Sterol binding by methyl-β-cyclodextrin and nystatin - comparative analysis of biochemical and...

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],

[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.


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.



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).



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.



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;



http://www.unichrom.com

http://www.unichrom.com

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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TaATG4 F: CTAGTGATGTCAACT

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Sterol binding by methyl-β-cyclodextrin and nystatin - comparative analysis of biochemical and...

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