Patch-clamp measurements on isolated stomatal protoplasts

Patch-clamp measurements on isolated guard cell protoplasts and vacuoles

by Raschke K., Hedrich R. (1989)

Klaus Raschke, Rainer Hedrich,


In Methods in Enzymol. 174: 312–330 – –


This chapter discusses the patch-clamp measurements on isolated guard cell protoplasts and vacuoles.

Guard cells can serve as models for other cells that export or import ions and break down carbohydrates to make organic acids for balancing cation fluxes. Such cells occur in roots, growing tissue, and in the pelvini of the leaves of plant species displaying nasty leaf movements.

It allows separate recordings of the activities of the transport systems in the plasmalemma and in the tonoplast.

Characterization becomes possible of electrogenic pumps, ion channels, and carders of very high turnover in any type of membrane. The basic requirement for the application of the technique is an absolutely clean membrane surface.

The chapter also describes the whole-vacuole configuration (analogous to whole cell) can be established by rupturing the membrane patch covering the tip of the patch pipette (starting from a vacuole-attached configuration).

The simultaneous requirement of CO2 and ABA for stomatal closure leads to the inference that ABA inhibits the expulsion of H(+) from guard cells

Common cocklebur (Xanthium strumarium)

Simultaneous requirement of carbon dioxide and abscisic acid for stomatal closing in Xanthium strumarium L.

by Raschke K. (1975)

Klaus Raschke, MSU/ERDA Plant Research Laboratory, Michigan State University, 48824, East Lansing, Michigan, USA.


In Planta 125: 243-259 – DOI: 10.1007/BF00385601 –


Open stomata of detached leaves of Xanthium strumarium L. closed only when carbon dioxide and abscisic acid (ABA) were presented simultaneously.

Three parameters of stomatal closing were determined after additions of ABA to the irrigation water of detached leaves, while the leaves were exposed to various CO2 concentrations ([CO2]s) in the air;

a) the delay between addition of ABA and a reduction of stomatal conductance by 5%,

b) the velocity of stomatal closing, and

c) the new conductance.

Changes in all three parameters showed that stomatal responses to ABA were enhanced by CO2; this effect followed saturation kinetics. Half saturation occurred at an estimated [CO2] in the stomatal pore of 200 μl l(-1).

With respect to ABA, stomata responded in normal air with half their maximal amplitude at [ABA]s between 10(-6) and 10(-5) M(+-)-ABA. The amounts of ABA taken up by the leaves during the delay increased with a power <1 (on the average, 0.67) of the [ABA] in the transpiration stream.

The minimal amount of ABA found to produce a stomatal response was about 1 pmol of (+-)-ABA per cm(2) leaf area, almost two orders of magnitude smaller than the original content of the leaves in ABA indicating that most of the endogenous ABA was in a compartment isolated from the guard cells.An interaction between stomatal responses to CO2 and ABA was also found in Gossypium hirsutum L. and Commelina communis L.; it was however much weaker than in X. strumarium.

Based on earlier findings and on the results of this investigation it is suggested that stomata close if the cytoplasm of the guard cells contains much malate and H(+). The acid content in turn is determined by the relative rates of production of malic acid (from endogenous as well as exogenous CO2) and its removal (by transport of the anion into the vacuole and exchange of the H(+) for K(+) with the environment of the guard cells).

The simultaneous requirement of CO2 and ABA for stomatal closure leads to the inference that ABA inhibits the expulsion of H(+) from guard cells.

Stomatal responses to pressure changes and interruptions in the water supply

Stomatal responses to pressure changes and interruptions in the water supply of detached leaves of Zea mays L.

by Raschke K. (1970)

Klaus Raschke

Botanisches Institut der Universität Giessen, D-63 Giessen, Germany

Michigan State University-Atomic Energy Commission Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48823


In Plant Physiol. 45: 415-423 –


Stomata of Zea mays L. respond to changes in hydrostatic pressure in the water supply of the leaves almost instantaneously and in all leaf parts simultaneously. Therefore, the leaf is a hydraulic unit.

The stomata are part of it and their aperture is controlled by the water potential in the water-conducting system. Stomatal aperture is not uniquely related to the relative water content of a leaf. The relation depends also on the humidity in the air and is different for the upper and the lower epidermis.

Phosphate translocator of isolated stomatal guard cell chloroplasts transports glucose-6-phosphate

Screen Shot 2018-11-12 at 12.59.44
Figure 1. Flow diagram of the isolation procedure for guard-cell chloroplasts. For details see “Materials and Methods.”


Phosphate translocator of isolated guard cell chloroplasts from Pisum sativum L. transports glucose-6-phosphate

by Overlach S., Diekmann W., Raschke K. (1993)

Susanne Overlach, Wilfried Diekmann, and Klaus Raschke

Pflanzenphysiologisches lnstitut und Botanischer Garten der Universitat Gottingen, Untere Karspüle 2, W-3400 Gottingen, Cermany


in Plant Physiol. 101:1201–1207 – PMID: 12231774 – PMCID: PMC160640

Screen Shot 2018-11-12 at 13.02.26
Figure 2. Electron micrographs of guard-cell protoplasts. Close association between chloroplasts (P) and mitochondria (M) in a guard-cell protoplast of P. sativum, as exhibited through EM. In the lower frame a microbody (MB) appears as well as a Colgi system. Bar = 0.5 ftm.


Chloroplasts were isolated from ruptured guard-cell protoplasts of the Argenteum mutant of Pisum sativum L. and purified by centrifugation through a Percoll layer.

The combined volume of the intact plastids and the uptake of phosphate were determined by silicone oil-filtering centrifugation, using tritiated water and [14C]sorbitol as membrane-permeating and nonpermeating markers and [32P]phosphate as tracer for phosphate.

The affinities of the phosphate translocator for organic phosphates were assessed by competition with inorganic phosphate.

The affinities for dihydroxyacetone phosphate, 3-phosphoglycerate (PGA), and phosphoenolpyruvate were in the same order as those reported for mesophyll chloroplasts of several species. However, the guard-cell phosphate translocator had an affinity for glucose-6-phosphate that was as high as that for PGA.

Guard-cell chloroplasts share this property with amyloplasts from the root of pea (H.W. Heldt, U.I. Flugge, S. Borchert [1991] Plant Physiol 95: 341-343).

An ability to import glucose-6-phosphate enables guard-cell chloroplasts to synthesize starch despite the reported absence of a fructose-1,6-bisphosphatase activity in the plastids, which would be required if only C3 phosphates could enter through the translocator.

Stomatal aperture and CO2



Temperature dependence of CO2 assimilation and stomatal aperture in leaf sections of Zea mays.

by Raschke K. (1970)

Screen Shot 2018-06-15 at 09.16.51


in Planta 91: 336–363. – doi: 10.1007/Bf00387507 –


CO2 exchange and air flow through the stomata were measured in leaf sections of Zea mays at temperatures between 7 and 52° and under optimal water supply. The results were summarized in polynomials fitted to the data.
In leaf samples brought from 16° and darkness into different experimental temperatures and light, CO2 assimilation has a maximum near 30°. Above 37° (in other experiments above 41°), net CO2 uptake stops abruptly and is replaced by CO2 evolution in light. If a 1-hr treatment with 25° and light is inserted between darkness and the experimental temperatures, the threshold above which the assimilatory system collapses shifts 3 degrees upwards, to 40° (or 44°); the decline of CO2 assimilation with high temperatures is less steep than without pretreatment; and the upper compensation point moves upscale by as much as 5 degrees.
Stomatal conductance for CO2 does not, in general, follow an optimum curve with temperature. Between 15 and 35° it is approximately proportional to net CO2 assimilation, indicating control by CO2; but above 35°, stomatal aperture increases further with temperature (and so does stomatal variability): the stomata escape the control by CO2 and above 40° may be wide open even if CO2 is being evolved. Stomatal conductance for CO2 below 15° may also be larger than would be proportional to CO2 assimilation.
Net CO2 assimilation and stomatal conductance at 25° were reduced if the leaf samples were pretreated with temperatures below approximately 20° and above 30°. Stomata were more sensitive to past temperatures than was CO2 assimilation.

Rubisco activity in stomata



Rubisco activity in guard cells compared with the solute requirement for stomatal opening 

by Reckmann U., Scheibe R., Raschke K. (1990)

Udo Reckmann, Renate Scheibe, Klaus Raschke,

Pflanzenphysiologisches Institut und Botanischer Garten der Universitat Gottingen, Untere Karspule 2, 3400 Gottingen, West Germany (U.R., K.R.),

Botanisches Institut der Universitat Bayreuth, Lehrstuhl Pflanzenphysiologie, Universitatsstrasse 30, 8580 Bayreuth, West Germany (R.S.)


in Plant Physiol. 92: 246–253 – 10.1104/pp.92.1.246 – 

[PMC free article] [PubMed] [Cross Ref] –


We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light.

Amounts of ribulose 1,5-bisphophate carboxylase/ oxygenase (Rubisco) were determined by the [14C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270.

The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99.

After exposure of guard-cell protoplasts to 14C02 for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate.

The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement.

The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.

The ABA contents of isolated stomatal protoplasts



Abscisic-acid contents and concentrations in protoplasts from guard cells and mesophyll cells of Vicia faba L.

by Lahr W., Raschke K. (1988)

Pflanzenphysiologisches Institut und Botanischer Garten, Untere Karspüle 2, D-3400, Göttingen, Federal Republic of Germany.

W. Lahr

Klaus Raschke, Georg-August-Universität Göttingen, Germany


in Planta  173: 528–531 – DOI: 10.1007/BF00958966 –

[Google Scholar] [CrossRef] [PubMed] –


The abscisic-acid (ABA) contents of isolated guard-cell protoplasts and mesophyll-cell protoplasts from Vicia faba were determined by high-pressure liquid chromatography followed by gas chromatography. The amounts of ABA found immediately after preparation of the protoplasts varied from 90 to 570 amol per guard-cell protoplast, and from 75 to 100 amol per mesophyll-cell protoplast. These contents correspond to concentrations between 36 and 230 μmol per liter in guard-cell protoplasts and between 2.7 and 3.3 μmol per liter in mesophyll-cell protoplasts.

During exposure of protoplasts to betaine concentrations of 0.3, 0.5, and 0.8 mol·l(-1) at 0° and 20°C for 30 min, ABA contents as well as the fractions of ABA that leaked into the medium remained constant for both protoplast types.

There was no evidence for net production of ABA in isolated protoplasts subjected to osmotic stress.

Shuttle of potassium and chloride between stomatal guard cells and subsidiary cells



Stomatal movement in Zea mays: Shuttle of potassium and chloride between guard cells and subsidiary cells

by Raschke K., Fellows M. P. (1971)

  • Klaus Raschke,
  • Margaret Pierce Fellows,

MSU/AEC Plant Research Laboratory, Michigan State University, East Lansing, USA


in Planta 101: 296-316 –


When stomates of Zea mays open K and Cl migrate from the subsidiary cells into the guard cells; when the stomates close both elements return to the subsidiary cells. Subsidiary cells function as reservoirs for K and Cl. Import of K and Cl into the guard cells and loss of both elements from the guard cells become observable 1 or 2 min after light is turned on or off, both when histochemical methods and the electron-probe microanalyzer are used for detection. Each stomatal complex of maize contains on the average 10±3×10-13 gram equivalents (eq) of K and 4±1×10-13 eq of Cl. Guard cells accumulate K in the light and CO2-free air at an average rate of 10×10-15 eq K per minute, and Cl at approximately half that rate.

Gain of the feedback loop involving CO2 and stomata



Gain of the feedback loop involving carbon dioxide and stomata.

by Farquhar G. D., Dubbe D. R., Rachke K. (1978)

Graham D. Farquhar, Dean R. Dubbe, Klaus Raschke,

Graham D. Farquhar,

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824.


in Plant Physiol. 62: 406–412 – PMID: 16660527 PMCID: PMC1092136 – DOI:

CrossRefPubMedGoogle Scholar –


The physiological and physical components of the feedback loop involving intercellular CO(2) concentration (c(i)) and stomata are identified.

The loop gain (G) is a measure of the degree of homeostasis in a negative feedback loop [the expression 1/(1-G) represents the fraction to which feedback reduces a perturbance]. Estimates are given for the effects of G on responses of stomata and c(i) to changes in ambient CO(2) concentration, light intensity, and perturbations in the water relations of a leaf.

At normal ambient CO(2) concentration, the gain of the loop involving stomatal conductance and c(i) was found to be -2.2 in field-grown Zea mays, -3.6 if plants of this species were grown in a growth chamber, and zero in well watered Xanthium strumarium in the vegetative state.