Sterol binding by methyl-b-cyclodextrin andnystatin – comparative analysis of biochemical andphysiological consequences for plantsJulia Valitova1, Albina Sulkarnayeva1, Ekaterina Kotlova2, Anastasia Ponomareva1, Fakhima K.Mukhitova1, Lyaisan Murtazina3, Irina Ryzhkina3, Richard Beckett4 and Farida Minibayeva1
1 Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, Kazan, Russia
2 Komarov Botanical Institute, Russian Academy of Sciences, St Petersburg, Russia
3 Institute of Organic and Physical Chemistry, Russian Academy of Sciences, Kazan, Russia
4 School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, Scottsville, South Africa
Keywords
glycoceramides; methyl-b-cyclodextrin;
nystatin; plant sterols; wheat roots
Correspondence
F. Minibayeva, Kazan Institute of
Biochemistry and Biophysics, Russian
Academy of Sciences, Lobachevsky Str. 2/
31, Kazan 420111, Russia
Fax: +7 8432927347
Tel: +7 843 2319045
E-mail: [email protected],
(Received 30 September 2013, revised 11
February 2014, accepted 19 February 2014)
doi:10.1111/febs.12761
The dependence of membrane function on its sterol component has been
intensively studied with model lipids and isolated animal membranes, but
to a much lesser extent with plant membranes. Depleting membrane sterols
could be predicted to have a strong effect on membrane activity and have
harmful physiological consequences. In this study, we characterized mem-
brane lipid composition, membrane permeability for ions, some physiologi-
cal parameters, such as H2O2 accumulation, formation of autophagosomal
vacuoles, and expression of peroxidase and autophagic genes, and cell via-
bility in the roots of wheat (Triticum aestivum L.) seedlings in the presence
of two agents that specifically bind to endogenous sterols. The polyene
antibiotic nystatin binds to endogenous sterols, forming so-called ‘nystatin
pores’ or ‘channels’ in the membrane, and methyl-b-cyclodextrin has the
capacity to sequester sterols in its hydrophobic core. Unexpectedly,
although application of both methyl-b-cyclodextrin and nystatin reduced
the sterol content, their effects on membrane permeability, oxidative status
and autophagosome formation in roots differed dramatically. For compari-
son, we also tested the effects of the antibiotic gramicidin S, which does
not bind to sterols but forms nonspecific channels in the membrane. Gram-
icidin S considerably increased membrane permeability, caused oxidative
stress, and reduced cell viability. Our results suggest that a decrease in the
sterol content is, in itself, not sufficient to have deleterious effects on a cell.
The disturbance of membrane integrity, rather than the decrease in the
sterol content, is responsible for the toxicity of sterol-binding compounds.
Introduction
Sterols are integral components of the membrane lipid
bilayer. The plasma membranes (PMs) of higher plant
cells contain a complex mixture of sterols [1], unlike
those in fungi and mammals, where ergosterol and
cholesterol, respectively, are the predominant sterol
forms. The predominant sterols in plants are b-sitos-
terol, campesterol, and stigmasterol, and the presence
of cholesterol has also been demonstrated [2]. At pres-
ent, plant sterols and their roles in cellular processes
are being intensively investigated. In the plant PM,
sterols are involved in the modification of membrane
fluidity and permeability [3], regulation of the activity
Abbreviations
Deff, effective hydrodynamic diameter; DW, dry weight; MbCD, methyl-b-cyclodextrin; PM, plasma membrane; f-potential, electrokinetic
potential.
FEBS Journal 281 (2014) 2051–2060 ª 2014 FEBS 2051
and distribution of membrane-bound proteins [4], and
the formation of detergent-insoluble polar regions of
increased metabolic activity [5,6]. Sterols can control
the positioning of membrane-bound proteins such as
receptors, enzymes, and components of signaling path-
ways [7,8]. An important property of sterols is their
high affinity for sphingolipids, which provides optimal
properties for interaction between these lipid species
[9]. Hydrophilic interactions occur between the hydro-
xyl groups of the sterols and the sphingolipid acyl
chains [10]. This enables sterols to fill the voids
between the sphingolipid acyl chains, resulting in a
high packing density and promoting the formation of
lipid microdomains. The association of proteins and
lipids with membrane microdomains has emerged as
an important regulator of signal transduction, protein
and membrane polarized intracellular sorting, and
cytoskeletal organization [11]. Therefore, a disturbance
of membrane sterols might have a strong impact on
membrane integrity and activity.
A common experimental approach for elucidating
the roles of membrane sterols is the binding of sterols
with agents such as nystatin and methyl-b-cyclodextrin(MbCD) (Fig. 1), which specifically bind to endoge-
nous sterols [12]. The polyene antibiotic nystatin has a
broad spectrum of antimycotic activity, and is used in
medicine to suppress the growth of pathogenic fungi
[13]. Several views regarding the possible mechanisms
of nystatin interactions with membrane lipids exist in
the literature. For example, it has been suggested that
nystatin membrane partitioning is affected by the
extent of regular sterol distribution, the so-called ‘ste-
rol superlattice’ model [14]. According to the other
view, nystatin can form so-called ‘nystatin pores’,
which consist of an equal number of molecules of anti-
biotics and sterols with hydrophilic hydroxyl groups
along the pore surface [15,16]. In microorganisms, nys-
tatin induces the loss of potassium ions, phosphates,
sugars, and other compounds, leading to cell death
[17,18]. Recently, we demonstrated the ability of nysta-
tin to bind to plant sterols, decreasing their level and
increasing membrane permeability for ions in wheat
roots [19]. Previously, we also showed other nystatin-
induced changes in root physiology, such as shifts in
membrane potential, rates of respiration, and heat pro-
duction [20]. Given these findings, we assumed that
binding of sterols by nystatin can cause toxicity and
eventually result in cell death in plants. However, the
precise mechanisms of nystatin toxicity remain uncer-
tain. The aim of the present study was to elucidate the
significance of sterol depletion for membrane integrity
and the physiological status of wheat roots. To under-
stand the physiological effects of sterol depletion, we
used two other agents that share some similarities with
nystatin but differ in the mechanisms of their mem-
brane binding. The polypeptide antibiotic gramici-
din S, like nystatin, can form channels in membranes,
owing to it binding to phospholipids but not sterols.
The other agent, MbCD, is known to bind to sterols
and cause sterol depletion, but it does not form chan-
nels. Cyclodextrins are cyclic glucose oligomers that
have the capacity to sequester lipophilic molecules in
A B
C
Fig. 1. Chemical structures of: (A) MbCD;
(B) gramicidin S; and (C) nystatin.
2052 FEBS Journal 281 (2014) 2051–2060 ª 2014 FEBS
Sterol-binding effects in plants J. Valitova et al.
their hydrophobic core [21]. The effects of MbCD are
based on the ‘host–guest’ interactions between oligo-
saccharides and sterols, and can cause sterol leakage
from cells [22].
The results obtained clearly indicate that a decrease
in sterol content is, in itself, not a determining factor
for the toxicity of sterol-binding agents. Rather, these
compounds are only toxic to plants when sterol bind-
ing is accompanied by an increase in membrane per-
meability for ions, leading to oxidative stress and loss
of cell viability.
Results
Effects of MbCD on lipid composition and the
formation of the complex between MbCD and
b-sitosterol
Incubation of excised wheat roots with 5 mM MbCDdecreased the contents of all major sterols by 50%
(Table 1; Fig. 2A). MbCD slightly raised the levels of
phospholipids, especially phosphatidylethanolamine,
diphosphatidylglycerol, and phosphatidylglycerol
(Fig. 2C), but significantly increased the total content
of glycoceramides after treatment for 2 h (Fig. 2B).
Molecular species of glycoceramides in roots treated
with MbCD did not change much as compared with
the untreated control (Table 2).
Using dynamic light scattering, we demonstrated the
formation of complexes between MbCD and b-sitos-terol. A solution of b-sitosterol on its own was charac-
terized by a monomodal pattern of size distribution
with a particle size of 115 nm, whereas the solution of
MbCD had a bimodal pattern with particle sizes of
3.7 nm and 290 nm (Fig. 3; Table 3). A solution of a
mixture of b-sitosterol and MbCD was characterized
by a bimodal pattern of size distribution, with particle
sizes of 0.7 nm and 4.5 nm. The electrokinetic poten-
tials (f-potentials) of particles in individual and mixed
solutions also differed. The f-potentials of the particles
in a b-sitosterol solution and a solution of MbCDwere �3 mV and �2.5 mV, respectively; in a mixed
solution of b-sitosterol and MbCD, the f-potential was�4.5 mV.
Effects of nystatin, MbCD and gramicidin S on
membrane permeability
Treatment of intact wheat seedlings with nystatin
greatly increased the permeability of membranes to
K+ in a concentration-dependent manner (Table 4).
Similarly to the application of nystatin, the application
of gramicidin S greatly enhanced K+ leakage from
roots. In contrast, MbCD did not induce marked
changes in the permeability of membranes to ions.
Neither gramicidin S nor MbCD changed the pH of
the incubation medium, whereas a high concentration
of nystatin induced alkalization (Table 4).
Oxidative stress, autophagy and cell viability in
wheat roots in the presence of nystatin, MbCD,and gramicidin S
Treatment of roots with MbCD did not change the
level of intracellular H2O2, whereas application of nys-
tatin and gramicidin S increased its content by up to
Table 1. Content of sterol molecular species (lg�g�1 DW) in wheat
roots after 2 h of treatment with 5 mM MbCD.
Sterol Control MbCD
Cholesterol 0.06 � 0 0.02 � 0
Campesterol 2.23 � 0.11 1.98 � 0.21
Stigmasterol 1.94 � 0.85 0.23 � 0.07
b-Sitosterol 6.81 � 0.27 4.08 � 0.61
Values are means � standard deviations, n = 3.
0
50
100
150
200
250
Control MβCD
Pho
spho
lipid
con
tent
[μg(
g D
W)–1
] PCPEPGDPGPIPS
A B C
0
2
4
6
8
10
12
14
16
Ste
rol c
onte
nt [μ
g(g
DW
)–1]
Control MβCD0
20
40
60
80
100
120
Gly
coce
ram
ide
cont
ent [
μg(g
DW
)–1]
Control MβCD
Fig. 2. The effect of MbCD on the total amount of sterols (A), the total amount of glycoceramides (B) and phospholipid composition (C) in
wheat roots. DPG, diphosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI,
phosphatidylinositol; PS, phosphatidylserine. Hatched bars represent lipids in MbCD-treated samples.
FEBS Journal 281 (2014) 2051–2060 ª 2014 FEBS 2053
J. Valitova et al. Sterol-binding effects in plants
three-fold and two-fold, respectively (Table 4). H2O2
accumulation induced by nystatin and gramicidin S
was accompanied by the upregulation of a gene encod-
ing a 37-kDa peroxidase (Fig. 4). Oxidative stress
induced by nystatin and gramicidin S was accompa-
nied by the formation of autolytic vacuoles (auto-
phagosomes), which are structures involved in the
Table 2. Content of molecular species of glycoceramides (%) in
wheat roots treated with 5 mM MbCD for 2 h.
Sphingoid
base Fatty acid
[M + Na+]
(m/z) Control MbCD
d18:2D4,8 16:0(OH) 736.5 25.3 � 1.4 21.2 � 3.7
d18:2D4,8 20:0(OH) 792.59 7.4 � 0.9 7.8 � 1.5
ND ND 798.59 17.7 � 3.5 17.5 � 3.2
ND ND 842.67 10.4 � 1.2 11.5 � 2.6
ND ND 846.64 9.6 � 0.7 10.0 � 0.7
t18:1D8 24:1(OH) 864.7 19.1 � 2.7 20.0 � 1.8
t18:1D8 24:0(OH) 866.7 10.5 � 0.5 12.0 � 0.7
Values are means � standard deviations, n = 3. ND, not deter-
mined.
0
5
10
15
20
0.1 1 10 100 1000 10000Deff (nm)
Deff (nm)
Deff (nm)
Inte
nsity
(%)
0
5
10
15
20
0.1 1 10 100 1000 10000
Inte
nsity
(%)
0
5
10
15
20
0.1 1 10 100 1000 10000
Inte
nsity
(%)
A
B
C
Fig. 3. Patterns of size distribution of particles of (A) 5 mM MbCD,
(B) 5 mM b-sitosterol and (C) the mixture of MbCD and b-sitosterol
at a ratio of 1 : 1 estimated by the intensity of light scattering in
0.2% dimethylsulfoxide/water solutions at 25 °C.
Table 4. The alterations in extracellular pH and K+ efflux, H2O2
accumulation and cell viability in roots after growing wheat
seedlings in the presence of nystatin, gramicidin S and MbCD for
12 h.
Treatment pH
K+ (leq�h�1�g�1
FW)
H2O2 (lM�g�1
FW)
Cell
viability
(%)
Control 5.9 � 0.1 0.5 � 0.0 6.3 � 0.0 100
Nystatin
(1 lM)
5.8 � 0.2 1.4 � 0.1 19.8 � 0.0 92
Nystatin
(10 lM)
5.9 � 0.1 2.3 � 0.1 – 64
Nystatin
(20 lM)
6.6 � 0.1 4.9 � 0.1 – 33
Gramicidin
S (1 lM)
5.9 � 0.1 1.4 � 0.1 11.2 � 0.0 87
MbCD
(5 mM)
5.9 � 0.0 0.5 � 0.0 6.0 � 0.1 98
FW, fresh weight.
Table 3. Deff values and f-potentials of particles.
Compounds Deff (nm) f-Potential (mV)
b-Sitosterol 115 � 1.8 – 3 � 0.3
MbCD 3.7 � 0.1 – 2.5 � 0.2
290 � 4 ND
b-Sitosterol + MbCD 0.7 � 0.1 – 4 � 0.5
4.5 � 0.3 ND
ND, not determined.
0
1
2
3
4
5
6
7
Rel
ativ
e le
vel o
f exp
ress
ion
(a.u
.)
Control
Gramicidin S (1 μM)
Nystatin (1 μM)
TaPOX TaATG4
Fig. 4. Relative level of expression of the 37-kDa peroxidase gene
TaPOX and the autophagic gene TaATG4 assessed with real-time
PCR after 12 h of treatment of roots with nystatin and
gramicidin S. a.u, arbitrary units.
2054 FEBS Journal 281 (2014) 2051–2060 ª 2014 FEBS
Sterol-binding effects in plants J. Valitova et al.
degradation of oxidized macromolecules and damaged
organelles. Autophagosomes appeared as bright-col-
ored puncta (Fig. 5C–F). The induction of autophagy
was also confirmed by the elevated expression level of
a gene encoding the autophagic protein ATG4
(Fig. 4). When nystatin was applied at a higher
(20 lМ) concentration, large aggregates were observed
(Fig. 5F). Treatment of seedlings with MbCD did not
cause the formation of autophagosomes (Fig. 5B).
Cell viability in roots treated with nystatin and
gramicidin S was low in comparison with the control
(Table 4). Nystatin at 20 lМ significantly reduced cell
viability (by 70%). Cell viability was not affected by
MbCD (Table 4).
Discussion
In the present work, the significance of sterol depletion
for membrane integrity and the physiological status of
roots of wheat seedlings was tested by examining the
effects of two sterol-binding agents, nystatin and
MbCD, and also the nonspecific channel-forming com-
pound gramicidin S. Membrane sterols are multifunc-
tional components that can stabilize membranes and
control the positioning of various membrane-bound
proteins [7,8]. Therefore, the changes in membrane
sterols might have a strong impact on membrane
integrity and activity, with deleterious physiological
consequences. The results presented here suggest that a
decrease in sterol content alone is not sufficient to
have deleterious effects on a cell. The toxicity of ste-
rol-specific agents for plants is only manifested when
sterol binding is accompanied by increased membrane
permeability to ions, oxidative stress, and loss of cell
viability.
Membrane lipids in wheat roots affected by
nystatin and MbCD
Previously, we showed in wheat roots that the binding
of sterols by nystatin decreases the level of sterols, in
particular b-sitosterol [from 10.62 lg�g�1 dry weight
(DW) in the untreated control to 3.39 lg�g�1 DW]
and campesterol (from 3.96 to 1.32 lg�g�1 DW), and
significantly (up to 70%) increases the glycoceramide
content [19]. In addition, ESI-MS/MS analysis has
demonstrated that nystatin alters the ratio of the
molecular species of glycoceramides. The present study
shows that another sterol-binding agent, MbCD, simi-
larly to nystatin, significantly reduces the level of total
sterols (Table 1; Fig. 2A). Zidovetzki and Levitan [22]
demonstrated that MbCD induces sterol leakage as a
result of the formation of a supramolecular complex
between MbCD and a sterol. Using dynamic light-
scattering we analyzed the patterns of size distribution
and f-potential of the particles of MbCD and b-sitos-terol in water solutions (Fig. 3; Table 3). Our observa-
tions suggest the formation of a stable supramolecular
complex between sterol and MbCD with a size and
f-potential distinct from those for individual com-
pounds. Previously, we demonstrated the formation of
a stable complex between b-sitosterol and nystatin
[19]. Roche et al. [23] have shown that MbCD signifi-
cantly decreases the total sterol content of a cell cul-
ture of tobacco BY2, but does not affect the
phospholipid content. In our experiments, the analysis
A B
D
C
E F
Fig. 5. Visualization of autophagosomes in
root cells with Lyso Tracker Red after 12 h
of treatment with MbCD and antibiotics
(nystatin and gramicidin S). (A) Control. (B)
MbCD (5 mM). (C) Gramicidin S (1 lM). (D)
Nystatin (1 lM). (E) Nystatin (10 lM). (F)
Nystatin (20 lM).
FEBS Journal 281 (2014) 2051–2060 ª 2014 FEBS 2055
J. Valitova et al. Sterol-binding effects in plants
of the phospholipid composition in roots in the pres-
ence of MbCD showed a slight increase in total
phospholipid content (Fig. 2C). Thus, the results pre-
sented here and the literature data demonstrate that
MbCD can specifically bind to phytosterols and cause
sterol depletion in plants, but does not affect the phos-
pholipids.
Although it does not affect phospholipids, binding
of MbCD to sterols affects sphingolipids by elevating
the level of glycoceramides (Fig. 2B), similarly to the
effect of nystatin [19]. However, unlike nystatin, which
changes the ratio of molecular species of glycocera-
mides, MbCD does not change these ratios (Table 2).
The existence of a special relationship between sterols
and sphingolipids is supported by the ability of sterols
to undergo van der Waals interactions with the
saturated alkyl residues of sphingolipids [10]. Such
interactions enable these two classes of membrane lipid
to achieve a high packing density, and facilitate the
formation of lipid microdomains [24]. Furthermore,
studies on yeast and animal cells have indicated that
the biosynthesis of sterols and sphingolipids is also
coordinated [25]. This likely to happen in plants; for
example, disturbance of the sterol biosynthesis path-
way in leek seedlings impairs the synthesis of complex
sphingolipids such as glucosylceramides [26]. Our data
provide further support for the interrelationship
between sterol and sphingolipid metabolism in plants,
which may have implications for the maintenance of
the relative ratios of these lipids, which are essential
for lipid raft formation and function [27].
Physiological consequences
Although they have similar effects in reducing sterol
content, nystatin and MbCD cause different physiologi-
cal responses in wheat roots. Surprisingly, sterol binding
by MbCD does not alter the permeability of membranes
to ions (Table 4), although increased membrane perme-
ability has been documented for rat liver lysosomes
treated with a similar concentration of MbCD [28]. In
contrast, nystatin-induced sterol binding in roots shar-
ply increases membrane permeability to K+, H+, and
SH-containing compounds, probably because of the for-
mation of ‘nystatin pores’ [29]. To better understand the
physiological effects of sterol binding, we used another
channel-forming compound, gramicidin S, which is a
cyclic polypeptide antibiotic produced by Bacillus brevis
that can form channels in lipid bilayers [30]. Gramici-
din S interacts with the polar regions of phospholipids
through electrostatic forces, but does not bind to mem-
brane sterols. Treatment of roots with gramicidin S,
similarly to treatment with nystatin, increases mem-
brane permeability to K+ (Table 4). Treatment of roots
with antibiotics, which increase membrane permeability
to ions, namely nystatin and gramicidin S, induces
oxidative stress as shown by H2O2 accumulation
(Table 4) and the upregulation of a gene encoding a 37-
kDa peroxidase, which, as we found previously, is the
most stress-sensitive peroxidase isoform in wheat roots
[31]. By contrast, the binding of sterols by MbCD,
which is not accompanied by increased membrane per-
meability, does not cause oxidative stress (Table 4).
Taken together, these data suggest that, in wheat roots
treated with sterol-binding compounds, increased mem-
brane permeability is caused by channel formation
rather than sterol depletion. Furthermore, increased
membrane permeability, rather than simply sterol deple-
tion, is a cause of oxidative stress in roots.
Further evidence for the mechanism of antibiotic tox-
icity comes from observation of the consequences of
oxidative stress at the cellular level. Oxidative stress
may cause the accumulation of oxidized proteins and
damaged organelles. Accumulated oxidized macromole-
cules are dangerous for cells, and it is therefore vital that
these are rapidly degraded. An effective mechanism of
degradation is a process called ‘autophagy’ – ’self-eat-
ing’ [32]. This process is characterized by the formation
of special vesicles, the autophagosomes, and the subse-
quent fusion of these vesicles with vacuoles for degrada-
tion. Previously, we demonstrated that oxidative stress
induces the intensive formation of autophagosomes in
wheat roots [33]. In the current work, oxidative stress in
roots treated with the channel-forming antibiotics
nystatin and gramicidin S was also accompanied by
intensive autophagosome formation as visualized with
Lyso Tracker Red (Fig. 5) and the stimulation of
expression of a gene encoding the autophagic protein
ATG4 (Fig. 4). This protein is a cysteine-containing
protease involved in the formation of autophagosomes
[34]. Therefore, the disturbance of membrane integrity
and the development of oxidative stress observed fol-
lowing nystatin and gramicidin S treatments result in
the initiation and progression of autophagy and, as a
consequence, decreased cell viability (Table 4). In yeasts
and mammalian cells, increased ion permeability of the
PM induced by various agents, including antibiotics and
antimicrobial peptides, is often accompanied by the
accumulation of reactive oxygen species and subsequent
programmed cell death [35]. In contrast, MbCD has no
effect on membrane permeability or the level of H2O2.
As a consequence, autolytic vacuoles do not form
(Fig. 5), and cell viability is maintained (Table 4). Thus,
our data confirm that sterol binding without changes in
membrane permeability does not lead to autophago-
some formation and does not reduce cell viability.
2056 FEBS Journal 281 (2014) 2051–2060 ª 2014 FEBS
Sterol-binding effects in plants J. Valitova et al.
Conclusions
In the present study, the significant differences in the
physiological and biochemical effects induced in wheat
roots by the application of the sterol-binding agents
nystatin and MbCD clearly demonstrate that decreases
in sterol content do not necessarily have deleterious
consequences. The toxic effects of nystatin are likely to
be caused by disturbance of membrane integrity and
induction of oxidative stress rather than the decrease in
sterol content. It remains uncertain why sterol binding
and depletion do not necessarily cause the disturbance
of membrane integrity and subsequent toxicity. How-
ever, the absence of obvious physiological and cellular
responses of roots to sterol depletion does not preclude
the possibility that changes occur at the molecular level.
In particular, these may involve the activity of genes
responsible for sterol biosynthesis, such as those encod-
ing sterol methyltransferases. It seems likely that a
reduction in sterol content will activate sterol biosyn-
thesis, and therefore, in future research, we plan to
study the activity of the genes involved.
Experimental procedures
Plant material
Wheat (Triticum aestivum L. cv. Kazanskaya Jubilejnaya;
Niva Tatarstana, Kazan, Russia) seedlings were grown
hydroponically in 0.25 mM CaCl2 at 22 °C for 4 days.
Depending on the experimental setup, excised or intact
roots were used. For lipid analysis, roots were excised from
seedlings and incubated with gentle agitation in 0.25 mM
CaCl2 without (control) or with 20 lM nystatin (Sigma, St
Louis, MO, USA) or 5 mM MbCD for 2 h. As nystatin
was dissolved in 0.2% dimethylsulfoxide, an appropriate
amount of dimethylsulfoxide was added to the control
incubation medium. The incubation media contained no
K+, and their pH was adjusted to 6.0.
Intact seedlings were used in the assessment of membrane
permeability to K+ and H+, the amount of H2O2, protease
activity, and cell viability, and the visualization of auto-
phagosomes. Seedlings were grown in 0.25 mM CaCl2 for
4 days, transferred into the test solutions (nystatin, MbCD,
and gramicidin S) for 12 h, and then used for measure-
ments.
Lipid extraction and analysis
Lipids were extracted with a mixture of isopropanol and
chloroform (1 : 1), according to Nichols [36] with modifi-
cations [37]. Individual phospholipids and sphingolipids
were analyzed with 2D TLC, according to Vaskovsky and
Terekhova [38] with modifications. The full description of
the methodology of lipid analysis has been published previ-
ously [19]. Briefly, molecular species of glycoceramides
were determined by estimating the value of m/z with an
MX5310 high-resolution time-of-flight mass spectrometer
with orthogonal input and an electrospray ion source
(ESI-TOF). Amounts of individual phospholipids and total
glycoceramides were determined with a densitometer
(DenSkan, Russia). Eluted sterols were evaporated to
dryness and, after sample preparation, analyzed with an
HP6890 gas chromatograph (Agilent, Palo Alto, CA,
USA) interfaced with an HP 5973 mass selective detector
(Agilent). Data were acquired and processed with HP-CHEM-
STATION software (Agilent). Sterol identification was
performed by use of the mass spectral library of the
GC-MS data system [39]. Quantification was performed
with the chromatographic software UNICHROM (http://www.
unichrom.com), with naphthalene as the internal standard.
Sizes and f-potentials of MbCD, sterols, andMbCD–sterol particles
The sizes [effective hydrodynamic diameter (Deff)] and
f-potentials of particles of MbCD, sterols and MbCD–
sterol in 0.2% dimethylsulfoxide/water solutions were stud-
ied by dynamic light scattering and electrophoresis with a
Zetasizer Nano ZS high-sensitivity analyzer (Malvern
Instruments, Malvern, UK) [40]. The procedure of sample
preparation ensured appropriate dust removal from the
solutions. The signals were analyzed with software provided
by manufacturer, and the error of measurements was not
greater than 5%.
Membrane permeability
The K+ content in the root incubation medium was mea-
sured with a Flapho 41 flame photometer (Carl Zeiss,
Germany). pH measurements were performed with a pH-
meter (Mettler Toledo, USA).
H2O2 content and cell viability
H2O2 content was measured spectrophometrically with
xylenol orange (Acros; k = 560 nm) [41]. H2O2 content was
calculated by use of a calibration curve. Cell viability was
determined by staining with Evans blue [42]. Roots were
stained with 0.25% Evans blue for 15 min, and washed
three times for 10 min each. Root tips were then cut and
washed with N,N-dimethylformamide for 1 h at room
temperature. The absorbance of the washed solution was
measured spectrophotometrically at k = 600 nm.
Fluorescence visualization of autophagosomes
Autophagosomes were visualized with the fluorescent dye
LysoTracker Red DND 99 (Invitrogen; kab = 577 nm;
FEBS Journal 281 (2014) 2051–2060 ª 2014 FEBS 2057
J. Valitova et al. Sterol-binding effects in plants
kem = 590 nm), as intensively stained puncta, with an
LSM 510 META confocal microscope (Zeiss, Germany)
equipped with an HeNe laser (543 nm, 60.0%). Back-
ground staining of cell walls, cytoplasm and the perinuclear
region is nonspecific with this dye.
Real-time PCR analysis of TaPOX and TaATG4
Total RNA was isolated with TRIzol reagent. To remove
DNA, the samples were treated with 0.1 units of DNaseI
(Fermentas) at 37 °C for 30 min. Reverse transcription
was performed with Moloney murine leukemia virus
reverse transcriptase (Sileks, Russia) and oligo(dT)18 prim-
ers (Sintol, Russia), according to the manual. RNA
concentration and purity were assessed with a Nano-
Drop ND-1000 spectrophotometer (Thermo Scientific,
USA), and the integrity was verified on a 1% agarose gel.
Real-time PCR was performed with an ICycler IQ 2 Mul-
ticolor Real-Time PCR Detection System (Bio-Rad, USA).
The templates were amplified three times at 95 °C for
3 min, and this was followed by 40 cycles of amplification
(95 °C for 10 s and 65 °C for 40 s). ADP-ribosylation fac-
tor (TaARF) and RNaseL inhibitor-like protein (TaRLI)
genes were used as reference genes [43]. The gene-specific
primers and TaqMan probes used for real-time PCR are
shown in Table 5. Quantification was performed according
to Pfaffl [44].
Statistics
The experimental data were processed by the method of
variation statistics. Biological experiments were repeated
three times, with triplicate analytical tests. The tables and
figures present mean values and standard errors. The statis-
tical significance of differences between the control and
treatment samples was assessed according to Student’s cri-
terion, with a confidence level of P = 0.95.
Acknowledgements
This study was carried out with financial support from
the Russian Foundation for Basic Research (Nos.
13-04-00865, 14-04-00205, and 14-04-31421), the Fed-
eral Program of the Ministry of Education and Science
of the RF (No. 8117 from 23 July 2012), and the
Program of the Presidium of Russian Academy of Sci-
ences ‘Molecular and Cellular Biology’, with support
from Higher Scientific Schools (No. 825.2012.4).
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