2008. Baker. Chlorophyll Fluoresncence, A Probe of Photosynthesis in Vivo

ANRV342-PP59-05 ARI 27 March 2008 1:28

Chlorophyll Fluorescence:A Probe of PhotosynthesisIn VivoNeil R. BakerDepartment of Biological Sciences, University of Essex, Colchester, CO4 3SQ,United Kingdom; email: [email protected]

Annu. Rev. Plant Biol. 2008. 59:89–113

The Annual Review of Plant Biology is online atplant.annualreviews.org

This article’s doi:10.1146/annurev.arplant.59.032607.092759

Copyright c© 2008 by Annual Reviews.All rights reserved

1543-5008/08/0602-0089$20.00

Key Words

carbon dioxide assimilation, electron transport, imaging,metabolism, photosystem II photochemistry, stomata

AbstractThe use of chlorophyll fluorescence to monitor photosynthetic per-formance in algae and plants is now widespread. This review exam-ines how fluorescence parameters can be used to evaluate changesin photosystem II (PSII) photochemistry, linear electron flux, andCO2 assimilation in vivo, and outlines the theoretical bases for theuse of specific fluorescence parameters. Although fluorescence pa-rameters can be measured easily, many potential problems may arisewhen they are applied to predict changes in photosynthetic perfor-mance. In particular, consideration is given to problems associatedwith accurate estimation of the PSII operating efficiency measuredby fluorescence and its relationship with the rates of linear electronflux and CO2 assimilation. The roles of photochemical and non-photochemical quenching in the determination of changes in PSIIoperating efficiency are examined. Finally, applications of fluores-cence imaging to studies of photosynthetic heterogeneity and therapid screening of large numbers of plants for perturbations in pho-tosynthesis and associated metabolism are considered.

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Heat loss: occurswhen excitationenergy withinpigments is lost asheat; often termednonradiative decayor thermaldeactivation

Excitation energy:energy within apigment moleculeafter a photon isabsorbed andgenerates an excitedstate of the molecule

QA: primaryquinone electronacceptor of PSII

Photochemicalquenching: resultsfrom using excitationenergy withinphotosystem II(PSII) to driveelectron transportfrom P680 to QA

Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 90BACKGROUND . . . . . . . . . . . . . . . . . . . 90PHOTOSYSTEM II

PHOTOCHEMISTRY . . . . . . . . . . 91Dark-Adapted State . . . . . . . . . . . . . . 91Light-Adapted State . . . . . . . . . . . . . . 95

RELATIONSHIP BETWEENPHOTOSYSTEM IIOPERATING EFFICIENCY,LINEAR ELECTRON FLUX,AND CO2 ASSIMILATION . . . . . 96

FACTORS THAT DETERMINEPHOTOSYSTEM IIOPERATING EFFICIENCY . . . . 98Photochemical Quenching . . . . . . . . 99Nonphotochemical Quenching. . . . 101

IMAGING OFFLUORESCENCE . . . . . . . . . . . . . . 104

INTRODUCTION

The use of chlorophyll a fluorescence mea-surements to examine photosynthetic perfor-mance and stress in algae and plants is nowwidespread in physiological and ecophysio-logical studies. This has come about owingto the development of a sound understandingof the relationships between fluorescence pa-rameters and photosynthetic electron trans-port in vivo and the commercial availability ofa range of affordable, easy to use portable flu-orimeters. Fluorescence can be a very power-ful tool to study photosynthetic performance,especially when coupled with other noninva-sive measurements such as absorption spec-troscopy, gas analyses, and infrared thermom-etry. This review examines how some keyfluorescence parameters can be used to as-sess photosynthetic performance in vivo andto identify possible causes of changes in pho-tosynthesis and plant performance; it is aimedat plant biologists who seek to use fluores-cence as a tool in their research. However,the underlying theoretical bases of fluores-

cence changes in vivo are complex and cor-rect interpretation of changes in fluorescenceparameters can often be difficult. Considera-tion is given to some problems associated withthe measurement of these parameters and theassumptions made when using these param-eters to evaluate changes in photosyntheticperformance.

BACKGROUND

Following the observation by Kautsky &Hirsch (55) that changes in fluorescence in-duced by illumination of dark-adapted leavesare qualitatively correlated with changes inCO2 assimilation, it became evident that un-der some circumstances fluorescence emis-sions in photosynthetic organisms could becorrelated to their photosynthetic rates (54,56, 77). Butler (21) developed a simple modelfor photosystem II (PSII) photochemistry inwhich photochemistry competes with the pro-cesses of fluorescence and heat loss for excita-tion energy in the pigment antenna of PSII(Figure 1). This model followed from theproposal that electron transfer from the re-action center chlorophyll of PSII (P680) tothe primary quinone acceptor of PSII (QA)quenches fluorescence (28), a process termedphotochemical quenching. Increases in therate of heat loss result in nonphotochemicalquenching of fluorescence. The model pre-dicts that PSII fluorescence emission couldbe used to monitor changes in photochem-istry, provided that the rate constants for flu-orescence and heat loss do not change (21).However, it is now well established that largechanges can occur in the rate constant for heatloss from the PSII antenna (61, 65). Con-sequently, to estimate PSII photochemistryfrom fluorescence, it is essential to determinethe fluorescence quenching that results fromboth photochemical and nonphotochemicalprocesses.

Separation of fluorescence quenching intophotochemical and nonphotochemical com-ponents was first achieved by the additionof 3-(3,4-dichlorophenyl)-1,1-dimethylurea

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(DCMU) to intact chloroplasts and Chlorellacells at points throughout the fluorescenceinduction curve (64, 66). DCMU inhibitselectron transfer from QA to the secondaryquinone acceptor of PSII (QB), which resultsin a rapid reduction of QA and an increasein fluorescence as photochemical quenchingis prevented. A slower increase in fluores-cence follows, which is associated with thedecay of nonphotochemical quenching. Un-fortunately, this DCMU technique is not suit-able for analyzing fluorescence quenching inleaves owing to the slow and uneven pen-etration of DCMU into leaf tissues. Also,the irreversibility of the DCMU inhibitionof electron transport makes the techniqueunsuitable for continuous measurements onindividual leaves. However, maximal QA re-duction in leaves in the light can be achievedby rapidly exposing leaves to a very large in-crease in light (17). This light-addition tech-nique is used to quantitatively determine thefraction of fluorescence quenching that isattributable to photochemical and nonphoto-chemical quenching processes (18). The de-velopment of fluorimeters that use weak mod-ulated measuring beams in which phase andfrequency decoding are used to detect fluo-rescence yield changes enabled the routine,nondestructive, quantitative determination ofphotochemical and nonphotochemical pro-cesses in leaves by the application of a brief(less than 1 s) saturating flash of light suffi-ciently intense as to maximally reduce the QA

pool in the sample (26, 102). The value of themodulated technique is that it provides a con-tinuous measure of the relative quantum yieldof fluorescence (101). This technique was usedto demonstrate that the quantum yield of PSIIphotochemistry of a leaf at a given actiniclight intensity can be estimated from the mod-ulated fluorescence yield prior to the appli-cation of the saturating flash and the maxi-mum modulated fluorescence yield during theflash (37). In the absence of photorespiration,which competes with CO2 assimilation for theproducts of electron transport, the quantumyield of PSII photochemistry is directly re-

Light

Photosystem II

Photochemistry

P680e–

QA

HeatChlorophyllfluorescence

Figure 1Simple model of the possible fate of light energy absorbed by photosystemII (PSII). Light energy absorbed by chlorophylls associated with PSII canbe used to drive photochemistry in which an electron (e−) is transferredfrom the reaction center chlorophyll, P680, to the primary quinoneacceptor of PSII, QA. Alternatively, absorbed light energy can be lost fromPSII as chlorophyll fluorescence or heat. The processes of photochemistry,chlorophyll fluorescence, and heat loss are in direct competition forexcitation energy. If the rate of one process increases the rates of the othertwo will decrease.

Nonphotochemicalquenching: occurswhen there is anincrease in the rate atwhich excitationenergy withinphotosystem II is lostas heat

Quantum yield(quantumefficiency) of aprocess: number ofmoleculesundergoing theprocess divided bythe number ofphotons absorbed bythe system

Actinic light: lightthat is absorbed bythe photosyntheticapparatus and willdrive electrontransport

lated to the quantum yield of CO2 assimilationby the leaf, φCO2 (37), thus allowing, undercertain conditions, the application of fluores-cence measurements to provide a rapid, non-destructive probe of CO2 assimilation. A listof the fluorescence parameters used in this re-view, their definitions, and comments on theirphysiological relevance are given in Table 1.

PHOTOSYSTEM IIPHOTOCHEMISTRY

Dark-Adapted State

When a leaf is kept in the dark, QA becomesmaximally oxidized and the PSII reaction cen-ters are referred to as being ‘open’, i.e., capa-ble of performing photochemical reduction ofQA. Exposure of a dark-adapted leaf to a weakmodulated measuring beam [photosyntheti-cally active photon flux density (PPFD) of ca.0.1 μmol m−2 s−1] results in the minimal levelof fluorescence, Fo (Figure 2). The intensityof the measuring beam must be nonactinic

www.annualreviews.org • Chlorophyll Fluorescence 91

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Table 1 Chlorophyll fluorescence parameters frequently used in studies of photosystem II photochemistry

Parameter Definition Physiological relevanceF, F ′ Fluorescence emission from dark- or

light-adapted leaf, respectively.Provides little information on photosynthetic performancebecause these parameters are influenced by many factors. F ′ issometimes referred to as Fs

′ when at steady stateFo, Fo

′ Minimal fluorescence from dark- andlight-adapted leaf, respectively

Level of fluorescence when QA is maximally oxidized (PSII centersopen)

Fm, Fm′ Maximal fluorescence from dark- and

light-adapted leaf, respectivelyLevel of fluorescence when QA is maximally reduced (PSII centersclosed)

Fv, Fv′ Variable fluorescence from dark- and

light-adapted leaves, respectivelyDemonstrates the ability of PSII to perform photochemistry(QA reduction)

Fq′ Difference in fluorescence between Fm

′

and F ′Photochemical quenching of fluorescence by open PSII centers.

Fv/Fm Maximum quantum efficiency of PSIIphotochemistry

Maximum efficiency at which light absorbed by PSII is used forreduction of QA.

Fq′/Fm

′ PSII operating efficiency Estimates the efficiency at which light absorbed by PSII is used forQA reduction. At a given photosynthetically active photon fluxdensity (PPFD) this parameter provides an estimate of thequantum yield of linear electron flux through PSII. Thisparameter has previously been termed �F/Fm

′ and φPSII in theliterature.

Fv′/Fm

′ PSII maximum efficiency Provides an estimate of the maximum efficiency of PSIIphotochemistry at a given PPFD, which is the PSII operatingefficiency if all the PSII centers were ‘open’ (QA oxidized).

Fq′/Fv

′ PSII efficiency factor Relates the PSII maximum efficiency to the PSII operatingefficiency. Nonlinearly related to the proportion of PSII centersthat are ‘open’ (QA oxidized). Mathematically identical to thecoefficient of photochemical quenching, qP .

NPQ Nonphotochemical quenching Estimates the nonphotochemical quenching from Fm to Fm′.

Monitors the apparent rate constant for heat loss from PSII.Calculated from (Fm/Fm

′) – 1.qE Energy-dependent quenching Associated with light-induced proton transport into the thylakoid

lumen. Regulates the rate of excitation of PSII reaction centers.qI Photoinhibitory quenching Results from photoinhibition of PSII photochemistry.qL Fraction of PSII centers that are ‘open’ Estimates the fraction of ‘open’ PSII centers (with QA oxidized) on

the basis of a lake model for the PSII photosynthetic apparatus.Given by (Fq

′/Fv′)(Fo

′/F ′)qT Quenching associated with a state transition Results from phosphorylation of light-harvesting complexes

associated with PSIIφF Quantum yield of fluorescence Number of fluorescent events for each photon absorbed

PPFD:photosyntheticallyactive photon fluxdensity

to ensure that QA remains maximally oxi-dized. If the period used for dark adaptationis not long enough QA may not become max-imally oxidized. Then a pulse of weak far-redlight, which preferentially excites photosys-tem I (PSI) and removes electrons from QA,should be applied prior to the measurements

of Fo. In some leaves (32) and algae (10) sig-nificant accumulation of reduced QA can oc-cur in the dark owing to nonphotochemicalreduction of plastoquinone by chlororespira-tion; the reduced plastoquinone must be re-oxidized by a pulse of weak red light beforemeasurement of Fo. If after reaching Fo the

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F'

Fm'

Fm

Fo'

Fq'

0 302010Time (min)

0.1 µmol photons m–2 s–1 0.1 µmol photons m–2 s–1

Baseline

685 µmol photons m–2 s–1

Measuring light alone

Saturating pulse, PS II closed

Actinic light, PS II partially closed

Far-red PS I light, PS II open

Variable fluorescence

Quenched fluorescence

Fm F

p

Fv

Fv'

Fo

Figure 2Fluorescence quenching analysis using modulated fluorescence. A dark-adapted leaf is exposed to variouslight treatments. The parameters denoted with a prime (′) are from the leaf exposed to actinic light. Theparameters without a prime are obtained from the leaf in the dark-adapted state. The different colors ofthe trace denote different light treatments. White: weak measuring light alone (0.1 μmol photonsm−2 s−1) that gives Fo. An important feature of this measuring beam is that its intensity must be lowenough so it does not drive significant PSII photochemistry. Yellow: saturating light pulse (≤1 s duration,>6000 μmol photons m−2 s−1) that gives Fm in darkness and Fm

′ in light. Blue: actinic light (685 μmolphotons m−2 s−1) that drives photosynthesis and gives F ′. Red: far-red light (30 μmol photons m−2 s−1

at 720–730 nm for 4 s) that excites photosystem I (PSI) preferentially, and thus oxidizes theplastoquinone and QA pools associated with PSII and gives Fo′. Orange: variable fluorescence calculatedas Fv = Fm – Fo from the dark-adapted leaf and Fv

′ = Fm′ – Fo

′ from the illuminated leaf. Green:fluorescence that is quenched from Fm

′ to F ′ by PSII photochemistry in the illuminated leaf, calculatedas Fq

′ = Fm′ – F ′. All parameters, except Fq

′, Fv, and Fv′, are measured from the baseline. Figure

reproduced from Reference 8, with permission.

leaf is now exposed to a short actinic pulse ofhigh PPFD (typically less than 1 s at severalthousand μmol m−2 s−1), QA will be maxi-mally reduced and the maximal fluorescencelevel, Fm, is observed (Figure 1). PSII re-action centers with reduced QA are referredto as being ‘closed’. The difference betweenFm and Fo is defined as the variable fluores-cence, Fv. The ratio of Fv/Fm can be used

Open center:photosystem II(PSII) reactioncenter in which theprimary quinoneacceptor of PSII,QA, is oxidized andcapable ofphotoreduction

to estimate the maximum quantum yield ofQA reduction, i.e., PSII photochemistry, fromthe simple model of Butler (21). The fluores-cence emission from a leaf, F, is defined byI.Aleaf .fractionPSII .φF , where I is the incidentPPFD on the leaf, Aleaf is the proportion ofincident PPFD that is absorbed by the leaf,fractionPSII is the fraction of absorbed PPFDthat is received by PSII and φF is the quantum


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Closed center:PSII reaction centerin which QA isreduced and unableto performphotochemistry

I: PPFD incident onthe leaf

Aleaf : proportion ofincident PPFD onthe leaf that isabsorbed by the leaf

fractionPSII : fractionof absorbed PPFDthat is received byPSII

yield of fluorescence. φF is defined by kF/(kF + kH + kPP), where kF , kH , and kP arethe rate constants for the decay of excitationenergy in PSII by fluorescence, heat loss, andphotochemistry, respectively, and P is the frac-tion of PSII reaction centers that are open. AtFo, PSII reaction centers are maximally open,P = 1, and the fluorescence quantum yield,φFo, is given by kF/(kF + kH + kP). At Fm, thePSII reaction centers are maximally closed,P = 0, and photochemistry cannot occur, thuskPP = 0 and the fluorescence quantum yield,φFm , is given by kF/(kF + kH ). Thus, φFv /φFm

is given by (φFm – φFo )/φFm = kP/(kF + kH +kP), which shows that this ratio estimates themaximum quantum yield of PSII photochem-istry. Assuming that I, Aleaf , and fractionPSII areconstant for measurements of Fo and Fm, thenFv/Fm can be used to estimate the maximumquantum yield of PSII photochemistry. Thissimple model requires a number of other as-sumptions that are not necessarily correct forall situations (15). For example, fluorescenceat both Fo and Fm is assumed to be emittedfrom a homogeneous system where all the ex-cited states of the chlorophylls are the same.Clearly this is generally not the case; conse-quently, Fv/Fm should not be considered toprovide a rigorous quantitative value of thequantum yield of PSII photochemistry (15).However, Fv/Fm does provide a very useful rel-ative measure of the maximum quantum yieldof PSII primary photochemistry; Fv/Fm valuesfor nonstressed leaves are remarkably consis-tent at ca. 0.83 (14).

When plants are exposed to abioticand biotic stresses in the light, decreasesin Fv/Fm are frequently observed. This issuch a widespread phenomenon that Fv/Fm

measurements provide a simple and rapidway of monitoring stress. Unfortunately, thereasons for stress-induced decreases in Fv/Fm

are often complex. Stressing photosynthetictissues in the light can result in increasesin nonphotochemical quenching processes,which decrease Fm. Such quenching maynot recover during a short period of darkadaptation, or even overnight, and results

in decreases in Fv/Fm (1, 2). However,identification of the intrinsic causes of suchdecreases can often be difficult. In many stresssituations increases in nonphotochemicalquenching can often be accompanied byphotoinactivation of PSII reaction centers,which then dissipate excitation energy asheat rather than as photochemistry (79).Photoinactivation can lead to oxidativedamage and loss of PSII reaction centers (4),both of which are associated with an increasein Fo (19, 90). However, caution must beexercised when attempting to interpret thesignificance of decreases in Fm or increases inFo that occur as a result of a stress treatments.These fluorescence levels are determinedboth by the physicochemical properties ofPSII and the optical properties of the leaf.Unfortunately, during many stress treat-ments, especially when changes in leaf waterstatus occur, the optical properties of theleaf can change markedly and modify Aleaf .Changes in fractionPSII can occur owing tochanges in thylakoid membrane structure andorganization. Such modifications will resultin changes in Fo and Fm that are independentof changes in φFo and φFm . In such situations,absolute changes in Fo and Fm cannot beused with confidence to indicate loss of PSIIreaction centers or increases in nonphoto-chemical quenching. However, when ratiosof fluorescence parameters, such as Fv/Fm,are considered, the influence of changes inAleaf and fractionPSII are canceled out andchanges in the ratio are indicative of changesin the ratio of quantum yields of the twoparameters; for example Fv/Fm is defined by(I.Aleaf .fractionPSII .φFv )/(I.Aleaf .fractionPSII .φFm )= (φFv /φFm ).

In many ecophysiological studies it is sug-gested that stress-induced decreases in Fv/Fm

imply that the photosynthetic efficiency of theleaves under ambient light conditions is com-promised. This is not necessarily the case, be-cause the quantum yield of PSII photochem-istry under ambient light may be considerablybelow the observed Fv/Fm value, which esti-mates the maximum quantum yield of PSII

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photochemistry, not the yield at which PSIIis operating under the ambient light (see be-low). The maximum quantum yield of PSIIphotochemistry is only achieved at very lowambient light levels.

Light-Adapted State

A leaf in continuous actinic light has a flu-orescence level termed F ′, which rises to themaximal fluorescence level, Fm

′, when the leafis exposed to a brief saturating light pulse thatmaximally reduces QA (Figure 2). A primenotation (′) used after a fluorescence param-eter indicates that the sample is exposed tolight that will drive photosynthesis, i.e., ac-tinic light. The difference between Fm

′ andF ′ is designated Fq

′ and results from quench-ing of Fm

′ by PSII photochemistry. The ra-tio Fq

′/Fm′ is theoretically proportional to the

quantum yield of PSII photochemistry priorto application of the saturating light pulse(37). Genty and coworkers empirically con-firmed this theory from mass spectromet-ric measurements of oxygen evolution (38).For leaves exposed to actinic light the quan-tum yield of PSII photochemistry is equiva-lent to the quantum yield of linear electronflux (LEF) through PSII reaction centers, andhereafter is referred to as the PSII operatingefficiency. Measurements of Fq

′/Fm′ provide a

rapid method to determine the PSII operat-ing efficiency under different light and otherenvironmental conditions; Fq

′/Fm′ has previ-

ously been termed �F/Fm′ and φPSII in the

literature.There are a number of potential sources

of error associated with measurements ofFq

′/Fm′, which can be important when eval-

uating changes in PSII operating efficiency.These sources of error can also be a problemwhen measuring dark-adapted Fv/Fm. The re-lationship between Fq

′/Fm′ and the true quan-

tum yield of PSII photochemistry can be af-fected if PSI contributes significantly to themeasurements of the fluorescence parameters(41, 57, 97). When using Fq

′/Fm′ to deter-

mine the quantum yield of PSII photochem-

LEF: linear electronflux

istry all the measured fluorescence is assumedto originate from PSII. Although this is truefor variable fluorescence, it is not the case forFo if fluorescence is monitored at wavelengthsabove 700 nm (70, 85). PSI is generally as-sumed to make a negligible contribution tofluorescence at wavelengths below 700 nm.Unfortunately, most commercial instrumentsmeasure a significant amount of fluorescenceat wavelengths above 700 nm. The PSI con-tribution to Fo at wavelengths above 700 nmhas been estimated at ca. 30% and 50% forC3 and C4 leaves, respectively (41, 97). Con-sequently, decreases in Fq

′/Fm′ will occur and

therefore give estimates of PSII operating ef-ficiency that are lower than the true values. AsPPFD increases Fq

′/Fm′ decreases (Figure 3),

but PSI fluorescence yield remains reasonablyconstant (25), thus the PSI contributions thatresult in depression of Fq

′/Fm′ and the con-

sequent errors that lead to underestimationof PSII operating efficiency become propor-tionally greater. Measurement of fluorescenceat wavelengths below 700 nm minimizes sucherrors by markedly reducing the PSI contri-bution to the signals (41, 97). However, mea-surements at these shorter wavelengths resultin an increase in the contribution of fluores-cence from the upper regions of the leaf be-cause the probability of reabsorption of emis-sions at the shorter wavelengths is greater thanfor emissions above 700 nm (71).

Another error can arise in estimations ofFq

′/Fm′ via the use of saturating light pulses

that induce multiple turnovers of PSII reac-tion centers, as is the case with most commer-cial instruments. Such saturating pulses canresult not only in the reduction of QA, but alsoin the reduction of plastoquinone to plasto-quinol. Plastoquinone, but not plastoquinol,is a quencher of chlorophyll fluorescence. Adecrease in plastoquinone during the applica-tion of the saturating light pulse will result in adecrease in quenching and an overestimationof Fm

′ that can be as large as 20% (62, 105).Such errors are significant only in leaves withhigh plastoquinone/plastoquinol ratios priorto application of the saturating light pulse,


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PPFD (µmol m–2 s–1)

0

NP

QNPQ

200 400 600 800 1000 1200

1.0

0.8

0.6

0.4

0.2

0

2.0

1.5

1.0

0.5

0

Fq'/F

m',

Fq'/F

v', F

v'/F

m',

qL

qL

Fq'/F

m'

Fv'/F

m'

Fq'/F

v'

Figure 3The responses of photosystem II (PSII) operating efficiency (Fq

′/Fm′), maximum PSII quantum

efficiency (Fv′/Fm

′), the fraction of the maximum PSII efficiency that is realized in the light (Fq′/Fv

′), thefraction of PSII reaction centers that are open (qL), and nonphotochemical quenching (NPQ) in a tobaccoleaf to increasing photosynthetically active photon flux density (PPFD). The leaf was kept in anatmosphere containing 100 μmol mol−1 CO2 and 2% O2 to reduce CO2 assimilation and eliminatephotorespiration, respectively. Data taken from Reference 63 with permission.

which is the case at very low light levels, andeven then overestimates of Fq

′/Fm′ will be less

than 10% (6).Fortunately, errors in the measurement of

Fq′/Fm

′ due to PSI fluorescence and plasto-quinone quenching are small in many cases.The frequently observed linear relationshipbetween Fq

′/Fm′ and the quantum yield of

CO2 assimilation with increasing light inleaves from a wide range of species in whichphotorespiration was absent or suppressed(e.g., 23, 24, 27, 29, 37, 39, 40, 51, 58, 59, 60)matches what is theoretically predicted (seebelow). Also, the yield of oxygen evolutionfrom PSII determined by mass spectrometryis linearly related to Fq

′/Fm′ (38). If large er-

rors in the measurement of Fq′/Fm

′ existed,then such linear relationships would not beobserved. However, it is possible that errorscould be more significant in leaves with un-

usual pigment or plastoquinone contents andcaution should be exercised in such situations.

RELATIONSHIP BETWEENPHOTOSYSTEM II OPERATINGEFFICIENCY, LINEARELECTRON FLUX, ANDCO2 ASSIMILATION

The operation of linear electron flux (LEF)from water through PSII and PSI to elec-tron acceptors requires similar electron fluxesthrough the reaction centers of both PSII andPSI. When the quantum yield of PSI pho-tochemistry and PSII operating efficiency aremeasured simultaneously over a range of lightintensities, linear relationships between thetwo parameters are observed frequently (34,35, 39, 43, 44, 45, 57). In mature C4 leaves,where CO2 assimilation is the main sink for

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ANRV342-PP59-05 ARI 27 March 2008 1:28

the products of LEF (i.e., ATP and NADPH)(29), the PSII operating efficiency should bedirectly related to the quantum yield of CO2

assimilation, φCO2 (37). Such linear relation-ships between the PSII operating efficiencyand φCO2 have been observed over a rangeof light intensities (37, 58, 59, 60, 103), overa range of atmospheric CO2 concentrations(37, 103), and during induction of photosyn-thesis when dark-adapted leaves are exposedto actinic light (37). When photorespirationis inhibited in mature C3 leaves by reduc-tion of the atmospheric oxygen from 21% to2% and CO2 assimilation is the only majorsink for ATP and NADPH, a linear relation-ship is also observed between PSII operatingefficiency and φCO2 (23, 24, 27, 39, 40, 51).These observations demonstrate that PSII op-erating efficiency is a very good monitor ofLEF.

In principle, the linear relationship be-tween PSII operating efficiency and LEFallows the use of Fq

′/Fm′ to estimate the

noncyclic electron transport rate throughPSII (ETR), where ETR = I.Aleaf .fractionPSII .(Fq

′/Fm′). As discussed above, care should

be taken when determining and interpretingFq

′/Fm′, but often difficulties arise in the ac-

curate determination of the other parametersinvolved in the estimation of ETR. Aleaf isfrequently assumed to be 0.84, i.e., 84% ofincident PPFD is assumed to be absorbed byleaves. This assumption may be reasonable formany mature green leaves, but is not alwaysthe case and large deviations from this valuecan frequently occur (30, 47, 53). Aleaf shouldbe measured using a integrating sphere with alight source similar to that used to drive pho-tosynthesis and a spectroradiometer or quan-tum sensor. Similarly, fractionPSII for leaves isfrequently assumed to be 0.5, which is unlikelyto be the case in many situations. AlthoughfractionPSII has been estimated for leaves, theprocedure is not straightforward and involvesnumerous assumptions (67, 68, 83). Anotherproblem is that leaves of many species ac-cumulate nonphotosynthetic pigments, suchas anthocyanins, which can markedly mod-

ETR: electrontransport ratethroughphotosystem II

ify not only Aleaf but also fractionPSII ; this isoften the case when leaves experience envi-ronmental stresses during development. Un-fortunately, commercial modulated fluorom-eters automatically calculate values of ETR byassuming that leaves have values of Aleaf andfractionPSII of 0.84 and 0.5, respectively, oftenleading to substantial errors in calculations ofETR. ETR values calculated by such instru-ments should not be used unless the assumedvalues of Aleaf and fractionPSII have been vali-dated for the leaves being measured. In caseswhere such validations have not been made,changes in Fq

′/Fm′ should be used only to de-

termine changes in the relative quantum yieldof LEF and not used to estimate differencesin ETR.

If the allocation of the ATP and electronsthat result from LEF to sinks other than CO2

assimilation is negligible or constant, thenPSII operating efficiency also provides a goodrelative measure of the quantum yield of CO2

assimilation. The relationship between thePSII operating efficiency and the quantumyield of CO2 assimilation (φCO2 ) is defined byφCO2 = (Fq

′/Fm′).fractionPSII . (1/k), where k is

the number of electron equivalents producedby LEF required to reduce one molecule ofCO2. For C3 leaves in which photorespirationis inhibited and other electron sinks are neg-ligible k is assumed to be 4. If k and fractionPSII

are constant then Fq′/Fm

′ is a good indica-tor of changes in φCO2 . In many cases k andfractionPSII will not remain constant betweentreatments and Fq

′/Fm′ should not be used to

monitor changes in φCO2 . The value of k isdependent upon the proportion of reductantsproduced by LEF used for CO2 assimilation.k will change when other sinks for these re-ductants change relative to CO2 assimilation.Differences in k occur in leaves at differentstages of growth and in response to environ-mental stresses. In C3 leaves large changes ink occur with changes in intracellular CO2 andO2 concentrations, which modify the relativerates of CO2 assimilation and photorespira-tion. The difficulties in the accurate determi-nation of k and fractionPSII preclude the use of


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estimations of φCO2 from Fq′/Fm

′ to calculateactual rates of CO2 assimilation from (I. Aleaf .φCO2 ). However, relative changes in rates ofCO2 assimilation can be evaluated from esti-mations of φCO2 provided that k and fractionPSII

are constant.A linear relationship between PSII operat-

ing efficiency and φCO2 is not found in manysituations. Linearity is lost if the proportionof electrons consumed by CO2 assimilationrelative to other metabolic processes changes.In such cases Fq

′/Fm′ should not be used to

estimate changes in φCO2 unless the relation-ship between Fq

′/Fm′ and φCO2 has been de-

termined for the particular system under in-vestigation. This is the case for C3 leaveswhen photorespiration is operating; the ratioof PSII operating efficiency to φCO2 increaseswith increasing photorespiratory activity rel-ative to carbon assimilation (39, 42, 45). Envi-ronmental stresses can induce large increasesin the PSII operating efficiency:φCO2 ratio.For example, when the leaves of some C4

species develop at suboptimal growth temper-atures, PSII operating efficiency:φCO2 is in-creased significantly (31, 36). These increasesare accompanied by increases in the levelsof antioxidants and activities of enzymes in-volved in scavenging reactive oxygen species,which suggests that an increased electron fluxto oxygen, relative to CO2 assimilation, is oc-curring via the Mehler reaction (31, 36). Simi-lar increases in PSII operating efficiency:φCO2

were observed in leaves of mangrove, a C3

species, growing at high temperatures in trop-ical Australia (22).

FACTORS THAT DETERMINEPHOTOSYSTEM II OPERATINGEFFICIENCY

PSII operating efficiency, Fq′/Fm

′, is given bythe product of two important fluorescence pa-rameters, Fv

′/Fm′ and Fq

′/Fv′ (37), where Fv

′ isequal to Fm

′ − Fo′ and is the variable fluores-

cence of the light-adapted leaf and Fo′ is the

minimal fluorescence level in the light whenQA is maximally oxidized (Figure 2). Fv

′/Fm′

estimates the maximum quantum yield of PSIIphotochemistry (hereafter termed maximumPSII efficiency) that can be achieved in thelight-adapted leaf when QA is maximally ox-idized. Consequently, this parameter can beused to assess the contributions of nonphoto-chemical quenching to changes in the PSIIoperating efficiency of leaves in the light.Fq

′/Fv′ provides an estimate of the fraction

of the maximum PSII efficiency that is ac-tually realized in the leaf under the environ-mental conditions during the measurement,and is hereafter termed the PSII efficiencyfactor. The PSII efficiency factor is nonlin-early related to the fraction of PSII reactioncenters with QA oxidized, i.e., the fraction ofPSII centers that are open, and is mathemat-ically identical to the frequently used coeffi-cient of photochemical quenching, qP. Fq

′/Fv′

is determined by the ability of the photosyn-thetic apparatus to maintain QA in the oxi-dized state, which is a function of the relativerates of QA reduction and oxidation. Determi-nation of Fv

′/Fm′ and Fq

′/Fv′ makes it possible

to assess whether changes in PSII operatingefficiency are attributable to changes in non-photochemical quenching or the ability of anexcited PSII reaction center to drive electrontransport.

Calculation of Fv′/Fm

′ and Fq′/Fv

′ requiresdetermination of Fo

′, which can often be dif-ficult. Fo

′ is usually measured by exposing theleaf to a pulse of weak far-red light, after re-moving the actinic light, to maximally oxi-dize QA (101). However, in many situationsmaximal oxidation of QA may not be achievedduring the far-red pulse and also nonphoto-chemical quenching can partially relax, thusresulting in an overestimation of Fo

′ (6). Thisproblem can be overcome by calculating Fo

′

from values of Fm′ at the point of measure-

ment and dark-adapted values of Fo and Fm

using Fo′ = Fo/[(Fv/Fm) + (Fo/Fm

′)] (92).Maxwell & Johnson (78) suggested that thiscalculation should not be used if leaves arestressed and significant photoinhibition hasoccurred. However, a problem exists onlyif Fm is measured after Fm

′ and recovery

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from photoinhibition occurs during the darkadaptation period prior to measurement ofFm (6).

The PSII operating efficiency of a leaf de-creases as PPFD increases owing to decreasesin both Fv

′/Fm′ and Fq

′/Fv′ (Figure 3). How-

ever, the relative contributions of these twoparameters can change markedly with increas-ing PPFD. Generally, increases in nonpho-tochemical quenching, indicated by decreasesin Fv

′/Fm′, saturate at much lower light levels

than decreases in Fq′/Fv

′, which demonstratesthat a decrease in the ability to oxidize QA, notan increase in nonphotochemical quenching,is the major factor that determines the largechanges in PSII operating efficiency at highlight intensities. Also, increases in the PSIIoperating efficiency during induction of pho-tosynthesis when a dark-adapted maize leaf isexposed to actinic light are primarily associ-ated with increases in Fq

′/Fv′ and not changes

in Fv′/Fm

′ (92). This finding demonstrates thatthe ability of processes downstream of PSII toutilize the products of LEF, rather than non-photochemical quenching, is most importantin the regulation of the induction of photo-synthesis in this leaf.

The rate of consumption of NADPH andATP are major factors that determine PSII op-erating efficiency in many situations. Changesin carboxylation efficiency, the rate of re-generation of ribulose 1,5-bisphosphate, thesupply of CO2 from the atmosphere to thesites of carboxylation via the stomata, pho-torespiration, and the rate of transport ofcarbohydrates out of the cell can all influ-ence the rate of NADPH and ATP utilization(Figure 4), and consequently the PSII oper-ating efficiency. Many environmental stressesimpact on CO2 assimilation, although thesites of photosynthesis limitation during thesestresses can be quite varied. Stress-induceddecreases in stomatal conductance, carbonmetabolism, and transport processes can alldecrease PSII efficiency. The specific mech-anisms by which a restriction in metabolicturnover can result in decreases in PSII oper-ating efficiency are not fully understood. In-

creases in NADPH and ATP decrease LEFand the rate of QA oxidation, which can bemonitored by decreases in Fq

′/Fv′. However,

acidification of the thylakoid lumen as ATPlevels increase also results in an increase innonphotochemical quenching and a decreasein Fv

′/Fm′ (see section on Nonphotochemical

Quenching, below).

Photochemical Quenching

An important factor in determining the prob-ability of PSII photochemistry is the redoxstate of QA, i.e., the fraction of PSII reactioncenters that are open and capable of pho-tochemistry. Frequently, the PSII efficiencyfactor (or the mathematically equivalent qP) isused to estimate the redox state of QA. Unfor-tunately, in most situations the relationshipbetween the PSII efficiency factor and thefraction of PSII centers in the open state isnot linear and consequently changes in Fq

′/Fv′

(or qP) cannot simply be used to estimate theredox state of QA. The relationship betweenthe PSII efficiency factor and the fraction ofopen PSII centers is only linear if there isnegligible excitation energy transfer amongindividual PSII complexes and associatedantennae. This is the ‘puddle model’ in whicheach PSII reaction center and its associatedantenna cannot transfer excitation energy tothe antennae of other PSII reaction centers. Itis widely accepted that this is not the case andexcitation in PSII antennae can be competedfor by a number of reaction centers (21,69, 72). If all the PSII reaction centers areconsidered to be embedded within a singleantennae matrix and are capable of receivingexcitation energy from antenna pigmentsthroughout the matrix (‘lake model’), then therelationship between Fq

′/Fv′ and the redox

state of QA is curvilinear (7, 52). However,the degree of curvilinearity is dependent notonly upon the fraction of PSII centers that areopen but also on the amount of light-inducednonphotochemical quenching that is occur-ring; for a fixed oxidation state of QA increases


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in nonphotochemical quenching decrease thecurvilinearity (7).

Assuming a lake model for PSII, the redoxstate of QA is linearly related to the fluores-cence parameter (Fq

′/Fv′)(Fo

′/F ′), which hasbeen termed qL (63). Consequently, if an ac-curate assessment of the redox state of the QA

pool is required then qL, and not Fq′/Fv

′ (or

qP), should be used. When the PSII operat-ing efficiency is modified by exposing leavesto a range of PPFDs, although the patterns ofchange of Fq

′/Fv′ and qL with increasing PPFD

are similar, values of qL are always lower thanfor Fq

′/Fv′ (or qP), and at high PPFDs qL values

can be almost half of Fq′/Fv

′ (63) (Figure 3).Consequently, large errors can occur when

Photosynthesis

Photorespiration

O2

O2+ 4H

Leaf

Mesophyll cell

Chloroplast

Thylakoid

PSI

2H+

2H

H+

H+

NADP + H+ NADPHFd

2H+N

O2+ 4H 2H

PQH

PQ

2

Translocation

SugarsCarbon metabolites

Carbon metabolites

Starch

Photosyntheticcarbon

reductioncycle

Other chloroplastmetabolism

CO2

CO2

CO2

CO2

Stomate

ATP ADP + Pi

PC

2H2O

ATP

ase

Cyt bfPSII

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estimating changes in the redox state of QA

using Fq′/Fv

′, rather than qL, especially at highlight intensities.

Nonphotochemical Quenching

Although Fv′/Fm

′ can be used to evaluate thecontribution of changes in nonphotochemicalquenching to changes in PSII operating effi-ciency, levels of nonphotochemical quench-ing are often assessed by the parameter NPQ.NPQ is calculated from (Fm/Fm

′) − 1 (13) andestimates changes in the apparent rate con-stant for excitation decay by heat loss inducedby light relative to this rate constant in thedark (65). Because NPQ compares nonphoto-chemical quenching from a dark-adapted leafat Fm to that at Fm

′ for the leaf exposed to ac-tinic light, NPQ values can only be comparedfor leaves that have similar nonphotochemicalquenching characteristics in the dark-adaptedstate, e.g., leaves with similar Fv/Fm values.Changes in NPQ are nonlinearly related toand rise to higher values than Fv

′/Fm′ for a

given change in nonphotochemical quench-ing (Figure 3). Consequently, changes inNPQ do not allow evaluation of the propor-tion of changes in PSII operating efficiencythat are attributable to changes in nonphoto-chemical quenching.

Nonphotochemical quenching in leavescan consist of three components: energy-dependent quenching, qE, photoinhibitory

quenching, qI , and state transition quenching,qT (65). Researchers have resolved nonpho-tochemical quenching into qE, qI , and qT

from analyses of the relaxation kinetics ofthese quenching components in the dark (49,98, 106). However, care must be taken whenattempting to quantify the contributions ofthese components because the characteristicsof their relaxation kinetics can vary as aresult of changing environmental conditionsimposed on leaves. Generally, in nonstressedleaves under moderate to saturating lightqE is the major component, and qI becomesprominent at light levels well in excess of thatrequired to saturate photosynthesis or whenstresses severely restrict the consumptionof reductants produced by photosyntheticelectron transport. Quenching associatedwith state transitions, qT , is important only atlow light levels, but can be very significant inalgae (3, 33). Development of qE is associatedwith quenching in the PSII antennae owingto the acidification of the thylakoid lumenresulting from electron transport (66). Thisacidification results in activation of violaxan-thin de-epoxidase (109) and protonation ofsome carboxylic acid residues of the PsbS,a protein associated with the PSII antennae(74, 75) (Figure 5). Protonation of PsbSand binding of zeaxanthin to PSII producesconformational changes in the antennaethat result in increases in the quantumyield of thermal dissipation of excitation

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 4Relationships between photosynthetic electron transport, carbon metabolism and transport, and CO2supply. Electron transport, driven by the excitation of photosystem I (PSI) and photosystem II (PSII),results in the reduction of NADP to NADPH and the accumulation of protons in the thylakoid lumen.The resulting proton motive force is used to make ATP by driving protons back across the membranethrough ATP synthase (ATPase). Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzesthe assimilation of CO2 with ribulose 1,5-bisphosphate (RuBP) in the carboxylation reaction of thephotosynthetic carbon reduction cycle in the chloroplast stroma. Stomata regulate the diffusion of CO2from the atmosphere to the sites of carboxylation. Other reactions of the photosynthetic carbonreduction cycle utilize NADPH and ATP to produce triose phosphates, which are required for thesynthesis of carbohydrates. NADPH and ATP are also used in a range of other chloroplast metabolicactivities, e.g., nitrogen and sulfur metabolism and lipid and pigment synthesis. Rubisco can also catalyzethe oxygenation of RuBP in the process of photorespiration, which also involves consumption ofNADPH and ATP by the photosynthetic carbon reduction cycle. Abbreviations: Cyt bf, cytochrome b6fcomplex; Fd, ferredoxin; PC, plastocyanin; PQ, plastoquinone; PQH2, plastoquinol.


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OH

HO

O

O

Violaxanthin

OH

HO

Zeaxanthin

Zeaxanthinepoxidase

Violaxanthinde-epoxidase

PSIIZH+

Low light

Increasing light

Acidification ofthylakoid lumen

High light

ActivationLow rate of heat lossLow q

E

High rate of heat lossHigh q

E

PSIIV

Protonation

PsbS

PsbS

Figure 5Mechanism of light-induced energy-dependent quenching of excitation energy in photosystem II (PSII).At low light that is limiting for photosynthesis a xanthophyll pigment, violaxanthin (V), is associated withthe PSII antenna and PSII has a low rate of heat loss and consequently a low level of energy-dependentquenching, qE, which is an important component of nonphotochemical quenching (NPQ). At higherlight intensities increased electron transport results in acidification of the thylakoid lumen. When thelumen pH drops below ca. 6 violaxanthin de-epoxidase is activated and converts violaxanthin tozeaxanthin (Z) and PsbS becomes protonated. The zeaxanthin associated with PSII is an efficientquencher of excitation energy in the PSII antenna and the rate of heat loss from PSII increases, whichincreases qE. When light intensity decreases deprotonation of PsbS occurs and zeaxanthin epoxidaseconverts zeaxanthin back to violaxanthin, which decreases qE.

energy (50, 65, 95). Photoinactivation ofPSII and zeaxanthin-related quenchingcan be involved in the development of qI

(65).

More detailed analyses of nonphotochem-ical quenching can resolve the excitationenergy fluxes into light-induced quenchingprocesses and non–light-induced quenching

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 6Imaging the heterogeneity of photosynthetic activities of leaves, individual cells, and chloroplasts.(a) Image of Fq

′/Fm′ for a mildly water-stressed leaf of a Japanese anemone (Anemone × hybrida)

collected from a local park on a warm and windy day and exposed to an actinic photosynthetically activephoton flux density (PPFD) of 200 μmol m−2 s−1. This image demonstrates the large heterogeneity inphotosynthetic activity across the leaf. The colored bar indicates the range of Fq

′/Fm′ values. (b) Image of

Fq′/Fm

′ of chloroplasts in pair of stomatal guard cells of an attached leaf of Tradescantia albiflora exposedto a PPFD of 250 μmol m−2 s−1. Values of Fq

′/Fm′, Fv

′/Fm′, and Fq

′/Fv′ shown for two individual

chloroplasts demonstrate the heterogeneity of photosynthetic activity between chloroplasts in similarcells; this is primarily attributable to differences in Fq

′/Fv′. (c--h) Images taken from a pair of guard cells

of an attached leaf of Commelina communis with the stomate open (c–e) and after closure by decreasing therelative humidity ( f–h). (c, f ) are reflected light images; (d, g) are images of Fm

′. (e, h) Images of Fq′/Fm

′at a PPFD of 150 μmol m−2 s−1 showing the large decrease in PSII operating efficiency that occurs onclosure of the stomata. ( j ) Reflected light image from an intertidal benthic biofilm collected from a saltmarsh mud flat at Colne Point, Essex, UK and (k) image of Fq

′/Fm′ from these cells demonstrating the

very large differences in the PSII operating efficiency between species. A number of different species canbe identified in the biofilm: Gyrosigma limosum (1); Euglena sp. (2); Plagiotropis vitrea (3); Pleurosigmaangulatum (4); and Navicula sp. (5). Images in (b–h) are taken from Reference 7, with permission; imagesin ( j ) and (k) are taken from Reference 94 with permission of copyright holder, American Society ofLimnology and Oceanography.

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ANRV342-PP59-05 ARI 27 March 2008 1:28

0.50.2

0.60.3

25 µm

1

2

3

54

k

d e

hgf

0.3 0.5

a

c

j

20 µm

b Fq'/F

m' = 0.40

Fv'/F

m' = 0.54

Fq'/F

v' = 0.73

Fq'/F

m' = 0.31

Fv'/F

m' = 0.59

Fq'/F

v' = 0.53


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processes (46, 63). The quantum yield ofquenching due to light-induced processes,φNPQ, can be calculated (63):

φNPQ = 1 − F ′q

F ′m

− 1Fm−F ′

mF ′

m+ 1 + F ′

qF ′

v.

F ′o

F ′ .(

FmFo

− 1) (1)

Because the sum of the quantum yields ofPSII photochemistry (φPSII), light-inducedquenching processes, and non–light-inducedquenching processes is assumed to equal 1, thequantum yield of non–light-induced quench-ing processes, φNO, can be estimated fromφNO = 1 – (Fq

′/Fm′) – φNPQ (63).

IMAGING OF FLUORESCENCE

The development of instruments capable ofimaging chlorophyll fluorescence has pro-vided a powerful tool to resolve spatial hetero-geneity of leaf photosynthetic performance(86, 91). Photosynthetic heterogeneity hasbeen identified in many situations, e.g., dur-ing induction of photosynthesis (20, 92), withchanges in carbohydrate translocation (80),at the onset of senescence (108), in responseto changes in leaf water status (82, 87, 107)(Figure 6a), chilling (48) and ozone (73)stresses, wounding (99), and infection withbacteria (12, 16) and fungi (100, 104). Non-imaging fluorescence measurements wouldoften not detect such heterogeneity. Imagingof appropriate fluorescence parameters canprovide information about the causes of theheterogeneity. During induction of photosyn-thesis in a maize leaf, large changes in the de-gree of heterogeneity of the PSII operatingefficiency occur (7). Similar patterns of het-erogeneity are found in the images of Fq

′/Fm′

and Fq′/Fv

′, which are not seen in the Fv′/Fm

′

images. Consequently, the heterogeneity is at-tributable to differences in the ability of cellsto oxidize QA, which results from an inabil-ity to consume NADPH and ATP in CO2

assimilation.

For C3 leaves in which photorespiration isinhibited, the mean PSII operating efficiency(determined from images of Fq

′/Fm′) is lin-

early related to φCO2 (determined from gasexchange), which allows quantitative visual-ization of the spatial distribution of photo-synthesis (40). From gas exchange measure-ments made in conjunction with fluorescenceimaging, Meyer & Genty (81) determinedthe relationship between PSII operating ef-ficiency and intercellular CO2 concentration(Ci) and constructed images of Ci from imagesof Fq

′/Fm′. This approach has made it possi-

ble to map the two-dimensional distributionof Ci across leaves to study the lateral diffu-sion of CO2 in leaf tissues (84). However, thisprocedure requires the assumption of spatiallyhomogenous light absorption across the leafarea under study, which may not be the case inmany leaves, such as when leaves have devel-oped under stress or have been infected withpathogens.

High-resolution imaging has been used toexamine the photosynthetic activities of sin-gle cells and even individual chloroplasts (93).The responses of electron transport in indi-vidual stomatal guard cells and adjacent mes-ophyll cells in intact leaves to changes in light,atmospheric CO2 concentration, and humid-ity have been studied by imaging Fq

′/Fm′ (71)

(Figure 6c–h). The isolation of individualchloroplasts from images of the guard cellsof Tradescantia albiflora exposed to a PPFD of250 μmol m−2 s−1 indicates that they showa wide range of mean Fq

′/Fm′ values, rang-

ing from 0.27 to 0.43 (7). Such differencesin the PSII operating efficiencies of individ-ual chloroplasts are primarily attributed todifferences in the ability to utilize ATP andreductants, not to differences in nonphoto-chemical quenching, because differences inFq

′/Fv′ are considerably greater than those for

Fv′/Fm

′ (7) (Figure 6b). Imaging has also re-solved large differences in photosynthetic per-formance among benthic diatom species inbiofilms (94) (Figure 6j,k). One problem inthe production of images of Fq

′/Fm′ of such

biofilms is that some of the cells can move

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between the time that the images of F ′ and Fm′

are captured, and therefore these cells mustbe moved within one image to allow themto be superimposed before calculation of theFq

′/Fm′ image (91, 94).

Fluorescence imaging can be used inscreening procedures to identify organismswith modified photosynthetic performance, ashas been done for algae (11, 88) and Arabidopsis(89) mutants. Perturbations of metabolic pro-cesses not directly involved in photosyntheticmetabolism often induce changes in fluores-cence parameters (9, 96), which can be usedto screen for such perturbations. The devel-opment of commercial fluorescence imaging

instruments that can image areas greater than100 cm2 allows the screening of large numbersof plants simultaneously. High-throughputscreening of metabolic perturbations in Ara-bidopsis seedlings can be achieved by grow-ing plants in the wells of 96-well microtiterplates (9) (Figure 7). Fluorescence imagingcan also be used to estimate leaf area andconsequently estimate growth; one applica-tion is the early growth of seedlings thathave planophile, nonoverlapping leaves, suchas Arabidopsis, from images of Fm. The totalarea from which the fluorescence is emittedis directly related to the leaf area that con-tains chlorophyll (9). However, for plants in

1 2 3 4 5 6 7 8 9 10 11 12

a

b

d

c

79.072.0

Figure 7High-throughput screening for metabolic perturbations in Arabidopsis. (a) Five-day-old Arabidopsis plantsin a 96-well plate 24 h after being treated with 0.4 (rows 5 and 11), 0.8 (rows 4 and 10), 4 (rows 3 and 9),and 8 (rows 2 and 8) mM Imazapyr, a herbicide that inhibits acetolactase synthase and consequently thesynthesis of branched chain amino acids. Untreated controls are in rows 1, 6, 7, and 12. (b) Images ofFv/Fm for these plants. (c,d ) Enlargements of the plants and images outlined by the yellow boxes in(a) and (b) respectively. Although differences in growth cannot be detected by visual observation, thereare very large differences in the images of Fv/Fm between the control and herbicide-treated plants. Takenfrom Reference 9 with permission of copyright holder, American Society of Plant Biologists.


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which the leaves overlap significantly or theleaves cannot be positioned normal to thecamera this relationship does not necessar-ily hold. In such cases examination of the re-

lationship between the area of fluorescenceand leaf growth is required before the useof fluorescence to screen for differences ingrowth.

SUMMARY POINTS

1. Dark-adapted Fv/Fm is a useful relative measure of the maximum quantum yield ofphotosystem II (PSII) photochemistry, but does not provide an accurate quantitativevalue of this quantum yield.

2. Fq′/Fm

′ is a useful relative measure of the quantum yields of PSII photochemistry andlinear electron flux through PSII.

3. Fq′/Fm

′ can be used to estimate the rate of linear electron transport. This requiresaccurate determination of the photosynthetically active photon flux density (PPFD)incident on the leaf, the proportion of incident PPFD that is absorbed by the leaf,and the fraction of absorbed PPFD that is received by PSII.

4. In certain circumstances Fq′/Fm

′ is a good indicator of changes in the quantum yieldof CO2 assimilation by the leaf, φCO2 , but it should not be used to estimate absoluterates of CO2 assimilation.

5. Many metabolic and physiological factors influence Fq′/Fm

′ by determining the rateof consumption of ATP and NADPH.

6. Fluorescence imaging can identify spatial heterogeneity of photosynthetic perfor-mance and offers new possibilities for understanding the operation and regulation ofphotosynthesis. Fluorescence imaging can also be used to image other physiologicalphenomena indirectly if they interfere with the operation of photosynthesis and itsassociated metabolism, e.g., herbicide effects and stomatal heterogeneity.

FUTURE DIRECTIONS

1. Chlorophyll fluorescence parameters can now be easily measured and provide usefulprobes of photosynthetic performance in vivo and the extent to which performanceis limited by photochemical and nonphotochemical processes.

2. Coupling of appropriate fluorescence measurements with other noninvasive tech-niques, such as absorption spectroscopy (5), gas exchange (76), and thermal imaging(107), can provide insights into the limitations to photosynthesis under given condi-tions.

3. Fluorescence imaging has great potential in future plant screening programs andother areas of applied plant physiology. The selection of appropriate fluorescenceparameters and careful calibration of their changes with key plant performance indi-cators is important. Once a satisfactory calibration has been achieved, fluorescencecan offer rapid, high-throughput screening. The use of automated sampling devicesin conjunction with increases in the areas than can be imaged will enhance the ratesof screening procedures even further.

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DISCLOSURE STATEMENT

The author is not aware of any biases that might be perceived as affecting the objectivity ofthis review.

ACKNOWLEDGMENTS

I would to thank the many colleagues with whom I have had discussions during the preparationof this review, in particular Steven Driever, Jeremy Harbinson, David Kramer, Tracy Lawson,James Morison, Phil Mullineaux, and Don Ort. Many of my studies using chlorophyll fluo-rescence have been supported by the Biotechnology and Biological Research Council and theNatural Environment Research Council in the UK.

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Annual Review ofPlant Biology

Volume 59, 2008Contents

Our Work with Cyanogenic PlantsEric E. Conn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1

New Insights into Nitric Oxide Signaling in PlantsAngelique Besson-Bard, Alain Pugin, and David Wendehenne � � � � � � � � � � � � � � � � � � � � � � � � � 21

Plant Immunity to Insect HerbivoresGregg A. Howe and Georg Jander � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 41

Patterning and Polarity in Seed Plant ShootsJohn L. Bowman and Sandra K. Floyd � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 67

Chlorophyll Fluorescence: A Probe of Photosynthesis In VivoNeil R. Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 89

Seed Storage Oil MobilizationIan A. Graham � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �115

The Role of Glutathione in Photosynthetic Organisms:Emerging Functions for Glutaredoxins and GlutathionylationNicolas Rouhier, Stephane D. Lemaire, and Jean-Pierre Jacquot � � � � � � � � � � � � � � � � � � � � �143

Algal Sensory PhotoreceptorsPeter Hegemann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �167

Plant Proteases: From Phenotypes to Molecular MechanismsRenier A.L. van der Hoorn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �191

Gibberellin Metabolism and its RegulationShinjiro Yamaguchi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �225

Molecular Basis of Plant ArchitectureYonghong Wang and Jiayang Li � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �253

Decoding of Light Signals by Plant Phytochromesand Their Interacting ProteinsGabyong Bae and Giltsu Choi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �281

Flooding Stress: Acclimations and Genetic DiversityJ. Bailey-Serres and L.A.C.J. Voesenek � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �313

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Roots, Nitrogen Transformations, and Ecosystem ServicesLouise E. Jackson, Martin Burger, and Timothy R. Cavagnaro � � � � � � � � � � � � � � � � � � � � � � �341

A Genetic Regulatory Network in the Development of Trichomesand Root HairsTetsuya Ishida, Tetsuya Kurata, Kiyotaka Okada, and Takuji Wada � � � � � � � � � � � � � � � � � �365

Molecular Aspects of Seed DormancyRuth Finkelstein, Wendy Reeves, Tohru Ariizumi, and Camille Steber � � � � � � � � � � � � � � �387

Trehalose Metabolism and SignalingMatthew J. Paul, Lucia F. Primavesi, Deveraj Jhurreea, and Yuhua Zhang � � � � � � � �417

Auxin: The Looping Star in Plant DevelopmentRene Benjamins and Ben Scheres � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �443

Regulation of Cullin RING LigasesSara K. Hotton and Judy Callis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �467

Plastid EvolutionSven B. Gould, Ross F. Waller, and Geoffrey I. McFadden � � � � � � � � � � � � � � � � � � � � � � � � � � � � �491

Coordinating Nodule Morphogenesis with Rhizobial Infectionin LegumesGiles E.D. Oldroyd and J. Allan Downie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �519

Structural and Signaling Networks for the Polar Cell GrowthMachinery in Pollen TubesAlice Y. Cheung and Hen-ming Wu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �547

Regulation and Identity of Florigen: FLOWERING LOCUS T MovesCenter StageFranziska Turck, Fabio Fornara, and George Coupland � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �573

Plant Aquaporins: Membrane Channels with Multiple IntegratedFunctionsChristophe Maurel, Lionel Verdoucq, Doan-Trung Luu, and Veronique Santoni � � � �595

Metabolic Flux Analysis in Plants: From Intelligent Designto Rational EngineeringIgor G.L. Libourel and Yair Shachar-Hill � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �625

Mechanisms of Salinity ToleranceRana Munns and Mark Tester � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �651

Sealing Plant Surfaces: Cuticular Wax Formation by Epidermal CellsLacey Samuels, Ljerka Kunst, and Reinhard Jetter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �683

Ionomics and the Study of the Plant IonomeDavid E. Salt, Ivan Baxter, and Brett Lahner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �709

vi Contents

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Alkaloid Biosynthesis: Metabolism and TraffickingJorg Ziegler and Peter J. Facchini � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �735

Genetically Engineered Plants and Foods: A Scientist’s Analysisof the Issues (Part I)Peggy G. Lemaux � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �771

Indexes

Cumulative Index of Contributing Authors, Volumes 49–59 � � � � � � � � � � � � � � � � � � � � � � � �813

Cumulative Index of Chapter Titles, Volumes 49–59 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �818

Errata

An online log of corrections to Annual Review of Plant Biology articles may be foundat http://plant.annualreviews.org/

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2008. Baker. Chlorophyll Fluoresncence, A Probe of Photosynthesis in Vivo

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