In vivo spectrophotometric analyses of stomata, stomatal guard cell protoplasts and epidermal cells of Vicia faba have shown that kaempferol 3,7-O-glycosides are localized in the vacuoles

In vivo– microspectrophotometric characterization of flavonol glycosides in Vicia faba guard and epidermal cells

Schnabl H., Weissenböck G., Scharf H. (1989)


J Exp Bot 37: 61–72 –


Cytological observations by fluorescence and U.V.-absorption microscopy together with in vivo spectrophotometric analyses of stomata, guard cell protoplasts and epidermal cells of Vicia faba have shown that kaempferol 3,7-O-glycosides are localized in the vacuoles. The alkaline-induced emission spectra recorded with guard and epidermal cells after NH4OH-treatment were identical, exhibiting an emission maximum at 525 nm; the spectra correlated with that of reference flavonols after exposure to NH4OH The excitation spectra of both cell types are typical of these flavonols showing two maxima at 290 nm and 390 nm. In agreement, two absorption maxima were recorded for guard cells at 270 nm and 330 nm, without alkali, which shifted bathochromically to 275 nm and 380 nm, respectively, after NH4OH treatment. The fluorescence intensity measured at 525 nm demonstrates a photostability in epidermal cells whereas it increases by a factor of about five with the excitation time up to 30 min in guard cells. For the latter, several possible processes are discussed.

Fusion of Different Plant Cell Protoplasts : STOMATAL GUARD CELL PROTOPLASTS

Electrically Stimulated Fusion of Different Plant Cell Protoplasts : MESOPHYLL CELL AND GUARD CELL PROTOPLASTS OF VICIA FABA

Scheurich P., Zimmerman U., Schnabl H. (1981)

Peter ScheurichUlrich ZimmermannHeide Schnabl,


Plant Physiology 67(4): 849-853 – DOI10.1104/pp.67.4.849


Cell fusion is induced between guard cell and mesophyll cell protoplasts of Vicia faba by electrical field application. The process of fusion is initiated by electrical breakdown of the cell membrane. Prior to the application of an external electrical field pulse which brings about reversible breakdown of the membrane, the cells (suspended in a low-conducting medium) are brought into close contact with one another by exposing them to an external alternating, nonuniform field (5 volts, electrode distance, 200 micrometers; 500 kiloHertz). During this process, they form “pearl chains” which may become sufficiently long to form bridges between the electrodes. The process is reversible as long as this voltage is not exceeded. Cell fusion is initiated as a result of an electrical field pulse of 50 microseconds duration and of sufficiently high intensity to induce reversible electrical breakdown of the membranes. The process of fusion is completed within 40 minutes or less in the case of guard cell protoplasts, as well as in the case of fusion between guard cell and mesophyll cell protoplasts. The fused cells are spherical in shape, if the fusion product consists only of two or three cells.

Properties of PEP carboxylase in desalted extracts from isolated guard cell protoplasts

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard cell protoplasts

by Schnabl H., Kottmeier C. (1984)

Institut für Botanik der Technischen Universität, Arcisstrasse 21, D-8000, München 2, Germany.


In Planta 162: 220–225 –


Properties of phosphoenolpyruvate (PEP) carboxylase (EC obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column.

The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K+ concentration within 2–3 min after homogenization of the guard-cell protoplasts.

The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K+ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the Km(PEP) and Vmax values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.

Availability of chloride affects the balance between KCl and potassium malate in guard cells

Availability of chloride affects the balance between potassium chloride and potassium malate in guard cells of Vicia faba L.

by Raschke K., Schnabl H. (1978)

Klaus Raschke, Heide Schnabl,


In Plant Physiol. 62: 84–87 – DOI:


Electron probe microanalysis for K and Cl and enzymic determination of malate were performed on epidermal strips of Vicia faba L. which had been incubated with 0.1 equivalent of K+ per liter in the absence or presence of Cl.

In the absence of Cl, iminodiacetate, a presumed impermeant zwitterion, served as anion. With no Cl in the medium, 91% of the K+ imported into the guard cells during stomatal opening was neutralized by malate production; import of Cl (presumably from the rest of the epidermal tissue) contributed 6%.

In the presence of Cl, 50% of the necessary negative charges were provided by malate synthesis, 45% by Cl import.

Stomatal opening was not obviously affected by the chloride concentration in the incubation medium, but malate production declined roughly linearly with the logarithm of [Cl] between 10−5 and 10−1 equivalent per liter.

Potassium chloride as stomatal osmoticum

Potassium Chloride as Stomatal Osmoticum in Allium cepa L., a Species Devoid of Starch in Guard Cells

by Schnabl H., Raschke K. (1980)


In Plant Physiol. 65(1): 88–93 – PMID: 16661151 PMCID: PMC440273 –


K(+) and Cl(-) contents of guard cells and of ordinary epidermal cells were determined in epidermal samples of Allium cepa L. by electron probe microanalysis; malate contents of the same samples were determined by enzymic oxidation.

KCl was, in general, the major osmoticum in guard cells, irrespective of whether stomata had opened on leaves or in epidermal strips floating on solutions. The solute requirement varied between 50 and 110 femtomoles KCl per micrometer increase in aperture per pair of guard cells. Stomata did not open on solutions of K iminodiacetate, presumably because its anion could not be taken up.

Stomata opened if KCl or KBr was provided. Taken together, the results indicate that the absence of starch from guard cells deprived them of the ability to produce malate in amounts of osmotic consequence and that the presence of absorbable Cl(-) (or Br(-)) was necessary for stomatal opening.

Previous nutrient supply of the plants determined whether the charges of K(+) in guard cells were completely balanced by Cl(-) or only partially. Addition of K(2)SO(4) to the nutrient solution reduced the participation of Cl(-) in stomatal ion transfer, even if epidermal strips of these plants were later exposed to KCl solution.

The anion supplying the charge complement in these cases is not known. Although malate appeared not to participate in stomatal ion transfer in onion, epidermal samples of this species did contain malate. Malate accumulated in the epidermis of leaves put into the light but disappeared from illuminated epidermal strips floating on solutions. In whole leaves, epidermal malate content was positively correlated with stomatal opening; in epidermal strips floating on solutions, the correlation was negative or absent.