ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2 : 17 - 20 
Sprejeto (accepted): 2003-10-30 
Inter- and intracellular distribution of modulated glutathione in 
plant tissues 
Maria MULLER, Bernd ZECHMANN, Andreja URBANEK & Gtinther ZELLNIG 
Institute of Plant Physiology, University of Graz, SchubertstraBe 51, 8010 Graz, Austria 
e-mail: maria.mueller@uni-graz.at 
Introduction 
Abstract. In the present study we want to summarize <lata dealing with the inter-
and intracellular distribution of glutathione in different plant tissues under various 
environmental conditions. Monochlorobimane fluorescence of reduced glutathione 
is described for light microscopical investigations and allows distinguishing between 
glutathione in cytoplasm and in the nucleus. Further, a specific antibody, which 
recognizes both the reduced and oxidized form of glutathione, was used in order to 
demonstrate glutathione in different cell compartments. The labelling showed 
different glutathione concentrations in the cell compartments with a high staining 
intensity in mitochondria. 
Keywords: Glutathione, immunolabelling, monochlorobimane, plant tissues, 
ultrastructure 
Glutathione (GSH) is considered to have a broad spectrum of functions in plants. It is a cellular 
protectant, signal substance and a major reservoir of non-protein reduced sulphur in plants. 
Glutathione is present in all organs, with different concentrations in the organs or within the same 
organ at different developmental stages (FOYER & RENNENBERG 2000), but thus far little is known 
about the GSH distribution in cellular compartments. Intercompartmental variations in glutathione 
concentration are supposed to be crucial in signalling processes. NocroR & al. (2002) discuss varying 
concentrations of glutathione in chloroplasts, which are reported between 8 % and 50 % of the leaf 
glutathione depending on the isolation media and the methods. 
Our study presents an overview of a histochemical and an immunocytochernical method for the 
determination of inter- and intracellular glutathione contents in plant tissues. 
Material and methods 
Plant material 
Epidermal cells of Allium cepa L. as well as leaves of Cucurbita pepo L. were used as described 
elsewhere (MDLLER & al. 1999 a, 2002 b) . 
18 Acta Biologica Slovenica, 46 (2), 2003 
Monochlorobimane (BmCl) labelling of thiols (modified according to MOLLER & al. 1999 a, 
2002 a). 
A stock solution of BmCl (Molecular Probes) was diluted in phosphate buffer, pH 7 .2, 
immediately prior to use to obtain the required concentration of 50 mM BmCl. All plant material 
was exposed to BmCI from 5 to 20 minutes (depending on the used material). After removing the 
staining solution, the samples were washed with buffer to remove excess staining solution. Single 
cells and single cell layers were investigated immediately under the fluorescence microscope. 
Immunogold-analysis (modified according to M0LLER & al. 2002 b) 
Small sections of the plant material were fixed according to a standard fixation protocol (cf. 
ZELLNIG & al. 2000) or in 0.5 % glutaraldehyde/2.5 % paraformaldehyde, dehydrated in a graded 
series of ethanol and embedded in LR-White resin. Ultrathin sections were incubated with an anti-
glutathione rabbit polyclonal lgG (Signature Immunologics, lnc.) followed by a 10 nm gold-labelled 
secondary antibody (British Biocell lnt.). The antiserum was shown to react selectively with 
glutathione, although it does not differentiate between the reduced and oxidized forms of glutathione. 
To confirm the selectivity of the immunolabelling in the present set of experiments, the tissue sections 
were either incubated with the primary antibody, which was exposed to 5 mM of GSH prior to the 
application, followed by the secondary antibody or treated with the secondary antibody alone. Both 
treatments resulted in a negative immunolabelling. 
Results and discussion 
The technique of histochemical glutathione tracing by monochlorobimane (BmCl) in fluorescence 
microscopy was developed for single cell layers (Allium epidermis - M0LLER & al. 1999 b) and for 
suspension ce]l cultures (root protoplasts of Allium - M0LLER & al. 1999 a). The method relies on 
conjugation of BmCl to GSH within intact tissues resulting in a fluorescent GS-bimane conjugate 
(Fig. 1). The GS-bimane fluorescence levels are well known as an indicator of GSH in living 
marnmalian tissues and are also described for plants (FRICKER & al. 2000, MEYER & FRICKER 2000, 
MEYER & al. 2001, SANCHEz-FERNANDEZ & al. 1997). A photoactivation of fluorescence from non-
conj11gated BmCI after UV excitation, as previously mentioned by FRICKER & al. (2000) was not 
observed. Further data dealed with the manipulation of the intensity of the fluorescence that is 
observed after modification of the glutathione status of the cells, either by application of oxidants 
(M0LLER & al. 1999 c) or by inhibiting glutathione synthesis by buthionine sulphoximine (M0LLER 
& al. 1999 a). For the tissue- and subcellular imaging of glutathione concentrations in sections of 
intact root tissues (Brassica oleracea), where the glutathione contents were manipulated by H
2
S 
fumigation, the staining procedure had to be adjusted (MOLLER & al. 2002 b). In the roots of Brassica, 
a sulphur demanding plant, the H
2
S treatment resulted in an increased fluorescence in the meristem 
cells, implying an enhanced glutathione content in the cytoplasm as well as in the nucleoplasm. 
These results showed the sensitivity of this method, making it possible to distinguish in the glutathione 
contents not only between different tissues but also in different cell compartments at the light 
microscopic level. 
With this background, it was of interest to collect more precise data on GSH localization and 
GSH concentrations in the cells by using transmission electron microscopy. The subcellular 
localization of glutathione was investigated with an indirect immunogold-labelling method. On all 
investigated cell sections gold particles were present after the immunoreaction, but the labelling 
intensity varied over a wide range between the compartments. Very low intensities of gold labelling 
were found in peroxisomes and the ER; high intensities could be detected in the mitochondria (Fig. 
2). The density of gold particles in chloroplasts and especially in the nuclei was lower (Fig. 2) than 
expected from either biochemical data (NocroR & al. 2002) or from staining with bimane derivates 
(M0LLER & al. 2002 b). 
M. Mtiller, B. Zechmann, A. Urbanek & G. Zellnig: Inter- and intracellular distribution of modulated... 19 
p 
cw 
Figure 2: Part of a mesophyll cell of Cucurbita pepo L. Electron micrograph showing the distribution 
of GSH in various compartments after immunolabelling; gold particles are present in the chloroplast 
(P), the nucleus (N) and in an increased number in the mitochondria (M). Bar= 1 mm. 
Conclusions 
We can conclude that with the use of the above-described methods it is possible to obtain data 
about glutathione concentrations in different compartments by light and electron rnicroscopy. But 
there is stili a need of quantification analyses of the total pool of glutathione and till now no systematic 
investigations ofthe subcellular expression of glutathione, and the distribution ofy-glutamylcysteine, 
the immediate precursor of glutathione, were done with plant tissues. Therefore the investigations 
have to be continued and intensified in this field. 
20 Acta Biologica Slovenica, 46 (2), 2003 
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