PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences

Rapid chlorophyll a fluorescence transient of Lemna gibba leaf as anindication of light and hydroxylamine effect on photosystem II activity

David Dewez, Nadia Ait Ali, Francois Perreault and Radovan Popovic*

Received 19th September 2006, Accepted 22nd January 2007First published as an Advance Article on the web 12th February 2007DOI: 10.1039/b613605b

Rapid chlorophyll fluorescence transient induced by saturating flash (3000 lmol of photons m−2 s−1)was investigated when Lemna gibba had been exposed to light (100 lmol of photons m−2 s−1) causingthe Kautsky effect or in low light intensity unable to trigger PSII photochemistry. Measurements weremade by using, simultaneously, a pulse amplitude modulated fluorometer and plant efficiency analyzersystem, either on non-treated L. gibba leaf or those treated with different concentrations ofhydroxylamine (1–50 mM) causing gradual inhibition of the water splitting system. When any leaf wasexposed to continuous light during the Kautsky effect, a rapid fluorescence transient may reflect currentactivity of photosystem II within the photosystem II complex. Under those conditions, a variation oftransition steps appearing over time was related to a drastic change to the photosystem II functionalproperties. This value indicated that the energy dissipation through non-photochemical pathways wasundergoing extreme change. The change of rapid fluorescence transient, induced under continuouslight, when compared to those obtained under very low light intensity, confirmed the ability ofphotosystem II to be capable to undergo rapid adaptation lasting about two minutes. When the watersplitting system was inhibited and electron donation partially substituted by hydroxylamine, theadaptation ability of photosystem II to different light conditions was lost. In this study, the change ofrapid fluorescence kinetic and transient appearing over time was shown to be a good indication for thechange of the functional properties of photosystem II induced either by light or by hydroxylamine.

Introduction

Illuminated leaf, after a dark adaptation, dissipates variablechlorophyll a fluorescence, a phenomena known as the Kautskyeffect.1,2 Fluorescence induction has been shown to have twodistinct kinetics in time: one as a rapid fluorescence rise closelyrelated to photosystem II (PSII) photochemistry, and regulatedby PSII electron donor and acceptor sides, and another known asthe slow fluorescence decay, lasting for several minutes dependingon the energy balance between PSII and PSI and also on theCO2 fixation process.3–5 When a dark-adapted plant is exposed tocontinuous illumination, such slow decay of variable fluorescenceyield has been shown to be closely related to the oxido-reductionstate of the plastoquinone pool.6 On the other hand, whena plant has been exposed to a short saturating flash (300–700 ms), the maximum fluorescence rise has been found to bestrongly dependent on the reduction state of PSII primary electronacceptor QA and, following on, the electron carriers QB andplastoquinones.7,8 Kinetics of rapid fluorescence induction in vivohave been recognized to indicate the functional state of the watersplitting system, the charge separation at the PSII reaction centerand the reduction state of the PSII electron acceptor side.9 Thefluorescence yield appearing up to 40–50 ls was considered as theF 0 fluorescence level indicating the state when all PSII reactioncenters are open.8,10 The variation of F 0 fluorescence was proposed

Department of Chemistry, Environmental Toxicology Research Center(TOXEN), University of Quebec in Montreal, 2101 Jeanne-Mance, Mon-treal, Province of Quebec, Canada H2X 2J6. E-mail: popovic.radovan@uqam.ca; Fax: (514) 987-4054; Tel: (514) 987-3000, ext. 8467

to be due to the change of the equilibrium transfer between PSIIantennae excitation and the PSII reaction center.11 Fluorescencekinetics from F 0 level to the maximum fluorescence yield FM

have different transitions reflecting the functional properties ofPSII complex. Fluorescence yield at transition J–I was seen tobe dependent on the reduction of QA and QB electron carriers.7,8

It has been also proposed for transition J–I to be dependenton the charge distribution between the water splitting systemand PSII primary QA and secondary QB electron acceptors.12

Such interpretation was supported by theoretical simulations ofdependency between PSII primary photochemistry and relatedelectron transport process.13,14 A fluorescence quenching effectduring I–P transition, seen as D dip, has been shown to indicatea dynamic equilibrium between PSII and PSI activity.15 Whena leaf is exposed to heat treatment, the appearance of anearly K step around 200–300 ls has been attributed to be anindication of the water splitting system inhibition.9,16,17 Accordingto this interpretation, loss of J–I–P transitions, i.e. when thewater splitting system was inhibited, is interpreted to indicate animbalance between PSII electron flow from acceptor and donorsides causing an accumulation of YZ

+ serving as PSII primaryelectron donor. Therefore, it has been proposed for the K stepto indicate the first reduction of QA occurring after the light-induced charge separation between P680 and Pheo.18 However,the quenching effect of the fluorescence yield following the Kstep was proposed to reflect an oxidation of QA

− via subsequentelectron acceptor.19 On the other hand, when the water splittingsystem of PSII in pea leaves was inhibited and then PSII electrontransport ability partially reconstituted by hydroxylamine, the

532 | Photochem. Photobiol. Sci., 2007, 6, 532–538 This journal is © The Royal Society of Chemistry and Owner Societies 2007

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quenching effect following the K step did not appear.17 Presently,the most information on the dependency between PSII activityand fluorescence transient is obtained when leaves have beendark-adapted and exposed to short illumination. However, animportant question should be posed: what is the fluorescencetransient response related to PSII activity when a plant is exposedto continuous different conditions of illumination? If one maycompare such studies with results obtained from a dark-adaptedplant, further understanding of how the rapid fluorescence tran-sient reflects PSII electron transport activity toward to PSI may beprovided. In this study, when Lemna gibba leaves were exposed todifferent conditions of illumination provided by continuous light, asaturating flash was induced periodically in order to investigate thechange of rapid fluorescence transient when PSII photochemistryoccurred. Furthermore, this study provides valuable informationon how the presence of a PSII exogenous electron donor, inthis case hydroxylamine (NH2OH), affects in vivo (intact leaf)the rapid fluorescence transient when the leaf is exposed tocontinuous illumination. Such results will provide also a furtherunderstanding of the dependency between the change of rapidfluorescence transient and PSII electron transport during the firstterm of continuous illumination (Kautsky effect).

Materials and methods

Biological material

The duckweed Lemna gibba was cultivated in an inorganic auto-claved growth medium (pH 6.5).20 Stock culture of L. gibba wasmaintained at 23 ◦C under a continuous illumination of 100 lmolphotons m−2 s−1 provided by cool white fluorescence lamps(Sylvania GRO-LUX F40/GS/WS, Drummondville, Canada).Triple-fronded L. gibba plants in the exponential growth phasewere used for experiments.

Rapid fluorescence induction kinetic and related parameters

The measurements of the rapid fluorescence induction from 10 lsto 1 s was done with a PEA fluorometer (Hansatech Ltd.,King’s Lynn, Norfolk, UK) by using a saturating flash when thephotosynthetic apparatus of L. gibba was undergoing differentillumination conditions (a modulated light of 1 lmol photonm−2 s−1 and an actinic light of 100 lmol photons m−2 s−1) inducedby a PAM fluorometer (Hansatech Ltd., King’s Lynn, Norfolk,UK). The PEA saturating flash was provided by an array of sixlight-emitting diodes giving a maximum emission at 650 nm withan intensity of 3000 lmol photons m−2 s−1. The fluorescence yieldat 50 ls was considered as F 0 value (F 50 ls) and the maximumfluorescence yield attained as FM.8,21 The fluorescence inductioncurves were plotted on a logarithmic time scale allowing for thevisualization of transitions. The determination of the ‘appearingtime’ for all rapid fluorescence transients was done by analyzingthe first derivative of the fluorescence induction curve.22

Evaluation of photosynthetic parameters related to fluorescenceyields or flux ratios within the PSII complex were based onthe theory of energy fluxes in biomembranes:23 The maximumquantum yield of primary photochemistry was determined asφPo = (FM − F 50 ls)/FM; the probability that a trapped excitonmoves an electron into electron transport chain beyond QA

− was

estimated as wo = 1 − [(F 2ms − F 50 ls)/(FM − F 50 ls)], whereF 2ms represents the fluorescence yield at 2 ms. The quantumyield of electron transport was evaluated as φEo = φPowo. Thequantum yield of energy dissipation not used into primaryphotochemistry was determined as φDo = 1 − φPo. The completenon-photochemical quenching of chlorophyll fluorescence yieldfor sample in light adapted state was evaluated as qCN = 1 −(FM(at F2, F3, F4, F5 or F6)/FM(at F1)).24

Hydroxylamine treatment

The stock solution of NH2OH was prepared in 100% ethanol. Thetreatment media was composed of growth medium with additionof NH2OH from a stock solution. The final concentration ofethanol in NH2OH treated and control samples was 0.15%. Atthis concentration of ethanol, no inhibition of photosyntheticprocesses was noticed by comparing control with and withoutethanol (data not shown). Six triple-fronded L. gibba were exposedto 1, 2, 5, 10 and 50 mM of NH2OH under static conditions (sameas growth conditions) during 30 min in darkness. Treatment of L.gibba plant was done in Petri dish containing 50 ml of growthmedium and appropriate NH2OH concentration. The controlsample was considered to have the same content, but with noaddition of NH2OH. Each experiment was conducted in triplicate.

Oxygen evolution measurement

For oxygen evolution measurements, three triple-fronded L. gibbaplants were placed in a gas-phase oxygen electrode chamber(LD2/3, Hansatech Ltd., King’s Lynn, Norfolk, UK). The rateof oxygen evolution was monitored by using an oxygen electrodedisc known as a Clark type polarographic sensor (Hansatech Ltd.,King’s Lynn, Norfolk, UK) at a light intensity of 100 lmol photonsm−2 s−1. The temperature of the oxygen electrode chamber wascontrolled at 23 ◦C. Calibration of the chamber and gas-phasemeasurement of oxygen evolution was done according to standardmethod.5

Fluorescence measurements protocol

For the investigation of rapid fluorescence transient during con-tinuous illumination, PAM and PEA fluorometers as an analyticalapproach were synchronised where all measurements were done onthe same L. gibba sample. An optical set up condition such as thiswas used in order to providing for reproducible and comparablefluorescence yields, by using the same sample in order to avoid thenormalization of fluorescence yields during measurements. Underdifferent light conditions provided by the PAM fluorometer, sixconsecutive PEA saturating flashes distributed, as presented inFig. 1, were used.

After being adapted for 30 min in darkness, L. gibba wasexposed to a weak modulated light unable to trigger primary PSIIphotochemistry for the 2 min before the first PEA saturating flashwas applied (F1). Two min following the first flash the Kautskyeffect was induced by PAM actinic light. Second (F2), third (F3)and fourth (F4) flashes were applied at maximal fluorescenceemission (FP), at exponential fluorescence decay and at steadystate of fluorescence emission of the Kautsky effect, respectively.The length of exposure between flashes was sufficient to avoid theadditional effect of a saturating flash on the change of fluorescence

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Fig. 1 Time of PEA fluorescence induction measurements when L. gibbaleaf was dark adapted and exposed to different light conditions. For moredetails, see Materials and methods.

yield under continuous illumination. Following the fourth flash theactinic light was turn off and after 2 min the fifth saturating flash(F5) was applied. Following 2 min after the fifth flash the far-redlight (735 nm) was on and after 2 min the sixth flash (F6) wasapplied. Such flash distribution should provide information onthe dependency between the change of fluorescence transient andPSII functional and structural properties when dark adapted leafwas exposed to different continuous illuminations.

Data analysis and statistics

Experiments were done five times per treatment using similar testconditions. Standard deviation and means were calculated for each

treatment. Significant differences between controls and treatedsamples were determined by the Tukey-test where p values < 0.05were considered significant.

Results and discussion

The changes in the rapid fluorescence transient when leaf isexposed to different light conditions

In this study, PAM was employed to provide different lightconditions permitting the investigation of the change of PSIIfunctional properties. Under such conditions, the change offluorescence transient was obtained from the same leaf samplepermitting a direct comparison of fluorescence yields (see Fig. 1).Kinetics of rapid fluorescence rise and transient obtained by F1represents a typical fluorescence induction obtained from darkadapted leaf (Fig. 2(A)) having the same steps J, I, D and M(as maximum fluorescence yield, FM) as reported earlier.8 TheD dip appeared to be evident either for F1 induced after darkadaptation or F5 after relaxation under very low energy obtainedby modulated light and for F6 when leaf was exposed to far-red light. However, when leaf was exposed to actinic light withan addition of a saturating flash (F2, F3 and F4), the D dipdid not appear, showing an imbalance of electron transport infavour of PSII compared to PSI. When a leaf was exposed tocontinuous actinic light for a few minutes, the decrease of themaximum yield of chlorophyll fluorescence at F3 and F4 indicatedthat some PSII reaction centers were converted into dissipativeform, not contributing to electron transport.25,26 It is known

Fig. 2 Rapid fluorescence inductions of 1 s when L. gibba leaf was dark adapted and exposed to different light conditions as presented in Fig. 1.Saturated flash was induced by the following order: F1, under modulated light; F2, F3 and F4, under continuous actinic light; F5, under modulated light;F6, under far-red illumination. (A) Control leaf and (B) leaf treated with 50 mM NH2OH. In the left panel, the six fluorescence induction curves arepresented on the same scale for comparison. Right panel, same fluorescence kinetics were separated sufficiently to compare only the form of fluorescencekinetics, therefore the scale order was ignored.

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that the plastoquinone pool contributing to proton flux regulatesenergy transfer from the light harvesting complexes to PSII. Theincrease in proton gradient within the interthylakoid space (lumen)will cause a decrease of energy transfer from light harvestingcomplex to PSII reaction center. This process is considered tobe a regulatory mechanism where excess energy excitation of PSIIlight harvesting complex is dissipated as heat.27–29 Therefore, itmay be considered that a conformational change of PSII, inducedunder continuous light, is likely to be the main reason for thedecrease of maximum fluorescence yield at step M, which indicateda decrease of PSII quantum yield φPo and an increase of quantumyield of energy dissipation φDo (Fig. 2(A), Table 1). We mayassume for the increase of F 0 by 40 and 10% respectively atF2 and F3 to be caused by the same effect. The increase of F 0

value during the initial phase of continuous illumination wasreported earlier and this change was interpreted to be due tothe conformational change at PSII induced by the dramatic shiftof equilibrium between photochemical and non-photochemicalprocesses. Such processes are known to be related to pH gradientformation and feedback by the synthesis of NADPH and ATP.24,30

After 2 min exposure to continuous actinic light, the decreaseof F 0 level reached its steady state as it was shown for F 0 valueat F4 (see Table 1). When F2, F3 and F4 were applied (in thepresence of actinic light), the change of variable fluorescence yieldfrom J to P step showed active PSII electron transport via QB

since the wo value, as indicator of electron transport beyondQA

−, was increased comparing to wo values obtained with F1and F5 (under omitted actinic light). However, the increase ofthe wo value for F6 (under far-red light) may indicate that far-red light also contributes to PSII as well as PSI electron transport.Therefore, under actinic light, quantum yield of electron transportfrom QA toward to plastoquinone pool was increased, as wasindicated by the increase in the φEo value for F2, F3 and F4(Table 1). Under those conditions we may expect that when theleaf is exposed to actinic light, the complete non-photochemicalquenching, indicated by QCN value, should be increased accordingto the proposed model of Rohacek and Bartak.24 Indeed, we foundthat QCN was increased by seven times (Table 1) under actinic light(F3 and F4) as compared, to leaf exposed to low light energy

(F1 and F5 under modulated light and F6 under far-red light).When the ratio between ATP, NADPH synthesis and CO2 fixationis not in equilibrium (during the Kautsky effect), it is knownthat proton concentration in the thylakoid lumen is increasedand consequently this increases the non-photochemical quenching(NPQ) effect.31 The initial increase of proton concentration in thethylakoid lumen will lead to inactivation of the oxygen evolvingcomplex resulting in accumulation of P680+.32,33 Under thoseconditions, the reduction of P680+ by alternative ways may takeplace. It is proposed that P680+ recombination with Pheo− orQA

− results in P680 formation by a non-radiative process causingheat dissipation.33,34 It has been shown that non-photochemicalenergy dissipation decreases the rate of QA reduction and PSIIquantum yield efficiency.35 Therefore, the non-photochemicalquenching reflects a regulatory mechanism involved in dissipationof excess absorbed light energy by thermal dissipation via PSIIlight harvesting antenna complex.36,37 This may explain why theQCN value was negligible or non visible when F1, F5 and F6were applied (Table 1). Since PSII electron transport and energydissipation were undergoing evident change, it was of interest toinvestigate the change of fluorescent transient appearing timewhen flashes were applied. We found that differences in theappearing time were evident when a flash was applied during leafexposure to actinic light, initiating active electron transport towardto PSI. However, when L. gibba leaf was exposed to differentlight conditions, the appearing time for step J was not affected(see Fig. 2(A) and Table 1). This should be expected since theappearance of step J was earlier found to be mostly related tothe reduction state of QA.8,23 On the other hand, it was shown forstep I to be strongly affected by plastoquinone pool reoxidation.18

Therefore, for the non-appearing D dip under F2, F3 and F4 (inthe presence of actinic light), one may interpret it to be causedby an electron transport activity in favour of PSII compared toPSI. Consequently to those conditions, we may assume for D dipto be masked. On the other hand, if a saturating flash was inducedwhen the leaf was exposed to low light energy (modulated light orfar-red light), steps I and D appeared at 30 and 45 ms, respectively.For the delay of step M appearing time (when F2, F3, F4 and F6were applied), this might be interpreted to be due to the oxidation

Table 1 Parametersa based on the rapid rise fluorescence induction measured when L. gibba was adapted 30 min to darkness. For more details, seeMaterials and methods, and also Fig. 3

PEA flashes after dark or light adaptation

Fluorescence parameters F1 F2 F3 F4 F5 F6

F 0 574 820 638 585 579 575FM 2568 2525 2299 2276 2521 2525φPo 0.78 0.68 0.72 0.74 0.77 0.77wo 0.48 0.63 0.68 0.69 0.48 0.63φEo 0.37 0.43 0.49 0.51 0.37 0.49φDo 0.22 0.33 0.28 0.26 0.23 0.23qCN 0.00 0.017 0.105 0.114 0.018 0.017

Appearing time s/msStep J 2 2–3 2–3 2–3 2 2Step I 30 N.d.b N.d.b N.d.b 30 30Step D 45 N.d.b N.d.b N.d.b 45 45Step M 200 250 425 425 215 355

a Data represent the mean of five experiments (p < 0.05) where coefficient of variation do not exceed 5%. b N.d.: the appearing time was not distinguishable.

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of plastoquinone pool under actinic or far-red lights. The effect of50 mM NH2OH on the change of rapid fluorescence rise inducedby six consecutive flashes (Fig. 2(B)) will be discussed in the sectionbelow concerning NH2OH as an inhibitor of water splitting systemsubstituting electron donation to PSII.

Rapid fluorescence transient of L. gibba leaf exposed tohydroxylamine

Exposure of L. gibba to 10 mM of NH2OH during 30 min in thedark was sufficient to induce an inhibition of the water splittingsystem (Fig. 3 and Table 2: oxygen evolution). It is known forNH2OH to liberate manganese from oxygen evolving complex andirreversibly inhibits electron transport between primary electrondonor Yz and P680+.38 When different concentrations of NH2OHfrom 1–50 mM were applied, a gradual decrease of fluorescenceyield induced by saturating flash was observed indicating theinhibition of oxygen evolution, shown by polarographic methodmeasurement (Fig. 3 and Table 2). We found that the decreaseof PSII quantum yield was linearly dependent (R2 = 0.9) to theinhibition of oxygen evolution induced with different treatmentof NH2OH concentrations. At 5 mM of NH2OH, the maximal

Fig. 3 Rapid chlorophyll a fluorescence transient of L. gibba leaf treatedwith 1 to 50 mM NH2OH during 30 min of dark adaptation priory tomeasurement. For more details, see Materials and methods.

fluorescence yield was quenched by half compared to the controlplant and the disappearance of the J step was followed by theappearance of a K step with a s ≈ 300 ls as an indication ofpartial inhibition of water splitting. When higher plants or algaewere exposed to heat treatment, the appearance of K transientwas related to the water splitting system inhibition.17–19 On theother hand, when PSII spinach membranes were exposed toTris treatment, causing Mn depletion of PSII oxygen evolvingcomplex, the appearance of a dip after J step was correlatedto an alteration in oxygen evolution. Under such conditions,the observed decrease in rate of J–P transition was consideredto be useful indicator of partial inhibition of oxygen evolutionactivity. At the same time, the appearance of a dip after Jstep was suggested to be an indication of manganese releasefrom oxygen evolving complex.39 However, this study did notprovide information on K transient probably because of differentmeasuring and experimental conditions. Our results concerningthe change of rapid transition induced by different hydroxylamineconcentration are in agreement with those previous reportedresults. The decrease of fluorescence yield at J step may indicate alower accumulation of QA

− due to the limitation of electron supplyby the water splitting system. However, a rapid fluorescence risewas still found to be present when L. gibba was treated with 10and 50 mM of NH2OH (Fig. 3) since PSII electron transport wassupported by NH2OH serving as an electron donor to P680+.40

From an early time, it has been reported that complete loss ofoxygen evolving capacity did not affect the photooxidation ofNH2OH serving as artificial electron donor to PSII.41 It wasshown for NH2OH to react with O2 evolving center of PSII inthe S1 state delaying the advance of H2O oxidation cycle by twocharge separations. During dark incubation of oxygen evolvingmembranes with high concentrations of NH2OH, Mn was releasedfrom its protein cluster of the oxygen evolving complex.42 Ithas also been shown by EPR study that, when PSII membranesreleased Mn complex by NH2OH, electron transport of PSII wasidentical compared to electron transport in untreated PSII. Therequirement of manganese for the photooxidation of NH2OH byPSII was necessary.43,44 Therefore, it may be interpreted that, whenoxygen evolution in L. gibba was inhibited by 10 and 50 mM

Table 2 The change in the rapid rise fluorescence parametersa when oxygen evolution of L. gibba was inhibited by NH2OH. L. gibba was treated withNH2OH during 30 min in the dark priory to measurements of the first 1 s fluorescence induction

[NH2OH]/mM

Fluorescence parameters 0 1 2 5 10 50

F 0 549 637 591 596 530 324FM 2494 2131 1729 1318 1147 621φPo 0.78 0.70 0.66 0.55 0.54 0.48wo 0.48 0.45 0.44 0.37 0.32 0.32φEo 0.38 0.31 0.29 0.20 0.17 0.15φDo 0.22 0.30 0.34 0.45 0.46 0.52O2 evolution/nmol ml−1 g−1 6.2 4.6 1.6 0.7 0.0 0.0

Appearing time s/msStep K N.d.b N.d.b N.d.b 0.3 0.3 0.3Step J 2 2 2 N.d.b N.d.b N.d.b

Step I 30 30 30 30 30 30Step D 80 85 90 100 150 200Step M 200 380 440 370 370 400

a Data represent the mean of five experiments (p < 0.05) where coefficient of variation do not exceed 5%. b N.d.: the appearing time was not distinguishable.

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of NH2OH, maintenance of rapid fluorescence rise will indicatean electron transport toward to QB, although with low efficiencyas indicated by the decrease of FM value by four times and φPo

was diminished from 0.78 to 0.48. It was reported that NH2OHis able to support electron transfer via QA and QB by an electrondonation to P680+ when water splitting system was inhibited in pealeaf by heat treatment.17 When the concentration of NH2OH wasgradually increased, we showed for wo and φEo values to be alsogradually decreased indicating a diminished electron transportbeyond QA, while an indicator of non-photochemical energydissipation, φDo was increased from 0.22 to 0.52. We interpretthe decrease of the F 0 value induced by high concentration ofNH2OH (50 mM) to be due to a conformational change of PSIIlight harvesting complex. The decrease of electron transport viaPSII induced by NH2OH changed the electron transport balancein favour of PSI, which is shown by the increase of appearing timefor step D from 80 to 200 ms and for step M from 200 to 400 ms(Table 2).

By using the same experimental protocol and conditions as usedfor non-treated L. gibba (Fig. 2(A)), we investigated the change ofrapid fluorescence transient under different light regime when L.gibba was treated with 50 mM of NH2OH (Fig. 2(B)). Under theseconditions, the fluorescence rise induced by F1 showed evident Iand M steps. Fluorescence yield at M level was lower comparedto I step, indicating for NH2OH to be a non-efficient electrondonor to PSII, since the maximum plastoquinone reduction ratewas not still achieved. In this case, lower fluorescence yieldat M level is also likely to indicate the rapid reoxidation ofplastoquinone probably due to active PSI electron transport.However, when F2, F3, F4 (in the presence of actinic light),F5 (under low energy modulated light) and F6 (under far-redlight) were applied, non-significant electron transport was initiated(Fig. 2(B)). We may interpret this discrepancy to be a lack ofmanganese within the PSII water splitting system since manganeseis required to restore PSII electron transport via hydroxylamineas a substitute of water splitting system electron donor to PSII.44

However, further investigation is necessary to understand the eventof non-recovery of the rapid rise of fluorescence dependent toPSII activity. However, we found that, under those conditions,the presence of transient K indicates that the PSII reactioncenters are most likely in the P680+ form, indicating negligiblePSII electron transport toward to plastoquinone.18 We shouldemphasize that the interpretation of our results obtained in vivoare in close agreement with previous studies by using isolated PSIImembranes.

Conclusion

When leaves are exposed to dark adaptation, rapid fluorescencetransient induced by a saturating flash is representative of PSIIfunctional properties. When PSII is going through the Kautskyeffect, the rapid fluorescence transient indicates the change ofthe equilibrium transfer between PSII antennae excitation andthe PSII reaction center, and also indicates a drastic variationof energy dissipation when PSII is transferred from active todissipative and inactive forms. At the same time, parameter wo,evaluated on the fluorescence yield above step J, indicates anincrease of electron transport beyond QA electron carriers. Valueof QCN, as a relative indication of energy dissipation through

non-photochemical pathways, was ongoing through an extremechange by showing an increase more than seven times. Such drasticchange of PSII functional properties was reflected by the variationof appearing time for transitions I–D–M. The activity of PSIprolonged the appearing time of fluorescence transients causingtransition I–D none distinguishable. Under those conditions, themaximal fluorescence yield indicated by step M, was delayed bymore than two times.

The change of rapid fluorescence transients induced undercontinuous light, when compared to those obtained under verylow light intensity, showed the ability of PSII for rapid adaptationduring 2 min of transition. When water splitting system wasinhibited and electron donation was partially substituted byNH2OH, the adaptation ability of PSII to different light conditionswas lost. On the other hand, the fluorescence yield at step Mwas decreased four-fold, indicating a poor electron donation ofNH2OH to PSII. Consequently, the PSII quantum yield wastwice lower and the non-photochemical dissipation increased bya similar amount. The delay of electron transport via PSII wasevident since the appearance of steps D and M was increased bydouble compared to control. The changes of fluorescence transientkinetics and their time of appearance in L. gibba leaf either treatedor not with NH2OH reflect the very rapid change of functionalproperties of PSII exposed to different light conditions.

List of abbreviations

ADP: Adenosine diphosphate; ATP: adenosine triphosphate;NADPH: nicotinamide adenine dinucleotide phosphate, reducedform; NH2OH: hydroxylamine; PAM: pulse amplitude modulated;PEA: plant efficiency analyzer; PSII: photosystem II; QA: quinoneA as the primary electron carrier within PSII; QB: quinone B asthe secondary electron carrier within PSII; PQ: plastoquinone.

Acknowledgements

This research was supported by the Natural Science and Engi-neering Research Council of Canada (NSERC) through GrantGP0093404 awarded to R. Popovic.

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