PhotosystemI I electron flowas ameasure for
phytoplanktongrossprimary production
FotosysteemI I elektronentransport alseenmaatvoor
debruto primaire produktie van fytoplankton
Promotor:
dr.W.J.Vredenberg
Hoogleraar indeplantenfysiologiemetbijzondere aandachtvoorfysische
aspecten
Co-promotor: dr.J.F.H.Snel
Universitair docent bijdeleerstoelgroep plantenfysiologie
Corine Geel
PHOTOSYSTEM I I ELECTRON FLOW AS A MEASURE FOR
PHYTOPLANKTON6ROSS PRIMARY PRODUCTION
Proefschrift
ter verkrijgingvandegraadvandoctor
opgezagvanderector magnificus
vandeLandbouwuniversiteitWageningen
dr.CM.Karssen
inhetopenbaar teverdedigen
opwoensdag 12november 1997
des namiddagstevier uur indeAula
' S * ; fïT-51/
Theworkpresented inthisthesiswasperformed attheWageningenAgricultural University,
Department of Plant Physiology,Arboretumlaan 4,6703 BDWageningen,TheNetherlands
Theinvestigationsweresupported bytheCornelusLelyfoundationandthe NationalInstituteof
Coastaland Marine Management(RIKZ)
Coverdesignfront page:HenkGeel
Coverdesignbackpage:comicstrip "Heinz"from RenéWindigandEddydeJong
ISBN 90-5485-789-7
BIBLIOTHEEK
LANDBOUWUNIVERSITEIT
WAGENINGEN
^NO?^ö|,z^y^
STELLINGEN
1 Deminimale fluorescentie van eukaryote algen iseengoede maat voor het
lichtabsorberend vermogen vanfotosysteem II, mits despectrale verdeling
van het fluorescentie-excitatielicht identiek isaan dievan het omgevingslicht.
Dit proefschrift.
2 Zolang eronduidelijkheid blijft bestaan overhetbruto ofnettokarakter van
fotosynthese metingen met behulpvan 14Cincubatie isheteenduidiger om
fotosynthese metingen tebaseren opmetingen van defytoplankton electronen
flux metbehulpvan fluorescentie.
Dit proefschrift.
Williams et al,JPlankton Res 18(10):1961-1974 (1996).
3 Dedoving van demaximale fluorescentie in hetdonkerbij gelijkblijvende
efficiëntie, zoals waargenomen door Ting en Owens,isbeter teverklaren met
een zogenaamde 'statetransition' dan metdedoor hen gesuggereerde
chlororespiratie.
Dit proefschrift.
Ting and Owens, Plant Physiol 101:1323-1330 (1993).
4 Het feit dat anno 1996slechts weinig mensen zich persoonlijk
verantwoordelijk voelen voorhetmilieu wordtgeïllustreerd door onder andere
detoename van hetprivé gebruikvanelektriciteit en auto,endetoename van
dehoeveelheid huishoudelijk afval.
Milieubalans, RIVM (1997).
5 Het gesuggereerde vertrek vanhethoofdkantoor vanPhilips uit Eindhoven
omdat Eindhoven zichtever vandefinanciële centrabevindt is een
ontkenning van demogelijkheden van dehuidige communicatie middelen aan
deontwikkeling waarvan Philips zelf bijgedragen heeft.
6 Deminimaliseringvanmineralenverliezeninderundveehouderij kantot
gevolghebbendatrunderenhethelejaaropstalmoetenblijven staan.Uit
oogpuntvandierwelzijn enhumanerecreatieisditeenongewenste
ontwikkeling.
7 Degenediezichergertaaneenanderisbezigdeanderteverbiedenwat hij/zij
zichzelfnietkantoestaan.
MarinusKnoope,Humanist6/7/8(1997).
8 Mensendielouterindegeestvanhetcollectivismegrootgebracht worden
kunnenonvoldoendehunindividualiteitontwikkelenenzullenalsreactie
hieropeerderegoïstischgedragvertonendanmensenbijwiehun
individualiteitwelvoldoendeontwikkeldis.
GuusKuijer, hetgeminachtekind.
9 Wietijdenshaarzwangerschapnietopdehoogtegesteldwilwordenvan
allerleirampscenario's aangaandezwangerschap,bevallingenhethebbenvan
jongekinderenkanhetbestehetgezelschapvanmensenmetjongekinderen
vermijden.
Stellingenbehorendebijhetproefschrift "PhotosystemIIelectronflow asa
measureforphytoplankton grossprimaryproduction".
Wageningen, 12november 1997.
CorineGeel
VOORWOORD
Ikwilgraag eenaantalmensenbedankenvoor hunbijdrage aandetotstandkoming vandit
proefschrift.Zowelvoor mijnwetenschappelijke alsvoor mijnpersoonlijkeontwikkeling hebikde
afgelopen 6jaren alseenleerzame,somsmoeizame,maarookleuketijdervaren.
Vooreerstwilikiedereenbedankenvoordegezelligheid,waardoor ikaltijdwelweerzinhadom
naarWageningen,envoortweekorteperiodes,naarZeelandaftereizen.
Voorwetenschappelijke entechnische ondersteuning wilikbedanken:deledenvande
fotosynthese groep,metnameJanSnel,WilmaVersluis,Wim Vredenberg ennatuurlijkVictor
Curwiel,TijmenvanVoorthuysenenGertSchansker;WillemBuurmeijervoor de programmatuur;
vandewerkplaatsJan vanKreelenRuthvander Laan;destudenten Marjola Maasv.d.Weijde,
KittyJansen (ikwacht nogsteedsopeenfoto!) enMartineSegers;mijnbroer Martinvooreen
deelvandefiguren;vandebegeleidingscommissie BartAlthuis,KeesPeeters,HansHofstraaten
JaccoKromkamp;uit Middelburg enJacobahaven:LouisPeperzak, MartinSteendijk,Arjen
Pouwer,VincentEscaravageenTheoPrins;for thedevelopment oftheXe-PAMfluorometer
whichopened manypossibilitiesfor myresearch project Iwanttothankdr Schreiber.
Gerritwilikbedankenvoor zijngeweldig relativeringsvermogen envoor alleeigenschappen die
hijinroyale matetentoonspreidt endieikweleensteweinigbezit.
CONTENTS
Listof abbreviations
1 General introduction
1.1 Primaryproduction inmarineecosystems
1.2Photosyntheticenergyconversion
1.2.1 Light reactions
1.2.2Darkreactions
1.3Regulationof PSIIelectronflow
1.3.1 Light harvesting
1.3.2 PSIIelectronflow
1.4Chlororespiration
1.5Measurement ofprimary production
1.5.1 Increaseinbiomass
1.5.2Carbonuptake,oxygenevolution
1.5.3 PSIIelectronflow
1.6Outlineofthethesis
1
2 Simple determination of photosynthetic efficiency and photoinhibition of Dunaliella
tertiolecta bysaturating pulsefluorescence measurements
2.1 Introduction
2.2 Measurement ofsaturating pulsefluorescence
2.3 Experimental application
2.4 Resultsanddiscussion
2.4.1 Measurement of dpps,, andqP
2.4.2 Determination of photoinhibition
2.4.3 Modificationsfor phytoplankton measurement
2.5 Conclusions
11
3 Estimation of oxygen evolution by marine phytoplankton from measurement of the
efficiency of photosystem IIelectron flow
26
3.1 Introduction
3.2 Materials andmethods
3.3 Results
3.4 Discussion
3.5 Conclusions
4 Measurement of phytoplankton electron flux using aXe-PAMfluorometer
4.1 Introduction
4.1.1 ComparisonoftheXe-PAMandthe PAMfluorometers
4.1.2 Phytoplanktonelectronflux
41
4.2 Materials andmethods
4.3 Resultsanddiscussion
4.3.1 Spectraldistributionoftheefficiencyof PSIIelectronflow
4.3.2 F0 asameasureof PSIIexcitation
4.3.3 Determination oftheefficiency of PSIIelectronflow
4.4Conclusions
5 The relation between carbonfixation and photosystem IIelectronflow in marine
phytoplankton
5.1 Introduction
5.2 Materials andmethods
5.3 Results
5.4 Discussion
5.5 Conclusions
6 Estimation of photosynthetic performance of marine phytoplankton in a model
ecosystem by means of chlorophyll fluorescence
6.1 Introduction
6.2 Materials andmethods
6.3 Results
6.4 Discussion
7 The minimal fluorescence inthe dark as a measurefor photosystem IIexcitation in the
light: Effects of chlororespiration and state transitions
7.1 Introduction
7.2 Materials andmethods
7.3 Results
7.4 Discussion
7.4.1 Effectsofchlororespirationonthefluorescenceyield
7.4.2 Effectsofstatetransitions onthefluorescenceyield
7.4.3 HowmuchdoesF0'(thelight adaptedstate) differfrom F0(thedarkadapted state)?
7.5 Conclusions
8 General discussion
8.1 Introduction
8.2 Fluorescencemeasurementsonphytoplanktonsamples
8.3 Therelation betweenPSIIelectronflow,oxygenevolutionandcarbonfixation
8.3.1 Oxygenevolutionandcarbonfixation
8.3.2 Minimalfluorescence, F0
8.4 Phytoplanktonelectronflux or phytoplankton carbonflux?
8.5 Recommendationsforfuture research
Summary
56
67
83
92
99
Samenvatting
References
CurriculumVitae
101
103
110
LIST OF ABBREVIATIONS
A
B
C
chl
D
DPFD
E
F
F0; F 0 '
FM; F M '
Fv
Jc
JE
JEL and JEM
J0
k
Kd
LHC
NSDP
nPS H
PCF
PEF
PFD
POF
PPC
PPE L and PPE M
PPO
PQ
PS
qN
qP
QA
SDR
(j)Po
(tppsn
Opsii
constant usedinthephotosynthesis model
constant usedinthephotosynthesis model
constant usedinthephotosynthesis model
chlorophyll aconcentration
initialslopeofphytoplankton carbonfluxasafunctionof phytoplankton
electronflux
daily photon flux density from 4 0 0 to 7 0 0 nm
constant used in the photosynthesis model
actual fluorescence in light adapted state
minimal fluorescence in dark adapted and light adapted state respectively
maximal fluorescence in dark adapted and light adapted state respectively
variable fluorescence (F M -F0)
rate of carbon fixation
rate of PSII electron flow
rate of PSII electron flow, determined in the laboratory and in the
mesocosm respectively
rate of oxygen evolution
constant used in the photosynthesis model
apparent diffuse light attenuation coefficient
light harvesting complex
net system daily production of oxygen in the mesocosm
number of PS II reaction centers
phytoplankton carbon flux
phytoplankton electron flux
photon flux density from 4 0 0 to 7 0 0 nm
phytoplankton oxygen flux
daily primary production measured as carbon fixation
daily primary production measured as photosystem II electron flow, using
laboratory data and mesocosm data, respectively
daily primary production determined from the oxygen concentration
plastoquinone
photosystem
non-photochemical quenching of fluorescence
photochemical quenching of fluorescence
primary electron acceptor of photosystemII
system day respiration
the photochemical yield of open PS II reaction centers
apparent quantum yield of photosystem II electron transport
absorption cross section of photosystemII
1 GENERAL I N T R O D U C T I O N
1.1 Primary production in marine ecosystems
Themarineecosystemcontains photoautotrophic andheterotrophic organisms.
Phytoplanktonandmacrophytes belongtothephotoautotrophic organisms andconvert
inorganic carbonintoorganic matter bymeansof photosynthesis.Thisorganic matter,whichis
thebasisofthefoodweb,isconsumed byheterotrophic organisms likefor exampleprotozoans,
shrimps,musselsandfishesandbymicrobeswhichdecomposeorganic materialbacktothe
basicsubstances likeC02,N03'andP043~.
Primaryproduction isdefinedastheincreaseinphytoplanktonorganic matter andcanbe
expressed intwoways.Grossprimary production istheincreaseinorganic matter includingthe
organic matter lostinrespiration.Netprimaryproductionistheorganicmatter producedminus
theorganic matter respired.Primaryproduction canbemeasuredastheincreaseinbiomassbut
canalso bederivedfromthechlorophyllcontent,photosynthetic carbonfixationoroxygen
evolution.
TheNorthSeaecosystem ingeneralcontainsthefollowing phytoplankton groups:Cyanophyta
(cyanobacteria);Chrysophyta,especiallythe Prymnesiophyceae(includingflagellatesand
coccolithophorids) andthe Bacillariophyceae (diatoms);Phyrophyta (dinoflagellates) and
Cryptophyta (Kirk 1994, Reidetal 1990,Hofstraat etal 1994b).Chlorophyta (green algae) are
onlyscarcelypresent intheNorthSea.Thecyanobacteriaarepresent inlarge numbers,butare
of minor importancefor primary production becauseoftheir smallsize.Bothdiatoms,flagellates
[P/iaeocyst/s) anddinoflagellates aremajor primary producers oftheNorthSeaecosystem.The
Cryptophytaareoccasionally significant componentsofthephytoplankton incoastalwaters (Kirk
1994).
1.2Photosynthetic energy conversion
Inphotosynthesis lightenergyisusedto convertwater (H20) andcarbondioxide (C02) into
carbohydrates:
C02+H20
~ 8 / i V > (CH20) + 02
Photosynthesis consistsoftwogroups of reactions:the light reactions andthedarkreactions.
Ineucaryotesthelight reactionstakeplaceatthethylakoid membranesinthechloroplast,the
dark reactionstake placeinthestromaofthe chloroplast.
1.2.1 Lightreactions
Thelight isabsorbed byantennapigments inthe light harvesting complexes (LHC) andthe
fluorescence
öFf-»0" -fc~
(0EC->z4p68Q
[CH20]
X
-f—±J
H,0-^
NADPH.
V2ÛJ
,ATP-
• H+
COj
*ADP+Pi
lumen
stroma
Figure 1.1. Simplifiedschemeofthephotosyntheticelectrontransport.Z:intermediaryelectrondonor
forP680+; pheo:pheophytin,intermediaryelectronacceptorofP680; Fe-S: Rieskeiron-sulphurprotein,
partofthecytochrome4 /complex; X: intermediaryelectronacceptorofP700; Fd: ferredoxin; CF0-CF,:
ATPasecomplex.Othersymbolsareexplainedinthetext.
excitation energyisdeliveredtotheP700andtheP680 reactioncentre molecules,imbeddedin
photosystem I(PS I)andII(PS II) respectively (Fig.1.1). Inthereactioncentreof PS IandPS II
thisexcitation energyinducesachargeseparation.AtPSIIthebound plastoquinone QAisthe
primary stableelectron acceptor.Aftertaking up2electronsfromQAand2protonsfromthe
stromathesecondary acceptorQBdissociatesfromitsbindingsiteat PS IIanddiffuses into
thylakoid membrane,becoming partofthemobileplastoquinone (PQandPQH2) pool.Upon
bindingof PQH2tothecytochrome Z»6/complex theprotonsarereleasedintothelumenandthe
electrons aretransferredtocytochrome / Fromcytochrome /theelectronsgotoplastocyanine
(PC)andarefinally usedto reduceP700+.P680+isreducedbyelectronsderivedfromthesplitting
ofwaterwhich results intotheliberationofprotons andoxygenattheoxygenevolvingcomplex
(OEC)of PS II.Theproton gradientwhichisformedisusedbytheATPasetoformATP.
ChargeseparationinPSIinitiatesaseriesofredox reactionswhichresult inthereductionof
the mobileelectron acceptorferredoxin.NADP+takesup2electronsfromferredoxin (transferred
byferredoxin NADP+reductase) and2protonsfromthestromatoform NADPH+ H+ (Hall and
Rao 1987).
Incontrasttolinearelectronflowas describedabove,incyclicelectronflowtheelectronsfrom
ferredoxinare nottransferredto NADP+butaretransferredto PQ,accompanied bythe uptake
of 2protonsfromthestroma per 2electrons.Theformed PQH2canbeoxidised,releasingthe
protons inthe lumenanddeliveringtheelectronsto cytochrome f.Cytochrome /"donatesthe
electrons againto PSIviaPC.Thestoichiometryincyclicelectronflow is 1proton translocated
perelectron.
Light harvesting complexesinphytoplankton
Light harvestingoccursbylight harvesting pigmentcomplexes.TheLHC ofhigher plants
contains chlorophyll aandbasthemainantennapigmentsandthethylakoidsareorganisedin
granastacks.Alotofalgaedifferfrom higher plantsintheir light harvesting pigments andinthe
organisation ofthe LHC.Ingreenalgaethe LHCiscomparabletothat inhigher plantsthoughthe
granastacking islessprominent. Inthe mainbrownalgalgroups (coccolithophorids,flagellates,
diatomsanddinoffagellates) thethylakoids aregrouped per 3.Coccolithophorids,flagellatesand
diatomscontaintheantennapigmentschlorophyll a,c,and c,andthecarotenoidfucoxanthin.
Dinoflagellates containchlorophyll aand ç,andthecarotenoid peridinin (Kirk 1994).The
carotenoidsfucoxanthinandperidininarebothefficient inlight harvesting (HaxoandBlinks
1950;Goedheer 1970; PrézelinandHaxo 1976).TheCryptophytacontainchlorophyll aand c2
andphycobilisomeswitheither phycoerythrins or phycocyanins aslight harvesting pigments.The
thylakoid membranes areorganized inlooselyassociatedpairs (Kirk 1994).Thethylakoidsof
the procaryotic Cyanobacteriaareinvaginations ofthecellmembrane.Cyanobacteriacontain
chlorophyll aandphycobilisomeLHCswhichcontainphycocyanin pigments (Kirk 1994).
Efficiencyof PSIIelectronflow
Theefficiency of PSIIelectronflow,asusedhere,isdefinedastheefficiencywithwhich
excitationenergyisusedfor photochemistry driving PSIIelectronflow. InFigure 1.2aschematic
drawingofthe processesoccurring atthe LHC andatthe PSIIreactioncentre,basedonthe
bipartite modelofButler (1978),isgiven.Light absorbed bythe LHCwillresult inanexcited
chlorophyll molecule.Thisexcitedstate (anexciton) canmovearoundbetweenchlorophyll
moleculesinthe LHC. Theexcitonreleases itsenergyviadifferent pathways:non-radiative energy
dissipation,e.g.heat release (rateconstant kD),radiativeenergydissipation,e.g.fluorescence
(rateconstant kp)or itcantransfer itsenergytothe PSIIreactioncentre (rateconstantkT).
Energydeliveredto the reactioncentrewill resultintheexcitationofthe reactioncentre
chlorophyll P.P* cantransfer anelectrontothe primaryelectron acceptorA,whichdetermines
the rateof primary photochemistry (rate constant kp).Bothstates P+-A" andP-A" (inwhichP+is
reduced bythe primary donor of PSII) arecalledclosed reactioncentres.Aslongasareaction
centre isclosed,it isnotableto perform another electrontransferto QA, e.g.kp iszero: P*
cannot perform photochemistry andwilltransfer itsenergybacktothe LHC(kj; P* canalso
relaxvianon-radiative energydissipation inthe reactioncentre (kd), buttherateconstant ofthis
process isverysmallmakingtheyieldneglectable under normalconditions.Inhibitionof
photochemistry willtherefore result inanincreaseofheat release,D,andfluorescence,F.The
fluorescenceyieldwhenallreactioncentresareopeniscalledtheminimalfluorescence,F0. The
fluorescenceyieldwhenallreactioncentresareclosediscalledthe maximalfluorescence, FM.
Figure 1.2. primaryreactionsofPS IIaccordingtothe
modelofButler (1978).Symbolsareexplainedinthetext.
Themodelassumesthat allrateconstantsareconstant,except kpwhichcanbecomezero ina
closed reactioncentre;later observations haveshownthatk,, canchangeinresponseto
acidificationofthethylakoid lumen (seesection 1.3.2). Morerecent researchhasshownthat
althoughthebipartite modelof Butler (1978) isbasedonaincomplete mechanism,onams
timescalethemodelgivesgood results (forareviewseeDau 1994).
Twopathwaysthat are not includedinthe Butler modelarecharge recombinationandcyclic
electronflowinPSII.ChargerecombinationfromthestateP+-A" backto P-Awasobservedto
occur at-196 °C(Butler etal 1973,Murataetal 1974) butat roomtemperature theforward
reaction ismuchfaster thanthe backreaction.Cyclicelectronflow inPSIIhasbeensuggestedto
proceedfromthestate P+-A"to P-Aviaelectronflowtothedonor sideof PSII(Prasiletal
1996).Thedetermination oftheefficiencyof PSIIelectronflow ishardlyinfluenced bycharge
recombination andcyclicelectronflow inPS II.
1.2.2Darkreactions
TheNADPHandATPformedinthelight reactions areusedto reducecarbondioxideto the
levelofcarbohydrate.Akeyreactioninthefixationofcarbondioxideisthe reactionofcarbon
dioxidewithribulose 1,5-bisphosphate (RuBP).Thisreactioniscatalysed bytheenzyme ribulose
1,5-bisphosphatecarboxylase oxygenase (rubisco) andit resultsintheformationof 2molecules
phosphoglycerate (PGA).
Dissipative dark reactions
Inthe presenceofoxygenrubiscocanalsoactasanoxygenasecatalysingtheoxygenationof
RuBPto 1PGAand 1moleculeof phosphoglycolate.Thisreactionistheinitiationof
photorespiration andisfavoured at highoxygenconcentrations and/or lowcarbondioxide
concentrations,andat hightemperatures. Phosphoglycolate isnot metabolised intheCalvin
cycle,but 2moleculesphosphoglycolate areconverted into 1molecule PGAunderthe
consumption of 3molecules0 2 andthe releaseof 1moleculeC02 (Tolbert 1979).Asinthe
photosynthetic light reaction,precedingtheformationof 2moleculesphosphoglycolate,2
molecules0 2 havebeenformedtheoverallstoichiometryofthephotosynthetic light reactions
andthe photorespiratory dark reactions is 1molecule0 2takenupper 1moleculeC02released.
Another non-productive pathwayisthe Mehler-reaction,alsonamedpseudo-cyclic electron
flow(Gimmler 1977). Inthe Mehler-reaction electrons (e) comingfromferredoxinare
transferredto oxygentoformthesuperoxide radical (402 + 4e~-> 402").Theelectrons needed
aregenerated inthephotosynthetic light reactions (2H20 -> 4e"+ 4H+ + 02).Thesuperoxide
radicalsare rapidlyconvertedtotheradicalH202and0 2 (4Cy + 4H+ ->•2H202+ 202) bythe
enzymesuperoxidedismutase.Thereforeatthispoint anuptakeofoxygenisfound (Asadaand
Takahashi 1987,Schreiber etal 1995a).Inchloroplasts H202isscavenged byascorbateforming
monodehydroascorbate (Foyer andLelandais 1993,Schreiberetal 1995a).Thisreactionis
catalysed byascorbate peroxidase.Themonodehydroascorbate whichisformed inthis reaction
isrecycled bymonodehydroascorbate reductase (theoverall reaction:2H202+ 4e"+ 4H+ ->•4
H20).Theelectrons neededfor reductionof monodehydroascorbate aregenerated inthe
photosynthetic light reactions (2H20-> 4e"+ 4H+ + 02). Inthetotal ofthese reactions 4
moleculesofoxygenand4moleculesof NADPHareformedbutalso4 moleculesofoxygenand
4 moleculesof NADPHareconsumed.Ithasbeensuggestedthattheprotontranslocation
associatedwiththeelectronflowto oxygenisimportantforthe regulationof photosynthesis
(Björkmanand Demmig-Adams 1995).Aspecialcaseofphotosynthetic regulation isthe
activation oftheCalvincyclewhichrequires atransthylakoid protongradient (Schreiber etal
1995a).
Nitrogen metabolism
Furthermore NADPHisnotonly usedincarbonassimilationbut,for example,nitrate reduction
intoammonium alsorequires NADPH.Ammoniumistheform usedinnitrogen metabolism (i.e.
aminoacidsynthesis).Fortheconversionof N03"to NH4+ 8electrons areneededwhichmight
represent asmuchasone-fourthoftheelectrons requiredforthefixationofC02 (Lindbladand
Guerrero 1993).
1.3 Regulation of PSIIelectron flow
1.3.1 Lightharvesting
Inhigher plantsandgreenalgaechangesinthelight environment result instatetransitions.
Thesestatetransitions involvechangesinenergydistributionfrom LHCII,themajor light
harvesting pigment proteincomplex,to PSIIandPSI.Thestatetransitions aremediatedby
phosphorylationof LHCII,resulting indisconnectionof LHCIIfrom PSIIandattachmentto PSI. A
reduced PQ-pool,dueto adisproportional excitationof PSII,causesactivation ofthekinase
whichphosphorylatesthelight harvesting complexconnectedtothe PSIIreactioncentre(LHC
II). Phosphorylated LHCIPsdisconnectfromthe PSIIreactioncentre,reducingtheamountof
excitationenergyavailablefor photochemistry inPSII(state II).Apermanently active
phosphatase dephosphorylates LHCII, resulting inthe reattachment of LCHIItothe PSIIreaction
centre inthecasethekinaseisinactivewhenthe PQ-poolisoxidised (state I)(Allen 1992).The
number of LHC'sdelivering excitationenergyto PSIIdeterminethefunctionalabsorptioncrosssectionof PSII.Atransitionfrom state IIto state Iistherefore accompaniedwithaparallel
increaseofthe minimalandthe maximalfluorescence (Malkinetal 1986).
Theregulationofenergydistribution inmarinephytoplankton species isasyet notas
extensively examinedasinhigher plantsthoughsomeaspects havebeendescribed inthe
literature. Forthediatom Phaeodactylumtricornutumitwasfoundthat 510nmlight,whichis
absorbed mainlybythefucoxanthinofthe major antennacomplex,isnotdelivered preferentially
to PSIIbut ismoreequallydistributed betweenthephotosystems (Owens 1986b).Theenergy
distribution betweenPSIIandPSI wassuggestedto beregulated byionicdistributions rather
thanbythe PQpool redox state.Furthermore,themechanism regulating energydistribution
betweenPSIIandPSIwasfoundto berather light intensity dependentthanlight quality
dependent (Owens 1986b). Regulationofenergydistributionto PSIandPSIIbystate
transitions independentofwavelengthbutdependentonthephotonfluxdensitywasalsofound
intheyellowgreenalgaPleurochlorismeiringensis(Bücheletal 1988,BüchelandWilhelm
1990). Inthebrownmacrophyte MacrocystisperiferaanincreaseofFM ofabout 20 %going
fromalight mainlyabsorbed byPSII(0.9W m"2540 nmlight) adaptedstateto alight mainly
absorbed byPSI(3.5W m'2700 nmlight,additionaltothelight II) adaptedstatewasfound
(Forketal 1991).Theeffect oflight IandIIonF0wasmuchsmaller.Thehalftimesofthe
transitions (about 10minfor astate I to IItransition,about 5minfor astate IIto Itransition) are
comparableto whatwasfoundfor higher plants (Allen 1992).Thetransitionfrom state Ito state
IIledto adecreaseof-196 °Cfluorescence of both PSIIandPSI.Forketal (1991) calculated
thedistribution of light IIbetweenPSIandPSIIandfoundanalmostevendistribution of lightII
to both photosystems inbothstates.Incyanobacteriastatetransitions dueto phosphorylationof
the phycobilisomes,werefoundto regulateenergydistribution (Allen 1992).Thetransition
however ismuchfaster thaninhigher plants (DominyandWilliams 1987). Recentresultssuggest
that statetransitions incyanobacteriaareaconsequence ofchangesintheintensityofthe
irradiance ratherthanthespectrum oftheirradiance (RouagandDominy 1994).
Theprocessofstatetransitions inhigher plants,greenalgaeandcyanobacteria isrelatively
wellunderstood.Theoccurrence andregulationofstatetransitions inimportant phytoplankton
groups (e.g.coccolithophorids,flagellates,diatomsanddinoflagellates) hasnotbeen
investigated ingreat detailasaretheconsequencesfor estimationof PSIIelectronflowfrom
chlorophyllfluorescence measurements.
1.3.2PSIIelectronflow
Atlowphotonfluxdensities (PFD)therateofphotosynthesis increasesalmost linearwiththe
photonflux density.Thisimpliesthat at lowPFDalltheabsorbed energyinprincipalcanbeused
for photochemistry withanearlyconstant efficiency.Athigh PFD'sthe rateofphotosynthesisis
saturated.Underthiscondition photochemistryworksatits maximalrateandexcessiveenergy
should bedissipatedto prevent damageonthephotosynthetic apparatus,inparticular PS II
(Critchley 1981). Besidesstorage inphotosynthetic productsthere are 3other waysto dissipate
energy:i) viaenergyconsumption inmetabolic processesthat donot result inenergystorage.
Anexampleisphotorespiration (Björkmanand Demmig-Adams 1995,section 1.2.2) butalso
Mehler reaction (seesection 1.2.2)issuggestedtofunctionin dissipation ofexcessiveenergy
(BjörkmanandDemmig-Adams 1995,Schreiber etal 1995a);ii) reemissionofphotonsas
fluorescence.However,theamountofexcitationenergywhichcanbedissipated influorescence
issmall (below4%ofthetotal) andcanbeneglectedformostpractical purposes;iii)thermal
dissipationoftheenergyinthepigment bed.Thisdissipation pathwayisveryimportantin
regulationofPSIIelectronflow (BjörkmanandDemmig-Adams 1995).
Inthelight aproton gradient acrossthethylakoid membraneisformed,whichinhigher plants
andgreenalgaeinducestheformation ofzeaxanthinfromviolaxanthin.Itissuggestedthat both
theproton gradient andthepresenceofzeaxanthininthylakoid membranes causeachangein
theconformationoftheassociationofthe pigment moleculesintheantennacomplex.This
conformational changeisresponsibleforanincreaseofthe heatreleaseintheantennacomplex
(viaanincreaseofkD)(Bilger andBjörkman 1994,BjörkmanandDemmig-Adams 1995).For
brownalgaeitissuggestedthat increasedheathreleaseintheantennacomplexiscorrelatedto
theconversionofdiadinoxanthintodiatoxanthin (Oilazolaetal 1994).Theincreaseofheat
release results inareductionoftheamountoffluorescence.Thereforefluorescence canbeused
to monitor bothchangesinphotochemistry andinheatrelease (Schreiber etal 1986,Krause
andWeis 1991).
1.4 Chlororespiration
Inchloroplasts of Chlamydomonasreinhardtii\n\\\W\or\ ofdark respiration (measured as an
increaseof ,802) duetoshort saturatingflasheswasfoundbyPeltier etal (1987). The
respirationwasinsensitivetoDCMU,aninhibitor ofphotosynthetic electrontransport,and
antimycinA,aninhibitor ofthecytochrome Z>6/complex,andwasinhibited bycyanide, an
inhibitor ofcytochrome oxidase.Thisendogenous respiratory process,whichwasnamed
chlororespiration,wassuggestedtomakeuseofanthylakoid NADPH-dehydrogenase,the PQpool,ahypotheticalcytochromeb/ccomplexandahypotheticalcytochrome oxidase (Peltierand
Schmidt 1991). Influorescence experiments itwasfoundthat antimycinAsignificantly increased
the redoxstateofPSIIacceptors indarkadapted Chlamydomonasreinhardtii(Ravenel and
Peltier 1991). NAPDH-dehydrogenase activityinthechloroplastwasconfirmed bySeidel-Guyenot
etal (1996) inP. meiringensisandbyCuelloetal (1995) inbarley.Thusfar,however,
cytochrome oxidaseactivity inthechloroplast hasnotbeendemonstrated.The electronflowin
thechlororespiratory pathwayissupposedtocreate atrans-thylakoidprotongradient. In P.
'tricornutumanincreaseofF0 andFM wasfoundafter additionofuncouplersofthe proton
gradient acrossthethylakoid membrane (CCCP andnigericin) andafter additionofantimycinAor
byanaerobiosis (TingandOwens 1993).ThisincreaseofF0 andFMwasexplainedbydissipation
or preventionofachlororespirationdependent pHgradient inthedark.ThedataofTing and
Owens(1993),however,suggest that FV/FM didnotincrease.Thiswouldbeexpectedtobethe
casewhenenergydependent quenching is involved.
Chlororespiration might decreasetheF0 andFM, observed inthedark,duetomembrane
energizationandit mightincreasetheapparent F0, observed inthedark,dueto reduction ofQA.
Therefore chlororespiration mightdisturb interpretation ofthefluorescence datawith respectto
photosynthesis.
1.5 Estimation of primary production
Primaryproductioncanbeestimated inseveralways,e.g.theincreaseinbiomass(cell
numbers,dryweight),biovolume,chlorophyll content,orviathedetermination ofthe
photosynthetic reactionbycarbonfixation,oxygenevolutionor byPSIIelectron transport.
1.5.11ncreaseinbiomass
Theincrease incellnumbers bycounting under amicroscope gives inpreciseinformationon
speciescomposition andcellnumbers.Cellscanbecounted more rapidlyainCoulter counter
which,however,cannot distinguish betweenphytoplanktoncellsandheterotrophic cells.In
Coulter counter measurementsthesphericequivalent diameter ofthecell isalsoestimatedand
thisinformation canbeusedto estimatethe biovolume.Withflowcytometry it ispossibleto
analyse individual cellsandusetheir opticalpropertiesfor identification.Inthisway
phytoplankton cellsweredistinguishedfrom heterotrophic cellsandaroughseparation ingroups
ofspecies hasbeenachievedbyHofstraat etal (1990).
Thechlorophyll concentration ofaphytoplanktonsampleisbestdetermined after extraction
viaabsorptionorfluorescence measurements.
Themeasured increaseinbiomassshould becorrectedfor thegrazing of heterotrophic
organisms. Furthermorefor allofthesemethods itshould benotedthat algaeareflexible
organisms andthesizeandgeometry ofanalgalcellisnot aconstant, neither isthechemical
compositionofthe cell.Therefore,the resultsobtainedwithmeasuring biomass increasedepend
bothonthemethodusedandonthephysiologicalstateofthealgaeinthesample.
1.5.2Carbonuptake, oxygenevolution
Anestimationofdetermining primary production ismeasuringthephotosynthetic conversion
ofinorganic carboninto organic matter (biomass).Thusfarthishasmainlybeendoneby
measuring carbon uptake (using labelled bicarbonate, l4C) oroxygenevolution.Oxygenevolution
measurementsgive net photosynthesis becausediscrimination betweenoxygenproduced atthe
oxygenevolvingcomplex of PSIIandoxygentakenupduring respiration (e.g.mitochondrial
respiration,photorespiration and Mehler reaction) cannot bemade.Carbonfixation
measurements canestimateeither grossor net photosynthesis depending ontheduration ofthe
incubationwith ,4C (Williamsetal 1996).
Net photosynthesis dataaccountfor photosynthetic and respiratory activity inthelightperiod.
Foraconversion ofthesedatainto primary productionthe respirationduringthenight period
should also betakeninto account.
/.5.3PSIIelectronflowbymeansofchlorophyllfluorescence
ThePSIIfluorescenceyielddependsontherateof photosynthesis. Basedonthis
phenomenon amethodwasdevelopedto measuretheefficiency PSIIelectronflow,(pPSI|,with
fluorescence parameters (Gentyetal 1989).TheefficiencyofPSIIelectronflowat anyirradiance
conditioncanbedeterminedfromtheactualfluorescence,F,measuredattheambient irradiance
andthemaximalfluorescence,FM', measuredwhenanadditionalmultipleturnover saturating
flashwasgiven:
I
Qpsii =
M
—
FM
(1.1)
Amorecomprehensive description ofthisderivation andofother parameters isgiveninChapter
2.Whentheefficiency of PSIIelectronflow isknown,the rateof PSIIelectrontransport canbe
calculated astheproduct of (p^,,andthephotonfluxdensityabsorbed byPS II.
1.6Outline of the thesis
InChapter 2thesaturating pulsemethodfor determining PSIIefficiency isreviewedand
appliedto cultures ofthegreenalga Dunaliella tertiolecta. Photochemical and non-photochemical
quenching andtheactualandmaximalefficiencyof PSIIweredetermined undervarying growth
light intensities.Efficiency of PSIIcorrelatedwellwiththegrowth ratesofthecultures. Itwas
concludedthatthesaturating pulsemethodispromisingfor determination of phytoplankton
growth,butthe lackofsensitivity ofthe PAM fluorometer,about afactor 1000too lowfor
application inthemarineenvironment,mightbeaseverelimitation.Afurther requirement isthe
establishment andcalibrationofthe relation between PSIIelectronflowandprimary production
ofthe major phytoplankton groups involved inmarine primaryproduction.
Chapter 3describes comparative measurements onthe relation between PSIIelectronflow
estimated as(p^,,timesthe photonfluxdensity (PFD) andphotosynthetic oxygenevolutionof5
marinealgalspecies,whicharemembersofthemainphytoplankton groups.Therelation
betweenoxygenevolution andPSIIelectronflowwasfoundto beapproximately linear at lowand
mediatephotonflux densities.Athighphotonfluxdensitiesthe relation isshownto benonlinear.Thenon-linearity isnot relatedto photorespiration butto aMehler-typeofoxygen
reductionor lightdependent respiration.Therelation betweenPSIIelectronflowandoxygen
evolution could bemodelled byincludinganextraoxygenuptaketerm inthemodelof Eilersand
Peeters (1988).Theinitialslopeoftherelation betweenthe rateof oxygenevolution andthe
rateof PSIIelectrontransport wasfoundto bespeciesdependent.Thisspeciesdependence
wassuggestedto berelatedto differences inPSIIabsorptioncross-section.
Theextremely sensitiveXe-PAMfluorometer, builtfor this project byDrU.Schreiber,is
employed inchapter 4to developamethodfor estimationoftheabsorption cross-section ofPS
IIusingtheminimalfluorescence F0. Theconcept ofphytoplanktonelectronfluxwasintroduced,
whichisthetotal amountof PSIIelectronflowinaphytoplankton sample.The relationship
betweenthephytoplankton electronfluxandthe rateofoxygenproduction ofthesame
phytoplankton samplewassimilarfor cultures of D. tertioledaandPhaeodactylumtricornutum.
InChapter 5thephytoplankton electronflux (PEF) iscomparedwiththephytoplankton carbon
flux (PCF) inculturesof 3marinealgalspecies.Therelation showedasimilar curvilinearityas
foundfor thecomparisonofthe rateofoxygenevolution andtherateof PSIIelectronflowin
Chapter 3.Theratioof PCF and PEF differed byafactor 1.5 whichissmallerthanthevariation
found inthe ratioofoxygen production andelectronflowinChapter 3.
InChapter 6 hard- andsoft-ware (Xe-PAMandhomebuild measuring unit;PPMONandPEF
concept) weretested underfieldconditions inasmalloutdoor artificial ecosystem (mesocosm)
where PSIIelectronflowwascontinuously monitoredfor threeweeks.2phytoplankton blooms
wereobserved andduringthese bloomsthe relation betweenPEFandPCFmeasured inthe
mesocosmwassimilartothat insamples measured inthe laboratory.
Chapter 7describes experimentsto assesaspectsof phytoplankton physiology,e.g.,
chlororespiration andstatetransitions,whichmightaffect theminimalfluorescence F0 and
consequentlytheestimationofPEF.Itisconcludedthat underourconditionstheerrorwasbelow
10%.
Finally,Chapter 8discusses prospectsandlimitations ofthefluorescence methodsweapplied
to determinetheprimary phytoplankton productioninamarineecosystem.Thedataobtained
withfluorescence measurements arediscussed inrelationtothoseobtainedfrom estimateson
primary carbonfixation or oxygenproduction.
10
2 SIMPLE DETERMINATION OF PHOTOSYNTHETIC
EFFICIENCY AND PHOTOINHIBITION OF ÙUNALIELLA
TERTIOLECTA BY SATURATING PULSE FLUORESCENCE
MEASUREMENTS
2.1 Introduction
Fluorescence measurements havebecomewidelyapplied inthedeterminationof
phytoplankton biomass.Basedonthe registrationoffluorescence emissionandexcitation
characteristics ofphytoplankton communities,taxacanberoughlyidentified (Yentschand
Yentsch 1979,YentschandPhinney 1985a,b,Hiltonetal 1989).Themeasurementof
chlorophyllfluorescence intensity hasfoundgeneralacceptancefortheestimation of autotrophic
phytoplankton biomass (Heaney 1978,Butterwick et al. 1982,FalkowskiandKiefer 1985).An
important advantageofthisapproach isthat insitumeasurements onphotosynthetic organisms
arepossible.Togain insight inthedistribution (bothhorizontally,over largeareas,andvertically,
inthewatercolumn) ofphytoplankton, insitumeasurementsareindispensable.
Itappears,however,that thecorrelationbetweenchlorophyll afluorescence intensityand
phytoplankton biomass,usuallyapproximated bychemicalmeasurement ofthetotal amountof
extracted chlorophyll a, is,inmanyinstances,notverygood (FalkowskiandKiefer 1985).In
particular,whenonetriesto correlatedataacquired underdifferent circumstances,e.g.at
different depths,indifferent water massesoratdifferent timesoftheday,thequantitative
agreement betweenthechlorophyll aconcentrations asdetermined byextraction andby
fluorescence maybepoor.Thisismainlyduetothefactthatthefluorescencewhichismeasured
predominantly stemsfrom chlorophyll amoleculesthat arepart ofthephotosynthetic apparatus
ofthe cell;thisapparatus isadynamicsystemwhichcanrespond rapidlyto changesinthe
environmentalcircumstances. Pollutant stress,light intensity andtemperature maythusstrongly
influencethefluorescenceyield (FalkowskiandKiefer 1985,Falkowskiet al. 1986, Lichtenthaler
andRinderle 1988).Thus,chlorophyll afluorescence intensity,whichcanbeexaminedby
determination oftheamountoffluorescence per moleculeofchlorophyll a(i.e.thefluorescence
quantumyield),canalsobeusedto obtaininformationontheconditionofthe photosynthetic
apparatus.Thismeansthat,inadditionto estimatesoftotal biomass,other information canbe
derivedfrom phytoplanktonfluorescencecharacteristics:estimatescanbeobtainedofthe
photosynthetic efficiency and,viathat,oftheprimary production andenvironmentalstress.These
aspectsareconsidered inmoredetailinthefollowingsection.
Thepresent paperwilldescribethe principlesandapplicationsofthesaturatingpulse
fluorescencetechniquefor determinationofphotosynthetic characteristics ofphytoplankton.This
method hasmainlybeenappliedto investigate higher plants,andcanbeusedto obtaina
quantitative estimateofthephotochemical efficiencyofthe photosynthetic system (Schreiber
1983,RengerandSchreiber 1986). Photoinhibitory effectsandtheamountsof photochemical
andnon-photochemical quenchingcanalsobedetermined (Schreiber etal 1986). Measurement
Publishedas:HofstraatJW, PeetersJHC, SnelJHF andGeel C (1994) MarEcolProgSer103:187-196
ofthephotochemicalefficiencycanbedonedirectly andissuitablefor in5/ii/application(Genty
etal 1989,FalkowskiandKolber 1990).Assuch,ithasimportant advantages overthe HCincorporation measurement,whichislargelyagreeduponasthestandard methodfor
determining photochemicalefficiencywhenthe phytoplanktonconcentration islow,asisgenerally
thecaseinmarineapplications (Peterson 1980,DringandJewson 1982). Forhigherplantsand
ineutrophiedsituations,oxygenmeasurements canalsobeapplied,whicharemuchmoreeasily
applicablefrom anexperimental pointofview (Bryanetal 1976). Determinationof
photoinhibitory effects andof photochemical andnon-photochemical quenching requires dark
adaptation prior tothemeasurement,andarehencelesssuitedfor dynamic insitu
measurements.
Experiments inthisstudyweredoneon Dunaliellatertioledalaboratory cultureswitha
dedicated,commercially availableinstrument thatwasdesignedfor measurement ofleaves
(Schreiber 1986).Theapplicability ofthesaturating pulsefluorescencetechniquefor marine
phytoplanktonwillbedemonstrated,andmodificationsthat arerequiredto maketheinstrument
suitablefor insitupurposeswillbediscussed.Firstly,however,theprinciples ofthesaturating
pulsefluorescence measurement aredescribed.
2.2 Measurement of saturating pulse fluorescence
Thefluorescencethat ismeasuredatambienttemperatures stemsalmost exclusivelyfrom
chlorophyll associatedwiththeantennaeofphotosystem (PS) II.Thefluorescent yieldof PSIis
lowbecause photochemistry hereisaparticularlyefficient competitor, inadditiontotheother
non-radiative decayprocesseslikethermalemissionandtripletformation,unless measurements
aredoneat lowtemperatures (Strasser andButler 1977,Briantaisetal 1986).Chlorophyll
associatedwiththephotochemical reactioncentresrepresents onlyaverysmallfractionofthe
total chlorophyll contentofphytoplankton and,inaddition,hasaverylowfluorescence quantum
yield.
Thefluorescenceyieldof PSIIchlorophyll,however,ishighlyvariable. Itisstrongly influenced
bythe physiologicalstateofthephytoplankton.Theexcitedstatesthat areproducedbylight
absorption canbedeactivated by4processes:(1) photosynthetic energyconversion, which
requiresenergytransfertothe reactioncenter andsubsequent electrontransport followedby
interactionwithPSI,(2) tripletformation, (3) radiationless processesand (4) radiativetransfer,
the processthat isaccompanied byfluorescence.Thefluorescenceemissionhenceprovides
informationonthe photochemical processesinPSII.Whenthephotochemical reactioncenters
are"open",i.e.availableto efficientlytraptheexcitedstateenergyandto beinvolvedin
photochemical energyconversion,theyieldofthe non-photochemical processes (2-4) willbe
low.On theother hand, whenthe reactioncentersareclosedthe photochemicalyield isverylow
andthenon-photochemical processesbecomeimportant.Thefluorescenceyieldthereforevaries
inverselywiththeyieldof photochemistry: itconsistsofaconstant part andavariablepart,which
isdetermined bythestateofthephotochemical reactioncenter (openorclosed).Thestateof
the reactioncentersisinfluenced byenvironmentalcircumstances (light history, nutrient status,
12
4 2>
3-
£ 1O -"
1
2
SP
qN=o
FVF„= ( - ^ >
q„=0
3
+AL
q N =0
_ (FM'-F)
qP= ( F M ' - F O ' )
4
1>q P >0
1>q N >0
_
SP
_ - , (FM'-FO 1 )
QN
(F^FoT
H
q P = 0
5
-AL
«
1>qN>0
+FR
1>qN>0
(FM'-F)
<t>p=
/vyy/i?2.1. Schematic representation ofthe saturating pulsefluorescence method (Van Kooten andSnel
1990). 5different statesofthe photosynthetic systemare definedinthecourseofafull experiment.At
the onsetofthe experiment the dark adaptedstates F0(1) andFH (2)are defined byusing modulated
measuring light (ML)only and anadditional saturating pulse (SP)respectively,forthe2parameters.
Next,thesampleisilluminatedwith actinic light (AL) andafluorescence induction (orKautsky) curveis
obtained. Inthesteady state F(3)andFM'(4)can be determined byagain measuringwith modulated
measuring light andanadditional saturating pulse,nowincombination withtheactinic light. Finally, the
actinic light sourceisextinguished andfar-red illuminationisappliedtoreach F0'(5).Todeterminethe
photochemical efficiency accordingtothe methodofGentyetal (1989) itissufficiently todetermineFM'
and Funder steady state conditions.
presence ofpollutant stress). Itisjust this dependence which makes simple, straightforward, in
s/Mluorescence intensity measurements difficult to useforphytoplankton chlorophyll
determinations.
Ontheother hand fluorescence measurements can beusedtomonitor the photosynthetic
processes.Thesaturating pulse method that isused inthis paper allows information onthese
processes tobeobtained byapplication ofarefined experimental scheme. InFigure 2.1 the
principles ofthe saturating pulse method areillustrated.Themethod isbased onthe application
of 3different excitation schemestothephytoplankton.The chlorophyll fluorescence ismeasured
using alowpower, modulated, light emitting diodeforexcitation.Thefluorescence inducedby
this probe beam isselectively registered byapplication ofphase resolved detection. Inaddition,
13
aconstant,variable intensity,whitelightsource maybeusedfor actinic illumination ofthe
sample.To investigatethe photosynthetic system,averyhigh intensity pulseofwhitelightmay
alsobeapplied,whichcansaturatethephotochemical reactioncenters.
Thefulltechnique showninFigure2.1 provides4usefulparameters (seealsoGentyetal
1989):
(1) Photochemical quenching,qP.Photochemicalquenchingoffluorescence iscaused bythe
transfer ofexcitedstateenergytothe photochemical reactioncenters,whereit isavailablefor
photochemical energyconversion.Indarkadapted phytoplanktonthe PSIIreactioncentersare
allopenandphotochemical quenchingismaximal.Thefluorescence intensityatthis pointis
designated F0. Whenashort pulseofintense light isgiven,allPSIIreactioncentersareclosed
withinafewhundred ms.Duringthepulsethefluorescence intensity reachesitsmaximumvalue
FM.Duringthe illuminationwiththeactinic lightsource,or under ambient light,qPcanbe
determined bymeasuringthenormaliseddifference betweenthemaximumandtheactual
fluorescenceyield:
FL-F
Qp = —;
:
(2.1)
whereFM'= themaximumfluorescence intensitythat isobtainedbysaturationofthe reaction
centers understeadystateconditions;F= the normalfluorescence intensity inthe steadystate;
andF0'= thefluorescence intensitythat isobtaineddirectly afterthe actinic lightsourcehas
beenswitchedoff; inaddition illumination byfar-red light isapplied,whichis predominantly
absorbed byPSIandresultsinreopening of intact PSIIreactioncentersbyreoxidationofthe
quinone acceptorQA.
(2) Non-photochemical quenching,qN. Non-photochemical quenching isusedto describeall
quenching processes of PSIIchlorophyllfluorescence processesthat arenot relatedto
photochemistry. It mainlyinvolvesnonradiative,dissipative,processesthat arefor instance
inducedbythebuild-up ofapHgradient acrossthethylakoid membrane ('ApH-quenching').
Non-photochemical quenching canonlybedeterminedwhenFMisavailablefrom dark-adapted
samples;it isdefinedasthenormaliseddecreaseofthemaximalfluorescenceyield,withrespect
tothedarkadaptedsituation:
F'
-F'
(3) Thephotochemicalyieldofopen PSIIreactioncenters,FV/FM. Thisratioequalsthe
product ofthe probabilities ofexcitationtransfer betweenantennaeand PSIIreactioncenter,
andviceversa (e.g.Butler 1978). Ifnon-radiativetransfer inthereactioncenterissignificantly
smallerthanthebacktransfertotheantennaepigments,theyieldofphotochemistry oftheopen
PSIIreactioncenters isgivenby:
14
_ FV _ FM
.
*'o - 7
—
F
0
(2 3)
-
—
Itappearsthat (|)p0isagood indicator of photoinhibition. Light induceddamagetothe PS II
reactioncenter leadsto loweringofthephotochemicalyieldofthe reactioncenters (Björkman
1987).Toassessphotoinhibition prolonged darkadaptation ofthesample mustbeapplied prior
tothe measurementtofully removetheeffects ofnon-photochemical quenching.Therecoveryof
the photosynthetic systemfrom photoinhibitory effects oftentakesalongtime,typicallymany
hours.
(4) Thephotochemicalefficiency of PSIIperabsorbed photon,or photonyield, <t>pS„. The
method isbasedontheassumptionthatthe photonyield isgivenbythe product ofthe effiency
ofanopen PSIIreactioncenter andthefraction ofopen reactioncenters (Gentyetal 1989):
.
F
M~F0
QPSII =
—
F
M
F
M~F
x
—
-,=
F
M ~ F0
F
M~F
JF
M
(2.4)
Sinceinsteadystatethefluxoftheelectrons inPSIandPSIIshould beequal, the photon
yieldof PSIIgivesagood estimation oftheefficiencyofthelinear electronflow inthe
photosynthetic apparatus.Theoverall rateofelectronflowJE[in umole(umol PSII)"1s"'],can
becalculated bymultiplyingthe photonyieldwiththe amount ofphotonsabsorbed byPS II:
J
E
=
^PSII°PSIIPFD
whereaKn = theabsorptioncrosssectionof PSII;andPFD= the photonfluxofthe
photosynthetically active radiation.Aswewerenotableto determine oPSI|directly,the (relative)
JEwasestimated bytheproduct 0 PSII •PFD.Thisisareasonableestimate:accordingto Kolberet
al (1988) theabsorptioncrosssectionof D.tertiolectadoes notchange bymorethan 14-17%
inthe light rangeof 20-200uE.
Thesaturating pulsefluorescent approachcanbeapplied inconjunctionwiththe 14C-method,
whichisconventionally usedfor primary production measurements inthe marineenvironment.An
important advantageofthefluorescencetechniqueoverthe ,4C-methodisthattheformer
measurement givesinformation ontheinstantaneous photosynthetic statusandcanbeapplied in
situ(i.e.,intheseawater ataparticular location,measurements,for instancetimeseries,canbe
performed).Themeasurement canbedonedirectly asit doesnot requiredarkadaptation.To
obtainabsolute rates,calibrationwith ,4C-incorporationmeasurements mayberequired.
Analternative opticalapproach,dubbed pump-and-probefluorescence,canbeusedto
measure propertiessimilartothosemeasuredwiththesaturating pulsefluorescence method
usedinthisstudy (FalkowskiandKiefer 1985,FalkowskiandKolber 1990).Themaindifference
betweenthetwomethodsisthe procedure usedtodetermine FM andFM', whichcanproduce
different results.Inparticular,asthepump-and-probe techniqueonlyusesasingle,short pump
pulseonly asingleturn-over ofthe PSIIreactioncentersiseffected,whichresults inreductionof
QAonly. Inthesaturating pulseapproachalonger intense pulseisapplied,whichresults intotal
15
(2.5)
reduction of QAandofthe PQ pool.Therefore FM andFM'ingeneralwillbesomewhat lower inthe
pump-and-probe measurement.Thetrendsdetermined byboth methods,however,maybe
similar. Forcomparison ofthe2methodsseeSchreiberet al (1995b).
2.3 Experimental application
Table 2.1. Photon flux densities of
photosynthetically active radiation
underwhichtheD. tertiolectacultureswere grown
Culture #
PFD (umolm"2s"1)
1
22.6
2
42.1
3
66.0
4
92.3
5
107.4
6
137.3
7
214.3
Thechlorophyte D.tert/o/edams grownat 15°Cinanf/2 medium (McLachlan,1975) under
a 14hlight/10 hdarkcycle.Thecultureswerekept inculturebottles,whichwereplacedin
plastic containerswithwhite,diffuse reflectingwalls,covered bysuitableneutraldensity (gray)
attenuationfiltersto obtain7different light intensities.Anincubator (Gallenkamp OrbitalINR401) equippedwithfluorescenttubesoperating atafulllight intensityof 300-400 umolm"2 s"1
wasused.Light intensities inthecontainersweremeasuredwithaPhotodyne88XLAspherical
sensor.TheintensitiesunderwhichtheculturesweregrownaregiveninTable 2.1.
Thegrowth rateofthecultureswasdetermined bymeansofaflowcytometer,especially
constructedfor usewithphytoplankton;thecytometer measuredtheconcentrationof particles
with redchlorophyllfluorescence (> 670 nm)under excitationwithanAr-ion laseroperatedat
529nm(HofstraatetaM990).
Saturatingpulsefluorescencemeasurementsweredonewithapulseamplitude modulation
fluorimeter (PAM,producedbyWalz,Effeltrich,FRG,asdevelopedbyU.Schreiber,seeSchreiber
1986).ThePAMconsistsof4units,thesettingsofwhichwereoptimized beforetheactual
experimentwasstarted.Firstly,themodulatedmeasuring lightintensity provided byapulsed
LEDinthe 101-EDunit ofthe PAM,peakingat650-660 nm,hadto bechosensoasto obtain
enoughsensitivity,yetto belowenoughto preventinduction ofsignificantvariablefluorescence.
16
1.0
,-s 0.8
3
S 0.6
c
(U
u
v>
% 0.4
O
3
0.2
0.0
r-
0
60
120
180
240
300
time (s.)
1.0
B
^0.8
S 0.6
c
«
IA
£
o 0.4
3
0.2
^IPI^^
0.0
I—
o
60
120
180
240
300
time (s.)
Figure2.2. Typical resultsofsaturating pulsefluorescence measurementsfor 2culturesofD. tertiolecta
grown under (A) low (66.0 umol m"2s') and (B)high (214.3 pmol m'2s"1) light intensity. Cultureswere
measured 17dafter commencingtheexperiment. Seetext andTable 2.2 formoredetails.
17
10
i
i
i
10
i
8
6
-
A
^<s
j
/ ü
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i
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o
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i
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i
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-
^^
2
/
CHJ
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0
Culture 2
i
()
i
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8
10
i
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i
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T-I^J
•Q
4
o
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E
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6
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i
8
-
t
12
4
-
f
^^
Culture 7
2
0
16
^*T§
,c
.£
20
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time (days)
i
l
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3
Culture 4
i
^ s ^
•Q
2
20
^r
w
0>
r
•v^
v
3
,C
i
16
^m
^"5
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6
i
12
-
time (days)
8
>~
5
-
o
20
Y7-V
xrv
<D
-
^ j S ^
(-J-J
-
i
12
rv——""""^
rT
x^
<D
-Q
8
^"S
E
i
Cj~^
6
time (days)
o
o
i
5o
•Q
E
i
8
^"55
o
o
i
•
i
i
i
4
8
12
16
20
time (days)
Figure2.3. IncreaseinD. tertiolectacellnumbersinthecultures,asdeterminedbyflowcytometry.Curvesareshownfor4typicalcultures;thecorrespondinglightintensitiesaregiveninTable
2.1.
Secondly,the actinic illuminationfromthe KL-1500lamp,controlled bythe PAM102 unit,was
chosento matchthetotal light intensityunderwhichthedifferent cultureshadbeengrown, as
determined byaUDTPIN-10photodiode.Thematchingwasaccomplished byapplyingthe
availablesettings oftheactinic light source incombinationwithanumber ofOrielneutraldensity
filters.Thespectral intensity distribution ofthe KL-1500light sourceandthat ofthe incubator
weremeasuredwithacalibrated,sphericalsensor, using 10nm full-width-at-half-maximum
bandpassfiltersto measuretheabsolute light intensityevery 20nm.Theintensity distributions
ofthetwo lightsourcesdid not matchcompletely,the KL-1500sourcebeing moreintenseinthe
orangeandred part ofthespectrum.Thirdly,theintense,saturating light-pulseoftheKL-1500
lightsource,controlled bythe PAM103 unit,wasoptimizedto ensurecompleteclosureofthePS
IIreactioncentersbyobservingthetime resolvedfluorescencesignalasobtained bythe
18
modulated measuring light.Atthesametimeactiniceffects resultingfromthesaturatingpulse
should beabsent;thiswasaccomplished byapplication ofa700 mspulseat anintensityofmore
than2000W rrf2. Finally,far-red illumination (> 730 nm)wasappliedto reopenthe PS II
reactioncentersfollowing asaturating pulse.Dataacquisitionandhandlingwereperformedby
thededicated program 'FLUORESC',developedatthedepartment of Plant Physiology,
WageningenAgricultural University.
Themeasuring procedurewasexecutedonadaily basisandcommencedexactly 3hafterthe
light inthe incubator wasswitchedon.Samplesweretakenfromtheflasksinthe incubator and
put inthe MKS-101cuvette (provided byWalz,Effeltrich,FRG)withminimallightexposure.
Subsequently,the measurementwasstartedwiththeactinic light intensityofthe PAMmatching
that towhichthesample hadbeenexposedinthe incubator. Duetothetransfertothe
measuringcuvettethesamplehadbeensubjectedto achangeinlightclimate.Hence,the
samplewasirradiatedwiththeactinic lightsourcefor 5minutesbeforetheactual measurement
ofFM'andF.Toconfirmthat afterthisadaptationtimethesteadystateconditionwasreached,
fluorescence and,at regular intervals,saturating pulsemeasurementsweredoneduringactinic
illumination.Followingthissequencetheactinic lightwasswitchedoffandanother seriesof
readingsoffluorescence andsaturating pulsefluorescencewasdone inthedark,to checkfor
possible measurement induced quenchingeffects. Forthemeasurements inthedarkthepulse
rateofthe LEDinthe 101-EDunitofthePAM,usedfor inductionofthefluorescence, was
changedfrom 100kHzto 1.6kHz,to prevent anyactiniceffects.
Inaseparate experiment,samplesweredarkadaptedfor 15minutesto removeanyenergy
dependent quenching andsubsequently FV/FMwasmeasuredto determine photoinhibition
effects.FV/FMwasindependent ofthechlorophyll aconcentration between0.5-40ug
chlorophyll/ml (unpublished results). Dilutesamples (lessthan 10 6 cells/ml,correspondingto
lessthan 1ugchlorophyll/ml) wereconcentrated bycentrifugationat 3,000gfor 5min.
Centrifugationdid notaffecttherateofphotosynthesis.
2.4 Results and discussion
2.4.1Measurementof(p^,,andqP
First,theapplicability ofthesaturation pulsemethodforthe measurement of <\>p andqpwas
studied;thismeasurement doesnot requiredarkadaptation ofthephytoplankton.Ascanbe
inferredfrom Equation (1),determination of FM',F andF0', allparameterswhichcanbeobtained
under steadystateconditions,sufficesfor determination of d^s,,andqP.
Thefluorescence induction kineticsobtainedfromtwoculturesthat hadbeengrownfor 17
daysunder lowandhigh light conditions,respectively,areshowninFigure2.2.Figure2.2A
showsthefluorescencetransientfor lowlight adapted phytoplankton (culture 3).Atthislight
intensitythesample isstillgrowingexponentially (seeFig.2.3).Thefluorescence induction
kinetics indicatethat the phytoplankton returnsto asteadystatealmost immediately after the
light pulse.Inconstrast,forthe highlight adapted culturesinthestationary phase (culture 7,
Fig.2.3) astrong reduction oftheeffect ofclosingthe reactioncenterswithasaturating pulseis
19
Table2.2.Photosyntheticcharacteristicsforthe2D. tertiolectaoiWwts showninFigure2.2
Culture #
PFD(pmolrrf2
qN
qP
<PPS ii
F V /F M
s"')
3
66.0
0.37
0.96
0.58
0.69
7
214.3
0.61
0.81
0.25
0.42
observedoncethesample isre-illuminated.Therelativelyshorttime inwhichthe phytoplankton
iskeptinthedarkpriortothemeasurementforthehighlightcaseappearsto besufficient to
alowsomerecuperation ofthelight-induced non-photochemical quenching.Also,forthehigh
lightsamplethesaturating pulseappearsto causesometransient quenchingeffects. The
difference betweenthesamplesgrownunder relativelylowandrelatively highlight regimesis
striking (seeTable 2.2).Thelowlight sample,whichisintheexponentialgrowth phase,showsa
significantly higher photochemical efficiencythanthehighlightsample,whichisinasteadystate.
Photochemicalquenching,whichcanalsobecalculatedfromthedirect measurement (under
actinic illumination),appearsto besomewhat higherforthelowlight adaptedsample.Both
observations indicatethat photosynthetic energyconversion ismoreefficient inthelowlight
situation (seealso Kolber etal 1990).Thecurve inFigure2.2B alsosuggests amuchhigher
contribution ofnon-photochemical quenching inthe highlight intensity case.However,asno
lengthydarkadaptationwasdone,datafor q Ncannot beconsidered conclusive.Residualnonphotochemicaleffectscanstillbepresent;darkadaptationwillremovetheseeffects,whilemore
persistent photoinhibitory effectswillremain.Non-photochemical (orenergy dependent)
quenchingwillbemoreimportantforthe highlight case,sothatthedifference inthevalues
obtainedfor qNgiveninTable2.2 willbemorepronouncedwhenthesamplesaredarkadpated
for alongertime.
Whencultures grownunder different light intensities arefollowedovertime,observations
similartothosementioned abovearemade.InFigure2.4Athedevelopment of (bp^,withtimeis
presented. Fivedaysaftertheculturehasbeenstarted,the 6cultures (cultures 2to 7,Table
2.1) hadmoreor lesssimilarvaluesfor (p^,, (0.64-0.74).Allculturesaregrowing exponentially.
After 17dacleardifference,depending onthe light intensity,hadbecomeapparent.Atthattime
(bP5ll rangesfrom 0.30-0.67. The2culturesgrown underthe lowest light intensities arestillin
exponential growth after 17d. Indeed,thevalueof (bpsll determinedfor thesecultures appeared
to havehardlydecreased.Cultures4and 5showexponential growth untilDay 13.Cultures6and
7 enterthestationary (growth) phasearound Day 11,thetime atwhich (bp^,startstodecrease.
(bps,,onlygivesanestimateoftheefficiency oftheelectronflow inPSII.Theoverall rateof
electronflow,JE,whichrepresentsthe photosynthetic activityofthe phytoplankton,canbe
calculatedbymultiplying tyKll withtheamountof photonsabsorbedbyPSII.Ifweassumeequal
absorption crosssectionsfor thedifferent cultures,the (relative) JE canbeapproximated bythe
20
1,0
i
1
1
r
12
14
0,8
0,6
0,4
0,2
0,0 4—m-\
f
120
Q
UL
Q.
90
*
w
0.
'S.
60
30
8
10
16
18
time (days)
Figure2.4. Developmentof(A)$KU and(B)(J)PSn •PFDwiththe
timeforD.thecultures2to7,grownunderlightintensitiesgiveninTable2.1. Culture2:(o); culture3:(•); culture4: (v);
culture5:(»);culture6:(•) andculture7: ( • ) .
product of (ppsiiandthe integrated intensityofthelight usedto culturethesamples.Figure 2.4B
showsthe product dips,,•PFDasafunctionofincubationtimefor thedifferent cultures. (J)PSn •
PFDisanapproximationofthelinear electronflow inPSII, assumingthattheabsorption cross
sectionisequalfor allcultures.Clearly,thehighlight culturesshowsignificantlyhigher overall
rates inthefirst phaseoftheexperiment.The2culturesgrownatthe lowest light intensities
showconstant JEduringthecourseoftheexperiment.
Arelation betweenthephotosynthetic activityandthegrowth rateofthe phytoplankton
cultures isexpected.Figure2.5 showsthe relationbetween 4>PSh•PFD(theestimatedelectron
transport) andthe measuredgrowth rateoftheculturesduringtheexponential growthphase.
Therelation isclear: at lowelectrontransport rates (i.e.,at light levelsbelow 100pmolm "2 s"1)
21
1,0
i
i
i
I
o
O
0,8
T3
o
o
0,6
2
-
o
0,4
o
-
•o
-
0,2 -
o
0,0
0
I
I
I
30
60
90
I
120
150
•»PS,,*™
Figure2.5. Relationbetweenct>PSH •PFD andD. tertiolecta
growthrate.Both(t>PS„andthegrowthrateweredeterminedas
averagesduringtheexponentialgrowthphase;N = cellnumber.
theexponentialgrowthrate ismoreor lessdeterminedbythe light intensity.Athigher lightlevels
thegrowth rateapproachesalimitingexponentialvalueofabout 1. Extrapolationtozero
electrontransport ((bPSn•PFD= 0) indicatesnogrowth,asexpected.
Theaboveclearlyindicatestheapplicabilityofthesaturating pulsefluorescencetechniquefor
theestimation ofthephotochemicalefficiencyof PSII,andhenceofthetotal photosynthetic
activityofphytoplankton.Theresultssuggestthatthisapproachcanbeausefulalternativefor
the ,4C-incorporationtechniquethat isnormallyusedto measure primary productivity (Peterson
1980,DringandJewson 1982). Indeed,Gentyetal(1989) havereportedverygood correlations
betweenthephotochemicalyieldasdetermined bythesaturating pulsetechniqueandthe
quantumyield ofC02-assimilationasdetermined bythe '"(^-incorporationtechnique.The
saturating pulsetechnique andthepump-probetechnique (Falkowskiand Kolber 1990) have
severaladvantages overthelatter method.Firstly,theydonot require radioactivesubstances.
Secondly,theycanbeapplieddirectly,without incubation asrequiredforthe ,4C-technique.
Therefore,theyarelesscumbersomeandmoreefficient andcanevenbeapplied insitu, sothat
the photosynthetic activityofphytoplankton canbemonitored,for example,over acertainperiod
oftime.On theother handthe ,4C-methoddirectly monitorsthe incorporation ofC02 inthe
phytoplankton andtherefore gives morestraightforward information onthe photosynthetic
energyconversion process.Thefluorescencetechniques describedheregivedirect information
onphotosynthetic electronflow,astheyarebasedsolelyonmeasurement of PSII fluorescence.
22
1,0
i
>
i
i
i
i
-
0,8
s 0,6
"•
0,4
-
-
0,2
0,0
i
i
i
6
8
10
i
12
i
14
i
16
18
time (days)
Figure2.6. DevelopmentoftheparameterFV/FM, representative
oftheextentofphotoinhibition,withtimeforD. tertiolectacultures2to7accordingtoTable2.1.Culture2:(o); culture3: (•);
culture4:(v);culture5:(•); culture6:(D) andculture7: ( • ) .
Withthesetechniques,therefore,it isnot possibleto distinguish betweenenergystorage inthe
Calvincycleandthat inother assimilation pathways (e.g.,N-reduction,photorespiration).Another
advantage ofthe ,4C-methodisthat itprovides atime-averaged (e.g.daily) rateofprimary
production,whichcanbeusefulfor certainapplications. Inthis respectthe 2approaches,the
fluorescence-based methodsandthe ,4C-methodarecomplementary. Presumably,infuture
applications onewouldperformmostmeasurementswiththefluorescencetechnique,butwould
takeseveralsamplesfor HC-incorporationanalysisto calibrate (bps,,intermsofbiomass
production.
2.4.2Determinationofphotoinhibition
Asindicatedabove,thephotoinhibition canbeestimatedfromthe ratio ofthevariableand
maximumfluorescence FV/FM= (FM-F0)/FM.Thisratio isameasureofthephotochemicalyieldin
open reactioncenters (Björkman, 1987). Dueto primarydamagetothePSIIreactioncenter,
thevariablefluorescence especially isreduced.Photoinhibition canbedeterminedonlywhenall
transient quenching processes havebeenallowedto relax.Hence,followingtransfer ofthe
samplesfromthe illuminated containers,thesamplesaredarkadaptedfor 15minutes,whichis
sufficient to removeanyphotochemicalor energy-dependent quenching.ThenF0andFM are
measured,the latter byusingashort pulseofsaturating light.
InFigure 2.6 FV/FM hasbeendeterminedfor cultures2to 7(Table2.1) duringthecourseof
23
theexperiment. Inthefirst phaseoftheexperiment,whenalltheculturesarestillgrowing
exponentially,differences inthe ratioaresmall:allculturesshowvaluesbetween0.73 and0.80.
However,after 12dofgrowth,significant differences areobserved.Inparticular theculturesthat
aregrownunder high lightconditionsstartto giveindications ofphotoinhibitory effects,the F V/FM
ratiodroppingto amere0.4for the 2culturesexposedtothe highestlightconditions.Atthe
sametimethegrowthcurvesfor theseculturesshowclear non-exponential growth behaviour,
andevenasmalldeclineincellnumbersforthelastdayoftheexperiment.Thefactthatin
exponentiallygrowing culturesFV/FH isalmostashighas0.8 suggeststhat evenatthe relatively
highcellconcentrations usedinthemeasurements,saturationoffluorescenceand reabsorption
effects arenot important. Forother phytoplankton speciessuchas Phaeodactylumtricornutum
and Rhodomonassp. valuesofFV/FM of0.7 andhigherwereobserved (Geelet al,tobe
published).FV/FM ishighlycorrelatedwith <\>KK, whichsuggeststhat chlororespiration should
haveonlyaminorinfluence.
TheexampleshowninFigure 2.6 illustratesthepotential ofthistechnique asasimplemeans
to monitor photoinhibitory effects inphytoplankton.Usingthesaturating pulsefluorescence
techniqueP/lcurvescaneasily bemade.Asthemeasurement isbasedonarelativeapproach,
the method appearsto beextremely robust.Therepeatibilityofthedeterminationof FV/FMis
better than 1%.However,duetothe insensitivityofthePAMfluorimeter appliedto
phytoplankton measurement,it iscrucialto implementcorrectionsfor theeffect of straylight.
Thiseffect cannot beneglected;it hasaparticularly strong influenceonthedetermination of F 0,
but it caneasily becorrectedfor.
2.4.3Modificationsforphytoplanktonmeasurement
Afinal problemofthesaturating pulsetechnique asrealizedinthePAM-fluorimeter isthatthe
instrument wasspecifically designedfor studiesonhigher plants.Therefore,it isnot sufficiently
sensitivefor studyofphytoplankton.Inourstudywefoundthat theconcentrationof D. tertio/ecta
shouldexceed 106cells ml"' inorderfor good resultsto beobtainedwiththePAM.Atthis
concentration <\>Kn canstill bedeterminedwitharepeatibility ofbetter than 5%,asthe
determination isarelativeoneandtherefore relatively robust. However,for applications onfield
samplesorfor diluteculturesthesensitivity is3to 4orders ofmagnitudetoo low.Inthe
laboratorythe rangeofthe instrument canbeextendedfor mostphytoplankton speciesby
centrifugation.Forfieldsamplesthisapproachcannot beapplied.However,thesensitivity ofthe
instrument canprobably beimprovedsignificantly byrelativelystraightforward modifications.The
present instrument hasbeendesignedfor applicationto plantsandleaves.Itusesexcitationin
the 650-660 nmregionanddetection >710 nm.Forphytoplankton,wherethe chlorophyll
concentration ismuchlower,excitation intheblueanddetection >670 nmwouldalreadyyielda
tremendous increase insensitivity. Inaddition,thevolumeofinteraction betweenlightand
phytoplankton isverysmall.For phytoplanktondispersed insurfacewater at relativelylow
concentration amuchlargervolumeofinteractionwouldalsoleadto asignificant increasein
sensitivity. Finally,someimprovements inthedataacquisitionandtransfer canbeimplemented.
Recentexperimentswithamodified PAMinstrument,incorporating thechangessuggested
above and,inaddition,apulsedXenonflashlampasexcitationsource haveshownthatthe
24
required improvements insensitivity canindeedberealized (Schreiber, personal
communication).AmodifiedPAMinstrument ispresentlybeingbuilt,andwillbeusedforfurther
study.
2.5 Conclusions
Thesaturating pulsefluorescencetechniqueappearsto beamethodthat canbesuccessfully
usedforthe non-destructive measurement ofimportant photosynthetic characteristics of D.
tertioleda.Itallowsforthesimpleanddirect measurement of photosynthetic efficiency,of
photoinhibitory effects andoffluorescencequenching processes.Forsomeparametersthe
samples must bedarkadaptedpriortothe measurement.Thesaturating pulsefluorescence
approachandthepumpandprobetechnique (FalkowskiandKolber 1990),seemto offer useful
extensionstotheconventionally usedtechniques. Forinstance uC-uptakemeasurements require
the useof radioactive material,supplytime-integrated photosynthetic dataandcannot beused
for insitumonitoring applications.Oxygenmeasurements,whichmaybeusedfor insitu
applications,arenotsufficientlysensitivefor marineuse.
However,severalimprovements stillhaveto bemadeinthedesignofthe PAM fluorimeter.
Firstandforemostthesensitivity ofthe instrument,whichwasdevelopedfor plant studies,must
beimprovedto enable measurement offieldsamples.Also,themeasurements donewiththe
saturating pulsetechnique haveto becomparedwiththoseoftheconventionaltechniques,as
hasalready beendone (withgood results) forplants.
25
3 E S T I M A T I O N OF OXYGEN E V O L U T I O N BY M A R I N E
PHYTOPLANKTON FROM MEASUREMENT OF T H E EFFICIENCY
OF PHOTOSYSTEM I I ELECTRON FLOW
3.1 Introduction
Chlorophyll afluorescence hasfoundwideapplication intheanalysisof photosynthesis (Krause
andWeis 1991). Usingpulseamplitude modulation (Schreiberetal 1986,Schreiberetal 1993)
or pumpandprobetechniques (Mauzerall 1972,FalkowskiandKiefer 1985),it hasbecome
possibleto determinetheefficiencyof PSIIelectrontransport.Agoodcorrelation betweenthe
efficiencyof PSIIelectrontransport andtheefficiencyofcarbonuptakewasdemonstrated in
barley andmaizeunder non-photorespiratory conditions (Gentyetal 1989).Thiscorrelation
might alsobeveryusefulinestimating carbonfixationor photosynthetic oxygenevolutionof
phytoplankton bymeansofchlorophyllfluorescence.Thefluorescence measurement only lastsa
fewsecondsandmight bespeciallysuitedfor insitumeasurementsto monitortheefficiencyof
PSIIundertheambient irradiance.Thisincontrastwithmeasurementsofoxygenevolutionor
carbonfixation onsampleswith lowphytoplanktonconcentrations,whichusuallyrequire large
samplesandlong light incubationtimes (2-6 hours) inalaboratoryset-up or inbottle-enclosures inthe naturalenvironment (HallandMoll 1975,Falkowskiand Kolber 1990).
Therelationship betweentheefficiency of PSIIelectrontransport (Ops,,)andtheefficiencyof
carbondioxide uptakehasbeenexaminedunder avarietyofconditions inmainlyhigher plants
andgreenalgae.Thelinear relationshipwasconfirmedinC4plants (KrallandEdwards 1990)
andinseveralC3plantsunder non-photorespiratory conditions (Harbinsonetal 1990). A
non-linear relationshipwasfoundinthepresenceof20%oxygeninC3plants (Harbinsonetal
1990).ÖquistandChow(1992) reported asinglecurvilinear relationship athighCO2levels
where photorespiration isreducedbutwhere Mehler-typeoxygen reductionstillmightoccur.
Morerecently,Gentyetal(1992) eliminatedthecontributions ofoxygenuptakeby
photorespiration and Mehlerreactionbymeasuring ,8 0 2 inmass-spectrometricmeasurementsin
beanleavesandconfirmedthe linear relationship betweenelectronflowand photosynthetic
oxygenevolution.Athighirradiance alarger reduction intheefficiencyofoxygenevolutionthan
intheefficiency of PSIIelectrontransport hasbeenfoundin Dunaliellatertiolecta(Falkowskietal
1986,Reesetal 1992).Thisphenomenonwassuggestedto becausedbycyclicelectronflow
around PSII(Falkowskietal 1986). Reeset al (1992) haveexcludedphotorespiration asa
possibleelectronsinklinkedto oxygenuptakeandproposednon-assimilatoryelectronflow,
either linearwholechainor cyclicaround PSII,to bethecauseofthe non-linearity.
Atverylowirradiances,anadditionalnon-linearity hasbeenobserved,whichhasbeen
explainedat least partly byrespiration (Oberhuber etal 1993) or bythepresenceof inactive
PSIIcentreswhich lowertheyieldofoxygenevolutionmorethantheyieldof PSIIelectron
transport (Hormannetal 1994).
Therateof photosynthetic oxygenevolutioncanbecalculatedfromtheestimated rateof PS II
Publishedas:GeelC,VersluisW andSnelJFH (1997) PhotRes51:61-70
2
closed
02<*.
od Y
1
open
\
f
l
,.-02
^k.
3
inhibited
si
Figure3.1. Thephotosynthesismodelshowingthethreestates
of the'photosyntheticfactories' (EilersandPeeters 1988). The
modification(dottedline)emphasizesoxygenconsumptiondrivenbylinearelectronflow.
electrontransport (tym •PFD) using oPS)l, theabsorptioncross-section of PSII.Theresultsof
KrallandEdwards (1990) suggestthattheabsorption cross-section isalmost identicalinthree
different C4plants (maize, Panicummaximum, P.miliaceum) but different inthe C3plant (wheat)
measured,althoughallplantswereculturedatthesameirradiance.Otherresearchersfoundthat
theeffective absorption cross-sectionof PSIIisdependent onthegrowth irradiance (Leyand
Mauzeral 1982,Kolber etal 1988) andcanchange rapidlyinfluctuating light bymeansofstate
transitions (Kroon 1994).Theeffective absorptioncross-section of PSIIfurthermoreis
dependent onthenutrient availability andtheC3species involved (Kolber etal 1988).Falkowski
and Kolber (1990) foundagoodcorrelation betweentheestimated rateof PS IIelectron
transport andthe rateofcarbonfixation inphytoplankton usingapumpandprobefluorescence
methodto estimatethe effective absorption cross-sectionof PSII.Thisclosecorrelation between
PSIIelectronflowandcarbonfixationsuggeststhat algalgrowthandPSIIelectronfloware
correlated aswell,whichwasdemonstrated ina Dunaliellatertioledalaboratory experiment
(Hofstraat etal 1994a).
Inorderto assesstheusefulnessofthefluorescence methodto estimateprimary production
inmarine phytoplanktonweexamined boththe rateof PS IIelectrontransport determinedby
fluorescence andthe rateofoxygenevolutioninseveralmarinealgaeundervaryinglight
conditions.Thedataweremodelledwithamodifiedversionofthemodelof EilersandPeeters
(1988) to accountfor oxygenconsumingprocesses.Therelationbetweenthe rateofPSII
electronflowandthe rateofoxygenevolutionwascurvilinear andisdependent onspeciesand
experimental conditions.Theobserved non-linearity at highirradiances isprobably notcausedby
photorespiration.
27
I
'
'
i
i
i
i
i
i
i
i
i
i
1
_
cCU
1.5 S
CU
0,4
"^^^^~^"
1
• — — _ _ _ _ ^
°~
~~~F
0,2
0,0
400
5
o
»•—
^~*
C '(0
O Ol'
o
1,0 =
- 0,5 ó
I
l
i
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i
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• 80
- B
CM
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T-
60 Z
o
o>
n.
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E
300
1—
•G E
Iele
ole
2,5 "=
ni
- 2,0 S
\ ^F
,;
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0,6
-e-
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i
A
0,8
CO
0.
'
200
Q.
55 |
o. 3 100
•
0
CM
20 O
i
0
i
i
500
1000
PFD (pmol m s )
o
i
1500
0
Figure3.2.A:The actual (o) and maximal (o)fluorescence levels andthe efficiency ofPSIIelectron
transport cj)psil ( • ) asafunction ofthe irradiance. B:Therate ofoxygenevolution (o) and the
estimated rateofPSIIelectron transport ( • ) asafunction ofthe irradiance.Theexperiment wasdone
with P.tr/cornutumat 20 °Cwithasingle sample usingtheXe-PAMfluorometer andthe Hansatech
DW2/2 oxygen electrode chamber. Growthtemperature: 20°C. Lines represent fits accordingtothe
photosynthesis model ofEilersand'/Meters(1988) adaptedtoallowfor electronflow driven oxygen
uptake (seetextformore details).
3.2 Materials and methods
The marine algae Phaeodadylum tricornutum, Dunaliella tertioleda, Tetraselmissp., Isochrysis
sp. andRhodomonassp. were kindly supplied byL.Peperzak ofthe National Institute of Coastal
and Marine Management. The algae were grown inunialgal batch cultures inf/2 medium at pH
28
i
'
i
'
i
'
i
'
'<• 50
f
ü
o 40
/^
j/
3 L
'
/ ^ * ^k
\
\
p
7
"5
1. 30
v ^
c
g
= 20
o
7
4)
>
/
g 10
O»
/
/
/
/
-
/
_//
^f
• J
S?
o
c»
./
/^
i
0
£P
- T
1
0
.
1
100
.
1
.
200
1
300
,
1
400
.
500
2 1
PSIIelectronflow(umolem" s' )
Figure3.3.Therelationshipbetweenoxygenevolutionand
estimatedPS IIelectrontransportinP. tricornutum. Dataare
fromFigure3.2Bandthelinerepresentsthefitaccordingtothe
modelofFilersandPeeters(1988) modifiedasdescribedin
Figure3.2.
7.4 (Madachlan 1975) inartificialseawater (35 gseasalts l"\ Sigmaseasalts).Thecultureswere
kept at 20 °Cor25 °Cand 100pmolm"2s"1continuous irradiance.Theyweremixedby
bubblingwithair enrichedinC02.Thecellswerekept intheexponentialgrowth phaseas
determined spectrophotometricallyat 680nm.
Thealgaewereharvested bycentrifugationandusedat afinalconcentrationof0.5-7 |jg
chlorophyll aml'1.Thealgaeweredarkadaptedfor 30minpriortothestart ofthe measurement.
Oxygenandfluorescenceweremeasured insteadystatewhichwasreachedafter 5to 10
minutes.Measurementsweremadeatthegrowingtemperature.Unlessstatedotherwise,anew
samplewastakenfor everymeasurement.
Oxygenandfluorescenceweremeasuredsimultaneously inaDW2/2oxygenelectrode
chamber (Hansatech,KingsLynn,Norfolk,UK).Oxygenmeasurementswerecontrolled usingthe
CBD1control box (Hansatech).Netoxygenevolutionwascalculatedfromthe rateofoxygen
increaseminusthedark respiration.Theefficiency of photosystemIIelectrontransportwas
determinedwithexcitation at 650 nmanddetectionabove700 nmusingthePAMChlorophyll
Fluorometer (HeinzWalz,Effeltrich,Germany) orwithbroadband excitationfrom 400-560nm
(Xenonflashlampplus 4mmSchott BG39) andemissiondetectedabove 650 nm(BalzerR65
plusSchott RG645) using aslightly modifiedversionoftheXe-PAMfluorometer (Schreiber etal
29
1993).The PAMandXe-PAMfluorometer gavesimilar resultsfor theefficiency of PSIIelectron
flow (Geelunpublished results).Thesignalto noiseratioofthe PAMmeasurementswas
comparabletothe measurements showninHofstraat etal (1994).Thesignalto noise ratioof
theXe-PAMmeasurementswasmuchhigher dueto thehighsensitivity oftheXe-PAM
fluorometer (Schreiber et al 1993).Usingthe PAM fluorometerthe LS2light source (Hansatech)
andneutral densityfilters (Schott) wereusedto illuminatethesampleandsaturating light pulses
(6000 nrnolm"2s"1)weregivenwithaFL-103 Fiber Illuminator (Walz).TheXe-PAMfluorometer
isequippedwithtwo halogenlampswhichwereusedfor actinicandsaturating light.The
saturating lightwasfilteredthrough a650 nmshort-passfilter (Balzers DTCyanspecial) and
wasusedatanintensity of 6000 umolm"2s"\ theactinic irradiancewasadjustedwithneutral
densityfilters. Lightsaturationwascheckedbycomparing FM intheabsenceandinthepresence
of DCMU. Thefluorescence measurementswerecorrectedfor abackground signalcausedby
scattering oftheexcitation light,filterfluorescenceor electricalartifactswhichsignificantly affect
the resultsat lowchlorophyllconcentrations. Fluorescence nomenclaturewasaccording toVan
KootenandSnel (1990).Theefficiency of PSIIelectronflowwascalculatedas (FM'-F)/FM'. The
rateofelectrontransport wasestimated bythe product oftheefficiencyof photosystem IIand
the photonfluxdensity (PFD).Thechlorophyll aconcentrationwasdetermined after extraction
with boiling ethanolaccording to Nusch (1980).Therates ofoxygenevolutionandelectron
transport werevariedbychangingtheirradiance. Photonfluxdensityfrom 400to 700 nmwas
measuredwithaSkyephotometer equippedwithaquantumsensor.
Theratesof PSIIelectronflowandoxygenevolutionwerefitted bythe phytoplankton
photosynthesis modelproposed byEilersand Peeters (1988).Themodelassumesthata
photosynthetic unit canexist inoneofthethreefollowingstates:open,closed orinhibited,
existing infractions x1,A^andx3,respectively (seeFig.3.1).Absorption of light byaunitin
state 1resultswithaprobability a inaphotochemical event leadingto oxygenproductionand
conversion into aclosed unit (state 2).Excitationoftheclosed stateresults inaninhibitedstate
(state 3) with probability ßwhichismuchsmallerthan a . Theclosedunit isreopened bythe
darkreactions associatedwithcarbonassimilationwitharateconstant y- Theinhibited stateis
converted intotheopenstatewith rateconstant 5. Theprobabilityfor recoveryof
photoinhibition 5 ismuchsmallerthantheprobabilityfor photosynthesis darkreactions \. A
systemofdifferential Equationscanbeobtainedfrom Figure 3.1:
dx,
dt
dx2
dt
-cc/x, +yx2 + ÔX3
(3.1)
alx. - (ß/ +y)x2
(3.2)
dx~
—- = ß/x, - ôx,
dt
Tosimplifythe equations, bx3andß/x,canbeomitted inthe Equations (3.1) and (3.2) (see
30
(3.3)
Eilers and Peeters 1988).Thesteady state solutionfor the rate of electronflow (JE) is given
by:
,
ayô7
=
E
2
(aß/ +aó7 +Y ô)
(3 4)
'
Substituting A- ß/(YÖ), ß= (a+ß)/(cxY) and C- or' the equation issimplified to:
J
E
_/
- ,iw, Z nw ^
(AI +BI+Q
(3.5)
Oxygen evolution datawerefitted using k\o account for the PSIIabsorption cross-section and
for the stoichiometryofoxygen evolution and one additional parameter ^describing oxygen
consumption.Apart of the enzymatic reactions leadingtoreopening (rate constant y) ofclosed
unitswas assumedtobeassociated with oxygen consumption.The ratio of the rate constantsof
oxygen uptake and oxygen productionwasassumedto be proportional to the redox stateof
ferredoxin.The redox state offerredoxin isinequilibrium withthe 'redox state' xzlxs of the
photosynthetic factories.
=(x2/xi)E
(3.6)
YOP
withYoc. YOP:r a t e constants of oxygen consumption and production, respectively and E, a
proportionality constant.
Furthermore,weassumed that the sumofthe rate constants wasconstant and equaltoy:
Yoc + yOP = y
(3.7)
With ß « c x and 5 « y thefollowing expression canbederived:
Xj=( — ) * !
(3.8)
The relative rate constants Yocand YOPc a n n o w De calculated.Net oxygen evolution (J0.net)is
the sumofoxygen production (Y 0 P) and oxygen consumption (Yoc)-Weassumed that oxygen
consumption wascaused by Mehler reaction.This reaction usesonly 2 electrons per oxygen
moleculewhereas oxygen production isaccompagniedwiththe releaseof4electrons per
molecule.Therefore net oxygenevolution isgivenas:
J
onel = kx2^lop-2yoc)
(3.9)
The experimentally observed rate of oxygen evolution (J0) cannow be described bythe following
equation:
J
o
=
kI(C - 2BEI)
;
(AI2 +BI +Q(BIE + Q
31
(3.10)
\
30
+HCO3"/
3. 20
o
E
/
/
;
\
-
/ /
Q.
o
o
(// control
o
oxygen evolution
7
/ S' /v
IE
ü
0
50
100
150
200
PSIIelectronflow(nmolem'2s"1)
Figure3.4.Therelationshipbetweenoxygenevolutionand
estimatedPS IIelectrontransport inP. tricornutumgrownat25
°Cwith(o) andwithout (•) additionof0.625Mm HC03". The
measurementsweredoneat25 °CusingthePAM Chlorophyll
Fluorometer,otherconditionsasinFigure 3.1.
withk. fractionofincident photonsabsorbedbyPSII,asdetermined bytheabsoluteabsorption
cross-section,dividedbythenumberofelectrons requiredforwatersplitting.
Thefitting procedurewasperformed inMathCad6.0p/us(MathSoft,Cambridge,Mass., USA)
andstartedwithfitting Equation (3.5) tothe PSIIelectronflowdataasafunctionof irradiance
byoptimizingthe modelparameters A,Band C. KeepingA,B, and Cconstant, A'wasoptimized
assuming alinearslopeofoxygenevolutionto irradiance at lowirradiances. Finally Fwas
optimized byfittingthetotalsetofoxygenevolutiondatato Equation (3.10),keepingallother
parameters constant.
3.3 Results
Thesteadystate ratesofoxygenevolutionandtheactual (F) andthemaximal(FM')fluorescence
levelshavebeendetermined inanumberofmarinealgaeover arangeoflight intensities.Figure
3.2A showstheefficiencyof PSIIelectrontransport andtheactualandthe maximalfluorescence
levelsasafunction ofthe irradiance in P.tricornutum. Thequantumefficiencyof PSIIelectron
transport isabout 0.68 inthe darkanddecreasesto about0.3 at 1560|jmol m "2s"'.Incontrast
32
f* 150
i
-
•
i
•
i
'
i
'
D
'M
Tetraselmis sp.
i
'
•
D. tertiolecta
Rhodomonas sp.
O)
'
P. tricornutum
• o
E
O
i
•
s.
"5 100
E
Isochrysis sp.
/
/
Q.
C
o
,
/ / /
Â. /
'••-»
•••"
^ ~ ~ ~ *
<V
/
3
>
50
<D
C
0)
D)
>.
X
o
2
0
0
100
200
300
400
500
PSIIelectron flow (ixmol e m"2 s"1)
Figure3.5.TherelationshipbetweenoxygenevolutionandestimatedPS IIelectron
transport inthemarinealgaeP. tricomutum(•), D. tertiolecta(o), Tetraselmissp.
(•), Rhodomonassp. (•) andIsochrysissp. ( • ) . Experimentsweredoneusingthe
PAM ChlorophyllFluorometer,otherconditions asinFigure 3.1.
to higher plantsnotonly Fbut alsoFM'increaseswhenadarkadaptedsample isilluminatedwith
upto about 100(Jmolm"2s"1.Thisisinlinewithobservationsfrom Falkowskietal (1986)who
foundhigher maximalfluorescencevaluesat intermediate irradiances (about 100pmolm "2 s'1)
comparedto lowirradiances (< 1pmolm"2s'1) inSkeletonema costatum, Thallasiosira weisflogii
and Isochrysisgalbanausingthe pumpandprobemethod.TingandOwens(1993) interpreted
comparable results in P.tricornutumasenergydependentfluorescence quenching.From 100to
1560 pmol m"2s"1both levelsarereduced.Thismeansthatfluorescencequenching analysisas
described earlier (Schreiber etal 1986,VanKootenandSnel 1990) isnot possibleasthe
maximalfluorescence levelinthe dark (FM)islowerthanthemaximalfluorescence inthe light
(FM') leadingto negative non-photochemical quenchingcoefficients.Themethoddescribedto
determinetheefficiency of PS IIfromtheactualandmaximalfluorescence levelsinthelight
(Gentyetal 1989) doesnotdependonthe maximalfluorescence inthedarkandistherefore
unaffected bythe increaseinFM'observedinFigure 3.2A.
33
Theestimated rateof PS IIelectrontransport andthe rateofoxygenevolution areshownin
Figure 3.2B asafunctionoftheirradiance.Therateofoxygenevolution increaseswith
increasing irradiance untilamaximumisreachedatabout800 pmolm"2s ' . Athigher irradiances
therateofoxygenevolution decreases.Theestimated rateof PSIIelectrontransport also
increases in P.tricornutummXU increasing irradiance butsaturation isnotyetobserved at 1560
umolm"2s"1.Thefitted linesindicatethatthemodified modelof Eilersand Peeters (1988) can
giveagooddescription ofboth PSIIelectronflowandoxygenevolutiondata.
Table3.I.Summaryoftheparametersresultingfromafitofthemodeltotheestimatedratesof PSII
electronflowandoxygenevolutionshowninFigure3.4.
<IW
f'(a)
A
B[~t)
s2
s
k
E
Rhodomonas sp.
0.80
0.828
1.29E-7
1.18E-4
0.424
0.210
Tetraselmis sp.
0.76
0.716
9.42E-7
7.20E-4
0.407
0.217
D. tertioleda
0.73
0.713
5.47E-7
5.14E-4
0.239
0.154
Isochrysis sp.
0.81
0.781
0
5.32E-4
0.442
0.545
P. tricornutum
0.64
0.637
6.18E-07
2.45E-4
0.225
1.16
2
'(tanmeasuredatlowlight (<15 pmolm" s')
Figure 3.3 showsthe relationship betweenoxygenevolution andtheestimated rateof PSII
electrontransport for P.tricornutum. Therelationship isapproximately linear at lowand
moderate photosynthetic ratescorrespondingto light intensities upto about 400 prnol m"2 s"1
(cf. Fig.3.2B).Athighphotosynthetic ratesthedecreaseofthe rateofoxygenevolution results
inanon-linear relationship at high irradiance.Asthisdecrease inthe rateofoxygenevolutionis
notaccompanied byadecrease inthe rateof PSIIelectrontransport,it mustbecausedby
oxygenconsuming processeslinkedto linear electronflow, e.g., Mehlerreactionor
photorespiration.
We examinedthe roleof photorespiration byadditionof bicarbonatetothemediumto
suppress oxygenation of ribulose 1,6-diphosphate.Theeffect ofadditional bicarbonateonthe
relation between PSIIelectrontransport andoxygenevolutionisshowninFigure 3.4.The
additionof bicarbonate results inbothahigher maximalrateofoxygenevolutionandahigher
maximalrate of PS IIelectrontransport.Theirradiance atwhichtherelation betweenthe rateof
PSIIelectrontransport andthe rateofoxygenevolutionbecomesnon-linear (about 250 umol
m"2s'\ not shown) is,however,notsignificantly influencedbythe HCO3" concentration.Similar
effectswereobserved in Tetraselmissp. (Geelunpublished results) andwetherefore conclude
that under our experimentalconditions photorespiration isnotthe major causeforthenon-linearity observedat highirradiance.
Figure 3.5 showsthe relation betweentherate ofoxygenevolutionandtheestimated rateof
34
'co
350
j
•
i
'
i
•_
j
•
A
CM
'E
4
300
Ol
A
ö
E
250
3.
200
5
O
«»-
150
c
o
o
<u
100
<D
w
/ /
100 j °
//
-
1
120
80
^<^^~-
T r ^ * ^ ^ ^
o
T,
-§É^°
60
40
o
en
3
o
E
3
CM
50
20
0
Q.
O
i
0
400
800
1200
0
1600
P F D ( n m o l m" 2 s"1)
'at
ÜE
o
O)
i
"5
E
o.
c
g
i
'
i
'
i
'
i
'
i
'
i
.s'
120
A
r
A
A
/'
80
n%Ja
D
p-:?'jy
o
>
<u
c
<u
at
>.
X
o
4>
c
40 h
„
.-•a-tr
0
O
A y*r~
o^.-0' o
n <
0
I
50
100
150
200
250
PSIIelectronflow (jimol m 2
300
i-
35C
s1)
Figure3.6.A: Therateofoxygenevolution (opensymbols)andtherateofPS II
electrontransport (closedsymbols)asafunctionoftheirradianceat 10°C (*,°),
15°C (H.D) and20 °C (A,A). Thesolidlines representthefits of PSIIelectron
flowandthedashed linesfitsofoxygenevolution.B:Therelationship between
oxygenevolution andestimated PSIIelectrontransport in P. tricornutum.
Symbols: 10°C (o), 15°C ( • ) and20 °C (A). Thecombinedfitsareshownas
lines: 10°C(
), 15°C(—•) and20 °C(
). Eachmeasurementwasdone
withaseparate sample ,other conditions asinFigure 3.1.
PSIIelectrontransport for P.tricornutumandfour other species.Atlowratesanapproximately
linear relationship betweenoxygenevolution andPSIIelectrontransport isobserved.Athigh
ratesthe relation ismorenon-linear. Ingeneralwefindsaturationofthe rateofoxygenevolution
whichisnot accompanied byasimultaneous saturationoftheestimated rateof PSIIelectron
35
1
60
50
-J\
*—
3 .
co
o
-\
-
-A
\
-
ro
o
fluorescence
•2- 40
•
10
,
0
0
200
400
600
PFD([jmol m"2s"1)
800
Figure3.7. Actual(opensymbols)andmaximum(closed
symbols)fluorescencelevelsasafunctionoftheirradianceat
5°C («.o), 15°C (B,D) and 25 °C ( A , A ) . Conditions asin
Figure 3.4.
transport.Theirradiance atwhichdeviationfrom linearity starts isdifferent for thedifferent algal
speciesexaminedandvariesfrom 200 nmolm"2s"1 inIsochrysis.to400 pmolm"2s~'in P.
tricornutum, about 500 [jmolm"2s"' in Tetraselmissp. and Rhodomonassp.andnosaturation
until 1000 pmolm~2 s~'inD.tertioleda.Table 3.1 summarizesthe resultsofthefitprocedures.
Thevalueofthe probability a ofphotochemistry inanopenunitisquitesimilarto thevalue of
thequantumefficiencyofPSIImeasuredatlowlight,albeit slightlysmaller inmostcases.
Assuming a » ß , Yequals B'. Table 3.1 showsthatthe rateconstant y for thedark
conversionofclosedtoopenunitsismuchmorespeciesdependent anddiffersalmostanorder
of magnitude between Rhodomonassp.and P.tricornutum. Theproportionality constant /rfor
the relation betweentheestimated rateofPSIIelectrontransport andthe rateofoxygen
evolutionisclearlyspecies-dependent andvaries byafactor of2betweenthespecies.
InFigure 3.6Atheeffect oftemperatureonthe ratesofPSIIelectrontransport and oxygen
evolutioninP.tricornutumisshown.Boththe maximalrateofPSIIelectrontransport andthe
maximalrateofoxygenevolution decreasewithdecreasingtemperature. Theinitialslopeofthe
relationbetweentheestimated rateofPSIIelectrontransport andthe rateofoxygenevolutionis
independent oftemperature (Fig.3.6B).Thelight dependenceof theactualandmaximum
fluorescence levels (FandFm')isshowninFigure 3.7 atvarioustemperatures.At15and25 °C
thefluorescence levelsF andFM'increaseatlowto mediumirradiances anddecrease again at
36
higher irradiances.Theirradiances atwhichthemaximaarereacheddecreasewithdecreasing
temperature;at 5°Cthe light-induced increaseof FM'isnolonger observed.Thetemperature
effects on F andFM result inanincreaseofthe FV/FMwithdecreasingtemperaturefrom0.61 at
25 °Cto 0.67 at 5 °C(Table 3.2).
Table3.2.TheFV/FM ofdarkadapted P.
tricornutumgrown at20 °Casafunctionofthe
incubationtemperature.F0andFMwere
measuredafter 30minutesdarkadaptationin
themeasuringcuvettewiththeXe-PAM
fluorometerusing 10replicatesamplesateach
temperature.
Temperature
(°Q
FV/FM±STD
5
0.671 ± 0.004
10
0.673 ± 0.004
15
0.645 ± 0.006
20
0.636± 0.006
25
0.612 ±0.006
30
0.550 ±0.012
3.4 Discussion
Undertheconditionsexamined here (normalandincreased HCO3"concentration,different
temperatures) wefound indifferent algalspeciesalinear relationbetweentherateofoxygen
evolution andtheestimated rateof PSIIelectrontransport at irradiances upto 200 ( Isochrysis
sp.)or 1000 [Jmolm~2 s"1(D.tertioleda), but mostlyto irradiances of about 400 umolm"2s"1.
Thisisinthe rangeofthetypicalmeanirradiances inaseawater column (Kirk 1994).
Furthermorethe rangeoflinearity extendsto anirradiancewhichis2to 5timeshigherthanthe
irradiance atwhichthespeciesweregrown.Thisrangeinwhichalinear relationshipwasfoundis
widerthaninReeset al (1992),whodemonstrated non-linearity inthe relation betweenoxygen
evolutionand PSIIelectrontransport at irradiances comparabletothoseatwhichthealgae
weregrown.
Thenon-linear relationship at highirradiances resemblesthe resultsof Harbinsonetal
(1990) and Kralland Edwards (1990) withhigher plants.Inexperiments usinghigher plant
thylakoids andartificialelectronacceptors Hormannetal (1994) havedemonstrated agood
relationship betweenthe ratio (FM'-F)/FM' andthequantumyieldofPSIIelectronflow. Fortheir
37
experimentsthismeansthatthe non-linearity isnotcausedbycyclicelectronflowwithin PSII,but
mustbeduetothe nonassimilatory electronflowassociatedwithoxygenuptake.Falkowskietal
(1986) suggestedthat cyclicelectronflowaround PSIImightexplainthedissimilarity betweenthe
normalizedflash-induced increaseinthefluorescenceyieldandthe normalized rateofoxygen
evolution inanumber ofeucaryotic algae.Intheir experiments,however,contributionsfrom
photorespiration or Mehler reactionwerenottakenintoconsideration.Inamorerecent
publication Prasiletal (1996) providedevidencefor uncoupling oflinear PSIIelectronflowand
oxygenevolution,buttheir conclusionswerebasedonfluorescence andoxygenmeasurements
using 150 |jsflashtrains resulting inasingleturnover per PSIIreactioncenter. Underthese
conditions PSIIphotochemistry andoxygenevolutioncanbeuncoupled,depending onthestate
ofthewatersplitting systemwhichwasnot measuredinthe light.Inourconditionsamultiturnover light-pulsewasusedto closethePSIIreactioncenter diminishingthedependence ofthe
redox-stateofthewatersplitting systematthestart ofthesaturating light pulse.We, therefore,
regardtheresults of Prasiletal (1996) notasconclusiveevidencefortheoccurrence ofcyclic
PSIIelectronflow in vivo. Ascyclicelectrontransport inPSIIisexpectedto occurwithinthe
reactioncentre,its kineticsshould besimilar inthylakoids or intactalgae.Thereforewethinkthat
the resultsof Hormannetal(1994) might beusedto dismisstheexplanationofcyclicelectron
transport inourexperiments.
Thenon-linear relationshipwasnotinfluencedbyaddition ofextraHCO{in our experiments
(Fig. 3.4) indicatingthat photorespiration isnot involved.Bicarbonate addition didstimulate
oxygenevolutionsomewhat,indicatingthat somephotorespiration waspresent incontrol
conditions (Fig.3.3).Whilephotorespirationwasreduced bytheincreasedcarbondioxide
concentration,the Mehler reactionshouldnot beaffected bytheaddition of bicarbonate.This
wouldfavour aroleofthe Mehler reactionwhichisdependent onreducedferredoxin.Asthe
detoxificationof reducedoxygenspecies results intheformationof molecular oxygen,this
detoxification processshouldnot beveryeffective underour conditions.Thisisinlinewiththe
resultsof Reeset al (1992) whofoundastronger decreaseinthequantumyieldofoxygen
evolutionthaninthequantumyieldof PSIIelectrontransport under non-photorespiratory
conditions in Dunaliella C9AA. Theysuggestedthattheir resultsgiveevidencefor
non-assimilatoryelectronflow. Resultsof Kanainplanktonfromestuarine surfacewatersandin
cyanobacteriaconfirmthepossibility ofincreased Mehler-reaction-oxygen-uptake athigh
irradiances (Kana 1990, Kana 1992, Kana1993) Anincreased Mehlerreactioncorrespondswith
amore reducedferredoxin,whichisincorrespondencewiththeextensionofthe modelwiththe
oxygenuptakecomponent. Extraoxygenuptake alsomight becausedbyanincreaseof
mitochondrial respiration inthe lightasWegeret al (1989) foundinthediatom Thallassiosira
weisflogii. Theyfoundthat light respirationwasashighas30%ofmaximalphotosynthesis.
Resultsof Beardalletal(1994) showanincreasedoxygenconsumptionjust after the light isput
out comparedto thedarkadaptedstate.Inthelineoftheseresultsour assumptionthat
mitochondrial respiration ratesareconstant atalllight intensities leadsto anunderestimationof
photosynthetic oxygenevolution at highirradiances,whichmaybethecauseof (part of) the
non-linearity betweenoxygenevolutionandPSIIelectronflow.However,non-linearitywasalso
found inthecomparisonof PSIIelectronflowdatawithshorttermcarbonfixationdataina
38
naturalseawatersample (CGeelunpublished results),whichsuggeststhat increased
mitochondrial respiration isnottheonlycauseofthe non-linearity.Thenon-linearity inthelow
light regionobserved byÖquistandChow(1992),Oberhuberetal (1993) andHormannetal
(1994) wasalsopresent inour measurements.Theunderlying mechanismofthis non-linearity
hasnotyetbeenelucidated.Inmicroalgaeratesoflight-dependent mitochondrial respiration
involvedinammonium assimilation might behighenoughto affecttherelation betweensteady
stateoxygenevolution andphotosynthetic electronflow (Wegeret al 1988).Theratioofthe
estimated rateofelectrontransport andthe rateofoxygenevolution isnotdependenton
temperature in P.tricornutum (Fig. 3.6) and Tetraselmissp.. Theseresultsarenot inagreement
withfindings in DunaliellaC9AAwhereastronger decrease inthequantumyieldofoxygen
evolutionthaninthequantumyieldof PSIIelectrontransport at lowtemperature hasbeen
reported (Reeset al 1992).
BothFV/FM andFM increasewithdecreasingtemperature (Table 3.2 andFig.3.7).An
explanation might befound inenergy-dependent quenchinginduced bychlororespiration whichis
believedto beassociatedwithprotontranslocation acrossthethylakoid membraneduring
oxidation ofthe plastoquinone poolattheexpenseofstromal NADPH(Peltier andSchmidt
1991).Thisproton gradient wassuggestedto bethecauseofthe lowFM andFV/FM levelsinP.
tricornutuminthedark (Ting andOwens 1993). Inourcasethe lower rateofenzymatic
processes at lowtemperatures might leadto adecreaseoftheactivityofthe chlororespiratory
pathwaywhichinturn might leadto alower proton gradient acrossthethylakoid membranein
thedarkadecreased energy-dependent quenchingandanincreasedvalueof FM and FV/FM.
However,inthe presenceofthe uncouplers,nigérianeor CCCPwedid notobserve higher F V/FM
values indicative of lessenergydependent quenching in P.tricornutum (CGeelseeChapter 7).
Thisobservation suggeststhat chlororespiration isnot involved inlowering FV/FMinP.
tricornutum. Alowertemperature might leadto alarger absorption cross-sectionofPSIIwhichin
turn coulddecrease PSI fluorescence contribution resulting inahigher FV/FM.Anincrease inaPSII
should leadto anincreased initialslopeoftheoxygenevolutionasafunction ofthe rateofPSII
electronflow. Basedonthe relation between PSIIelectronflowandoxygenevolution (Fig.3.6B)
thechange intheabsorptioncrosssectioncannot belarge.
Atlowlight,the ratioof estimated PSIIelectrontransport andoxygenevolution, k, variesa
morethanafactor 2betweenthe different speciesexamined (Fig.3.5andTable 3.1). Theratio
betweenthe estimated PSIIelectrontransport andtheoxygenevolution mightvary lessthana
factor 2whenthe rateof PSIIelectronflow iscalculated astheproduct oftheefficiency ofPSII
electrontransport,the irradiance andtheabsorption cross-sectionofphotosystemII, oKn. As
photosynthetic electrons arenot onlyusedincarbon reduction but alsointhe reductionof
nitrateto ammonium,the nitrogen sourcealsomight affect theslopeofthe relation betweenthe
rateof PSIIelectrontransport andtherate ofoxygenevolution (Wegeret al 1988, Holmesetal
1989, HuppeandTurpin 1994). Foraccuratepredictions of phytoplankton oxygenevolutionor
carbonfixationfrom insituchlorophyllfluorescence measurementsfrequent calibrations inthe
laboratorywilltherefore berequiredto determinethe ratio betweentheestimated rateof PS II
electronflowandoxygenevolutionor carbonfixation.Themodelof EilersandPeeters (1988),
asmodified here,canprovideagoodtoolto estimateoxygenevolution at highirradiancewhere
39
the relation isnon-linear.
3.5 Conclusions
Therelationship betweenphotosynthetic oxygenevolution andPSIIelectronflowasestimated
bysimplechlorophyllfluorescence measurements isshownto benon-linear inanumberof
marinealgae.At lowandmoderateirradiancestherelationship isalmost linear andoxygen
evolution andestimated PSIIelectronflowarewellcorrelated.Therelationship ismore non-linear
at highirradiances andthenon-linearity issuggestedto berelatedto oxygenconsumption other
thanphotorespiration asit isnot affected byadditionof bicarbonate.Therelationship canbe
described byaphotosynthesis modelmodifiedto allowfor oxygenuptake.Therelationship
betweenoxygenevolutionandestimatedPSIIelectronflowunder limitinglightisshowntobe
not dependent ontemperature between 10and20°C.
40
4 MEASUREMENT OF PHYTOPLANKTON ELECTRON FLUX
U S I N G A X e - P A M FLUOROMETER
4.1 Introduction
InChapter 3the rateof PSIIelectronflow,estimatedasthe product oftheefficiency of PS II
electronflowandtheambient photonfluxdensity,hasbeencomparedwiththe rateofoxygen
evolution per amountofchlorophyll inseveralmarinealgalspecies.Foreachspeciesadifferent
relationwasfound.Therefore it isnot possibleto predictthephotosynthetic oxygenevolutionof
aphytoplanktonsamplefrom asinglemeasurement oftheefficiencyof PSIIelectronflow.For
thedetermination oftheabsolute amountofPSIIelectronflowofaphytoplankton samplethe
functional absorption cross-section of PSIIhasto bedetermined aswell.Another problem inthe
determination ofthe phytoplankton electronflux,PEF,isthelowsensitivity ofthePAM
fluorometerwhichprevents itsusefor fluorescence measurements onmarine phytoplanktonat
theconcentrationswhicharefound inthenaturalenvironment.Anewfluorometer wasdeveloped
whichusesaXenonflash lampfor excitation (Schreiber etal 1993).Thisapparatus,theXe-PAM
fluorometer, ismuchmoresensitivethanthe PAM fluorometer,especiallywhenit isusedwiththe
PPMONprogramwhichwasdedicatedfor controllingtheoperation ofthedatalogger andthe
analysisoftheoutput data.Also,duetothewhiteexcitation lightoftheXenonflashlampweare
ableto estimatealsotheabsorption cross-section undercertainconditions. InthisChapterwe
haveexaminedthe possibilitiesandlimitationsofthissystemandtheconditions underwhich
correct measurements oftheefficiency of PSIIelectronflowandthefunctional absorptioncrosssectionof PSIIcanbemade.
4.1.1ComparisonoftheXe-PAMandthePAMfluorometers
ThePAMfluorometer
ThePAMfluorometer hasbeendevelopedfor plantstudies (Schreiber 1986). Itusesred
excitationfrom alight emitting diode (LED)whichpeaksatabout 650 nm.Measuring light above
680 nmisrejectedwithashort passfilter (Balzers DTCyan).Fluorescence emissionismeasured
withaphotodiodedetector whichisscreened byalongpassfilter (1 mmSchott RG9)that
transmits lightabove700 nm.Stray lightfromthe LEDandfromtheactinic light,andlow
wavelengthfluorescencegenerated bytheactinic light isrejected bythe long passfilter.
Fluorescenceoriginatingfromthemodulated measuring light isselectiveseparatedfrom allother
fluorescence components bythewindowamplifier usedinthe pulse-amplitude-modulation
technique.The650 nmmeasuring lightexciteschlorophyll awhichispresent inPSIIand PS I
andchlorophyll bwhichmainly resides inPSII.Thefluorescence emergesfrom both PSIIandPS
I.PSIIfluorescence peaksat about 680 nm.Thefluorescenceyield of PSIisnotvariable andit
peaksat about 730 nm(Holzwarth 1990, KrauseandWeis 1991) butthefluorescenceyieldis
muchlowerthanthefluorescenceyieldof PSII.Thereforestillmostofthefluorescence measured
withthe PAMfluorometer originatesfrom PSII.Althoughfluorescence isonlydetected at
41
Saturation
Puises
Emission
Filters
.
V
Fiberoptics Mirrored
i
Cuvette
Photodiode
Detector
&
.1i
Wïï
>
s~
Quartz
Rods
Pulse
Preamplifier
Excitation
Filters
Actinic
Light
Flash Driver
&
Xe-Fiash
lamp
Detector
Unit
Synchronous
Amplifier
Main
Control
Unit
Signal
Registration
Figure4.1. Experimentalset-upoftheXe-PAM fluorometerinitsstandardopticalconfiguration. ModifiedfromSchreiberetal(1993).
wavelengths above700 nmthesetup issufficiently sensitivefor higher plants.
TheXe-PAMfluorometer
ThecustomversionoftheXe-PAMfluorometer (Schreiber etal 1993) developedfor this
project, usesaXenonflashlampfor excitation andaphotodiode detector for detection.The
white measuring lightoftheXenonlampinour set-up isusually restrictedto arangeof400 to
560 nmbyacolourfilter (Schott BG39,4mm) andfluorescence canbedetectedfrom 650nm
upwards.Thedetector isprotectedfromactinicandmeasuringlightbyacombinationofa
dichroicfilter (Balzers R65) andalong passfilter (Schott R6645).Thenewfluorescence
excitation anddetection range improvedthesensitivityoftheapparatus significantly by2ways:i)
boththeexcitation anddetection rangeareenlargedallowing more pigmentsto beexcited:
chlorophyll isexcited intheSoret band,butalsoaccessorypigments likethe carotenoids
fucoxanthin andperidininandthe phycobilisomepigment phycoerythrin (Kirk 1994) areexcited;
furthermore nearlythewholechlorophyllfluorescence emissionbandismeasured;ii) interference
ofexcitation light andfluorescence detection isminimized.
TheXenonflash lampcanonly properly operate atafrequency belowor equalto 128Hzwhich
42
ismuchlowerthanthefrequencies usedwiththe LEDinthe PAM fluorometer. However,asthe
flash intensityoftheXenonlampishigherthanthat ofthe LED,theXe-PAMgivesahigher
fluorescencesignalandisthusmoresensitive.Itmustbekept inmind,however,that asingle
measuring pulseshould notexcite morethanafewpercent ofthePSIIreactioncentres inorder
to prevent actiniceffects.
TheXe-PAMisequippedwithquartz rodswhicharemuchmoreefficient inlight transmission
thanthefibre optics usedinthePAM.TheXe-PAMisnowoptimizedfor usewithalgaland
chloroplast suspensions inafluorescencecuvettewith 1mleffectivesamplevolume.Thequartz
rodsfor excitation anddetectionarepositioned at anangleof90° reducingtheamountof
measuring lightthat reachesthedetector. Forthesamereasonthequartz rodfortheactinic
light andthesaturating light pulseisalsopositioned at anangleof 90 ° withthedetector (Fig.
4.1).
Eventhoughtheexcitationanddetectionwavelengths canbewellseparated usingan
appropriatefilter setandtheconstructionofthemeasuringset upisoptimizedfor minimalstray
light,abackground signalmayisusuallymeasured.Thesignalfrom algalsamplescanbe
correctedfor thisbackground signalbyzeroingthesignalinablanksamplewiththezero
compensation connectedto the measuring lightswitch.
Thespectral compositionofthe measuringlightwillinfluencethebackground signal.Blue
measuring lightitselfwillhardlycontributetothe backgroundsignalasthedetector usually
measures light above650 nm.However,iftheoptical pathcontainsfluorescing substancesother
thanchlorophyll,blue measuring light mayleadtofluorescenceemissionnot relatedto
chlorophyll.Redmeasuring lightismoredifficultto rejectwiththelongpassfilter andstray light
willtherefore contributetothe background signal,butontheother handredlightwillhardly
excitateother fluorescingsubstances.
WiththeXe-PAMvariablefluorescence canbemeasuredonsampleswithachlorophyll
concentration aslowas0.02 ugr' (Schreiber et al 1993) whereasthePAMfluorometer requires
achlorophyll concentration ofabout 1ugml"1for measurablefluorescencetraces (Chapter 3).
4.12 Phytoplanktonelectronflux
Theabsolute phytoplankton electronflow inanalgalsampleisdependent onseveral
parameters ofthealgal photosystemsandoftheenvironment. First,theambient photonflux
densitywill poseanupper limittothe rateof PSIIelectrontransport.ThePEFofanalgalsample
isdetermined bythecapacityofthealgaeto absorbthis lightwhichisdescribed bythe
functional absorption cross-sectionof PSII,0PSn,andtheoverlapbetweenthespectrumofthe
absorptioncross-section andthespectrum oftheambient photonfluxdensity.Furthermorethe
PEFisdetermined bythecapacityofthealgaeto usetheexcitationenergyfor electron
transport, described by( j ) ^ andbythenumberofPSIIreactioncentres,nP5l|,present inthe
sample.
43
4.2 Materials and methods
Culturingthealgae
Themarinealgae PhaeodactylumtricornutumandDunaliellatert/o/ectamre growninbatch
cultures inf/2 mediumat pH7.4 (Machlachlan 1975) inartificialseawater (35 gseasalts l"\
Sigmaseasalts).Thecultureswerekept at 20 °Cand 100 umolm"2s"1continuous irradiation.
Theywere mixedbybubblingwithair.
Preparationofthephytoplanktonsamples
Samplesusedto determinethemaximalefficiencyof PSIIelectrontransport weredark
adaptedfor about 15minprior tothemeasurement. Light adaptationofsamplesto measurethe
steadystateefficiency of PSIIelectronflow lastedabout 10min.Concentration ofthealgae,
whennecessary,wasachieved bycentrifugation andresuspensioninfresh medium. All
measurementswerecarried outat 20°C.
Fluorescence measurementswithaXe-PAMfluorometer
ThemeasurementswerecarriedoutwithaXe-PAMfluorometer (seealsoChapter 3) ata
modulationfrequency of 16Hz.Thefluorometer wasusedincombinationwithaPC equipped
withadatalogger (Keighleymodel 576).Adedicated program PPMON (Phytoplankton
Photosynthesis MONitor, LovoanSoftwareand Education,Wageningen,The Netherlands)
controlledtheoperationofthedatalogger andtheanalysisoftheoutput-data. Fluorescence
parametersweredetermined asdescribed inChapter 3.
ThePPMONprogram
PPMON,written inTURBOPASCAL6.0,isadedicated programforfluorescence and/or
oxygenmeasurements.Applicationsforfluorescence measurement aredescribed hereonly.
Severalparameterscanbecontrolled bytheprogram:thetimeatwhichthemeasuring lightis
put on,the number ofsaturating lightpulses,thelengthofthesaturating light pulse,the
repetitionfrequencyofthesaturating light pulseandthetimewhentheactiniclight isput onor
off.Thebaselineisdetermined beforethemeasuringlight isputon.TheFlevelisdetermined
during atime intervalof0.2 swhichends0.1 sbeforethesaturating light pulse isturnedon.
Themaximalfluorescence isdetermined during eachsaturating light pulse.Theperiod over
whichthebaselineandtheFlevelaredetermined canbechosen.Furthermorethe program
enablesaveraging dataandcanmakecorrectionsfor blancsamples.Theprogram isableto
repeat agiven procedure over along period resulting insemi-continuous measurements.The
program calculates<\>K]I fromthe baseline,the FlevelandtheFM' level.
Determinationoffluorescence excitationandemissionspectra
ExcitationandemissionspectraofF0 andFMweremeasuredusingfilter combinationsshown
inTable 4.1. Asinglesamplewasusedto measureacompleteexcitationspectrum.Emission
spectraof F0 andFM weremeasuredbyvaryingthedetectionwavelength.Excitationwas
performedwith broadbandmeasuring light (400to 560 nm,BG39,Schott).Detectionwas
44
performed at 670 nm (670 nm bandpass filter, 10 nm bandwidth, Balzers), 680 nm (680 nm
bandpass filter, 20 nm band width,Oriel), 705 nm (705 nm bandpass filter 15 nm band width,
Balzers), 730 nm (730 nm bandpass filter, 20 nm bandwidth,Oriel) and 750 nm (750 nm
bandpass filter, 11 nm band width, Balzers).
Table4.1. Description of excitation and detection wavelengths and applied colour filters
Numbered filters are specified asfollows: I:BG39;Il RG645; III: 430 nm bandpass filter, 12
nm bandwidth;IV:475 nmbandpassfilter, 6 nmbandwidth;V: 530 nmbandpassfilter, 10
nm bandwidth;VI:550 nm bandpass filter, 7 nm bandwidth;VII:625 nm bandpassfilter,9
nmbandwidth; IX:705 nmbandpassfilter; 15nmbandwidth;X:R65,dichroicfilter; XI: B42,
dichroic filter; XII: G49/59, dichroic filter; XIII: 655 nm shortpass filter; XIV: 650 rm
bandpass filter, 20 nm bandwidth; XV: 680 nm bandpass filter, 10 nm bandwidth. Filtee
weresupplied bySchott (I and II), Balzers (IIItoXII) andOriel (XIIItoXV).
Excitation
Detection
band (nm)
filters
band (nm)
filters
400-560
I
>650
X II
430
III;XI;I
>650
X II
480
IV; I
>650
X II
530
V; XII; I
>650
X II
550
VI;XII,I
>650
X II
625
VIII;XIII
6801
X; II; XV
625
VIII;XIII
7052
X; II; IX
650
XIV; XIII
705
X; II; IX
1
Thisdetection rangewasusedforD. tertiolecta.
Thisdetection rangewasusedforP. tricornutum.
2
Determination of phytoplankton electron transport and oxygen evolution
Sampleswere dark adapted for 30 min.NaHC03was added after the dark adaptation to a
final concentration of 0.625 mM. Oxygen andfluorescence measurements were done as
described in Chapter 3for the Xe-PAMfluorometer. The chlorophyll aconcentration was
determined as described in Chapter 2.
Determining the effect of the measuring light intensity on the efficiency of PS IIelectron flow
The measuring light intensity wasvaried by using neutral density filters.Samples were diluted
to a concentration of 10 |jg chlorophyll aper ml and dark adapted.At each measuring light
intensity aseries of 5 or 10 measurements was made using 2 samples.The control
measurement was done on the non-diluted sample.
45
0,8
i
a
D
0,6
i
D
i
D
i
i
a
D
*
3
*
s
LL.
0,4
•
P. tricornutum
a
D. tertiolecta
400-560 nm
i
0,2
4()0
i
450
500
i
i
550
600
1
650
700
excitation wavelength (nm)
Figure4.2. Measuredefficiency ofPS IIelectronflowinthedarkasafunctionofwavelengthofthe
measuringlight. DataareshownforD. tertiolectaand P. tricornutum. Eachserieswasrepeated2 (D.
tertiolecta) or4[P.tricornutum) times.Errorbarsrepresentthestandarddeviation.
Determining theeffect ofthesaturating light pulseontheefficiency of PSIIelectronflow
Theeffect oftheintensity ofthesaturating light pulseontheobserved maximalfluorescence
wasexamined indarkadaptedsamples.Foreachintensity 4samplesweremeasuredand
averaged.Thesaturating light pulses lasted 1s.Theeffect ofthelengthofthesaturating light
pulsewasexamined indarkadaptedsamples usinga 10ssaturating light pulse.Theaverageof
10measurements ondifferent samplesweretaken.Thesaturating light pulses lasted 10s.The
comparison oftheeffect ofthesaturating light pulseandoftheaddition of DCMU wasexamined
insampleswhichweredarkadaptedfor 45 min.DCMU wasaddedinthedarkatafinal
concentration of 10 pM. Foreachexperiment 3samplesweremeasuredandaveraged.
4.3 Results and discussion
Thephytoplankton electronflux inasample isdetermined bytheambient photonflux density,
thesizeandoptical properties ofthe light harvesting complexesofeachphotosystem,the
numberof PSIIreactioncentreswhicharecapablefor electrontransport andtheefficiencyof
the photosystems inconvertingtheexcitation energy intoelectrontransport.Thisisdescribed in
46
Equation(4.1)
PEF =npsuPFDA f10°IAa)
oPSII(X) <bpsn(X)d{l)
J400
(4.1)
With PEF:phytoplankton electron flux; nPSI|:thenumber ofPSII reaction centres; PFDA: the
ambient photon flux density (umol m"2 s"'); lA(Â):thespectral distribution function ofthe ambient
light; ( ^ „ ( Â ) : thespectral distribution function ofthe PSIIabsorption cross-section and (bPS
„(A): thespectral distribution function ofthe efficiency ofPSIIelectron flow (electronper
photon).
4.3.1 Spectral distribution of the efficiencyof PSII electron flow
The efficiency ofPSIIelectron flow was tested onitsdependency onthe wavelength ofthe
measuring light. Ifthe efficiency ofPSIIelectron flow isindependent onthe wavelength ofthe
2,0
Dunaliella
0,8
1,5
s
A
1,0
0,5
I
2ä
1i
1
1,0
0,5
0,0
0,4
m
1,0
0,8
1,5
s
s
u.
0,2
m
Phaeodactylum
0,6
Â
X
670
ïh
1
ïh
HI
r
I
680
705
730
0,6
-V
0,4
0,2
750
detectionwavelength(nm)
Figure4.3.Measured minimalfluorescence (white boxes) and maximal efficiencyofPSIIelectronflow(dashed boxes) inthe dark asafunctionofthe
emissionwavelength. Dataareshownfor/?, tertiolectaand P. tricornutum.
Error bars representthe standard deviation.Sampleswereconcentrated20
times bycentrifugation.
47
measuring light Equation (4.1) canbesimplified bytaking (j^,, outthe integralfunction.In
Figure 4.2 FV/FM obtainedwith broad bandmeasuringlight (400-560 nm) iscomparedwiththat
obtainedwith narrow bandmeasuring lightof430,480, 530, 550,625 and650 nm.In D.
500
I
I
I
I
I
I
I
A
400
o
'in
,- 300
o
f 200
"5
E
3; 100
o
o
o
o
o
o
o
o
O
—i
0
0«» •
I
I
0
I
I
I
I
I
50 100150200250300350
JE
250
I
I
I
I
I
I
B
200
•
150
°
•
•o°°
LL.
O
100
0_
•o
50
0 - 8b
i
0
i
2
i
4
i
6
i
8
i
10 12
PEF
F/gure4.4.A:oxygenevolutionperchlorophyll(^)asafunctionof PS
IIelectronflowperPS IIreactioncentre(4).P. tricornutum. O;D.
tertiolecta:%. B:phytoplanktonoxygenevolution(P0F)asafunction
ofphytoplanktonelectronflux(PEF).SymbolsasinA.
48
tertiolectaallFV/FM valuesare invariablewithinthestandard deviation exceptforthevalue
measured at measuring light of625 nm.InP.tricornutumwe usedahigher detectionwavelength
(at 705 insteadof680 nm)for the625 excitation.Thisresulted inanFV/FM valueinvariablefor
thewavelength bandsused.Previousexperimentsonthequantumyieldfor oxygenevolution
(moloxygenper molphotons absorbed) inthealga Chlorellashowedsomespectral dependency
asalower quantumyieldwasfoundatabout 480 nmthanfor theotherwavelengths,whichwas
assignedto absorption ofcarotenes (Lawlor 1993). However,fluorescence measurements are
moredirectly relatedtothatfractionoftheabsorbed photons asonlythe absorbed photonsthat
reachthe chlorophyll poolcontributeto FV/FM. OnthebasisofourFV/FMmeasurementswe
concludethat <bKn isnotdependent ontheexcitationwavelength oncethe excitation reachesthe
chlorophyll pool,both in D.tertiolectaandinP. tricornutum.
Wealsoexaminedthe relation between cbPSI|andthewavelength rangeof fluorescence
detection (Fig.4.3). Both in D.tertiolectaandin P.tricornutum^^ isindependent onthe
detection range.Gentyetal (1990) foundahigher maximalefficiencyof PSIIelectronflowat
690 nmascomparedto 730 nminbarleyandmaize.Adecrease inFV/FM at higher detection
wavelengthswasfound bytheseauthorsto beaccompaniedwithanincrease inthe relative
amountof non-quenchableminimalfluorescence at 730 nm.Inaleaf reabsorption of PS II
fluorescenceemissionisrelatively highandthiswill leadto arelatively largecontribution ofnon
variable PSIfluorescence atwavelengths above700 nm.Asour algalsamplesaremuchmore
dilutethe process of reabsorption doesnotaffecttheapparent emissionspectrum.InFigure4.3
alsothe minimalfluorescence isshown.Theminimalfluorescencewashighestfrom 680to 730
nmfor D. tertiolectaandfrom 705 to 730 nmfor P. tricornutum. This iscomparablewiththe
roomtemperaturefluorescenceemissionspectraof P. tricornutum'found byGoedheer (1973)
andOwens (1986a).
Asunder our conditionsthe measuredefficiency of PSIIelectronflowisindependent onthe
excitationwavelengththe spectral distributionfraction <t>psn(A)isconstant andEquation (4.1)
canbesimplifiedto:
PEF =npsu 4>pOTPFDA J™ 0Iß.) opsiß) d(X)
(4 . 2)
4.3.2F0asameasureofPSIIexcitation
Inthis paragraphwewillfocusonthenumber of PSIIreactioncentres,n^,,,andthespectral
distributionfunctionof boththeambient irradiance andthefunctionalabsorptioncross-section.
We suggestthatthe product ofthesetermscanbeestimatedfrom F0 whichcanbedescribedby
anEquationsimilartoPEF:
F
o =npsn 4>F0uPFDM
f ™°W
< W À ) d(X)
(4.3)
J 400
With(t)Fo:the efficiency bywhichabsorbed photons leadto minimalfluorescence (photonper
photon); PFDM:thephotonfluxdensityofthe measuring light and lM(A):thespectral distribution
function ofthe measuring light.
49
Inthecasethatthespectral distributionfunctions lA(À)andlM(A)are identical,the integral
term canbeeliminatedfrom Equations (4.2) and (4.3):
PEF =FApsnPFD
°™
l
A
$F'PFD.
V
M
^
Thespectral distributionfunctionoftheambientlightandthemeasuring light aredependent
onthe lightsource.Inour experimentswiththeXe-PAMfluorometertheambient light issupplied
withahalogen lampwhichhasacontinuouswhitespectrum.Themeasuring light oftheXe-PAM
fluorometer isaXenon-flash lamp.Thislampalsoshowsacontinuouswhitespectrum,but it
normally isfilteredwithabluegreencolourfilter (BG39).Thereforethespectraoftheambient
lightandthe measuring light,although not exactlythesame,arenottoo different.
d)Foisgenerally assumedto beconstant. Inthecasethatthe measuring light photonflux
density iskept constant,thesetwo parameters canbetakentogether toformtheconstant m =
((t)Fo* PFDM)"\ Thereforethe relationof Equation (4.4) canbesimplifiedto thefollowing
Equation:
PEF =mFQ<bps[{PFDA
( 4. 5)
Equation (4.5) isonlyvalid iftheamountoffluorescencewhichisdetected isproportional to
theamount offluorescenceemissionunder allconditions. Iftheopticalpathwayofthe
measurement ischanged Equation (4.5) isnotvalidanymore.Theamount of light absorbedby
thesample isnot relevant aslongastheopticalpathsfor actinicandmeasuring lightaresimilar.
Theproduct of m•F0 canberegarded asameasurefortheamountof light intercepted byPSII
inthesample:
mF
o
=n
psu [70°7 A/ À ) < W * ) d(X~)
(4.6)
J400
Tochecktheapproximation described byEquation (4.5) wehavedonemeasurements on
boththeelectronflux per PSII,JE,andthephytoplanktonelectronflux,PEFandcomparedthem
with respectivelytheoxygenevolutionper unitofchlorophyll,J0,andwiththe phytoplankton
oxygenflux,POF(Fig.4.4). InP.tricornutummuchhigher J0valuesarefoundatcomparableJE
valuesthan in D.tertioleda.InthecomparisonofJEwithJ0it isimplicitly assumedthatthe
number of PSIIreaction centres isproportional totheamount ofchlorophyll a. Thisisonlytrue
wheneachreactioncentre isassociatedbyaconstant amountofchlorophyll aunderall
conditions.Thishasbeenshownnotto betrue (Gallagher etal 1984,Wilhelm 1993).D.
tertioledacontains chlorophyll aandbasphotosynthetic allyactive pigments, P. tricornutum
contains chlorophyll aand candfucoxanthin (OwensandWold 1986). Inthe right panelofFigure
4.4the phytoplankton oxygenfluxandthe phytoplanktonelectronflux arecomparedfor both
species.Theadapted method described inEquation (4.5) leadsto similar relations between
oxygenevolution andelectrontransport inbothspecies.Thereforeweconcludethattheuseof
theminimalfluorescence,towhichallphotosynthetic allyactivepigments contribute,leadstoa
better estimateforthe amountof PSIIreactioncentresthanchlorophyll a.
50
1
'
' <' ' i
i
1.0
u.s 0.8
i »
/ / / i//y///////////////////.
//A//7777|S^/r////////////
LL
C
£ 0.6
Q.
Q.
01
0.4
V.i.
l
l
0.1
1
2
measuring light intensity (nmol m" s"1)
Figure4.5. FV/FM asafunctionofmeasuringlightintensityinP. tricornutum. The grey
boxindicatesthetruec|3RS„value± 5 %.
4.3.3Determinationoftheefficient ofPSIIelectronflow
Effectofmeasuring light intensity onvariation inefficiencyofPSIIelectronflow
Foracorrect determination oftheefficiencyofPSIIitisessentialtoconsidertheeffect ofthe
intensityofthemeasuring light intensity.Ahighmeasuring light intensitywill result inahigher
fluorescence signalandtherefore inahigher signaltonoise ratiothanalowmeasuring light
intensity. However,asthe measuring lightisabsorbed bythephotosystemsitalsochangestheir
reduction state.Thereforethe measuringlight intensity should belowenough notto changethe
reduction stateofPSIInoticeable.TheefficiencyofPSIIelectronflowisgivenasafunctionof
themeasuring light intensityfor Phaeodactylumtricornutum(Fig.4.5).Atlowmeasuring light
intensities$Kn stabilizesatabout 0.67.Athighmeasuring lightintensities lowervaluesofcj>ps„
arefound.Thisdecreaseisduetothe reduction ofthe PQpool,causedbythe measuring light.
Thetrue efficiencyofPSIIelectronflowwasdeterminedwithhighprecision inanon-diluted
sample.Thegreybox representsthe areaofthis cj>ps,,plusorminus 5%.Itcanbeseenthatthe
51
2,0
I
'
I
- ^ 1,0
LL
E
LL
w
I
j
'
TI
yjJlr^rt~Yi __
1,5
o
iL
'
'f
0,5
f
D.tertiolecta
0,0
I
I
1,5
o
LL
^
1,0
LL
i
'
.f
I
I
i
'
I
I
'
,>-§--£-—~Ï
i
I
I
ï i
-
E
LL
~
0,5
6
P.tricornutum
0,0
.
0
i
.
2000
i
4000
.
i
6000
PFDp(iimol m V )
Figure4.6. Normalizedfluorescence ((FM-F0)/F0)asafunctionoftheintensityofthesaturatinglightpulse.Samplesareaveraged4(D. tertioleda) or 3
[P.tricornuturri) times.
intermediate measuring light intensities (betweenabout0.05and0.25nmolm"2s"1)givethe
bestvaluesfor§Kr Itshouldbenotedthatthelower limitofmeasuring light intensities leading
to acceptable (tpps,, valuesisalsodeterminedbytheconcentration ofthesample.Furthermoreit
shouldbenotedthat inmanycasesthemeasurementsarenotrestricted bythevariationinthe
measurement itselfbutbythebiologicalvariation betweensamples.
Effect ofsaturating light pulseonthedetermination ofefficiency ofPSIIelectronflow
Foracorrect determinationofthe maximalfluorescence levelthesaturating light pulseshould
beintenseenoughandlast long enoughtocauseacomplete reductionofthe PQ-pool (Tingand
Owens 1992).Ontheother handtoohighanintensityoflight mightinduce undesired
52
^
2
D.tertiolecta
32
g>
'>.
<D
4
Q)
O
(O
(U
3
o
c
o
^
2
P.tricornutum
4,
f *
O
5
10
15
*
20
Time (s)
/y<7i//i?4 7 Effectofdurationofasaturatinglight pulseonthemaximal fluorescence.Durationofthesaturatinglightpulse:10s.Dataareshownfor/?.
tertiolectaandP. tricornutum. Measuringlighton:T andoff: J.Saturating
lightpulseon: IIandoff: It.Thesaturatinglightpulsewasgivenwithacontinuouswhitelampandashutter.Curvesaretheaverageof 10measurements
withdifferentsamples
fluorescence quenching inthephytoplankton (KrauseandWeis 1991). InFigure 4.6the maximal
fluorescence isshownasafunction oftheintensity ofthesaturating light pulse. Both inD.
tertiolectaandP.tricornutumsaturation isreachedatabout 3000 pmolm"2s''. ForP.
tricornutumpreviously ahigher levelof 6000 pmolm"2s"' wasfoundto benecessary (Tingand
Owens 1992).
InFigure 4.7thefluorescence behaviour during along saturating light pulseisshownforD.
53
^
1
1
1
1
1
1
$
o
"ro
I
\
o
jrescence yield (a.u)
0,3
•
l
,
"*"«**î~U "® r A
yyvi^MWAMAAwvA
«r* -
0,0
1
I
0
2
I
I
4
6
I
8
10
12
Time (s)
Figure4.8. EffectofDCMU onmeasuredfluorescenceoff. tricornutum.The
saturatinglightpulsewasgivenwithacontinuouswhitelampandashutter.
tertioledaand P.tricornutum. D.tertioledashowsamaximumat 0.5 safter thestart ofthe
saturating light pulseandahighquenchingof 50%in 10safterwards.Whenthesaturating light
pulse isturned off thefluorescencealmostimmediately decreasestothe minimalfluorescence
levelwhichwasattained beforethesaturating lightpulse. P.tricornutumshowsaverystable
maximalfluorescence,exceptfor asmalldecreasebetween0.5 and 1safter theonset ofthe
saturating light.Whenthesaturating light pulseisturned offthefluorescencedecreases rapidly
to alevelalmost 2timeshigherthanthe minimalfluorescence,increasessomewhatandfinally
decreasesveryslowlyto theminimalfluorescence.Theinductionofquenching duringthe
saturating light pulseasfound inD.tertioledashould beavoidedwhenmore measurementsof
(bpsll haveto bemadeonthesamesample.Itaffectstheenergizationofthealgalsampleand
thusaffectstheobserved (bpSn.Although P.tricornutumûoes notshowthisquenching duringa
longsaturating light pulse,thefluorescence patternfound after thesaturating light pulsehas
turned off suggeststhat in P.tricornutumthesaturating light pulsealsoinducedsomechanges
inthe photosynthetic system.Undertheconditions usedhere (continuouswhite lampanda
shutter) aduration ofthesaturating light pulseof 0.5 sisenoughto reachsaturation.Whenthe
saturating light pulseisprovided byturning onahalogenlampalongertime isneeded asthe
lampitself takesabout 300 msto reachthe maximalphotonfluxdensity.
Saturation of photosynthesis bythesaturating light pulsewascheckedwith DCMU. DCMU
bindsto theQBbinding placeonPSIIandinhibits linear electrontransport through PSII(Krause
andWeis 1991). Closingof PSIIreactioncentresoccursalready atlowlight intensities inthe
presenceof DCMU.Anexample ofafluorescencetracewithandwithout DCMUisshowninFigure
4.8.TheDCMUsampleshowsahigher staystatefluorescencethanthesamplewithoutDCMU
suggestingthatthemeasuring lightalreadycausesanoticeable reductionofQAinthe presence
54
of DCMU. Alsosaturation of photosynthesis duetothesaturating light pulseisfaster inthe
presenceof DCMU.Boththesamplewithandwithout DCMU reachthesamemaximal
fluorescence.Thereforeweconcludethat inthisexperimentthe photonfluxdensityofthe
saturating light pulsewashighenoughto reachsaturation.
4.4 Conclusions
Inthis chapterwehaveshownthatwithcertainlimitations it ispossibleto determinethe
phytoplankton electronfluxinanalgalsample.Theefficiency of PSIIelectronflowisindependent
ontheexcitationwavelength undertheconditions used.Theaccuracy inthedetermination of(pps
i,isdependent onthelight intensity ofthe measuringlightwhichshould behighenoughfor a
goodsignalto noiseratio andlowenoughto minimizeactiniceffectsofthemeasuring light.The
accuracywithwhich tyKn canbedetermined isdependent onthe intensity andthedurationof
thesaturating light pulse.Theintensity ofthesaturating lightpulseoftheXe-PAMwasfoundto
behighenoughfor saturationof photosynthesis ofthealgaeunder our conditions.The
relationship between PSIIelectronfluxandoxygenevolution expressedonachlorophyll basis
differed byafactor 4inthediatom P.tricornutumandthegreenflagellateD.tertiolecta. Applying
Equation (4.5) asimilar relationship betweenPEFandthe POFwasfoundforthediatomP.
tricornutumandgreenflagellate D. tertiolecta.
55
5 THE RELATION BETWEEN CARBON FIXATION AND
PHOTOSYSTEMI I ELECTRON FLOW I N MARINE
PHYTOPLANKTON
5.1 Introduction
InChapter 3acurvi-linear relationship betweenphotosynthetic oxygenevolutionandPSII
electronflowwasfound inseveral marinealgae.Thenon-linearity,whichwasmostobviousat
highphotonfluxdensities,wasshownnotto berelatedto photorespiration andcouldbe
describedwithaphotosynthesis modelmodifiedtotakeoxygenconsumption intoaccount. Inthe
marineenvironmentwherethephytoplankton content isvery low ,4Cfixation measurementsare
moresuitedto determine photosynthesis thanoxygenevolution measurements.Thisisbecause
themeasurement of 14Cfixation ismuchmoresensitivethanthemeasurement ofoxygen
evolution byusing oxygenelectrodes.Photosynthetic oxygenevolutiondatainanaturalseawater
samplewillbecontaminated bythe respirationof bacteriaandzooplankton. I4Cfixation
measurements inphytoplankton might result ingrossor net photosynthesis depending onthe
durationoftheincubation.Therecyclingof respiratory carbondioxidewasfoundto behigh
(Williamsetal 1996). Photosynthetic oxygenevolution isalmostdirectly coupledto electron
transport at PSIIbutthecoupling between PSIIelectronflowandphotosynthetic carbonfixation
ismore indirect.Carbonfixation isdependent ontheATPand NADPHwhichareformed bylinear
electronflow intheelectrontransport chaininthe photosynthetic membrane.Carbonreduction,
however, isnottheonlysinkfor ATPandNADPH.Thereforethe relationship betweencarbon
fixationand PSIIelectronflow might beexpectedto bemorecomplexthanthe relationship
betweenoxygenevolution and PSIIelectronflow (Kroonet al 1993).Sothe nextstep inour
researchwasto examinethe relation betweencarbonfixationandPSIIelectronflow.
InChapter 3resultsofthe relation betweentheefficiency of PSIIelectronflowandthe
efficiency of carbonfixation inhigher plantshavebeendescribed.Underoptimalgrowing
conditions andnon-photorespiratory conditionstheyfoundalinear relationship betweenthe
efficiency of PSIIelectronflowandtheefficiency ofcarbonfixation (Gentyet al. 1989;Harbinson
et al 1990, KrallandEdwards 1990,Oberhuber etal 1993). Photorespiration ledto anon-linear
relationship betweentheefficiency of PSIIelectronflowandtheefficiency of carbonfixation
(Harbinson etal 1990, KrallandEdwards 1990).Cyclicelectrontransport wasshownto be
unimportant under conditionswere linear electronflowisnotsuppressed (Harbinson etal 1990,
Prasilet al 1996). However, more recent resultswith maizeshowedamuchlargervariability in
the relation betweentheefficiency of PSIIelectronflowandtheefficiency ofcarbonfixation
(Baker etal 1995). Fromtheseexperimentswithoutdoors growing maizeBaker et al. suggested
that others sinksthancarbon reduction mightbeimportant underthesenon-optimal growing
conditions.Apossiblesink might bethe Mehlerreactionassuggested byÖquistandChow
(1992)(see alsoChapter 3).Using ,8 0 isotopes Kana(1990,1992,1993) indeedfoundan
increasedoxygenuptakeinthe lightcomparedtothedarkinphytoplanktonfrom estuarine
56
surfacewatersandincyanobacteria.Astheoxygenuptake inthecyanobacteriawasinhibitedby
DCMUheattributed ittothe Mehler-reaction.Another sinkfor photosyntheticallyformedATPand
NADPHmight benitrate reduction (HuppeandTurpin 1994). Furthermore anincreased
mitochondrial respiration inthelight comparedtothedarkaswassuggested byWegeretal
(1989) andBeardallet al (1994) mightexplainpart ofthe non-linearity inthe relation between
PSIIelectronflowandcarbonfixation.NewexperimentsfromXueetal (1996) showthat light
induces bothanincrease inoxygenconsumption andadecrease incarbondioxideproduction.
Thiswould leadto adifferent relation betweencarbonfixationand PSIIelectronflowthan
betweenoxygenevolution andPSIIelectronflow.
Inmarine phytoplankton samplesfromtheAtlantic ocean FalkowskiandKolber (1990) and
Kolber andFalkowski (1993) havefound areasonablecorrelation betweenthe rateofcarbon
fixationandthe rateof PSIIelectronflow.Theyestimatedtheproduct ofthenumber of closedPS
IIreaction centresandtheeffective absorption cross-sectionof PSIIwiththe pumpandprobe
fluorescence methodto calculatethe rateof PSIIelectronflow.Thevariationtheyfound inthe
relation betweenthe rateofcarbonfixation andthe rateof PSIIelectronflowissomewhat
smallerthanthevariationfound inthe resultsof Bakeretal (1995).
We determined bothcarbonfixation andPSIIelectronflow in3different marinealgae.The
phytoplankton electronflux,PEF, wasestimated bythe product oftheefficiency of PSIIelectron
flow, (bps,,, the photonfluxdensity,PFD,andtheminimalfluorescence ofthealgae inthedark, F0.
Thephytoplankton carbonflux,PCF, wasdetermined directly ascarbonfixation per m"3.We also
examinedwhether apreincubationofthealgaeat agivenphotonflux densities did influencethe
relation betweencarbonfixationandPSIIelectronflow.
Ingeneralwefoundthatthe relation betweenPCF andPEFresemblesthe relationbetween
oxygenevolutionandPSIIelectronflowasfound inChapter 3.Therelation betweenPCFandPEF
at lowphotonfluxdensities ismoreor lessthesamefor all3species.
5.2 Materials and methods
Culturing conditions
Diluteandnutrient-replete,non-axenic monocultures of Isochrysissp. andPhaeocystissp.
macroflagellates (Prymnesiophyceae),andSkeletonema costatum(Bacillariophyceae),were
grown inf/2 mediumat 10°C and69 pmolm"2s"1ina12:12light-dark cycle.Atthestart ofthe
experiments,thecultures hadbeeninthe lightfor 6hours.
Incubation ofthe phytoplankton samples
120mlphytoplankton sampleswereincubated inflasksat 8discrete photonflux densities (0,
11, 24, 54, 136,281, 664 and 1530 umolm"2s"1) at 12°C inathermostatedincubator.Ateach
photonflux density9sampleswereincubatedofwhich8sampleswereusedfor thecarbon
fixation measurements (Fig.5.1).Allsampleswereput intheincubator att = 0. NaH,4C03was
addedatt = 0to sample 1.Att = 0.5 (hr) sample 1 washarvestedandtreatedforthe
determination ofthecarbon incorporation inorganicsubstances (seebelow).Thecarbonfixation
57
PCF sample 1
Phytoplankton Caiibon Flux
PCF sample 2
PCF sample 3
PCF sample 4
PCF sample 5
PCF sample 6
PCF sample 7
PCF sample 8
PHytoplankton Electron Flux
PEF sample
—O-
-o
-o-
-o-
-o
1
2
-o-
-o-
-o—
3
Incubation time (hours)
F/gure5.1. Incubationschemeof9samplesforcarbonfixationmeasurementsandfluorescence
measurements.Foreachphotonfluxdensity8samplesforcarbonfixationwereputintheincubatorat
t=0. ForPS IIelectronflowmeasurements 1 sampleperphotonfluxdensitywasputintheincubator.
Carbonfixationwasmeasuredintheperiodsindicatedwithagreybox.PS IIelectronflowwas
determinedatthetimesindicatedwithO.Aftersubsampling2ml theflaskwiththesamplefor PS II
electronflowmeasurementswasputbackintheincubator.
insample 1ofeachphotonfluxdensitywasestimatedduringtheperiodfromt = 0tot = 0.5.
Alsoatt = 0.5 NaH,4C03wasaddedto sample2.Sample2wassubsequently harvested att = 1
andtreated assample 1, etcetera.TheshadedboxesinFigure 5.1 representtheperiod inwhich
the incorporationof NaH,4C03wasoccurringfor allsamples.Forthefluorescence measurements
onlyoneflaskper photonfluxdensity (samplenumber 9) wasused (Figure 5.1). On the
indicatedtimes 2mlwastakenoutoftheflaskto measuretheefficiencyof PSIIelectron flow.
Theflaskwasput backintothe incubatorto continuetheincubation.Thesamplingand
measurement oftheefficiency of PSIIelectronflowoccurred inthemiddleofeachcarbonfixation
period.
Carbonfixation
Phytoplankton sampleswereincubatedat8 photonfluxdensities asdescribed before.The
incubationwith NaH14C03lasted0.5 hoursfor eachsample.Afterthisperiodthesamplewas
takenout oftheincubator, inorganic carbonwasremovedbyacidificationandthecarbon
58
incorporation inorganic substanceswasmeasured (Escaravageetal 1996).The phytoplankton
carbonflux,PCF, wascalculatedfromthe ,4CuptakeinBqm"3 s\
Fluorescence measurements
TheXe-PAMfluorometerwasusedasdescribed inChapters 3and4.Fluorescence
parametersweredetermined asdescribed inChapter 3.
Forthemeasurement oftheefficiency of PSIIelectronflowinthelightthe phytoplankton
sampleswereincubated asdescribed before.The2mlsamplesweretransferred rapidlyfromthe
incubatortotheXe-PAMcuvette,readaptedtotheincubationirradiancefor another 30seconds
anddppsn wasmeasured.
Thephytoplankton electronflux,PEF,wascalculatedastheproduct oftheefficiency ofPSII
electronflow,ct>psM, the photonfluxdensity,PFD,andtheminimalfluorescenceofthedark
adaptedsample,F0, determined atthebeginning oftheexperiment (seealsoChapter 4).
Inorderto establishtheextentofthephotoinhibitory damageofthesamplesincubated inthe
light at 1530 |jmolm 2 s"1these2mlsamplesweredarkadaptedfor onehourat 18°Cafter the
measurement.Thenthe maximalefficiency of PSIIelectronflowofthesesampleswasmeasured
andcomparedwiththe maximalefficiency measuredbeforethe lightincubation.
Modelling
The PEFwasfittedtothephotosynthesis modelof EilersandPeeters (1988) yieldingthreefit
parametersAE,BE andCE. ThePCFwasfitted separatelytothesamemodelyieldingthe
fitparametersA,-,BcandCc. Thefitting procedurewasperformed inMathcad 6.0+ (MathSoft,
Cambridge,MA,USA)using aleast squaresmethod.
5.3 Results
InFigure 5.2 PEFandPCF, measuredafter various preincubationtimes,areshownfor the3
species.Thedifferences inmaximalPEFandPCF betweenthese 3species isdependent onthe
specific activity ofthespeciesandontheconcentrationofthealgae inthesample.At lowto
intermediate photonfluxdensities (0to about 200 pmolm"2s"1)PEFincreases linearwiththe
photonflux density inall 3species.Intheincubationsfrom0to 0.5 hour andfrom 0.5 to 1hour
PEFisnotsaturated atthehighest photonfluxdensityinIsochrysis. Inthelonger incubationsa
saturation levelisreachedatabout 300 pmolm'2 s'. InPhaeocystisthe PEFreachesa
saturation levelatabout 300 pmolm'2s"1for allincubationperiods.InS. costatumsaturationof
PEFdoes notoccur inthis rangeof photonfluxdensities.ThePCF inthese 3speciesalsoshows
alinear increasewiththephotonfluxdensityatlowphotonfluxdensities.ThePCFin Isochrysis
saturates atabout 650 pmolm"2s"1intheshorttimeincubations butafter longer incubations
saturation occursalreadyat 300 pmolm"2s"1or less.InPhaeocystissaturationof PCF isaround
300 [Jmolm"2s~' independent oftheincubation period.Saturation of PCF inS. costatumoccurs
at about 650 [jmolm 2 s"' after shorttimeincubations andbetween 300 and650 pmolm"2 s"1
after longer incubation periods.Both PEFandPCF areindependent ontheduration ofthe
59
O
500
1000
1500 0
500
2
1000
1500
1
PFD (u.molm" s" )
Figure5.2. Phytoplanktonelectronflux(PEF,left panel) andphytoplanktoncarbonflux(PCF,right
panel) asafunctionofthephotonfluxdensity.Incubationperiodfrom0to 30min:0;from30to60
min:D; from60to90min:A;from90to 120min:v;from 120to 150min:• ; from 150to 180
min:• ; from 180to210min:A;from240to270min:• . SomeofthePEF datameasuredatthe
twohighestirradiancesinIsochrysismà Phaeocystisvtetediscardedastheywerenotreliabledueto
therelativelylowsignaltonoiseratio.
incubation,exceptforthe incubationfrom 0to 0.5 hrwhichshowshighervaluesfor PEFandPCF
comparedtothelater experiments.
Thedataof Figure 5.2 suggestthatthe relation betweenthe PCF andthe PEFiscomplexfor
all 3species.Asat lowto intermediate photonflux densities bothPCF andPEFincrease linearly
withthe photonflux density,the relation betweenPCF andPEFwillbelineartoo.At high photon
fluxdensitiesthislinearity islost.
Incarbonfixationmeasurements atwo hours incubation period isquitecommonforfield
60
lil
a.
1000
500
1500 0
2
1000
1500
1
PFD (urnol m" s" )
Figure5.3. Phytoplanktonelectronflux (PEF,leftpanels)andphytoplanktoncarbonflux(PCF, right
panels) asafunctionofthephotonfluxdensity.Eachdatapointistheaverageofdatafromsamples2
to4(seeFig.5.1)fromthedatapointsfrom0.5 to2hoursofincubation. Thefitofthesedatapointsto
themodelofEilersandPeeters(1988) isgivenbythesolidline.
measurements (Escaravageetal 1996). InFigure 5.2 it canbeseen,however,thatthedata
fromthefirst incubation perioddifferfromthedataoftheother periods inalmosteach
experiment.Thereforewechooseto omitthedatafromthefirst incubation periodandaverage
PEFand PCF datafrom 0.5 to 2hrincubation periods (Fig.5.3). Inmostcasesthefitted lineis
withinthestandard deviation ofthedatapoints.ThePEFcould bedeterminedwithalow
standard deviation at lowto medium photonfluxdensities,butthestandard deviation increases
somewhatat higher photonfluxdensities.IngeneralthestandarddeviationinthePCFvaluesis
higherthaninthe PEFvalues.Thefitsofthe modelof EilersandPeeters (1988) to theaverage
PEFand PCF areshown bythesolidline.
61
1400
i
l
i
-
1200
~ 1000
-
1/
co
"
/
800
/
£ 600
O
°- 400
•
200 0
~
Jp~
Jtf^
-dr
i
i
0
10
20
P E F(r.u.)
Figure5.4. Phytoplanktoncarbonflux(PCF)asafunctionof
phytoplanktonelectronflux(PEF).DatafromFig.S3.Isochrysis.
• ; Phaeocystis. O; S. costatum. D. Thefitofthemodelof
EilersandPeeters(1988)tothesedatapointsisgivenbythe
solidline.
Tocomparetherelation betweenPCF andthe PEF ofthe 3specieswehaveplottedthelinear
rangeof PCF asafunctionof PEFofthese 3species inFigure 5.4. Itshould benotedthatPCF
and PEFarephotosynthetic outputs per m"3sampleandthereforedependent ontheamountof
algaeinthesample.Thephotonflux density rangeoverwhichlinearitywasfound (aswas
deducedfrom Fig.5.3,notshownhere) isdifferentfor all3species:Isochrysis. linearuntilabout
650 [jmol m"2s"1;S. Costatum. linear untilabout 280 pmolm"2s"';Phaeocystis. linear untilabout
140 pmolm'2 s"'.Atcomparable PEF valuesthefit of thedataofS. Costatumshowssomewhat
higher PCF valuesthanthefitsofthedataof Isochrysisand Phaeocystis.
Wewereinterested inthedegreeof photoinhibition occurring after incubatingthe
phytoplankton samplesat 1528 pmolm~2 s"'.Table 5.1 showsboth d^s,,,FV/FM andF0for each
speciesasafunctionoftheincubation period.Bothinisochrysisand inPhaeocystistheefficiency
of PSIIelectronflow inthe light decreased rapidlyto undetectablevalues.Themaximalefficiency
of PSIIelectronflow,measuredafter onehourdarkincubation,islowered bytheincubation but
muchhigherthanthe efficiency of PSIIinthe light,indicatingthatthere arestillactive PS II
reaction centres inthesesamples.S. costatumalsoshowedalowO^,, inthe light,butthe effect
onFV/FM wasnot aslargeasfor theother 2species.Inisochrysistheminimalfluorescence starts
to decreaseafter 1hour incubationatthehighestphotonfluxdensityto alevelof 55%ofthe
initialF0after 4hours.JnPhaeocystistheminimalfluorescence increasesfromt = 0tot = 1.5
62
hoursanddecreases afterwards to 75 %ofthe initialF0.InS. costatumtheminimalfluorescence
doesnotchangeduetothe incubation inthelight.Thevariablefluorescence measuredafter
darkadaptationafter 4hr incubation inthelight correspondsto respectively 5,11 and 30
percent ofthevariablefluorescence measuredatt = 0for Isochrysis, Phaeocystisand5.
costatumrespectively (datanotshown).
5.4 Discussion
Withthe 3algalspecies usedhere [Isochrysis, Phaeocystismû S. Costatum) wefound afairly
linear relation betweenthe PCF andthe PEFatlowto mediumphotonflux densities (Fig.5.2 and
5.3). Deviationfrom linearity occurred at highphotonfluxdensities.Applicationofthe
photosynthesis modelof EilersandPeeters (1988) givesasatisfactoryfit ofboth PEFandPCF
asafunction ofthe photonfluxdensity.Therelationbetween PCF andPEFislinear untilabout
140umolm~2 s"1inPhaeocystis, untilabout 280 umolnr2 s"1inS. Costatumanauntilabout 650
umolm"2s"1inisochrysis. For PhaeocystisandS. Costatumthesephotonfluxdensities aremore
or lessequivalenttotheirradiancewerecarbonfixation startsto saturate (lk). Forisochrysisthis
photonfluxdensity issomewhathigherthan lk.Thisrangecorresponds reasonablywiththe
rangeofthetypicalmeanphotonflux densities inaseawater column (Kirk 1994).Thelinear
range between PCF and PEFextendsto aphotonfluxdensitywhichis2to 10times higherthan
the photonfluxdensityatwhichthespeciesweregrown.Thiscoversalarger rangeaswhatwas
foundfor the linear relation betweenoxygenevolution andPSIIelectronflow inChapter 3(2to 5
timesthegrowth irradiance) andismuchlargerthanreported byReeset al (1992) whofound
non-linearity betweentheefficiencyof oxygenevolutionandtheefficiencyof PSIIelectronflow
evenatgrowth irradiances.
Thedifference inPCFversusphotonflux densityandPEFversus photonflux density resultsin
anon-linearity between PCF andPEF.Thenon-linearity might becaused byseveralfactors.
Previousexperimentswith marinealgae (Chapter 3) haveshownthatthe non-linearity isnot
caused byphotorespiration.Thereforewedonotexpectthat intheseexperiments
photorespiration isinterfering either.Alternatively anincreasedactivityoftheMehler-reaction at
high photonfluxdensities might leadto adecreaseincarbonfixationaslessNADPHisformed,
leadingto anon-linearity inthe relationship betweencarbonfixation and PSIIelectronflow.
Resultsfrom Kanawith planktonfrom estuarinesurfaceswatersandincyanobacteriaconfirmthe
possibility of anincreased Mehler-reaction at highirradiances (Kana 1990,1992,1993).Weger
et al.(1989) found ahigher mitochondrial respiration inthelightthaninthedark.This
phenomenon isalsosuggested bythe resultsof Beardaletal (1994) whofound anincreased
oxygenconsumption directly after alight incubation.Whencarbonfixation measurementsgive
netphotosynthesis data (Williamsetal 1996) thenon-linearity between PEFandPCFinour
experiments might becaused byanincreased mitochondrial respirationat high photonflux
densities.Ontheother handinexperimentsofXueetal (1996) the increaseinoxygen
consumption inthe lightcomparedtothedarkwasaccompanied byadecrease incarbondioxide
release.Accordingtotheseresultsweshould not necessarily expectthesamerelationfor carbon
63
dioxide fixation as for oxygen evolution. We are not able to compare the PEF values of the
oxygen evolution measurements as shown in Chapter 4 with the carbon fixation measurements of
this Chapter as the fluorescence measurements were not done under the same conditions.
Table 5.1. Actual and maximal PS II efficiency (<))„ „ and f /£ respectively) after varying
incubation periods at 1528 umol m"2 s ' . FV/FMwas measured after 1 hour dark adaptation at 18
°C. n.d stands for not detectable which means that tyK „ was lower than 0 . 1 . £ is given as % of
the F0level at t = 0.
Isochrysis
Time
(min)
Phaeocystis
S. costatum
<t>PSII
FV/FM
F0
<J>PSII
Fv/FM
Fo
4>PSII
F v /F„
Fo
0
-
0.694
100
-
0.651
100
-
0.690
100
10
0.180
0.683
107
n.d.
0.440
108
0.178
-
-
30
0.148
0.434
107
n.d.
0.324
120
0.124
0.449
103
60
n.d.
0.345
105
n.d.
0.296
125
0.104
0.412
94
90
n.d.
0.570
91
n.d.
0.284
115
0.132
0.402
104
120
n.d.
0.283
66
n.d.
0.267
99
0.138
0.416
110
150
n.d.
0.138
68
n.d.
0.291
92
0.150
0.434
98
180
n.d.
0.135
76
n.d.
0.256
91
0.163
0.467
106
240
n.d.
0.144
55
n.d.
0.219
75
0.143
0.397
102
Another cause of the non-linearity at high photon flux densities might be a shift in the
metabolic pathways from carbon fixation to more nitrogen reduction (Huppe and Turpin 1994).
Nitrate reduction was found to increase with increasing photon flux densities (Weger and Turpin
1989), though it is not clear wether the increase in nitrate reduction is comparable to, or larger
or smaller than the increase in carbon fixation with increasing photon flux densities. The nitrogen
source (ammonium or nitrate) was found to have a large effect on the amount of fixed carbon
(Williams et al 1979).
All 3 species suffered from a high amount of photoinhibition when incubated at the highest
photon flux density as can be concluded from the decrease in the maximal efficiency of PS II
electron flow (Table 5.1). After 4 hr of incubation FV/FM is 0.144, 0.219 and 0.397 in respectively
Isochrysis, Phaeocystis and 5. costatum. In Isochrysis the F 0 has decreased to 55 % of the initial
level after 4 hr of incubation. In Phaeocystis the minimal fluorescence increases during the first
1.5 hr and decreases somewhat afterwards. In S. costatum the decrease in FV/FM is caused by a
decrease in the maximal fluorescence. Schansker (1996) found a maximal efficiency of PS II
electron flow measured with fluorescence of 0.44 in samples were oxygen evolution was inhibited
completely. The variable fluorescence of these samples was equal to about 1 2 % of the variable
64
fluorescence incontrol leaves.Heattributedthisvariablefluorescenceto PSIIreaction centres
withaninactiveoxygenevolutionsystemaccordingtothe resultsof Gierschand Krause (1991)
andofVanWijkand Krause (1991). Inourexperimentswith Isochrysis, Phaeocystisand5.
costatumtbe variablefluorescence after four hoursofphotoinhibition correspondsto 5, 11and
30% respectivelyoftheinitialvariablefluorescence.Accordingtotheinterpretationof
Schansker (1996) the number of reactioncentres,whichareinactiveinoxygenevolution,isnot
largerthan respectively 5,11 and 30%.However,asthe PCFdatahavenotdecreased markedly
during 4hoursof photoinhibition,wesuggestthat therearenoreactioncentres inactivein
carbonfixation at allinthesealgalspecies.Thisisastriking differencewithpealeaves,spinach
thylakoids and Valerianellalocustaprotoplasts examined bySchansker (1996), Krauseetal
(1990) andVanWijkand Krause(1991) respectively.
InChapter 3wecompared electronflowcalculatedasthe product of <$>Kn andthe irradiance
withoxygenevolutioncalculatedonachlorophyll abasis.Inthiscasechlorophyll aisthe major
photosynthetic pigment.Thisoxygenevolution ratescanbeusedasameasurefor electronflow
per 'photosynthetic unit'. However,mostalgalspeciescontainmorephotosynthetic pigmentsand
the ratio of chlorophylltotheother photosynthetic pigments isvariable (FalkowskiandKiefer
1985, Kirk 1994).Thereforewehavesuggestedthat chlorophyll aisnotauniquemeasureofa
'photosynthetic unit'.Thiscomparison infact resulted indifferent slopesofthe relation between
oxygenevolutionand PSIIelectronflowforthedifferent speciesweexamined.InChapter 4we
proposed that the PEFcanbeestimatedfromtheminimalfluorescence,theefficiency of PS II
electronflowandthephotonfluxdensity. Indeedwefound asimilar relation betweenthePEF
andthe phytoplankton oxygenflux (POF)for D. tertiolectaandP.tricornutum. InthisChapterwe
appliedthis methodtothecomparisonof PEFandPCFwith 3different algalspecies.All 3algal
speciesshowmoreor lessthesamerelationfor PCF asafunctionof PEF(Fig.5.4) butsome
variation inthis relationship isleft.Thisvariation inthe relation betweenPCFandPEFis
somewhat smallerthanthevariationfoundinthe ratioofcarbonfixation and PSIIelectronflowin
thedataof Falkowskiand Kolber (1990) andKolber and Falkowski (1993) whousedthepump
andprobe methodto calculate PSIIelectronflow.
Weusedtheminimalfluorescence atthebeginning oftheexperiment to calculatethePEF
duringthecompleteexperiment.However,astheexperiment lasted4 hours,the minimal
fluorescence might havechangedduringtheexperiment,dueto growthor photoinhibition (atthe
high photonflux densities). Forthe incubations atthehighest photonfluxdensity F0hasbeen
determined (Table 5.1). For Isochrysisand Phaeocystisadecreaseoftheminimalfluorescence
wasfoundof 55and75 %respectivelyafter 4hrof incubation.However,(\>Kn wasnot
detectablyfor these datapointsandwecannotcalculatetheeffect ofthedecreaseofF0onthe
PEF.InS. costatumtbeminimalfluorescencedidnotdecreasecomparedtothevalueatt = 0.
Thereforethe PEFinS. costatumcalculatedforthehighest photonflux density isnotchangedby
photoinhibition orgrowth.Theeffects ofgrowthandphotoinhibition ontheminimalfluorescence
shouldalso bereflected inthe PCF data.However,asalreadyconcluded before,thevariationin
the PCFdatacould not beassignedtothe lengthofthetotal incubationtime.Thereforewe
suggestthatthere isnoeffect ofgrowthor photoinhibition onthe PEFdatainthis experiment.
Theuseofthe minimalfluorescenceasameasurefor the product ofthe number of PS II
65
reactioncentre andtheabsorptioncross-section suffersfrom onelimitation:theminimal
fluorescence measured inthedarkdoesnotaccountfor light inducedchangesinthe absorption
cross-section.Theselight inducedchangesintheabsorption cross-sectionareimportant in
cyanobacteria (WilliamsandAllen 1987,Geeletal 1994) but muchlessisknownaboutthe
occurrence intheeukaryoticalgae.
5.5 Conclusions
Therelation betweencarbonfixation andPSIIelectronflow isshownto bealmost linear atlow
andmediumphotonflux densities inseveralmarinealgae.Athighphotonflux densitiesthe
relationship isnon-linear.Theminimalfluorescence seemsto beareasonabletool inestimating
the product ofthe numberof PSIIreactioncentresandtheabsorptioncross-sectionwhichis
necessaryto calculatethe PEF.Whenelectronsinksotherthancarbonfixation (forexample
nitrate reduction) areinvolvedthiswillinfluencethe relation betweenPEFandPCF.
Thevariation inthe relation between PCF andPEFissomewhatsmallerthanthevariationfound
inthe ratio of carbonfixationandPSIIelectronflow inthedataof Falkowskiand Kolber (1990)
and Kolber andFalkowski (1993) whousedthepumpandprobemethodto calculatePS II
electronflow.
66
6 E S T I M A T I O N OF P H O T O S Y N T H E T I C PERFORMANCE OF
M A R I N E PHYTOPLANKTON I N A MODEL ECOSYSTEM BY
M E A N S OF CHLOROPHYLL FLUORESCENCE
6.1 Introduction
Inmarineecosystemssolar energyisphotosynthetically converted intochemicalenergy byits
phytoplankton.Thischemicalenergyintheform oforganic matter isthebasisofthefoodweb
andthe rest ofthecommunity isusingtheenergybyrespiration.Theratioofphotosynthesis and
respiration (P/R-ratio) isanimportant parameter. Inmanyaquaticecosystemsthis P/R-ratiocan
becalculated bymeasuring oxygenproductionaswellasoxygenconsumption.Inthe marine
environment,withitslowchlorophyllcontent,however,thesemeasurementsare notfeasibleas
thesensitivity oftheoxygenmeasuring methodistoo lowto detectthesmallchanges inoxygen
concentration. Measurement of photosynthetic carbonfixationby 14Cincorporationand
respiration by ,4Creleaseisamoresensitivemethod,but canonlybeappliedinbottle
enclosures inthenatural environment or inanincubator simulatingthis environment.
Inrecentyearschlorophyllfluorescence hasemergedasamethodto estimate phytoplankton
photosynthesis. Photosynthetic electronflow,whichisthe basisfor carbonfixationandoxygen
production,canbedetermined measuringtheefficiencyof PSIIelectrontransport,tyKU,andthe
absorptioncross-section,am, bymeansoffluorescence usingthepumpandprobemethod
(Falkowskiand Kiefer, 1985).Therateof PSIIelectronflowisthencalculatedastheproductof
4>PSII.Ops,,,andtheambient photonflux density,PFD.Falkowskiand Kolber (1990) foundagood
correlation betweentherateof PSIIelectronflowandcarbonfixation inmarinephytoplankton.
Schreiber et al (1986) introducedthesaturating pulsemethodto measuretheefficiencyof
PSIIelectronflow.Thesaturatingpulsemethodnormallyusesamultiple-turnover saturating
flash,whichreducestheplastoquinone pool.Inthe pumpandprobemethodashort saturating
flashisusedwhichleadsasingleturnover inthePSIIreactioncentreresulting inthetemporary
reduction ofQA.Duetofluorescence quenching bytheoxidizedplastoquinone pool,themaximal
fluorescence levelmeasuredwiththe pumpandprobe methodwillbelowerthanthelevel
measuredwiththesaturating pulsemethod (Schreiber etal 1995b).Thereforetheefficiencyof
PSIIcalculatedfromthepumpandprobedatawillgenerally belowerthantheefficiency ofPSII
measuredwiththesaturating pulsemethod.
Inhigher plantsandgreenalgaetherelationship betweentheefficiency ofPSIIelectronflow,
determined bythesaturating pulsemethod,andtheefficiencyofcarbonfixationor oxygen
evolution hasbeenexaminedunderavariety ofconditions. Ingeneralalinear relationshipwas
foundalthoughthe relationship isinfluencedbyprocesses likephotorespiration (Gentyetal
1992), Mehler reaction (ÖquistandChow 1992),cyclicelectronflowaround PSII(Falkowskietal
1986,Reesetal 1992) andbythepresenceofinactivePSIIcentres (Hormannetal 1994).For
amoreprecisedescriptionof recent resultsseeChapter 3.
Underseveralgrowing andpreilluminationconditions Heinzeetal (1996) foundalinear
67
relation betweentheefficiency of PSIIelectronflowandtheefficiency ofoxygenproduction inthe
greenalgaScenedesmusobliquus. Inanumber of marinealgaewefoundalinear relation
between PSIIelectronflowdeterminedwiththesaturating pulsemethodandoxygenevolutionat
lowto intermediate irradiances (Chapter 3).Athigher irradiancestheyfoundelectronflowwhich
isnotconnectedwithparalleloxygenevolution.
Inadditionto itsuseto determinethe phytoplankton photosynthetic activityfluorescenceis
alsoused to estimate phytoplankton biomasswhichisusuallyapproximated bythetotalamount
ofchlorophyll a (Heany 1978,Butterwicketal 1982,Falkowskiand Kiefer 1985).Therelation
betweenfluorescence andthechlorophyll aconcentration,however,isdependent onthespecies
composition,thegrowing conditions ofthesampleandthe photosynthetic processes.The
minimalfluorescence measured inthedark,F0, isnotdependent onphotosynthetic processes
andthereforewillgivethe best possiblecorrelationwithchlorophyll aconcentration.
Herewepresentamethod basedoninsituchlorophyllfluorescence measurementsto
estimateprimaryproduction inamodelecosystem.Wehavemeasured PSIIelectronflowby
meansoffluorescence,carbonfixation andoxygenevolution inanartificial ecosystem ina
mesocosmcontainer andexaminedtheir relationships. Duetothe relatively high phytoplankton
concentration inthemesocosmtheoxygendeterminationwassensitiveenoughto calculatethe
dailyoxygenproduction.Theirradiancedependencyofcarbonfixationwasmeasured.We also
measuredtheirradiance dependency of PSIIelectronflow,JE,determined asthe product ofthe
efficiency of PSIIelectronflowandthe photonfluxdensity,JE= (pPS// •PFD, inthe mesocosmand
inthe laboratory. Phytoplankton electronflux,PEF, wasestimated bythe product ofJEandthe
minimalfluorescencewhichwasmeasured inthe mesocosm.Theminimalfluorescencewasused
asameasureof both biomassandPSIIabsorption crosssection.Photosynthesisversus
irradiance curvesof PSIIelectronflowandcarbonfixationwerefittedtothe modelof Eilersand
Peeters (1988) resulting in 3parametersA,BandC. Theseparameterswereusedto integrate
theprimary production overthewater columnandovertheday.
Dailyprimary production calculatedfrom PSIIelectronflowshowed reasonable correlationwith
carbonfixation.Thecorrelationwithoxygenevolution ishighest inthepeakofthealgalblooms.
Insitumeasurements doneinthe mesocosmcontainer result inthesameprimary productionas
thelaboratory measurementsfor PSIIelectronflow.
6.2 Materials and methods
Mesocosm
Theexperimentswerecarried outfrom May 19toJune 13 1994 inamesocosmconsistingof
a 3000 Iblack polyethylenetank (height 3m,0 1.2 m)(Fig.6.1).Atthe bottom asediment
container of 150I wasplaced.Thewaterwasmixedwitharotating mixer (mixingtime ca10min)
Thecontainer wascoveredwithadiffusor screen leadingto amorehomogeneous light gradient
inthewater column, withanintensityatthesurfaceof 74 %oftheavailablesunlight andan
increased apparent lightattenuation.Themixingandthe lightconditions inthemesocosm
reproducedtheconditions of a 10m water columninthe Dutchcoastalwaters (Peeters etal
68
lightdiffusor
drivingchainn l
n^mi
I
• • • . • . . M . l l . . . l .
D n
|
motor
+Seawater(100l/d)
+ Nutrientsolution
A
s.
Peristaltic
pump
(©
JWwuMm MMMpit
3m
grazingchamber
•1.2m
•
AutomatedMeasurement Unit
Oxygen
Fluorescence
pH
Conductivity
Irradiance
Figure 6.1.Schematic view ofthe mesocosm container
69
1993). Onthefirst dayoftheexperimentthecontainerwasfilledwithseawater. Subsequently
thecontainerwascontinuously refreshedwithseawaterataflushing rateof 1001day"1,resulting
inadilution rateof0.033 day"1.Inorganic nutrientswereaddedcontinuouslyfromstock
solutionstothecontainer: 7.68 mmolday"1dissolved inorganic nitrogen (N0 3 ),0.63 mmolday"1
dissolvedinorganic phosphateand4.71 mmolday"' silicate.Thewaterfromthecontainerwas
flowedthrough abenthos chamber atarateof 1001day',wherethealgaeweregrazed by 16
mussels.Fluorescence,oxygen,temperature,conductivity,andpHwerecontinuously monitored
viaabypass.Thecontainerwascooledbysprayingseawater ontheouterwallofthetank. Fora
moreprecisedescription seeEscaravageetal (1996).
Irradiance
Photonfluxdensity (PFD)wasmeasuredcontinuouslywithaKippandZonenSolar integrator
incombinationwithalight sensor andaveragedper hour. Lightattenuation inthewater column
wasmeasured 3timesaweekwithaLiCordatalogger LI-1000connectedwithaLiCorSPAQUANTUMsphericalsensor.Theapparent attenuationcoefficient (Kdinm"1)wascalculatedfrom
ln(iyi0) = -Kd•zusing linear regression.I0isthedaily irradianceatthesurfaceandL,isthe
incident irradiance at zmeters.
Chlorophyll
Chlorophyll a(inmgm"3) wasanalysedthreetimesaweekbyreversed phaseHPLCmethod
(Escaravage etal 1996) after extractionaccordingto GieskesandKraay (1984).The
continuously measuredfluorescence datawhichweremeasuredinthebypassofthemesocosm
containerwerescaledwiththeestimatedchlorophyll aconcentration (chl).Onthe intermediate
dayschlorophyll awascalculatedfromthefluorescencedataandthecalibrationto chlorophylla.
Carbonfixation
Theirradiancedependence ofthephytoplankton carbonflux,PCF, inumol m"3s ' ,was
measured (by ,4C-incubations) twiceaweek.Samplesofthemesocosmweretakenat 7a.m.and
the incubationfor carbonfixationstarted at 8a.m..Thesampleswereincubatedfor 2hourswith
185kBqMC-bicarbonatein 120mlat8discrete PFDvaluesintherangefrom0-1628 umolm"2
s"' inathermostatedincubator. Thetemperaturewasadaptedtothemesocosmtemperature.
After incubationthereactionwasstopped byadditionof lugol.Carbonincorporation inorganic
substances wasmeasuredafter inorganic carbonwasremoved byacidification (Escaravageetal
1996).Thenormalized rateofcarbonfixation,Jc,wascalculatedasPCF/chl.
Oxygenproduction
Theoxygenlevelofthe mesocosmwater andofthewaterthat leavingthebenthoschamber
wasmeasuredautomaticallyat regular intervals.Theincreaseinoxygenmeasured inthe
mesocosmfrom sunrise untilsunset iscalledthenetsystemdaily (oxygen) production NSDP.It
isdetermined byphotosynthesis andrespirationfrom phytoplankton and heterotrophic
organisms inthemesocosm,musselrespiration inthebenthoschamber anddiffusion ofoxygen
from andtotheatmosphere. Thedecreaseinoxygenfrom sunset untilsunriseiscalledthe
70
Output Signal
Î
Xe-PAM
Main Control Unit
''
Saturation
Pulses
Xe-Flash
Lamp
Optica Filter|
Optical Filter
tl
Flash Driver&
Synchronous Amplifier
Photodiode
Detector
&
Pulse
Preamplifier
Optical Filter!
Figure6.2.Themeasuringschemeandset-upoftheXe-PAM fluorometer.
system night respiration.It isdetermined bythe respiration ofallorganisms anddiffusionfrom
andto the atmosphere.Assumingthat respiration duringthenight isequalto respiration during
the daythesystemday respiration (SDR) iscalculated.Dailyprimary oxygenproduction (PPO) is
calculatedasNSDP+ SDR+ diffused oxygen (Prinsetal 1995).
Fluorescence
Fluorescence measurementsweredonewithaXe-PAMfluorometer. TheXe-PAMfluorometer
(Walz,Effeltrich) usesexcitation pulsesto discriminatethefluorescencegenerated bythe
measuring lightfromthefluorescencegenerated bythe background light (Schreiber et al 1993).
AXenon-flash lamp,filteredthrough abluegreenfilter (6 mmBG39)wasusedfor excitation,and
aphotodiode detector placed behindtwolong-passfilters (dichroicfilter R65,Balzersand
RG645,Schott) wasusedfor detection.Saturating light pulsesof 1secondduration anda
photonfluxdensityof ± 6000 pmolm"2s~'PARweregivenwithahalogen lamp.Thesaturating
light pulseswerefilteredthrough a650 nmshort-passfilter (Balzers DTCyanspecial).The
measurementswerecarried out at amodulationfrequencyof 16Hz.TheXe-PAMfluorometer
wasusedincombinationwithaPC equippedwithadatalogger (Keithley model 576). A
dedicated program PPMON(Phytoplankton Photosynthesis MONitor, LovoanSoftwareand
71
Education,Wageningen,TheNetherlands) controlledtheoperationofthedatalogger andthe
analysisoftheoutput-data. Fluorescence nomenclaturewasaccordingtoVanKootenandSnel
(1990).Theefficiency of PSIIelectronflowwasdetermined as(FM'-F)/FM'.
Forthecomparisonoffluorescence dataobtained inlaboratory experimentswithdata
obtained insitu,mesocosmsampleswereincubated inthelaboratory underthesameconditions
asfor carbonfixation.Theefficiency of PSIIelectronflowwasmeasured insubsamples of 1mlin
theXe-PAMcuvetteafter 10minutes, 1hour and2hours incubation (onday 11and29). The
efficiency of PSIIelectronflowafter 1and2hourswasalmostthesameasafter 10minutes.On
day 18and24theefficiency of PSIIelectronflowwasmeasuredafter 10minutesincubationin
thecuvette,atthesame8 irradiances.PSIIelectronflow,JEL,wasestimated asthe productof
cppsn andthe imposed PFD.Phytoplanktonelectronflux,PEFL,wasestimated bytheproductof
(bps,,,PFDandF0(seeChapter 4).
Inthe mesocosmcontainer thefluorescence measurementsweredone2cmbelowthewater
surface.Fibreopticsandquartz rodswereusedtoconductthe lightfromtheXe-PAM
fluorometertothewater sampleinthecontainer andback (Fig.6.2).Theperspexfibre holder
wassituated onthenorthsideofthecontainer to minimize lightattenuation.Simultaneous
measurements oftheefficiency of PSIIelectronflowandphotonfluxdensity were performed
every 30minutes inthemesocosmundertheprevailing lightconditions.Thephotonflux density
wasmeasured abovethewater surface underthediffusor screenwithamodified Hansatech
fluorescence detector probe (FDP/2) equippedaneutraldensityfilter andaPARfilter
(transmissionfrom 400to 700 nm).Theoutputwasamplified byafluorescence detector control
box (FDC) anddigitized bythedatalogger.Thephotodiode datawerecalibrated usingthe
global irradiancedata.Theminimalfluorescence,F0wasdetermined byaveraging measurements
from 0a.m.to 4a.m.PSIIelectronflow,JEM,wasestimatedasthe product of$m andthe
ambient PFDatthetimeofthemeasurement. Phytoplankton electronflux,PEFM,wasestimated
bythe product of (pps,,,PFDandF0(seeChapter 4).
Modelling
Thephotosynthesis modelof EilersandPeeters (1988) describesthephotosynthesis process
with photosynthetic factorieswhichcanexist inanopen,aclosedor aninhibited state.This
modelresults inasteadystatesolutionforthe rateof photosynthetic production(P):
I
2
(AI +BI +Q
where Iisthephotonfluxdensity andA,BandC areconstants.
Parameters characteristicfor thephotosynthesis irradiance curvecanbederivedfromA,Band
C: the initialslope a = C'; the maximumproduction Pm = (B + 2(ACf}'\ the irradianceat
whicha andPm intersectlk = C/(B+ 2(ACf).
For aderivation andexplanationofthisformulaseeEilersand Peeters (1988) andChapter
3.Thefitting procedurewasperformed inMathCad 5.0+ (MathSoft,Cambridge,Massachusetts,
USA)using aleast squaresmethod.
Fittingthecarbonfixation,JCn (thecarbonfixation permgchlorophyllfor eachdaynonwhich
72
(6.1)
^
ü
80
"À
60
~~
i
i
40
200
o
/ /
20
•
100
/ /
0 CV O i •
B
Q
U- 20
O.
Q
i
f
\
/
•
i »
^'
*
•L
10
/
o' r^
i
^
1
1
1
1
10
15
o
600
o.
Q
400
u
200
•
/\.~
\ '
• '
0
800
A*
\ i\J:''\ '•
'd^
11
i
/
O
:
i
•'/
•
*
*
. /
1
1
0
_c
CM
o io 9
8
/
i . . _ . ...
5
v
1
1
20
25
3
0
Time (days)
Figure6.3.A:chlorophyll (mgm"3): o ;theminimalfluorescence (r.u.):• ; B:dailyphotonfluxdensity
(molm2d"1):— ; theproductofchlorophyllcontentandirradiance*;C: oxygenconcentration (mgV)
solidline.
carbonfixationwasmeasured),to Eqn.1ledtotheoptimized modelparametersA^, BCnand
CCn.Foreachhour carbonfixation inthewater columnwasestimated bycombiningthe hourly
averaged photonflux densityatthesurface,thelight attenuation inthewater column,Kd, andchl
withthe model parametersA^, BCnandQ-n asdescribed inEilersand Peeters (1988). Daily
primary production incarbonfixation,PPC, wasestimated bysummation ofthecarbonfixation
dataofeachhour oftheday.As,4C-incubationswerecarried outonlytwiceaweekon
73
1
400
'
i
'
0,8
o
LL
ü.
<
S
f
\ / r^00^/ 1
300
0,6
co
0.
-e-
Uj/w
200
<\
0.4
D
Ui
—>
0,2
100
i .-*
0
()
5
i
i
10
15
i
K>
a l l
j 0,0
20
Time ofday (hours)
F/gure6.4. Exampleofone dayofmeasurements inthe mesocosm:the efficiency ofPSII
electron flow: D;the photon flux density (umol m"2s"'): A ;the estimated rateofPSIIelectron
flow:— , data ofday 13.
intermediate days thefit parameters A, Band Cofthe nearest day were taken forcalculationof
the daily primary production.
PSIIelectron flow data from themesocosm,JEM, and thePSIIelectron flow data fromthe
laboratory, JEL,were also fitted to Eqn. 1 leading tomodel parameters A En M , BEnMand CEnM and
A E n \ BEnLand CEnLrespectively. For each hour PSIIelectron transport inthe water column was
estimated bycombining thehourly averaged photon flux density atthe surface,thelight
attenuation inthe water column, Kd and F0values with themodel parameters. Daily primary
production inPSIIelectron flow, PPEM and PPEL respectively, was estimated bysummation ofthe
PSII electron flow data ofeach hour ofthe day. AsPSIIelectron flow laboratory measurements
were notcarried outevery day on intermediate days thefit parameters A, Band C ofthe nearest
day were taken forcalculation ofthe daily primary production.
6.3 Results
Figure 6.3illustrates the results ofthe experiment. The chlorophyll aconcentration, chl,
displays apattern associated with two algal blooms. The first maximum occurred around day10
and thesecond maximum around day 26 (Fig. 6.3A). Both blooms mainly consisted of diatoms:
Nitzschia de/icat/ssima and Chaetoceros sp\n the first bloom; Rhizosoleniasp. Eucampia
zoodiacus, Nitzschia seratia and the flagellate Phaeocystis sp. inthesecond bloom.The minimal
74
i
i
!
i
150
•
A
•
•
125
E
m
100
-
•y. •
->
-
75
•
•
7
50
-
f9
-
25
0
I
I
I
100
200
300
2
400
500
1
PFD (|imol m~ s" )
i
i
200 -
i
I
0,15 . f *
(0
B
0,12
2^
150 UJ
•
—
' •——-—
O
ra
0,09
*//
100 -
Z
0,06
O
E
O
E
3
ü
-J
50 0,03
•
0 0
i
i
i
500
1000
1500
-
0,00
PFD (jamol m"2s"1)
Figure6.5. A: TheestimatedrateofPSII electronflowasafunctionoftheirradiance,dataof
Figure6.4.ThesolidlinerepresentsthefitsaccordingtothemodelofEilersandPeeters(1988)
(AE13M= 4.3-10"6,BE13M= 2.0-103,CE13M= 1.53).B:theestimatedrateofPSII electronflow
(o) andtherateofcarbonfixation,J^,(|jmolmg chl"1s"')(») asafunctionoftheirradiance, determinedinthelaboratory,datafromday 11(A=„ L = 2.96-106,BE1,L= 9.98-10"4,CE,„L= 1.44,
A-,, = 3.34-10"3,B c „ = 4.33,Q,, = 1304). '
fluorescence,F0, displaysasimilar patternasthechlorophyll aconcentration.The proportionality
betweenF0 andchlindicatesthat,under our conditions,F0isareasonable measurefor the
chlorophyllcontent.
75
Figure6.6. Phytoplanktoncarbonflux,PCF,(pmolm s )asafunctionofthephytoplankton
electronflux(PEFL)(bothmeasuredinthelaboratory),dataofrespday 11, 18,24and29. The
solidlines,D, representtheinitialslopesdeterminedfromthefirst 5Datapointsineach graph.
ValueofD: day 11: 4.5-10"4;day18:5.8-10'4;day24:8.5-10"4;day29:5.6-10"4.
Figure 6.3B showsthedailyphotonflux density,DPFD,andthe"primaryproduction"
approximated here (but seelater) astheproduct ofchlorophyllconcentration andphotonflux
density.This primary productionshowsrelationwiththetwoalgalbloomswhichwerefound inthe
chlorophyll andfluorescencedata (Fig.6.3A)
Figure 6.3Cshowsthechangesintheoxygenconcentrationofthe mesocosm.Theoxygen
concentration increasesduring bothalgalblooms.Theincrease isaboutthesamefor both
blooms.
Figure 6.4 showsthetimecourseofthe photonfluxdensityandtheefficiency of PSIIelectron
flow measuredjust belowthewatersurfaceinthe mesocosmatday 13. Duringthetwo periods
ofhigh irradiancetheefficiency of PSIIelectronflowdropped below0.4.Thevariation inthett>PSM
76
l
1
900
I
I
i
900
-i
LU
Q.
600 Q-
LU
Q.
Q- 600
•
0
-
300
0
1"
5
l
i
10
15
<
\-rO<rü"^
i
20
25
300
u
30
Time(days)
Figure6.7. Primaryproductioninthemesocosmintegratedoverdepthandtimecalculatedfrom
electronflowdata:usingthefitsofthemesocosm(o) andlaboratory (•).
values measuredduring thenightgiveanideaontheaccuracy (approximately 5%) ofthe
applied method.Therateof PSIIelectronflowintheupperwater layerwascalculated (JEM= PFD
* dppsn)andshowsthat inthehigh irradiance periods PSIIelectronflowissomewhat higher but
disproportional withthat inthelowirradianceperiods.
Fromthe PFDandJEMtimeseriesthelightdependenceof PSIIelectronflowwasmodelled (figure
6.5A) to estimatethe parametersAE13M,BE13MandCE13MusingtheEilersandPeeters
photosynthesis model.Figure 6.5A showsthatthedataaresatisfactorily described bythe model
although eachdatapoint represents anothertimeoftheday.Thereforechanges inthelight
dependenceof photosynthesis dueto light adaptationdid notoccur onatimescaleof 15hour.
Anexampleofthe light responseofcarbonfixation,Jc,and PSIIelectronflowasmeasuredin
thelaboratory, JEL,isshowninFigure6.5A (dataofday 11).Thecarbonfixation dataandthe
PSIIelectronflowdatawerefitted separatelyyieldingA^,,,BC11, Q n for carbonfixationand
AE,,L,BEn \ CEi11Lfor PSIIelectronflowonday 11.
Thedistribution ofdatapointsaroundthefit asshowninFigure6.5A andinFigure 6.5Bis
representative forthe mesocosmexperiment.Theirradiance rangeinthe mesocosm experiments
issmallerthan inthelaboratory experiments.Onsomedaysthisirradiance rangecoversonlythe
irradiance dependent part ofthephotosynthesis irradiancecurve.ThedeterminationofCwhich
describestheinitialslope,C=or1 isnotdisturbed.However,mistakesmight bemadeinthe
determination ofAand B values,whicharenecessaryfor acorrect determination of lkand Pm.
Theirradiance dependencyforthe irradiancewhichisexperiencedonthat daycanbedescribed
anyhowwiththemodel.Therefore alsothedailyprimary productioncanbecalculated.Inthe
77
900
- A
PPE
chl'DPFD
900
LU
Q_ 600
a.
10
15
20
Time(days)
Figure6.8.A: primary production inthe mesocosmcalculated from electronflowdata,
usingthefits ofthe mesocosm (PP?): — a — ; primary production estimated asthe
product ofchland PFD:
B:PPË*asinFigure 6.8A: — o — ; primary productionin
carbonfixed,PPC,(mol m"2d"1):—; primary oxygen production inthe mesocosm,PPO,
(mol nr2 d"1):
C:PPC/PPEM*103:—; PPO/PPEM*103:
;chrDPFD/PPE':—.The
initial slopes offour experiments onthe relation between PSIIelectron flow and carbon
fixation atthe indicated days asshowninFigure 6.6, D*1Cr: • .
laboratory experiments the irradiance range ishigh enough toguarantee correct determination
of allfit parameters.
Data points of phytoplankton carbon flux, PCF,and associated phytoplankton electron flux,
PEFL measured onday11,18, 24and 29are shown inFigure 6.6. ForlowPEFLvalues there
appears tobealinear relationship. Itsslope,called D,can beconsidered asa calibration
parameter related totheyield ofcarbon flux perPSIIelectron flux.
78
InFigure 6.7 thedailyamount of PSIIelectronflpwasdetermined bothfromthemesocosm
measurements andfromthelaboratory measurements isshown.Several peaksarefound,the
firsttwo (day9and 12) duringthefirst algalbloom,thelast one (day 29) inthesecondalgal
bloom.Thedataof PSIIelectrontransport derivedfrom mesocosmand laboratory
measurements showclosesimilarity.Thereforewecometotheconclusionthat underthese
modelling conditionsthe //?5/ft/fluorescence measurements inthe mesocosmcontainer givea
good alternativetothe incubator measurements.
InFigure 6.8A PPEandchl*DPFDarecompared.Figure6.8Bshowsthedailyamountsof
fixedcarbon,PPC,andthedailyamountsof oxygenproduction,PPO.Bothshow3maxima,2in
thefirst algal bloom,(day 9,12) and 1inthesecond,(26/27). Inthefirst bloomoxygen
productionvaluesareabit higherthancarbonfixationvalues.Inthesecondbloomcarbon
fixationvaluesareabit higherthanoxygen productionvalues.Duringtheblooms PPE(Fig.
6.8B) showsbest similaritywiththe PPOdata,asbothshow2maximaofaboutthesameheight.
Betweenthe 2bloomshowever PPOstaysrelatively highandPPEdecreasesto almostzero.This
ismore inagreementwiththe PPC data.
Figure 6.8Cshowsthe ratio of PPC andPPEduringtheexperiment.Theratiovariesfrom 0.2
to 1. Duringthesecond bloomsomewhat higher PPC/PPEvaluesarefoundthanduring thefirst
bloom.Thevaluesofthe ratio D(PCF/PEF1)at limiting light,asdeterminedfromthedatain
Figure 6.6,are ingoodagreementwiththe ratios PPC/PPEobserved inthemesocosm(Fig.
6.8C). Figure 6.8Calsoshowstheratio of PPOandPPE duringtheexperiment.This ratiovaries
from 0.3 to 2.6 duringtheblooms.Inbetweenthe 2blooms PPO/PPEishigher,dueto thehigh
PPOvalues inthis period.Theratio ofchl*DPFDandPPEshowsarelatively constantvalue
duringthe 2bloomsandhighervalues inbetweenthe2blooms.
6.4 Discussion
Laboratory measurements
Phytoplanktonelectronflux,PEF,determinedfromfluorescence measurements givesa
reasonable measureof phytoplankton carbonflux,PCF, (Fig6.6).Thereforeweconcludethat
fluorescence measurements giveamanageablealternativetothecarbonfixationmeasurements,
withtheadvantagethat ahighertime-resolution canbereached.InChapter 3the relation
betweentheestimated rateof PSIIelectronflow,JE,andthe rateofoxygenevolution,J0,ina
seriesof singlespeciesexperimentswascomparedandwefoundthatthe relationshipwas
speciesdependent.Wesuggestedthatthiswaspartlydueto differences inlight absorptionand
that measurement ofthe PSIIabsorption crosssectionmightovercomethis problem.Herewe
estimated the light intercepted byPSII,i.e.,theproduct ofthenumber of PSIIreactioncentra,
nPSI|,andthe PSIIabsorptioncrosssection,aKn, byF0(seeChapter 4).Aswefoundagood
correlation between phytoplankton electronfluxandphytoplanktoncarbonflux (Fig.6.6),we
concludethatthisestimationwhichisbasedontheassumptionthat F0 isproportional tothe
product ofthe number of PSIIreactioncentraandthe PSIIabsorptioncrosssection,isjustified.
Becauseofthe resemblance ofresults onPPEMandPPEL(Fig.6.7) weconcludethatthe
79
incubation method andthe insitumethodgivecomparable resultsforthe longtimescale
measurements asusedhere.
Correlation of chlorophyll aconcentrationandminimal fluorescence
Therelation betweentheminimalfluorescenceandthechlorophyllconcentration (Fig.6.3A)
changesduringtheexperiment,especiallyduringthe riseanddecreaseofthefirst algalbloom.
Thischange isprobablycausedbyaccessoryphotosynthetic pigmentsother thanchlorophyll
whichaffecttheabsorption cross-sectionandcontribute moretotheminimalfluorescence inthe
declineofthefirst bloomthanintheriseofthis bloom.Themainaccessory photosynthetic
pigmentwill havebeenfucoxanthinasitisthemost important accessoryphotosynthetic pigment
ofthe diatomswhichweredominatingtheecosystemduringtheentireexperiment.Thereforethe
variation inthe relation betweenminimalfluorescence andchlorophyll concentration isprobably
notattributableto achangeinthepresenceof algaeofdifferent pigment classes.Acomparable
variation inthe relation betweenchlorophyllfluorescence andtheamountofextracted chlorophyll
hasbeenfound previously (Heany 1978,Butterwick etal 1982, Falkowskiand Kiefer 1985).
Estimation of primary productionfrom chlorophyll aconcentration andirradiance
The ratio betweentheproduct ofchland DPFDandPPEisrelativelyconstant duringthe
blooms but increasessubstantially inthe period betweenthetwo blooms (days 17-21)(Fig.
6.8C). Dueto celldegradationtheamount of inactivechlorophyll isexpectedto behigher inthis
period.Theproduct ofchland DPFDtherefore overestimates primary production inthisperiod.
PSIIelectronflowasameasurefor carbonfixation
Thedailyamountsoffixedcarboninthesecondbloomarehigherthanthefirst one(Fig.
6.8B),eventhoughtheamount ofchlorophyll islowerinthisperiod (seeFig.6.3A).Thehigh
carbonfixation inthesecond bloommight beexplained bythepresenceof moreaccessory
pigments other thanchlorophyll (e.g.fucoxanthin) inthesecond bloomcomparedtothefirst
bloom.Asthesephotosynthetically activepigmentswillcontributeto F0, thenalsoahigher
phytoplankton electronflux inthesecond bloomcomparedtothefirst oneisexpected.
Phytoplankton electronflux,however, reaches roughlythesamemaximaduring thetwo blooms
(Fig. 6.8B) andthereforethehighcarbonfixation inthesecond bloom isnot dueto more
accessory pigments.Thisvariation inPPC/PPEduringthegrowthperiod (Fig.6.8C) is
comparabletothevariationfoundintheratio ofcarbonfixationand PSIIelectronflowinthedata
of Falkowski (1990). Bakeretal (1995) foundthatthe ratioof PSIIefficiency andthequantum
yieldof carbonfixation inmaizeishigher under suboptimal growingconditionsthanunder
optimal growing conditions.Theysuggestthat another,yet unknown,electronsinkmightbe
involved under suboptimal growing conditions.Otherstudiesonhigher plants (OquistandChow
1992,Gentyet al 1992) showlessvariation inthe ratioof PSIIquantumyieldandthequantum
yieldofoxygenevolution.
Thehigh PPC/PPEratio inthesecondbloomcomparedtothat inthefirst onewouldsuggest
that oxygenconsuming (i.e.photorespiratory andMehler) reactionsor electronconsuming
reactions (i.e.theconversionof N03"to NH4+) areimpaired inthesecondbloom.
80
Photorespiration andMehler reactionareprobably alsoresponsiblefor thedeviationfrom
linearity inthe relation betweencarbonfixation andPSIIelectronflowwhichisfound athigher
irradiances (Fig.6.6). Thedailyirradiance (Fig.6.3B) andthehourlyaveraged photonflux
densities (data not shown) giveusno reasonto suggest thatthese reactionswould bemore
probable inthefirst bloomthaninthesecond.
Theratio D,i.e. PCF/PEF1 at limiting light asdetermined under laboratory conditions,
representsthemaximalefficiencyofcarbonfixedper PSIIelectrontransported.Atthismaximal
efficiency processes likephotorespiration andMehler reactionplayaminor,orat leastconstant
role.Theratios D determined attheindicated days (Fig.6.6) comparewellwith PPC/PPE(Fig.
6.8C). Inviewofthese resultsweconcludethat photorespiration andMehler reactionareoflittle
consequencetothevariation inthe ratioof PPC andPPEinthisexperiment.Thereforewejoin
thesuggestion of Bakeretal (1995) that another,yet unknown,electron acceptor mightbe
involved inthevariationof PPC/PPE.Apossiblecandidatefor electronconsumption isthe
conversionofnitrateto ammonium (HuppeandTurpin,1994). Inthisexperiment,however,we
havenodataonnitrate conversion anditseffect onthe PPC/PPEratio.
Estimation of primary productionfrom insituoxygenmeasurements
Theoxygenproduction ishigherthanthecarbonfixation inthefirst bloom but lower inthe
second (Fig.6.8B). Intheabsenceof non-assimilatorypathwayscarbondioxidefixationis
coupledwith PSIIoxygenproduction inaoneto oneratio.ThisratiodecreaseswhenNADPHis
usedfor theconversion ofammoniumto nitrate insteadoffor carbonfixation.Thereforewe
wouldexpectthat oxygen production isaslargeas,or largerthancarbonfixation andwe
concludethat duringthesecond bloom (after day23 ) either anoverestimationofcarbon
fixation or anunderestimation ofoxygenproduction hasoccurred.Theoxygen methodis
restricted byitssensitivity andbythecorrections onehasto maketo calculategrossoxygen
production.Weassumedthatthe respiration duringthedayisidenticaltothe night respiration.
HoweverWegeretal (1989) and Beardalletal (1994) foundenhanced respiration inthe light.
Theuncertainty intheoxygendataisreflected bytheunexpectedly highoxygenevolution levels
whicharefound betweenthe 2algal blooms (day 17-21).Theserelatively highoxygenevolution
levelsare not consistent withthe PSIIelectronflowdataandthecarbonfixation data (Fig 6.8B).
Thechlorophyll data (Fig.6.3A) andtheamountofalgalcells (data notshown) inthis period
alsopredict lowphotosynthesis levels.Duringthealgal bloomsthechanges inoxygen
concentrations arelarger andthecalculationswillbelesssensitiveto errors intheestimationof
respirationanddiffusion.
Concluding remarks
Measurement ofchlorophyllfluorescence iseasyandcanbedone insitu. Theadvantageis
that onecanmeasure under prevailing conditions,inour casenutrient limitation.Thetime
resolution israther highasonemeasurement onlylastsafewseconds.Thisiscontraryto the
carbonfixation measurementswhichlast relatively long andmustbedone inclosedcontainers.
Both methods,however,needanindependent measurement of lightdependenceandlight
environmentfor theextrapolationtotheecosystem level.Theproduct ofchlandDPFDisalso
easilydone but inperiodswith relatively highamountsof inactivechltheprimary productionis
overestimated.Theoxygenmethodhastheadvantagethat ityieldsanecosystem parameter, but
it isnotverysensitiveandespecially inperiodswithlowproduction ratessmallerrors inthe
correctionfor diffusionandrespiration haveasignificant effect onthe result.
82
7 T H E M I N I M A L FLUORESCENCE I N T H E DARK AS A
MEASURE FOR PHOTOSYSTEM I I E X C I T A T I O N I N T H E
LIGHT:
E f f e c t s of chlororespiration and state transitions
7.1 Introduction
InChapter 4wehaveshownthat under certainconditionstheminimalfluorescence (F0)can
beusedto estimatetheproduct ofthe number of photosystem II(PSII) reaction centres andthe
absorption cross-section.Fromtheambient PFDandthis product PSIIexcitationcanbe
estimated.AsF0 cannot bemeasured inthelight,weusedF0 inthedarkto estimate PS II
excitation inthelight.Thismethodgaveagood relationship betweenthephytoplankton electron
fluxandthephytoplankton carbonflux at lowand medium irradiances aswasshowninChapters
4-6. InChapter 3itwasalsoshownthatthemaximalvalueofFM inPhaeodadylumtricornutum
wasobserved inlowlight insteadof inthedark.ThisincreaseinFM inlowlightcomparedtothe
darkwasalsofoundfor theother speciesdescribed inthisthesis (datanotshown) andisabout
10% inour conditions.Thisincrease inFM might beexplainedeither byastatetransitionfrom
state IIto state I,or bychanges inspill-overfrom PSIIto PSI,or byenergy dependent
fluorescence quenching inthedarkwhichisinhibited inlowlight.Asignificant statetransition
during light adaptationwould implythattheminimalfluorescence,F0, islowerthantheminimal
fluorescence inthe light adaptedstate,F0'.Incaseofenergydependent quenching causedby
chlororespiration,assuggested byTingandOwens(1993),F0'isobservedwhichislowerthan
the F0under non-energized conditions,butthiseffect might bemaskeddueto reductionofQA by
chlororespiration. Inthis chapterwelooked attheeffect ofconditionswhichmodulate
chlororespiration andstatetransitions ontheapparent F0inP. tricornutum, aspecies relatively
wellexaminedwith respectto regulation of light harvesting andchlororespiration,to see ifthe
assumption iscorrect that F0measured inthedarkisagoodmeasurefortheminimal
fluorescence inthelight.
7.2 Materials and Methods
Growthof Phaeodadylumtricornutum, oxygenevolution,fluorescence measurements andthe
determination offluorescence parametersweredoneasdescribed inChapter 3. Fluorescence
measurementsweredonewiththeXe-PAMfluorometer.F0 isdefined astheminimalfluorescence
observed indarkness inthepresenceofonlymeasuring light.F0'isthe minimalfluorescence
observed inanylight adaptedstate.Experimentsweredoneat 20 °Candoxygensaturation
unless indicatedotherwise.
Theoxygencontent ofthesampleswasloweredwithglucose/glucose oxidase.Inhibitorsof
chlororespiration (antimycinA,CCCP andnigericin,dissolved inethanol) wereaddedto the
83
samples 5minutesbeforethestartofthemeasurement. Finalconcentrations (inuM)were:
antimycinA: 50;CCCP:5;nigericin:5.Thefinalethanolconcentration never exceeded 1%.
Samplesmeasuredat 5and 10°Cwereadaptedtothattemperaturefor 30 minutes inthedark
prior to measurement. Far red light intensitywasmeasured inW m"2andconverted to photon
flux density bycalculation.
Table 7.1.Effects of temperature, oxygen concentration, antimycin A, CCCPand nigericin onthe
minimal (F0) andthe maximal (Ç, )fluorescence andonthe maximalefficiency of PSIIelectronflew
(FV/FM) of P. tricornutum. Alldataareexpressed relativeto the control measurements at 20 °C and
an oxygen content of 100 % of air saturation without further additions. The oxygen data wee
determined from the experiment presented in Figure 7.1.The FV/FMof the control sampleswas
between0.64 and0.70.
condition (numberof
experiments)
FM (± std)
(r.u.)
F0(± std)
(r.u.)
Fv/F„( ±std)
100
100
100
10°C(10)
1.02 ±0.03
1.14 ±0.03
1.06 ±0.01
5°C(10)
1.09 ±0.04
1.21 ±0.04
1.06 ±0.01
22% 02(4)
0.94 ± 0.01
1.02 ±0.01
1.05 ±0.01
4% 02(3)
0.89 ± 0.01
0.91 ± 0.02
1.01 ±0.01
control
backte20 %0 2 (1)
92
88
97
+ antimycinA(16)
1.08 ±0.18
1.04 ±0.09
0.98 ± 0.06
+ CCCP (15)
0.95 ±0.13
1.05 ±0.11
1.04 ±0.03
+Nigericin (11)
1.05 ±0.16
1.08 ±0.13
1.01 ±0.02
7.3 Results
InChapter 3itwasshownthat Fv/FM inP.tricornutumincreasedwithdecreasing temperature
andwesuggested thatthisphenomenon might beinterpreted astheinhibitionof
chlororespiration at lowtemperatures.Table7.1 showsthat loweringofthetemperaturefrom 20
to 10°Cleadsto anincreaseofFv/FM, causedbyanincreaseof FM. Thistemperature effect
might beexplained bythe removalofchlororespiration but aswellbyachange inenergy
distributionviaspill-over.Afurther loweringofthetemperatureto 5°Cledto anincreaseof both
F0andFM, buttheFv/FM didnotincreaseanyfurther (Table7.1).TheincreaseofF0andFM can
not beexplained bythe removalof energydependent quenching asthat should result inan
increaseof Fv/FM aswell.Theobservedcongruentual increaseof both F0 andFM isbetter
84
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Time (minutes)
Figure7.1. Effectofloweringtheoxygenconcentration (#, O) onF0 (D,
• ) , FM ( • , o) andFV/FM ( T , V).Theoxygencontentwasloweredwith
glucose/glucoseoxidase;20unitsofglucoseoxidasein20piwereaddedto
2mlalgalsuspensionattimezero.Glucosewassubsequentlyaddedin
amountsof0.125umolin2.5 ulatthetimesindicatedbythethinarrows.
After25min,whenanoxygenconcentrationof4%ofairsaturationwas
reached,thecuvettewasopenedtolettheoxygencontentincreaseagain
(heavyarrow).TheopensymbolsrepresentthedatapointsusedinTable 7.1.
explained byastatetransitionfrom state Ito stateII.
Apossible involvement of chlororespiration anditseffect onboth F0, FM andFv/FM inthe
temperature experimentswasexaminedbyusingknowninhibitors ofchlororespiration.As
oxygenisasubstrate,loweringtheoxygencontent ofthesamplesshould diminish
chlororespiration (Bennoun 1982).Theoxygenconcentration inthealgalsampleswaslowered
usingtheglucose/glucose oxidasesystem.Thereactionwasstarted bythe injection ofsmall
amountsof glucoseintothecuvette.F0 slightlydecreased andFM showedaverysmallincrease
byloweringtheoxygencontentfrom 100%to 22 %of air saturation,leadingto asmall
increase ofFv/FM (Fig.7.1, Table7.1). Belowanoxygencontent equivalent to 22 %ofair
saturation FM decreased.Atanoxygencontentof4%ofair saturation both F0 andFMwhere
smallerthanat 100%ofairsaturation,butFv/FM wasaboutthesameasat 100%ofair
saturation (Fig.7.1, Table7.1).ThedecreaseofFMwasnot readilyreversible uponthe
readditionof oxygenandthedecreaseof F0 appearsto relax (Fig.7.1, Table7.1).Theeffectson
F0andFM ledto anFv/FM whichwasslightly lowerthanatthe beginning oftheexperiment (Fig.
7.1, Table7.1).Theabsenceof recoveryof bothFM andFv/FM uponincreasingtheoxygen
85
Figure7.2.Theeffectof
farredlightonthesteady
stateandmaximal fluorescenceyieldofP. tricornutum. Thefarredlight
wasobtainedfroma halogenlampshieldedbya
715nmlongpassfilter
resultinginaphotonflux
densityof6umolm"2s"\
Integratedmeasuring
lightintensity3.5 nmolrrf
2 1
s" . Flashfrequency8
Hz. Abbreviation:FR: far
redlight.
concentration isnotwellunderstood yet.At 10 °Caloweringoftheoxygencontent did not
causeaneffect onFv/FM (data notshown).Thissuggeststhat,ifchlororespiration ispresent,
theeffect ofchlororespiration onfluorescence isabsentat 10CC.
Tomodulate apossible proton gradient inthedarkweaddedtheinhibitor of
chlororespiration,antimycinA,andtheuncouplers CCCP andnigericinto darkadaptedsamples
of P. tricornutum.AntimycinA,CCCP andnigericingaveonlysmalleffects onF0, FM andFv/ FM
(Table7.1),whichinviewofthe relatively largestandarddeviation (Table7.1), canhardlybe
consideredto besignificant.Asmallincreaseof F0 isfoundfor antimycinAandnigericin,
whereasCCCP decreases F0. TheFM increasesfor alladditions,leadingto asmalldecreaseofFv/
FMinthecaseof antimycinA,asmallincreaseof Fv/FM inthecaseofCCCP andalmost noeffect
onFv/FM inthecaseof nigericin.We did notfindenergydependentfluorescence quenching in
the dark.Thereforeweconcludethat inthedarkadaptedstatetheprotongradient, ifpresentat
all, isnot largeenoughto induceenergydependent quenching.Ourresults aredifferentfromthe
results ofTing andOwens(1993).Theyfoundasmallbut significant increaseof both F0 and FM
using antimycinA,CCCP, nigericin andanaerobiosis.However,theydidtheir experiments after a
preilluminationwithoffar redlight of 10Wattm'2of 704 nm.Thereforeweexaminedtheeffectof
far red light onsteadystatefluorescence F,FM' and(bPSn.InFigure 7.2 itcanbeseenthatthe
fluorescence inlowfar redlight (6 pïnolm 2 s') islowerthanindarkness (ingeneralabout3
%). Thedecreaseofthe minimalfluorescence inthe presenceoffarred light suggeststhat inthe
darkthe PQ-poolissomewhat reduced.Figure 7.3 showsthedependencyof F0,FM' and(bPSnon
86
C
(0
0
50 100 150 200 250 300 350 400
PFDof thefarred light (urno!m"2s"1)
Figure7.3.ThesteadystatefluorescenceF (O),maximalfluorescence FM'
(O) andtheefficiencyofPS IIelectronflow(•) asafunctionofthephoton
fluxdensityofthefarredlight.Datapointsaretheaverageof2measurements.Oneseriesofphotonfluxdensitieswasmeasuredusingasingle sample.F andFM' weredeterminedafter 3minlightadaptationafterwhich the
photonfluxdensitywasincreased. Thefarredlightandmeasuringlightwere
suppliedasinFigure7.2.
thephotonfluxdensityofthefarred light.ConclusivewithFigure 7.2 F0slightlydecreased inlow
farred lightand remainsconstant at higher photonfluxdensities.FM' and^>PS„ bothshowan
increaseat lowphotonfluxdensitiesandadecreaseatat higher photonflux densities.The
maximumof FM' wasfoundatabout 35 prnolm"2s"1far redlight.Thisincreaseof FM'mightbe
explained both byastate IIto state Itransitionor bytheelimination ofenergydependent
fluorescence quenching betweendarknessand 35 pmolm"2s'. At 65 [Jmolm"2s"1offarred light,
whichisapproximatelythephotonfluxdensityusedbyTingandOwens (1993),wefounda
highervalueofbothFM' andct>psn comparedtothevaluemeasuredindarkness.Thedecreaseof
FM'at higher photonfluxdensities isprobablydueto energydependentfluorescencequenching.
Theresponsible protongradient mightbecausedbyPSIdrivencyclicelectronflow,or bylinear
electronflowdrivenbyfarredlightdeliveredtothePSIIreactioncenteraswell.
Figure 7.4 showsthefluorescencetransientsofadarkadapted P.tricornutumsample
illuminatedwithfarred light (245 (Jmolm"2s"1) Incontrasttothesteadystate results shownin
Figure 7.2 wherethephotonflux densitywasincreasedstepwisewithout dark adaptation
87
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/yi7we7 4 Theeffectsoffarredlightontheminimal fluorescenceofP. tricornutum. Thefarredlight (245 pmol m"2
s"1)wassuppliedasinFigure7.3.Integratedmeasuring
lightintensity4.3 nmolm"2 s'1.Flashfrequency4Hz. Abbreviation:FR: farredlight.
between2succeeding photonfluxdensities,thefarred lightlight ledto animmediateincreaseof
fluorescence.Thisincrease might beexplained byatransitionfrom state IIto state I,althougha
statetransition intermsof phosphorylation isnotverylikelyasthefluorescenceriseisalmost
instantaneous.Whenthefarred light isturned offthefluorescenceyieldshowsatransient
increasefollowed byaslower decreasetothefluorescence levelatthebeginning ofthe
experiment.Thisphenomenon might beexplainedbyatemporaryoverreduction ofthe PQ-pool•
causedbyanunequaldistribution ofexcitonsjust after thefarred lightwasputoff. However,this
would implicatethattheverylowintensity of measuringlight (inthisexperimentthephotonflux
densityofthe measuring lightwas4.3 nmolm"2s"1)isableto induce reductionofthe PQ-poolin
P.tricornutum. Thesuggestionthatevenmeasuring lightofsuchlowphotonfluxdensities might
influencethe measurement isconfirmedbythe resultsshowninTable7.2.Thistableshowsthat
evenatverylowphotonfluxdensitiesthe FV/FM isdependent onthemeasuring light.
7.4 Discussion
7.4.1Effectsofchlororespirationonthefluorescenceyield
Ourmeasurements indicatetheabsenceofaprotongradient inthedark inP. tricornutum'as
the FV/FM inthe presenceofthe uncouplersCCCP andnigericinisnotdifferentfromthe control
(Table7.1).AlsoantimycinA,suggestedto beaninhibitor ofchlororespiration (Raveneland
Peltier 1991) did not affectthe minimalandmaximalfluorescence inthedark (Table7.1).
Thereforeweconcludethat underour conditions F0 isnotaffected byenergydependent
quenching inthedark.Theseresults mightatfirst glanceseemto beincontradictionwiththe
resultsofTing andOwens (1993).TingandOwens(1993) foundfluorescencequenching inthe
dark,whichcouldbeinhibited byCCCP, nigericin,antimycinAandanaerobiosis.Theyconcluded
88
Table 7.2. Maximal efficiency of PS
II electron flow (FV/FM) of P. tricornutum as a function of the photon
flux density of the measuring light.
Results are the average of 10 measurements. Photon flux density
(PFD) is given as the integrated
measuring light intensity. Flash
frequency was 16 Hz.
PFD (nmol m"2s"1)
FV/FM
17
0.65
6.9
0.66
3.6
0.67
2.0
0.70
that a proton gradient, caused by chlororespiration, was present in the dark. Our experiments
with the sensitive Xe-PAM fluorometer indicate that the FV/FMof P. tricornutum\s very sensitive
for light as the FV/FMincreased from 0.65 to 0.70 upon decreasing the measuring light from 17
nmol m"2 s"1 to 2 nmol m"2 s"1 (Table 7.2). It is very well possible therefore that the measuring
light, or the farred light used to oxidize QA"prior to the measurements, may have induced energy
dependent quenching in the experiments of Ting and Owens (1993).
What is the evidence for chlororespiration driving energy dependent quenching? Fluorescence
quenching in the light in the presence of DCMUwas found by Owens (1986b) and Caron et al
(1987) and the quenching was intensity dependent (Ting and Owens 1993). The quenching
disappeared after addition of NH4CI (Caron et al 1987) and CCCP (Ting and Owens 1993)
suggesting that energy dependent quenching is involved. The quenching was also inhibited by
antimycin A which was attributed to the inhibition of a chlororespiratory pathway which involves
the quinones of the PQ-pool but not the cytochrome b 5 f complex (Ravenel and Peltier 1992).
However, as noted by Ting and Owens (1993), a proton gradient in the light can also be induced
by cyclic electron around PS I. Furthermore, Oxborough and Horton (1987) showed that
antimycin A affects the relation between energy dependent quenching and the proton gradient.
Such an effect of antimycin A might be involved in the removal of the fluorescence quenching as
found by Caron etal (1987) and by Ting and Owens (1993). Relaxation of fluorescence
quenching was also found under anaerobiosis (Ting and Owens 1993), but those results could
not be confirmed here.
From the arguments above it seems obvious that a réévaluation of the contribution of
chlororespiration to energy dependent quenching in the dark is necessary.
89
7.4.2Effectsofstatetransitionsonthefluorescenceyield
Farredlight (245 umolm"2s"1)immediatelyledto anincreaseofthefluorescence inadark
adaptedsampleof P. tricornutum(Figure 7.4). Turningoffthefar redlight ledto atemporary
increaseofthefluorescence,followed byasubsequent decrease.Thistransient atfirst sight
resemblesastatetransitionfromstate IItostate I,involving movementof LHCIIfrom PSItoPS
II.Therateofthesechangesis ,however,muchfasterthanmight beexpectedwhenthe
transition involves phosphorylation anddephosphorylation of LHCII. Halftimesfor relaxationof
fluorescence quenching dueto statetransitions of 5to 8minutesinbarleywerereported by
QuickandStitt (1989) andbyHortonandHague(1988),respectively.Amechanism involving
regulationofenergydistribution inP.tricornutumbyionicdistributions independently ofthe
redox stateofthe PQ-pool,assuggested byOwens(1986b), mightseemmorecompatiblewith
the rateofthesechanges.Theimmediateincreaseuponilluminationwithfar redlight probablyis
partially causedbyreductionofQA bythefar red light.Theincreaseandsubsequent decreaseof
thefluorescenceyieldwhenthefar redlightwasturned off canbeexplainedbyatemporary
overreduction of PSII,causedbytheunequalenergydistributionwhenthefar redlight isturned
off.Thisimpliesthat measuring lightof4.3 nmolm 2 s"1alreadyisactinic.Thisisinagreement
withthe lightdependenceof FV/FM (Table7.2).Acceptingthatthemeasuring lightissomewhat
actinic,isiteasyto interpretthedecreaseof F0found inFigure7.2 and7.3 underweakfar red
lightasanoxidation ofQA".
Astatetransitionfrom state IIinthedarkto state Iinthelightwould implicatethat inthedark
the PQ-pool ismorereducedthaninthelight.Thismight becausedbythefluorescence
measuring light (seeTable7.2).Adecreaseoftheamountofspill-over betweendarknessand
lowlightwouldimplicatethat inthedarkadaptedstateenergydistributiontowards PSIis
favoured.
TheincreaseinFM' foundwithfarredlight (Fig.7.3) andwithwhitelight (Chapter 3) also
suggests atransitionfrom state IIinthedarkto state Iinthe lightor adecreaseoftheamount
ofspill-overfromdarkto lowlight.AstheincreaseinFM' withfar redlightisfoundatalower
photonfluxdensitythanwithwhitelight (about 35 pmolm~2 s"1comparedto about 100 pmolm"2
s') wesuggestthatthestatetransitionorspillover in P.tricornutumisboth intensityand
wavelength dependent.Thisisdifferentfromthestatetransitions inPleurochlorismeiringensis
whichwereshownto beonlydependent ontheintensity oftheactiniclight (BüchelandWilhelm
1990).
7.4.3HowmuchdoesF0'(thelightadaptedstate)differfromF0 (thedarkadaptedstate)?
Figure 7.2 showsthatfar redlight leadsto asmalldecrease (about 3%) oftheapparentF0,
probablydueto oxidation ofthePQpool byPSI.WhenF0ismeasuredinthedarkonlyasmall
overestimationofthe realF0 inthedarkismade.Inthelightadaptedstateastatetransition ora
changeinspill-over mightoccur,leadingto higherfluorescence levels inthe light adaptedstate.
Therefore anunderestimation oftheF0 inthelightmightbemadebyusingthe F0 measuredin
thedark.Basedonour observations ontheincreaseofthe maximalfluorescencewethinkthat
theunderestimation doesnotsurpass 10%inthecaseofstatetransitionandevenlessinthe
caseofspill-over. Furthermoreoneshouldnotethattheeffect of reductionofthe PQpooland
90
theeffect ofstatetransitionswork intheopposite directionsotheoveralleffect isexpectedto be
somewhatsmallerthan 10%.
7.5 Conclusions
We were notableto demonstrate energydependent quenching inthedark in P. tricornutum
andthe PQ-poolwaslargelyoxidized.Thissuggeststhat chlororespirationwasnotactiveinour
samplesandthereforewecanexcludeanyprominent effect ofchlororespiration ontheminimal
fluorescence inthedark inourexperiments.
Thehigher maximalfluorescence inmediumwhiteandinlowfar red lightcomparedtothe
darkissuggestedto becaused byastatetransition involvingachangeinabsorptioncrosssectionof PSIIfrom state IIto state Ior byadecreaseintheamountofspill-over.Themaximal
underestimation of F0'byusingthe F0 is 10%.TheF0 measured inthedark-adapted stateis
therefore agoodestimatefor theF0 inthelightadaptedstate.
91
8 GENERAL D I S C U S S I O N
8.1 Introduction
Ourgoalwasto explorethe possibility ofdetermining primary production inamarineecosystemwithchlorophyllfluorescence measurements insteadofoxygenevolutionorcarbonfixation
measurements.Thefirst stepwasto overcomethe methodological limitations inthedeterminationofthe rateof PSIIelectronflow inphytoplanktonsamples.Thisledtothedevelopment ofthe
Xe-PAMandtotheconcept ofphytoplankton electronflux (PEF)to describe PSIIelectronflowin
phytoplankton samples.Atthesametimethe relation betweenPSIIelectronflowandprimary
production determinedascarbonfixation or oxygenevolution hadto bedescribed,first insingle
speciesexperiments,but lateralso innaturalphytoplankton samples.Thisthesis mainlydescribesthe relationbetweenPSIIelectrontransport determinedwithfluorescence andphotosyntheticoxygenevolutionor carbonfixation.Withthis informationonthe relation betweenthephytoplankton electronfluxandphytoplankton carbonflux (or phytoplankton oxygenflux)theprospectsandlimitationsofeachmethodcanbeoutlined resulting insomerecommendationsfor
futureresearch.
8.2 Fluorescence measurements on phytoplankton samples
InChapter 2acorrelationwasfoundbetweengrowthandthe rateof PSIIelectrontransport
(determined as(pPSn•PFD) inDunaliella tertiolectagrowninbatchculturesatanumber of light
intensities usingaPAM101fluorometer (Fig.2.5). Boththeactualandthemaximalefficiencyof
PSIIelectronflowdecreasedattheendoftheexponential growthphase (Fig.2.4A and 2.6).
Thisstudyalsodemonstratedthemainshortcomings ofthe PAM101fluorometerfor marine
applications:i) lackofsensitivity;ii) unfavourable excitation anddetectionwavelength. Two
researchlineswerefollowed:onewasto compare PSIIelectronflowwithother methodsfor
determining photosynthesis likeoxygenevolution andcarbondioxidefixation andto extendthe
researchto algalgroupswithother lightharvesting complexes (seenextparagraph);the other
wasto develop afluorometer withanincreasedsensitivity.ThePAM101fluorometer, asapplied
inChapter 2,canonlybeusedwithsampleswhichhaveahighchlorophyll density.
InChapter 4wehavedescribedtheXe-PAMfluorometer whichwasdevelopedfor our purpose
byProf U.Schreiber andwhichhasamanyfoldhighersensitivityandismuchmoreversatilethan
the PAM101fluorometer (Schreiber etal 1993;Chapter 4).Thiswasreachedbyanumberof
measures:i) useofaXenonflashlampasanexcitationsource,enablingexcitation intheVIS-UV
wavelength region;ii) improveddetection rangetypicallyfrom650 nmto 750 nmwhichcovers
mostofthe mainchlorophyllfluorescence band;iii) improvedopticalefficiencythroughtheuseof
quartz rodsfor lighttransmission;iv) rapidchangeofexcitationanddetectionwavelengthsby
meansofopticalfilters.Theapparatuswasmostsensitivewhenexcitation anddetectionwereat
a relativeangleof 90 ° andlightwasconducted tothemeasuringcuvettewithquartz rods.In
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themesocosmmeasurementsdescribed inChapter 6wehadto dealwith 2problems:i) the
timing oftheXenonflash lampandthesynchronous pulseamplifierwasfoundto betemperature
dependent.Thisledtothemeasurement of negativeF0valuesinsomenights;ii) incoming solar
radiation at bright dayswasintenseenoughto saturatethesensitivedetector. Thisdidcause
non-linear behaviour resultinginerrorsinthemeasurementoftheefficiencyofPSIIelectron
flow,whichweresometimes hardto discover or recognize.Thereforethedetector preferentially
hadto beplacedataangleof90 ° relativeto incomingsolar radiationto preventsaturation.
Theuseof afilter whichdoes nottransmitfar redlight andheatradiationalsomight help.Beside
thedevelopment ofthefluorometer,thedevelopment oftheprocesscontrolanddataacquisition
program PPMON wasimportantfor accuratemeasurementsat lowchlorophyllconcentrations.
ThePPMONprogramcontainsproceduresfor controlandsimultaneously registration offluorescenceandoxygenmeasurements,for averaging measurements andfor semicontinuousmonitoringoftheefficiency of PSIIelectronflow.Forthispurposeanewcuvettefor simultaneous
oxygenandfluorescence measurementswasdeveloped.Themonitoring procedure ofthe
PPMONprogram provedto bevery usefulintheexperimentsofChapter 6wheretheefficiencyof
PSIIelectronflowwasmonitored inamesocosmcontainerfor aperiod of 3weeks.Furthermore
the PPMONprogram could beusedto averagemeasurements andcorrectthemwithblanc
samples,whichwasvery usefulat lowchlorophyll concentrations.Thecombination oftheXe-PAM
fluorometer andthe PPMONprogramwassensitiveenoughfor monitoring phytoplankton electronflux inamesocosmandtherefore itappearsto besensitiveenoughfor monitoring phytoplanktonconcentration andactivityincoastalwaters.
8.3 The relation between PSIIelectron flow,oxygen evolution andcarbon fixation
8.3.10xygen evolutionandcarbonfixation
Comparingtheapparent PSIIelectronflow per photosystem II,JE, withphotosynthetic oxygen
evolution per chlorophyll a, J0,wefoundthatthe relationwasnearly linear atlowto medium
irradiancesfor thespeciesexamined (Fig.3.3,3.4, 3.5 and 3.6B).Thecellsweregrownata
photonfluxdensityof 100pmolm"2s"1andlinearitybetweenJEandJ0wasfoundbelow400 umol
nr2 s'. Asimilar relationwasfoundcomparing phytoplanktonelectronflux,PEF, with phytoplankton oxygenflux,POF,(Fig.4.4) orwith phytoplanktoncarbonflux,PCF,(Fig.5.2, 5.3 and 6.6).
IntheexperimentsofChapter 5thelinear relationbetween PEFandPCFwasfoundatirradianceswhichwereabout 4timeslargerthanthegrowthirradiance (about 280 and69 umolm"2 s"1
respectively).
Athigherirradiancesnon-linearitywasfoundbothintherelationbetweenJEandJ0(Chapter
3) and inthe relation betweenPEFandPOF(Chapter 4) or PCF(Chapters 5and6). InChapter3
thenon-linearity betweenJEandJ0wasshownnotto becausedbyphotorespiration andwe
suggestedthat aMehlertypeof reactionor increased mitochondrial respiration at highirradiancesasthe mostprobableexplanation.Thenon-linearity betweenPEFandPCF isalso attributed
tothese processes asour carbonfixation measurements mostprobablygive net photosynthesis
data (seeWilliamsetal 1996).Asimilar relation betweenJEandJ0asfoundfortheseeukaryotic
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specieswasfoundfor thecyanobacterium SynechocystisPCC6803(Geeletal 1994).
Eveninthe linearpart ofthe relationtheratioofJ0andJEwasspeciesdependent (Fig.4.5).
Toaccountfor differences inpigmentcompositionandPSIIabsorption crosssectionweintroducedtheconceptofthephytoplanktonelectronflux.InChapter4 itwasshownthatthecomparisonof PEFwithPOFyieldedasimilar ratiofor both Phaeodactylumtricornutumanà Dunaliella
tertiolecta(Fig.4.4).Theratioof PCF andPEF(Fig.5.4) wasabout 1.5 timeslarger in Skeletonema costatumthan intheother 2species.Thisvariationintheratio of PCF andPEFissomewhat lessthanwasfound intheratio ofJ0andJEintheexperiments ofChapter 3 (Fig.3.5),though
largerthanthevariation inthe ratioofPOF andPEFinChapter 4 (Fig.4.4). Inalltheseexperimentsweworkedwithexponentiallygrowingalgaewhichallgrewonnitrate.Thisisnotthecase
intheexperimentsofChapter6.ThemesocosmiscontinuouslysuppliedwithN03"but asalgal
decompositiontakesalsoplacealso NH4+ mightbemoreor lessavailableanddegradation
product ofchlorophyll might bepresent.Thesephenomenamighteffecttheratio of PSIIelectron
flowandcarbonfixation.Twoknowneffectsarethenitrogensourceusedfor metabolismandthe
contamination of F0(both:seelater),butotheryet unknowneffect mightalso beimportant.
Indeed,inChapter 6thevariation inthe ratioofthedailyprimary production measuredas
carbonfixationandthedaily primary production measuredasPSIIelectronflowwaslargerthan
inthelaboratory experiments andwasshowntovarybyafactor 4 (Fig.6.8C).TheparameterD
(the ratioof PCF andPEF,both measured inthelaboratory) variedbyafactor 2 (Fig.6.8C),
thoughthevariation inthe ratio ofJcandJEdeterminedfromthesame4experimentswastwice
aslarge (datanotshown).Thereforeweconcludethat bothfor algaegrownunder controlled
laboratory conditionsandunder uncontrolled outdoor conditionstheF0 concept yieldssomewhat
better resultsthanthecomparisonofJ0or JcandJE.However,under uncontrolled outdoor
conditionsthevariation inthecomparison of PSIIelectronflowandcarbonfixation isexpected
to belarger,thoughthis isnot causedbythemeasuring method but bythephysiological stateof
thealgae.
ComparisonofcarbonfixationwithPSIIelectronflowshouldtakeintoaccountthat NADPHis
notonlyusedincarbonreductionbutalsofor sulfateandnitrogenreduction.Thereforethe
relation betweenPEFandPCF alsodependsontherateofthispathwayrelativetotherateof
carbon assimilation.Assumingthattheaverageelementalcompositionofthe phytoplankton is
constant andequalsthe Redfield ratio (GeiderandOsborne 1992),it canbecalculatedthat
reductionof N03"to NH4+ requires about 25 %ofthe NADPHgenerated byPSIIelectron
transport (Lindblad andGuerrero 1993).Whenboth N03"and NH4+ arepresent,normally NH4+is
preferentially metabolized.Whentheconversionof N03"to NH4+ isassumedto beindependentof
the NADPHsupplybythelight reactions,andthereforeindependent oftheirradiance,achange
inthe ratio of PCF andPEFmight beexpecteddueto changesintheavailability of N03"and
NH4+.This,however,canonlyexplainasmallpart ofthevariations inthe ratio of PCF andPEF.
Another causeforthevariationintheratioof PCFandPEFmightresultfromthecontaminationofF0bynon-photosyntheticfluorescingsubstances. Non-photosyntheticfluorescingsubstanceswill result inanapparent increaseofF0.However,theapparent increaseinF0isaccompanied
byanapparent increaseof F andFM', whichleadsto adecreaseoftheapparent ct>PS„-Theeffect
ofthedecreaseof (|)pS„counteractstheincreaseinF0inthecalculationofthe PEF, butdoesnot
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cancelitcompletely.Theuseof phytoplankton electronfluxasameasurefor carbonfixation
requires afrequent calibrationofboththeF0measurement andthe ratio of PCF andPEF.
Tomodelour dataon PSIIelectronflowandoxygenevolutionweextendedthephotosynthesismodelof EilersandPeeters (1988) withanextraoxygenuptakecomponentwhichwas
assumedto beproportional totheproportion of reducedferredoxin,asubstrate ofoxygen
reduction.Themodelgivesasatisfactory descriptionofthe relation betweenJEandJ0atthecost
ofanincreased number of parameters.Thisprovedto beaproblem inthe PEFandPCF measurementsofChapter 5and6.Themodified modelwithits 5independent parameters couldnotbe
appliedbecausetheincubator did not allow morethan7lightintensities.Furthermorethe PSII
electronflowdatawhichwereobtained inthemesocosmcouldnot becomparedwiththe carbon
fixation dataobtained inthelaboratoryastheirradianceswerenotthesame.Insteadthedata
wereanalysedseparatelywiththe unchangedmodelof EilersandPeeters (1988) whichuses
only 3parameters.Forabetter fitting procedure,orwhenmoreinformationabout kineticsis
needed,PEFand/or PCF shouldbemeasuredat more lightintensities.
Both modelsarelesssuitedto gainmoreinformation onthephysiologicalstateofthephytoplankton.ThemodelstreatthePSIandPSIIreactioncentreasoneentity,butfor analysisof
phytoplankton electronflux it ismoreinterestingto describeeventsoccurring atthe PSIIreactioncentre.Themainproblemofthemodelisthat PSIIefficiency isnot regulated byheatdissipationintheantenna,asreflected inenergydependentquenchingbutonlybyPSIIclosureand
photoinhibition. Incorporation ofregulationofPSIIefficiency bymaking PSIIexcitation ( a •I)
dependent onheatdissipationwillmakethismodelmuchmorecomplex,
8.3.2Minimalfluorescence,F0
InChapter 4itwasshownthatthe relation betweenPEFandPOFwasthesamefor P. tricornutumandD.tertiolecta(Fig.4.4).TheuseofF0 asanestimateof PSIIexcitationwasshownto
becorrectwhencertainconditions aremet:thespectral distributionofthe measuring lightand
theactinic lightshouldbeidenticalandthe measuring lightphotonflux densityandallother
optical properties should bekeptconstant inoneseriesof measurements.Whenthisisnot
possible it isrecommendedto measurethespectrumandthePFD,sothat different measurementscanbecalibratedto eachother.
Thusfar PEFwascalculatedusingF0, theminimalfluorescence inthedarkadaptedstate
insteadof F0', the minimalfluorescence inthelightadaptedstate.Thereasonfor thisisthatF0'
isextremely difficult to measure.Thefactthatthe maximalfluorescence inlowlight ishigherthan
inthedark (Fig.3.2A) andtheincreaseof FV/FM whenthetemperature decreases (Table 3.2)
gaveusreasonto assumethat F0 might beinfluenced by chlororespiration orstatetransitions
whichcould leadto anerror intheestimationofF0'. Chlororespiration hasbeensuggestedto
leadto aprotongradient (TingandOwens 1993), resulting inaquenching of F0,but italso
leadsto areductionofQAresulting inanincreaseofapparentF0. Chlororespiration mightthus
either increasetheapparent F0 or decreasetheapparent F0, depending onthe redoxstateofQA
andthe protongradient inthedarkrespectively. Energydependentfluorescencequenching,
whichmight havebeeninducedbychlororespirationwasnotfound (Table7.1), butthe induction
of astatetransition or achangeinspillover betweendarknessandlowlight isquitelikely(Fig.
95
7.3 and 7.4).
Astatetransitionwillleadto anincreaseofF0'comparedto F0, whichincaseof P. tricornutumwouldleadto anunderestimation of F0'ofabout 10%.Whenspill-over isresponsiblefor the
increaseinFM the underestimation ofF0'willbesmallerthan 10%.
Intheprokaryotic cyanobacteriaareductionofthe PQ-poolinthedarkandadarkto light
statetransition isfoundwhichisrelatedto respiratory electronflowacrossthethylakoidmembraneinthedark (Scherer etal 1988).Statetransitions incyanobacteriaarefoundto bequite
large:anincreaseofthe maximalfluorescence ofabout 85 %wasfound inAnacystisby Dominy
andWilliams (1987),asimilareffect canbeseeninfluorescencetracesof Synechococcus'm
Schreiber etal (1993) andinSynechocystisPCC6803\n Geeletal (1994).Theeffect ofthe
statetransition onthe relation betweenPSIIelectronflowandoxygenevolutionor carbon
fixation mighttherefore expectedto belarge.TheresultsofGeelet al (1994),however,showed
asimilar relationbetweenJEandJ0for SynechocystisPCC6803asfor theeucaryotic algae
described inChapter 3.IntheecosystemoftheNorthSeacyanobacterianormallyhardlycontributeto primary production.Thereforethevariation inPEFdetermined onNorthSeaphytoplankton samples isexpectedto benot morethanabout 10%.
Itwasfound inChapter 7that inP.tricornutuminthedarkeventheverylowintensity ofthe
measuring light oftheXe-PAMmight causeasmallreductionofQA(Fig.7.2 and7.3).The
reduction ofQAleadsto asmalloverestimationofF0'(about 3 %).
WehavenotdeterminedtheseeffectsonF0for other species.Thoughweexpectthatfor the
speciesdescribed inthisthesistheeffectwillbeofthesamesizeasfoundfor P. tricornutum'as
theyallcontainamoreor lesssimilarlyorganized photosynthetic system (seeChapter 1).
Thusfarwetreatedthealgalsamplesasifallcellsbehavethesame.However,incasethata
sample contains bothasmallpopulationofalgaewithahighFV/FM (F0 = 10,FM = 33,FV/FM =
0.70) andadominant specieswithalowFV/FM(F0 = 90,FM = 150,FV/FM= 0.40) itcanbe
calculatedthatthe measured FV/FM ofthemixtureofboth (F0= 100,FM = 183,FV/FM= 0.45) is
higherthantheweighted mean(FV/FM= 0.1 •0.70 + 0.9 •0.40 = 0.43).Thiseffect isonly
important inextremecasesasdescribed here.Ingeneralonlyaminor influenceofthiseffecton
thecalculated PEFisexpected.
8.4 Phytoplankton electron flux or phytoplankton carbon flux?
Thescientific choice betweenmeasurements of primary production basedon measurements
of PEFor PCFinmyopinion should beachoiceonaphysiological basis.Both methodshave
shownto havetheir ownlimitations inthepredictionof primary production.Though it canbe
concludedthatthevariation inPEFmeasurements issmallerthanthevariation inPCF measurements (Fig. 5.3).
PSIIactivity canbedetermined /ns/tuwlth chlorophyllfluorescence measurements.The
fluorescence measurements canalsobedonesimultaneouslywith ,4Cincubations incarbon
fixation measurements allowingthe measurementsto becarriedout under identicalconditions.
ThePEFisameasureof PSIIactivity inasample butthe PEFgivesnoinformation aboutthe
96
natureoftheelectronsinks.Part ofthe reducingequivalentswillbeutilisedinassimilatory
processes,e.g.,carbonfixationor nitrogenassimilation andapartwilldrivedissipative reactions
likephotorespiration andMehler-reaction.
Forcarbonfixation measurementssampleshaveto betakenout ofthephytoplanktonsystem
for arelatively long incubation (inthisthesiscarbonfixation measurements onnaturalphytoplankton sampleslasted 2hours,Chapter 6) to reachsufficient sensitivity. Measurements ofthis
lengtharefoundto givemostly netcarbonfixation (seeWilliamsetal 1996) Carbonfixation
measurements donotgiveinformation ondissipative reactions likephotorespiration and Mehlerreaction butareameasureoftheincreaseofthephytoplanktonbiomassduringthelightperiod.
At lowto medium irradiances,wherethedissipative reactionsonlyplayaminor role,agood
correlation betweenPEFandnet photosynthesis determined asPCFwasfoundandfluorescence
measurements might beusedto predict carbonfixation.
Independent ofthe relation between PEFandPCF, <\>KK isagood parameterfor determining
effects ofenvironmental conditions,e.g.toxicsubstances (herbicides),excessivelight(photoinhibition) onthe phytoplankton andF0isagood parameterfor estimation chlorophyll ofphytoplankton.
8.5 Recommendations for future research
Inour experimentswewerenot ableto identifythecauseofthe non-linearitywhichwasfound
at high irradiances inthe relation between PSIIelectronflowandcarbonfixation or oxygen
evolution. Itisveryinteresting,though,to identifytheprocessesthat areinvolved inthe
dissipation ofexcessexcitationenergyat high irradiances,andtheir roleinthefunctioningof
algae. Furthermore agooddescription ofthis non-linear relation isessentialfor thecalculationof primary production.Themodelof EilersandPeeters (1988),usedinthisthesis,isnot
easily modified intoasimplemodelthat incorporatesfluorescence quenchingto modelthe
relation between PSIIelectronflowandoxygenevolution orcarbonfixation.Forthat purpose
anewmodelwillhaveto bedeveloped.
Inthecalculationofthe phytoplankton electronfluxF0 isanessential parameter.Therefore
more researchontheeffects ofstatetransitions or achangesinspill-over onF0isneeded.
Phytoplankton photosynthesis istheoutput of manyindividuals.Thusfarwehaveregarded
phytoplankton samplesasifthe individuals haveidenticalproperties.However,this isnotthe
case.Theimportance ofthis phenomenon innatural phytoplankton samples isworthexamining. Itmight beexaminedwiththe PHYTO-PAMfluorometerwhichisdeveloped byKolbowski
andSchreiber (1995).ThePHYTO-PAMfluorometer usesdifferent lightemitting diodesfor
excitation andmakesit possibleto distinguish betweenalgalgroupswithdifferent light
harvesting pigments.Themethod might bepromisingfor measurement ofthecontributionof
greenalgae,cyanobacteriaanddiatomsto phytoplankton electronfluxand,perhaps,inthe
separation of periphytonand macrophytePSIIactivity insitu.
Thecurrent equipment canmeasurevariablefluorescence onlaboratory sampleswitha
chlorophyll concentration aslowas0.02 ugI"1(Schreiber etal 1993).Atthese lowconcentra-
97
tionsthanacalibrationwithareferencesampleisrequiredto subtract background signalsfor
accurate determination of F andFM'.Inexperimentswithdilutesuspensions itwasdifficult to
obtainareliable reference sample.Thuswhenwantsto increasethesensitivity,theavailability
ofagood reference system isrequired.
Whenoutdoor measurements haveto bedonewithanapparatus basedontheXe-PAM
fluorometerthefollowing shouldbeconsidered:i) thermostattingtheequipment becausethe
synchronization oftheXenonflashlampandthesynchronous pulseamplifier wastemperature
dependent; ii) optimal locationofemitter anddetector with respectto eachother andwith
respecttotheambient light;iii) needfor anaccuratedetermination of F0inoutdoorsmeasurements;iv) provisionsto accountfor automated measurements;v) Finallyoneshouldconsidertheuseofcombinedspectralabsorptionandfluorescence.Withsuchanapparatus it
might bepossibleto measurethe lightenvironment,determinethecompositionofthephytoplanktonsampleandmeasurethephotosynthetic activity.
98
SUMMARY
Saturating pulsefluorescence measurements,wellknownfromstudies of higher plantsfor
determination of photosystemII(PSII) characteristics,wereappliedto culturesofthegreenalga
Dunaliellatertiolecta (Chapter 2).Theactualefficiency of PSII(cppsu), the maximalefficiencyof
PSII(FV/FM),andboth photochemical andnon-photochemicalfluorescencequenchingwere
determinedfor cultures of D. tertiolectagrowing undervarying light intensities.Therateof PS II
electronflow (JE),estimated astheproduct of (bPSnandthe photonflux density (PFD),appeared
to correlatewellwithgrowth ratesdeterminedfor the D. tertiolectacultures.Theresults indicated
thatthesaturating pulsefluorescence method maybesuccessfully usedto determine
photosynthetic characteristics of phytoplankton.However,anincreaseofsensitivity byafactor
1000wasfoundto beneededfor theapplicationofthistechniqueto insitumeasurements.
Conditionswereoutlinedwhichhaveledtothedevelopment oftheXe-PAMfluorometerwitha
manyfoldhighersensitivity.
Therelation betweenphotosynthetic oxygenevolution (J0,expressedasoxygen production
per chlorophyll a)andJEwasinvestigatedfor themarinealgae Phaeodactylumtricornutum, D.
tertiolecta,Tetraselmissp.,Isochrysissp.andRhodomonassp.byvaryingtheambientPFD
(Chapter 3).At limiting lightalinear relationwasfound inallspecies.At PFD'sapproaching light
saturation linearitywaslost.Theobserved non-linearity at high PFD'sismost probably not
caused byphotorespiration but byaMehler-typeofoxygenreduction.Therelationship couldbe
modelled byincluding aredox-state dependent oxygenuptake.Thelinear range betweenJEand
J0extendsto aPFDwhichis2to 10timeshigherthanthePFDatwhichthespeciesweregrown.
Theratio ofJEandJ0inthelight-limited rangeisspeciesdependent andrelatedto differences in
absorption cross-sectionof PSII (oPSn).TheratioofJEandJ0inthe light-limited range isnot
dependent ontemperature.FV/FM wasfoundto betemperature dependentwithanoptimumnear
10°C inthediatom P. tricornutum.
Thephotosynthetic electronflux inaphytoplankton sample (PEF)wasshownto dependon
theproduct ofJE( = 4)PSn•PFD),oKn andthenumber of PS II(n^Jin thesample (Chapter 4). A
mathematical expressionwasderivedwhichrelatestheminimalfluorescence (F0)to n^,,and o^
H undertheconditionthatthespectraldistributionoftheambientlightandthemeasuring light
areidentical.Thisconditioncanbeapproximated measuring F0withtheXe-PAMfluorometer.The
experimentalconditions underwhichthe relationship between PEF,<ppsilandF0 isvalid,were
examined.Themaximalvalueof (J^H (FV/FM)wasshownto beindependent onthewavelength
underthe measuringconditions.Theapparent FV/FM dependsontheintensity ofthe measuring
lightandthedurationandintensity ofthesaturating light pulse.Itisshownthat,under certain
conditions,theminimalfluorescencecanbeusedasameasureof PSIIexcitation inthe light.F0,
obtainedwiththebroad bandexcitation lightofafilteredXenonflash lamp,thuswasusedasa
measurefor theproduct ofn^,,andaKn. Therelationship between PEFcalculatedwiththis
expression andnetoxygenevolution (phytoplankton oxygenflux,P0F,expressed asoxygen
production per samplevolume) wasfoundto besimilar inthediatom P.tricornutumandthe
greenalgaD. tertiolecta. ThereforeweconcludethattheuseofPEF asameasurefor POFyields
99
better resultsthantheuseofJEfor J0.TheXe-PAMfluorometer wasfoundto besensitiveenough
for coastalapplications.
Therelation between PEFandcarbonfixation (phytoplankton carbonflux,PCF, expressedas
thecarbondioxidefixed per samplevolume)wasexamined inculturesof Isochrysissp.,
Phaeocystissp.macroflagellatesandSkeletonemacostatum(Chapter 5).TheF0usedto
calculate PEFwasmeasuredatthestart oftheexperiments.Boththe PFDandthedurationof
the incubationwerevaried.Asfound beforefortherelation betweenJEandJ0,the relation
between PEFandPCFwasalsoapproximately linear at limiting lightanddeviatedfrom linearityat
saturating light.Thelinear rangebetweenPCF andPEFalsoextendsto aPFDwhichis2to 10
timeshigherthanthe PFDatwhichthespeciesweregrown.Thelengthofthe incubationdidnot
affect PEFandPCF exceptforthehighest PFD(1530 [Jmolm2 s_1).ThedeclineofFV/FM ofthe
samples irradiated atthehighest PFDshowedafastcomponentwithin 30minutes incubation
andaminor slowcomponent,indicatingthat photoinhibitionwasinduced inthefirst 30minutes.
PEF,PCF,oxygenproductionandtheir relationshipswerefurthermore examinedduring
phytoplankton development inamesocosmatthefieldstation (Chapter 6). PEFwascalculated
from F0, (bps,,andthe PFDwhichwerecontinuously monitoredfor threeweeksinthe upperwater
layer ofthemesocosm.Inaddition PEFandPCFwereestimatedfrom laboratory measurements
onsamplestakenfromthe mesocosms.Dailyprimary production inthe mesocosm,measuredas
either PSIIelectronflow (PPE) or carbonfixation (PPC),wascalculatedusingaphotosynthesis
model. Dailyphotosynthetic oxygenevolution (PPO)wascalculatedfromchanges intheoxygen
concentration overtheday.Intheperiod betweenthe2bloomsthe rationof PPOandPPE was
higherthanduringthepeakoftheblooms.Theratioof PPC andPPEwasmuchmoreconstant.
Ingeneral PPEgaveareasonable measureof both PPC andPPO.
Theeffects of chlororespiration andstatetransitions onF0weredetermined inthediatomP.
tricornutum(Chapter 7). Inhibitionofchlororespiration byantimycinAoranaerobiosis didnot
affect FV/FM. Theobserved F0wasinsignificantly (8%) increased uponaddition ofantimycinAand
slightlydecreased uponilluminationwithfarredlight (6 pmolmz s"1).Theseeffects mightbe
attributedto chlororespiratory activity,butcould aswellbecausedbyreductionofQAbythe
weakfluorescence measuring light.Additionofthe uncouplers CCCP or nigericindid not increase
themaximalfluorescence (FM).Thedatashowthat under our conditions reductionofQA and
energy dependent quenching inthedarkbychlororespiration do not occur in P. tricornutum.
Light-induced increases inFM, therefore,aresuggestedto becausedbystatetransitions. Useof
theF0to estimate photoncapture inthelight might leadto anunderestimation ofthe PEF. The
error isestimatedto benot morethanabout 10%ascalculatedfromthe increaseinmaximal
fluorescence inthelight.
Thepresentworkillustratesthatthefluorescence pulsemethodisareliabletechniqueto get
insight intothephotosynthetic performance andgross primary productionofthepopulationof
algalcells inamarineecosystem.
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SAMENVATTING
Fluorescentiemetingen metbehulpvandeverzadigende pulsmethode,eenveelgebruikte
methodevoor debestuderingvandeeigenschappenvanfotosysteem II(PSII) bij hogere
planten,zijntoegepast opculturenvandegroenealg Dunaliellatertiolecta(Hoofdstuk 2).Zowel
deactueleefficiëntievanPSII(cbpsil ),demaximaleefficiëntievanPSII (FV/FM),alsde
fotochemische enniet-fotochemischefluorescentiedovingzijnbepaaldaanculturenvanD.
tertiolectadieonderverschillende lichtconditiesgroeiden.Desnelheidvanelektronentransport
door PSII (JE),berekendalshetproduktvan(pPSI|endefotonfluxdichtheid (PFD),correleerde
goed metdegroeisnelheidvandeculturen.Deresultaten latenziendatdeverzadigende puls
methodeookbijfytoplanktontoegepast kanwordenomfotosynthetische eigenschappenvastte
stellen.Deopdatmoment beschikbare apparatuurwasechtereenfactor 1000te ongevoelig
voor insitumetingen.Voorwaardenwerdenopgesteld diemedegeleidhebbentot de
ontwikkelingvaneenXe-PAMfluorometer meteenverhoogdegevoeligheid.
Derelatietussenfotosynthetische zuurstof produktie (J0,uitgedrukt alszuurstof produktieper
hoeveelheid chlorofyl a)enJEisonderzocht indemarienealgen Phaeodactylumtricornutum, D.
tertiolecta,Tetraselmissp.,Isochrysissp.enRhodomonassp.door delichtintensiteit tevariëren
(Hoofdstuk 3).Bijlimiterend lichtwerdeenlineairerelatiegevondenvoor allesoorten.Bij
verzadigend lichtveranderde derelatie.Hetgebiedwaarover lineariteitwerdgevonden is2tot
10maalgroter dandePFDwaaronder dealgenopgegroeidzijn. DeverhoudingtussenJEenJ0
bijlimiterend lichtwasafhankelijkvandesoort engerelateerd aanverschillen indeabsorptie
doorsnedevanfotosysteem II(öps,,).Deniet-lineariteitdiebij hoge PFD'sgevonden iswordt
waarschijnlijk door eenzuurstof reductievanhet Mehler-typeveroorzaakt ennietdoor
fotorespiratie. DerelatietussenJEenJ0kanbeschrevenwordenmeteenfotosynthesemodel
waarineenzuurstof opname isopgenomendieafhankelijk isvanderedoxtoestandvan
ferredoxine.Indediatomee P.tricornutumbleekdat FV/FM afhankelijkwasvandetemperatuur en
optimaal bij 10°C.
Defotosynthetische elektronenfluxvaneenfytoplankton monster (PEF) isgedefinieerdals
het produktvanJE( = (b^,, •PFD),oK„ enhetaantalaanwezigePSIIreactiecentra (n^,,)
(Hoofdstuk 4).Dooreenwiskundige afleiding blijkt hetmogelijkomPEFteschatten metbehulp
van(fppsiiendeminimalefluorescentie (F0),diebeidenmetdeXe-PAMfluorimeter bepaald
kunnenworden.Deexperimenteleconditiesonderwelkedezerelatiegeldigiszijnonderzocht.
Demaximale$Ktl (FV/FM) bleekonafhankelijktezijnvandegolflengtevanhet meetlicht.De
schijnbare FV/FM hangtafvandeintensiteitvanhetmeetlichtenvandeduur enintensiteitvande
verzadigende lichtpuls.Onderbepaaldecondities blijkt F0 gebruiktte kunnenwordenalseen
maatvoor PSIIexcitatie inhet licht.F0, verkregenmetbreedbandig excitatie lichtvandeXenonlampwerd gebruikt alseenmaatvoor het produktvanoKK ennPSn.Erwerdeenzelfde relatie
tussendeuitgerekende PEFendezuurstof produktievanhetsample (fytoplanktonzuurstofflux,
POF,uitgedrukt inzuurstof produktie pervolume) gevondenindediatomee P.tricornutum'en de
groene alg D.tertiolecta. Daaromconcluderenwedat PEFeenbetereschatting geeftvandePOF
danJE van J0.
101
PEFenkoolstoffixatie (fytoplanktonkoolstofflux,PCF,uitgedrukt inkooldioxyde gefixeerd per
volume) zijnbepaaldinculturenvandeflagellaat Isochrysissp., vandemacroflagellatenvan
Phaeocystissp.envandediatomeeSkeletonema costatum(Hoofdstuk 5). DePEFwerd
uitgerekend metbehulpvanF0 dieaandestartvandeexperimenten iniederesoort bepaald
was.Zoweldelicht intensiteit,alsdeduurvandebelichting zijngevarieerd.Zoalseerder
beschrevenvoor derelatietussenJEenJ0,bleekderelatietussenPEFenPCFbijlimiterendlicht
bijbenadering lineairtezijnenbijverzadigend lichtaftewijkenvandezelineariteit.Hetgebied
waarover lineariteitwerdgevondenwasookhier 2tot 10maalgroter dandePFDwaarbijde
algenzijnopgegroeid.Deduurvandeincubatie hadweiniginvloedopderelatietussenPCF en
PEFbehalve bijdehoogste lichtintensiteitenwaarmetnameindeeersteincubatie periodevan0
tot 0.5 uur hogerewaardenvoorzowelPCF alsPEFwerdengevondendanindelatere
incubaties. DeafnamevanFV/FMvandesamplesdiemet 1530pïriolm"2s"1warenbelicht
vertoonde eensnellecomponent inheteerstehalf uurvandeincubatieendaarnaeen
langzamerecomponent. Ditduidtaandatindeeerste 30minutenfotoinhibitie geïnduceerdis.
PEF,PCF enzuurstof produktiezijnonderzocht inmarienfytoplankton ineenmesocosm
(Hoofdstuk 6).PEFiszowelinlaboratorium metingenalsininsitumetingenindemesocosm
bepaald. PCFwerdaanmesocosmmonsters inhetlaboratorium bepaald.Dedagelijkse primaire
produktie uitgedrukt inzowelelektronentransport (PPE) alskoolstoffixatie (PPC)werden
uitgerekend metbehulpvaneenfotosynthese model.Dagelijksezuurstof produktie (PPO)werd
uitgerekend aandehandvandedagelijkseveranderingen indezuurstof concentratie.Tussende
2 bloeiperiodenvanhetfytoplanktonwasdeverhoudingtussenPPOenPPEhoger dantijdens
depiekvanieder bloei.DeverhoudingtussenPPCenPPEwasconstanter. Inhetalgemeengeeft
PPEeenredelijke maatvoor zowelPPC alsPPO.
Deeffektenvanchlororespiratie en"statetransities"opF0 zijnonderzocht indediatomeeP.
tricornutum(Hoofstuk 7). Remmingvanchlororespiratie door antimycineAofdoor anaerobiose
hadgeeneffect op FV/FM.DewaargenomenF0namnietsignificanttoe inaanwezigheidvan
antimycineA,ennamenigszins aftijdens belichting metverrood licht (6 pmolm"2s"1).Deze
effektenzoudendoor chlororespiratieveroorzaakt kunnenworden,maar kunnenook uitgelegd
wordenalsreductievanQAdoor hetfluorescentiemeetlicht.Toevoegingvandeontkoppelaars
CCCP ennigericine leiddeniettot eentoenamevanFM. Dezedatalatenziendat inonze
experimenten met P.tricornutumreductievanQA enfluorescentiedovingdoor energetisatievan
hetthylakoid membraaninhetdonker,beidenveroorzaakt door chlororespiratie,niet
voorkomen.DelichtgeïnduceerdetoenamevanFM wordt daaromverklaard door hetoptreden
vaneen"statetransitie". HetgebruikvanF0 omdeexcitatievanPSIIinhet lichtteschattenkan
leidentot eenonderschatting vanPEF.Defout isnietgroter dan 10%,gebaseerd opde
toenamevandemaximalefluorescentie inhetlicht.
Hetonderzoek heeft latenziendatdefluorescentie puls methodeeengoedinstrument isvoor
debepalingvandefotosynthese capaciteit enbruto primaire produktievandealgenineen
marieneecosysteem.
102
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CURRICULUM VITAE
ColineGeelwerdop4juli 1967geboren inNuenen.In 1985 behaaldezijhetVWO diplomaaan
het Rythoviuscollegete Eersel.Daarnabegonzijmetdestudie MoleculaireWetenschappenaan
de Landbouwuniversiteit teWageningen.Hetdoctoraal examenmetfysisch-chemischeoriëntatie
werdbehaald in 1991.Dehoofdvakkenwaren:fysische plantenfysiologie bijdr.J Snelen
industriële microbiologie bij M.Smith.Innovember 1991 iszijbegonnenalsAIOaande
toenmaligevakgroep Plantenfysiologisch OnderzoekvandeLandbouwuniversiteit teWageningen
dienugefuseerd ismetdevakgroep Plantenfysiologie. Hetonderzoeksproject 'energieconversie
infytoplankton'werdgefinancierd door het Rijksinstituutvoor KustenZee (RIKZ)endeComelus
Lelystichting enheeft geleidtot dit proefschrift.
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