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Plant Physiology and Biochemistry 48 (2010) 16e20

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Plant Physiology and Biochemistry

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

Research article

Chlorophyll a fluorescence study revealing effects of high salt stresson Photosystem II in wheat leaves

Pooja Mehta, Anjana Jajoo*, Sonal Mathur, Sudhakar BhartiSchool of Life Sciences, Devi Ahilya University, Indore 452017, M.P., India

a r t i c l e i n f o

Article history:Received 26 February 2009Accepted 26 October 2009Available online 10 November 2009

Keywords:Fluorescence induction kineticsPhotosystem IISalt stress

Abbreviations: Chl, chlorophyll; CS, cross-section;beyond QA; Fo, Fm, Fv, minimum,maximum and variablstate; RCs, reaction centers; TRo, energy trapping flux by* Corresponding author. Tel.: þ91 731 2477166; fax

E-mail address: anjanajajoo@hotmail.com (A. Jajoo

0981-9428/$ e see front matter � 2009 Elsevier Masdoi:10.1016/j.plaphy.2009.10.006

a b s t r a c t

In order to study the effects of high salt stress on PS II in detached wheat (Triticum aestivum) leaves, theseedlings were grown in Knop solution and temperature was 20 � 2 �C. Detached leaves were exposed tohigh salt stress (0.1e0.5 M NaCl) for 1 h in dark and Chl a fluorescence induction kinetics was measured.Various parameters like Fv/Fm, ABS/RC, ETo/TRo, performance index and area over the florescence curvewere measured and the energy pipeline model was deduced in response to salt stress. Our results showthat the damage caused due to high salt stress is more prominent at the donor side rather than theacceptor side of PS II. Moreover the effects of high salt stress are largely reversible, as the acceptor sidedamage is completely recovered (w100%) while the recovery of the donor side is less than 85%. Based onour results we suggest that in response to high salt stress, the donor side of PS II is affected more ascompared to the acceptor side of PS II.

� 2009 Elsevier Masson SAS. All rights reserved.

1. Introduction

A high salt stress affects 7% of the world's land area, whichamounts to 930 million ha [28] and is a major factor limiting thecrop production [4,17]. High salt is one of the major abiotic stressesaffecting the plant productivity because of its negative effects onthe plant growth, ion balance and water relations [2,12,19]. Saltstress leads to a decrease in the efficiency of photosynthesis [21]and is known to influence the chlorophyll content of the plantleaves [8,14]. PS II is more sensitive to all types of stresses ascompared to PS I [3]. However, photosynthesis may decrease by thedirect effect of salt stress on photosynthetic electron transport [5].Salt stress involved the known component of osmotic stress andionic toxicity [13]. Salinity reduces plant growth through bothnonspecific (osmotic) effects and ion specific mechanisms [29]. Bya two phase model of salt injury proposed by Munns et al. [18],where growth is initially reduced by osmotic stress and then by Naþ

toxicity, it is difficult to assess with any confidence the relativeimportance of the twomechanisms to yield reduction because theyoverlap. Previous studies indicate that salt tolerance is a develop-mentally regulated, stage specific phenomenon because tolerance

ETo, electron transport fluxe fluorescence in dark-adaptedPS II centers.: þ91 731 4263453.).

son SAS. All rights reserved.

at one stage of plant development is not necessarily correlated withtolerance at other stage [5]. Under salt stress there is loss in chlo-rophyll protein (47 kDa) and a core membrane linker protein94 kDa that can attach phycobilisome to thylakoid [10]. Effects ofsalt stress in cyanobacterium Spirulina platensis [27] showeda decrease in PS II mediated oxygen evolution activity and anincrease in PS I activity. It was ascribed to changes in the thylakoidmembrane protein profile which led to the decreased energytransfer from light harvesting antenna to PS II. Salt adapted cellscan maintain a high conversion efficiency of excitation energythrough the down regulation of PS II RCs [7].

Chl a fluorescence analysis has proven to be a sensitive methodfor the detection and quantification of changes induced in thephotosynthetic apparatus. Chl a fluorescence intensity of dark-adapted photosynthetic organisms follows a characteristic varia-tion with time after the onset of illumination. This effect is wellknown as “Kautsky effect” [15]. In fluorescence induction curve, attheminimal fluorescence Fo all the reaction centers are open and atmaximal fluorescence Fm all the reaction centers are closed. Alloxygenic photosynthetic materials investigated so far show poly-phasic fluorescence rise consisting of a sequence of phases denotedas O, J, I and P [26]. The O-J-I-P transient is defined by a certain timeduring the induction kinetic: O, first measurement at onset ofillumination, J at about 2ms, I at about 30ms and P at about 500ms[22]. A method has been developed for the quantification of OJIPfluorescence transients known as JIP test [11]. With this test, it ispossible to calculate several phenomenological and biophysicalexpressions of PS II [23,25,26]. The JIP test is thus a powerful tool for

P. Mehta et al. / Plant Physiology and Biochemistry 48 (2010) 16e20 17

the in vivo investigation of the behavior of PS II function, includingthe fluxes of absorption, trapping and electron transport [25,26].

Other Chl a fluorescence parameters have also been used tostudy high salt induced damage to PS II. By measuring 77 K fluo-rescence emission spectra in dark grown wheat leaves under highsalt conditions, it was shown that salt stress inhibits the chlorophyllaccumulation by restraining several steps in porphyrin formation[1]. Delayed fluorescence measurements in Arabidopsis thalianaseedlings have also proved to be useful as a marker for detectingdamage caused by salt stress [30].

In the present work we have investigated the effects of highsalt stress on Chl a fluorescence induction kinetics in wheat leaves.On the basis of various parameters from fluorescence inductioncurves like Fv/Fm ratio, performance index (PI), area over thecurve etc. and an energy pipeline model, we have tried to explainthe effects of high salt stress on energy absorption and energydissipation. We have concluded that the effects of high salt aremore pronounced at the donor side of PS II as compared to theacceptor side of PS II.

Fig. 2. Changes in the percentage of variable fluorescence (Vj), efficiency of forwardelectron transfer (ETo/TRo) beyond QA

� and area over fluorescence curve in response tohigh salt stress in wheat leaves. Recovery was measured after keeping 0.5 M NaCltreated leaf in distilled water for 24 h.

2. Results and discussion

Polyphasic chlorophyll a fluorescence transient was measuredto evaluate the effects of high salt stress on the photochemicalefficiency of PS II. The OJIP transient represents the successivereduction of electron transport pool of PS II [11]. The intensity offluorescence in the OJIP transient decreased with increase in NaClconcentration, as shown in Fig. 1. An increase in salt concentrationcauses a significant decrease in the minimal fluorescence (Fo),variable fluorescence (Fv) and maximal fluorescence (Fm) (data notshown). A decrease in the fluorescence yield of leaves can beattributed to an inhibition of electron flow at oxidizing site of PS II[7]. The decrease in Fm and fluorescence at J, I, P may be due to tworeasons, first by inhibition of electron transport at the donor side ofthe PS II which results in the accumulation of P680þ [11,20] andsecond due to a decrease in the pool size of QA

�. Area over thefluorescence induction curve between Fo and Fm is proportional tothe pool size of the electron acceptor QA on the reducing side of PSII. If the electron transfer from reaction center to quinone pool isblocked, this area will be dramatically reduced. As compared tocontrol leaves the area over the fluorescence curve was decreasedby 78% in 0.5 M NaCl treatment (Fig. 2). This decrease in area over

Fig. 1. The OJIP Chl a fluorescence transient curve (log time scale) in wheat leavesexposed to various concentration of NaCl for 1 h dark.

the fluorescence curve with increase in NaCl concentrationsuggests that high salt stress inhibits the electron transfer rates atthe donor side of PS II. Fv/Fm ratio was not affected significantly inhigh salt treatment. In 0.5 M NaCl treated leaves the Fv/Fm ratiodecreased by only 4% (Table 1). ABS/RC i.e. effective antenna size ofan active reaction centers, is calculated as a total number of photonsabsorbed by chl molecules of all RCs divided by total number ofactive RCs. It is influenced by ratio of active/inactive RCs. Withincrease in NaCl concentration the value of ABS/RC increased,which results decrease in antenna size of active RCs (Table 1). Inorder to see that whether high salt induced damage caused to PS IIis irreversible or reversible, we immersed the stressed leaves(0.5 M) in distilled water for 24 h and then measured the fluores-cence induction curves. It was observed that Fm was increased by44% i.e. similar to control. The area over the curve was increased by80% in recovered leaves (Fig. 2). Our results show that acceptor sidedamage was recovered completely while damage at the donor sideof PS II was recovered more than 80% (Fig. 2). The rapid decline inphotosynthesis under NaCl stress is reversible and specific toosmotic stress, where as the slow decline is irreversible and specificto ionic stress [30].

To localize the action of high salt stress in electron transportchain on acceptor side of PS II the kinetics of relative variablefluorescence (Vj) was calculated. Vj is equivalent to (Fj-Fo/Fm-Fo).Fj is the fluorescence at J step i.e. at 2 ms, relative variable fluo-rescence (Vj) at 2 ms for unconnected PS II units, equals to thefraction of closed RCs at J step expressed as proportion of the total

Table 1Changes in the ratios i.e. Fv/Fm, ABS/RC in response to various concentration of NaClexposed wheat leaves. The stress was given to wheat leaves for 1 h in dark. The datashows mean value � S.D. Numbers in parenthesis show the normalized value.

NaCl Conc. (M) ABS/RC Fv/Fm

Control 1.39 � 0.033 (100) 0.855 � 0.003 (100)0.1 1.57 � 0.124 (113) 0.849 � 0.007 (99)0.2 1.59 � 0.085 (114) 0.845 � 0.004 (99)0.3 1.60 � 0.071 (115) 0.844 � 0.017 (98)0.4 1.67 � 0.087 (119) 0.834 � 0.008 (97)0.5 1.71 � 0.087 (123) 0.828 � 0.003 (96)Recovery 1.39 � 0.002 (100) 0.851 � 0.001 (99)

Fig. 3. The change in performance index in wheat leaves exposed to various concen-tration of NaCl for 1 h. Inset shows the inhibition of PI values caused by 0.5 M NaCl andits recovery when kept in distilled water.

P. Mehta et al. / Plant Physiology and Biochemistry 48 (2010) 16e2018

number of the RCs that can be closed [9]. Efficiency with whicha trapped exciton can move an electron in to the electron transportchain further than QA

� (Jo, which is calculated as ETo/TRo) was alsomeasured. Increase in the value of Vj by 29% and a decrease in thevalue of Jo by 26% (Fig. 2) in 0.5 M NaCl treatment revealed a lossof QA

� reoxidation capacity and an inhibition of electron transport atthe acceptor side of PS II [6] and also beyond QA

�.The most popular parameter of JIP test is the performance index

(PI). The photosynthetic performance index is an indicator ofsample vitality. It is the combined measurement of the amount ofphotosynthetic reaction centers (RC/ABS), the maximal energy fluxwhich reaches to the PS II reaction centers and the electrontransport at the onset of illumination, PI can be calculated as

PIABS ¼ RC=ABS_� FPo=ð1� FPoÞ �JEo=ð1�JEoÞWhere FPo, is the exciton trapped per photon absorbed and JEo, isthe probability that an electron can move further than QA

�. We havedistinguished the effects of high salt stress on individual compo-nents of PI and found that the density of the reaction centersinitially decreased by 12% and at 0.5 M NaCl treatment it wasdecreased by 17% (Table 2). FPo/(1 � FPo) was not affected much upto 0.3 M NaCl treated leaves while its value decreased by 15% and18% in 0.4 M and 0.5 M NaCl. Since Fv/Fm ratio was not decreasedsignificantly with high salt stress it can be said that high salt stressdid not influence the number of quanta absorbed per unit time. Theratio JEo/(1 � JEo) decreased with increase in NaCl concentrationand became 57% of the control (Table 2), suggesting that the effi-ciency of the forward electron transport rates were decreased.These results are in accordance with the data of ET/TR (Fig. 2). Withincrease in NaCl concentration a significant decrease in the value ofperformance index was observed and its value became half of thecontrol in 0.5 M NaCl treatment (Fig. 3).

The derived parameters can also be visualized by means ofdynamic energy pipeline leaf model of the photosynthetic appa-ratus [16,24] (Fig. 4). The leaf model deals with the phenomeno-logical energy fluxes (per cross-section) [6]. Electron transport ina PS II cross-section (ETo/CS) deals with the reoxidation of reducedQA via electron transport over a cross-section of active and inactiveRCs [9]. A decrease in the electron transport per excited cross-section (ETo/CS) due to inactivation of reaction center complexewas observed after the treatment with various NaCl concentrations.Density of the active reaction centers (RC/CS) indicates that thenumber of active RCs in PS II cross-section (indicated as open circlesin Fig. 4) decreased with increase in salt concentration. A decreasein RC/CS ratio reflects that the active RCs are converted into inactiveRCs. ABS/CS is the number of photons absorbed by an excited PS IIcross-section [9]. A decrease in the energy absorbed per excitedcross-section (ABS/CS) was observed indicating that the energyabsorption efficiency of PS II was decreased with increase in saltconcentration.

Table 2Changes in the parameters of performance index inwheat leaves exposed to variousconcentration of NaCl for 1 h in dark. The data shows mean value � S.D. Numbers inparenthesis show the normalized value.

NaCl Conc.(M)

The density of reactioncenters RC/ABS

The efficiency oflight reactionFPo/(1- FPo)

The efficiency ofbiochemical reactionJEo/(1- JEo)

0 0.719 � 0.01 (100) 5.89 � 0.12 (100) 1.12 � 0.03 (100)0.1 0.636 � 0.06 (88) 5.62 � 0.28 (95) 0.84 � 0.01 (75)0.2 0.628 � 0.03 (87) 5.45 � 0.19 (93) 0.84 � 0.06 (75)0.3 0.625 � 0.03 (87) 5.41 � 0.69 (92) 0.74 � 0.08 (66)0.4 0.602 � 0.03 (84) 5.02 � 0.26 (85) 0.73 � 0.06 (64)0.5 0.598 � 0.09 (83) 4.81 � 0.11 (82) 0.64 � 0.12 (57)Recovery 0.715 � 0.001 (99) 5.69 � 0.03 (96) 1.10 � 0.00 (98)

Salt stress involves osmotic as well as ionic components. Inorder to differentiate between osmotic and ionic effects caused byhigh salt, we carried out experiment with 1 M sucrose. Osmotically,1 M sucrose should behave as 0.5 M NaCl. Hyperosmotic conditionscause a decrease in the volume of cytoplasm via efflux of waterthroughwater channel and reversibly inactivate the photosyntheticmachinery [2]. In contrast, after high salt treatment Naþ ions leaksinto the cytoplasm through Kþ/Naþ channel but there is noshrinkage of cytoplasm [2]. Our result shows that the treatment ofwheat leaveswith 1M sucrose caused a decrease in the efficiency oflight reaction FPo/(1 � FPo), rate of biochemical reaction (JEo/(1 � JEo)) and thus Performance index (PI) by 11%, 20% and 30%respectively (data not shown). The effects due to 1 M sucrose wererecovered totallywhen the leaveswere immersed in distilledwater.In comparison, the effects observed in these parameters in 0.5 MNaCl were much higher (Table 2, Fig. 3) and the effects were nottotally reversible. It suggests that the effects observed in oursamples treated with 0.5 M NaCl exhibit both the osmotic and ionicaspects of NaCl. The initial reversible effects may be ascribed to theosmotic aspects while the later, irreversible effects may be becauseof the ionic aspects of NaCl [2]. Our results are in contention withearlier studies in A. thaliana where measurement of delayed fluo-rescence in high salt stressed seeds demonstrated that the rapiddecline in photosynthesis under NaCl stress is reversible andspecific to osmotic effects, where the slow decline is irreversibleand specific to ionic stress [30].

Thus we conclude that high salt stress inhibits the electrontransport rates at the donor (w75%) as well as at the acceptor side(w25%) of PS II. As compared to acceptor side, the donor side of PS IIis significantly affected. Inactive PS II centers increased withincreasing salt concentration. Complete recovery of the damagecaused at the acceptor side was observed, while damage to donorside could be recovered by more than 80%.

3. Materials and methods

3.1. Plant material: wheat (Triticum aestivum)

Lok-1 cultivar of wheat was used for experiments. Wheat seedswere allowed to germinate in a photosynthetically active photonflux density (PPFD) of 300 mmol m�2s�1, 20 �C and a photoperiod of20/4 h light/dark. They were then transferred to petriplates

Fig. 4. Energy pipeline leaf model of phenomenological fluxes (per cross-section, CS) in control in wheat leaves exposed to various concentrations of NaCl for 1 h. The value of eachparameter can be seen in relative changes in width of each arrow. Active RCs are shown as open circles and inactive RCs are closed circles.

P. Mehta et al. / Plant Physiology and Biochemistry 48 (2010) 16e20 19

containing Knop solution. The seeds were daily replenished withdistilled water. The plantlets were grown up to two leaf stages andthen NaCl treatment was given to them. The measurements wereperformed on cut leaves (detached leaves). The leaves were kept indark (30 min) before treatment and NaCl stress was also given indark.

3.2. High salt (NaCl) treatment

High salt stress of different concentrations i.e. 0.1 M, 0.2 M,0.3 M, 0.4 M and 0.5 M NaCl was given to the floating leavesegments for 1 h. The measurements were performed two inchesaway from the tip and base i.e. in the middle portion on the abaxialsurface of the leaves. Thirty measurements were taken for eachtreatment.

3.3. Measurement of fluorescence induction kinetics

The chlorophyll a (Chl a) fluorescence induction kinetics wasmeasured at room temperature using a Plant Efficiency Analyzer(PEA, Hansatech, King's Lynn, Northfolk, England). Excitation lightof 650 nm (peak wavelength) from array of three light e emittingdiodes is focused on the surface of the leaf to provide a homoge-nous illumination. Light intensity reaching the leaf was3000 mmol m�2s�1 which was sufficient to generate maximalfluorescence for all the treatments. The fluorescence signal isreceived by the sensor head during recording and is digitized in thecontrol unit using a fast digital converter. Energy pipeline modelwere prepared using the software Biolyzer HP 3 (the chlorophyllfluorescence analyzing program by Bioenergetics Laboratory,University of Geneva, Switzerland).

Acknowledgements

Financial support for Indo-Russian Project (INT/ILTP/B-6.27) toAJ from DST- RAS is thankfully acknowledged. PM thanks CSIR forthe senior research fellowship [09/301/(0019)09/EMR-I]. We thankProf. Reto Strasser for gifting the Biolyzer programme.

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