ISSN 1062�3604, Russian Journal of Developmental Biology, 2014, Vol. 45, No. 1, pp. 41–51. © Pleiades Publishing, Inc., 2014.Original Russian Text © L.R. Nigmatullina, N.I. Rumyantseva, Yu.A. Kostyukova, 2014, published in Ontogenez, 2014, Vol. 45, No. 1, pp. 50–62.

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INTRODUCTION

Tripeptide glutathione (γ�glutamyl�cysteinyl�gly�cine) is the major redox buffer in animal cells, inwhich it is present in two forms—reduced (GSH) andoxidized (GSSG). In plants, the major contribution tothe intracellular redox buffer is made by ascorbate anddehydroascorbate. However, the function of glu�tathione in the plant cells is as important and diverse asin the animal cells: glutathione is directly involved inthe protection of cells from oxidative stress, function�ing as a scavenger of superoxide anion, hydroxyl radi�cal, and singlet oxygen. It also serves as a substrate forantioxidant enzymes, such as glutathione�S�trans�ferase, glutathione peroxidase, and dehydroascorbatereductase. Glutathionylation is one of the mostimportant posttranslational modifications of proteinsthat protects the thiol�containing proteins from oxida�tive damage and, due to the reversibility of the reac�tion, contributes to the modulation of their intracellu�

lar activity (Dixon et al., 2005). Glutathione isinvolved in the cell cycle regulation in plants, althoughthe mechanism of this regulation is not completelyelucidated (Sanchez�Fernandez et al., 1997; Vernouxet al., 2000). In plants, glutathione is the major sourceof reserve sulfur, which is particularly important dur�ing the embryonic development of plants (Foyer et al.,2001). It was shown that glutathione is involved in theregulation of various morphogenetic processes both invivo and in vitro (Kerk et al., 2000; Jiang et al., 2003;Tybursky et al., 2010).

The formation of a disulfide bond during the oxida�tion of two glutathione molecules yields the oxidizedglutathione, GSSG. The latter, accumulated in animalcells as a result of oxidative stress, is toxic and caninduce apoptosis (Filomeni et al., 2005). It should benoted that the induction of apoptosis is determined bythe level of GSH depletion rather than by excess ROS(Franco et al., 2007). In animal cells, the GSH/GSSGratio reflects the development of stress and is used asan additional indicator for diagnosing various pathol�ogies. It was shown that, in plants, the content of GSH

Effect of D,L�Buthionine�S,R�Sulfoximine on the Ratio of Glutathione Forms and the Growth of Tatar Buckwheat Calli

L. R. Nigmatullina, N. I. Rumyantseva, and Yu. A. KostyukovaKazan Institute of Biochemistry and Biophysics, Kazan Scientific Center, Russian Academy of Sciences,

ul. Lobachevskogo 2/31, Kazan, Tatarstan, 420111 Russiae�mail: nat_rumyantseva@mail.ru

Received May 7, 2013; in final form, September 9, 2013

Abstract—We studied the intracellular content of reduced (GSH) and oxidized (GSSG) glutathione, glu�tathione reductase activity, glutathione�S�transferase, and ascorbate peroxidase in morphogenic and non�morphogenic Tatar buckwheat calli during the culture cycle as well as under the treatment with D,L�buthion�ine�S,R�sulfoximine (BSO), an inhibitor of γ�glutamylcysteine synthase, the first enzyme of glutathione bio�synthesis. We found that, during passaging, cultures only slightly differed in total glutathione content;however, the content of GSH was higher in the morphogenic culture, whereas the content of GSSG washigher in the nonmorphogenic culture. In the morphogenic callus, the glutathione�S�transferase activity was10–20 times higher and the glutathione reductase activity was 2–2.5 times lower than in the nonmorphogeniccallus. Under the treatment with BSO, the decrease in the GSH content in the morphogenic callus was tem�porary (on day 6–8 of passage), whereas that in the nonmorphogenic callus decreased within a day andremained lower than in the control throughout the entire passage. In the morphogenic callus, BSO did notaffect the content of GSSG, whereas it caused GSSG accumulation in the nonmorphogenic callus. Thesedifferences are probably due to the fact that, in the BSO�containing medium, glutathione reductase is acti�vated in the morphogenic callus and, conversely, inhibited in the nonmorphogenic callus. Although BSOcaused a decrease in the total glutathione content only in the nonmorphogenic culture, the cytostatic effectof BSO was more pronounced in the morphogenic callus. In addition, BSO also had a negative effect on thedifferentiation of proembryonic cell complexes in the morphogenic callus. The role of the glutathione redoxstatus in maintaining the embryogenic activity of cultured plant cells is discussed.

Keywords: Fagopyrum tataricum (L.) Gaertn., morphogenic callus, nonmorphogenic callus, GSH, GSSG,glutathione reductase, glutathione�S�transferase, D,L�buthionine�S,R�sulfoximine, somatic embryogenesis

DOI: 10.1134/S1062360414010056

MECHANISMS OF CELL PROLIFERATION AND DIFFERENTIATION

Abbreviations: BSO—D,L�buthionine�S,R�sulfoximine; PECC—proembryonic cell complex; ROS—reactive oxygen species;PMSF—phenylmethylsulfonyl fluoride.

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also decreases in different types of stress, whereas thecontent of GSSG, conversely, increases (Ruiz et al.,2002). Resistant genotypes of plants are characterizedby a high content of GSH; exposure to various stres�sors leads to the activation of enzymes of the ascor�bate/glutathione cycle in them.

Plant cell cultures exhibit different ability to regen�erate plants in vitro. Therefore, depending on themanifestation of this ability, they are divided into mor�phogenic and nonmorphogenic cultures. The mor�phogenic cultures that are most often used in bothbasic research and biotechnological developments arethe so�called embryogenic lines, which are character�ized by a high frequency of regeneration activity. Themain route of morphogenesis in these lines is thesomatic embryogenesis. Typically, embryogenic lineshave a “nodular” phenotype, which is characterizedby the presence of proembryonic cell complexes(PECCs) or, according to another terminology, proem�bryogenic masses (Finer et al., 1994). Depending on thespecies and even variety of culture, PECCs may be rep�resented by proembryos or even globular embryoswhose developed was stopped by addition of auxin tothe culture medium. A significant mass of the embryo�genic callus cultures is formed by loose nurse tissue (or“soft” callus), which is formed as a result of looseningthe preexisting PECCs. This tissue is metabolicallyactive; however, its cells are not able to actively divide,in contrast to PECC cells, and ultimately age and die.We have shown that, in the morphogenic callus of theTatar buckwheat (Fagopyrum tataricum (L.) Gaertn.),which is a typical nodular culture, the reproductioncycle of PECCs is maintained, which is triggered bycallus transfer to the new environment with2,4�dichlorophenoxyacetic acid (Rumyantseva et al.,2003, 2004). The morphogenic calli of the Tatar buck�wheat retain their ability for morphogenesis for severalyears of cultivation; the nonmorphogenic clones occurin them rarely and are immediately characterized by aloose phenotype and the absence of PECC, as well asby polyploidy, rapid growth, and inability for any typeof differentiation. We have previously shown that,unlike the morphogenic cultures, the nonmorpho�genic cultures are characterized by a high content ofhydrogen peroxide and an increased level of lipid per�oxidation, a low catalase activity and a high superox�ide dismutase activity (Kamalova et al., 2009); theycontain a smaller amount of phenols and differ in thequalitative composition (Sibgatullina et al., 2012). Weassumed that cultures capable of regeneration shouldhave a high level of antioxidant protection, whichmakes it possible, on the one hand, to protect thegenetic apparatus of cells from the damage caused byROS and, on the other hand, to effectively control theredox regulation of signaling.

In this study, we investigated the effect ofD,L�buthionine�S,R�sulfoximine (BSO), an inhibitorof γ�glutamylcysteine synthase, the first enzyme of the

glutathione biosynthesis, on the glutathione redox sta�tus and the growth of Tatar buckwheat calli.

MATERIALS AND METHODS

This study was performed with the morphogenicand nonmorphogenic calli of the Tatar buckwheatFagopyrum tataricum (L.) Gaertn. The morphogeniccallus was obtained from immature embryos asdescribed earlier (Rumyantseva et al., 1989, 1992,1998; Lukina et al., 1999). Line 1�8 of the Fagopyrumtataricum (L.) morphogenic callus was obtainedin 2005 and maintained in vitro for 8 years. The mor�phogenic culture had a typical nodular morphotypeand consisted of PECCs and “soft” callus. The mor�phogenic calli retained morphology, diploid chromo�some number, and ability to undergo somatic embryo�genesis and gemmogenesis in culture for a long time(several years) (Rumyantseva et al., 1989, 1998). Thenonmorphogenic callus of line 1�8p consisting only ofthe parenchymal cells was selected as a clone formedon the morphogenic callus. The nonmorphogenic cal�lus differed from the morphogenic one by loose struc�ture, high growth rate, significant genetic variation(chromosome numbers from n to 8n), and the com�plete loss of the ability to undergo morphogenesis.Callus cultures were maintained in an incubator at25 ± 0.5°С in the dark on the RX callus inductionmedium (Rumyantseva et al., 1998). The morpho�genic calli were able to form somatic embryos in the MShormone�free medium and buds in the RX mediumsupplemented with 6�benzylaminopurine and indoleacetic acid. The nonmorphogenic and morphogeniccalli were passaged every 2 and 3 weeks, respectively.The callus culture growth was assessed by an increase inwet tissue weight per certain periods of time.

The content of total, reduced, and oxidized glu�tathione was determined according to Zhang et al.(1996). The total glutathione content was evaluated inthe color reaction with the formation of a complex of5,5'�dithiobis�2�nitrobenzoic acid and GSH. TheGSSG content was assessed using 2�vinylpyridine,which binds to GSH and masks its. Briefly, a piece ofcallus tissue (250 mg) was homogenized in 1 mL of5% 5�sulfosalicylic acid. The homogenate was centri�fuged at 10000 g for 5 min, and the resulting superna�tant was divided into two parts. One part was mixedwith 375 µL of 0.5 M sodium phosphate buffer(pH 7.5) and 12 µL of distilled water (this sample wasused to determine the total glutathione content). Thesecond part of the supernatant was mixed with 375 µLof 0.5 M sodium phosphate buffer (pH 7.5) and 12 µLof 2�vinylpyridine (97% 2�vinylpyridine stabilizedwith 0.1% ethyl 4�tert�butylcatechol) to mask thereduced glutathione. The reaction mixture contained600 µL of 0.18% Na�EDTA in phosphate bufferedsaline, 100 µL of 0.16% NADPH, 200 µL of 0.12%5,5'�dithiobis(2�nitrobenzoic acid), 2 µL of glu�tathione reductase (0.76 U), and 100 µL of the super�

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EFFECT OF D,L�BUTHIONINE�S,R�SULFOXIMINE 43

natant. The optical density was measured at 412 nmfor 60 s at 2.5 s intervals using a Lambda 25 spectro�photometer (Perkin Elmer, United States). The con�centrations of GSH and GSSG were calculated usingthe calibration curves built for the known concentra�tions of GSH and GSSG. The optical density of GSHwas calculated as the difference between the opticaldensity of the total glutathione and GSSG.

Glutathione reductase (EC 1.6.4.2) activity wasdetermined according to Verlan (2008). A callus tissuepiece (250 mg) was homogenized in extraction buffercontaining 50 mM К2НРО4, 4% polyvinylpyrroli�done, 0.1 mM Na�EDTA, 0.3% Triton X�100,1.3 mM ascorbic acid, and 1 mM PMSF. The homo�genate was centrifuged at 11000 g for 10 min. Thesupernatant was transferred to a new Eppendorf tubeand stored in a container with ice. The reaction mix�ture contained 1.91 mL of the measurement buffer(100 mM Tris and 1 mM Na�EDTA; pH was adjustedto 8.0 with HCl), 20 µL of 0.4% NADPH, and 50 µLof the extract. The reaction was initiated by the addi�tion of 20 µL of 3% GSSG. The control cell contained1.93 mL of the measurement buffer and 50 µL of theextract. The activity of glutathione reductase wasdetermined by the change in absorbance at 340 nmdue to NADPH oxidation for 3.5 min at 1 s intervalswith a Lambda 25 spectrophotometer (Perkin Elmer,United States). The enzyme activity was calculatedusing the extinction coefficient for NADH+ at 340 nm(6.22 mM–1 cm–1).

Glutathione�S�transferase (EC 2.5.1.18) activitywas determined according to Habig et al. (1974) by therate of formation of glutathione�S�conjugates betweenGSH and 1�chloro�2,4�dinitrobenzene. Briefly, a cal�lus tissue piece (350 mg) was homogenized in anextraction buffer containing 50 mM К2НРО4, 4%polyvinylpyrrolidone, 0.1 mM Na�EDTA, 0.3% Tri�ton X�100, 1.3 mM ascorbic acid, and 1 mM PMSF.The homogenate was centrifuged at 12000 g for 5 min.The supernatant was used for analysis. The reactionmixture contained 2.5 mL of 0.1 M potassium sodiumphosphate buffer (pH 6.5), 0.2 mL of 0.015 M GSH,and 0.1 mL of the supernatant. The reaction was initi�ated by the addition of 0.2 mL of 0.015 M 1�chloro�2,4�dinitrobenzene. The activity of glutathione�S�transferasewas determined by the change in absorbance at 340 nmdue to the formation of glutathione�S�1�chloro�2,4�dinitrobenzene for 1.5 min at 1 s intervals with aLambda 25 spectrophotometer (Perkin Elmer, UnitedStates). The enzyme activity was calculated using theextinction coefficient for glutathione�S�1�chloro�2,4�dinitrobenzene at 340 nm (9.6 mM–1 cm–1).

Ascorbate peroxidase (EC 1.11.1.7) activity wasdetermined according to Verma et al. (2003). Briefly, acallus tissue piece (150 mg) was homogenized in50 mM potassium sodium phosphate buffer (pH 7.8)supplemented with 1 mM PMSF, 1 mM ascorbic acid,and 1% polyvinylpyrrolidone. The homogenate was

centrifuged at 12000 g for 5 min, and the resultingsupernatant was used for measurements. The reactionmixture contained potassium sodium phosphate buffer(pH 7.0), 0.2 mM ascorbic acid, 0.2 mM Na�EDTA,and the supernatant. The reaction was started by add�ing 20 µM hydrogen peroxide. The optical density wasmeasured at 290 nm for 120 s at 1 s intervals with aLambda 25 spectrophotometer (Perkin Elmer, UnitedStates). The amount of the enzyme that changed theabsorbance of the reaction mixture by 0.001 opticaldensity units per unit time was taken as one unit ofenzyme activity.

Protein content was determined by the binding toCoomassie Brilliant Blue G�250 (CBB G�250, Sigma,United States) according to Bradford (1976).

Cell viability was determined spectrophotometri�cally using Evans Blue dye as described by Castro�Concha et al. (2006). Five hundred microliters of0.025% dye was added to 150 mg of callus tissue andincubated at room temperature for 15 min. The cellswere then washed from the dye with distilled water and1 mL of 1% sodium lauryl�Na in 50% ethanol wasadded. The solution with cells was incubated in a waterbath at 60°C for 30 min. Then, it was centrifuged at12500 g for 5 min, and the optical density of the super�natant was measured at 600 nm with a Lambda 25spectrophotometer (Perkin Elmer, United States).Callus tissue (150 mg) that was boiled for 20 min ortwice frozen in liquid nitrogen and then thawed wasused as a control (100% dead cells).

Mitotic index was calculated using squashed calluspreparations prefixed in Clark fixative and stainedwith propionic lakmoid. For simultaneous stainingand maceration, material was boiled for 5–10 s in thedye, after which squashed preparations in 45% lacticacid were prepared. The preparations were analyzedunder a Jenamed microscope (Carl Zeiss, Germany),photographed using an AxioCam MRc5 digital cam�era equipped with AxioVision Rel. 4.6 software, andthen processed using Adobe PhotoShop 7.0 software.

For histological studies, tissue pieces were fixed in2.5% ethyl glutaraldehyde in phosphate buffer andpostfixed in 1% OsO4. The tissue was dehydrated in aseries of increasing alcohol concentrations, acetone,and propylene oxide and then embedded in Epon. Toprepare histological samples, semithin sections pre�pared using an ultramicrotome (LKB, Sweden) werestained with 1% toluidine blue, examined under aJenamed microscope (Carl Zeiss, Germany), andphotographed using an AxioCam MRc5 digital headwith AxioVision Rel. 4.6 software.

In experiments, the inhibitor of glutathione biosyn�thesis BSO was preliminarily sterilized through a Milli�pore ultrafilter (pore diameter, 0.22 µm) and added at aconcentration of 0.1 mM to the culture medium. Calluspieces were weighed, placed in a medium with theinhibitor, and cultured in an incubator.

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Data were processed by the methods of mathemat�ical statistics using Microsoft Office Excel 2003. Thearithmetic mean error was indicated as the scatter ofthe experimental data; the sampling fraction error wasindicated for the calculation of the mitotic index.

Reagents. In this study, we used thiamine�HCl,pyridoxine�HCl, nicotinic acid, 2,4�dichlorophenoxy�acetic acid, indoleacetic acid, naphthylacetic acid,kinetin, casein hydrolysate, Na�EDTA, lacmoid, agar�agar, PMSF, glutathione reductase (0.76 U) frombaker’s yeast, 5�sulfosalicylic acid, and Coomassie Bril�liant Blue CBB G�250 from Sigma Aldrich (UnitedStates); NADPH, GSH, GSSG, and polyvinylpyrroli�done from AppliChem (Germany); 5,5'�dithiobis�2�nitrobenzoic acid, 1�chloro�2,4�dinitrobenzene,2�vinylpyridine (97% 2�vinylpyridine stabilized with0.1% tert�butylcatechol), and OsO4 from Alfa Aesar(United States); Triton X�100, L,D�buthionine�S,R�sul�foximine, and sodium lauryl sulfate from Acros Organics(Belgium); and ascorbic acid and Tris from Panreac(Spain). Other reagents were produced in Russia.

RESULTS AND DISCUSSION

1. Effect of BSO on the Growth Morphogenic and Nonmorphogenic Callus of the Tatar Buckwheat

The growth of the nonmorphogenic callus of theTatar buckwheat can be described by an S�shapedcurve (Fig. 1a). Callus cells began to divide as early ason the second day after their transfer to a new culturemedium. They intensely divided until day 7 and thenbegan to stretch, which was observed until day 10–12.The stage of stretching was followed by the stage ofaging: the culture growth reached a plateau on day 14,which was ultimately ended by the cell death onday 16–18. It should be noted that the nonmorpho�genic calli are extremely sensitive to the time of trans�fer to a new medium, because even at a slight delay in

the timing of transfer (several few days), the growth ofcalli can be either very weak (in the form of growth ofonly some areas) or absent at all.

For the morphogenic culture, the S�shaped curve isnot characteristic, which can be attributed to the lackof well�expressed stage of degradation and death ofculture (callus may start to grow in a new medium evenafter 2–3 months of storage in the same medium).

Previously, it was shown that, during the culturecycle, the morphogenic callus has several peaks ofmitotic activity, which is associated with PECC for�mation cycles (Rumyantseva et al., 1998). The mor�phogenic cultures grow more slowly compared to thenonmorphogenic ones (4–5 times by the growth ofwet biomass (Fig. 1b)).

BSO is used as an inhibitor of GSH biosynthesis,whose content is reduced as a result of inhibition of theactivity of γ�glutamylcysteine synthase. Both in ani�mals and plants, the γ�glutamylcysteine synthase geneknockout causes embryonic lethality (Shi et al., 2000;Cairns et al., 2006). Under the influence of BSO, thegrowth of the biomass of the nonmorphogenic calluson day 14–16 of cultivation lagged behind the control(Fig. 1a), whereas the growth of the morphogenic cal�lus did not differ from the control (Fig. 1b). It shouldbe noted that the viability of cells of the nonmorpho�genic callus was the same (85%) as in the control.Importantly, despite the absence of differences in thebiomass growth, BSO affected the formation ofPECCs on the morphogenic callus, significantlyreducing their number (Figs. 2a, 2b). Figure 2b showssporadic PECCs on the BSO�containing medium.Histological analysis performed on day 5 of cultivationin the BSO�containing medium showed that growthand differentiation of PECCs in the morphogenic cal�lus were disturbed (Figs. 2c, 2d). It can be seen in his�tological sections that PECCs in the calli grown in theBSO�containing medium were significantly smaller

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Fig. 1. Effect of BSO on the growth of (a) nonmorphogenic and (b) morphogenic callus of the Tatar buckwheat in (1) RX mediumand (2) RX medium + 0.1 mM BSO.

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EFFECT OF D,L�BUTHIONINE�S,R�SULFOXIMINE 45

than in the control and were formed by a small numberof meristematic cells. Such PECCs are visually indis�tinguishable on the callus surface (Fig. 2d). Morpho�logically, PECCs grown in the BSO�containingmedium corresponded to the newly formed complexesin the medium without the inhibitor on the second dayof cultivation. On day 5 of cultivation, PECCs in thecontrol culture were visually distinguishable structuresformed by different types of cells and had a size of 0.5–1 mm (Fig. 2c). It is noteworthy that, on the histolog�ical sections, the cells of the soft callus grown in thepresence of BSO were somewhat larger than in thecontrol, which can be explained by the intensificationof vacuolation of cells by BSO. However, BSO had nosignificant effect on the histological characteristics ofthe nonmorphogenic callus, although measurementsshowed that the length of its cells increased by almost20% (data not shown).

According to the published data, the homeostasisof the cell redox potential is the key regulator of the

cell fate both in mammals and in plants, and adjust�able changes in the increase or accumulation of oxida�tion and reduction signals can markedly affect the cellcycle progression (Sanchez�Fernandez et al., 1997;Vernoux et al., 2000; Belmonte et al., 2005; Maughanet al., 2006; Reichheld et al., 2007). It is known thatanimal cells are characterized by a low level of glu�tathione in the G1 phase of the cell cycle. An increasein the GSH content is required for the passage of theG1/S phase of the cell cycle by cells (Kerk et al.,2000). Since the depletion of glutathione caused byBSO may affect the cell cycle passage and cell division,we tested the effect of BSO on the mitotic index inboth cultures (for 7 days of culturing). In the nonmor�phogenic callus, a significant difference in the mitoticindex decrease was observed starting from 3 days; onthe second day, the culture still actively divided in boththe experiment and control (Fig. 3a). Even on day 7 ofculturing, approximately 1% of cells continued todivide in the nonmorphogenic callus (Fig. 3a),

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Fig. 2. Effect of BSO on the morphology and histology of the morphogenic callus of the Tatar buckwheat: (a) exterior view of themorphogenic callus grown in RX medium, (b) exterior view of the morphogenic callus grown in RX medium + 0.1 mM BSO,(c) histology of PECCs in RX medium, (d) histology of PECCs in RX medium + 0.1 mM BSO. Arrows indicate PECCs.

(c) (d)

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whereas not more than 0.44% of cells continued todivide in the morphogenic callus (Fig. 3b). In themorphogenic callus, BSO suppressed cell division asearly as after one day of cultivation (Fig. 3a). Thus, wehave found that BSO at a concentration of 0.1 mMaffects cell division in both callus cultures, exerting acytostatic effect. Earlier, Sanchez�Fernandez et al.(1997) showed that the depletion of glutathione afterthe addition of 1 mM BSO to the growth mediumreduced the mitotic index in A. thaliana roots, whereasthe addition of 0.25 mM exogenous GSH increased it.We have shown that, in the cultured cells of the Tatarbuckwheat, the cytostatic effect was exerted by a muchlower concentration of the inhibitor. Apparently, theinhibition of cell division explains the fact that thenumber of PECCs in the morphogenic callus was sig�nificantly lower than in the control by day 7 of cultiva�tion in the BSO�containing medium. The absence ofdifferences in the biomass growth of the morphogeniccallus in the medium with and without the inhibitorwas probably due to an increased vacuolization andhydration of cells.

2. Dynamics of the Content of Glutathione and Antioxidant Enzymes during Passage in Cultures

with Different Morphogenic Activity

In general, the total glutathione content in themorphogenic and nonmorphogenic calli differed onlyslightly (Fig. 4c) and ranged over a passage within400–600 µM per gram dry weight. The main differ�ences were observed in the ratio of the content of thetwo forms of glutathione, GSH and GSSG, in cells(Figs. 4a, 4b, 4d). We have also identified some char�acteristic features in the dynamics of the GSH contentduring passage of both callus cultures (Fig. 4a).

The transfer of the nonmorphogenic callus to afresh culture medium was accompanied by a slightincrease in the GSH content, which was observed untilday 6 of passage (Fig. 4a) and was apparently associ�

ated with the division of callus cells. It was shown thatGSH is required for maintaining cell division inA. thaliana root meristem (Sanchez�Fernandez et al.,1997). GSH was detected in actively dividing initialcells but was absent in the slowly dividing cells of thequiescent center (Jiang et al., 2003). The GSH con�tent in the cells gradually decreased on day 14 of culti�vation (on average, 330 µM per gram dry weight) andthen drastically increased on day 16 (1050 µM pergram dry weight). In another experiment in which thecallus cultivation time was increased to 18 days (datanot shown), we again observed a sharp increase in theGSH content (up to 800 µM per gram dry weight) onday 15 and a drastic decrease (200 µM per gram dryweight) on day 18 of cultivation. During passage, theGSSG content in the nonmorphogenic callus cellsvaried from 150 to 250 µM per gram dry weight,increasing to 280–400 mM per gram dry weight onday 16–18. It can be assumed that such fluctuations inthe content of GSH and GSSG were due to cell agingand death, because the content of GSH upon agingdecreases whereas the content of GSSG, conversely,increases (Groten et al., 2006). The sharp increase inthe GSH content on days 15–16 of cultivation is diffi�cult to explain; possibly, it is associated with a decreasein the activity of enzymes that remove glutathionefrom the cell. This period apparently corresponds tolimitation of the culture for the substrate (e.g., sulfate)and its starvation. A similar effect was described byPellny et al. (2009) for A. thaliana suspension culture.The authors of that study showed that the GSH con�tent in the cells, which increased in the exponentialgrowth phase, dramatically decreased when the cul�ture growth was stopped. The exponential growth ofthe culture and GSH accumulation was extended afterthe addition of fresh medium to the cultured cells.

The GSH/GSSG ratio during the passage of thenonmorphogenic callus was higher on days 1–6 of cul�tivation (up to 2.8), then decreased to 1 on day 14 and,

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Fig. 3. Effect of BSO on the mitotic index of (a) nonmorphogenic and (b) morphogenic callus of the Tatar buckwheat grown in(1) RX medium and (2) RX medium + 0.1 mM BSO.

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EFFECT OF D,L�BUTHIONINE�S,R�SULFOXIMINE 47

after a sharp and short�term increase on day 15–16(up to 4), it dropped to 0.48. In addition to line 1�8p,we also studied the content of two glutathione forms intwo other lines (1�10p and 1�5p) of the nonmorpho�genic callus of the Tatar buckwheat (data not shown).Similarly to line 1�8p, the GSH/GSSG ratio in theselines during the passage was very low compared to themorphogenic callus and ranged from 1 to 2.

In the morphogenic callus, the GSH content dur�ing the passage increased on the second day of cultiva�tion, decreased on day 6, and then increased again onday 8 (Fig. 4a). This sequential increase, decrease, andanother increase in the GSH content was observed intwo independent experiments and is reliable. Probably,this dynamics in the GSH content is associated withthe PECC formation cycle. On days 2–4 of cultiva�tion, enhancement of secretory processes, formationof trichomes, and loosening of preexisting PECCs wasobserved (Rumyantseva et al., 2004). It is known that

the secretion of proteins (Chakravarthi et al., 2004)and the formation of trichomes (Gutierrez�Alcalaet al., 2000) depend on the GSH synthesis. Theenhancement of secretion is correlated with the begin�ning of cell division and the initiation of new PECCs.As already mentioned, a high intracellular GSH con�tent is characteristic of proliferating cells and mer�istems; in senescent cells, the GSH content decreases,whereas the GSSG content increases. The decrease inthe GSH content on day 6 was probably due to the ter�mination of secretory process, which correlates to thedeath of trichomes. The subsequent increase in theGSH content was due to the division and differentiationof the newly formed PECCs. It is important to notethat, in different morphogenic callus lines, the PECCformation cycle was characterized by the same dynam�ics of changes in the GSH content (data not shown).

The GSH contents during the culture passage was,on average, 416 µM per gram dry weight (except for

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2 4 6 8 10 12 14 16Cultivation time, days

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2 4 6 8 10 12 14 16Cultivation time, days

1

2(d)

3

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300200

500

700

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1100

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SG

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120

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800

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2 4 6 8 10 12 14 16Cultivation time, days

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Fig. 4. Dynamics of (a) reduced and (b) oxidized glutathione, (c) total glutathione content, and (d) GSH/GSSG ratio in the cellsof morphogenic and nonmorphogenic callus of the Tatar buckwheat. Designations: (1) RX medium, morphogenic callus;(2) RX medium + 0.1 mM BSO, morphogenic callus; (3) RX medium, nonmorphogenic callus; (4) RX medium + 0.1 mM BSO,nonmorphogenic callus.

48

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NIGMATULLINA et al.

day 6). It should be noted that the content of GSSG inthe morphogenic callus hardly changed during thepassage, remaining approximately two times lowerthan in the nonmorphogenic callus and only slightlyincreasing by the end of the passage (Fig. 4b). TheGSH/GSSG ratio during the entire passage of themorphogenic callus was maintained at a high level (2.8to 5.5) (Fig. 4d).

The study of the activity of glutathione reductase,ascorbate reductase, and glutathione�S�transferaseshowed that the activity of glutathione reductase in thenonmorphogenic callus was higher than in the mor�phogenic callus, whereas the activity of ascorbatereductase and glutathione�S�transferase was higher inthe morphogenic callus (Figs. 5a, 5b, 5c). A very large(10 to 20�fold) difference in the activity of glu�tathione�S�transferase in the two cultures is also worthmentioning. Interestingly, the activity of glutathionereductase and glutathione�S�transferase in the mor�phogenic callus correlated with the increase and

decrease in the GSH content in the cells. The activity ofascorbate peroxidase did not show such a relationship.

3. Effect of BSO on the Content of Different Forms of Glutathione and the Activity of Antioxidant Enzymes

in the Cells with Opposite Morphogenic Capacity

In the presence of 0.1 mM BSO, the GSH contentdecreased in both the morphogenic and nonmorpho�genic callus compared to the control (Fig. 4a). How�ever, the decrease in the GSH content in the morpho�genic callus was observed on days 6–8 of cultivationand it was temporary, whereas the GSH content in thenonmorphogenic callus decreased already after oneday and remained lower than in the control through�out the entire passage. In the morphogenic callus cells,the GSH content on days 2 and 14 of cultivation in theBSO�containing medium was higher than in the con�trol. Importantly, BSO did not affect the GSSG con�tent in the morphogenic culture cells, whereas in the

00.02

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Fig. 5. Effect of BSO on the activity of (a) ascorbate peroxidase, (b) glutathione reductase, and (c) glutathione�S�transferase inthe nonmorphogenic and morphogenic callus of the Tatar buckwheat. Designations: (1) RX medium, morphogenic callus;(2) RX medium + 0.1 mM BSO, morphogenic callus; (3) RX medium, nonmorphogenic callus; (4) RX medium + 0.1 mM BSO,nonmorphogenic callus.

RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY Vol. 45 No. 1 2014

EFFECT OF D,L�BUTHIONINE�S,R�SULFOXIMINE 49

nonmorphogenic callus cells an increase in the GSSGcontent was observed starting from the third day. TheGSH/GSSG ratio in the morphogenic callus grown inthe BSO�containing medium varied from 2 to 6 and ondays 1, 2, and 14 was higher than in the control. In thenonmorphogenic callus, the GSH/GSSG ratiodecreased to 1.3 already after one day of cultivation inthe BSO�containing medium and did not exceed0.5 starting from day 6 of cultivation (Fig. 4d).

In the nonmorphogenic callus, an increased activ�ity of ascorbate peroxidase in the BSO�containingmedium was detected starting from day 6 of cultiva�tion, although it still remained several times lower thanin the morphogenic callus (Fig. 5a). The activity ofascorbate peroxidase in the morphogenic callusdecreased only on the second day of cultivation; onother days of cultivation, it remained the same as inthe control or even increased (day 14).

The activity of glutathione reductase in the mor�phogenic callus grown in the BSO�containingmedium increased on days 6, 14, and 16 of cultivation;on other days, it was comparable to the control level(Fig. 5b). Conversely, the activity of glutathionereductase in the nonmorphogenic callus decreasedalready after 2 days of cultivation but was still higherthan in the morphogenic callus.

The activity of glutathione�S�transferase in thenonmorphogenic callus, which was low in generalcompared to the morphogenic callus, was inhibited asearly as on the first and second days of cultivation inthe BSO�containing medium and then did not differfrom the control. In the morphogenic callus, the sup�pression of the activity of glutathione�S�transferase inthe BSO�containing medium was observed tempo�rarily, on days 6–8 of cultivation (Fig. 5c). Glu�tathione�S�transferases catalyze the formation of con�jugates of glutathione with a wide range of hydropho�bic, electrophilic, and cytotoxic substrates, which arethen transported by the ATP�dependent pump GS�Xto the vacuole. Many glutathione�S�transferases func�tion as GSH�dependent peroxidases and catalyze thereduction of organic hydroperoxides. It was shownthat transgenic A. thaliana plants overexpressing theglutathione�S�transferase gene are more resistant tooxidative stress induced by paraquat, blossom earlierthan the control plants, and have a greater number ofleaves (Gong et al., 2005). In some studies, the corre�lation between the expression of glutathione�S�trans�ferase genes and the ability of cultured cells to undergosomatic embryogenesis was identified (Kuriyamaet al., 2002; Galland et al., 2001, Imin et al., 2001).Apparently, the overexpression of glutathione�S�transferase not only ensures the protection against thedestructive effects of ROS but also modulates signalingin cultured cells. It is known that the signalingresponse to an increased level of hydrogen peroxide,which triggers morphogenetic responses in cells,includes an increased calcium mobilization, protein

phosphorylation, and gene expression (Neill et al.,2002).

CONCLUSIONS

Thus, our study has shown:(1) In Tatar buckwheat cultures with opposite mor�

phogenic activity, the total glutathione content isapproximately the same. However, the GSH contentwas higher in the morphogenic culture, whereas theGSSG content was higher in the nonmorphogenicculture. Thus, the morphogenic calli have a largereserve of GSH, which may be involved in the protec�tion against inactivation of enzymes and reduction ofdehydroascorbate or directly reduce ROS.

(2) During passage, the GSH content changes inboth the morphogenic and nonmorphogenic cultures.In the morphogenic callus, the GSH dynamics is asso�ciated with the PECC formation cycle, whereas anincrease in the GSH content in the nonmorphogeniccallus was associated with the end of the stationarygrowth phase and preceded the culture death. In themorphogenic callus, the content of GSSG during pas�sage hardly changed, remaining approximately twotimes lower compared to the nonmorphogenic callus.

(3) An exposure to BSO resulted in a decrease inthe total glutathione content only in the nonmorpho�genic culture, which might testify to the suppression ofγ�glutamylcysteine synthase activity and decrease inthe GSH synthesis. Nevertheless, BSO had a cyto�static effect on cells of both cultures, and this effectwas more pronounced in the morphogenic callus. Itcannot be ruled out that this effect in the morphogeniccallus cells may be associated with a decrease in theascorbate content and, respectively, an increase in thedehydroascorbate content. It is known that dehy�droascorbate inhibits division in plant cells (Jianget al., 2003). It was also shown that dehydroascorbateand its degradation product oxalate can cause loosen�ing of cell walls and enhance cell vacuolation (Linet al., 1991), which we observed in both cultures.

(4) Judging by the fact that, under exposure toBSO, the content of GSH in the morphogenic culturedecreased very slightly (only on day 8) and can evenincrease (days 2 and 14), it can be assumed that GSSGin the morphogenic callus grown in the BSO�contain�ing medium is actively reduced. This reduction isprobably catalyzed by glutathione reductase, becausethis enzyme is activated in the callus cells grown in theBSO�containing medium. In the nonmorphogenicculture grown in the BSO�containing medium, con�versely, glutathione reductase is inhibited.

(5) The high activity of glutathione�S�transferases,detected by us in the morphogenic cultures of theTatar buckwheat, is also characteristic of the embryo�genic cultures of other plants. Glutathione�S�trans�ferases are protein markers of somatic embryogenesisand allow cells to effectively resist oxidative stress

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NIGMATULLINA et al.

(Sharifi et al., 2012). It can be assumed that, at least inpart, the high content of GSH in the morphogeniccultures is required for efficient functioning of glu�tathione�S�transferase.

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Translated by M. Batrukova