Contents lists available at SciVerse ScienceDirect
Journal of Plant Physiology
j o ur nal homepage: www.elsev ier .com/ locate / jp lph
hysiology
amage and protection of the photosynthetic apparatus from UV-B radiation. I.ffect of ascorbate
nelia G. Dobrikovaa, Vassilena Krastevaa,b, Emilia L. Apostolovaa,∗
Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl.21, Sofia 1113, BulgariaDepartment of Biophysics and Radiobiology, Faculty of Biology, Sofia University, ‘St. Kliment Ohridski’, 8, Dragan Tzankov Boulevard, 1164 Sofia, Bulgaria
r t i c l e i n f o
rticle history:eceived 16 March 2012eceived in revised form8 September 2012ccepted 4 October 2012
a b s t r a c t
In this work, the effect of the exogenously added ascorbate (Asc) against the UV-B inhibition of thephotosystem II (PSII) functions in isolated pea thylakoid membranes was studied. The results reveal thatAsc decreases the UV-B induced damage of the donor and the acceptor side of PSII during short treatmentup to 60 min. The exogenous Asc exhibits a different UV-protective effect on PSII centers in grana andstroma lamellae, as the effect is more pronounced on the PSII� centers in comparison to PSII� centers.
Data also suggest that one of the possible protective roles of the Asc in photosynthetic membranes is themodification of the oxygen-evolving complex by influence on the initial S0–S1 state distribution in thedark.
Depletion of the ozone layer in the atmosphere results inncrease of solar UV-B (280–320 nm) photon flux in the biosphere.V-B radiation has many direct and indirect effects on plant cells,
ncluding damage of nucleic acids, proteins, lipids and pigments,esulting in impaired chloroplast function, inhibition of the pho-osynthesis and decreased protein synthesis (Jansen et al., 1998;ollosy, 2002; Vass et al., 2005; Jordan, 2010). Photosynthetic inhi-ition is associated primarily with damage of photosystem II (PSII)Nedunchezhian and Kulandaivelu, 1991; Jansen et al., 1998; Melis,999; Vass et al., 2005; Vass, 2012). The tolerance to UV-B dependsn the balance between a variety of damage reactions and bothepair and acclimation responses (Jansen et al., 1998).
UV-B radiation also causes oxidative stress through the genera-ion of reactive oxygen species (ROS), which in turn cause enhancedipid and protein oxidation (oxidative damage) (Hideg and Vass,
996; He and Häder, 2002; Gao and Zhang, 2008; Gill and Tuteja,010; Pospísil, 2012). To cope with oxidative stress, plant cells haveeveloped highly efficient defense mechanisms involving both
Abbreviations: A, initial oxygen burst amplitude; Asc, ascorbate; BQ, 1,4-enzoquinone; PMA, phosphomolybdic acid; PSII, photosystem II; QA, primaryuinone electron acceptor of PSII; QB, secondary quinone electron acceptor of PSII;i , redox state i of the water-oxidizing complex; Y3, amplitude of the flash-inducedxygen yield after third flash.∗ Corresponding author. Tel.: +359 2 979 26 21; fax: +359 2 971 24 93.
enzymatic and non-enzymatic constituents (Munné-Bosch andAlegre, 2002; He and Häder, 2002; Hollosy, 2002; Vass et al., 2005).The non-enzymatic antioxidants are generally small molecules,such as ascorbate (Asc) and glutathione, which are found in thy-lakoid lumen and stroma of chloroplasts (in the aqueous phase),while lipophilic antioxidants as �-tocopherol and �-carotene arein the lipid matrix, associated with protein domains in thylakoidmembranes (He and Häder, 2002; Munné-Bosch and Alegre, 2002;Munné-Bosch et al., 2005). All mentioned molecules are centralcomponents of plant antioxidant defenses limiting ROS accumu-lation. It has also been demonstrated the significance of Asc forthe antioxidant defense system and for the interplay betweenhydrophilic and lipophilic antioxidants in chloroplasts (Munné-Bosch and Alegre, 2002). Asc is the most abundant low-molecularweight antioxidant in plant cells and it plays a crucial role in com-plex antioxidation processes by direct scavenging of the producedROS and as a co-substrate of many enzymes which detoxify ROS(Conklin, 2001). He and Häder (2002) demonstrated significantprotection of Asc against the UV-B-induced oxidative stress, lipidperoxidation, DNA breaks, photosynthetic damage, etc. In addi-tion, Asc has a major role in photoprotection as a cofactor andco-substrate for violaxanthin de-epoxidase in the xanthophyll cycle(Müller-Moulé1 et al., 2002; Jahns et al., 2009). Recently it hasbeen shown the physiological role of Asc as PSII electron donor
in heat-stressed leaves lacking active oxygen-evolving complexes(Tóth et al., 2011).
Photosensitizers that absorb in the UV-B range and that havebeen proposed to be potential target sites of UV-induced damage
f PSII are the quinone electron acceptors (QA and QB), the catalyticn cluster and the redox-active tyrosines (Vass et al., 1996; Jansen
t al., 1998; Vass, 2012). Since the degree of damage caused by UV- exposure should be strongly dependent on the efficiency of UVrotection and repair, the aim of this study was to verify the effectf different concentrations of the exogenously added Asc on thenhibition of PSII functions during UV-B irradiation of isolated thy-akoid membranes. To achieve this goal, we investigated the Ascrotection against UV-B damage of the oxygen-evolving activityf thylakoid membranes by a rate (Joliot-type) and a concentra-ion (Clark-type) electrode as well as the energy transfer betweenigment–protein complexes in pea thylakoid membranes.
aterials and methods
reparation of thylakoid membranes
Thylakoid membranes were isolated from 2-week-old peaPisum sativum L. cv. Borec) as described previously (Dobrikovat al., 2000). The plants were grown at controlled conditionsnder light intensity 150 �mol photons m−2 s−1 with 16 h light/8 hark photoperiod. The thylakoid membranes were re-suspended
n a measuring buffer containing 40 mM HEPES (pH 7.6), 10 mMaCl, 5 mM MgCl2, 400 mM sucrose for all measurements. The
otal chlorophyll concentration was determined by the method ofichtentaler (1987).
V-B treatment of isolated thylakoid membranes
Thylakoid membranes were suspended in a buffer containing:0 mM HEPES (pH 7.6), 10 mM NaCl, 5 mM MgCl2 and 400 mMucrose at a chlorophyll concentration 500 �g mL−1, forming a
mm thin suspension layer at continuous stirring. The membranesere illuminated for different times (0–60 min) in a Petri dish at
0 ◦C with maximal UV-B emission at 315 nm from a Cole-Parmerltraviolet lamp (Model 3UV-34). Spectrum of the UV lamp usedor the experiments is shown in Fig. 1. The range of the emissionf the UV-B lamp was from 280 nm to 340 nm. The intensity ofadiation, measured with a Radiometer/Photometer IL 1400A, was9.2 W m−2. The control samples (non-irradiated) were kept in dim
ight at 20 ◦C for different times. Ascorbate (Asc, Fluka) was added tohe thylakoid suspension in different concentrations (0–1000 �M)efore the UV-treatment.
hotochemical activity of photosystem II (PSII)
Photochemical activity of PSII was measured polarographicallyith a Clark-type electrode (Model DW1, Hansatech, Instruments
td., King’s Lynn, Norfolk) in temperature-controlled cuvette at0 ◦C, using saturated white (achromatic) light. The PSII activity waseasured by the rate of oxygen evolution in the presence of exoge-
ous electron acceptors 1,4-benzoquinone (BQ, Sigma–Aldrich)r phosphomolybdic acid (PMA, Sigma–Aldrich). The reactionedium contained: 20 mM MES (pH 6.5), 10 mM NaCl, 5 mMgCl2, 400 mM sucrose and an exogenous acceptor (0.2 mM BQ
r 0.1 mM PMA with 10 �M DCMU). The chlorophyll concentrationas 25 �g mL−1.
xygen evolution measurements
Oxygen flash yields and initial oxygen burst were measured
sing a custom-built polarographic oxygen rate electrode a Joliot-ype described by Zeinalov (2002). The working potential of theathode was −800 mV and the time constant of the electrode wasess than 2 ms. Polarograph sensitivity was 1.5–3 V �A−1.
Fig. 1. Spectral distribution of the applied UV-B radiation. A Cole-Parmer ultravioletlamp (Model 3UV-34) was used as UV-B light source.
Thylakoid membranes were suspended in a medium withoutartificial electron acceptor containing: 40 mM HEPES (pH 7.6),10 mM NaCl, 5 mM MgCl2 and 400 mM sucrose. The chlorophyllconcentration was 150 �g mL−1 in 100 �L sample volume andformed a layer of 2 mm height. Samples were pre-illuminatedwith ca. 20 flashes and then dark adapted for 5 min beforemeasurements. Oxygen flash yields were induced by saturat-ing (4 J) and short (t1/2 = 10 �s) periodic flash sequences with650 ms dark spacing between the flashes. The initial oxygenburst was recorded during irradiation with continuous white light(400 �mol photons m−2 s−1).
According to the non-cooperative mechanism proposed by Koket al. (1970) the cooperation of five (intermediate redox) oxidationstates of oxygen-evolving complex (S0–S4) in the same PSII centersis required for production of one oxygen molecule. The initial S0–S1state distribution in the dark was determined by the least squaredeviations fitting to the theoretically calculated yields according toKok’s model with the experimentally obtained oxygen flash yields.
Low-temperature (77 K) chlorophyll fluorescence
Low-temperature (77 K) chlorophyll fluorescence measure-ments were performed in a cylindrical quartz cuvette in a mediumcontaining 40 mM HEPES (pH 7.6), 10 mM NaCl, 5 mM MgCl2,400 mM sucrose at chlorophyll concentration 20 �g mL−1. The sam-ples were quickly frozen by plunging them in liquid nitrogen.Fluorescence spectra were recorded from 600 nm to 780 nm usingJobin Yvon JY3 spectrofluorimeter equipped with a red-sensitivephotomultiplier (Hamamatsu R928) and a liquid nitrogen device.The width of the exciting and measuring slits was 4 nm. The datawere digitized by an in-built A/D converter and transferred to
IBM-compatible computer for further analysis. The chlorophyllfluorescence was excited either at 436 nm (Chl a) or at 472 nm(Chl b).
A.G. Dobrikova et al. / Journal of Plant Physiology 170 (2013) 251– 257 253
Fig. 2. Effect of 400 �M Asc on the UV-B inhibition of the steady-state oxygenevolution of pea thylakoid membranes in the presence of artificial electron accep-tors 1,4-benzoquinone (BQ) or phosphomolybdic acid (PMA). The values of theoxygen evolution are normalized to the non-irradiated control membranes. Meanvalues ± SE were calculated from 3 to 4 independent experiments. Statistical dif-fwm
S
t(t
R
Eo
Uatled(fcwpniabn
dooomcn6tta
Fig. 3. The amplitude of the oxygen flash yields after the third flash (Y3) and theinitial oxygen burst amplitude (A) under continuous irradiation with white lightafter UV-B treatment of thylakoid membranes with and without 100 �M Asc. Opensymbols indicate the addition of Asc. The values of the oxygen evolution are repre-sented as a percentage of the value in non-irradiated control thylakoid membranes.
erences (P < 0.05) were found between control and UV-B-irradiated membranes asell between the thylakoid membranes with and without Asc after 60 min treat-ent.
tatistical analysis
Results are expressed as means values ± SE. Data were subjectedo analysis of variance by ANOVA (VassarStats). Treatment effectsof UV-B and/or Asc) were considered significant at P < 0.05 usinghe least significant difference (LSD).
esults
ffect of the Asc on the UV-B induced inhibition of photosyntheticxygen production
In order to assess the protective effect of Asc against theV-B inhibition of the electron transport via the primary (QA)nd secondary (QB) quinone electron acceptors we measuredhe PSII-mediated electron transport (steady-state oxygen evo-ution) with a Clark-type electrode in the presence of twoxogenous acceptors (1,4-benzoquinone (BQ) and phosphomolyb-ic acid (PMA)). It is known that BQ accepts electrons from QBH2O → QA → QB → BQ) (Vermaas and Arntzen, 1983) while PMArom QA (H2O → QA → PMA) (Barr et al., 1975). Using various con-entrations (0–1000 �M) of Asc, the maximum protective effectas found at a concentration 400 �M (0.8 �M Asc per �g chloro-hyll) and higher concentrations had almost the same effect (dataot shown). The addition of 400 �M Asc to the control (non-
rradiated) thylakoid membranes stimulates the electron-transportctivity in the presence of both exogenous acceptors (BQ or PMA)y 20% in comparison to thylakoid membranes without Asc (dataot shown).
Inhibition of the steady-state oxygen evolution after UV-B irra-iation of thylakoid membranes for 30 and 60 min in the presencef 400 �M Asc is shown in Fig. 2. Data show that the inhibitionf the PSII-mediated electron transport is bigger in the presencef the exogenous acceptor BQ than in the presence of PMA, whicheans stronger UV-B inhibition of the electron transport via QB in
omparison to that via QA. The UV-B treatment up to 30 min causesegligible changes in the electron transport via QA, while after the
0 min of treatment it is inhibited to about 22% (Fig. 2). At the sameime, the oxygen evolution in the presence of the exogenous accep-or BQ (accepted electrons from QB) is inhibited by about 17 and 35%fter 30 and 60 min irradiation, respectively (Fig. 2). These results
Mean values ± SE were calculated from 3 to 4 independent experiments. Statisticaldifferences (P < 0.05 for Y3 and P < 0.01 for A) were found at the comparison betweenthylakoid membranes with and without Asc during the UV-B treatment.
show that short UV-B treatment (up to 30 min) does not influencethe primary (QA) quinone electron acceptor and/or its environment.On the other hand, data reveal that Asc in concentration 400 �Mcompletely protects oxygen evolution during 30 min UV-B treat-ment of the thylakoid membranes and decreases the UV-inducedinhibition of the oxygen evolution during 60 min treatment in thepresence of both exogenous acceptors (BQ or PMA).
For more detailed study of the protective role of Asc on the oxy-gen evolving complex against UV-B damage, we used a Joliot-typeoxygen rate electrode with flash and continuous illumination. Inthis case, the measurements were performed without exogenousacceptors i.e., electrons are accepted from the plastoquinone (PQ) inthylakoid membranes. The maximal amplitude of the oxygen flashyields obtained after the third flash, Y3 (Figs. 3A and 4A) was usedto estimate the UV-B-induced damage of the functionally activePSII centers evolving oxygen by non-cooperative Kok’s mechanism(PSII� in grana domains), while the oxygen burst amplitude, A(Figs. 3B and 4B) was used to assess all functionally active PSII cen-ters (PSII� in grana and PSII� in stroma domains) (see in Ivanovaet al., 2008). The results clearly indicate a stronger UV-B inhibi-tion of the oxygen flash yields than the oxygen burst amplitudes
for thylakoid membranes without exogenous Asc (Fig. 3). It is alsoseen that the oxygen flash yields (Y3) and the amplitudes of oxygenburst decrease rapidly with increasing irradiation time in the range0–60 min. A faster rate of photoinhibition was registered within the
254 A.G. Dobrikova et al. / Journal of Plant Physiology 170 (2013) 251– 257
Fig. 4. Effect of 400 �M Asc on the UV-B inhibition of the oxygen flash yields as a function of the flash number in dark-adapted (for 5 min) thylakoid membranes (A) ando B treao (solidA of thy
fittitwotKseB
oUpIe4
ltttsAitAo(s
tnr
xygen burst kinetics under continuous irradiation (B). (A) Control (circles) and UV-f Asc. (B) Control and UV-B irradiated membranes for 60 min in the presence of Ascsc on the oxygen evolution parameters (Y3 and A) during 60 min UV-B irradiation
rst 20 min for the flash-induced oxygen yields (Fig. 3A) and withinhe first 40 min for the oxygen burst under continuous illumina-ion (Fig. 3B). The rate of photoinhibition decreases with furtherncreasing the irradiation time. The observed stronger inhibition ofhe flash-induced oxygen yields during UV irradiation, comparedith the oxygen burst amplitudes (Fig. 3), could be a consequence
f the destruction of the grana domains and/or the inhibition ofhe active PSII� centers evolving oxygen through non-cooperativeok’s mechanisms (giving oxygen flash yields). Similar higher sen-itivity to UV radiation of PSII� in comparison to PSII� centers wasstablished in previous studies (Tevini and Pfister, 1985; Yu andjörn, 1996; Ivanova et al., 2008).
Examining the effect of different concentrations (0–1000 �M)f Asc on the kinetic parameters of the oxygen evolution duringV-B irradiation showed that the minimum Asc concentration thatrovides a detectable protection against UV-B damage is 100 �M.
t was observed that this concentration of Asc reduces the harmfulffect of UV-B radiation on the oxygen evolution parameters up to0 min treatment, but has no effect after 60 min treatment (Fig. 3).
The effect of different concentrations of Asc on the oxygen evo-ution parameters (Y3 and A) during 60 min UV-B irradiation ofhylakoid membranes is presented in Fig. 4 insets. It can be seen thathe protective effect of Asc increases very rapidly with increasinghe concentrations between 100 and 400 �M, and does not changeignificantly up to 1 mM Asc (Fig. 4 insets). Data also show thatsc at concentration 400 �M and above decreases the 60 min UV
nhibition of the flash oxygen yields by 14% (Fig. 4A inset) and ofhe oxygen burst amplitudes by 20% (Fig. 4B inset). Therefore, thesc protection against UV-B irradiation is more effective for thexygen burst (Fig. 4B inset) compared to the oxygen flash yieldsFig. 4A inset), which indicates more pronounced effect of Asc fortroma situated PSII� centers than for PSII� in grana domains.
The obtained kinetic parameters of the oxygen evolution of thehylakoid membranes in the absence and presence of an exoge-ous Asc during UV-B irradiation confirm the expectation that UV-Badiation like UV-A (Turcsányi and Vass, 2000; Ivanova et al., 2008)
ted (squares) thylakoid membranes for 60 min. Open symbols indicate the addition lines) and without Asc (dotted lines). Insets: The effect of different concentrations
lakoid membranes.
leads to increase of the active PSII centers in the initial S0 state(Table 1). On the other hand, the addition of Asc to non-irradiatedthylakoid membranes also leads to increase of the centers in the ini-tial S0 state (Table 1). The significant changes of misses and doublehits were not detected after Asc addition to controls (Table 1).
The induction curves registered under continuous illuminationexhibit biphasic exponential decay after the initial oxygen burst(Fig. 4B). Previously it was suggested (see in Ivanova et al., 2008)that the ratio of the amplitudes of the fast and slow components(A1/A2) corresponds to the ratio of functionally active PSII� to PSII�centers. Our results show that this ratio increases after addition ofAsc to the control (non-irradiated) membranes and decreases afterUV-B irradiation (Table 1), which is due to a decrease of the fastamplitude, A1 (i.e., the substantial loss in sharp oxygen burst afterUV-B irradiation, Fig. 4B), originating from PSII� centers in granadomains (see Ivanova et al., 2008). The ratio A1/A2 after 60 min UVtreatment of thylakoid membranes with 400 �M Asc is higher incomparison to those without an exogenous Asc (Table 1).
Results suggest that the Asc has a protective effect against UV-Binduced damage of the oxygen evolution measured by an oxygenrate electrode, as the effect is more pronounced for PSII� (operat-ing by the cooperative mechanism) in comparison to PSII� centers(operating by the non-cooperative Kok’s mechanism).
Effect of the Asc on the UV-B induced changes in low-temperature(77 K) chlorophyll fluorescence emission spectra
Analysis of the low-temperature (77 K) chlorophyll fluorescenceemission spectra was made to evaluate the energy transfer betweenchlorophyll–protein complexes during UV-B treatment. The fluo-rescence emission spectra of isolated thylakoid membranes hadthree bands: at 685 nm and 695 nm related to PSII and the one at
735 nm related to PSI (Krause and Weis, 1991).To assess the influ-ence of the UV-B radiation on the 77 K chlorophyll fluorescence,the fluoresce ratios F735/F685 and F695/F685 were calculated. Theratio F695/F685 was used to estimate the energy transfer between
A.G. Dobrikova et al. / Journal of Plant Physiology 170 (2013) 251– 257 255
Table 1Effect of Asc on the UV-B induced changes of the initial dark distribution of PSII centers in the S0 and S1 states (S1 (%) = 100 − S0), misses (˛), double hits (ˇ) and the ratioof functionally active PSII� to PSII� centers (A1/A2). Treatments were made on isolated pea thylakoid membranes for 60 min in measuring buffer. Mean values ± SE werecalculated from 4 to 5 independent experiments. Values in the same column followed by a different letter are significantly different at P < 0.05 (estimated by ANOVA).
Thylakoid membranes S0 (%) (%) (%) A1/A2
Control 20.4 ± 0.6 a 20.2 ± 0.6 ab 3.9 ± 0.4 a 2.45 ± 0.11 aUV-B 32.7 ± 2.1 b 23.7 ± 1.1 b 7.1 ± 1.3 b 1.19 ± 0.08 b
18.8
14.4
cbce1f7lrct
eTwAipTwt(obtrtb
D
PtscQ2rpPt(o
TEc
Control + 400 �M Asc 24.9 ± 0.3 c
UV-B + 400 �M Asc 35.5 ± 1.5 b
hlorophyll–protein complexes in the LHCII–PSII complex. It haseen suggested that the fluorescence ratio F735/F685 (i.e., PSI/PSII)ould be used to examine the variations in the redistribution ofxcitation energy between both photosystems (Krause and Weis,991) and it is a sensitive probe for monitoring the effect of stressactors in higher plants (Agati et al., 1995). Having in mind that7 K chlorophyll fluorescence spectra reflect maximal fluorescence
evel (Yamane et al., 1997) as well as the strong influence of the UVadiation on the variable fluorescence (Larkum and Wood, 1993), itould be proposed that UV-B induced damage of PSII also influenceshe F735/F685 fluorescence ratio.
Data presented in Table 2 show that F735/F685 and F695/F685mission ratios increase in UV-B treated thylakoid membranes.he observed changes in the ratio F735/F685 (at 436 nm excitation)ere about 30% without exogenous Asc and 41% with addition ofsc during 60 min exposure of the thylakoid membranes to UV-B
rradiation. There were no significant differences when chloro-hyll a (436 nm) or chlorophyll b (472 nm) were excited (Table 2).he smaller changes after UV treatment (between 9 and 14%)ere observed for the ratio F695/F685 (Table 2), which indicates
hat UV-B radiation influences predominantly the ratio F735/F685energy redistribution between the both photosystems). On thether hand, the emission ratio F735/F685 for control thylakoid mem-ranes slightly decreases in the presence of the 400 �M Asc, whilehe ratio F695/F685 does not change by the action of Asc (Table 2). Theesults presented in Table 2 reveal that the UV-induced increase ofhe ratio F735/F685 in thylakoid membranes with exogenous Asc isigger in comparison to the membranes irradiated without Asc.
iscussion
Here, we present evidence showing the UV-B inhibition of theSII functions and the defense role of Asc in the response of the pho-osynthetic apparatus to UV-B stress. It is known that UV-sensitiveites in the PSII complex are the redox-active tyrosines and the Mnluster on the donor side, and quinone electron acceptors (QA andB) on the acceptor side (Jansen et al., 1998; Vass et al., 2005; Vass,012). However, the suggestions about primary targets of UV-Badiation in the PSII are still controversial. Some investigators sup-osed that the primary cause for the UV-induced inhibition of the
SII function is the inactivation of the electron transport betweenhe Mn cluster and the redox-active tyrosines on the donor sideHideg et al., 1993; Vass et al., 1996, 1999; Hakala et al., 2005), whilethers claimed that it is the quinone electron acceptors (Renger
able 2ffect of Asc on the 60 min UV-B-induced changes on 77 K fluorescence emission ratios (Falculated from 3 to 4 independent experiments. Values in the same column followed by
Thylakoid membranes �exc. = 436 nm
F735/F685 F695/F
Control 1.36 ± 0.02 a 0.85 ±UV-B 1.76 ± 0.02 c 0.93 ±Control + 400 �M Asc 1.21 ± 0.02 b 0.89 ±UV-B + 400 �M Asc 1.71 ± 0.08 c 1.04 ±
± 0.9 a 4.4 ± 0.8 a 3.56 ± 0.16 c± 0.8 c 7.9 ± 0.6 b 1.72 ± 0.09 d
et al., 1986; Trebst and Depka, 1990; Melis et al., 1992). Our datareveal that the UV-B radiation influences less the primary (QA) thansecondary (QB) quinone acceptors or their environments (Fig. 2).The stronger effect on QB could be due to specific destruction ofthe reduced quinone by the UV-B light or structural changes in QB-binding site (Hideg et al., 1993; Vass et al., 1999). On the other hand,the results obtained with an oxygen rate (Joliot-type) electrodeshow stronger inhibition of the oxygen evolution (Figs. 3 and 4)than those obtained with a concentration (Clark-type) electrode(Fig. 2). The observed more pronounced UV-B inhibitory effect onthe oxygen evolution of thylakoid membranes in the absence ofan exogenous acceptor (Figs. 3 and 4) in comparison to that inthe presence of the artificial acceptor BQ (Fig. 2) could originatefrom different oxidation–reduction properties of PQ and BQ and/orfrom a difficult interaction between the QB and PQ. Impairment inthe function of PQ could also not be excluded (Melis et al., 1992).In addition, the UV irradiation of thylakoid membranes for 60 mincauses only 22% decrease of PSII activity when PMA (accepting elec-trons from QA) was used as acceptor (Fig. 2), while without artificialacceptors (only PQ) the oxygen-evolving parameters (Y3 and A) arereduced by over 70% (Fig. 3). These findings indicate that the accep-tor side of PSII is more sensitive to the UV-B radiation, which isassociated with damage of the QB quinone acceptor or modificationof its surroundings (Vass et al., 1999, 2005; Vass, 2012), as well asimpairment of PQ (Melis et al., 1992). In line with these findings,we can suppose the stronger UV-B impairment on the acceptor sidethan on the donor side of PSII.
However, the decrease in the photosynthetic activity appearedto be a complicated response to UV-B irradiation. The oxida-tive damage of proteins, lipids and pigments (Jansen et al., 1998;Hollosy, 2002; Vass et al., 2005; Jordan, 2010) also contributes tothe decrease in thylakoid membranes function and alterations inthe organization of the membrane complexes. It has been suggested(Sfichi et al., 2004) that one of the primary responses to UV-B irra-diation is the modification of the LHCII size and organization. Basedon previous data indicating the strong effect of UV radiation on thevariable fluorescence (Larkum and Wood, 1993), may be explainedthat the UV-B-induced increase of the fluorescence ratio F735/F685is a consequence of the damage of PSII photochemistry (Table 2). Inaddition, the altered structure of thylakoid membranes and/or dis-
connection of LHCII from PSII as a response to UV-B stress (Brandleet al., 1977; Renger et al., 1986) could also lead to increase of thisratio (Table 2) by influencing the energy redistribution betweenPSII and PSI similarly to the action of UV-A radiation (Ivanova
735/F685 and F695/F685) of isolated pea thylakoid membranes. Mean values ± SE were a different letter are significantly different at P < 0.05 (estimated by ANOVA).
�exc. = 472 nm
685 F735/F685 F695/F685
0.01 a 1.13 ± 0.02 a 0.85 ± 0.01 a 0.02 a 1.46 ± 0.03 b 0.94 ± 0.04 a
0.02 a 1.02 ± 0.04 a 0.85 ± 0.03 a 0.02 b 1.39 ± 0.06 b 0.97 ± 0.09 a
t al., 2008). This probably is a protective mechanism of the pho-osynthetic apparatus against UV-B damage, which is thought toe essential for short-term acclimation of the plants to the stressactors (Carlberg et al., 1992).
The obtained induction curves under continuous illuminationFig. 4B) exhibit biphasic exponential decay after the initial oxygenurst, which could be explained by the existence of two parallelechanisms (cooperative and non-cooperative) for oxygen pro-
uction (Zeinalov and Maslenkova, 1996; Zeinalov, 2009). It hasupposed that the cooperative mechanism is realized by diffusionf oxygen precursors within the different oxygen evolving centersmainly PSII� centers) while the non-cooperative Kok’s mechanisms realized by PSII� centers. It has also suggested that the ratio A1/A2orresponds to the ratio of functionally active PSII� to PSII� centerssee in Ivanova et al., 2008). The proposed UV-B induced structurallterations in thylakoid membranes (Brandle et al., 1977; Rengert al., 1986) in turn may lead to observed changes in the A1/A2 ratioTable 1). The UV-B induced decreasing of this ratio (Table 1) coulde associated with inactivation of PSII� centers and/or the conver-ion of PSII� to PSII� centers. It is known that the PSII� centersn grana regions are especially sensitive to UV-B radiation (Yu andjörn, 1996) and a great part of these centers is blocked, leading tohe observed by us bigger inhibition of oxygen flash yields than ofhe amplitudes of oxygen burst (Figs. 3 and 4).
The protective effect of the exogenous Asc against UV-B inducedamage of photosynthetic oxygen evolution was also investigated
n the present study. Our data reveal that the exogenous additionf Asc diminishes the UV-induced impairment of both the acceptorFig. 2) and the donor side (Figs. 3 and 4) as the degree of damageepends on the concentration of the Asc added to the thylakoidembranes before UV-B exposure (Fig. 4 insets). Increased con-
entrations of Asc do not exhibit full protective effect with respecto PSII activity. The defense effect increases up to 400 �M Asc andecomes almost constant above this concentration (Fig. 4 insets).e and Häder (2002) have also suggested that the antioxidants (likesc) cannot completely prevent photochemical activity of PSII fromV-B induced inhibition.
The addition of Asc to control thylakoid membranes leads tolightly increasing of the oxygen evolution and the A1/A2 ratio (Fig. 4nd Table 1) indicating an increase of the ratio of functionally activeSII� to PSII� centers, evolving oxygen by the non-cooperative andhe cooperative mechanism, respectively. Taking into account thebove as well as the increased amount of the PSII centers in the S0tate (Table 1) can be assumed that the exogenous Asc most proba-ly alters the PSII organization in the control membranes before theV treatment, which leads to increase of the functionally active PSIIenters in grana domains (PSII�) operating by the non-cooperativeok’s mechanism. The results reveal that during UV-B irradiationsc prevents in different extent the PSII centers situated in granand stroma domains, as it appears to be more effective in protectionhe PSII� centers in stroma lamellae (Figs. 3 and 4).
It has been supposed that one of the reasons for the UV-inducednhibition of oxygen evolution is the direct absorption of UV lighty Mn ions in Mn (III) and Mn (IV) oxidation states (see in Vass et al.,005). The oxidation state of the Mn clusters in S0 state (Mn2+, Mn3+,n4+, Mn4+) is lower by one oxidizing equivalent than in S1 state
Mn3+, Mn3+, Mn4+, Mn4+) (Hoganson and Babcock, 1997). In thearkness only the S0 and S1 states are stable and the increased S0opulation means the reduction of one Mn3+ to Mn2+. Therefore,
t could be proposed that increased amount of PSII centers in thenitial S0 state after addition of Asc (Table 1), accompanied withecrease of Mn (III), is one of the possible reasons for the decreased
ensitivity of the photosynthetic apparatus to UV radiation in theresence of Asc. These observations imply another possible role ofsc in the photosynthetic membranes: protection of the Mn clusterf oxygen-evolving complex from donor-side induced UV damage.
Physiology 170 (2013) 251– 257
Protection by direct scavenging of ROS produced during donor-sideinduced photoinhibition could not be excluded (Conklin, 2001; Gilland Tuteja, 2010).
In conclusion, our data suggest that the exogenous addition ofAsc to the thylakoid suspension before UV-B irradiation (up to60 min) decreases the UV-B induced damage on the donor andacceptor side of PSII, as well as influences the ratio of the func-tionally active PSII� to PSII� centers and the initial S0–S1 darkdistribution by modification of the Mn cluster.
Acknowledgements
We thank Prof. Y. Zeinalov for helpful discussion and comments.This work was supported by the Bulgarian Academy of Sciences.
References
Agati G, Mazzinghi P, Fusi F, Ambrosini I. The F685/F730 chlorophyll fluorescenceratio as a tool in plant physiology: response to physiological and environmentalfactors. J Plant Physiol 1995;145:228–38.
Barr R, Crane FL, Giaquinta RT. Dichlorophenylurea-insensitive reduction of silico-molybdic acid by chloroplast photosystem II. Plant Physiol 1975;55:460–2.
Brandle JR, Campbell WF, Sisson WB, Caldwell MM. Net photosynthesis, electrontransport capacity and ultra structure of Pisum sativum L. exposed to ultraviolet-B radiation. Plant Physiol 1977;60:165–8.
Carlberg I, Bingsmark S, Vennigerholz F, Larsson UK, Andersson B. Low temperatureeffects on thylakoid protein phosphorilation and membrane dynamics. BiochimBiophys Acta 1992;1099:111–7.
Conklin PL. Recent advances in the role and biosynthesis of ascorbic acid in plants.Plant Cell Environ 2001;24:383–94.
Dobrikova AG, Morgan RM, Ivanov AG, Apostolova E, Petkanchin I, Huner NPA, et al.Electric properties of thylakoid membranes from pea mutants with modifiedcarotenoid and chlorophyll–protein complexes composition. Photosynth Res2000;65:165–74.
Gao Q, Zhang L. Ultraviolet-B-induced oxidative stress and antioxidant defense sys-tem responses in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana. J PlantPhysiol 2008;165:138–48.
Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stresstolerance in crop plants. Plant Physiol Biochem 2010;48:909–30.
Hakala M, Tuominen I, Keränen M, Tyystjärvi T, Tyystjärvi E. Evidence for the role ofthe oxygen-evolving manganese complex in photoinhibition of photosystem II.Biochim Biophys Acta 2005;1706:68–80.
He Y-Y, Häder D-P. UV-B-induced formation of reactive oxygen species and oxidativedamage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acidand N-acetyl-l-cysteine. J Photochem Photobiol B 2002;66:115–24.
Hideg E, Sass L, Barbato R, Vass I. Inactivation of photosynthetic oxygen evo-lution by UV-B irradiation: a thermoluminescence study. Photosynth Res1993;38:455–62.
Hideg E, Vass I. UV-B induced free radical production in plant leaves and isolatedthylakoid membranes. Plant Sci 1996;115:251–60.
Hoganson CW, Babcock GT. A metaloradical mechanism for the generation of oxygenfrom water in photosynthesis. Science 1997;277:1953–6.
Hollosy F. Effects of ultraviolet radiation on plant cells. Micron 2002;33:179–97.Ivanova PI, Dobrikova AG, Taneva SG, Apostolova EL. Sensitivity of the photo-
synthetic apparatus to UV-A radiation: a role of light-harvesting complexII – photosystem II supercomplex organization. Radiat Environ Biophys2008;7:169–77.
Jahns P, Latowski D, Strzalka K. Mechanism and regulation of the violaxanthincycle: the role of antenna proteins and membrane lipids. Biochim Biophys Acta2009;1787:3–14.
Jansen MAK, Gaba V, Greenberg BM. Higher plants and UV-B radiation: balancingdamage, repair and acclimation. Trends Plant Sci 1998;3:131–5.
Jordan BR. Effects of UV-B radiation on plants: molecular mechanisms involved inUV-B responses. In: Pessarakli M, editor. Handbook of plant and crop stress. 3rded. Boca Raton, FL: CRC Press, Taylor and Francis Group; 2010. p. 565–76.
Kok B, Forbush B, McGloin M. Co-operation of charges in photosynthetic O2 evolu-tion. I. A linear four step mechanism. Photochem Photobiol 1970;11:457–75.
Krause GH, Weis E. Chlorophyll fluorescence and photosynthesis: the basics. AnnuRev Plant Physiol Plant Mol Biol 1991;42:313–49.
Larkum AWD, Wood WF. The effect of UV-B radiation on photosynthesis and res-piration of phytoplankton, benthic macroalgae and seagrasses. Photosynth Res1993;36:17–23.
Lichtentaler HK. Chlorophyll and carotenoids: pigments of photosyntetic biomem-branes. Methods Enzymol 1987;148:350–82.
Melis A. Photosystem-II damage and repair cycle in chloroplasts: what modulatesthe rate of photodamage in vivo? Trends Plant Sci 1999;4:130–5.
Melis A, Nemson JA, Harrison MA. Damage to functional components and partialdegradation of photosystem II reaction center proteins upon chloroplast expo-sure to ultraviolet-B radiation. Biochim Biophys Acta 1992;1100:312–20.
Müller-Moulé1 P, Conklin PL, Niyogi KK. Ascorbate deficiency can limit violaxanthinde-epoxidase activity in vivo. Plant Physiol 2002;128:970–7.
Zeinalov Y. Is the concept of photosynthetic units verified? Z Naturforsch2009;64c:459–75.
A.G. Dobrikova et al. / Journal of
unné-Bosch S, Shikanai T, Asada K. Enhanced ferredoxin-dependent cyclic elec-tron flow around photosystem I and alpha-tocopherol quinone accumulation inwater-stressed ndhB-inactivated tobacco mutants. Planta 2005;222:502–11.
unné-Bosch S, Alegre L. Interplay between ascorbic acid and lipophilic antioxi-dant defences in chloroplasts of water-stressed Arabidopsis plants. FEBS Lett2002;524:145–8.
edunchezhian N, Kulandaivelu G. Evidence for the ultraviolet-B (280–320 nm)radiation induced structural reorganization and damage of photosystem IIpolypeptides in isolated chloroplasts. Physiol Plant 1991;81:558–62.
ospísil P. Molecular mechanisms of production and scavenging of reactive oxygenspecies by photosystem II. Biochim Biophys Acta 2012;1817:218–31.
enger G, Voss M, Gräber P, Schulze A. Effect of UV irradiation on different partialreactions of the primary processes of photosynthesis. In: Worrest C, CaldwellMM, editors. Stratospheric ozone reduction, solar ultraviolet radiation and plantlife. Berlin: Springer-Verlag; 1986. p. 171–84.
fichi L, Ioannidis N, Kotzabasis K. Thylakoid-associated polyamines adjust the UV-Bsensitivity of the photosynthetic apparatus by means of light-harvesting com-plex II changes. Photochem Photobiol 2004;80:499–506.
evini M, Pfister K. Inhibition of photosystem II by UV-B radiation. Z Naturforsch1985;40c:129–33.
óth SZ, Nagy V, Puthur JT, Kovács L, Garab G. The physiological role of ascorbateas photosystem II electron donor: protection against photoinactivation in heat-stressed leaves. Plant Physiol 2011;156:382–92.
rebst A, Depka B. Degradation of the D-1 protein subunit of photosystem II in
isolated thylakoids by UV light. Z Naturforsch 1990;45c:765–71.
urcsányi E, Vass I. Inhibition of photosynthetic electron transport by UV-A radiationtargets the photosystem II complex. Photochem Photobiol 2000;72:513–20.
ass I. Molecular mechanisms of photodamage in the photosystem II complex.Biochim Biophys Acta 2012;1817:209–17.
Physiology 170 (2013) 251– 257 257
Vass I, Kirilovsky D, Etienne A-L. UV-B radiation-induced donor- and acceptor-sidemodifications of photosystem II in the cyanobacterium Synechocystis sp. PCC6803. Biochemistry 1999;38:12786–94.
Vass I, Sass L, Spetea C, Bakou A, Ghanotakis DF, Petrouleas V. UV-B-induced inhi-bition of photosystem II electron transport studied by EPR and chlorophyllfluorescence. Impairment of donor and acceptor side components. Biochemistry1996;35:8964–73.
Vass I, Szilárd A, Sicora C. Adverse effects of UV-B light on the structure and functionof the photosynthetic apparatus. In: Pessarakli M, editor. Handbook of photo-synthesis. 2nd ed. Boca Raton, FL: CRC Press, Taylor and Francis Group; 2005. p.827–43.
Vermaas WFJ, Arntzen CJ. Synthetic quinones influencing herbicide bindingand photosystem II electron transport. The effects of triazine-resistance onquinone binding properties in thylakoid membranes. Biochim Biophys Acta1983;725:483–91.
Yamane Y, Kashino Y, Koike H, Satoh K. Increases in the fluorescence Fo level andreversible inhibition of photosystem II reaction center by high-temperaturetreatments in higher plants. Photosynth Res 1997;52:57–64.
Yu S-G, Björn LO. Differences in UV-B sensitivity between PSII from grana lamellaeand stroma lamellae. J Photochem Photobiol B 1996;34:35–8.
Zeinalov Y. An equipment for investigations of photosynthetic oxygen productionreactions. Bulg J Plant Physiol 2002;28:57–67.
Zeinalov Y, Maslenkova L. Mechanisms of photosynthetic oxygen evolution. In: Pes-sarakli M, editor. Handbook of photosynthesis. New York: Marcel Dekker; 1996.p. 129–50.
