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Plant Physiology and Biochemistry 81 (2014) 74e83

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Reduced glutamine synthetase activity plays a role in control ofphotosynthetic responses to high light in barley leaves

Marian Brestic a,*, Marek Zivcak a, Katarina Olsovska a, Hong-Bo Shao b,c, HazemM. Kalaji d,Suleyman I. Allakhverdiev e,f

aDepartment of Plant Physiology, Slovak Agricultural University, Tr. A. Hlinku 2, 949 76 Nitra, Slovak RepublicbKey Laboratory of Coastal Environmental Processes & Ecological Remediation and Key Laboratory of Coastal Biology & Bioresources Utilization, YantaiInstitute of Coastal Zone Research (YIC), Chinese Academy of Sciences (CAS), PR Chinac Institute of Life Sciences, Qingdao University of Science & Technology, Qingdao 266042, PR ChinadDepartment of Plant Physiology, Faculty of Agriculture and Biology, Warsaw Agricultural University SGGW, Nowoursynowska 159, 02-776 Warsaw, Polande Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, Moscow 127276, Russiaf Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia

a r t i c l e i n f o

Article history:Received 1 November 2013Accepted 3 January 2014Available online 12 January 2014

Keywords:BarleyGlutamine synthetasePhotoinhibitionPhotorespirationChlorophyll fluorescenceNon-photochemical quenching

* Corresponding author. Tel.: +421 37 6414 448.E-mail addresses: marian.brestic@uniag.sk,

(M. Brestic), marek.zivcak@uniag.sk (M. Zivcak).

0981-9428/$ e see front matter � 2014 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2014.01.002

a b s t r a c t

The chloroplastic glutamine synthetase (GS, EC 6.3.1.2) activity was previously shown to be the limitingstep of photorespiratory pathway. In our experiment, we examined the photosynthetic high-light re-sponses of the GS2-mutant of barley (Hordeum vulgare L.) with reduced GS activity, in comparison to wildtype (WT). The biophysical methods based on slow and fast chlorophyll fluorescence induction, P700absorbance, and gas exchange measurements were employed. Despite the GS2 plants had high basalfluorescence (F0) and low maximum quantum yield (Fv/Fm), the CO2 assimilation rate, the PSII and PSIactual quantum yields were normal. On the other hand, in high light conditions the GS2 had much highernon-photochemical quenching (NPQ), caused both by enhanced capacity of energy-dependentquenching and disconnection of PSII antennae from reaction centers (RC). GS2 leaves also maintainedthe PSII redox poise (QA

�/QA total) at very low level; probably this was reason why the observed pho-toinhibitory damage was not significantly above WT. The analysis of fast chlorophyll fluorescence in-duction uncovered in GS2 leaves substantially lower RC to antenna ratio (RC/ABS), low PSII/PSI ratio(confirmed by P700 records) as well as low PSII excitonic connectivity.

� 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Plants convert the light energy into chemical energy (ATP andNADPH) through the photosynthetic electron transport on thyla-koidmembranes in chloroplast. This energy can be used for fixationof CO2 in Calvin cycle. However, plants are exposed to extremelyvariable conditions (e.g. excess of incident light, CO2 shortage dueto closed stomata, variable temperature, stresses) inevitablycausing imbalances between supply and demand of energy atdifferent levels. The excess of energy can result to damages ofmolecular structures in chloroplast (photoinhibition). Therefore,the complex of precisely regulated protective mechanisms acts tokeep the functionality of the photosynthetic apparatus (Melis,1999; Allakhverdiev et al., 2008; Vass, 2012).

marian.brestic@gmail.com

son SAS. All rights reserved.

The photorespiration is generally considered a “safety valve” ofphotosynthesis (Osmond and Grace, 1995). The photorespiratorypathway starts with the oxygenase reaction catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme (Rubisco), pro-ducing glycollate-2-phosphate, which is then metabolized to formthe Calvin cycle intermediate glycerate-3-phosphate. During thismetabolic process, ATP and reducing equivalents are consumed.Thus, the pathway represents the energy sink, which enables toprevent the over-reduction of the photosynthetic electron trans-port chain, especially under stress conditions leading to low CO2assimilation rates (Wingler et al., 2000; Foyer et al., 2009).

Although photorespiration includes many metabolic stepswhich are performed across chloroplasts, mitochondria and per-oxisomes, several studies suggest that the rate-limiting step is thereassimilation of ammonia catalyzed by chloroplastic glutaminesynthetase (GS2) (Wallsgrove et al., 1987; Häusler et al., 1994a).Kozaki and Takeba (1996) have demonstrated that a transgenictobacco plant over-expressing chloroplastic glutamine synthetasehad increased photorespiration capacity.

Abbreviations1

ACO2 CO2 assimilation rateChlF Chlorophyll FluorescenceGS glutamine synthetaseGS2 chloroplastic glutamine synthetaseLED light emitting diodeLHC light harvesting complexNPQ non-photochemical quenchingP700 primary electron donor of PSI (reduced form)P700þ primary electron donor of PSI (oxidized form)PAR photosynthetic active radiationPQ plastoquinonePSI Photosystem IPSII Photosystem IIQA primary PSII acceptorqE pH dependent energy dissipationRuBP Ribulose 1,5-bisphosphateWT Wild TypeDpH Transthylakoid pH gradient

M. Brestic et al. / Plant Physiology and Biochemistry 81 (2014) 74e83 75

In plants, GS2 together with ferredoxin-dependent glutamatesynthase (Fd-GOGAT) plays a major role in re-assimilation ofammonium liberated in mitochondria by the glycine decarbox-ylase, in the pathway known as glutamine synthetase/glutamatesynthase (GS/GOGAT) cycle in chloroplasts. Moreover, the productof this cycle, glutamate, is required for one of the peroxisomaltransamination reactions. The GS/GOGAT cycle runs in chloroplastand it is directly associated with photosynthetic electron transportas it consumes electrons from taken from reduced ferredoxin at theacceptor side of photosystem I (PSI) and ATP (Hodges et al., 2013).

Thus, the activity of GS2 is essential for the process of photo-respiration and decrease in the GS2 enzymatic activity leads tomultiple effect, including decrease of ammonium re-assimilation,accumulation of metabolic intermediates due to interruption ofphotorespiratory cycle beyond Rubisco as well as by the direct ef-fects on the redox poise of electron carriers in chloroplast, as it wasdemonstrated on GS2 mutant studies (Wingler et al., 2000).

Mutants of the photorespiratory cycle have contributed signifi-cantly to the understanding of this biochemical pathway and itslinks to other physiological processes (Leegood et al., 1995;Somerville, 2001; Reumann and Weber, 2006). The changes inphenotypes of these mutants compared to wild type have beenassumed to be due to the depletion of photosynthetic carbon andnitrogen cycle intermediates and perhaps to the accumulation oftoxic photorespiratory intermediates (Cousins et al., 2008). Whilethe mutants with completely reduced activity of particular photo-respiratory enzymes (i.e. the homozygous photorespiratory mu-tants) are not able to survive at ambient CO2 concentrations, theheterozygous mutants with only partially reduced enzyme activ-ities can be grownwell in air (Wingler et al., 2000). The GS2mutantof barley was shown to have normal rates of photosynthesis inmoderate light and ambient CO2, but lower rates when photo-respiratory flux was increased in high light and low CO2 (Häusleret al., 1994a; Wingler et al., 1999). Wingler et al. (2000) suggestthree ways how the reduced photorespiratory enzyme activity mayaffect photosynthesis in mutant plants. First, an impairment of therecycling of the carbon in the photorespiratory pathway couldresult in a depletion of Calvin cycle metabolites. The supply of

1 See Table 1 for other symbols representing chlorophyll fluorescence and P700parameters.

glutamine to a GS2 mutant of barley restored photosynthetic ac-tivity (Blackwell et al., 1987). However, the pools of RuBP wasshown to be almost unaffected by mutation of GS-2 (Leegood et al.,1995; Wingler et al., 1999).

The second possible effect could be an impairment of photo-respiratory nitrogen re-assimilation resulting in a decline in the leafnitrogen and protein content. In the GS2mutants, the problemwithNH3 loss can be expected (Häusler et al., 1994a). However, this ef-fect can be partially diminished by alternative pathway, where themutants bypass the normal photorespiratory pathway by oxidativedecarboxylation of glyoxylate and formation of serine fromformate. The advantage of this alternative photorespiratorypathway is the absence of NH3 loss (Häusler et al., 1996). The lowerleaf protein content was shown be a factor of lower importance inGS2 mutants (Wingler et al., 2000).

The third possible constraint affecting photosynthesis could beaccumulation of photorespiratory metabolites having a feedbackeffect on Calvin cycle activity (Leegood et al., 1995, 1996). NH3accumulation has probably only negligible direct negative effect onphotosynthetic electron transport (Blackwell et al., 1987). Accu-mulation of serine is also unlikely to inhibit photosynthesis(Wingler et al., 2000). Themore probable is the regulatory feedbackeffects of some metabolites on enzyme activities, e.g. changesglyoxylate content in GS2mutants influenced the activation state ofRubisco (Campbell and Ogren, 1990).

In our study, we have examined photosynthetic responses of GS2mutant of barley with reduced activity of chloroplastic glutaminesynthetase, employingmainly the non-destructive biophysical tools.In addition to conventional saturation pulse method of chlorophyllfluorescence (Schreiber, 1986) measured simultaneously with gasexchange or P700 absorbance, we applied also the analysis of fastchlorophyll afluorescence induction. This analytical tool can serve asa valid examination of environmental effects on the photosyntheticapparatus (�Ziv�cák et al., 2008; Kalaji et al., 2012; Bresti�c et al., 2012).Our results indicate that the mutation of the enzyme not directlyassociated with conversion of light energy led to surprisingly sig-nificant modifications of structure and function of photosystems.

2. Materials and methods

2.1. Plant material

As a plant material, the genotypes of barley (Hordeum vulgare L.)were used. We examined the “wild type” barley cv. Kompakt(hereinafter labeled WT) and GS2-mutant of barley, i.e. the het-erozygous photorespiratory mutant with reduced activity of chlo-roplastic glutamine synthetase (GS2, EC 6.3.1.2), provided by P. Lea,University of Lancaster, UK. This mutant was obtained by crossingmutant deficient in chloroplastic GS activity with non-mutantbarley line. The heterozygous mutant were characterized withapp. 66% GS2 activity compared to WT (Häusler et al., 1994b).

2.2. Cultivation of plants

In outdoor experiments, the plants were grown in the pots (18 lpots with soil substrate and 40 plants per one pot) under naturallight. Plants werewatered twice a day (according to their demands)and supplied by inorganic fertilizers.

In laboratory experiments, the plants were cultivated in middle-size pots (4 l) in standard peat substrate with neutral pH (9 plantsper pot). The pots were regularly irrigated and occasionally fertil-ized using standard liquid fertilizer with micronutrients. Plantswere grown in a growth chamber with artificial light provided byfluorescent tubes (Osram Fluora) with maxima in red and bluespectral region; the incident PAR at leaf level was app. 250 mmol

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photons m�2 s�1. The photoperiod was 14 h light/10 h dark withlight intensity reduced by half during the first and the last hour oflight period. Temperature ranged between 20 �C at night and 25 �Cduring the light period.

2.3. The chlorophyll fluorescence measurements of daily course

The measurements of chlorophyll a fluorescence outdoors wereperformedwith a portable MINI-PAM fluorometer (Walz, Germany).The firstmeasurements of F0 and Fmweremade before sunrise usingdark leaf clips. Thereafter themeasurements of the same parameterswere made from sunrise to sunset in 1-h intervals. The initial (F0)fluorescence level represented by all PSII RC as open was measuredby sufficiently low modulated light of 0.1 mmol photons m�2 s�1 inorder not to induce any variable fluorescence. The maximal (Fm)fluorescence represented by all PSII RC as closedwere determined inthe dark adapted leaves before sunrise using a leaf clip holder (Model2030-B, Walz, Germany) by applying total 0.8 s saturation pulse of8000 mmol photons m�2 s�1 intensity. Then the plants were illumi-nated by natural daily light. The chlorophyll fluorescence intensity ofnaturally illuminated leaf (F0) was thereafter recorded and the nextsaturating pulses of 8000 mmol photons m�2 s�1 were applied todetermine the maximal fluorescence level (F0m) in the light adaptedleaves. Saturating light pulses were applied through the fibre-opticcable that was oriented under 60� angle to the leaf surface.

2.4. Measurements of incident light

The diurnal environmental fluctuations in 5-s intervals weremonitored, and the datawere averaged each 5min by the automatic

Table 1Measured and calculated chlorophyll fluorescence and P700 parameters.

Parameters Name and basic physiological int

Measured or computed inputs for calculation of fluorescence and P700 parametersF, F0 Fluorescence emission from darkF0 Minimum fluorescence from darkFm, Fm0 Maximum fluorescence from darFv ¼ Fm � F0 Maximum variable fluorescenceF00 Minimum fluorescence from lighP P700 absorbance at given light inPm, Pm0 Maximum P700 signal measured

in dark (Pm) or light adapted sta

Chlorophyll fluorescence parameters derived from the saturation pulse analysis (SchreibeFv/Fm ¼ 1 � (F0/Fm) Estimated maximum quantum efFPSII ¼ (Fm � F0)/Fm0 Estimated effective quantum yielNPQ ¼ (Fm � Fm0)/Fm0 Non-photochemical quenching oqP ¼ (Fm0 � F0)/(Fm0 � F00) Coefficient of photochemical queQA�/QA(total) ¼ 1 � qP Excitation pressure; the ratio of r

FNO ¼ 1/[NPQ þ 1 þqL(Fm/F0 � 1) Quantum yield of non-regulatedFNPQ ¼ 1 � FPSII � FNO Quantum yield of pH-dependent

PSI parameters derived from the saturation pulse analysis of P700 absorbance (KlughammFPSI ¼ (Pm0 � P)/Pm Estimated effective quantum yielFND ¼ P700

þ /P700(total) ¼ P/Pm Fraction of overall P700 that is oFNA ¼ (Pm � Pm0)/Pm Fraction of overall P700 that is o

Basic values and JIP-test parameters derived from the fast chlorophyll fluorescence inductGovindjee, 2011)

Ft Fluorescence level at time tF0 ¼ F50ms Minimum fluorescenceFm ¼ FP Maximum fluorescence (the meaVt ¼ (Ft � F50ms)/(Fm � F50ms) Relative variable fluorescence atdV/dt0 Initial slope of fast fluorescence iArea Area above the OJIP curve betwejETo ¼ 1 � VJ Probability with which a PSII trapJREo ¼ 1 � VI Probability with which a PSII trap

(to PSI electron acceptors)dREo ¼ (1 � VI)/(1 � VJ) Probability with which e- from QABS/RC ¼ (dV/dto/VJ)(1/4Po) Apparent antenna size of active P

micro-meteorological station Datalogger LI-1400 (LiCor, USA) withcompatible horizontally oriented sensors of photosyntheticallyactive radiation e LI-190SA (other parameters like global radiation,air temperature and relative humidity, soil temperature and leaftemperature and leaf water potential were also recorded, but are notpresented here).

2.5. Simultaneous measurements of gas exchange and chlorophyllfluorescence

The light response curve was recorded using a Ciras-2 (PP-sys-tems, UK) with simultaneous measurement of chlorophyll fluo-rescence. Before the measurements, plants were exposed toambient light in the growth chamber for at least 30 min. Immedi-ately before the measurements, plants were dark adapted for20min in dark box and for app. 3min in themeasuring head. The F0and Fm values were then determined using saturation flash(6500 mmol photonsm�2 s�1) and the actinic light provided by lightunit with red and blue LEDs (initial induction at 130 mmol photonsm�2 s�1 was applied to open stomata and start-up photosyntheticmachinery) was switched on. When the steady state with openstomata was reached, gradually increasing light in steps (5 min foreach step) from 0 to 1200 mmol photons m�2 s�1) were applied,with measurements of CO2 and H2O followed by saturation pulseand far-red pulse for F00 determination. Within the measuring headthe following conditions were maintained: leaf temperature 20 �C,reference CO2 content 380 ppm, and ambient air humidity. A rangeof measured and calculated fluorescence parameters were used inthe analysis (Table 1).

erpretation

- or light adapted leaf, respectivelyadapted leaf, (PSII centers open)

k or light adapted leaf respectively (PSII centers closed)from dark adapted leaft adapted leaftensityusing saturation light pulse following after short far-red pre-illuminationte.

r, 1986; Genty et al., 1989; Kramer et al., 2004)ficiency (yield) of PSII photochemistryd (efficiency) of PSII photochemistry at given PARf Fmnching based on the “puddle” model (i.e., unconnected PSII units)educed QA to total QA poolenergy dissipation in PSIIenergy dissipation in PSII

er and Schreiber, 1994)d (efficiency) of PSI photochemistryxidized in given state due to a lack of electrons coming from electron donors12

xidized in given state by saturation pulse due to a lack of electron acceptors12

ion (Strasser and Srivastava, 1995; Strasser et al., 2000, 2010; Stirbet and

sured “peak” FP value)time t, (VJ, VI at 2 ms, 30 ms)nductionen F0 and Fm and the Fm asymptoteped electron is transferred beyond QA

ped electron is transferred from reduced QA beyond PSI

B will be transferred beyond PSISII RC (zChlAnt/ChlRC)

Fig. 1. The daily course of chlorophyll fluorescence values in leaves of wild type (fullsymbols) and GS2 mutant of barley (empty symbols) illuminated by incident sunlightoutdoors. The circles represent maximum fluorescence measured during saturationlight pulses in leaves illuminated by incident natural light (Fm0); the smaller trianglesrepresent the fluorescence intensity measured in leaves illuminated by incident light,recorded just before saturation pulse (F0). The filled area represents the intensity ofincident PAR at the leaf level (see Materials and methods).

M. Brestic et al. / Plant Physiology and Biochemistry 81 (2014) 74e83 77

2.6. Simultaneous measurements of P700 redox state andchlorophyll fluorescence

The P700 redox statewas measured with a Dual PAM-100with adual wavelength (830/875 nm) unit, following Klughammer andSchreiber (Klughammer and Schreiber, 1994). Saturation pulses(10,000 mmol photons m�2s�1), intended primarily for determina-tion of chlorophyll fluorescence parameters, were also used for theassessment of P700 parameters. As with gas exchange measure-ments, analyzed plants were first exposed to ambient light ingrowth chamber for at least 30 min; immediately before themeasurements, plants were dark adapted for 20 min in a dark boxand for app. 2 min in themeasuring head. After determination of F0,Fm and Pm, the induction curve at light intensity similar to ambient(130 mmol photons m�2 s�1) was used for induction of photosyn-thesis. After a steady-state was reached, a rapid light curve was

Fig. 2. Parameters derived from the simultaneous measurements of CO2 assimilation andassimilation; (B) actual quantum yield of PSII photochemistry; (C) the photochemical querecorded after pre-illumination at moderate light intensity to open stomata. Before records

initiated (light intensities 21, 45, 78, 134, 224, 347, 539, 833, 1036,1295, 1602, 1960 mmol photons m�2 s�1; duration of illumination ateach light intensity was 30 s). In all chlorophyll fluorescence re-cords, the correction for PSI fluorescence was done by method ofPfundel (Pfundel, 1998).

2.7. Measurements and analyses of fast chlorophyll fluorescenceinduction

ChlF induction curve was obtained by using Handy-PEA fluo-rimeter (Hansatech Instruments Ltd., UK). First, we measuredfluorescence transient in leaves kept in darkness for 30 min; thiswas our control. Then, we applied HL (see above: the protocol); andfluorescence transient was measured 1, 5, 10, 15 and 30 min afterrecovery from light. Fast fluorescence transients (the OJIP phase,where O stands for origin (minimal fluorescence); J and I are in-flections, and P stands for the peak, maximum fluorescence), thusobtained, andwere analyzed by the so-called “JIP test” (Strasser andSrivastava, 1995; Strasser et al., 2000, 2010; Stirbet and Govindjee,2011). The measured and calculated JIP parameters are in Table 1.

2.8. Determination of photosynthetic pigments

The segments from the mature, fully expanded WT and GS2leaves were homogenized using sea sand, MgCO3 and 100% acetoneand then extracted with 80% acetone. After 2-min centrifugation at2500 rpm, absorbance of the solution was measured, by a VISspectrophotometer (Spekol 11, Carl Zeiss, Jena, Germany), at470 nm, 647 nm, and 663 nm, with a correction for scattering,measured at 750 nm. The concentrations of Chla, Chlb and carot-enoids (Car) per leaf area unit were determined, using the equa-tions of Lichtenthaler (Lichtenthaler, 1987). Five leaves of eachgenotype were analyzed.

2.9. Photoinhibitory treatment

The photoinhibitory treatments reported here were realized inlaboratory conditions (temperaturew 23 �C, ambient CO2 content).Themiddle parts of leaves (which were measured) were exposed to

chlorophyll fluorescence in leaves of wild type and GS2 mutant of barley. (A) CO2

nching; (D) the non-photochemical quenching of Fm. The light response curves were, leaves were illuminated for 5 min at each light level.

Fig. 3. The values of parameters derived from simultaneous measurements of chlorophyll a fluorescence and P700 absorbance in leaves of WT and GS2. (A) Effective quantum yieldof PSII (VPSII); (B) the effective quantum yield of PSI (VPSI); (C) the quantum yield of regulated non-photochemical quenching in PSII (VNPQ); (D) the quantum yield of the PSI non-photochemical quenching caused by the donor-side limitation, i.e. the fraction of overall P700 that is oxidized in a given state (VND); (E) the fraction of energy captured by PSIIpassively dissipated in the form of heat and fluorescence (VNO); (F) the quantum yield of the PSI non-photochemical quenching caused by the acceptor-side limitation, i.e. thefraction of overall P700 that cannot be oxidized in a given state (VNA); (G) the non-photochemical quenching (NPQ); (H) the photochemical quenching (qP). The rapid light curveswere obtained after previous induction at moderate light; the duration of each interval with a given light intensity was 30 s. (see Materials and methods for details). The averagevalues � standard errors from 4 plants are presented.

M. Brestic et al. / Plant Physiology and Biochemistry 81 (2014) 74e8378

PAR intensity at the leaf level w1200 mmol photons m�2s�1 pro-vided by external halogen source with white polychromic light,tested by light meter Li-Cor LI-250A. Before and after photo-inhibitory treatment, the chlorophyll a fluorescence (and P700)was measured at exactly the same position on leaf by saturationpulse method using Dual-PAM-100 fluorimeter 1, 5, 10, 15 and30 min after recovery from light. The calculated parameters aredefined in Table 1. In other samples, the analogical measurementswere done using Handy PEA continuous flurimeter (Hansatech,Germany) as described below.

3. Results

During the sunny and warm day, the chlorophyll fluorescencemeasurements were done regularly in leaves illuminated by naturalsunny light to record the daily course of chlorophyll fluorescenceyields (Fig. 1).

The results clearly indicate that the photosynthetic responses tohigh light in GS2 barley mutant are substantially different to WT. Inaddition to high F0 level and low Fv/Fm in GS2, the early decrease ofFm0 below the F0 level induced by themoderate light intensities wasalso quite unusual. Such a decrease was not observed inWTeven inmidday. It is evident that the photochemical activity in GS2 leaves

in high light conditions was strongly suppressed in favor of thenon-photochemical processes. Hence, the reduced activity ofchloroplastic glutamine synthetase in mutant plants influencesstrongly the conversion of energy, making the PSII necessarilymuch more endangered by the photoinhibition in high light con-ditions compared to WT (this issue will be further discussed).

To reveal the causes of atypical expression in mutant plants,another experiment was carried out in laboratory conditions.Despite the significantly lower maximum quantum yield of PSIIphotochemistry (Fv/Fm value) in GS2 mutant, simultaneous mea-surements of CO2 assimilation and chlorophyll fluorescence atgradually increasing light intensities (photosynthetic lightresponse curves, Fig. 2) showed only insignificant differences bothin assimilation rate as well as in actual PSII quantum yield (FPSII).However, we observed significantly higher photochemicalquenching parameter qP (Fig. 2C), as well as higher non-photochemical quenching (Fig. 2D) in GS2 plants compared to WT.

Similar protocol (the rapid light curves) of simultaneous mea-surements of chlorophyll fluorescence and photosystem I absor-bance (reflecting changes of P700 redox status) confirmed previousresults, as we found insignificant differences in PSII and PSI quan-tum yields only (Fig. 3 A, B), but substantially higher qP and NPQ(Fig. 3G, H).

Fig. 4. (A) The relationship between the redox poise of PSII and PSI. The reduction level of primary PSII electron acceptor (QA�/QA total) was estimated as 1 � qP (it represents also

the excitation pressure at PSII); the level of oxidation of PSI donor side (P700þ/P700 total) is equal to parameter FND (both qP and FND were presented in Fig. 3). (B) The relationshipbetween the excitation pressure at PSII (QA

�/QA total, equal to 1 � qP) and the values of non-photochemical quenching (NPQ). (C) The relationship between the P700 oxidation(P700þ/P700 total) and the values of NPQ.

M. Brestic et al. / Plant Physiology and Biochemistry 81 (2014) 74e83 79

At high light, we observed slightly higher PSI donor side limi-tation in GS2 indicating lower electron supply from PSII. Weobserved in GS2 also significantly lower values of FNO and FNA(Fig. 3E, F). The first one represents the fraction of absorbed light byPSII that was not used by photochemistry nor dissipated by regu-lated non-photochemical quenching. The second parameter (FNA)quantifies the PSI acceptor side limitation, i.e. the level of reductionof PSI electron acceptors.

The values of some PSII and PSI parameters obtained in Fig. 3were mutually correlated to uncover more the reason of differ-ences between WT and GS2 (Fig. 4).

The photochemical quenching qP reflects the fraction of openreaction centers with QA in fully oxidized state (Krause et al., 1982).Hence, 1 � qP represents the fraction of closed reaction centerswith a reduced form QA

�. It represents also the PSII redox poise;moreover, 1 � qP serves as an estimate of the ‘Excitation Pressure’(Weis et al., 1987). The relationship of the PSII and PSI redox poise

Fig. 5. (A) The fast chlorophyll a fluorescence kinetics measured in dark adapted leaves. Econtains first phase of transient (0e2 ms, O-J-phase) on regular time scale. The bottom inserplotted also on the regular time scale. (B) The mathematical normalization of initial phasevariable fluorescence in time t, Vt ¼ (Ft � F0)/(Fm � F0). The plot contains 20 measurements inderived from the fast chlorophyll a fluorescence transient. The mean values of individual pashowed by dashed line. The mean values recorded in GS2 mutant of barley are shown in relatGS2 are indicated (*** statistically significant difference, ns e non-significant difference; AN

(supply of electrons by PSII vs. demand for electrons at PSI) un-cover shift of the balance between excitation of PSII and PSI(Fig. 4A). Compared to WT, in GS2 leaves the P700þ accumulatedmore quickly than QA

�; it means that at the same light intensity, thePSII release less e- than PSI. The relationship between excitationpressure and NPQ (Fig. 4B) show also different trends, with muchhigher level of NPQ at much lower excitation pressure in GS2leaves. Moreover, there is obviously biphasic increase of NPQ. Inboth GS2 and WT the first phase of step increase of NPQ starts atexcitation pressure (1 � qP) value w 0.15 and saturates at w 0.4(NPQ w 1.9 in WT and w3.0 in GS2). Then, in GS2 the secondsignificant rise started, while in WT there were only signs ofincreasing. We suggest this biphasic character can be attributed totwo different mechanisms contributing to non-photochemicalquenching (this issue will be discussed below). The relationshipbetween PSI redox poise (P700þ/P700 total) and NPQ (Fig. 4C)show almost linear trend (except for the final phase of the curve).

ntire 1 s record is plotted on logarithmic time scale (the main plot). Upper insertiontion shows two initial phases of chlorophyll a fluorescence rise (0e30 ms, O-J-I-phase)of chlorophyll a fluorescence transient (0e0.3 ms) i.e. the plot of Vt/V0.3ms ratio (Vt eGS2 and 20 measurements in WT. (C) The radar plot of selected biophysical parametersrameters recorded in wild type (Kompakt, WT) were used as a reference (equal to 1),ive units (related to wild type). The statistically significant differences between WT andOVA with Tukey HSD post hoc test, a ¼ 0.01). The parameters are defined in Table 1.

M. Brestic et al. / Plant Physiology and Biochemistry 81 (2014) 74e8380

This is probably because the redox status of P700 modulates theproton gradient (DpH) between stroma and lumen in thylakoids bytriggering the cyclic electron flow (Munekage et al., 2004). Theinitial slope of the curve for GS2 was higher than in WT (Fig. 4C),supporting the idea of higher capacity of DpH dependentquenching.

In the next step, we have analyzed the rapid chlorophyll a ki-netics record obtained at continuous illumination by strong actiniclight pulse for 1 s (Fig. 5A).

It is evident that F0 was much higher in GS2 leaves, while the Fmwas similar to WT. The insertions in graph also indicate the differ-ences in shapeof the curve: the steeper initial slope (in time0e2ms),but slower increase of the fluorescence intensity in the next step (2e30 ms) in GS2 leaves compared to WT. Another difference is evidentwhen the initial phase is mathematically normalized (Fig. 5B). Thesubstantial difference in sigmoidal character of the curves indicatesmuch lower excitonic connectivity among PSII units in GS2 leavescompared to WT (Strasser and Stirbet, 2001; Stirbet, 2013). Byapplying themathematical model designed by Strasser (Strasser andSrivastava, 1995; Strasser et al., 2000, 2010; Stirbet and Govindjee,2011) we calculated several biophysical parameters, presented herein relative values (Fig. 5C). In addition to significant differences in F0and Fv/Fmmentioned evenbefore, the analysis uncovereddifferencesbetween GS2 andWT in several other parameters.

The number of active reaction centers per absorbed light unit(RC/ABS) was in GS2 only half of those in WT. The value of theprobability that trapped electron will be transported beyond theprimary electron acceptor QA (jPo) was lower in GS2, indicatinglimitation at PSII acceptor side. But the lower number of RCs andPSII donor side limitation was balanced by enhanced electrontransport between PSII and PSI (indicated by parameters dREo),probably due to lower PSII/PSI ratio (Schansker et al., 2005; Ceppiet al., 2012) and hence, the overall probability that trapped elec-tron will move through PSII to PSI electron acceptors was higher inGS2 than inWT. As the area above fluorescence curve indicating thetotal pool of PSII electron acceptors was the same in WT and GS2,we may suggest that the low number of reaction centers is at least

Fig. 6. The relaxation kinetics of (A) PSII quantum yield (FPSII) and (B) non-photochemical quenching (NPQ) after 60 min of illumination by white actinic lightwith intensity 1200 mmol photons m�2 s�1. The measurements were done with fluo-rescence imaging system (Imaging-PAM, Walz, Germany).

partially balanced by enhanced PSI-PSII electron transport. Despiteanomalous shape of OJIP-transient caused by low number of reac-tion centers compared to the antenna size, the electron transportcapacity seems to be retained.

The enhanced non-photochemical quenching, low excitationpressure at normal level of CO2 assimilation rate allows us to as-sume that GS2 is expected to be more resistant to photoinhibitionthan WT. However, the results of photoinhibitory treatment fol-lowed by measurements of relaxation kinetics (Fig. 6) showed thatthe resistance to the short period of high light was in GS2 similar toWT plants. The relaxation kinetics of NPQ (Fig. 6 B) suggests thatthe increase of NPQ in GS2 was really caused by well-regulateddissipation of excessive light. Despite higher non-relaxed NPQ af-ter 30 min in the dark, the relaxed NPQ in GS2 was two timeshigher than in WT. The same was valid for the rapid relaxation ofFv/Fm and NPQ (after 1 min in the dark) indicating much higher qEin GS2 compared to WT. Thus, the relaxation kinetics confirmedmuch higher level of photoprotection in GS2, which was, however,not resulted to higher level of resistance to photoinhibition than inWT.

When we look at detail of relaxation of Fo and Fm values in darkafter photoinhibitory treatment (Fig. 7), we can see much higheramplitude between values in 1 min and 30 min of darkness in GS2than in WT e this amplitude was hidden when we have looked atFv/Fm relaxation only. It is interesting that, after photoinhibitorytreatment, the high F0 of GS2 plants fell down at the level ofWTandrelaxed slowly, but after 30 min of dark recovery, the F0 level in GS2was still below the initial F0 level. In contrary, in WT the F0 fell alsoslightly below initial F0 level, but during recovery F0 got early abovethe initial F0 level. We suggest that the energy dissipating compo-nents (such as zeaxanthin) are inactivated in GS2 much moreslowly and even after 30 min there is still present evident F0quenching, which is not the case of WT.

The analysis of photosynthetic pigments realized before expo-sition of plants to high light conditions (Table 2) indicate also

Fig. 7. The relaxation kinetics of F0 and Fm values following after 60 min of illumi-nation by white actinic light with intensity 1200 mmol photons m�2 s�1. The lonelysymbols left represent the dark adapted values of F0 and Fm measured just before lightexposition. The long dashed lines represent F0 and Fm values of WT; the dotted linesrepresent F0 and Fm levels of GS2. The measurements were done with fluorimeterHandyPEA (Hansatech, UK).

Table 2The leaf photosynthetic pigment content and ratios.

Parameter WT GS2

Chlorophyll a (mg m�2) 383.8 � 14.5a 304.1 � 27.3b

Chlorophyll b (mg m�2) 150.7 � 10.1a 145.9 � 15.7a

Total chlorophylls (mg m�2) 534.5 � 24.5a 449.9 � 42.9b

Total carotenoids (mg m�2) 82.3 � 5.4a 79.3 � 7.0a

Chlorophyll a to b ratio 2.57 � 0.08a 2.10 � 0.06b

Chlorophyll to carotenoid ratio 6.52 � 0.14a 5.69 � 0.27b

Mean values � SE from 5 replicates are presented. Letters indicate significant dif-ferences at P < 0.05 according to Tukey HSD test.

M. Brestic et al. / Plant Physiology and Biochemistry 81 (2014) 74e83 81

significant differences between WT and GS2 in chlorophyll a andtotal chlorophyll content, but not in chlorophyll b or total carot-enoid content. Consequently, in GS2 leaves, the chlorophyll a to bratio as well as chlorophyll to carotenoid ratio were significantlylower compared to WT.

4. Discussion

GS2 mutant with reduced activity of chloroplastic glutaminesynthetase is expected to reduce the rate of photorespiration,because glutamate is lacking for the transamination of glyoxylate toglycine (Kozaki and Takeba, 1996). The mutant we used belongs tothe group of heterozygous mutants of barley, in which photo-respiratory flux is restricted by reduced photorespiratory enzymeactivities. The GS2 mutants of barley has usually normal rates ofphotosynthesis in moderate light and ambient CO2 (Häusler et al.,1994a; Wingler et al., 1997); this was confirmed also by our mea-surements. It was previously shown that the reduced GS activity ledoften to decrease of CO2 assimilation and PSII electron transportrate, but mostly in photorespiratory conditions (Häusler et al.,1994a; Wingler et al., 1999; Streb et al., 1998). This was probablythe case of our measurements in natural sunlight (Fig. 1), where theFm0 decrease evidently goes along with low PSII quantum yield andETR. It was previously concluded that the negative effect of reducedGS activity acts mainly through accumulation of toxic metabolicintermediate products (Wingler et al., 2000). The CO2 assimilationrate in GS2 leaves was not below WT in our experiments (Fig. 2);anyway, it may be also caused by the fact that the measurementswere done in dark relaxed leaves in laboratory conditions (plantsspent some time in conditions with elevated CO2) and this mightnot be the case in the conditions where the plants were normallygrown, especially if some stress conditions occurred.

As mentioned before, the GS2 mutation is expected to affect theprocesses related to the dark phase of photosynthetic process.Therefore, it is surprising to what extent it affects the parametersrelated to the primary conversion of light energy (PSII and PSI-related parameters). The light and dark processes are inter-connected mainly through ATP and NADPH. Thus, decrease ofphotorespiration rate may lead to decrease of ATP and NADPHconsumption, leading to decline in linear electron transport be-tween PSII and PSI. Anyway, the photosynthetic rate is generallyvery variable during the day, depending strongly on CO2 availabilitydue to the openness of stomata. Therefore, the relatively smalldecrease of electron transport expected through the partialdecrease of photorespiration due to GS2 mutation may hardlyexplain the major residual effects, which we have observed.

One of the most typical properties of GS2 plants is the excessivenon-photochemical quenching (NPQ values frequently exceeding4), which exceeds the values typically found in annual grass species,even exposed to severe stress (Zivcak et al., 2013), such high valuesare more typical for evergreen woody plants. Parameter (NPQ) re-flects the decrease of fluorescence yield due to energy dissipatingprocesses in PSII. In fact, in NPQ value can be involved several

components. The “energy dependent” quenching (qE) is the fast-relaxing, delta pH-dependent component of NPQ. The low pH ofthylakoid lumen activates qE by protonating the protein PsbS (Liet al., 2000) and by xantophyll cycle (Demmig-Adams and Adams,1992). The qT component is relaxing more slowly and is associ-ated with state transitions, but it is more typical for algae and lowlight conditions. The slow relaxing qi component can be causedeither by the photoinhibition (accumulation of photodamaged RCs)or slow relaxing photoprotective responses. Our results indicatethat, in GS2, both fast relaxing component (qE) and slow relaxingdissipative processes contributing to qi were higher than in WT.The bi-phasic trend of NPQ increase (Fig. 4B) suggests that there areat least two major processes contributing to NPQ. The first oneseemed to be the typical DpH dependent component of NPQassociatedwith inter-conversions of xanthophylls (Demmig-Adamsand Adams,1992). This component seemed to be linearly correlatedwith P700 redox status and the trigger of cyclic electron flow. Onthe other hand, the second phase (we estimate its contribution tototal NPQ at maximum light intensity to w10% in WT and w30% inGS2) can be well explained by disconnection of PSII antenna com-plexes from RCs. Decreases in F0 (clearly evident in GS2, Fig. 7) arethought to be indicative of a photoprotective process by means of adisconnection of PSII from the LHCs, whereas sustained increases inF0 are associated with photoinhibitory damage (Demmig-Adamsand Adams, 1992). Recently, the several molecular studies werepublished uncovering the role of the protein kinases in protectionagainst high light, where the antenna complexes are released fromPSII RCs, but unlike the state transitions, they are not moved to PSI(Fristedt and Vener, 2011). This mechanism was found in grassspecies, like barley, but it was rare in dicots (Chen et al., 2013). Thecontribution of LHCs disconnection to total NPQ can well explainalso slow-relaxing component of NPQmentioned above, which wassignificant in GS2, but hardly distinguished in WT.

This all, however, does not explain the reasonwhy the excessiveNPQ is present in GS2 plants. The important information broughtthe analysis of rapid chlorophyll a fluorescence transient aboutsubstantially lower values of RC/ABS. This parameter reflects, infact, the ratio between the number of RCs and size of antennacomplexes; in other words, it represents the estimate of the ratiobetween the number of chlorophyll molecules in RC and thenumber of chlorophyll molecules in PSII antenna complexes(Stirbet and Govindjee, 2011). This assumption is supported also bythe results of photosynthetic pigment analysis, as chlorophyll a to bratio was significantly lower in GS2 plants (Table 2). The chlorophylla to b ratio in LHC of PSII was shown to be app. 1.33 (Tanaka andTanaka, 2011); thus, increase in LHC to RC ratio would lead todecrease in chlorophyll a to b ratio. A low value of RC/ABS in GS2leaves can be caused by decrease of the number of RCs as well as byincrease of PSII antenna size compared to wild type. As the chlo-rophyll b content was the same in GS2 andWT, we can assume thatthere is the similar size of PSII antenna complexes, but lowernumber of reaction centers in GS2 leaves compared to WT.

To uncover the phenomena observed in GS2 leaves, we havesearched for the analogical observations inwell defined conditions.Such results were found for samples treated by the lincomycin; thecompound recently commonly used in the photoinhibition studies.Lincomycin specifically inhibits the protein synthesis in the chlo-roplast (Mulo et al., 2003) and on the short run it avoids the repairof D1 protein in photoinhibited leaves without direct effect onelectron transport (Okada et al., 1991). Similarly to our GS2 samples,Gaspar et al. (2006) in maize leaves treated during the chloroplastdevelopment by licomycin found high F0 value, light induceddecrease of F0 below initial F0 value; excessive and slow relaxingNPQ and reduced amount of reaction centers, but unaffected an-tenna composition. In addition, the partial lack of fully functional

M. Brestic et al. / Plant Physiology and Biochemistry 81 (2014) 74e8382

core complexes led to a high amount of not properly organizedantenna complexes containing numerous monomeric LHCII unitsthat contribute to increase of fluorescence quenching.

In parallel to these findings, we can speculate that the reason ofthe atypical photosynthetic expression of GS2 can be the insuffi-ciency in synthesis and/or repair of PSII reaction centers that lead toimbalance between number RCs and PSII antenna complexes.Another argument indirectly proving the analogy with lincomycintreated plants is the substantially lower PSII excitonic connectivityindicated by the difference in sigmoidal shape of early phase of fastchlorophyll induction curve (Fig. 5B), in accordance with recentknowledge on excitonic connectivity (Stirbet, 2013; Tsimilli-Michael and Strasser, 2013). Provided that the GS2 plants has alsoimproperly organized antenna complexes with a high number ofmonomeric LHCs (similarly to those examined by Gaspar et al.(2006) in lincomycin treated leaves), the lower PSII excitonic con-nectivity have to be logically present in GS2, as the completesigmoidal induction has beenwell fitted by the model of connectedtetramers (Laisk and Oja, 2013). Anyway, this hypothesis need to beexperimentally verified at the molecular level before it is possibleto draw conclusions.

The discussion above raises another important question,whether the decrease of activity of glutamine synthetase can leadto these accompanying phenomena. In this regard, Takahashi et al.(2007) reported the results of the study carried out on photo-respiratory mutants of Arabidopsis with different mutations (notGS2). Based on the observation they concluded that an impairmentof the photorespiratory pathway accelerates photoinhibition ofphotosystem II by suppression of repair but not acceleration of thedamage. They found that the suppression of the repair process wasdue to inhibition of synthesis of D1 protein at the level of trans-lation. The link between impaired photorespiration and inhibitionof protein synthesis can be attributed to the interruption of Calvincycle (Takahashi et al., 2007; Takahashi and Murata, 2005). How-ever, this was not the case of our experiment, as the net carbonassimilation did not differ between WT and GS2; moreover, theRuBP decrease, which can limit Calvin cycle, was not observed inGS2 barley mutants even in conditions causing the decrease ofphotosynthetic rate (Leegood et al., 1995; Wingler et al., 1999).Similarly, the content of aminoacids was in GS2 barley mutantssimilar to wild type, even in photorespiratory conditions, whichindicates no direct effects of GS2 mutation on primary metabolismof nitrogen (Wingler et al., 2000). Alternatively, photorespirationcan cause extensive production of hydrogen peroxide (H2O2) orother ROS at the PSI acceptor side, which would inhibit the meta-bolic processes, such as synthesis or repair of PSII components(Nishiyama et al., 2006, 2011; Allakhverdiev and Murata, 2004;Murata et al., 2007). Despite some studies show a negligible pro-tective effect of photorespiration in stress conditions (Bresti�c et al.,1995), our recent results support the idea that the full functionalityof photorespiratory pathway, especially the activity glutaminesynthetase, is crucial for proper photoprotection (Kozaki andTakeba, 1996).

Further, we can hypothesize that the GS2 plants enhanced thephotoprotective systems to decrease the level of PSII photodamage.As the level of the photoinhibition was found to be linearly relatedto excitation pressure level (Kornyeyev et al., 2010), for plant en-dangered to photoinhibition, the maintenance of low excitationpressure is crucial to survive. Our results encourage us to believethat thanks to high NPQ and high number of electron carriers, therelease of electrons from reduced number of PSII reaction centers toPSII acceptor side was low enough to keep the excitation pressureand reduction level of PSI acceptor side at the level minimizing therisk of the oxidative damage of photosynthetic complexes(Takahashi et al., 2009).

In conclusion, despite the normal CO2 assimilation rate, GS2plants had a very specific responses to high light at the level of PSIIphotochemistry: an excessive non-photochemical quenching, highphotochemical quenching (and low excitation pressure), a low PSIIreaction centers to antenna ratio, lower PSII to PSI ratio, as well assignificantly lower excitonic connectivity among PSII unitscompared to non-mutant plants. We suggest that the anomalousphysiological expressions of GS2 were caused by decrease in syn-thesis and/or repair of molecular components of PSII due to thedecrease of photorespiratory activity. This in turn could result in alow number of reaction centers, large and not properly organizedlight harvesting complexes and to enhanced non-photochemicaldissipation of light energy to keep a low excitation pressure. Weconsider the GS2 mutant of barley as an interesting photosyntheticmodel, worthy of further examination as it can provide a newknowledge on the photorespiration functions, but also on redoxsignaling, photoprotection, repair or organization of light harvest-ing complexes.

Acknowledgment

This work was supported by the European Community underthe project no. 26220220180: “Construction of the “AgroBioTech”Research Centre”. SIA was supported by grants from the RussianFoundation for Basic Research (No: 13-04-91372, 14-04-01549,14-04-92690) and by Molecular and Cell Biology Programs of theRussian Academy of Sciences.

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