ISSN 1021�4437, Russian Journal of Plant Physiology, 2011, Vol. 58, No. 2, pp. 324–329. © Pleiades Publishing, Ltd., 2011.Original Russian Text © V.D. Kreslavskii, V.Yu. Lubimov, L.M. Kotova, A.A. Kotov, 2011, published in Fiziologiya Rastenii, 2011, Vol. 58, No. 2, pp. 262–267.

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INTRODUCTION

Induction of defense antioxidant systems by planttreatments with inhibitors of gibberellin synthesis,retardants chlorocholine chloride (CCC) and cholinechloride (CC) [1–4], and also with ABA and cytoki�nins increases plant tolerance to various stress factorsleading to oxidative damages [5–7].

Earlier, it has been found that treatment with CCCincreased tolerance of PSII in common bean com�pletely expanded leaves to UV�B stress and short�termheating [3]. Improved tolerance of the photosyntheticapparatus (PA) was explained by CCC�induced acti�vation of the antioxidant system in the leaves.

In addition, CCC and CC could change the con�tent of ABA in plant leaves, and this effect dependedlargely on the plant developmental phase, doses ofused compounds, light intensity, and many other fac�tors. Thus, Kur’yata et al. [8] showed that CCCincreased the free ABA content in raspberry leaves. Incontrast, Wang and Xiao [9] observed a decrease in thefree ABA content in potato leaves under the influenceof CCC.

The enhanced synthesis of ethylene, the increasedcontent of ABA and sometimes of cytokinins could

also protect PA and plants against negative stressoreffects [5–7, 10]. Thus, a rapid and strong increase inthe ABA level in wheat and cucumber plants duringthe early period of high hardening temperatures per�mitted a conclusion that this hormone is involved inthe development of plant tolerance to elevated tem�perature [7].

The objective of this work was to study the effects ofCCC on the contents of ABA, cytokinins, and gibber�ellins, and also pigments absorbing UV�B and hydro�gen peroxide during the development of PSII toler�ance to UV�B�induced stress in common bean plants.

MATERIALS AND METHODS

Common bean (Phaseolus vulgaris L., cv. Berbuk�skaya) seeds were imbibed in water for 14 h and thengerminated in petri dishes on moistened filter paper at20 ± 1°C under low light (5 W/m2). After the appear�ance of first true leaves, seedling roots were immersedin the 1.6 mM CCC solution or water for 24 h. There�after, the roots were washed from CCC with water fourtimes, and seedlings were planted in plastic vessels(18 × 25 cm2 in area and 12 cm in height filled withvermiculite) and grown at a 12�h photoperiod underDRLF�400 lamps (60 W/m2 PAR) and 23/20°С(day/night). In 6 days, true leaves were excised, placedin enamel bath on moistened filter paper, adapted todarkness for 1 h, and subjected to 7�h irradiation ofUV�B at the intensity of 0.45 W/m2 (11.3 kJ/m2).

Effect of Common Bean Seedling Pretreatment with Chlorocholine Chloride on Photosystem II Tolerance to UV�B Radiation, Phytohormone Content, and Hydrogen Peroxide Content

V. D. Kreslavskiia, V. Yu. Lubimova, L. M. Kotovab, and A. A. Kotovb

a Institute for Basic Problems of Biology, Russian Academy of Sciences, Institutskaya ul. 2, Pushchino, 142290 Russia;e�mail: vkreslav@rambler.ru

b Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia Received March 16, 2010

Abstract—In true leaves of common bean (Phaseolus vulgaris L., cv. Berbukskaya) 11�day�old seedlings, pre�treatment with 1.6 mM chlorocholine chloride (CCC) resulted in the improved photosystem II (PSII) toler�ance to UV�B radiation, an increase in the contents of cytokinins, ABA, and H2O2, but a decrease in the con�tent of gibberellins. It is suggested that development of increased PSII tolerance to UV�B under the influenceof CCC is partially related to the increase in the contents of ABA, cytokinins, and UV�absorbing pigments inthe leaves of pretreated plants.

Keywords: Phaseolus vulgaris, abscisic acid, tolerance to UV�B radiation, photosystem II, chlorocholinechloride.

DOI: 10.1134/S1021443711020087

Abbreviations: CCC—chlorocholine chloride; GA1—gibberellin 1;MeOH—methanol; PA—photosynthetic apparatus; PhH—phy�tohormones; PBST—phosphate�buffered saline containing Tri�ton X�100 (0.01 M sodium�phosphate + 0.15 M NaCl + 0.05%Triton X�100); Z—zeatin; ZR—zeatin riboside.

RESEARCHPAPERS

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EFFECT OF COMMON BEAN SEEDLING PRETREATMENT WITH CHLOROCHOLINE 325

Control nonirradiated plants were kept in the similarbath in darkness. Then, leaves were retained in dark�ness or under continuous light (30 W/m2). Immedi�ately before and after UV�B irradiation and also dur�ing subsequent 48 h, PSII activity was assayed andleaves were harvested for measuring the contents ofUV�absorbing compounds and Н2О2. Phytohormone(PhH) contents were measured before and after UV�irradiation and also in 36 h after irradiation. Disksexcised from leaves were fixed and stored in liquidnitrogen.

UV�B was produced using an LOS�2 system (SKB,Pushchino) on the basis of DKSSh�1000 lamp and theinterference filter with the maximum of 302 ± 12 nm.In some experiments on UV�B influence on growthparameters, we used an irradiator Electronika UFO�03�250N (Russia) on the basis of erithemic DRP�250lamp; UV�B was isolated using the interference filterwith the maximum of 302 ± 12 nm and UFS�1 glassfilter.

Chlorophyll fluorescence was measured with a sin�gle beam fluorometer. To excite fluorescence, the leafwas fixed on a holder and illuminated with a KGM�100 lamp through a SZS–22 glass filter at 45° to theleaf surface. Light intensity was 30 W/m2. Fluores�cence was recorded after 20�min adaptation to darkness,using an interference filter with λm = 685 ± 10 nm, anMDR�2 monochromator, and a FEU�119 light detec�tor. Fluorescence induction curves were recorded witha rapid Endim 322�01N self�recorder (VEB MA, Ger�many). Parameters of variable fluorescence of Chl a,described in detail in [11], were measured.

The intensity of UV�B was measured with an S�100spectrometer (Solar Laser Systems, Minsk) and anRTN�10 thermoelement; visible light intensity wasmeasured with Yanishevskii pyranometer.

To study possible reasons for CCC protectiveaction against UV�B radiation, the contents of cytoki�nins, ABA, and gibberellins were analyzed.

Isolation and purification of zeatin (Z), zeatinriboside (ZR), GA1, and ABA before their quantifica�tion by the ELISA method were performed asdescribed earlier [12, 13]. Leaf disks (0.5–0.9 g fr wt)were ground in liquid nitrogen and extracted with 6 mlof 80% methanol with antioxidants (200 mg/l of buty�lated hydroxytoluene (Sigma, United States) and100 mg/l of ascorbic acid) at 4°С for 20 h on theshaker. The extract was purified from lipids and chlo�rophylls using a minicartridge with 50 mg of divinyl�benzol stirol sorbent Porolas TM (Omskchimprom,Russia) and evaporated to water residue under the fluxof nitrogen at 40°С. To remove phenolic compounds,the sample was re�dissolved in 2 ml of 0.05 M K�phos�phate buffer (pH 3.0) and passed through the minicol�umn with 1 ml of polyvinylpolypyrrolidone (Sigma),which was then washed with 6 ml of the same buffer.Phytohormones (PhH) from the obtained eluate were

concentrated on the Amprep C18 minicolumn (300 mg,Amersham, England) attached to the first minicol�umn. PhH were successively eluted with the AmprepC18 minicolumn with the mixtures of methanol(MeOH) and 0.2 N aqueous acetic acid: 5.5 ml of 20%MeOH (Z + ZR), 4 ml of 25% MeOH (GA1), and,finally, 4 ml of 40% MeOH (ABA). During fraction�ation, each PhH fraction was concentrated using aminicartridge with Porolas TM attached to theAmprep C18 minicolumn. Minicartringes werewashed with 5 ml of H2O; PhH was eluted with 5 ml of100% MeOH, and fractions were evaporated undervacuum at 35°С up to dry residue. GA1�containingfractions were methylated with the solution of diazo�methane in diethyl ether. The yield of PhH, as assessedusing standards from Sigma, was 85% for ZR, 95% forZ, 85% for GA1, and 99% for ABA. Since PhH losseswere small, they were not taken into considerationduring PhH content determination.

ELISA determination of ZR, GA1 methyl ester,and ABA was performed as described earlier [14] in96�well flat bottom polystirol Immulon Dynatechmicrotiter microtiter plates (M129B, Dynatech Labo�ratories, United States). Hormone–protein conjugate(~0.1 μg/ml) (ovalbumin modified with lysine as aspacer and covalently bound with ZR, GA1, or ABA)in 0.05 M Na�carbonate buffer (pH 9.7) was prelimi�narily absorbed in the wells [14]. After washing withPBST (phosphate�buffered saline, pH 7.4, containing0.01 M Na�phosphate, 0.15 M NaCl, and 0.05% Tri�ton X�100), 100 μl of the sample diluted in PBST and100 μl of rabbit anti�ZR, anti�GA1 serum diluted withPBST in 200000 and 100000 times, respectively, oranti�ABA affinity purified antibody diluted in200 times with PBST containing 1% ovalnumin wasadded [14], and incubation was performed at 4°С for2 h. The tested hormone competed for antibody bind�ing with the hormone–protein conjugate absorbed onthe plate; thus, the content of antibody fixed on theplate was inversely depended on the hormone contentin the sample. Thereafter, the plates were washed withPBST, and 200 μl of secondary anti�rabbit antibodyconjugated with horseradish peroxidase (GamaleyaResearch Institute of Epidemiology and Microbiol�ogy, RAMS) diluted with PBST containing 0.5% oval�bumin (1 : 500) was added to each well; and plates wereincubated at 37°С for 1 h. After washing with PBSTand then H2O2, peroxidase activity was assessed in thereaction with substrate buffer (0.06 M Na�phosphatebuffer, pH 5.0, 0.03% H2O2, and о�phenylenediamine(0.4 mg/ml)) performed at 37°С for 30 min, and chro�mophore content was measured at 492 nm using aTitertek Multiscan MCC�340 plate reader. Immu�noreactivity of trans�Z was 40% from that of trans�ZR,and their total content was calculated in ZR equi�valent.

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To determine hydrogen peroxide pool, leaf disks(50–100 mg) were kept in liquid nitrogen for 2–3 min.Frozen material was transferred in 0.4 ml of 2 M TCAand ground. The homogenate was transferred in thecentrifuge tube with 0.05 M K�phosphate buffer bythree washing (1 ml each). 100 mg of activated char�coal was added to 3.5 ml of obtained homogenate toabsorb pheophytin and carotenoids, and centrifuga�tion was performed at 10000 g for 20 min. The super�natant was decanted and titrated with 2 M KOH up to

pH 7.0. The content of Н2О2 was determined in 100 μlof the extract by the method of bioluminescence in thesystem 5 × 10–6 M peroxidase : 5 × 10–4 M luminol(1 ml in volume) [15]. The Н2О2 content in controlplant leaves was 1.1 ± 0.1 μmol/g fr wt.

The content of UV�absorbing pigments was deter�mined by the method of Mirecki and Teramura [16].To this end, 15–25 leaf disks 10 mm in diameter (5disks from each leaf) were kept in acid MeOH (MeOH :water : HCl, 78 : 20 : 2) at 4°С for 24 h. Then opticaldensity was measured at 320 nm with an M�40 spec�trophotometer (Carl Zeiss, Germany); the amount ofUV�absorbing pigments was expressed in rel.; unitsper leaf dry weight.

Tables and figures present mean values and theirstandard errors (SE). Difference significance was eval�uated by the Student’s t criterion at P = 5%.

RESULTS

In the leaves of 6�day�old seedlings treated with1.6 mM CCC, PSII activity was lower than in controlnon�treated leaves (figure, curves 1 and 2). Leaf irradi�ation with UV�B resulted in a strong reduction in thePSII activity; subsequent leaf exposure to darkness for48 h further reduced PSII activity (figure, curve 4). InUV�B irradiated leaves of plants pretreated with CCC,PSII activity declined to a lesser degree (figure, curve 3).

Pretreatment with CCC decreased the content ofGA1�like compounds to 33.6% but increased contentsof ABA and Z + ZR to 192 and 125%, respectively(Table 1). In UV�B�irradiated seedlings, leaves main�tained reduced content of GA1�like compounds andelevated content of Z + ZR. Irradiated leaf exposure todarkness reduced whereas their exposure to lightincreased the content of Z + ZR in the leaves of CCC�treated plants. In control leaves, the level of Z + ZRincreased in darkness and remained almost unchangedin the light. As a result, the ratio of Z + ZR in theleaves pretreated and control plants after their keepingin the light increased up to 141%, whereas in darkness

Fv/

Fm

0.8

0.7

0.6

0.5

0.40 20 40 60

1

2

3

4

Time after the start of irradiation, h

Effect of common bean seedling UV�B irradiation andtreatment with 1.6 mM CCC on PSII photochemicalactivity.(1) Non�irradiated leaves of seedlings non�treated withCCC; (2) non�irradiated leaves of seedlings treated withCCC; (3) and (4) irradiated leaves of seedlings treated andnon�treated with CCC, respectively. Means of three repli�cations and their standard errors are presented.

Table 1. Effect of pretreatment with CCC and UV�B irradiation on the levels of ABA, GA1�like compounds, and cytokinins(Z + ZR) in true leaves of 11�day old common bean plants, pmol/g dry wt

Treatment

ABA GA1�like compounds Z + ZR

no treatment with CCC

treatment with CCC %* no treatment

with CCCtreatment with CCC %* no treatment

with CCCtreatment with CCC %*

Before irradiation 818 ± 81 1571 ± 204 192 10.34 ± 0.23 3.47 ± 0.05 33.6 3.75 ± 0.1 4.7 ± 0.1 125

0 h 568 ± 45 612 ± 51 108 6.46 ± 0.34 2.35 ± 0.1 36.4 3.64 ± 0.2 4.4 ± 0.2 121

36 h in darkness 886 ± 91 449 ± 71 52 4.88 ± 0.70 1 ± 0.1 20.5 4.77 ± 0.1 4.0 ± 0.2 84

36 h in the light 1841 ± 205 704 ± 143 38 2.95 ± 0.34 1 ± 0.5 34 3.75 ± 0.1 5.3 ± 0.7 141

Notes: Immunoreactivities of fractions containing Z + ZR and GA1�like compounds were expressed in equivalents of zeatin riboside andGA1, respectively. 0 and 36 h are times after UV�B irradiation. Means of three replications and their standard errors are presented.

* The ratio of hormone content in CCC�treated to CCC�non�treated plant leaves.

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it decreased to 84%. As distinct from cytokinins, UV�Birradiation reduced markedly the content of ABA inthe leaves of both control and CCC�pretreated plants.However, in the latter case, a decline in the ABA con�tent was much stronger. As a result, the ratio of ABAamount in the leaves of CCC�pretreated seedlings tothat in control ones reduced after irradiation from 192to 108%. Later, during 36�h dark and light exposure,the content of ABA in control increased substantiallyas compared with its level after UV�B irradiation andbecame much higher than in pretreated plants. Thiswas manifested in a decrease in the aforementionedratio of ABA to 52 and 38% in darkness and light,respectively.

The measurement of morphometric indices char�acterizing CCC retardant effect showed that 1.6 mMCCC suppressed growth of stems and leaves simulten�ously with the increase in their specific dry weight(Table 2). CCC also increased the content of UV�absorbing pigments in the leaves (predominantly fla�vonoids) [16] and Н2О2 pool. UV irradiation resultedin Н2О2 accumulation in the leaves of both treated andnon�treated with CCC seedlings; however, in theleaves of pretreated with CCC seedlings, an increase inthe Н2О2 content was much less.

DISCUSSION

Plant capacity of resistance to stress�induced dam�age to PA is critical for plant survival and the mainte�nance of their productivity. One of the ways forimproving PA stress�tolerance is application of retar�dants. Retardant types and their action on plantgrowth and development are described in somereviews [1, 17]. The mechanism of their action on PAtolerance to stresses is less studied. The enhancedantioxidant activity of the leaves in retardant�treatedplants plays a great role in PA and whole plant toler�

ance [3, 4, 18]. It might be determined by retardant�induced formation of Н2О2 and other ROS.

Several reasons may determine the increase in theН2О2 content. ABA accumulation is often accompa�nied by the development of oxidative stress and anincrease in the Н2О2 content [19–21]. A decrease inthe photosynthetic activity, which was observed afterthe treatment in the CCC concentration of 1.6 mM(figure), could result in the Н2О2 accumulation [22].In its turn, the development of oxidative stress resultsin the increase in the antioxidant potential in plantleaves, i.e., activation of antioxidant enzymes and thepool of low�molecular�weight antioxidants, whichmay be one of the reasons of the lower Н2О2 level inthe leaves at the combined action of UV�B and CCCas compared to that in irradiated but not pretreatedwith CCC plants.

Other reasons for plant improved stress�toleranceunder the influence of retardants may be changes inthe cell membrane lipid composition, accumulation ofUV�absorbing pigments and phytohormones, ABAand cytokinins [4, 8]. In fact, plant pretreatment withCCC resulted in the elevated content of ABA andcytokinins by 92 and 25%, respectively (Table 1). Anincreased level of cytokinins was maintained duringUV�irradiation, and it increased by 40% for 36 h in thelight after irradiation. These results are in agreementwith the data described in [23] that CCC increased thecontent of cytokinins in the first leaves of greeningwheat seedlings.

During UV�irradiation, the content of ABA in theleaves of plants both treated and non�treated withCCC declined strongly. After 36�h exposure of non�treated with CCC leaves to light or darkness, the levelof ABA increased markedly, whereas in CCC�treatedleaves it decreased in darkness and was almostunchanged in the light. These data show that less tol�erant non�treated with CCC plants accumulated the

Table 2. Effect of pretreatment with CCC and UV�B irradiation on morphometric indices, the content of leaf pigmentsabsorbing UV�B (A320), and H2O2 pool in the leaves of 11�day�old common bean plants

IndexNo treatment with CCC Treatment with CCC

no irradiation UV�B irradiation no irradiation UV�B irradiation

Stem length, cm 27.5 ± 3.4 – 12.4 ± 2.1 –

Leaf area, cm2 23 ± 4 – 9.5 ± 1.4 –

Leaf dry wt, % fr wt 8.8 ± 0.5 8.9 ± 0.5* 9.8 ± 0.5 9.6 ± 0.7

OD (A320) units/g dry wt 1.00 ± 0.06 1.23 ± 0.06 1.34 ± 0.01 1.34 ± 0.01

H2O2, % of control 100 ± 7 144 ± 10 128 ± 10 123 ± 9

Notes: Detached leaves were irradiated with UV�B for 7 h, determined morphometric indices; in subsequent 48 h in darkness, samples weretaken for the analysis of pigments and H2O2. The content of H2O2 in control was 12.5 ± 1 µmol/g dry wt. Means of three replicationsand their standard errors are presented.

* Insignificant difference between UV�B�irradiated and non�irradiated leaves (P > 0.05).

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stress hormone ABA more intensively. At the sametime, plants, which accumulated twice more ABAbefore irradiation under the influence of CCC, toleratedUV�B stress easier.

The development of plant stress tolerance is oftenassociated with their suppressed growth and metabo�lism. Treatment with the retardant CCC resulted inreduced stem height and leaf area as compared withnon�treated plants (Table 2), whereas plant toleranceto UV�irradiation increased (figure). Retardation ofstem growth could be explained by a decrease in thegibberellin level, whereas reduction in the leaf area, bythe action of inhibitors, ABA in particular. It is worthmentioning that ABA, like cytokinins, induces thesynthesis of not only heat shock proteins but also someother stress proteins, dehydrins in particular. Dehy�drins protect photosynthetic proteins against denatur�ing and aggregation and thylakoid membranes, againstinactivation [5, 24].

At moderate latitudes, the proportion of UV�Bcomprise only 3–4% of total amount of UV falling onthe plant. In the period of growing of various cultures,the day dose of UV�B radiation is not great and usuallycomprises 2–12 kJ/m2 (the highest dose correspondedthat applied in our experiments) [25]. Nevertheless,this component of solar radiation is most active in theinhibition of plant growth and photosynthesis. Itmight be supposed that CCC�induced accumulationof ABA, cytokinins, and UV�absorbing pigments, andalso activation of the antioxidant system result in theimproved PSII tolerance to UV radiation.

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