was used to investigate the role of zeaxanthin in the stomatal response to blue light.
Neither dark-adapted nor light-treated guard cells or mesophyll cells of the npql mutant contained detectable zeaxanthin. In contrast, wildtype guard cells had a significant zeaxanthin content in the dark and accumulated large amounts of zeaxanthin when illuminated.
The well-documented red light enhancement of blue light-stimulated stomatal opening, in which increasing fluence rates of background red light result in increased response to blue light, was used to probe the specific blue light response of Arabidopsis stomata.
Stomata from the npql mutant did not have a specific blue light response under all fluence rates of background red light tested. On the other hand, stomata from leaves of hy4 (cry 1), an Arabidopsis mutant lacking blue light-dependent inhibition of hypocotyl elongation, had a typical enhancement of the blue light response by background red light.
The lack of a specific blue light response in the zeaxanthinless npql mutant provides genetic evidence for the role of zeaxanthin as a blue light photoreceptor in guard cells.

The calcium message in stomata



Visualisation and measurement of the calcium message in guard cells

by Fricker M. D., Gilroy S., Read N. D., Trewavas A. J. (1991)

Department of Plant Sciences, University of Oxford, UK.


In: Molecular Biology of Plant Development. W. Schuch and G. Jenkins, editor. Cambridge University Press, Cambridge –

Symp Soc Exp Biol. 45: 177-190 –


We have applied several novel technologies to investigate the role of cytosolic free calcium [Ca2+]i in signal transduction in guard cells of Commelina communis L.

Fluorescence ratio imaging and photometry together with the fluorescent Ca2+ indicator Indo-1 were used to directly visualise and measure dynamic spatial and temporal changes in [Ca2+]i in response to various exogenous stimuli.

More subtle manipulation of the Ca2+ signal transduction pathway was achieved through the use of photoactivateable, caged Ca2+ and caged inositol-1,4,5-triphosphate (InsP3) released directly into the cytoplasm of the guard cell after microinjection. In these experiments, changes in [Ca2+]i were simultaneously monitored with the fluorescent Ca2+ indicator, Fluo-3.

Resting levels of [Ca2+]i (100-200 nM) increased in response to elevated [Ca2+]e, lowering [K+]e, application of the ionophore A-23187 or cytosolic release of either Ca2+ or InsP3 from their caged forms. Stomatal closure was triggered if [Ca2+]i increased above a threshold of about 600 nM.

Abscisic acid (ABA) had little effect on [Ca2+]i in the majority of cells studied, being elevated in only a minority of cells investigated. However, stomatal closure occurred in all cases after ABA application. This suggests that ABA acts through both Ca(2+)-independent and Ca(2+)-dependent pathways.

The imaging data revealed a substantial heterogeneity in [Ca2+]i within the guard cell. Cytoplasmic regions, particularly near the nucleus, often showed marked elevations and sometimes oscillations. The origin and kinetics of the Ca2+ fluxes leading to the dynamic spatial patterns is discussed along with several new approaches directed towards identification of the source of the Ca2+.

These methods include optical sectioning and 3-D reconstruction of both the endomembrane system and [Ca2+]i in living guard cells using confocal microscopy.

Overall, our data is consistent with multiple sources for [Ca2+]i, including uptake across the plasma membrane and InsP3- or Ca(2+)-induced Ca2+ release from internal stores.

Stomatal opening in isolated epidermal strips



Stomatal opening in isolated epidermal strips of Vicia faba. II. Responses to KCl concentration and the role of potassium absorption

by Fischer R. A., Hsiao T. C. (1968)

Laboratory of Plant-Water Relations, Department of Water Science and Engineering, University of California, Davis, California 95616.


in Plant Physiol. 43: 1953–1958 -DOI:

CrossRef | PubMed


This paper reports a consistent and large opening response to light + CO2-free air in living stomata of isolated epidermal strips of Vicia faba. The response was compared to that of non-isolated stomata in leaf discs floating on water; stomatal apertures, guard cell solute potentials and starch contents were similar in the 2 situations. To obtain such stomatal behavior, it was necessary to float epidermal strips on dilute KCl solutions. This suggests that solute uptake is necessary for stomatal opening.

The demonstration of normal stomatal behavior in isolated epidermal strips provides a very useful system in which to investigate the mechanism of stomatal opening. It was possible to show independent responses in stomatal aperture to light and to CO2-free air.