Polyphasic fluorescence Lazar_2006a.pdf

CSIRO PUBLISHINGwww.publish.csiro.au/journals/fpb Functional Plant Biology, 2006, 33, 930

Review:

The polyphasic chlorophyll a uorescence rise measured under highintensity of exciting light

Dusan Lazar

Palacky University, Faculty of Science, Department of Experimental Physics, Laboratory of Biophysics,tr. Svobody 26, 771 46 Olomouc, Czech Republic. Email: [email protected]

Abstract. Chlorophyll a uorescence rise caused by illumination of photosynthetic samples by high intensity ofexciting light, the OJIP (OI1I2P) transient, is reviewed here. First, basic information about chlorophyll auorescence is given, followed by a description of instrumental set-ups, nomenclature of the transient, and samplesused for themeasurements. The reviewmainly focuses on the explanation of particular steps of the transient based onexperimental and theoretical results, published since a last review on chlorophyll a uorescence induction [Lazar D(1999) Biochimica et Biophysica Acta 1412, 128]. In addition to old concepts (e.g. changes in redox states ofelectron acceptors of photosystem II (PSII), effect of the donor side of PSII, uorescence quenching by oxidisedplastoquinone pool), new approaches (e.g. electric voltage across thylakoidmembranes, electron transport throughthe inactive branch in PSII, recombinations between PSII electron acceptors and donors, electron transport reactionsafter PSII, light gradient within the sample) are reviewed. The K-step, usually detected after a high-temperaturestress, and other steps appearing in the transient (the H and G steps) are also discussed. Finally, some applicationsof the transient are also mentioned.

Keywords: uorescence induction, G step, H step, K step, model, OJIP (OI1I2P) transient, theory.

I Chlorophyll a uorescence and the F0 and FM levels

The quantum yield of chlorophyll (Chl) a uorescencein a solution (where excitation energy transfer andphotochemistry do not occur) is 2035% (Latimer et al.1956; Weber and Teale 1957) and this uorescence has alifetime of620 ns (Muller et al. 1969;Avarmaa et al. 1977;Pfarrherr et al. 1991; Brody 2002). In contrast, the quantumyield of Chl a uorescence from the photosynthetic apparatusis only 28% (from open to closed reaction centres ofphotosystem II, RCII; Latimer et al. 1956; Trissl et al. 1993)with an average lifetime of 300 ps (for open RCII; Keuperand Sauer 1989; Marder and Raskin 1993; Briantais et al.1996; Gilmore et al. 1996) and 1.6 ns (for closed RCII;Keuper and Sauer 1989; Marder and Raskin 1993; Brody2002). Under physiological conditions, uorescence signalduring uorescence rise (FLR) is assumed to originatemainly from photosystem II (PSII) [reviewed by Govindjeeet al. (1986); Krause and Weis (1991); Dau (1994)].Contribution of photosystem I (PSI) to the overalluorescence signal during FLR at room temperature is1520% (Strasser and Butler 1977; Wong and Govindjee1979; Stahl et al. 1989; Roelofs et al. 1992; Trissl et al. 1993)

Abbreviations used: See Table 1 for a complete list of abbreviations used.

and its uorescence is assumed to have a constant level;uorescence from PSI contributes only to minimaluorescence, F0, [however, see also Ikegami (1976) andByrdin et al. (2000); section II.4.2.10]. But at emissionwavelengths greater than 700 nm the contribution of the PSIuorescence to F0 can be up to 3055% (Pfundel 1998;Gilmore et al. 2000; Franck et al. 2002). Nevertheless, in arst approximation, which is well accepted in photosynthesisresearch, FLR is understood to originate from PSII.

Minimal uorescence, F0, is dened as the uorescencewhen all RCIIs are open, i.e. when the rst quinone electronacceptor of PSII, QA, is oxidised [see also Vredenberg(2000); Strasser and Stirbet (2001), and section II.4.2.3 foralternative approaches]. As Butler (1977, 1978) postulatedthat every uorescence signal comes from Chls of lightharvesting antennae (LHA), F0 is the uorescence signalcoming from excited Chls of LHA before the excitationsreach RCII (Mathis and Paillotin 1981). Owens (1996)suggested that F0 level is a consequence of the transferequilibrium: an equilibrium between the formation of theexcited states among all the light harvesting pigments andP680 and utilisation of the excited states for reversible

CSIRO 2006 10.1071/FP05095 1445-4408/06/010009

10 Functional Plant Biology D. Lazar

Table 1. List of abbreviations used in the text

Abbreviation Denition

A515, A520 Absorbance signal measured at 515 or 520 nm, respectivelyADRY Accelerator of the deactivation reactions of the enzyme Y in OECChl ChlorophyllCP43, CP47 A 43-kDa, 47-kDa Chl containing inner LHAs of PSIIcyt CytochromeDBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-bezoquinoneDCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea; diuronDF Delayed uorescenceF0 Minimal Chl a uorescenceFI Chl a uorescence induction (both the FLR and uorescence decay)FI Chl a uorescence signal at the I step in FLR measured under high intensity of exciting lightFJ Chl a uorescence signal at the J step in FLR measured under high intensity of exciting lightFK Chl a uorescence signal at the K step in FLR measured under high intensity of exciting lightFLR Chl a uorescence riseFM Maximal Chl a uorescenceFNR Ferredoxin-NADP+ oxidoreductaseFP Fluorescence parameterFV Variable uorescence (=FM F0)FV /FM Maximal quantum yield of PSII photochemistryHQ 1,4-benzenediol; hydroquinoneLED Light-emitting diodeLHA Chl containing light harvesting antenna(e)LHCII Chl containing light harvesting complex of PSIIMCA Metabolic control analysisMV MethylviologenNADP Nicotinamide adenine dinucleotide phosphateNH2OH HydroxylamineO, K, J ( I1 i), I ( I2), H, G, P Particular steps in FLR measured under high intensity of exciting lightOEC Oxygen-evolving complexP680 Primary electron donor in PSIIA3P680* Triplet excited state of P680P700 Primary electron donor in PSIA

PAM Pulse amplitude modulationPBQ 1,4-benzenedione; p-benzoquinonePC PlastocyaninPEA Plant efciency analyserPheo Primary electron acceptor in PSII, pheophytin, localised in D1 proteinPheo2 Pheophytin localised in D2 proteinPQ PlastoquinonePSI Photosystem IPSII Photosystem IIQ2 A component of PSIIQA The rst quinone electron acceptor in PSIIQB The second quinone electron acceptor in PSIIRCII Reaction centre of PSIIRRP Reversible radical pairRubisco Ribulose-1,5-bisphosphate carboxylase / oxygenaseSi (i= 0, 1, 2, 3) S-states of OECT820 Transmission signal measured at 820 nmTMPD N,N,N,N-tetramethyl-p-phenylenediamineTris Tris(hydroxymethyl)aminomethaneTSTM Three-state trapping modelVI Relative variable uorescence at the I stepVJ Relative variable uorescence at the J stepYZ Tyrosine 161 of D1 protein

AWhat is denoted as P680 and P700 is generally thought to be the primary electron donors in PSII and PSI, respectively, but recent resultsindicate that the primary electron donors in both PSII and PSI are probably accessory chlorophylls and not P680 and P700, respectively.For more details see van Mourik (2004), Novoderezhkin et al. (2005), and Groot et al. (2005) for PSII and Muller et al. (2003)and Holzwarth et al. (2005a, b) for PSI.

The polyphasic chlorophyll a uorescence rise Functional Plant Biology 11

primary photochemistry (i.e. charge separation and chargerecombination; Laible et al. 1994; see also Lazar 2003).Maximal uorescence FM is dened as the uorescencewhen all the RCIIs are closed, i.e. when all QA isreduced (Vredenberg 2000; Strasser and Stirbet 2001, andsection II.4.2.3 for alternative approaches). Even if chargeseparation (i.e. formation of P680+Pheo from P680*Pheo)can occur in the closed RSII, its rate constant is 36 timessmaller [see Lazar (2003) for a summary of the rate constantsaccording to different authors] than in the case of open RCII.Further, in the case of the closed RCII, the rate constant ofthe charge recombination (i.e. formation of P680*Pheo fromP680+Pheo) is higher than in the case of open RCII. Thechanges in the rate constants of the charge separation andrecombination lead to increased accumulation (lifetime) ofthe excited states in the closed RCII when compared withopen RCII; this leads to higher uorescence emission in theclosed RCII than in the open RCII.

II The polyphasic chlorophyll a uorescence rise:OJIP (OI1I2P) transient

The OJIP (OI1I2P) FLR is reviewed in this section.A summary of the instrumental set-ups for the measurementof the FLR is given rst, followed by a description of thenomenclature used for the FLR and of the samples usedfor the measurements. Finally, particular steps of the FLRare explained in detail. Additional information can be foundin related reviews (Govindjee et al. 1986; Krause and Weis1991; Dau 1994; Govindjee 1995; Joshi and Mohanty 1995;Mohammed et al. 1995; Schreiber et al. 1995; Lazar 1999;Rohacek and Bartak 1999; Samson et al. 1999; Chaerle andVan Der Straeten 2000, 2001; Maxwell and Johnson 2000;Rohacek 2002; Sayed 2003; Baker and Rosenqvist 2004;Oxborough 2004a). Readers are also referred to a recentlypublished book Chlorophyll a uorescence: a signatureof photosynthesis (Papageorgiou and Govindjee 2004), inwhich the state of the art, related to the FLR, is reviewedextensively (e.g. Govindjee 2004; Schreiber 2004; Strasseret al. 2004; Vredenberg 2004).

II.1 Instrumental set-ups

Three conditions must be guaranteed by an experimentalset-up to measure and distinguish the polyphasicFLR: a high (saturating) intensity of exciting light(300010000 mol photonsm2 s1), a fast beginningof illumination of a sample, and a fast time resolution ofdetected uorescence signal. The rst condition is realisedby high intensity light sources (halogen and xenon lamps,light emitting diodes, lasers) and the second conditionby using a fast enough shutter or shutter-less set-up.A fast enough analogue / digital converter must be usedto ensure a satisfactory time resolution of the detecteduorescence signal.

The rst results on the measurements of the FLR underhigh intensity of excited light were published by Morin(1964) and Delosme (1967). Both these investigators usedlaboratory made set-up where a fast enough shutter wasrealised by using a gun whose 22 long rie bullet blew apartametal plate thatwas between the sample and the illuminatingxenon lamp, serving as a light source. More user-friendlyinstruments and commercial uorometers have recentlyreplaced such a dangerous set-up. Ruth (1990, 1991)used a laboratory-made uorometer with a heliumneonlaser as a light source and acousto-optic modulator (Braggcell) as a shutter, ensuring a fast enough beginning of theillumination, to measure the FLR. However, the laser used byRuth did not provide a sufciently high intensity of excitinglight. Another laboratory-made uorometer was used for themeasurement of the FLR by Pospsil and Dau (2000, 2002).In this uorometer, light emitting diodes (LED) were usedas a light source, which also ensures a fast beginning ofthe illumination.

Ulrich Schreiber and co-workers (Schreiber 1986;Schreiber et al. 1986; Schreiber and Neubauer 1987;Neubauer and Schreiber 1987) published the rstmeasurements of the FLR under high intensity of excitinglight, using a commercial uorometer (Pulse AmplitudeModulation, PAM 101103; Walz, Germany) where ahalogen lamp serves as a light source and a mechanicalshutter (full shutter opening within 800 s) ensures a fastbeginning of the illumination. In 1991 and 1992 the rstFLR measurements with another commercial uorometer,the PEA (Plant Efciency Analyser; Hansatech, England)uorometer were published by Strasser and Govindjee(1991, 1992). There is no shutter in the PEA uorometerand a fast enough beginning of the illumination and theillumination itself is achieved by LEDs. The same set-up forthe measurement of the FLR is also used by the Double-Modulation Fluorometer (Photon Systems Instruments,Brno, Czech Republic). This uorometer even enablesmeasurements of the FLR with time resolution of 100 nsduring extremely strong (200 000 mol photonsm2 s1)short (up to 50100 s) saturating light ash, the so-called ash uorescence induction (Nedbal et al. 1999;Koblzek et al. 2001).

The uorometers most often used for the measurementsof the FLR, the PAM and PEA uorometers, however,use different approaches for the detection of uorescencesignal. In the PEA uorometer, continuous illumination of asample is used to induce photosynthetic electron transport(i.e. as actinic light), but also serves to assess the stateof a sample via its uorescence signal (i.e. as measuringlight). In the PAM 101103 uorometer, pulse-modulatedmeasuring light and continuous actinic light are separated.Hence, photosynthetic electron transport (and closure ofRCIIs) is induced by continuous illumination, but the stateof the sample is probed by short (1 s) measuring light


ashes placed 10 s apart (during the FLR measurement).By measuring the uorescence signal during each individualmeasuring ash as well as a few microseconds thereafterand then subtracting the two signals, the uorescenceexcited by the actinic light is eliminated. Therefore, asintensity of themeasuring light is constant, PAMuorometersmeasure uorescence yield, irrespective of the uorescenceintensity excited by actinic illumination. Fluorescence yieldmay vary by about a factor of ve (between all RCIIsbeing open or closed), whereas there is no limit foruorescence intensity, as it is proportional to intensity ofexcitation light.

The different approaches used by PAM and PEAuorometers for the measurements are reected in theway the F0 level is measured or calculated. In the PEAuorometer, the F0 level is not measured but calculatedfrom experimental data; FLR data points in the range of80120 s are tted by a linear function and then extrapolatedto time zero, whose uorescence signal is considered as theF0 level. The F0 level determined in this way is within10% of the uorescence signal measured at 4050 s ofthe FLR (e.g. Strasser et al. 1995; Susila et al. 2004).Therefore, the FLRs measured by PEA uorometer areusually presented starting from 4050 s (the O level).Using different Chl concentrations (acetone extracts, whereexcitation energy transfer and photochemistry do not occur)and sample thicknesses, Susila et al. (2004) pointed out thatthe uorescence signal detected by the PEA uorometer, isdistorted by the uorometer up to 50 s but starting fromthis time to the end of the measurement period, the detected

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Fig. 1. FLRs measured with dark-adapted barley leaves (part A; curve a, no treatment; curve b, leaf incubated in 32mM DCMU solution for5 h; data from Lazar et al. 1998) and tobacco leaf (part B, no treatment; data from Lazar 1999) by PEA and PAM uorometer, respectively, underhigh intensity of exciting light [3400 mol photonsm2 s1 of red light (A) and 9000 mol photonsm2 s1 of white light (B)]. The same curveas in main part B is presented on a logarithmic time-axis in the inset of part B. The O, J (I1), I (I2), and P (M) steps are labelled.

uorescence signal is constant. In contrast, the PAM101103uorometer measures the F0 level by the measuring ashesbefore the onset of actinic illumination. Although the energyof the individual measuring ashes is very high, their shortduration and repetition with enough time between two ashes(625 s for the F0 measurement) ensure that integral energyof measuring ashes is very small and does not cause anysignicant photochemical electron transport in the sample.However, it is necessary to establish that true F0 is beingmeasured by checking it at decreased light intensity of themeasuring ashes.

II.2 Nomenclature

All steps in the FLR can be clearly revealed only whenlogarithmic presentation of time axis is used. In the followingtext, the term step is used for a hump or a wave or a peakthat appears visually at a given time in the FLR. A transientor a phase is used in the following text as a part of theFLR that starts at a step and is completed in another step.The J (I1) and I (I2) steps usually appear at 23ms and3050ms, respectively (see Fig. 1A, curve a). The O stepstands for F0 and the P step represents FM usually reachedat 200500ms. The J and I notation of particular stepsin the FLR is based on the original work by Strasser andGovindjee (1991, 1992). However, the two steps appearingbetween the F0 and FM levels were denoted earlier as I1 andI2 byU. Schreiber and co-workers (Schreiber 1986; Schreiberet al. 1986; Schreiber and Neubauer 1987; Neubauer andSchreiber 1987; see Fig. 1B). Strasser et al. (1995) establishedequivalencies between the J and I1 steps and the I and I2 steps


since (i) the steps appear at the same time in theFLR (comparecurve a in Fig. 1A and the inset in Fig. 1B) and (ii) the relativeheights of the J and I1 steps on the one hand and of the Iand I2 steps on the other hand have the same light intensitydependencies. Delosme (1967) denoted by the small letter ithe only step he measured between F0 and FM at 2ms; itseems to be equivalent to the J ( I1) step. As the FLR isnow often measured by PEA, the OJIP notation is usedin the text.

As the initial OJ transient reects primary photochemicalreactions (see below), it was called the photochemical phaseof the FLR (Delosme 1967; Neubauer and Schreiber 1987;Strasser et al. 1995). The main feature of this phase is thatthe initial slope and relative height of the phase stronglydepends on the intensity of the exciting light (see e.g. Strasseret al. 1995; Tomek et al. 2001). In contrast, subsequentJIP transient cannot be speeded up by further increasein the intensity of exciting light (see e.g. Strasser et al.1995; Tomek et al. 2001) and it was called the thermalphase of the FLR (Delosme 1967; Neubauer and Schreiber1987) because it depends on the temperature of measurement(within physiological range).

Under certain conditions, additional steps, the K, G, andH steps, can appear in the FLR. These steps are described indetail later under separate sections.

II.3 Samples

Chlorophyll a uorescence rise can be measured withany photosynthetic organism, but it has been measuredmostly with whole leaves, mosses, algae, cyanobacteria,chloroplasts, and thylakoid membranes. FLRs from thesesamples measured at room temperature are usuallycharacterised by typical OJIP transients, as mentionedabove (but see also section III.2).While a detailed explanationof theOJIP transientmeasuredwith these usual samplesis given in the following section, a short description ofmeasurements of the FLR with unusual samples and theresults are briey summarised now.

When the FLR is measured with PSII membranes, theI step is not present (Pospsil and Dau 2000, 2002; Herediaand De Las Rivas 2003). Exploration of the FLR measuredwith PSII membranes led to new suggestions for the originof the particular steps in the FLR (see sections II.4.2.2and II.4.2.12). Fluorescence quenching by the oxidisedplastoquinone (PQ) pool (see section II.4.2.4 for moredetails) is more pronounced in PSII membranes than inmore structurally organised samples (thylakoid membranes,chloroplasts, leaves) (Kurreck and Renger 1998; Kurrecket al. 2000; Pospsil and Dau 2000, 2002). J. Kurreck andco-workers (Kurreck and Renger 1998; Kurreck et al. 2000)explained this nding by greater afnity of PQ moleculesfor LHA to form a quenching complex in the case ofPSII membranes than in the case of the more structurallyorganised samples.

The FLR was also measured with aggregates of lightharvesting complex of PSII (LHCII) and with trimeric PSIto explore the generation of uorescence quenchers from thetriplet states of chlorophyll (Barzda et al. 2000) and quantumyield of uorescence in PSI with initially reduced or oxidisedprimary electron donor in PSI, P700 (Byrdin et al. 2000; seealso section II.4.2.10), respectively.

II.4 Explanation

In this section possible explanations of the particular steps intheOJIPFLR, suggested in the literature, are summarised,separately for the photochemical and thermal phases of theFLR. It seems that each of these explanations separatelycannot explain particular steps of the FLR and probably allof the processes described below occur simultaneously andaffect the steps of the FLR to some extent.

II.4.1 The photochemical phase

II.4.1.1 Accumulation of reduced QA with QBbeing oxidised

Duysens and Sweers (1963) had already proposedthe existence of a quencher Q that was removed asChl a uorescence rose. According to the suggestion byDelosme (1967), the photochemical phase (OJ transient)corresponds to the destruction of a quencher Q which is theprimary reactant of the photoreaction II in photosynthesis.Using present notations, Q should be QA. In agreementwith this suggestion, a working hypothesis was introducedby Strasser and Govindjee (1992) proposing that the J stepreects light-driven accumulation of QA with QB, thesecond quinone electron acceptor in PSII, being oxidised,that is, J QAQB state.

A main experimental proof that the OJ FLR representsaccumulation of only reduced QA comes from measurementswith 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), aninhibitor of electron transport between QA and PQ moleculesof the PQ pool [Oettmeier and Soll 1983; Trebst and Draber1986; Trebst 1987; Shigematsu et al. 1989; DCMU inhibitsthis reaction by binding to the QB pocket of PSII (Velthuys1981)]. When a sample is treated with DCMU, the FLRmeasured at high intensity of exciting light is characterisedby a steep uorescence increase, reachingmaximal saturationlevel at approximately the position of the J step measured inthe sample without DCMU (Strasser et al. 1995; Lazar et al.1998, 2001; Tomek et al. 2001; compare curves a and b inFig. 1A). Theoretical simulations of the FLR either with orwithoutDCMUalso conrmed the suggestion that it ismainlyQA that accumulates in the reduced state in the position of theJ step (Stirbet and Strasser 1995a, b, 2001; Stirbet et al. 1995,1998; Lazar et al. 1997b, 2005b; Lazar and Pospsil 1999;Strasser and Stirbet 2001; Tomek et al. 2001; Lebedeva et al.2002; Lazar 2003; Zhu et al. 2005). It is important to realisethat when DCMU is not present, the QA may not be fully


reduced in the J step of the FLR because electrons from QAare continuously transferred to QB and further on towardsPSI. Therefore, when DCMU is not present, the J step mayrepresent only a partial and not full accumulation of QA.

II.4.1.2 The donor side of PSII

Using different treatments (Tris, high temperature, ADRY-reagents, NH2OH, pH) that inhibit donor side of PSII, thephotochemical phase of the FLR was shown to be partiallycontrolled by the donor side of PSII (see e.g. Schreiber andNeubauer 1987; Bukhov et al. 2004). The conclusion thatthe donor side of PSII can affect the photochemical phaseof the FLR was also made on the basis of measurement ofthe FLR under low intensity of exciting light (Hsu 1993;Lavergne and Leci 1993) and using the ash uorescenceinductionmeasurements (Koblzek et al. 2001). However, theeffect of the donor side of PSII on photochemical phase ofthe FLR found by Hsu (1993) and Lavergne and Leci (1993)was determined on the basis of assumption of a differentuorescence quenching in different redox states (the S-states)of oxygen evolving complex (OEC) and is therefore related tothe donor side uorescence quenching and not to the ratesof the S-state transitions of OEC as such.

Also theoretical simulations of the FLR revealed an effectof the donor side on the photochemical phase of the FLR(Stirbet et al. 1998; Lazar and Pospsil 1999; Lazar 2003).As inhibition of the donor side of PSII results in appearanceof the K step in the FLR, the effect of the donor side of PSIIon the FLR is discussed more extensively in section III.1.

II.4.1.3 Excitation energy transfer among PSIIs

Theoretical calculations and simulations of the FLRindicated that excitation energy transfer among PSIIs alsoaffect the photochemical phase of the FLR (Lavergne andLeci 1993; Stirbet et al. 1998; Lazar 2003; Zhu et al. 2005).

II.4.1.4 Electric voltage across thylakoid membranes

Using simultaneous recordings of the FLRand absorbancechanges at 515 nm (A515, reecting electric voltage acrossthylakoid membranes), Schreiber and Neubauer (1990)found that A515 has a maximum (there is a maximum inlight-induced electric voltage across thylakoid membranes)approximately at the position of the J step. Schreiber andNeubauer (1990) suggested that the electric voltage favoursformation of P680 triplet excited state (3P680*). Formationof 3P680* as such may lead to lower uorescence emissionbut energy of 3P680* can be further quenched either in RCIIor in LHA; this may lead to uorescence quenching at theJ step. Similarly, using different frequencies of sinusoidalmodulation of light source intensity, Dau et al. (1991)found that a time constant of uorescence signal equals atime constant of A520, showing that the uorescence signalincreases when electric voltage across thylakoid membrane

increases. Subsequent analysis of the measured data revealedthat formation of electric voltage across thylakoid membraneis responsible for 7% of the photochemical phase of FLR(Dau et al. 1991).

II.4.1.5 Electron transport through the inactivebranch in PSII

Schreiber (2002, 2004) has discussed a hypotheticalmechanism to be responsible for the uorescence quenchingat the J step. He proposed that in PSII with QA, an electrontransport through the inactive branch in PSII can occur,that is, an electron may be transferred from P680 to Pheolocalised in D2 protein of PSII (Pheo2) followed by anelectron transport from the reduced Pheo2 to QB or QB.The proposed P680Pheo2QB(QB) electron transportis assumed to be highly inefcient due to high yield ofback non-radiative recombination. The proposed electrontransport as such and the assumed high yield of back non-radiative recombination cause a uorescence quenching atthe J step, which results in uorescence at the J step beingsmaller than at the P step of the FLR. Although it has alreadybeen shown that QB can be reduced via the inactive branch inmutants of anoxygenic photosynthetic bacteria Rhodobactercapsulatus and Rhodobacter sphaeroides (Kirmaier et al.2003; Breton et al. 2004; Wakeham et al. 2004; Frolov et al.2005; Paddock et al. 2005), a consideration of the inactivebranch to affect the J step of the FLR as described above wasonly hypothetical.

As QB is involved in the mechanism mentioned above,it was also called the QB-quenching mechanism (Schreiber2002). However, further mechanisms in whichQB is involvedcan be also called QB-quenching mechanisms as Schreiber(2004) has discussed in more details. According to this view,the results of O Prasil, ZS Kolber, and PG Falkowski, asdescribed in section II.4.2.1, which, however, have not beenaccepted by others, may be considered as a manifestation ofa QB-quenching mechanism. Further research is necessary totest this hypothesis.

II.4.1.6 Recombination between PSII electronacceptors and donors

Goltsev and co-workers (Goltsev and Yordanov 1997;Goltsev et al. 2003, 2005; Zaharieva and Goltsev 2003)used a phosphoroscope method and theoretical simulationsof delayed uorescence (DF) and the FLR to study effects ofrecombinations between PSII electron acceptors and donorsleading to DF detected in the range of 350 s5.5ms (forreview on DF see Tyystjarvi and Vass 2004). The authorsfound that the time at which the rst peak (denoted as I1)in the DF intensity appears, corresponds with the time ofa maximal rate of the FLR during the OI phase measuredunder low intensity of exciting light (Goltsev and Yordanov1997; Goltsev et al. 2003; Zaharieva and Goltsev 2003). Theorigin of the I1 in the DF is probably in recombination of QA


with YZ+ when QB is singly reduced (Goltsev and Yordanov1997; Zaharieva and Goltsev 2003; Goltsev et al. 2005) andthe lifetime of YZ+QAQB is determined by the rate ofelectron transport from QA to QB (Goltsev et al. 2005).

II.4.1.7 Effects of other processes

Lazar (2003)made a detailed analysis of theFLRbymeansof theoretical simulations of the FLR. For these simulations,Lazar used amodel that was obtained by combination of threeexisting models for the description of energy and electrontransport steps in PSII: (i) the reversible radical pair (RRP)model (Breton 1983; van Grondelle 1985; Schatz et al. 1988;Leibl et al. 1989; Roelofs et al. 1992) describing energyutilisation leading to primary photochemistry, i.e. chargeseparation, recombination, and stabilisation (see Dau 1994for a review); (ii) themodel ofKok et al. (1970) describing thefunction of the donor side of PSII, i.e. that the Mn cluster ofOEC undergoes a cycle through its four S-states; and (iii) thetwo-electron gate model (Bouges-Bocquet 1973; Velthuysand Amesz 1974; Crofts and Wraight 1983) describing thefunction of the acceptor side of PSII, i.e. that QB, unlike QA,is a two-electron acceptor. Therefore, this model (almost)completely included reactions on both the donor and theacceptor side of PSII.

By changing values of particular rate constants or initialconcentrations of states in the model, theoretical simulationsby Lazar (2003) indicated that the photochemical phase ofthe FLR is also affected by non-photochemical uorescencequenching by P680+ and by oxidised PQ pool, by chargerecombination between P680+ and QA, by initial stateof OEC, and by rate of electron transport from YZ toP680+ (for further details, see Lazar 2003). Theoreticalsimulations by other authors also produced similar results(Stirbet et al. 1998; Zhu et al. 2005).

II.4.1.8 The QB-reducing / non-reducing heterogeneityof PSII

All previous explanations were made assuming PSIIto be homogeneous. However, it is well documented inthe literature that heterogeneity of PSII exists (reviewedby Lavergne and Briantais 1996). One type of the PSIIheterogeneity is with respect to the ability of PSII to reduceQB. In this way PSIIs can be divided into two parts:(i) PSIIs that can reduce QB, the QB-reducing PSII, and(ii) PSIIs that cannot reduce QB, the QB-non-reducing PSII(Graan and Ort 1984, 1986; Whitmarsh and Ort 1984; Melis1985; McCauley and Melis 1987; Chylla and Whitmarsh1989; Lavergne and Leci 1993). With respect to this typeof PSII heterogeneity, the photochemical phase was foundto reect accumulation of QA of the QB-reducing but alsoof the QB-non-reducing PSIIs (Hsu 1992a, b; Lazar et al.1997b; Strasser and Stirbet 1997; Tomek et al. 2001, 2003;Lazar 2003).

II.4.2 The thermal phase

II.4.2.1 Accumulation of reduced QB in additionto reduced QA

According to the suggestion by Delosme (1967),the thermal phase (JIP transient) corresponds to thedestruction of a quencher R. Using present notations,R should be PQ, either bound to PSII as QB or free inthylakoid membranes. In agreement with this suggestion,as in the case of the photochemical phase of the FLR (seesection II.4.1.1), Strasser and Govindjee (1992) suggested aworking hypothesis proposing that the I and P steps reectlight-driven accumulation of QB and QB2, respectively, inaddition to the accumulation of QA, that is, I QAQBstate and PQAQB2 state.

Although an accumulation of particular redox forms ofPSII at the time of the appearance of the I and P stepsin the FLR was conrmed only by theoretical simulations(Stirbet and Strasser 1995a, b, 2001; Stirbet et al. 1995, 1998;Lazar et al. 1997b; Strasser and Stirbet 2001; Tomek et al.2001; Lebedeva et al. 2002; Lazar 2003; Zhu et al. 2005),assignment of the I and P steps to the redox forms seemsto be reasonable, at least as the rst approximation. Even ifseveral different models were used for the simulations, theirresults are, in general, the same as for the accumulation ofparticular redox forms.

In agreement with the above-mentioned interpretation ofthe FLR are the unpublished results of O Prasil, ZS Kolber,and PG Falkowski, obtained by site-directed mutants of thegreen alga Chlamydomonas reinhardtii, with substitution ofAla251 in the QB-pocket of the D1 protein, which affectsthe afnity of the PQ molecules for D1. These unpublisheddata may suggest that the thermal phase of the FLR is relatedto occupancy of the QB-site by the PQ molecule and by acapacity of QB to deoxidise QA (see also Samson et al.1999). In agreement with this hypothesis, Yaakoubd et al.(2002) found, by measuring FM induced by a single turnoverash or by continuous illumination with samples treated withDCMU and exogenous PQs, that oxidised QB is responsiblefor 56% of the thermal phase of the FLR.

II.4.2.2 Protonation of QB2

Consistent with the explanation given in the previoussection are the results of Heredia and De Las Rivas(2003), using PSII membranes treated with 1,4-benzenediol (hydroquinone, HQ) and with 1,4-benzenedione(p-benzoquinone, PBQ). Heredia and De Las Rivas foundthat the addition of reduced and protonated quinones (HQ,PBQH2) resulted in uorescence quenching of the verylast part of the thermal phase of the FLR. As they usedPSII membranes for the measurements, which do notshow the I step in FLR (see section II.3), they labelled theuorescence level that is unquenched by the quinones as theH level. However, this H uorescence level seems to be


different from the H step (peak) described in section III.2and from a hump H described in section II.4.2.12. Herediaand De Las Rivas interpreted the JH transient to representthe reduction of QB to both QB and QB2 and theHP transient to represent the protonation of QB2. Aneffect of protonation of QB2 was also considered intheoretical simulations of the FLR (Lazar et al. 1997b;Stirbet et al. 1998).

II.4.2.3 The donor side of PSII

An inhibition of the donor side of PSII suppresses thethermal phase of the FLR (Schreiber and Neubauer 1987;Pospsil and Dau 2000; Bukhov et al. 2004). A partialinhibition of OEC, caused either by various treatments thatdeplete components on the donor side of PSII, or hightemperature treatment of PSII membranes (which lack theI step; see section II.3), led to a correlation between a rateconstant of the JP transient of the FLR (see section II.3)and a steady-state rate of oxygen evolution (Pospsil andDau 2000). Since a partial inhibition of the acceptor side ofPSII, caused by addition of subsaturating concentrations ofDCMU, resulted in a correlation between the rate constantand the steady-state rate of oxygen evolution too, therate constant of the JP transient may be considered anindicator of the extent of electron ow from water to PQmolecules generally (Pospsil and Dau 2000). An effect ofthe inhibition of OEC on the thermal phase of the FLRwas also revealed from theoretical simulation of the FLR(Lazar 2003).

Participation of the donor side of PSII in the JI phaseof the FLR was discussed by Vredenberg et al. (2005) onthe basis of a theoretical analysis of the three-state trappingmodel (TSTM) formulated by Vredenberg (2000) [see alsoStrasser and Stirbet (2001); Vredenberg (2004)]. In theTSTM, the open (PheoQA), semi-open(closed) (PheoQA),and closed (PheoQA) states of RCIIs are dened. Asmaller rate constant of P680+ reduction by YZ in thehigher S-states of OEC [summarised by Lazar (2003)]results in a lower efciency of closing of the semi-openRCIIs that leads to a slow FLR during the JI phase(Vredenberg et al. 2005).

II.4.2.4 Fluorescence quenching by the oxidisedPQ pool

As inhibition of OEC leads to a lack of electrons forthe reduction of the PQ pool, the pool remains oxidised,and acts as a uorescence quencher (Vernotte et al. 1979;Hsu and Lee 1995; Kramer et al. 1995; Kurreck and Renger1998; Kurreck et al. 2000; Haldimann and Tsimilli-Michael2005). This uorescence quenching by the oxidised PQ poolis suggested to lead to the suppression of the thermal phase(Pospsil and Dau 2000; Bukhov et al. 2004). Also usingthe pump and probe method and ash uorescence inductionmeasurements, the thermal phase was suggested to reectthe removal of the uorescence quenching by the oxidised

PQ pool (Samson and Bruce 1996; Koblzek et al. 2001).However, measurement of FM, induced by single turnoverash or by continuous illumination, in samples treated withDCMU and by exogenous PQs, revealed that uorescencequenching by the oxidised PQ pool is responsible for only25% of the thermal phase of the FLR (Yaakoubd et al. 2002).Therefore a question arises as towhichpart of theFLR reectsthe uorescence quenching by the oxidised PQ pool. AsDCMUand chemicals that accept electrons from the acceptorside of PSI (both keep the PQ pool oxidised) suppress the IPphase of the FLR, it is the IP phase that reects uorescencequenching by the oxidised PQ pool (Neubauer and Schreiber1987; Schreiber et al. 1989). An effect of the uorescencequenching by oxidised PQ pool on the FLR generally wasalso considered in theoretical simulations of the FLR (Stirbetet al. 1998; Lazar 2003; Zhu et al. 2005).

Toth et al. (2005) have recently found that if intact leavesare treated with DCMU carefully, the FM level is the samein the DCMU-treated leaves as in the controls (i.e. untreatedleaves). This fact implies that the FM level is not sensitiveto the redox state of the PQ pool and also that removalof the uorescence quenching by the oxidised PQ pool, asdiscussed above, is not responsible for the thermal phaseof the FLR, at least in intact leaves. As discussed by Tothet al. (2005), the FM level is lowered in leaves treated withDCMUby a not-so-careful procedure because the treatmentprobably causes a damage of PSII enabling a better contact ofoxidisedPQmolecules from thepoolwith excited chlorophyllmolecules in LHA. Therefore, Toth et al. (2005) suggestedthat extent of the PQ pool quenching expressed as loweringof the FM level could provide a tool to access the intactnessof the PSII protein complex and quality of the isolationprocedure used.

II.4.2.5 The Q2 component

Samson and Bruce (1996) suggested that the thermalphase of the FLR could also reect a reduction of acomponent, labelled as Q2. The term Q2 was rst usedby Joliot and Joliot (1977, 1979) as a putative electronacceptor, which needs more than one ash to be reducedin the presence of DCMU; it was part of an alternativeelectron pathway of very low quantum efciency. However,instead of dening the putative electron acceptor, Lavergneand Rappaport (1998) suggested that measured data can beexplained by charge recombination between P680+ andQA.Nevertheless, consideration of the charge recombination hadalmost no effect on the simulated thermal phase of the FLR(Lazar 2003).

II.4.2.6 Cytochrome b559

Lazar et al. (2005b) experimentally showed that anincrease of the amount of initially reduced cytochrome (cyt)b559 in DCMU-treated thylakoid membranes resulted in anincrease of the FM level. Theoretical simulations, where cytb559 was assumed to accept electrons from Pheo and donate


electrons to P680+ (in the presence of DCMU), furthersupported this experimental nding. Therefore, with respectto denition of the Q2 component, as mentioned above,such a cyt b559 could be identied as the Q2 component.The reduced Pheo from work of Lazar et al. (2005b) neednot be the Pheo localised in the D1 protein of PSII but itcan be Pheo2 localised in the D2 protein of PSII, whichis a part of the inactive electron transport branch inPSII (see section II.4.1.5) and is closer to cyt b559 thanPheo in D1 as known from the structure of PSII (Zouniet al. 2001; Kamiya and Shen 2003; Ferreira et al. 2004).Further, Schreiber and Neubauer (1987) had suggested thatif uorescence quenching by P680+ (Butler 1972; Mauzerall1972; Sonneveld et al. 1979; Deprez et al. 1983; Shinkarevand Govindjee 1993; Bruce et al. 1997) causes quenching ofuorescence signal at the J step, the reduction of P680+ byan alternate electron donor, e.g. by cyt b559 or a carotenoid,could result in uorescence increase during the thermal phaseof the FLR.

II.4.2.7 Fluorescence quenching in lightharvesting antennae

On the basis of measurements of uorescence decaysafter an application of single or multiple turnover ash, andtting of the experimental data by a model, Vasilev andBruce (1998) suggested that removal of the uorescencequenching in LHA of PSII is responsible for thethermal phase of the FLR. Moise and Moya (2004a, b)reached a similar conclusion on the basis of their phaseand modulation uorometry measurements: variable andtransitory uorescence quenching occurs during the thermalphase, and the quenching results from a conformationalchange in a LHA of PSII and the LHA of PSII, where theconformational changes occur is CP47.

II.4.2.8 Changes in yield of delayed(recombination) uorescence

Schreiber and Krieger (1996) suggested anotherinterpretation for the thermal phase of the FLR. Theseauthors assumed that uorescence signal during the FLRconsists of both the prompt uorescence and DF, the formeroriginating directly from the excited states and the latterindirectly after the formation of the excited states, viaradiative charge recombination between P680+ and Pheo(nanosecond range; for review on DF see Tyystjarvi and Vass2004). Schreiber and Krieger (1996) further suggested that agradual removal of non-radiative loss of the charge-separatedstate occurs in PSII in the P680+PheoQA state resultingin an increase of the yield of DF that leads to an increaseof uorescence signal during the thermal phase of the FLR.The origin of changes in the DF was discussed by the authorsto be connected to changes in positive charges stored at thedonor side of PSII.

Goltsev and co-workers (Goltsev and Yordanov 1997;Zaharieva and Goltsev 2003; Goltsev et al. 2003, 2005)studied an effect of recombinations between PSII electronacceptors and donors by a phosphoroscope methodand theoretical simulations of the DF and FLR (seesection II.4.1.6). The authors found that the time at whichthe second peak (denoted as I2) in the DF intensity appears,corresponds with the time of a maximal rate of the FLRduring the IP phase measured under low intensity ofexciting light (Goltsev and Yordanov 1997; Goltsev et al.2003; Zaharieva and Goltsev 2003). The origin of the I2in the DF is probably in recombination of QA with YZ+when QB is doubly reduced (Goltsev and Yordanov 1997;Zaharieva and Goltsev 2003; Goltsev et al. 2005) and thelifetime of YZ+QAQB2 is determined by the sum ofthe rate constants of electron transport from the S-statetransitions of OEC to YZ+ and of exchange between thereduced and oxidised PQ molecules in the QB-site of PSII(Goltsev et al. 2005).

II.4.2.9 Electron transport reactions after PSII

When the FLR is measured, in the green alga Chlorella,under low intensity of excitation light, a dip D usuallyappears after the I step (Munday and Govindjee 1969a).It was suggested that appearance of the dip is related tofunction of PSI (Munday and Govindjee 1969b; Schreiberet al. 1971; Satoh and Katoh 1981; Hansen et al. 1991).However, using theoretical modelling of the FLR measuredunder low intensity of excitation light, Baake and Strasser(1990) and Baake and Schloder (1992) showed that inclusionof electron transport reactions occurring after the PQ pool[cyt b6 / f complex, plastocyanin (PC), PSI] in a modeldoes not improve a t of the model to a part of the FLRaround the position of the dip D of the experimental FLRcurves measured with leaves. Also other authors assumedthe electron transport reactions occurring after the PQ poolin theoretical simulations of the FLR (Goltsev and Yordanov1997; Lebedeva et al. 2002).

New information about the electron transport chainafter the PQ pool has been obtained by simultaneousmeasurement of the FLR and transmission changes at 820 nm(T820), which should reect changes in redox state ofPC and P700. Schreiber et al. (1989), R. J. Strasser andco-workers (Strasser et al. 2001; Schansker et al. 2003,2005) showed that T820 has a minimum exactly at theposition of the I step in the OJIP FLR measured withleaves. On the basis of 2,5-dibromo-3-methyl-6-isopropyl-p-bezoquinone (DBMIB) and methylviologen (MV) actionon the FLR and T820 signal, Schansker et al. (2005)suggested that a transient limitation at the acceptor side ofPSI leading to a trafc jam of electrons transiently formedin the electron transport chain are responsible for the IPtransient of the FLR (cf. Munday and Govindjee 1969b).It is likely that this limitation on the PSI side can berst caused by inactive ferredoxin-NADP+ oxidoreductase


(FNR) and consequently by the inactiveCalvinBenson cycle(Schansker et al. 2003).

II.4.2.10 Fluorescence coming from PSI

Observations of direct uorescence from PSI has beenrecently reviewed by Itoh and Sugiura (2004). Here, I presenta discussion of the effect of PSI reactions on OJIP FLR.Applying saturating light pulses of varying lengths andmeasuring the T820 signal with leaves treated with MVand using far-red background illumination (to avoid anylimitation at the acceptor side of PSI), Schreiber et al. (1989)found that the electron transport chain is reduced within50ms; that is at a time when the I step appears in theFLR. On the basis of this result and of measured changesin the T820 signal, as mentioned above, the IP transient inthe FLR was suggested to reect a reduction of P700 andof PSI acceptor side caused by a limitation at the acceptorside of PSI (Munday and Govindjee 1969b; Schreiber et al.1989). Further, Ikegami (1976) found that when P700 isreduced by dithionite in P700-enriched particles, the particlesupon illumination (by blue light) showed an increase inuorescence signal (detected at 694 nm) at room temperature.This result together with the suggestion mentioned aboveled Ulrich Schreiber and co-workers (Schreiber et al. 1989;Schreiber 2002) to suggest that the IP transient of the FLRcould reect an increase in uorescence signal coming fromPSI. More recently, Byrdin et al. (2000) also found thatthe trimeric PSIs with initially reduced PSI by ascorbate(i.e. P700 being initially present) show a FLR (excited byHeNe laser at 633 nm and detected at wavelengths above665 nm) at room temperature with variable uorescenceFV (= FM F0) equal to 12 5% of FM. However, toprove that the IP transient of the FLR reects exclusivelyuorescence signal coming from PSI, direct comparisonof the FLRs measured at different emission wavelengthsis necessary.

II.4.2.11 Light gradient within a sample

As any photosynthetic sample used for measurementsof the FLR curves represents layers of pigments, a lightgradient within the sample, in addition to other opticaleffects, is created along the light path. The gradient thenresults in the excitation of particular sub-layers of the sampleby light of different intensities and the detected overalluorescence signal is a sum of the signals coming fromthe sub-layers. Assuming this simple rationale and usingdifferent sample preparations, Hsu and Leu (2003) suggestedthat the I step in the FLR originates in the abaxial layer(s)of the sample (when it is illuminated from the adaxialside). As it is the photochemical phase of the FLR thatis very sensitive to the intensity of exciting light (seesection II.2), the I step in the overall FLR should in factreect the photochemical phase of the FLR coming fromthe abaxial layer(s) of the sample (Hsu and Leu 2003).

Franck et al. (2005) also suggested a participation ofuorescence signals coming from particular layers of thesample to overall uorescence signal of the FLR on the basisof measurements of uorescence emission spectra at roomtemperature during the FLR with different samples and usingtheoretical simulations of the spectra. On the basis of thetheoretical simulations, Franck et al. (2005) also concludedthat as the time elapses from the onset of excitation, themeasured uorescence signal during the FLR originates fromdeeper layers.

Even though an effect of the light gradient withinthe sample on the overall FLR was explored by bothexperimental measurements and theoretical simulations(Susila et al. 2004), it was not proven that the light gradientis responsible for the appearance of the I step in the FLR.However, if the FLR is used as an analytical tool to accessinformation about photosynthetic function, care must beexercised in interpreting the data (Hsu and Leu 2003; Susilaet al. 2004). Susila et al. (2004) surmised that the lightgradient within the sample can signicantly affect the resultsof the JIP test (see section V.1) mainly in the case when theconcentration of Chls is changed when a plant is stressed.

II.4.2.12 Electric voltage acrossthe thylakoid membrane

When the FLR is measured with PSII membranes theI step is missing in the FLR, in contrast to more intactsamples where the I step is present (see section II.3).However, PSIImembranes do not form closed compartments,which would enable the formation of electric voltage acrossthe membrane. These facts, together with the existingcoincidence in the times of the I step appearance in theFLR (at 3050ms) and the formation of a peak in lightinduced electric voltage across the thylakoid membrane (at2050ms but with different samples, for reviews seeBulychev and Vredenberg 1999; Vredenberg 2004), ledPospsil and Dau (2002) to suggest that the I step reectslight induced changes in electric voltage across thylakoidmembranes. Application of valinomycin with potassiumions, led to a short-circuit with respect to the membranevoltage and the I step disappeared from the FLR measuredwith thylakoid membranes. Similarly the appearance of ahump, labelled as H (note: this hump is different fromthe H uorescence level described in section II.4.2.2.2 andfrom the H peak described in section III.2), in the FLRbut measured under low intensity of exciting light withBryopsis chloroplasts was related to changes in light-inducedmembrane voltage (Satoh and Katoh 1981).

However, appearance of the I step in FLR and a peak inlight-induced electric voltage across thylakoid membranes inthe same time as assumed by Pospil and Dau (2002) is incontrast to results of Schreiber and Neubauer (1990), whofound that the light-induced electric voltage across thylakoidmembranes has a maximum at the J step and not at the


I step of the FLR (see section II.4.1.4). Further, Schreiberand Neubauer (1990) suggested that increase of uorescencesignal during the thermal phase of the FLR reects a loweringof uorescence quenching driven by a relaxation of themembrane voltage, where the uorescence quenching occursby a mechanism as described in section II.4.1.4.

In addition to the participation of 3P680* as describedin section II.4.1.4, there are two other mechanisms relatedto changes in electric voltage across thylakoid membranesthat affect changes in uorescence emission directly andindirectly. In the direct mechanism, formation of the electricvoltage causes a decrease in Gibbs free-energy difference,G0, between the excited states in RCII and the chargeseparated state (P680+Pheo): the decrease inG0 leads to adecrease in the rate constant of primary charge separation andincrease in the rate constant of primary charge recombination,both resulting in an increased accumulation of excitedstates leading to increased uorescence emission (Dau et al.1991; Dau and Sauer 1991, 1992). In connection to theindirect mechanism, Graan and Ort (1983) found that amarked stimulation of the electron transport rate occurredwhen chloroplasts were treated with valinomycin, and theyexplained this effect by an increased rate of plastoquinoloxidation. Therefore, when valinomycin is not present alight-induced electric voltage across the thylakoid membraneis formed, causing a decrease in the rate of plastoquinoloxidation that leads to an increased accumulation of reducedQB and QA that results in an increased uorescenceemission. Similarly, Goltsev and Yordanov (1997) assumeda decrease in the rate constant of plastoquinol oxidationcaused by an intra-thylakoid space acidication in theoreticalsimulations of the FLR. It is evident from the above textthat both the direct and the indirect mechanisms involvechanges in the rate constants of electron transport steps.Therefore it is clear that when a light-induced electric eldis present, rate constants of all the electron transport stepswill be changed and will somehow contribute to changesin uorescence emission. Such an approach was used byLebedeva et al. (2002), who assumed a dependence ofthe rate constants of all the electron transport steps inthylakoid membranes on the light-induced electric voltageacross thylakoid membranes in theoretical simulationsof the FLR.

An effect of electrogenic events across thylakoidmembranes on the FLR has been extensively studied byWFJ Vredenberg and AA Bulychev (Bulychev and Niyazova1989; Vredenberg 2000; Bulychev and Vredenberg 2001;Vredenberg and Bulychev 2002, 2003). Bulychev andVredenberg (2001) found that electrogenic events generatedin PSI, when PSII photochemical activity was absent(caused by NH2OH treatments), affect the PSII. Thus,Vredenberg and Bulychev (2002) suggested the so-called thephotoelectrochemical control of uorescence yield: when thephotochemical activity of PSII is absent at the I step of

the FLR (assuming that all the electron acceptors in PSIIare already reduced), the subsequent IP transient is due tochanges in uorescence yield caused by electric events acrossthe thylakoid membrane (see also Vredenberg 2004).

An effect of light induced changes in pH or theelectric voltage across thylakoidmembranewas also includedin models, which simulated FLR curves for differentintensities of exciting light (Goltsev and Yordanov 1997;Lebedeva et al. 2002).

II.4.2.13 The heterogeneity in the rate of PQpool reduction

All the explanations, thus far discussed in this review,were made assuming PSII to be homogeneous. As alreadymentioned above (see section II.4.1.8), PSII is heterogeneousin many aspects. One aspect of the PSII heterogeneity is inthe rate of reduction of particular PQ pools (Joliot et al.1992; Kirchhoff et al. 2000). In addition to a fast-reducedPQ pool localised mainly in thylakoid grana, there alsoexists a slowly reduced PQ pool localised mainly in stroma-exposed thylakoid membranes. To incorporate this type ofheterogeneity into explanation of the FLR, the JI andIP transients of the thermal phase were suggested to reecta reduction of the fast and slow PQ pool, respectively(Strasser et al. 1995;Barthelemy et al. 1997; Schreiber 2002).Further, Bukhov et al. (2003) found that addition of a weakconcentration ofN,N,N,N-tetramethyl-p-phenylenediamine(TMPD) leads to a clearer appearance of the I step in the FLRmeasured with thylakoid membranes. Since Bukhov et al.(2003) used TMPD as an electron acceptor from the reducedPQ pool (see also Joly et al. 2005), the resolution of the I stepresulted from a decreased reduction of both fast and slowlyreducing PQ pool. Similarly, theoretical simulations of theFLR revealed that assuming a slowly reducing PQ pool in amodel leads to an appearance of a step in the thermal phaseof the FLR (Lazar 2003).

II.4.3 A summary

The results presented in the previous sections suggest thatmany events may affect the nal shape of measured FLR.Some of the conclusions were based on precise experimentsand their detailed analyses but some of them were onlyfrom hypotheses. It is not yet possible to say denitivelywhich mechanisms really (or only) contribute to the nalshape of the FLR and which do not. It is highly likelythat all the mechanisms mentioned contribute to the FLR tosome extent.

One way to quantify the extent by which a particularmechanism contributes to a measured quantity is to form amathematical model describing all the involved mechanismsand evaluate the extent to which a mechanism contributesto the measured quantity by means of exact mathematicalformulae. Such an approach is common in mathematicalmodelling of metabolic pathways and is known as metabolic


control analysis [MCA; for reviews on MCA, see Fell(1992); Visser and Heijnen (2002)]. In MCA, in additionto other coefcients, the control coefcient C, which exactlyquanties the extent to which a particular model parameter(concentration or activity or rate constant) controls a selectedvariable of the model (concentration or ux), is calculated.MCA has already been used for exploration of several plantmetabolic pathways, e.g. the CalvinBenson cycle (Poolmanet al. 2000) and the malate valve (Fridlyand et al. 1998),but it has also been used for exploration of uorescencedata related to photosynthetic function; a simple MCA-likemethod was used for the analysis of changes in oscillations insteady-state uorescence signal caused by high temperaturetreatment (Lazar et al. 2005c) and MCA was used forthe analysis of changes in FM of the DCMU-FLRs dueto changes in initial redox state of cyt b559 (Lazar et al.2005b; see section II.4.2.6). Therefore, it is a challenge forfuture work to construct a mathematical model of the FLR,which would include all suggested mechanisms and evaluatecontributions of particular mechanisms to FLR by meansof MCA.

III The K, H, and G steps in the uorescence rise

III.1 The K step

When samples are treated with high temperature, a newstep at 300400 s, denoted as K, appears in FLR (Guisseet al. 1995a, b; Srivastava et al. 1997; Lazar and Ilk 1997;Lazar et al. 1997a; Strasser 1997; see Fig. 2, curve b). Reto

GH

d

b

c

a

O

P

I

P

PJJ

KK

O

1 0234

1

2

3

4

5

Log time (s)

Fluo

resc

ence

inte

nsity

(rela

tive u

nits)

Fig. 2. Chlorophyll a uorescence rise measured with dark-adaptedpea leaves (curve a, no treatment; curve b, leaf incubated at 47Cin water for 5min), potato leaf (curve c, leaf incubated at 44C inwater for 13min), and with lichen Umbilicaria hirsuta (curve d, notreatment) by PEA uorometer under high intensity of exciting light[3400 mol photonsm2 s1 of red light; data from Lazar 1999 andcourtesy of P. Ilk and M. Bartak (curve d)]. The O, K, J, I, H, G, and Psteps are labelled.

Strasser and his co-workers (Guisse et al. 1995a; Strasser1997) suggested that the appearance of the K step reectsan inhibition of OEC, probably together with an inhibitionof the acceptor side of PSII (Lazar et al. 1999). Moregenerally, Strasser (1997) suggested that the K step ariseswhen the rate of electron ow from P680 to the acceptorside of PSII exceeds the rate of electron ow from thedonor side of PSII to P680. Srivastava et al. (1997) andLazar and Pospsil (1999) found that the appearance ofthe K step reects changes in the energetic connectivitybetween PSIIs.

As the K step reects an accumulation of reduced QA(Strasser 1997), the OK rise is also the photochemicalphase of FLR. However, the photochemical phase of theFLR measured at room temperature (the OJ transient) doesnot simply move to shorter times (the OK transient) whenmeasured with high temperature treated samples becauseboth the K and J steps can be measured together undercertain conditions (Guisse et al. 1995a, b; Srivastava et al.1997; see Fig. 2, curve c). This fact suggests that the Kand J steps might reect two different phenomena (Guisseet al. 1995a, b; Srivastava et al. 1997). Therefore, the sourceof the K step is expected to be present even in unstressedsamples but for dynamic reasons it does not appear as aclear step in the FLR (Guisse et al. 1995a, b; Srivastavaet al. 1997; Strasser 1997). The same conclusion can alsobe drawn from positions of peak accumulations of excitedstates simulated on the basis of a theoreticalmodel of the FLR(Lazar 2003).

III.2 The H and G steps

The OJIP FLR is not a typical property of allphotosynthetic organisms under standard conditions. TheP step, measured at high intensity of excitation light atroom temperature, was found to be split into two steps inthe FLR in foraminifers (Tsimilli-Michael et al. 1998a, b),zooxanthellae (Hill et al. 2004), lichens (Bukhov et al. 2004),and in lichens and lichenised algae (Ilk et al. 2006; see Fig. 2,curve d). To continue with the previous OKJI notation,the two steps were labelled as H and G (Tsimilli-Michaelet al. 1998a, b).

On the basis of simultaneous measurements of FLR andT820 signal with different samples treated with differentchemicals, Ilk et al. (2006) found that uorescence decreasefrom the H step to a dip between H and G steps is causedby a removal of limitation on the acceptor side of PSI,probably caused by light-induced activation of FNR (seesection II.4.2.9) or cyclic electron ow around PSI or Mehlerreaction, resulting in a transient reoxidation of reduced PQpool (and QA). Following uorescence increase from thedip between H and G steps to the G step is then caused bysubsequent PQ pool (and QA) re-reduction. The PQ pool re-reduction is not associated with cyclic electron ow aroundPSI and is probably caused by inability of the cyt b6 / fcomplex to rapidly reoxidise the PQ pool (Ilk et al. 2006).


In light of the discussion above, the H step mentionedhere seems to be different from the H uorescence level andfrom the hump H described in sections II.4.2.2 and II.4.2.12,respectively. Thus, a different nomenclature is ultimatelyneeded to describe this H step.

IV Statistical properties of parameters determinedfrom the uorescence rise

IV.1 Presentation of parameters determinedfrom the uorescence rise

Statistical evaluation of FLR data is not routine in theliterature and when it is used, it is only to fulll standardrequirements for the data presentation. The values of F0, FM,FV /FM parameters [and also other uorescence parameters(FPs) determined from FLR] are very often presented bymeans of the mean and standard deviation (or standard error)in the literature (see e.g. Bjorkman and Demmig 1987).But to present any parameter by means of the mean andstandard deviation (or standard error), the distribution of theparameters data should be Gaussian. However, the FPs (F0,FJ, FI, FM, FV, VJ, VI, FV /FM, etc.) generally do not haveGaussian distribution (Lazar and Naus 1998; Lazar et al.2003, 2005a, 2006). Therefore, presentation of data bymeansof the mean and standard deviation (or standard error) isnot appropriate and it masks the real data distribution of theFPs. The use of median, quartiles, and maximal and minimalvalues better describes a real situation of data distribution(Lazar and Naus 1998). Further, because the FPs of theFLR generally do not have Gaussian distributions, the non-parametric test should be used for statistical comparisons ofthe FPs rather than the parametric tests, which are based onthe assumption of Gaussian distribution of data.

IV. 2 Changes in statistical distributions of uorescenceparameters caused by stress

Generally, statistical distribution (histogram) of a FP,measured under non-stressed conditions, is not Gaussianbut skewed to a side (Lazar and Naus 1998; Lazar et al.2003, 2005a, 2006). When plant material suffers from stress,changes in statistical distributions of the parameters occurand these changes depend on the extent of the stress.For example, the FM level, measured with barley leavesupon high temperature treatment, is skewed to the left formeasurements at room temperature (i.e. most of the FMvalues are high, on the right in the histogram, but there arealso some smaller FM values, on the left in the histogram),then symmetrical (44C), then skewed to the right (51C),and nally symmetrical again (65C) (Lazar et al. 2005a).The observed changes in distributions of FM can be wellexplained in the sense of changes in functional heterogeneityof PSIIs, assuming that a stress leads to a malfunction ofPSII and causes a decrease in the FM level (Lazar et al.2005a). However, complete changes in the distributions asdescribed above can be observed only when plant material

suffers progressively fromweak to severe stress (Lazar 2005):when a stress is not severe, not all stages of change in thedistribution can be observed, aswas the case formeasurementwith pumpkin leaves that suffered from senescence andfungal infection (Lazar and Naus 1998) or with wheatleaves that suffered from senescence and other stresses(Lazar et al. 2003).

Changes in distributions of FPs, determined from FLR,caused by stress, as described above, are also accompaniedby changes in variances of measured data and the changesin variances are different for different FPs (Lazar et al.2003, 2005a). The different variances of the FPs can beindicative of different processes described by the FPs (Lazaret al. 2005a). The difference in variances of the FPs canbe used for an evaluation of mutual independence of twoFPs and, thus, also of mutual independence of processesdescribed by the FPs (Lazar et al. 2003, 2005a). Forexample, it was found, for the case of a high temperaturestress, that for almost all combinations of two basic FPs(F0, FK, FJ, FI, FM), there is an increase in mutualindependence of the FPs in the temperature range 4451Cand therefore also an increase in mutual independence ofprocesses characterised by the FPs in this temperature range(Lazar et al. 2005a).

Lazar et al. (2006) showed that a detailed analysis ofchanges in statistical distributions of FPs could be usefulfor early detection of plant stress. To obtain a large amountof experimental data for a given FP (necessary for detailedanalysis), they measured FI with leaf segments under alow intensity of exciting light with an imaging uorometer.Selected FP of control (no stress) and stressed samples(stress mainly by a dehydration of the segments in thiswork) were compared by classical statistical comparison(Mann-Whitney test; compares values of the medians) andby statistical comparison of shapes of distributions of theFPs (two-sample Smirnov test). The authors found thatexamples exist in which statistically signicant differenceis not revealed by the classical statistical comparison (forgiven critical level) but statistically signicant difference isrevealed by comparisons of distributions (for the same criticallevel). It implies that the shape of statistical distribution ofa FP is more sensitive to stress than the median of the FPand that the comparison of changes in shapes of statisticaldistributions of FPs is therefore more suitable for earlydetection of plant stress than a classical statistical comparison(Lazar et al. 2006).

V Applications of the uorescence rise

V.1 The JIP test for the so-called vitality screening

With the main goals to measure several hundreds of samplesper hour and to provide a manual that non-specialistscan execute the measurements and obtain results in astandard form, a test, known as the JIP test, was formulatedby Strasser and Strasser (1995) for vitality screening


upon changing environmental conditions. The test waslater produced as a free software Biolyzer (Laboratoryof Bioenergetics, University of Geneva, Switzerland;http://www.unige.ch/sciences/biologie/bioen/jipsoftware.html; veried 12 September 2005). The basis of the test ismeasurement of the FLRs with PEA uorometer followedby an analysis of measured curves. In this JIP test, basicparameters evaluated from the FLR curves, such as F0, FM,uorescence signals at 100 and 300 s and at the J, I andP steps, are used for subsequent calculation of differentparameters that are somehow related to energy and electronuxes in PSII and therefore to photosynthetic functiongenerally (see Strasser et al. 2004).

The JIP test seems to be very popular now; when asearch term jip and test and uorescence was used inthe Web of Science search engine, 31 articles were listed(search performed on 7 July 2005) published during aperiod from 1997 to 2005. For example, choosing onlyfrom ten of the most recently published articles, theJIP test was used for study of effects of ambient v.reduced UV-B radiation on Salix arctica plants (Albertet al. 2005), comparison of ozone foliar symptoms inwoody plant species (Bussotti et al. 2005), quanticationof the photosynthetic performance of phosphorus-decientSorghum plants (Ripley et al. 2004), assessment of stressconditions in Quercus ilex L. leaves (Bussotti 2004),description of effect of salinity stress on PSII in Ulvalectuca (Xia et al. 2004), exploration of low temperaturetolerance of tobacco plants (Parvanova et al. 2004),exploration of ozone action on Mediterranean evergreenbroadleaves and on woody plant leaves (Paoletti et al.2004; Gravano et al. 2004), phenotyping of dark- and light-adapted barley plants (Oukarroum and Strasser 2004), anddetection of draught and salinity tolerance chickpea varieties(Epitalawage et al. 2003).

Although the JIP test is very often used now, the resultsshould be taken with care, especially in the case when theconcentration of Chls is changed during stress (Susila et al.2004; see section II.4.2.11).

V.2 Remote sensing and pattern recognitionin precise agriculture

There is a rapidly growing interest for plant identication inagriculture practice with the aim of recognising cultivatedplants from weeds. Then, using a sprayer mounted on atractor, herbicides and other agrochemicals could be appliedselectively and precisely. This approach would result inprecision farming or precision agriculture, the terms that areused very often now.

Precision agriculture requires a tool that enables remotesensing of the plants and simultaneous recognition. Oneway to determine the plant species is to measure FLRs,which are characteristic for given species and can be usedas ngerprints. Application of sophisticated mathematical

procedures (the genetic algorithms and neural networksclassiers) then would enable recognition of particularspecies on the basis of their ngerprints. Fluorescencesignal can be also measured by a CCD camera froma large area (i.e. measurement of uorescence imaging;for reviews see Chaerle and Van Der Straeten 2000,2001; Nedbal and Whitmarsh 2004; Oxborough 2004a, b),enabling fast and effective remote sensing (for a reviewsee Moya and Cerovic 2004). Therefore, it seems thatchlorophyll uorescence signal is the tool needed forprecision agriculture.

The rst results of the pattern recognition usinguorescence techniques came from measurements ofuorescence induction (FI) by a PAM uorometer (i.e. withlow time resolution, but with both FLR and uorescencedecay being measured). A special design of illuminationroutine, given to dark-adapted samples, consisted ofapplication of red light of moderate intensity, far-red light,high intensity white light, and dark intervals (Tyystjarvi et al.1999). However, this type of data collection required a longtime, which conicts with the need for rapid data captureduring tractor movement in eld conditions. Therefore,FLRs measured with leaves without dark adaptation andusing a PEA uorometer were also used for subsequentanalysis by the same research group (Keranen et al. 2003).Importantly, the analysis based on the PEA data hadabout the same or, in some cases, even better accuracy ofcorrect identication as the analysis based on the PAM data(Keranen et al. 2003).

When FLR or FI is used for pattern recognition, wholemeasured curves are not used but, instead, only somefeatures of the curves are analysed. Currently, the featuresused are slopes and y-axis intercepts of regression linesto experimental FLR or FI data in selected time intervals.The best selection of the time intervals is therefore veryimportant to obtain features that are species-specic andtherefore usage of which would result in best patternrecognition. This problem was addressed by Codrea et al.(2003, 2004), who used an optimiser (genetic algorithm)to tune the endpoints of the time intervals to improve thepattern recognition.

V.3 In silico photosynthesis to understand photosyntheticregulations and limitations future prospects

A summary of possible origins of the steps in the FLR, givenin sections II.4 and III, clearly shows that there exist severalregulatory mechanisms that can ne tune photosyntheticperformance, evenduring the veryrst secondof illuminationof photosynthesising organisms.What is important is that notonly the function of PSII can be reected in the FLR but alsoa function of the electron transport chain within thylakoidmembranes and a means of subsequent electron usage byFNR and the CalvinBenson cycle. Therefore, in principle,all the photosynthetic reactions, involved in different


levels of organisation, should be somehow reected in theFLR or FI.

Prolonged light illumination and consideration of higherlevels of organisation lead to very large numbers ofdifferent regulatory mechanisms involved in different levelsof organisation. One way to understand the complexity ofoperating machinery is to use mathematical modelling of allthe events, i.e. to form in silico photosynthesis.Mathematicalmodelling is therefore an important tool for the betterunderstanding of the explored processes. For recent reviewson mathematical modelling of plant metabolic pathways, seeGiersch (2000) and Morgan and Rhodes (2002).

At present there is no global model of photosynthesis(but see http://www.e-photosynthesis.org for a developingproject; veried 12 September 2005) that would satisfactorilydescribe all the related phenomena measured by respectivequantities. However, several detailed models describingparticular photosynthetic events and regulations have beensuggested. For example, in addition to exploration of theOJIP FLR,models describing electron transport reactionswithin thylakoid membrane were also used for theoreticalexploration of the electron transport chain as such (Berry andRumberg 2001), ash and continual light induced oxygenevolution (Kana et al. 2002), dependence of uorescencesignal on linearly increasing temperature of the sample(Kouril et al. 2004), and forced uorescence oscillations(Nedbal et al. 2005). It is therefore a challenge for futureworkto construct a mathematical model of whole photosynthesis.Such model would be used, for example, in revealing theregulatory mechanisms that are not directly accessible orunderstandable from the available experimental data andfor nding the limitations of photosynthesis under differentexperimental conditions.

When limitations of photosynthesis under differentconditions are found, they can be used in an effortto increase photosynthetic efciency and crop yieldby means of specic mutations. However, engineeringmutants, which lead to increased efciencies and yields,remains a long-term goal. Sinclair et al. (2004) showed,by means of theoretical calculations, that assumed50% increase in mRNA production responsible for thesynthesis of small and large subunits of ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), the keyenzyme in CalvinBenson cycle, would lead only to37% increase in Rubisco content, 33% increase inlight-saturated leaf photosynthetic rate, 30% increasein photosynthetic rate of isolated plant, 18% increase inaccumulated crop mass, 6% increase in grain yield withadditional nitrogen supply, but possibly a 6% decrease ingrain yield without additional nitrogen supply. Therefore itseems that there may not be much room to increase efciencyand yield of natural photosynthesis. Thus, efforts should befocused on the construction of an articial photosynthesis toserve our increasing consumption demands.

VI Conclusions

This review has summarised the current understanding ofthe FLR measured under high intensity of exciting light, theOJIP transient. The review also included a discussionof other steps, K, H, and G, which appear in the FLRunder certain conditions. Some specic applications of theFLR were also mentioned. Although as the current textimplies, many interpretations have been suggested for theparticular steps of the FLR, yet this phenomenon is notfully understood. Further research is necessary to fullyunderstand the relationship of the OJIP transient tophotosynthetic reactions.

Acknowledgments

This work was nancially supported by the Ministry ofEducation of the Czech Republic by a grant numberMSM 6198959215. This review was also a part of theHabilitation Thesis of the author (Lazar 2005). I thankProfessors Govindjee, Ulrich Schreiber, and Reto J. Strasserfor their valuable comments that have improved thispresentation. In addition, Govindjee has also edited parts ofthe manuscript.

References

Albert KR, Mikkelsen TN, Ro-Poulsen H (2005) Effects of ambientversus reduced UV-B radiation on high arctic Salix arctica assessedby measurements and calculations of chlorophyll a uorescenceparameters from uorescence transients. Physiologia Plantarum124, 208226. doi: 10.1111/j.1399-3054.2005.00502.x

Avarmaa R, Soovik T, Tamkivi A, Tonissoo B (1977)Fluorescence lifetimes of chlorophyll-a and some relatedcompounds at low temperatures. Studia Biophysica Berlin 65,213218.

Baake E, Strasser RJ (1990) A differential equation model forthe description of the fast uorescence rise (OIDP-transient)in leaves. In Current research in photosynthesis. Vol. II.(Ed. M Baltscheffsky) pp. 567570. (Kluwer Academic Publishers:Dordrecht)

Baake E, Schloder JP (1992) Modelling the fast uorescence rise ofphotosynthesis. Bulletin of Mathematical Biology 54, 9991021.doi: 10.1016/S0092-8240(05)80092-8

Baker NR, Rosenqvist E (2004) Applications of chlorophylluorescence can improve crop production strategies: an examinationof future possibilities. Journal of Experimental Botany 55,16071621. doi: 10.1093/jxb/erh196

Barthelemy X, Popovic R, Franck F (1997) Studies on the OJIPtransient of chlorophyll uorescence in relation to photosystem IIassembly and heterogeneity in plastids of greening barley. Journalof Photochemistry and Photobiology. B, Biology 39, 213218.doi: 10.1016/S1011-1344(97)00012-2

Barzda V, Vengris M, Valkunas L, van Grondelle R, van Amerongen H(2000) Generation of uorescence quenchers from the triplet statesof chlorophylls in the major light-harvesting complex II from greenplants. Biophysical Journal 39, 1046810477.

Berry S, Rumberg B (2001) Kinetic modeling of the photosyntheticelectron transport chain. Bioelectrochemistry 53, 3553.doi: 10.1016/S0302-4598(00)00108-2


Bjorkman O, Demmig B (1987) Photon yield of O2 evolution ofchlorophyll uorescence characteristics at 77K among vascularplants of diverse origins. Planta 170, 489504. doi: 10.1007/BF00402983

Bouges-Bocquet B (1973) Electron transfer between the twophotosystems in spinach chloroplasts. Biochimica et BiophysicaActa 314, 250256.

Breton J (1983) The emission of chlorophyll in vivo. Antennauorescence or ultrafast luminescence from reaction centrepigments. FEBS Letters 159, 15. doi: 10.1016/0014-5793(83)80405-0

Breton J, Wakeham MC, Fyfe PK, Jones MR, Nabedryk E(2004) Characterization of the bonding interactions of QB uponphotoreduction via A-branch or B-branch electron transfer inmutantreaction centers from Rhodobacter sphaeroides. Biochimica etBiophysica Acta 1656, 127138.

Briantais J-M, Dacosta J, Goulas Y, Ducruet J-M, Moya I(1996) Heat stress induces in leaves an increase of theminimum level of chlorophyll uorescence, F0: a time-resolvedanalysis. Photosynthesis Research 48, 189196. doi: 10.1007/BF00041008

Brody SS (2002) Fluorescence lifetime, yield, energy transfer andspectrum in photosynthesis, 19501960. Photosynthesis Research73, 127132. doi: 10.1023/A:1020405921105

Bruce D, Samson G, Carpenter C (1997) The origins ofnonphotochemical quenching of chlorophyll uorescence inphotosynthesis. Direct quenching by P680+ in photosystem IIenriched membranes at low pH. Biochemistry 36, 749755.doi: 10.1021/bi962216c

Bukhov NG, Govindachary S, Egorova EA, Joly D, Carpentier R (2003)N,N,N,N-tetramethyl-p-phenylenediamine initiates the appearanceof a well-resolved I peak in the kinetics of chlorophyll uorescencerise in isolated thylakoids. Biochimica et Biophysica Acta 1607,9196.

Bukhov NG, Egorova EA, Govindachary S, Carpentier R (2004)Changes in polyphasic chlorophyll a uorescence induction curveupon inhibition of donor or acceptor side of photosystem IIin isolated thylakoids. Biochimica et Biophysica Acta 1657,121130.

Bulychev AA, Niyazova MM (1989) Modelling of potential-dependingchanges of chlorophyll uorescence in the photosystem 2. Biozika34, 6367.

Bulychev AA, Vredenberg WJ (1999) Light-triggered electricalevents in the thylakoid membrane of plant chloroplasts.Physiologia Plantarum 105, 577584. doi: 10.1034/j.1399-3054.1999.105325.x

Bulychev AA, Vredenberg WJ (2001) Modulation of photosystem IIchlorophyll uorescence by electrogenic events generated byphotosystem I. Bioelectrochemistry 54, 157168. doi: 10.1016/S1567-5394(01)00124-4

Bussotti F (2004) Assessment of stress conditions in Quercus ilex L.leaves by OJIP chlorophyll alpha uorescence analysis. PlantBiosystems 138, 101109. doi: 10.1080/11263500412331283708

Bussotti F, Agati G, Desotgiu R, Matteini P, Tani C (2005)Ozone foliar symptoms in woody plant species assessed withultrastructural and uorescence analysis. New Phytologist 166,941955. doi: 10.1111/j.1469-8137.2005.01385.x

Butler WL (1972) On the primary nature of uorescence yieldchanges associatedwith photosynthesis.Proceedings of theNationalAcademy of Sciences USA 69, 34203422.

Butler WL (1977) Chlorophyll uorescence: a probe for electrontransfer and energy transfer. In Encyclopedia of plant physiology.Photosynthesis I, volume 5. (Eds VA Trebs, M Avron) pp. 149167.(Springer-Verlag: Berlin)

Butler WL (1978) Energy distribution in the photochemical apparatusof photosynthesis. Annual Review of Plant Physiology and PlantMolecular Biology 29, 345378.

Byrdin M, Rimke I, Schlodder E, Stehlik D, Roelofs TA (2000) Decaykinetics and quantum yields of uorescence in photosystem I fromSynechococcus elongatus with P700 in the reduced and oxidizedstate: are the kinetics of excited state decay trap-limited or transfer-limited? Biophysical Journal 79, 9921007.

Chaerle L, Van Der Straeten D (2000) Imaging techniques and theearly detection of plant stress. Trends in Plant Science 5, 495501.doi: 10.1016/S1360-1385(00)01781-7

Chaerle L, Van Der Straeten D (2001) Seeing is believing: imagingtechniques to monitor plant health. Biochimica et Biophysica Acta1519, 153166.

Chylla RA, Whitmarsh J (1989) Inactive photosystem II complexes inleaves. Plant Physiology 90, 765772.

Codrea CM, Aittokallio T, Keranen M, Tyystjarvi E, Nevalainen OS(2003) Feature learning with a genetic algorithm for uorescencengerprinting of plant species. Pattern Recognition Letters 24,26632673. doi: 10.1016/S0167-8655(03)00109-0

Codrea CM, Aittokallio T, Keranen M, Tyystjarvi E, Nevalainen OS(2004) Genetic feature learning algorithm for uorescencengerprinting of plants. Lecture Notes in Computer Science 2936,371384.

Crofts AR, Wraight CA (1983) The electrochemical domainof photosynthesis. Biochimica et Biophysica Acta 726,149185.

DauH (1994)Molecularmechanism and quantitativemodels of variablephotosystem II uorescence. Photochemistry and Photobiology 60,123.

Dau H, Sauer K (1991) Electric eld effect on chlorophyll uorescenceand its relation to photosystem II charge separation reactions studiedby a salt-jump technique. Biochimica et Biophysica Acta 1098,4960.

Dau H, Sauer K (1992) Electric eld effect on the picoseconduorescence of photosystem II and its relation to the energetics andkinetics of primary charge separation.Biochimica etBiophysicaActa1102, 91106.

Dau H, Windecker R, Hansen U-P (1991) Effect of light-inducedchanges in thylakoid voltage on chlorophyll uorescence ofAegopodium podagraria leaves. Biochimica et Biophysica Acta1057, 337345.

DelosmeR (1967) Etude de linduction deuorescence des algues verteset des chloroplastes au debut dune illumination intense. Biochimicaet Biophysica Acta 143, 108128.

Deprez J, Dobek A, Geacintov NE, Paillotin G, Breton J (1983) Probinguorescence induction in chloroplast on a nanosecond time scaleutilizing picosecond laser pulse pairs.Biochimica et Biophysica Acta725, 444454.

DuysensLNM,SweersHE (1963)Mechanismof the two photochemicalreactions in algae as studied by means of uorescence. In Studieson microalgae and photosynthetic bacteria. (Ed. Japanese Societyof Plant Physiologists) pp. 353372. (University of Tokyo Press:Tokyo, Japan)

Epitalawage N, Eggenberg P, Strasser RJ (2003) Use of fast chlorophylla uorescence technique in detecting drought and salinity tolerantchickpea (Cicer arietinum L.) varieties. Archives des SciencesGeneve 56, 7993.

Fell DA (1992) Metabolic control analysis: a survey of its theoreticaland experimental development. Biochemical Journal 286,313330.

Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004)Architecture of the photosynthetic oxygen-evolving center. Science303, 18311838. doi: 10.1126/science.1093087


Franck F, Juneau P, Popovic R (2002) Resolution of the photosystem Iand photosystem II contributions to chlorophyll uorescence ofintact leaves at room temperature. Biochimica et Biophysica Acta1556, 239246.

Franck F, Dewez D, Popovic R (2005) Changes in the room-temperatureemission spectrum of chlorophyll during fast and slow phases of theKautsky effect in intact leaves. Photochemistry and Photobiology81, 431436. doi: 10.1562/2004-03-01-RA-094.1

Fridlyand LE, Backhausen JE, Scheibe R (1998) Flux control of themalate valve in leaf cells. Archives of Biochemistry and Biophysics349, 290298. doi: 10.1006/abbi.1997.0482

Frolov D, Wakeham MC, Andrizhiyevskaya EG, Jones MR,van Grondelle R (2005) Investigation of B-branch electron transferby femtosecond time resolved spectroscopy in a Rhodobactersphaeroides reaction centre that lacks the QA ubiquinone.Biochimica et Biophysica Acta 1707, 189198.

Giersch C (2000) Mathematical modelling of metabolism. CurrentOpinion in Plant Biology 3, 249253.

Gilmore AM, Hazlett TL, Debrunner PG, Govindjee (1996)Photosystem II chlorophyll a uorescence lifetimes and intensity areindependent of the antenna size differences between barleywild-typeand chlorina mutants: photochemical quenching and xanthophyllcycle-dependent nonphotochemical quenching of uorescence.Photosynthesis Research 48, 171187. doi: 10.1007/BF00041007

Gilmore A, Itoh SS, Govindjee (2000) Global spectral-kinetic analysisof room temperature chlorophyll a uorescence from lightharvesting antenna mutants of barley. Philosophical Transactionsof Royal Society of London Series B-Biological Sciences 335,114.

Goltsev V, Yordanov I (1997) Mathematical model of promptand delayed chlorophyll uorescence induction kinetics.Photosynthetica 33, 571586.

Goltsev V, Zaharieva I, Lambrev P, Yordanov I, Strasser R (2003)Simultaneous analysis of prompt and delayed chlorophyll auorescence in leaves during the induction period of dark tolight adaptation. Journal of Theoretical Biology 225, 171183.doi: 10.1016/S0022-5193(03)00236-4

Goltsev V, Chernev P, Zaharieva I, Lambrev P, Strasser RJ (2005)Kinetics of delayed chlorophyll a uorescence registered inmilliseconds time range. Photosynthesis Research 84, 209215.doi: 10.1007/s11120-004-6432-2

Govindjee (1995) Sixty-three years since Kautsky: chlorophyll auorescence. Australian Journal of Plant Physiology 22, 131160.

Govindjee (2004) Chlorophyll a uorescence: a bit of basicsand history. In Chlorophyll a uorescence: a signature ofphotosynthesis. (Eds GC Papageorgiou, Govindjee) pp. 142.(Springer: Dordrecht)

Govindjee, Amesz J, Fork DC (Eds) (1986) Light emission by plantsand bacteria. (Academic Press: Orlando, FL)

Graan T, Ort DR (1983) Initial events in the regulation of electrontransfer in chloroplasts. The role of the membrane potential. Journalof Biological Chemistry 258, 28312836.

Graan T, Ort DR (1984) Quantitation of the rapid electron donorsto P700, the functional plastoquinone pool, and the ratio ofthe photosystems in spinach chloro

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