ISSN 1021-4437, Russian Journal of Plant Physiology, 2018, Vol. 65, No. 2, pp. 168–176. © Pleiades Publishing, Ltd.

, 2018.
Original Russian Text © E.V. Pradedova, O.D. Nimaeva, A.B. Karpova, N.V. Semenova, A.L. Rakevich, V.N. Nurminskii, A.V. Stepanov, R.K. Salyaev, 2018, published in
Fiziologiya Rastenii, 2018, Vol. 65, No. 2, pp. 101–110.

RESEARCH PAPERS

Glutathione in Intact Vacuoles:

Comparison of Glutathione Pools in Isolated Vacuoles, Plastids,
and Mitochondria from Roots of Red Beet1
E. V. Pradedovaa, *, O. D. Nimaevaa, A. B. Karpovac, N. V. Semenovaa, A. L. Rakevichb,
V. N. Nurminskiia, A. V. Stepanova, and R. K. Salyaeva
aSiberianInstitute of Plant Physiology and Biochemistry, Siberian Branch, Russian Academy of Sciences,
Irkutsk, 664033 Russia
b
Irkutsk Branch of the Institute of Laser Physics, Siberian Branch, Russian Academy of Sciences, Irkutsk, Russia
c
Faculty of Biology and Soil Science, Irkutsk State University, Irkutsk, Russia
*e-mail: praded@sifibr.irk.ru
Received March 20, 2017

Abstract⎯Proportions between oxidized and reduced glutathione forms were determined in vacuoles isolated
from red beet (Beta vulgaris L.) taproots. The pool of vacuolar glutathione was compared with glutathione
pools in isolated plastids and mitochondria. The ratio of glutathione forms was assessed by approved meth-
ods, such as fluorescence microscopy with the fluorescent probe monochlorobimane (MCB), high-perfor-
mance liquid chromatography (HPLC), and spectrophotometry with 5,5'-dithiobis-2-nitrobenzoic acid
(DTNB). The fluorescence microscopy revealed comparatively low concentrations of reduced glutathione
(GSH) in vacuoles. The GSH content was 104 μM on average, which was lower than the GSH levels in mito-
chondria (448 μM) and plastids (379 μM). The content of reduced (GSH) and oxidized (GSSG) glutathione
forms was quantified by means of HPLC and spectrophotometric assays with DTNB. The glutathione con-
centrations determined by HPLC in the vacuoles were 182 nmol GSH and 25 nmol GSSG per milligram pro-
tein. The respective concentrations of GSH and GSSG in the plastids were 112 and 6 nmol/mg protein and
they were 228 and 10 nmol/mg protein in the mitochondria. The levels of GSH determined with DTNB were
1.5 times lower, whereas the amounts of GSSG were, by contrast, 1.5–2 times higher than in the HPLC
assays. Although the glutathione redox ratios depended to some extent on the method used, the GSH/GSSG
ratios were always lower for vacuoles than for plastids and mitochondria. In vacuoles, the pool of oxidized glu-
tathione was higher than in other organelles.

Keywords: Beta vulgaris, vacuoles, glutathione, mitochondria, plastids

DOI: 10.1134/S1021443718020048

INTRODUCTION condition of organisms, their cells, and cellular com-

In plant and animal organisms, glutathione
(γ-glutamyl-cysteinyl-glycine) is involved in multiple
biochemical processes, such as changes in oxidation
state of molecules, protein folding, signal transduc-
tion, detoxification, etc. [1–4]. Owing to its nucleop-
hilic properties and relative stability, glutathione is an
extremely active redox cofactor for diverse redox reac-
tions [5]. Because of high antioxidant activity and
large content of glutathione in cells and biological flu-
ids, this antioxidant serves as the main redox agent in
aerobic organisms [4]. Therefore, the physiological
1 Abbreviations:
ABC transporters—ATP-binding cassette trans-
porters; DTNB—5,5′-dithiobis-2-nitrobenzoic acid; GSB—glu-
tathione S-bimane, conjugate of glutathione with S-monochloro-
bimane; GSHtot—total amount of glutathione; GSH—concen-
tration of reduced glutathione; GSSG—concentration of oxidized
glutathione form; MCB—monochlorobimane.
partments is often assessed from the total amount of
glutathione (GSHtot) that represents the sum concen-
tration of its reduced (GSH) and oxidized (GSSG)
forms [1].
The content of GSH and GSSG is affected by var-
ious factors [1, 2, 5]. Changes in the GSH/GSSG
ratio represent a useful indicator of the redox state of
cells and whole organisms. The GSH/GSSG ratio
contains information on the resistance of organisms to
the action of stress factors [1, 4, 5]. However, the
application of this indicator is limited due to variable
glutathione content and its redox state in various
organisms and even in various cells and tissues of the
same organism. For example, the main pool of gluta-
thione in plants is concentrated in cells of photosyn-
thetic and storage tissues. Furthermore, the distribu-
tion of glutathione among intracellular structures is

168
 GLUTATHIONE IN INTACT VACUOLES: COMPARISON OF GLUTATHIONE POOLS
also uneven. The highest concentrations were found in
mitochondria, cytosol, and chloroplasts [1, 5]. The
central vacuole in plant cells is known to perform the
storage function. One could expect the accumulation
of glutathione in the vacuolar compartment, but the
experimental results showed either the absence or very
low concentrations of glutathione in vacuoles [1].
Nevertheless, the accumulation of glutathione in vac-
uoles was noted in some cases under the action of
stress factors [6]. At the same time, the selective trans-
port of GSSG into the vacuole by ABC transporters of
the vacuolar membrane (tonoplast) has been estab-
lished [3, 4]. Based on these facts, it was proposed that
the active accumulation of GSSG by central vacuoles
under stress conditions facilitates the maintenance of a
high GSH/GSSG redox ratio in the cytosol [3, 4, 6–8].
The presence of glutathione in the vacuolar com-
partment was considered until now to be tissue- and
species-specific, because information on its content in
vacuoles of different organs and tissues of various
plants was quite limited [1, 8]. In this connection, the
present work aimed at studying the pool of glutathione
in the vacuoles red beet (Beta vulgaris L.) roots. It
seemed worthwhile to compare the glutathione pool in
vacuoles with its pools in other cell compartments that
are characterized by a high content of glutathione. As
already noted, the highest glutathione concentrations
in plant cells were observed in mitochondria and chlo-
roplasts [1].
MATERIALS AND METHODS
Plant material. Experiments were performed with
roots of red beet (Beta vulgaris L., cv. Bordo) in the
period of physiological dormancy. Beetroots were
grown on the experimental plot of Siberian Institute of
Plant Physiology and Biochemistry (Siberian Branch
of the Russian Academy of Sciences) and stored at 4°C
throughout the winter period.
Isolation of vacuoles. Vacuoles from the main root
tissue were isolated with a modified macromethod [9].
The purity of the fractions was checked using an
NU-2E light microscope (Carl Zeiss, Germany) and
biochemical assays of marker enzymes specific of mito-
chondria, plastids, cytosol, etc. [10, 11].
Isolation of plastids. The plastids were isolated with
a conventional method [10]. Since the cells of red beet
roots contain mostly leukoplasts, a Percoll density gra-
dient (20, 30, 50, and 70%) was used to fractionate the
organelles at the final stages of isolation [10, 11]. The
plastid fraction of the highest purity was collected at
the interface between 30 and 50% Percoll layers.
Isolation of mitochondria. Mitochondria were iso-
lated by a conventional method [10]. The isolated
organelles were purified using a Percoll density gradi-
ent (18, 23, and 35%). The fraction enriched with
intact mitochondria was collected at the interface
between 23 and 35% percoll layers [10].
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 65
169
Fluorescence microscopy. The dye monochlorobi-
mane (MCB) interacts with GSH to form a fluores-
cent conjugate (GSB). The formation of GSB in iso-
lated organelles was observed by means of fluores-
cence microscopy. Two microscopes with different
facility levels were used: the MicroTime 200 (Pico-
Quant Gmbh, Germany) and the Axio Observer Z1
(Carl Zeiss, Germany). The confocal laser scanning
fluorescence microscope MicroTime 200 was suitable
only for observations with vacuoles. This microscope
permitted long-term imaging of individual vacuoles in
an autonomous mode. The Axio Observer Z1 fluores-
cent microscope equipped with AxioCam MRm
monochrome digital camera was used to determine
GSB formation at a limited time interval in all the
organelles examined (vacuoles, plastids, and mito-
chondria). The absorbance (extinction) was measured
at a wavelength of 375 nm, and emission was recorded
at 470–520 nm [12]. The isolated organelles were
incubated at room temperature for 1, 5, 10, 20, 30, 40,
and 60 min. The vacuoles were incubated in the
medium containing 500 mM KCl, 150 mM sucrose,
1 mM EDTA, and 10 mM Tris–HCl (pH 7.4); the
incubation medium for plastids contained
25 mM KCl, 250 mM sucrose, 1 mM EDTA, and
50 mM Tris–HCl (pH 7.2). The mitochondria were
incubated in the medium containing 300 mM sucrose,
1 mM EDTA, 0.1% BSA, and 10 mM Tris–HCl
(pH 7.5). In all the treatments, except for control con-
ditions, the incubation media were supplemented with
0.1 mM MCB (Sigma, United States). In one supple-
mentary treatment, the vacuoles were incubated in the
presence of both MCB and 10 mM Na3VO4, an inhib-
itor of the tonoplast ABC transporters [13]. The opti-
cal images were obtained in an automated mode under
identical times of signal acquisition and at equal set-
tings for the microscope used. The images were ana-
lyzed using the tools of AxioVision (Rel.4.8) and
ImageJ software. Glutathione concentrations were
calculated from the calibration curves plotted for each
experiment using GSH of chemically pure grade
(Sigma) at concentrations of 10, 25, 50, 100, 250, and
500 μM and 0.1 mM monobromobimane (MBB,
Sigma).
Glutathione extraction. Glutathione was extracted
after mixing the isolated organelles with a cooled solu-
tion containing 5% HPO3, 0.1% HCOOH, and
1 mM EDTA at a ratio of 1 : 2 (v/v) [14]. For compari-
son, glutathione was determined in root tissue extracts.
The tissue was homogenized at a ratio of 1 : 6 (w/v) in
the same solution under cooling with liquid nitrogen.
The extracts were centrifuged for 20 min at 4°C and
14500 g.
HPLC. High-performance liquid chromatography
was carried out after additional purification of the
samples. The extracts were applied to Sep-Pak C18
cartridges [14]. Before that, the cartridges were
washed out first with the concentrated CH3OH and
No. 2 2018
170 PRADEDOVA et al.
then with 0.5% HCOOH. The volume of applied sam-
ple did not exceed 1 mL. Thereafter, the cartridges were
washed with HCOOH-acidified deionized water and
then eluted with a mixture of 5% CH3OH and
0.5% HCOOH. The eluate was dried out using a
rotary evaporator IKA HB10 basic (WENK LabTec
GmbH, Germany). Measurements were carried out
with a Milikhrom A-02 liquid microcolumn chro-
matograph (Russia) under the following conditions:
2 × 75 mm column; ProntoSil 120-5-C18 AQ0838
sorbent (reversed phase); eluent A: 0.2 M LiClO4 and
0.005 M HClO4; eluent B: CH3CN; 40°C column
temperature; 0.34 s time constant; 210 nm wavelength;
100 μL/min flow rate; gradient chromatography (the
gradient from 5 to 100% CH3CN within 10 min). The
concentration was calculated from the calibration
curves plotted for standard concentrations of chemi-
cally pure GSH and GSSG (Sigma).
Spectrophotometric assay of glutathione is based on
the interaction of GSH and 5,5'-dithiobis-2-nitrobenzoic
acid (DTNB), called the Ellman reagent. The reaction
produces a chromophore, 5-thio-2-nitrobenzoic acid
(TNB). We used the conventional method [15]. In
order to block SH groups, 2-vinylpyridine (2%) was
used. The TNB formation was recorded with an Immu-
noChem-2100 (High Technology, United States) flat-
bed photometer at a wavelength of 405 nm [15].
Glutathione concentration determined by
MCB-based fluorescence microscopy was expressed
in micromoles per liter (μM) GSH, whereas concen-
trations determined by spectrophotometric assays and
HPLC were expressed in nanomoles of GSH or
GSSG per 1 mg protein. The protein concentration
was determined by the Bradford method [16]. The
total concentration of glutathione (GSHtot) in the
samples was determined by summing the concentra-
tions of GSH and GSSG.
All experiments were repeated five times on aver-
age, with three assays per treatment. Tables and graphs
show the mean values and their standard errors. Sig-
nificant differences between treatment means were
analyzed with the Student’s t-test for independent
samples.
RESULTS
Several methods for the determination of glutathi-
one have been developed to date. Quantitative meth-
ods include the HPLC-based and spectrophotometric
enzymatic in vitro analyses as well as the methods
based on the enzyme immunoassays and laser (confo-
cal or two-photon) scanning microscopy for in situ
measurements. However, none of these methods pro-
vides complete information because of the limitations
inherent to these methods [1, 2, 17–19]. These limita-
tions complicate the comparison and analysis of the
results. Various research groups expressed glutathione
concentrations in different units, which also hampered
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
the comparison of the results. For the same organs of
identical plant species, glutathione concentrations
were presented in units of nmol/g fr wt; nmol/mg pro-
tein; mmol/cell, etc. [1, 6, 8, 12, 14]. In order to
extend our knowledge on the glutathione content in
isolated organelles of red beetroots, we applied three
commonly used methods.
Fluorescence Microscopy with the Use
of Monochlorobimane
MCB permeates comparatively easy into the cell
and cell compartments where the enzyme glutathione
S-transferase (GST) ensures rapid reaction of MCB
with the thiol group of GSH, thus producing the fluo-
rescent conjugate GSB [20]. Thus, the presence of
GSH can be detected by MCB method on the condi-
tion that the fluorescent probe penetrates readily into
the cell compartments and GST activity is sufficiently
high. Our previous studies revealed the presence of
GST in the vacuoles of red beet taproots [11]. The flu-
orescence intensity of isolated vacuoles incubated with
MCB was found to increase, which implies the pene-
tration of the dye into the organelles and interaction of
MCB with the vacuolar GSH. Although isolated vac-
uoles represented an adequate system for the detection
of GSH with the fluorescent probe, the fluorescent
assay had some specific features to be kept in mind.
For example, the maximum fluorescence intensity
corresponding to the nearly complete interaction of
MCB with GSH was only observed after prolonged
incubation of vacuoles with MCB (≥40 min), whereas
10 min incubation was sufficient in the case of isolated
mitochondria and plastids. Another feature of isolated
vacuoles is their attachment to the glass slide, which
restricted their mobility. It was thus possible to exam-
ine individual organelles from the first stage of auto-
fluorescence assay during the whole period of incuba-
tion of vacuoles in the presence of MCB. Figure 1
shows the result of a representative experiment. In
addition, not all vacuoles retained their integrity
throughout the experiment, and the GSH quantities in
stable vacuoles differed substantially. Table 1 lists the
GSH concentrations determined in this experiment. It
should be noted that fluorescence of MCB per se did
not change during long incubation (40–60 min) under
our experimental conditions. No changes were
detected in the reactivity of this compound.
In studies with whole cells, the inhibitors of tono-
plast ABC transporters were commonly applied to
prevent the transfer into the vacuole of GSB formed in
the cytosol [12]. The tonoplast ABC transporters, like
some animal ABC transporters, are inhibited by vana-
date (sodium orthovanadate, Na3VO4) [1, 13]. There-
fore, in a series of experiments, we treated the vacuoles
with 10 mM Na3VO4. It should be mentioned, how-
ever, that isolated vacuoles are deprived of any external
source of GSH and that the vacuolar amounts of ATP
are insufficient for functioning of ABC transporters.
Vol. 65 No. 2 2018
 GLUTATHIONE IN INTACT VACUOLES: COMPARISON OF GLUTATHIONE POOLS 171

(a) (b) (c) (d) (e) (f)

1 4 4 4 4 4 1 4
1 1 1 1
2 5 2 5 2 5 2 5 2 5 5
7 7 7 7 7 7
3 6 6
3 6 3 6 6 6
[vacuoles][A.U.] [vacuoles][A.U.] [vacuoles][A.U.] [vacuoles][A.U.]
1 42.7 1 44.1 1 56.1 1 94.6
4 39.7 4 44.4 4 51.7 4 73.5
5 23.7 5 24.9 5 30.2 5 45.3
50 µm 6
7
27.9
26.2
6
7
30.2
36.8
6
7
35.1
71.5
6
7
44.9
101.7 50 µm

Fig. 1. Determination of GSH in isolated vacuoles by microscopy with the fluorescence probe monochlorobimane (MCB).
(a) Selection of the “experimental area of interest” in the analyzed sample, light microscopy; (b) autofluorescence of organelles,
fluorescence microscopy; (c) fluorescence after 5 min incubation with MCB (0.1 mM); (d) fluorescence after 20 min incubation
with MCB; (e) fluorescence after 40 min incubation with MCB; (f) morphological view of vacuoles in the analyzed area at the
end of the experiment, light microscopy. Numbers 1–7 enumerate individual isolated vacuoles. Arbitrary units (A.U.) are relative
units representing the fluorescence intensity.

The accumulation of GSB in individual vacuoles due
to the active transport across the tonoplast of conju-
gates formed in other vacuoles and released into the
medium seemed unlikely. In accord with these expec-
tations, the inhibitor added to the incubation medium
did not affect the average values of GSH concentra-
tion (Fig. 2). However, vanadate was found to destabi-
lize the tonoplast. By the end of the experiment, only
30% of all initially observed organelles were retained.
When glutathione was detected in isolated mito-
chondria and plastids, these organelles were incu-
bated in MCB-containing or MCB-free solutions for
1, 10, 20, 30, and 40 min. We found that 10-min
incubation was sufficient. A longer incubation did
not lead to a statistically significant increase in f luo-
rescence intensity. Figure 3 shows the results of a rep-
resentative experiment.
Table 2 summarizes the data obtained with this
method. The highest GSH content was found in the
mitochondria. The concentration of GSH in the plas-
tids was slightly lower, and the concentration of GSH
in the vacuoles was approximately fourfold lower than
in mitochondria.
Table 1. Glutathione content in individual vacuoles
Concentration of GSH
Vacuole no.
in individual vacuoles, μM
1 135.4
2 Integrity lost
3 Integrity lost
4 87.2
5 55.6
6 42.8
7 194.8
Data in table are the results of a representative experiment illus-
trated in Fig. 1.
Determination of Glutathione with Ellman’s Reagent
The determination of glutathione with Ellman’s
reagent is currently one of the most popular methods,
because the oxidized and reduced glutathione forms
can be quantified [17–19]. A substantial drawback of
this method is that the content of GSH and GSSG can-
not be determined simultaneously. The quantification
of GSSG requires the pretreatment of the analyzed
sample. Another disadvantage of this method is that the
reaction of GSSG reduction is cyclic, which lowers the
precision of GSSG determinations [19]. Thus, only
approximate estimations of GSSG content and GSHtot
are possible [17, 18]. In the examined samples, the
amount of GSSG was relatively high (Table 3). The
highest concentration of GSSG was observed in vacu-
oles (20% of vacuolar GSHtot content), while markedly
lower relative concentrations of GSSG were noted in
plastids (10.8%) and mitochondria (9.9%). The esti-
mated GSH concentrations were comparable with the
literature data [1]. The highest content of GSH was typ-
ical of mitochondria and the lowest content was
recorded in plastids. The concentrations of GSH in
vacuoles and plastids were similar. However, the total
GSH content in vacuoles was higher than GSHtot con-
tent in plastids because of the high vacuolar GSSG con-
centration. It should be noted that the glutathione con-
tent was noticeably lower in the organelles than in the
tissue extracts (Table 3). Based on the concentrations
of GSH and GSSG, the glutathione redox ratios were
quantified. The lowest ratio was attributed to vacu-
oles (2.9), while the highest to mitochondria (8.1).
These values reflect a comparatively high pool of oxi-
dized glutathione in vacuoles and a relatively large pool
of reduced glutathione in mitochondria.
Determination of Glutathione by HPLC
HPLC is considered to be a sensitive and rather
specific method for quantification of various com-

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 65 No. 2 2018

172 PRADEDOVA et al.

150 the procedure of sample pretreatment and can lead to

the losses in total content of glutathione.
The GSH concentrations determined by HPLC in
our study, in contrast to expectations, were slightly
higher than those obtained with the DTNB method.
At the same time, the GSSG content was 1.5–2 times
Glutathione content, µM

100 lower (Tables 3 and 4). Despite these obvious differ-

50
0 man’s reagent, the concentrations of GSHtot in vacuoles
Control
Fig. 2. Content of GSH in isolated vacuoles as determined
by microscopy with the fluorescent probe monochlorobi-
mane (MCB). In the control treatment, the incubation
medium contained 0.1 mM MCB; in the treatments with
an inhibitor, the incubation media contained 0.1 mM MCB
and 10 mM Na3VO4, an inhibitor of the tonoplast ABC
transporters.
pounds. The reverse phase HPLC with UV detection,
unlike the spectrophotometry with DTNB reagent,
allows simultaneous determinations of GSH and
GSSG without preliminary enzymatic reduction [21].
However, the additional purification step complicates
ences, the general trend remained consistent with the
other method data. The highest GSSG concentra-
tion occurred in vacuoles (10% of vacuolar GSHtot)
and the lowest GSSG content was observed in mito-
chondria (3.9%). At the same time, the largest con-
tent of GSH was in mitochondria and the lowest GSH
content was in plastids, like it was found with Ellman’s
reagent. In addition, the highest glutathione redox
ratio, GSH/GSSG (23.7) was found for mitochondria;
it markedly exceeded the GSH/GSSG ratios in other
materials (7.4, 19.8, and 13.9 for vacuoles, plastids, and
tissue extracts, respectively). Like it was found with Ell-
Inhibitor
and mitochondria were similar. The tissue extracts were
characterized by the highest GSHtot content, in consis-
tency with previous experiments.
DISCUSSION
Determination of glutathione in isolated organelles
is a widely applied approach, but it is potentially threat-
ened by the partial loss of low-molecular-weight com-
pounds during procedures of separation and purifica-
tion of organelles [22]. According to a shared notion,
the most reliable data on GSH content in organelles can
be obtained by studying whole cells with fluorescence
microscopy and fluorescent dyes [1, 12]. The results of

10 µm (b) 10 µm 10 µm (d) 10 µm
(a) (c)

II

10 µm 10 µm (c) 10 µm 10 µm
(a) (b) (d)

Fig. 3. Determination of glutathione in isolated mitochondria (I) and plastids (II) with the fluorescent probe monochlorobimane
(MCB). (a) The sample viewed under light microscopy; (b) autofluorescence of organelles; (c) sample viewed under light micros-
copy; (d) fluorescence after 10-min incubation of organelles in the presence of 0.1 mM MCB.

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 65 No. 2 2018

 GLUTATHIONE IN INTACT VACUOLES: COMPARISON OF GLUTATHIONE POOLS 173

Table 2. Glutathione content in isolated organelles established by fluorescence microscopy with monochlorobimane
Glutathione concentration, μM
Redox form of glutathione vacuoles plastids mitochondria
GSH 103.9 ± 30.6 378.8 ± 34.2 447.9 ± 52.1
Data in table are mean values and their standard errors (n = 50). Differences between the means for various organelles are significant
at P < 0.001.

Table 3. Glutathione content in beetroot organelles determined with Ellman’s reagent

Redox form and redox Glutathione concentration, nmol/mg protein
ratio of glutathione vacuoles plastids mitochondria tissue extract
GSH 90.2 ± 13.1 80.5 ± 1.9 136.8 ± 6.2 179.2 ± 11.5
GSSG 30.5 ± 7.3 10.8 ± 0.5 16.8 ± 5.4 25.2 ± 9.8
GSHtot 151.2 ± 27.7* 102.1 ± 2.9 170.4 ± 16.9* 229.6 ± 31.1
GSH/GSSG 2.9 7.4 8.1 7.1
GSSG, % 20.1 10.5 9.9 10.9
Data in table are mean values (n = 8) and their standard errors. Differences between the treatment means are significant at P < 0.001.
* Differences are significant at P < 0.05.

Table 4. Glutathione content in beetroot organelles determined with HPLC

Redox form and redox Glutathione concentration, nmol/mg protein
ratio of glutathione vacuoles plastids mitochondria tissue extract
GSH 182.3 ± 31.9 112.8 ± 7.1 227.5 ± 35.8 378.4 ± 51.2
GSSG 24.5 ± 0.8* 5.7 ± 0.7 9.6 ± 1.1 27.1 ± 6.7*
GSHtot 231.3 ± 33.5* 124.2 ± 8.5 246.7 ± 33.9* 432.6 ± 64.6
GSH/GSSG 7.4 19.8 23.7 13.9
GSSG, % 10.6 4.6 3.9 6.3
Data in table are mean values (n = 5) and their standard errors. Differences between the treatment means are significant at beetroot < 0.001.
* Differences are statistically insignificant.

such studies suggest that this technique quantifies the
content of GSH in various cell compartments, except
for the central vacuole [12]. Determinations of GSH
in the vacuoles in situ are hampered by the vacuolar
sequestration of GSB that is formed in the cytosol and
carried across the tonoplast by the tonoplast ABC
transporters [1, 3, 4]. In some studies, the presence of
glutathione in vacuoles of whole cells was detected
with MCB; however, these results were interpreted as
being due to the accumulation of GSB of cytosolic ori-
gin [3, 4, 7, 12]. The limitations inherent to whole cells
prompted us to perform an investigation with isolated
vacuoles.
Fluorescence microscopy with MCB as a thiol
probe clearly demonstrated the presence of GSH in
the analyzed organelles. This method is considered
to be the most specific, because the rate of nonenzy-
matic conjugation of MCB with thiol group of GSH
is very low, while the GSTs bind specifically MCB to
GSH [12]. At the same time, HPLC and spectropho-
tometry with DTNB allow researchers to quantify
both the GSH and GSSG pools as well as the
GSH/GSSG redox ratio, i.e., to assess the glutathi-
one redox state in each compartment.
Our study with red beet taproots revealed the larg-
est amounts of GSH in mitochondria. According to
HPLC and spectrophotometry with DTNB, the low-
est GSH content was typical of plastids, whereas fluo-
rescence microscopy with MCB reported the lowest
GSH content in vacuoles. It is possible that the accu-
racy of the results in experiments with the fluorescent
probe was influenced by the structural features of vac-
uoles. As already mentioned above, the fluorescence
intensity of isolated vacuoles was subject to a slow
increase, in contrast to stable fluorescence levels in
isolated plastids and mitochondria. One explanation
of this slow increase is the tonoplast permeability to
MCB. It is possible that the rate of dye accumulation
inside the organelles was also influenced by their size.
The average diameter of isolated vacuoles (50 μm) is

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 65 No. 2 2018

174 PRADEDOVA et al.
an order of magnitude larger than the average diameter
of mitochondria and leukoplasts (2–4 μm). Another
possible reason is the low activity of GST. According
to our earlier study, the vacuolar GST had rather high
activity at neutral pH, while the activity decreased
substantially under weakly acidic conditions [11]. It is
reasonable to suppose that the formation of GSB con-
jugates proceeds slower in vacuoles than in other
investigated organelles, because the vacuolar pH is low
compared to the pH levels in other organelles, which
are closer to the pH optimum of GST functioning. In
our view, the slow increase in fluorescence of vacuoles
should be taken into account when the compartmen-
talization of glutathione in whole plant cells is consid-
ered. It is probably because of these circumstances
that some researchers were unable to detect GSH in
vacuoles using MCB [12].
On the whole, the results obtained by various
methods of glutathione determination were compara-
ble. They demonstrated the presence of glutathione in
vacuoles of storage parenchyma in dormant red beet
roots. However, the presence of GSH in vacuoles was
not proven for all plant organs and tissues. For exam-
ple, immunohistochemical assays revealed either the
lack or insignificant amounts of vacuolar GSH in the
leaves and roots of Arabidopsis thaliana, Nicotiana
tabacum, and Cucurbita pepo [8, 23, 24]. The authors
explained these observations by rapid degradation of
glutathione after its entry into the vacuole [23, 24]. On
the other hand, some researchers reported the pres-
ence of glutathione in vacuoles. The concentration of
vacuolar GSH in A. thaliana leaves determined by
HPLC ranged from 613 to 733 μM; the GSH concen-
trations in plastids and the cytosol were 2.5–3.0 and
3.0–3.5 mM, respectively [22]. The micromolar con-
centrations of vacuolar glutathione established in the
present work agree with the results of an earlier
study [25]. In a different investigation performed with
mesophyll cells of A. thaliana, the vacuolar concentra-
tion of GSH determined by the spectrophotometric
method was 17.6 nmol/g fr wt, and the estimates based
on MCB method gave the value of 30 μM [6]. The
GSH concentration in the vacuoles of mesophyll cells
of N. tabacum was approximately 20 μM (100 times
lower than in chloroplasts) and corresponded to 17%
of the total GSH content in the cells [25]. Other plant
materials have also been examined. For example, in
protoplasts isolated from the mesophyll of Hordeum
vulgare leaves, the content of GSH and GSSG in
vacuoles determined by means of DTNB method was
0.9 and 12%, respectively, of the total cellular gluta-
thione [7]. In Pisum sativum leaves, the proportion of
vacuolar glutathione determined with HPLC was as
high as 30% of the cellular glutathione content [26]. In
A. thaliana plants, the content of vacuolar GSH with
respect to its total amount was 5.4% under control
conditions and increased sharply to 26% after subject-
ing the plants to oxidative stress. At the same time, the
content of glutathione in other compartments (cyto-
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
sol, mitochondria, peroxisomes, and nuclei) was found
to decrease upon oxidative stress [6]. In the same
study, it was noted that the glutathione levels in cell
compartments vary depending on the units of mea-
surement. Thus, the highest concentrations of GSH
determined with MCB were found in mitochondria
(6.1 mM) and the lowest levels were recorded in vacu-
oles (0.03 mM). However, a different distribution pat-
tern was observed when the content of GSH was
expressed in nmol/g fr wt: the highest content of GSH
(89.6 nmol/g fr wt) was found in the plastids, whereas
GSH contents in vacuoles and mitochondria were sim-
ilar (17.6 and 23.5 nmol/g fr wt) [6]. In our present
study, the glutathione content in vacuoles, plastids,
and mitochondria also varied significantly depending
on the method applied; variations were especially
noticeable when the content was expressed in different
relative units.
The concentrations of GSH in vacuoles, chloro-
plasts, and mitochondria were previously compared
for several plant species [6, 8, 22]. In those studies
where occurrence of GSH in vacuoles was reported,
its amounts in vacuoles were much lower than in
plastids. The plastids together with the cytosol are the
main locations where GSH is synthesized in plant
cells. According to available data, these organelles
contain up to 60–70% of the total cellular glutathi-
one [5, 22, 25, 27]. According to other data, the
amount of glutathione in chloroplasts may be much
less than in the cytosol and mitochondria [6]. For
example, in P. sativum leaves, the chloroplasts con-
tained only 10% of the total cellular glutathione [17].
Thus, the issue of high GSH content in plastids
remains controversial [25]. Apparently, variable data
on GSH content are due to multiple factors, includ-
ing the stressed plant conditions. In A. thaliana
plants subjected to oxidative stress, a slight increase
in the concentration of glutathione in plastids was
noted [6].
As mentioned above, the physiological conditions of
the organism, its cells, and cell compartments can be
assessed by taking into account GSHtot together with
the concentrations of GSH and GSSG forms [1, 4].
Since the organs and tissues of an individual plant dif-
fer in the content of glutathione, there are certain dif-
ficulties in comparing GSHtot, even in plants of the
same species. In some cases, the percentage of GSH
and GSSG concentrations in GSHtot provides a more
reliable characteristic. The content of GSH in plant
organs usually equals 90–99%, and the content of
GSSG is in the range of 1–10% [1]. In fact, the per-
centage of GSSG varies greatly depending on environ-
mental conditions, developmental phase, and func-
tional characteristics of organs and tissues. This per-
centage is sometimes used as a measure of the redox
state. Thus, in young P. sativum plants, the foliar GSSG
content accounted for 12–14% of GSHtot, and this
fraction in roots was 20%. During the dark period,
Vol. 65 No. 2 2018
 GLUTATHIONE IN INTACT VACUOLES: COMPARISON OF GLUTATHIONE POOLS
these values increased to 18 and 31% for the leaves and
roots, respectively [17]. By contrast, in Zea mays seed-
lings, the percentage of GSSG was higher in coleop-
tiles (24–32%) than in roots (2.3–7.6%) [28]. In sugar
beet main root, the GSSG percentage varied from 16
to 24% throughout the growth period under various
growing conditions [29]. The results of the present
study show that the percentage of GSSG in physiologi-
cally dormant main root of red beet (6 and 10% as
determined by HPLC and spectrophotometric meth-
ods, respectively) is significantly lower than in the sugar
beet main root sampled during the growth period.
In organelles of red beet main root, the percentage of
GSSG was relatively low in mitochondria and plastids,
but it was rather high in vacuoles. In the case of intact
organelles, the self-oxidation of glutathione may occur
during the isolation procedure. However, noncatalytic
chemical oxidation involves the deprotonation of the
thiol form (GSH) to thiolate (GS–) and depends on the
pH conditions. Since pKa values of GSH are close to
9.0, only 1% of GSH is in its deprotonated form at
pH 7.2 (per time unit). This percentage becomes sig-
nificantly lower in the acidic compartments, e.g., in
vacuoles (pH 5.5) [30]. A high pool of vacuolar GSSG
may result from the catalytic oxidation or inadequate
catalytic reduction of glutathione in vacuoles.
The GSH/GSSG redox ratio is the most popular
indicator of metabolic activity and stress occurrence in
cell compartments and whole organisms. This ratio is
considered to be a more precise measure of plant resis-
tance to the action of stress factors than the extent of
GSHtot [1, 4, 5, 7]. In our study, the glutathione redox
ratio in the vacuoles of beetroots was lower than that in
plastids and mitochondria, irrespective of the method
of determination. This conclusion is consistent with the
results obtained previously by other researchers. The
vacuoles isolated from protoplasts of H. vulgare leaves
were also characterized by a high content of GSSG and
by the diminished GSH/GSSG ratio (1.3), which was
much lower than the GSH/GSSG ratio of 17.0 typical
of protoplasts [7].
The high content of GSSG in vacuoles is probably
due to the fact that the tonoplast ABC transporters
are predominantly engaged in the transport of the
oxidized glutathione form [1, 27]. There are two
notions concerning physiological role of GSSG
transport into the vacuole. According to the first one,
GSSG is excreted from the cytosol to maintain redox
homeostasis [27]. The second notion is that the
GSSG molecule is less reactive than GSH and,
therefore, is more suitable for transport [1]. Despite
the predominant accumulation of GSSG by vacu-
oles, the concentration of GSH in the vacuoles of red
beet main root was significantly higher than the con-
centration of GSSG. Apparently, the glutathione
transported into the vacuoles undergoes redox con-
versions that affect its redox state.
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 65
175
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Translated by A. Bulychev
Vol. 65 No. 2 2018