Plant Physiology and Biochemistry 80 (2014) 75e82

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Research article

Effects of exogenous 24-epibrassinolide on the photosynthetic membranes under non-stress conditions Anelia G. Dobrikova*, Radka S. Vladkova, Georgi D. Rashkov, Svetla J. Todinova, Sashka B. Krumova, Emilia L. Apostolova Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev, St. 21, Sofia 1113, Bulgaria

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 December 2013 Accepted 22 March 2014 Available online 31 March 2014

In the present work the effects of exogenous 24-epibrassinolide (EBR) on functional and structural characteristics of the thylakoid membranes under non-stress conditions were evaluated 48 h after spraying of pea plants with different concentrations of EBR (0.01, 0.1 and 1.0 mg.L
1). The results show that the application of 0.1 mg.L
1 EBR has the most pronounced effect on the studied characteristics of the photosynthetic membranes. The observed changes in 540 nm light scattering and in the calorimetric transitions suggest alterations in the structural organization of the thylakoid membranes after EBR treatment, which in turn influence the kinetics of oxygen evolution, accelerate the electron transport rate, increase the effective quantum yield of photosystem II and the photochemical quenching. The EBRinduced changes in the photosynthetic membranes are most probably involved in the stress tolerance of plants. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Brassinosteroids Calorimetric transitions Chlorophyll fluorescence Oxygen evolution Pea plants Thylakoid membranes

1. Introduction Brassinosteroids (BRs) are a class of plant steroid hormones involved in several physiological and morphological processes, as well as in the responses to environmental stresses via activation of different protective mechanisms (Bajguz and Hayat, 2009). Exogenous application of very low (picomolar to nanomolar) BRs concentrations influences multiple plant growth and development processes, including cell elongation and division, pollen tube growth, ethylene biosynthesis, senescence and enzyme activation,  while micromolar concentrations cause inhibitory effects (see Çag et al., 2007; Sasse, 2003). Apparently requirement for an optimal BR level is needed for proper plant growth and development. BRs are also proposed to regulate photosynthesis; specifically 24epibrassinolide (EBR) application was shown to significantly

Abbreviations: BRs, brassinosteroids; Chl, chlorophyll; DSC, differential scanning calorimetry; EBR, 24-epibrassinolide; ETR, electron transport rate; Fv/Fm, maximum quantum efficiency of PSII photochemistry in dark-adapted state; Fv0 /Fm0 , maximum quantum efficiency of PSII photochemistry in light-adapted state; LHCII, lightharvesting complex II; PAM fluorescence, pulse-amplitude-modulated fluorescence; NPQ, non-photochemical quenching; PSI, photosystem I; PSII, photosystem II; qN, non-photochemical quenching coefficient; qP, photochemical quenching coefficient; qL, fraction of “open” PSII centers estimated on the basis of PSII lake model, FPSII, effective quantum yield of PSII photochemistry. * Corresponding author. Tel.: þ359 2 9792603; fax: þ359 2 9712493. E-mail address: [email protected] (A.G. Dobrikova). http://dx.doi.org/10.1016/j.plaphy.2014.03.022 0981-9428/Ó 2014 Elsevier Masson SAS. All rights reserved.

increase photosynthetic rate in plants by increasing the Rubisco activity (Xia et al., 2009a; Yu et al., 2004). In addition to their growth regulatory activities, the role of BRs in protecting the photosynthetic apparatus from environmental stress has been widely studied in the last years. Many works have demonstrated that exogenously applied EBR enhances plant tolerance to abiotic stresses such as drought, salinity, heavy metals, extreme temperatures, oxidative stress, etc. (Bajguz and Hayat, 2009; Fariduddin et al., 2014; Janeczko et al., 2011; Kagale et al., 2007; Ogweno et al., 2008, 2010; Sharma et al., 2013; Xia et al., 2009b; Yuan et al., 2012; Zhang et al., 2013; and Refs. therein). BR-enhanced stress tolerance is associated with the regulation of reactive oxygen species (ROS) metabolism and the increase in the antioxidant enzyme activity, as well as with elevated content of ascorbic acid, glutathione, carotenoids, abscisic acid, etc. (Bajguz, 2009; Liu et al., 2011; Mazorra Morales et al., 2014; Ogweno et al., 2008, 2010). Recent studies demonstrated that EBR could alleviate the detrimental effects of different stresses on the plant growth by improving photosynthesis in leaves, mainly due to upregulation of the levels of pigment, protein and proline content, increased expression of various oxidative stress marker genes, improved functioning of photosystem II (PSII) and photochemical activity associated with photosystem I (PSI) (Janeczko et al., 2011; Sharma et al., 2013; Yuan et al., 2012; Zhang et al., 2013). Although the effects of exogenous application of EBR on plants have been extensively investigated in the last years (Janeczko et al.,

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2011; Ogweno et al., 2008; Sharma et al., 2013; Yu et al., 2004; Yuan et al., 2012; Zhang et al., 2013), the mechanisms of the EBR action on the photosynthetic apparatus in plants are still far from being completely understood. In order to clarify the mechanisms of its action on photosynthetic membranes, we focus on the EBR-induced effects (at different concentrations of this hormone) on the structural and functional characteristics of thylakoid membranes under non-stress conditions, 48 h after EBR spraying of pea plants. The EBR action has been characterized by using polarographic oxygen rate electrode, pulse-amplitude-modulated (PAM) chlorophyll fluorescence, light scattering and differential scanning calorimetry (DSC), as well as by determination of the redox changes of P700 and low-temperature (77 K) chlorophyll fluorescence characteristics. The changes in the functional and thermodynamic parameters are discussed in terms of alteration in the structural organization of the thylakoid membranes. This study could improve our understanding of the mechanisms regarding adaptive responses of plants to abiotic stress. 2. Material and methods 2.1. Plant growth and treatment Pea plants (Pisum sativum L. cv. RAN1) were grown as a hydroponic culture under 10- h-light/14-h-dark cycle at 20e25  C. Pea plants (12 days old) were sprayed with 0.01, 0.1 or 1 mg.L
1 24epibrassinolide (SigmaeAldrich) or distilled water (control) as in Ogweno et al. (2008). Thylakoid membranes were isolated 48 h after EBR treatment as described in Ivanova et al. (2008). For all measurements the membranes were resuspended in buffer containing 40 mM Hepes (pH 7.6), 10 mM NaCl, 5 mM MgCl2 and 400 mM sucrose to appropriate concentrations. The total chlorophyll (Chl) concentration was determined by the method of Lichtenthaler (1987). 2.2. Oxygen evolution measurements Oxygen flash yields and initial oxygen bursts were measured using a custom-built polarographic oxygen rate electrode (a Joliottype) described by Zeinalov (2002). The working potential of the cathode was
800 mV, the time constant of the electrode was less than 2 ms and polarograph sensitivity was 1.5 V.mA
1. The Chl concentration was 300 mg.ml
1 and sample volume was 100 ml forming a 2 mm thick layer. Samples were pre-illuminated with ca. 20 flashes and then dark adapted for 5 min before the measurements. Oxygen flash yields were induced by saturating (4 J) and short (t1/2 ¼ 10 ms) periodic flash sequences with 650 ms dark spacing between the flashes. The initial oxygen burst was recorded during irradiation with a saturating continuous white light (400 mmol m
2 s
1). The decay kinetics after the initial oxygen burst were analyzed using Microcal Origin software implementing two exponential decay fitting. Kok’s model parameters (initial S0eS1 state distribution in the dark, misses (a) and double hits (b)) were determined by least square deviations fitting of the experimentally obtained oxygen flash yields with the theoretically calculated yields according to the Kok’s model (Kok et al., 1970). 2.3. Pulse-amplitude-modulated chlorophyll fluorescence PAM chlorophyll fluorescence traces were recorded with a PAM fluorometer (model 101/103, Walz, Effeltrich, Germany) as described in Vladkova et al. (2011). The Chl concentration of the thylakoid membranes and the applied white actinic light intensity were the same as for the oxygen evolution measurements (see

Section 2.2.). The samples (detached leaves or suspension of isolated thylakoid membranes) were dark adapted (for 20 or 15 min, respectively) at room temperature before the measurements. The minimal (F0) and maximal (Fm) fluorescence yields in darkadapted state were estimated by applying very weak red (0.056 mmol m
2 s
1, 650 nm) and saturating white (2700 mmol m
2 s
1, 0.8 s duration) light, respectively. The actinic light (AL, 400 mmol m
2 s
1) was applied for 6 min in combination with saturating pulses per 1 min. The following parameters were used for determining the photosynthetic characteristics of the various samples: Fv/Fm ¼ (FmeF0)/Fm e maximal quantum efficiency of PSII photochemistry in dark-adapted state, Fv0 /Fm0 ¼ (Fm0 eF00 )/Fm0 e maximal quantum efficiency of PSII photochemistry in lightadapted state, qP ¼ (Fm0 eF0 )/Fv0 e photochemical quenching coefficient, wherein F0 is the fluorescence level in light-adapted state, qL ¼ qP .F00 /F0 e parameter reflecting the photochemical quenching according to the lake model for the PSII photosynthetic apparatus, FPSII ¼ (Fm0 eF0 )/Fm0 e effective quantum yield of PSII photochemistry, ETR ¼ PPFD .FPSII. 0.5 e overall PSII electron transport rate, wherein PPFD is the photosynthetically active photon flux density, qN ¼ (FveFv0 )/Fv and NPQ ¼ (FmeFm0 )/Fm0 e parameters characterizing the non-photochemical quenching of variable and of maximal chlorophyll fluorescence, respectively (for more details see Baker, 2008). The PAM chlorophyll fluorescence derived parameters were calculated from 3 to 4 independent experiments. 2.4. Measurements of oxidation-reduction kinetics of P700 The redox state of P700 was determined on detached leaves using a PAM-101/103 modulated fluorometer (Walz, Effeltrich, Germany) equipped with an ED-800T emitter-detector. The oxidation-reduction kinetics of P700 was determined by illumination of the dark adapted (15 min at room temperature) detached leaves with far-red (FR) light supplied by a photodiode (102-FR, Walz, Effeltrich, Germany). The redox state of P700 was assessed by measuring the FR light-induced absorbance changes around 830 nm (DA830). 2.5. Low temperature (77 K) chlorophyll fluorescence measurements Low temperature (77 K) fluorescence emission spectra of isolated thylakoid membranes were recorded upon 436 nm excitation from 600 nm to 780 nm using Jobin Yvon JY3 spectrofluorimeter equipped with a red-sensitive photomultiplier (Hamamatsu R928) and a liquid nitrogen device. The widths of the exciting and measuring beam slits were 4 nm. The samples were resuspended to a final Chl concentration of 20 mg.ml
1 in a buffer containing 40 mM Hepes (pH 7.6), 10 mM NaCl, 400 mM sucrose supplemented or not with 5 mM MgCl2 and were quickly frozen by plunging into liquid nitrogen. 2.6. Light scattering The light scattering at 90 of isolated thylakoid membranes was monitored at room temperature in 1 cm glass cuvette using Jobin Yvon JY3 spectrofluorimeter. The excitation and measuring wavelengths were set to 540 nm. Thylakoid membranes were washed and resuspended to a final Chl concentration of 5 mg.ml
1 in a buffer containing 40 mM Hepes (pH 7.6), 10 mM NaCl, 400 mM sucrose supplemented or not with 5 mM MgCl2. Both stacked (with 5 mM MgCl2) and unstacked (without 5 mM MgCl2) thylakoid membranes were dark adapted for 30 min before measurements.

A.G. Dobrikova et al. / Plant Physiology and Biochemistry 80 (2014) 75e82

2.7. Differential scanning calorimetry (DSC) The DSC scans in the temperature range between 20  C and 100  C were recorded with a heating rate of 0.5  C min
1 with a high-sensitivity differential scanning microcalorimeter (DASM1, Biopribor, Pushchino). The transition temperatures, Tm, (temperatures at the maxima of the successive transitions in the excess heat capacity curve) and the calorimetric enthalpy of the transitions, DHcal, (the area under the excess heat capacity curve) were determined by mathematical deconvolution of the thermograms applying the built-in Gauss function in Origin.

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Table 1 Effect of different EBR concentrations on the ratio of fast to slow operating PSII centers (A1/A2), the initial dark distribution of PSII centers in the S0 state (S1 (%) ¼ 100 e S0), misses (a) and double hits (b). The thylakoid membranes were isolated 48 h after the EBR treatment. Mean values  SE are calculated from 5 to 6 independent experiments (*P < 0.05, **P < 0.01). EBR concentration (mg.L
1)

A1/A2

0 0.01 0.1 1.0

2.45 2.02 1.91 1.34

   

0.20 0.16 0.09* 0.10**

S0 state (%)

a (%)

   

16.4 19.9 19.4 20.3

22.9 26.2 27.1 28.7

1.2 0.8* 1.5* 2.2*

b (%)

   

0.8 0.5* 0.8* 1.4*

4.6 4.9 5.0 5.3

   

0.2 0.3 0.3 0.6

2.8. Statistical analysis The statistical differences among the means were determined using a two-tailed paired Student’s t-test. Values of P < 0.05 were considered as significant differences between the control and treatments. 3. Results 3.1. Photosynthetic oxygen evolution The oxygen evolution data for thylakoid membranes isolated from EBR-treated pea plants are presented on Fig. 1. The amplitudes of both flash-induced oxygen yields (Fig. 1A) and initial oxygen burst under continuous light (Fig. 1B) gradually decrease with the increase of the EBR concentration (0.01, 0.1 and 1.0 mg.L
1). EBRinduced decrease in the oxygen flash yields in comparison to the control (Fig. 1A) indicates a decrease in the oxygen-evolving PSIIa centers (situated in grana domains), which are functioning by the non-cooperative Kok’s mechanism (Kok et al., 1970). The amplitude (A) of the initial oxygen burst (Fig. 1B) is proportional to all functionally active oxygen-evolving centers (i.e. both PSIIa in the grana and PSIIb in the stroma domains). The decay kinetics after the oxygen burst are fitted with two exponential decay functions and the ratio A1/A2 of the obtained amplitudes for the fast (A1) and the slow (A2) components, which corresponds to the ratio of functionally active PSIIa to PSIIb centers (see Apostolova et al., 2006; Ivanova et al., 2008), estimated for the different treatments is presented in Table 1. It decreases with the increase in EBR concentration,

which is due to a decline of the fast amplitude, A1 (due to substantial loss in the sharp oxygen burst) and an increase of the slow amplitude, A2 (i.e. the slow component of the decay kinetic); a slow-down of the decay kinetics is observed in all EBR-treated samples. These results indicate a decrease of the functionally active PSIIa centers in grana core and an increase of the PSIIb centers in stroma lamellae caused by the EBR treatment. In addition, treatment with different EBR concentrations leads to higher (by 12e25%) integrated areas under the induction curves (Fig. 1B) and the maximum value (25%) was observed for samples treated with 0.1 mg.L
1 EBR. This indicates an increase in the evolved oxygen, which is confirmed by a rise (about 20%) of the PSII photosynthetic activity in the presence of exogenous acceptor (pbenzoquinone) from 80 to 96 mmol O2 mg.Chl
1.h
1 for untreated and treated with 0.1 mg.L
1 EBR samples, respectively. Detailed analysis of the oscillation patterns (Fig. 1A) was also done using the Kok’s model (Kok et al., 1970), which assumes cooperation of five intermediate oxidation states of the oxygenevolving complex (S0eS4) for a single PSII center for the production of one oxygen molecule. The calculated values for the initial population of the S0 state in darkness, misses (a) and double hits (b) show that the EBR application leads to increased misses, double hits, and amounts of PSII centers in the initial S0 state (Table 1). 3.2. PAM chlorophyll fluorescence To characterize the effects of EBR treatment on the efficiency of primary photochemical processes in PSII we carried out parallel

Fig. 1. Oscillation patterns of the flash-induced oxygen yields (A) and oxygen induction curves registered under continuous illumination (B) of thylakoid membranes. The thylakoid membranes were isolated 48 h after the spraying of plants with different EBR concentrations or distilled water. The measuring buffer contained 40 mM Hepes (pH 7.6), 10 mM NaCl, 5 mM MgCl2 and 400 mM sucrose. The polarographic sensitivity was 1.5 V. mA
1.

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PAM chlorophyll fluorescence measurements on both leaves and isolated thylakoid membranes and found similar results for both studied objects. Fig. 2 shows the effect of EBR treatment on the PAM chlorophyll fluorescence characteristics of the thylakoid membranes. It is seen that the EBR treatment does not change the maximal quantum efficiency of PSII photochemistry in darkadapted state Fv/Fm, (Fig. 2) as well as the maximal quantum efficiency of PSII photochemistry in light-adapted state (Fv0 /Fm0 ) and the non-photochemical quenching parameters qN and NPQ (data not shown). These results indicate that EBR treatment does not change the efficiency of excitation energy capture by open PSII reaction centres (Fv0 /Fm0 ), as well as the thermal dissipation in the antenna complexes (qN, NPQ). On the other hand, the coefficient of photochemical quenching qP (Fig. 2), its equivalent qL (not shown) according to the lake model for the PSII photosynthetic apparatus (see in Refs. Baker, 2008), the effective quantum yield of PSII photochemistry FPSII (Fig. 2) and the electron transport rate through photosystem II (ETR, not shown) are increased by w25% (at 0.01 mg.L
1 EBR) and by w 35% (at 0.1 mg.L
1 EBR) as compared to the control values (Fig. 2). At 1 mg.L
1 EBR, the values of the treated thylakoids are close to those of the control samples. In the present experimental conditions, we also found that the values of qL are proportional to those of qP upon varying the EBR concentrations. 3.3. Oxidation-reduction kinetics of P700 In order to characterize the effect of different EBR concentrations on the PSI function in vivo we measured steady-state P700 photo-oxidation (P700þ) by FR light-induced absorbance changes around 830 nm (DA830). These changes are caused by the oxidation (rise) of P700 to P700þ by FR light and the subsequent dark reduction (decay) after turning off the FR light, representing the photochemical activity of PSI (Klughammer and Schreiber, 2008; Savitch et al., 2011). The obtained relative amounts of P700þ (DA830) for control and EBR-treated leaves are shown in Table 2. It is seen that the treatment with 0.1 mg.L
1 EBR (shown in Fig. 3) has no significant effect on DA830 (Table 2). Similar results are also observed for the rate of PSI mediated electron transport (DCPIPH2 / MV) after the EBR application (data not shown).

Fig. 2. Effect of 24-epibrassinolide treatment on the PAM chlorophyll fluorescence characteristics of thylakoid membranes isolated from pea leaves 48 h after spraying with different EBR concentrations. PAM-characteristics: Fv/Fm e maximal quantum efficiency of PSII photochemistry in dark-adapted state, qP e photochemical quenching coefficient, FPSII e effective quantum yield of PSII photochemistry. Thylakoid membranes isolated from non-treated leaves served as a control. The data represent mean of three independent experiments  SE shown by vertical error bars (*P < 0.05, **P < 0.01).

Table 2 Effect of different EBR concentrations on the FR light-induced steady-state oxidation of P700 (DA830, P700þ) and the rate constants of P700þ dark reduction (k1 and k2) in pea leaves, measured 48 h after EBR treatment. Mean values are expressed in arbitrary units  SE and are calculated from 3 to 5 independent experiments (*P < 0.05). EBR concentration (mg.L
1)

DA830

0 0.01 0.1 1.0

34.06 34.75 35.93 32.55

k1 (s
1)

   

0.75 0.82 0.33 0.87

1.30 1.38 1.60 1.43

   

0.07 0.13 0.06* 0.20

k2 (s
1)

0.134 0.160 0.175 0.125

   

0.004 0.011 0.011* 0.015

The subsequent dark reduction kinetics of P700þ after turning off the FR light were fitted by two exponential decay components, fast (rate constant k1) and slow (rate constant k2). The calculated values of the rate constants for control and EBR-treated leaves (Table 2) show that only 0.1 mg.L
1 EBR increases significantly (P < 0.05) the rate constants of P700þ dark reduction relative to the control. 3.4. Low-temperature chlorophyll fluorescence The 77 K fluorescence emission spectra were measured in order to assess the effect of EBR on the distribution of excitation energy between the main chlorophylleprotein complexes of the photosynthetic apparatus. The spectra of control and EBR-treated thylakoid membranes have two clearly expressed maxima at 685 nm (F685) and 735 nm (F735), which are related to PSII and PSI emission, respectively (Krause and Weis, 1991). The fluorescence ratio F735/ F685 is used as a sensitive indicator for the energy redistribution between the two photosystems. We measured 77 K chlorophyll fluorescence spectra in low-salt buffer (without MgCl2) and in high-salt buffer (with 5 mM MgCl2), causing unstacking and stacking of the thylakoid membranes, respectively. It is known that unstacking of grana lamellae increases the energy flow from the PSII antenna complex toward the PSI complex, resulting in a considerable increase in the F735/F685 ratio, while upon membrane stacking this ratio decreases (Gross and Prasher, 1974). The data revealed that in the high-salt buffer F735/F685 ratio is greater for thylakoid membranes from 0.1 mg.L
1 EBR-treated plants in comparison to the control, while for the other two examined concentrations this ratio remains similar to the control

Fig. 3. Kinetic traces of the time course of P700 photo-oxidation (DA830) in control and EBR-treated (0.1 mg.L
1 for 48 h) pea leaves.

A.G. Dobrikova et al. / Plant Physiology and Biochemistry 80 (2014) 75e82

79

(Table 3). This very likely reveals small enhancement of the energy transfer (spillover) from PSII towards PSI complexes after the treatment with 0.1 mg.L
1 EBR. During the stackeunstack transition (high-salt to low-salt conditions) the increase of the F735/F685 ratio is the lowest for thylakoid membranes isolated from 0.1 mg.L
1 EBR-treated plants, in comparison to the other studied samples (Table 3). 3.5. Light scattering at 540 nm To further characterize the changes in the degree of grana stacking of thylakoid membranes after EBR treatment, we also measured the light scattering at 540 nm for thylakoid membranes isolated from control and EBR-treated pea leaves. Previously, it has been shown that the light scattering around 540 nm arises from the presence of granal structures (Wollman and Diner, 1980). The Sst/ Sun ratio (relative light scattering where Sst and Sun are light scattering intensities of stacked and unstacked thylakoid membranes), which can be used as a criterion for the degree of membrane stacking (Gross and Prasher, 1974; Stoichkova et al., 2006; Wollman and Diner, 1980), was estimated. The relative changes in the scattered light for the control and EBR-treated thylakoid membranes before and after addition of MgCl2 are presented in Table 3. It is seen that the values of the Sst/Sun ratio for thylakoid membranes after EBR treatment are lower by 13%, 21% and 4% for 0.01, 0.1 and 1.0 mg.L
1, respectively (Table 3), implying that EBR application results in a slight unstacking of thylakoid membranes (i.e. decreased extent of grana stacking or lower number of stacked membranes in the grana). The decrease in the Sst/Sun ratio is statistically significant only upon treatment with 0.1 mg.L
1 EBR. 3.6. DSC measurements The thermodynamic properties of isolated thylakoid membranes from control and EBR-treated (0.01, 0.1 and 1.0 mg.L
1) pea leaves were investigated by differential scanning calorimetry (DSC). DSC profiles possessed seven major endothermic transitions labeled as A, B, C, D, E, F and G in the 30e90  C range (Fig. 4), corresponding either to structural changes or to the denaturation of the main pigmenteprotein complexes of thylakoid membranes (see Dobrikova et al., 2003; Krumova et al., 2010). The calculated temperatures and enthalpies of the endothermic transitions obtained by mathematical deconvolution of the endotherms, using Gaussian fit, are given in Table 4. The deconvolution of the heat sorption curves results in two peaks for the transition denoted B with different transition temperatures (Tm) and enthalpies (DHcal) (Table 4). The comparison of DSC endotherms recorded for thylakoid membranes from control and EBR-treated pea leaves (0.01e

Table 3 Effect of different EBR concentrations on the 77 K fluorescence emission ratios (F735/ F685) and the relative light-scattering changes at 540 nm (Sst/Sun) in isolated thylakoid membranes. Stacked and unstacked thylakoids were obtained by resuspension in 40 mM Hepes (pH 7.6), 10 mM NaCl and 400 mM sucrose medium supplemented or not with 5 mM MgCl2, respectively. Thylakoid membranes were isolated from control and EBR sprayed pea leaves 48 h after the treatment. Mean values are expressed in arbitrary units  SE and are calculated from 5 independent experiments (*P < 0.05). EBR concentration (mg.L
1)

F735/F685 Stacked

0 0.01 0.1 1.0

1.28 1.30 1.36 1.27

   

0.01 0.05 0.02* 0.03

F735/F685 Unstacked 2.40 2.30 2.16 2.35

   

0.05 0.18 0.08* 0.10

Increase in F735/F685 (%)

Relative light scattering (Sst/Sun)

87 77 59 85

1.85 1.61 1.47 1.78

   

0.20 0.14 0.11* 0.13

Fig. 4. DSC thermograms of isolated thylakoid membranes from control and EBRsprayed (0.1 mg.L
1 for 48 h) pea leaves. Buffer/buffer baseline is subtracted from each scan. Membranes were suspended in buffer containing 20 mM Hepes (pH 7.6), 5 mM MgCl2, 10 mM NaCl and 400 mM sucrose at a chlorophyll concentration of 2 mg.ml
1.

1.0 mg.L
1) shows that the EBR application induces very distinct effects on the thermograms e either a reduction or an increase in the calorimetric enthalpies of the transitions labeled A, B, C, D and F, which are not accompanied by significant changes in the respective Tm of the transitions. The thermal transitions E and G seem to be unaffected by the EBR treatment (Table 4). The observed decrease in the enthalpy of the transitions A and B2 (DHcal, Table 4), corresponding to changes in the macroorganizations of the major lightharvesting complex of PSII (LHCII) (Dobrikova et al., 2003), is the strongest at 0.1 mg.L
1 EBR (Table 4); DSC scans of control and 0.1 mg.L
1 EBR-treated thylakoids are compared on Fig. 4. 4. Discussion It is known that at non-stress conditions various BRs can enhance the net photosynthetic rate (CO2 assimilation rate) and Rubisco activity in plant leaves (Hayat et al., 2000; Xia et al., 2009a; Yu et al., 2004). It has also been shown that the EBR-induced enhancement of photosynthetic capacity (stimulation of photosynthesis) has maximum effect at 0.1 mg.L
1 EBR on both net photosynthetic rate (PN) and effective quantum yield of PSII photochemistry (FPSII), which is suggested to reflects the increased demand in the Calvin cycle for ATP and NADPH (Ogweno et al., 2008; Xia et al., 2009a; Yu et al., 2004). In the present study, effects of the exogenously applied EBR on pea plants were investigated with the aim for more detailed understanding of its action on the photosynthetic apparatus. Our PAM chlorophyll fluorescence data on both leaves and isolated thylakoid membranes showed that EBR treatment does not change the maximal quantum efficiency of PSII photochemistry in dark-adapted and light-adapted states (Fv/ Fm, Fv0 /Fm0 ) and the non-photochemical quenching parameters (qN and NPQ), but enhanced the effective quantum yield of PSII photochemistry (FPSII), and stimulated the electron transport and photochemical quenching (qP and qL). Having in mind the physiological relevance of these parameters (Baker, 2008), we can conclude that EBR treatment of pea leaves enhances the ability of the photosynthetic apparatus to maintain QA in the oxidized state (qP), increases the proportion of the “open” PSII reaction centers (qL), improves the efficiency at which light absorbed by PSII is used for QA reduction (FPSII) and accelerates the overall electron transport through PSII (ETR). The present results for the effect of EBR on Fv/Fm, qP, FPSII of pea plants are in agreement with previous studies

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Table 4 Thermodynamic parameters derived from the heat sorption curves of isolated thylakoid membranes from control and EBR sprayed pea leaves after mathematical deconvolution of the DSC thermograms. Tm ( C) is the transition temperature and DHcal (mcal.mg
1 Chl) is the calorimetric enthalpy of the transitions. EBR (mg.L
1)

Peak area

A

B1

B2

C

D

E

F

0

Tm DHcal Tm DHcal Tm DHcal Tm DHcal

47.5 12.7  0.6 48.5 3.8  0.9 48 2.7  1.0 45 6.2  1.2

56 2.5  55 2.4  55.8 3.2  55.6 2.9 

61.5 17  1.4 61.3 14.6  0.5 61.1 12.6  0.8 61 15.5  1.1

65 3.3  65.2 2.9  65.6 2.3  66 3.2 

69.5 7.3  69.5 6.5  69.5 4.6  70 7.5 

74.5 10.2  1.1 74.5 10  0.5 74.6 11.7  1.0 74.6 12  1.3

82.2 7.4  82.1 7.6  82.1 9.6  82 9.9 

0.01 0.1 1.0

0.5 0.7 0.5 0.8

on EBR-treated cucumber (Xia et al., 2009a; Yu et al., 2004), tomato (Ogweno et al., 2008) and melon plants (Zhang et al., 2013). The EBR-induced stimulation of the effective quantum yield of PSII photochemistry observed here for pea leaves occurs without changes in the efficiency of excitation energy capture by open PSII reaction centers (Fv0 /Fm0 ) and in the thermal dissipation at antenna complexes, as it has been reported previously for cucumber leaves (Xia et al., 2009a; Yu et al., 2004). PSI activity, monitored by the absorbance changes in FR-induced P700 oxidation (DA830), revealed that the relative amount of P700þ in EBR-treated leaves was nearly not affected in comparison to the control ones, while the rate constants of the dark reduction kinetics were significantly increased only at 0.1 mg.L
1 EBR (Table 2). It has been proposed that the two phases of the dark reduction kinetics of P700þ after turning off the FR light originate either from two different electron donor systems or are due to reduction of two different pools of PSI located in different domains of the thylakoid membranes (Albertsson, 1995). A reduced electron carrier in the cyclic electron transport, for example ferredoxin, might play the role of a donor system capable of a fast electron donation, whereas the slow donor could be a reduced pyridine nucleotide in the chloroplast stroma (Endo et al., 1997; Havaux, 1996). It is known that PSI located in the grana margins (grana periphery) and in the stroma lamellae are different in their ability to reduce ferredoxin (Wollenberger et al., 1995). According to the model of Albertsson (1995) the linear electron transport occurs in the grana, while cyclic electron transport is restricted to the stroma lamellae. The rapidly operating pathway could be driven by enzymes located in the stroma lamellae, whereas enzymes mediating the slow pathway are in grana thylakoids (Bukhov et al., 2002). The rate constants of the dark reduction showed an increase in EBR-treated leaves in comparison to the control and this increase is higher for k2 than for k1, which indicates that EBR influences more the slow component of the decay kinetics (i.e. linear electron transport in the grana periphery). This could be probably a result from changes in the grana organization. On the other hand, the increased fast component (k1) indicates also an increase of the cyclic electron transport around PSI in the stroma lamellae. The data obtained for flash-induced oxygen yields (Fig. 1A) revealed that EBR treatment leads to a decrease of the functionally active PSIIa centers in the grana domains, as well as to an increase in the amounts of PSII centers in the initial S0 state (Table 1), which is related to lower oxidation state of the Mn cluster in the oxygenevolving complex (see in Ivanova et al., 2008). Taken together with the decreased ratio of functionally active PSIIa to PSIIb centers (A1/ A2 ratio, Table 1), these observations indicate changes in the organization of the PSII complexes (see Apostolova et al., 2006), most probably accompanied by EBR-induced decrease in grana size, as it was established by the relative light scattering changes (Table 3). The Sst/Sun ratio, which is a criterion for the degree of stacking of the thylakoid membranes (Wollman and Diner, 1980; Stoichkova et al., 2006), decreases in membranes from EBR-treated leaves in

0.4 0.3 0.3 0.3

0.3 0.4 0.7 0.4

G 0.9 0.4 0.6 0.9

89.3 2.7  89.5 2.6  89.2 2.5  89 2.8 

0.3 0.5 0.1 0.2

comparison to the control (Table 3); the highest effect was observed upon 0.1 mg.L
1 EBR application. The proposed EBR-induced structural alterations in the grana domains of the thylakoid membranes were also supported by the cation-dependent changes in the 77 K chlorophyll fluorescence spectra. It has been demonstrated that Mg2þ ions regulate lateral segregation of the PSII and PSI complexes in isolated thylakoid membranes and cause the cation-induced changes in chlorophyll fluorescence, which have been claimed to be an indicator of membrane stacking (Barber, 1980; Stoichkova et al., 2006). Our data demonstrate that during stackeunstack transition (from high to low-salt medium) the F735/F685 ratio is increased only to a smaller extent after the EBR treatment with various concentrations in comparison to the control, although to a different degree (Table 3). This indicates partial unstacking of the thylakoid membranes and the maximal effect was observed in samples treated with 0.1 mg L
1 EBR. Previous studies have shown that one of the primary responses to different stresses is lateral reorganization of the main membrane complexes, followed by unstacking of thylakoid membranes (Ches et al., 2000; Yuan et al., 2012). In addition, our recent study (Dobrikova et al., 2013) has demonstrated that EBR application modifies UV-B-induced alterations of the energy distribution between the main pigmenteprotein complexes in pea thylakoid membranes. Since it is known that the PSIIa centers are especially sensitive to UV radiation and that the susceptibility to stress factors is directly influenced by the degree of membrane stacking (Anderson and Aro, 1994; Ivanova et al., 2008; Yu and Bjorn, 1996), we suggest that the EBR-induced structural reorganizations in thylakoid membranes are needed in order to ensure the structural flexibility required for their adaptability to the changes in the environmental conditions. Our recent study on the pigment content of pea leaves showed that the EBR treatment with 0.1 mg.L
1 (2.10
7 M) causes an increase in the Chl a and carotenoids content in leaves and has almost no effect on Chl b content, which results in slight increase of the ratio Chl a/b compared to non-treated leaves (Dobrikova et al., 2013). Similar results were obtained by Ref. Sharma et al. (2013) after treatment with 10
7 M EBR as compared to the control. Having in mind that the Chl a/b ratios correlate with the degree of thylakoid stacking (Anderson and Aro, 1994), then the slightly increased Chl a/b ratio after the EBR treatment once again supports our assumption that the EBR treatment most probably causes partial unstacking of pea thylakoid membranes. In line with our hypothesis for EBR-induced changes in the structural organization of thylakoid membranes, electron micrographs of plant cells in cucumber seedlings treated with 0.1 mM EBR revealed that in the control the grana and stroma regions are well discernible, whereas in chloroplasts from EBR-treated plants the grana formation is less well defined (Xia et al., 2009b; Yuan et al., 2012). Our recent study on Arabidopsis mutants with enhanced BR signaling also revealed changes in the overall architecture of thylakoids and modified macroorganization of the photosynthetic complexes in the plants

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with endogenously modified BR level and perception in comparison to the wild type plants (Krumova et al., 2013). DSC is often used as a tool for assessing the thermal stability of the main photosynthetic complexes and for detecting structural changes related to them (for a review, see Krumova et al., 2010). It has been suggested that the endothermic transitions in the thylakoid membranes reveal characteristic structural transitions of the pigmenteprotein complexes (Smith et al., 1989; Dobrikova et al., 2003; Krumova et al., 2010). Previously, several thermal transitions in thylakoid membranes were resolved (Dobrikova et al., 2003; Krumova et al., 2010; Smith et al., 1989; Thompson et al., 1989). It has been demonstrated that the heat-induced disassembly of the chiral macrodomains of LHCII complex contributes significantly to some of the calorimetric events in isolated thylakoid membranes, including those of thermo-optical nature: unstacking of the membranes, lateral disassembly of the chiral macrodomains (transition A) and monomerization of LHCII trimers (for further details, see Dobrikova et al., 2003). Other events shown to contribute to transition A are PSII core monomerization and oxygen-evolving complex destabilization (reviewed in Krumova et al., 2010). The second main transition at around 60  C (with two overlapping transitions) has been assigned to the PSII core denaturation and the monomerization of LHCII trimers (Dobrikova et al., 2003; Krumova et al., 2010; Thompson et al., 1989). The endothermic transitions C, D and G, observed previously in PSIenriched fragments (stroma lamellae), have been assigned to PSI core degradation (Krumova et al., 2010), while the transitions at around 70e75  C originates from the denaturation of the LHCII proteins (Dobrikova et al., 2003; Krumova et al., 2010; Smith et al., 1989). Based on all of the above mentioned assignments, it could be proposed that the EBR-induced strong diminishment of the A and B2 endothermic peaks at around 48 and 61  C (i.e., DHcal values presented in Table 4), respectively, most probably indicates decreased amounts of oligomeric and trimeric forms of the main LHCII in the membranes. The presented data also reveal that this decrease is the strongest at 0.1 mg.L
1 EBR (Table 4), implying that the decrease of the LHCII-containing chiral macrodomains as well as the unstacking of the thylakoid membranes after EBR treatment is the most pronounced at this concentration. The sum of the calorimetric enthalpies of the transitions denoted D and E (Table 4), ascribed to LHCII proteins denaturation (Krumova et al., 2010), is not changed significantly after the EBR treatment indicating no changes in the amount and stability of LHCII monomers in the membranes. The total calorimetric enthalpies of the DSC thermograms after the application of different EBR concentrations were lower than that of the control, and the smallest changes was observed at a concentration of 1.0 mg.L
1 (Table 4). This suggests different specific heat capacities of the proteins constituting the thylakoid membranes after EBR treatment. The results from the DSC study provide the first evidence for exogenous EBR-induced alterations in the thermodynamic parameters of photosynthetic membranes under physiological (nonstress) conditions. The structural reorganizations revealed by us most probably ensure structural stability, required to preserve the integrity of the membranes and the entire organelle, and flexibility needed to allow the adaptability of the system and for different enzymatic activities (Zer et al., 2003). 5. Conclusion The results of this study show that under non-stress conditions the effects of EBR treatment on the structural organization and functionality of thylakoid membranes strongly depend on its concentration. Among the three different concentrations of EBR (in the

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range 0.01e1 mg.L
1) the most pronounced effect on the photosynthetic apparatus was exerted upon 0.1 mg.L
1 EBR treatment. This concentration was very effective in increasing the effective quantum yield of PSII and in stimulation of the electron-transport rate in pea thylakoid membranes. Further, the application of EBR was found to influence the kinetics of oxygen evolution, decreases the ratio of functionally active PSIIa to PSIIb centers and increases the amount of PSII centers in the most reduced S0 state. The observed changes in the energy redistribution between the two photosystems, the light scattering and the thermal stability of EBRtreated thylakoids strongly suggest structural reorganizations of the main pigmenteprotein complexes and partial unstacking of the membranes after EBR treatment with 0.1 mg.L
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