Regulationofphotosyntheticelectronflowinisolatedchloroplastsby
bicarbonate,formateandherbicides
Reguleringvanfotosynthetischelektronentransportingeïsoleerde
chloroplastendoorbicarbonaat,formiaatenherbiciden
CENTRALE LANDBOUWCATALOGUS
0000 0089 6213
Promotor:
dr.W.J.Vredenberg
hoogleraarindeplantenfysiologie,
metbijzondereaandachtvoordefysischeaspekten
Co-promotor: dr.ir.J.J.S.vanRensen
wetenschappelijk hoofdmedewerker
J. F.H.Snel
Regulationofphotosynthetic electron
flowinisolatedchloroplastsby
bicarbonate,formateandherbicides
Proefschrift
terverkrijgingvandegraadvan
doctor inde landbouwwetenschappen,
opgezagvanderector magnificus,
dr.C.C.Oosterlee,
inhetopenbaarteverdedigen
opwoensdag26juni1985
desnamiddagstevieruurindeaula
vandeLandbouwhogeschoolteWageningen
'6fJ< 711 bfZ-aï
BIBLIOTHEEK
DE-R
LAHDHOUWJ:OGVSCHOOÏ,
WAGENINGEN
^ M o g ^ > wVf
Stellingen
1. De procedure die door Badger en Canvin gebruikt is om het in dit proefschrift
beschreven bicarbonaateffekt op het elektronentransport in fotosysteem II aan
te tonen, is daarvoor inadequaat.
Badger, M.R., D.T. Canvin. 1981. Oxygen uptake during photosynthesis
in C , and C^ plants. In: Photosynthesis IV: Regulation of Carbon
Metabolism (G. Akoyunoglou, ed.), pp. 151-161, Balaban International
Science Services, Philadelphia, Pa.
2. Op grond van de in dit proefschrift beschreven effekten van formiaat op het
fotosynthetisch elektronentransport kan deze verbinding aangeduid worden met
de term "inhibitory uncoupler", zoals deze door Moreland voor herbiciden is
geïntroduceerd.
Moreland, D.E. 1980. Mechanisms of action of herbicides. Ann. Rev.
Plant Physiol. 31: 597-638.
Dit proefschrift, hoofdstuk 4.
3. Het onvermeld laten van het belang van de adenylaatkinase aktiviteit bij het
bepalen van de flits-geïnduceerde ATP synthese leidt gemakkelijk tot de
misvatting dat deze adenylaatkinase aktiviteit niet relevant is voor de betreffende meting.
Schreiber, U., S. Del Valle-Tascon. 1982. ATP synthesis with single
turnover flashes in spinach chloroplasts. FEBS lett. 150: 32-37.
k. Bij de interpretatie van de kinetiek van de licht-geïnduceerde potentiaalverschillen over het thylakoidmembraan in intacte chioroplasten dient ook de
invloed van de fosfaatpotentiaal, en daarmee het koolzuurmetabolisme, in
beschouwing genomen te worden.
Bulychev, A.A. 1984. Different kinetics of membrane potential
formation in dark-adaptated and pre-illuminated chloroplasts. Biochim.
Biophys. Acta 766: 647-652.
LAifDKOUWKOG! SCHOOL
WAGKMNGEN
5. De veronderstelling van Schuurmans dat de flits-geïnduceerde ladingsscheiding
in chloroplasten pas na enkele millisekonden resulteert in een gedelokaliseerde
transmembraanpotentiaal is niet houdbaar.
Schuurmans, 3.3. 1984. Dynamic aspects of photosynthetic energy
transduction. Dissertatie, Vrije Universiteit, Amsterdam.
Van Kooten, O., F.A.M. Leermakers, R.L.A. Peters, W.3. Vredenberg.
1984. Indications for the chloroplast as a tri-cornpartment system:
micro-electrode and P515 measurements imply semi-localized
chemiosmosis. i n : Advances in Photosynthesis Research (C. Sybesma,
ed.), Vol. I I , Martinus N i j h o f f / D r . W. 3unk Publishers, The Hague.
6. De optische en elektrische eigenschappen van de nieuwe typen lichtgevende
dioden (LED) maken deze LED's bij uitstek geschikt voor gebruik als moduleerbare lichtbron in het fotobiologisch onderzoek.
Ruraïnski, H.3., G. Mader. 1980. L i g h t - e m i t t i n g diodes as a light
source in photochemical and photobiological work, i n : Methods in
Enzymol., Vol. 69, pp. 667-675.
7. De export van, in het westen verboden, herbiciden (o.a. DDT) naar de landen
van de derde wereld is een onverteerbare zaak.
8. De grote rol die het toevalselement speelt in het praktisch gedeelte van
het huidige rijexamen vraagt om het opnemen van een additionele r i j v a a r d i g heidstest in een r i t - s i m u l a t o r .
9. De simpele visie van de nederlandse overheid op mogelijke consequenties van
de introduktie van het luchtafweersysteem " P a t r i o t " op het machtsevenwicht
in Europa staat in schril contrast t o t de gecompliceerde aard van dit wapensysteem.
Eerste situatierapport over de vervanging van het Nike luchtverdedigingssysteem. Tweede Kamer, vergaderjaar 1983-1984, 18000 hoofdst X,
nr 17.
10. De mening van Chodorow dat door een grotere betrokkenheid van beide seksen
bij de opvoeding van kinderen de maatschappelijke verschillen tussen mannen
en vrouwen verkleind kunnen worden, verdient een grotere aandacht in het
sociale overheidsbeleid.
Chodorow, N. (1980). Waarom vrouwen moederen. Feministische
Uitgeverij Sara, Amsterdam.
J.F.H. Snel
Regulation of photosynthetic electron flow in isolated chloroplasts by
bicarbonate, f o r m a t e and herbicides
Wageningen, 26 juni 1985
CONTENTS
page:
Voorwoord
Abbreviations
Chapter 1 General introduction
ii
iii
1
Chapter2 KineticsofthereactivationoftheHill
reactioninC02-depletedchloroplastsby
additionofbicarbonate intheabsence
andinthepresenceofherbicides.
18
Chapter3 Réévaluationoftheroleofbicarbonate
and formateintheregulationofphotosyntheticelectron flowinbrokenchloroplasts.
32
Chapter4 Effectsofformateandbicarbonateon
photosynthetic flowintheabsenceand
thepresenceofuncoupler.
49
Chapter5 Stimulationofthe flash-inducedtransmembranepotential inC02-depletedbroken
chloroplastsbyadditionofbicarbonate.
64
Chapter6 Effectsofbicarbonate and formateon
photosynthetic electron flowinisolated
intactchloroplasts.
76
Chapter 7 General discussion
98
Appendix1
105
Summary
113
Samenvatting
117
Publications
121
Curriculumvitae
123
VOORWOORD
Veelpersonenhebbenopdeéénofanderemanierbijgedragen
aan het tot standkomenvanditproefschrift;henwil ikhier
bedankenvoorhunmedewerking.Eenaantalvanhenheeftechter
een zo duidelijk stempel gedrukt op ditproefschrift,datik
debijdragenvandezemensengraagapartwilvermelden.
Allereerstwil ik hierderolvermeldenvanmijnco-promotor, JacquesvanRensen, zonder wiens inspanningenditonderzoek zeker niet tot stand gekomen zou zijn.BesteJacques,
jouw gedegen aanpak, jouw kritische opmerkingen enprettige
begeleidinghebbenermedevoorgezorgddathetonderzoekvoorspoedig verlopen is. Ook de rol van mijn promotor,
WimVredenberg, mag hier nietonvermeld blijven.BesteWim,
tijdens hetverloop vanhetonderzoek,maarzekerookbijhet
totstandkomenvanditproefschrift,hebjijmijgedwongenom
de resultaten en interpretaties daarvan kritisch tebekijken
ennauwkeurigteformuleren.
Verder wil ik hier ook debijdragen van "mijn" doctoraal
studenten vermelden.André, jijwas de eerste en jehebtje
metveelenthousiasme gestortophetwerkenmet "levend"materiaal en jouw onderzoekheeftgeleidtoteenbeterbegripvan
de rolvan bicarbonaat en formiaat.Dirk,jouwonderzoekmet
intactechloroplasten toonde,voorheteerst,aandathet"bicarbonaat-effect"ookinintactechloroplastenoptreedt.Free,
jij hebt geprobeerd deinteractie(s)tussenbicarbonaat,formiaat enherbicidenverderoptehelderen,hetgeenjeslechts
ten dele gelukt is in de kortetijddiejeeraankonbesteden.
Olaf,Robenook,gedurendekortetijd,WimVermaas,jullie
hebben erdoor jullieenthousiasme enstimulerendediscussies
eenleukeenvruchtbare tijdvangemaakt.
Als laatste wil ik nog demedewerksters van de afdeling
Tekstverwerking, met nameMarjonvanHunnik,bedankenvoorde
vlotteenaccurateverwerkingvanditproefschrift.
ii
LISTOFABBREVIATIONS,SYMBOLSANDTRIVIALNAMES
"518
ADP
ATP
ATP-ase
atrazine
azido-atrazine
BQ
BSA
Chi
DCMU
DHAP
Diuron
DNOC
DTE
E
m
EDTA
EPR
F6P
FBP
FBP-ase
FeCy
FNR
G1P
AG
ATP
GAL3P
GMCD
HEPES
i-dinoseb
absorbance at518nm
adenosine-5'-diphosphate
adenosine-5'-triphosphate
ATP-hydrolase/synthase
2-chloro-4-(ethylamino)-6-(isopropylamino)s-triazine
2-azido-4-(ethylamino)-6-(isopropylamino)s-triazine
1,4-benzoguinone
bovine serum albumine
chlorophyll
3-(3,4-dichlorophenyl)-l,1-dimethylurea
dihydroxyacetonephosphate
seeDCMU
dinitro-o-cresol
1,4-dithioerythritol
midpointredoxpotential
ethylenediaminetetraacetate
electronparamagnetic resonance
fructose-6-phosphate
fructose-1,6-bisphosphate
fructose-1,6-bisphosphatephosphatase
potassium ferrihexacyanate
ferredoxin-NADP oxidoreductase
glucose-1-phosphate
phosphorylgrouptransferpotentialofATP
formationreaction
glyceraldehyde-3-phosphate
gramicidin
4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid
2,4-dinitro-6-isobutylphenol
in
K
î
«m
K
r
K
r
kD
MES
MV
NADP
NADPH
"H
OEC
r
430
680
P
700
P
i
PGA
P-glycolate
Pheo
pmf
P
PQ
PQH 2
PSI
PSII
OB
QB-protein
RPP-pathway
RU5P
RUBISCO
RUBP
simeton
IV
dissociation constant
inhibitorconstant
apparentinhibitorconstant
Michaelis constant
reactivationconstant
apparentreactivationconstant
kilodalton
2-(N-morpholino)-ethanesulfonicacid
methylviologen
nicotinamide-adeninedinucleotide (oxidized)
nicotinamide-adeninedinucleotide(reduced)
Hill coefficient
oxygenevolvingcomplex
secondary acceptorofphotosystemI
primaryelectrondonorofphotosystemII
primaryelectrondonorofphotosystemI
orthophosphate
3-phosphoglycerate
2-phosphoglycolate
pheophytin
protonmotive force
plastoquinone
plastoguinol
photosystemI
photosystem II
primaryacceptorplastoquinone ofphotosystemII
secondaryacceptorplastoquinone ofphotosystemII
QB-bindingprotein
reductivepentosephosphatepathway
ribulose-5-phosphate
ribulose-1,5-bisphosphatecarboxylase/
oxygenase
ribulose-1,5-bisphosphate
2-methoxy-4,6-bis(ethylamino)-1,3,5-triazine
tricine
"Hill
vmax
N-(2-hydroxy-l,1-bis(hydroxymethyl)ethyl)
glycine.
Hillreactionrate
maximalHillreactionrate
secondarydonorofphotosystem II
Small arrows mark the point of time at
which a single turn-over actinic light
flashisgiven.
Open arrows indicate thepoint of time at
which continuous actinic light is switched
on.
Closed arrows indicate the point of time
at which the continuous actinic light is
turnedoff.
CHAPTER1
GENERAL INTRODUCTION
This Chapter presents abrief survey of some aspects of
photosynthesis inplants.Onlytheaspectthatareofrelevance for a good comprehension of the effects ofbicarbonate,
formate and herbicides onplantphotosynthesiswillbecovered. Inthis Chapterthereaderwillbereferredtorecentreviewarticles formoredetailed information.
Photosynthesis
in plants
Inplantphotosynthesis carbondioxideisfixedintheform
ofcarbohydrates.Starchisthemajorstorageproduct.Inhigherplants thisprocesstakesplaceinspecialorganelles,the
chloroplasts. A chloroplast consistsoftwocompartments:the
stromamatrix and the thylakoid lumen.The stroma matrix is
bound from the cytoplasmbytwocloselyassociatedmembranes,
the chloroplast envelope, and separated from the thylakoid
lumenbythethylakoidmembrane(1).
Inthe stroma matrix the reduction of C0 2 to sugarphosphates and the subsequentconversion intostarchtakesplace.
These processes,which proceed in the dark,require reducing
equivalents inthe form ofNADPH and chemical energy inthe
formofATP.NADPHandATP aresuppliedtothestromabylightdriven processes which take place atthethylakoidmembrane.
These processes serve thereductionofNADP andthephosphorylationofADP.Allenzymesrequired forthereductionof C0 2
arelocated inthestroma.
Thethylakoidmembranecontainstheapparatusnecessary for
the conversion of lightenergy intochemical energy (ATP)and
reducing equivalents (NADPH). Lightisabsorbedbythephotosynthetic antenna pigment systemsandtheassociated reaction
centers and its energy isusedtowithdrawelectrons fromwater.The electrons are transferred toNADP via an electron
transfer chain.Electron transport fromwatertoNADP isassociated with proton translocation across the thylakoidmembraneresulting inaprotonelectro-chemicalpotentialdifference across the thylakoid membrane.This protonpotential is
the driving force forthephosphorylation ofADPtoATP.Posphorlylation is catalyzed by the H -ATP-ase,whichisamembrane-spanning protein complex present inthe thylakoidmembrane.
Photosunthetic
CO2-reduction
carbon
in
metabolism
plants
Chloroplasts are able toreduce C0 2 tosugarphosphatesin
the Reductive Pentose Phosphate-pathway (RPP-pathway, other
names are:Calvin cycle,Benson-Calvin cycle, Photosynthetic
Carbon Reduction cycle). The enzymes for theRPPpathway are
located in the chloroplaststroma.TheRPPpathwaycanbedivided inthreestages(2):
a.fixationofCO,. A single C02-acceptormolecule,ribulose1,5-bisphosphate (RUBP), iscarboxylated yieldingtwomoleculesof3-phosphoglycerate (PGA).Thisreactioniscatalyzedbytheenzymeribulose 1,5-bisphosphatecarboxylase/oxygenase (RUBISCO).Thisenzymalsocatalyzestheoxygenation
ofRUBP.
b. reductionofPGA. The next stepisthereductionofPGAto
triosephosphates, mainly dihydroxyacetone phosphate (DHAP)
andglyceraldehyde 3-phosphate (GAL3P).Thisprocessrequlr-
esATP and NADPH. The overall reduction ofPGA totriose
phosphate ishighly reversible and subject to control by
energychargeandconcentrationofendproducts,
c.regenerationofRUBP.Finallytriosephosphates areconverted to RUBP in a complicated series of reactions. Inthis
conversion five molecules of triosephosphate yield three
molecules of ribulose-5-phosphate (RP5P).RU5PisphosphorylatedbyATP formingRUBP.
Foreverythreemolecules ofC0 2 fixedonemoleculetriosephosphate isformed.Thistriosephosphatecanbeusedtostore
carbon in the form of starch,butitcanalsobeusedtoincrease the concentration ofintermediates intheRPP-pathway.
Inthisway the RPP-pathway becomes auto-catalytic:byfixation of C0 2 theconcentrationofacceptormolecules (RUBP)is
increased which in turnwill increasetherateoffixationof
C0 2 . Theauto-catalyticnatureoftheRPP-pathwayallowsarapid response from adark-situation,wheretheconcentrationof
intermediates,is low, toastateinthelightwhereC02-fixationcanproceed rapidly (2).
In several plant speciesanadditionalC02-fixationmechanism exists,but thismechanism onlyhasasatemporarystorage function.Ultimately C0 2 isfixedandreduced intheRPPpathway. Formore detailsthereaderisreferred toe.g. ref.
4.
Apart from starch and glucose, triosephosphates appearto
be amajor endproduct ofphotosynthetic carbonreductionin
chloroplasts.AccordingtoHeberandHeldt (3)thechloroplast
mightbeconsidered asanorganellethatimportsphosphateand
exportstriosephosphate (mainlyDHAP). Bothimportofphosphate
and export of triosephosphate are catalyzed bythesame(exchange)carrier:thephosphate translocator.Thisenzymewhich
is located in theenvelope,catalyzestheexchangeof1triosephosphate for1phosphate (seeref.3formore details).
C02-fixation through the RPP-pathway is regulated bymany
factors (see e.g. refs. 3,4 forreviews).Oneofthesefac-
tors is of special interestwithrespecttothetopicofthis
thesis. This factor is the regulation ofcarbon flowthrough
the RPP-pathway bypH-dependentmodulationoftheactivityof
several key enzymes (3,4 ) .Saltsofweak acidse.g.acetate,
formate, bicarbonate and glyoxylatehavebeenshowntoaffect
thepHinthestroma (5,6 ) .Thusatneutral ormoderatelyalkalinepH values of the suspension medium theseweak acids,
caninhibitcarbonreduction.
Photorespiration
At ahigh 0 2 /C0 2 ratio RUBISCO functions as an oxygenase
instead of a carboxylase; in fact C0 2 and0 2 compete forthe
samebinding site at the enzyme (7).WhenRUBPisoxygenated
PGA and phosphoglycolate arethereactionproducts.Afterdephosphorylation ofphosphoglycolate in the stroma, glycolate
canbeoxidized intheperoxisome.Thisoxidationiscatalyzed
by glycolate oxidase andtheproducts areglyoxylate and H 2 0 2
(8). Under normal conditions two molecules ofglyoxylateare
converted to 1moleculeofC0 2 and1moleculeofglycerate in
a seriesofperoxisomal andmitochondrial reactions (8).Under
conditions inwhich theconversion ofglyoxylate toglycerate
isblocked, glyoxylate can reactwith H 2 0 2 , yielding formate
and C0 2 (9).Althoughthereactions involved inphotorespiration have been characterized, the physiological function of
photorespiration isstillpoorlyunderstood (8,9,10).
Photosynthetic
electron flow in plants
Most components of the photosynthetic electron transport
chaininhigherplants andeucaryotic algaearelocatedwithin
three major protein complexesthatareembedded inthethylakoidmembrane (11,12).Twooftheseproteincomplexes contain
chlorophyll andareknownasthePSIandthePSIIproteincom-
plexes.Thethirdmajorproteincomplexisknownasthecytochromeb 6 -f complex. Thesecomplexesareintrinsic,membranespanning,thylakoidmembraneproteincomplexes (11,12).Electronflowbetweenthesemajorproteincomplexesismediatedby
thesmallerelectroncarriersplastoquinone (actuallyahydrogen carrier), plastocyanin and ferredoxin (11).Thesemobile
carriers arelocated eitherwithinthelipidbilayer (plastoquinone)oratitssurface (plastocyaninandferredoxin),see
e.g. ref.11.
Forthesakeofconcisenessthissectionwilldeal further
onlywith those aspects ofphotosynthetic electron flowthat
arerelevanttothetopicofthisthesis.Thereaderisreferredtoref.13forseveralrecentsurveysofother interesting
aspectsofphotosynthetic electronflow.
Electron
flow in photosystem
II
Light absorbedbychlorophylls located intheantennaof
PSIIistransferredtothereactioncenterchlorophyllofPSII
(1*680)intheformofexcitons (14).Theprimaryeventofelectron flow inPSII isthegeneration ofthesinglet excited
*
*
state (P68o)upon arrivalofanexcition. P Ô S O candonateits
electron via atleastoneintermediate acceptor,pheophytin
(16), to the first "stable"electron acceptor ofPSII, Q a ,
+
creatingthestate [P 680 *Pheo]«Q Awithin2nsafterexcitation
(15, 16).AsP680andQ A arelocatedatoppositesidesofthe
thylakoid membrane, electron transfer from Pg 8 0 toQ.isaccompanied byatransmembrane chargeseparation (17).Recombinationofchargesinthestate [P68o*Pheo] 'Oil isnormallypre+
ventedbyreductionofP6sob v thesecondarydonorofPSII,Z.
This electron donation to V680occurswithinseveralps(15),
which is much faster thantherate ofrecombination inthe
state [Peso-Pheol-Q"(15).
After donationofanelectrontoPeso» z is left inthe
state Z .Z canbereduced bytheoxygen evolving complex
(OEC)which ultimately derivesitselectrons fromH 2 0. Inthe
OEC water is decomposed into protons,whichareliberatedin
the thylakoid lumen (17),molecularoxygenandelectrons.According to the Kok-scheme,theOECcanbeinoneoffivedifferentoxidationstates,generallyknownasthes-states(18).
Theoverallrateconstant foroxygenevolutionhasbeenreported to be around 1000 s~ (18).One important featureofthe
Kok-scheme is that all OEC's are assumedtooperateindependently fromeachother,i.e.nochargetransferoccursbetween
OEC's(18).
*
After the reduction ofPheobyP 6 8 o,Q» isreducedbyPheo
within200ps (15,16).UponreductionQ~,aspecialplastoquinone that accepts onlyoneelectron,staysinthesemiquinone
anion form (Q~)for 0.2 ms to1.2ms,dependingontheredox
state of the secondary electron acceptor Q_ (19).Q~isnormallynotprotonated (21).0_,whichisalsoaspecialplastoguinone, appears to functionasatwo-electrongate (19, 20).
Only after receiving a second electron from Q., Q B isfully
reduced to Q B ~ andbecomesprotonated (21).Theseprotonsultimately come from the stroma of thechloroplast.Q„appears
to be abound plastoquinone species. Inthe oxidized andin
the fully reduced form Q B is assumed to equilibrate rapidly
with theplastoquinones thatresideinthelipidbilayer;the
2-
semi-reduced forms ofQ_ (Q„ and,perhaps,Q B )are firmly
bound to abinding siteatthePSIIproteincomplex (21).The
identityoftheQß-bindingsiteisnotexactlyknown;probably
a32kDprotein,theQß-protein,isinvolved (12).Theoverall
half-time forplastquinone (PQ)reductionisabout2-3ms(11).
Proton uptake, as measured by alkalinization of the outer
aqueous phase, ishowever much slower (17),anenigma which
has notbeen solvedyet.Formoredetailsaboutthereactions
involving quinones the reader is referred to e.g. ref.22.
ForauxiliaryelectronacceptorsanddonorsofPSIIthatappear
tobepresent,someunderspecialconditions,seerefs.23and
18.
Electron
flow from PSII toNADP
Freelydiffusingplastoquinol (PQH2)arises fromthedissociationofQ B H 2 , aplastoquinol speciesboundtotheQß-protein.
PQH 2 canbindtothecytochromeb 6 -fcomplexwhichisaplastoquinol-plastocyanin oxidoreductase (seeref.19forareview).
AfteroxidationofPQH 2 theprotons arereleased inthethylakoid lumen (17).TherateofoxidationofPQH 2 andtheconcomittentreleaseofprotonsappeartobepH-dependent;theoxidationofPQH 2 isthemajorrate-determining step inphotosynthetic electron flow with ahalf-time of about 15ms(11).
There is apool of about 7freelydiffusingPQmoleculesper
electrontransferchain(11).
Plastocyanin, a copper containing protein located atthe
inner surface of thethylakoidmembrane,istheimmediatedonortoP7oo/ thereactioncenterchlorophyllofPSI(11).
ExcitationofP700 andsubsequentelectrontransfertoP430'
anopticallydetected acceptorspecies,resultsinatransmembranecharge separation (17).P43o» thesecondaryelectronacceptorofPSImaybeabound ferredoxinspecies(11).
Freelydiffusing ferredoxin,reducedbyPSI,canbeoxidizedin,atleast,threeways(24):
a.FerredoxincanbeoxidizedbyNADP viatheenzyme ferredoxin-NADP oxidoreductase (FNR).Electron flowfromwaterto
NADP isnamed linearelectronflow.
b. Ferredoxincanbeoxidizedbyoxygeninanon-enzymaticreaction. Electron flow fromwaterto0 2 isnamedpseudo-cyclicelectronflow.
c.Ferredoxin canbe oxidizedbycomponents ofthecytochrome
b 6 -f complex, resulting in acyclic electron flow around
PSI without reduction ofNADP but associatedwithproton
translocation acrossthethylakoidmembrane.
Artificial
electron
donors and
acceptors
Avarietyofartificial electronacceptors andelectrondonors have been used,sometimes incombinationwith inhibitors
of electron flow, to characterize various partsoftheelectron transport chain. Inthisthesisonlytheelectronacceptors ferricyanide (FeCy),benzoquinone (BQ)andmethylviologen
(MV)wereused.MVandFeCyhavebeenreported toacceptelectrons mainly from PSI,whileBQcanacceptelectrons fromthe
intersystem electron transport chain. A detailed review on
artificial electrondonorsandacceptors isgiveninref.25.
Energy conversion
bxj
thylakoids
Photosynthetic electron transport inthylakoids iscoupled
to phosphorylation of ADP ("photophosphorylation"). Under
steady state conditions themechanism ofcouplingofelectron
flow tophotophosphorylation appears to be explainedbestby
thechemiosmotictheorypostulatedbyMitchell (26).According
to this theory theelectrochemical protongradientacrossthe
thylakoid membrane, generated byelectron flow,isassumedto
be the high-energy intermediate that drives thephosphorylationofADP.
Theelectrochemicalprotongradient,orprotonmotive force
(pmf), contains two components:thetransmembraneprotongradientandtheelectricalpotentialdifference acrossthethylakoidmembrane.Thetransmembranepotential isgeneratedbythe
charge separations inPSI and PSII (17)and perhaps alsoin
thecytochromeb 6 -fcomplex (20).Theprotongradientiscaused by the liberationofprotons inthethylakoid lumenduring
water oxidation inthe OEC and the translocation of protons
across the thylakoid membrane accompanying the reductionand
subsequentoxidationofPQ.Thetransmembranepotential isdissipated by the movementofionsacrossthethylakoid membrane
(27); duringcontinuous illuminationthemembranepotential in
8
thylakoidshasbeen reported tobeonlyafewmillivolts(27,
see however ref. 17).Muchmoreimportantforphotophosphorylation under steady state conditions is theproton gradient
acrossthethylakoidsmembrane (17).Duringcontinuousillumination the contribution of the proton gradienttotheproton
motive forcemaybemorethan95%(17).Undernon-phosphorylating conditions theproton flux across thethylakoidmembrane
is small compared to the fluxes of other ions andtherefore
protons hardly contribute to the dissipation ofthemembrane
potential (17).However whenphotophosphorylation is taking
place,theadditionalproton fluxthroughtheATP-asedoescontribute significantly to thedissipationofthetransmembrane
potential (17).Thedissipationoftheelectrical fieldcanbe
greatlystimulatedbyionophores andprotonophores.Theproton
gradientcanalsobedissipated,bothbyprotonophores andpermeantbuffers.
Role of bicarbonate
Properties
in
photosynthesis
of bicarbonate
in aqueous
solutions
Whenbicarbonate isdissolved in an aqueoussolution,the
followingreactionsoccurs(28):
2H + +C0 3 " ^ H + +HCO3 ^ H 2 C0 3 5f£:H 2 0+ C0 2
(1)
(2)
(3)
(4)It
CO 2 + O H "
Thereactions (1)and (2),whichinvolveprotonation,arevery
fast and for ourpurposesonlytheequilibrium constantsneed
to be considered. Reactions (3)and (4)aremuchslower;dissociation ofH 2 C0 3 toH 2 0 and C0 2 occurwitharateconstant
of 20 s"1 (28).However,whenbicarbonate is dissolved, the
combined half-times of reactions (3)and (4)(inseries)and
reaction (4)yield anoverallhalf-time fortheconversionof
HCO3 to C0 2 of~ 15 satpH7.4.Thishalf-time ispH-dependent; atpH 5 it is only0.9 s (28).ThepKofreaction1is
10.35andthepKofthecombined reactions2,3and4 is6.35.
A main part ofthisthesisdealswithexperiments inwhich
reactionsofisolatedthylakoidswerestudiedstudiedindependence of [C02] and [HCO~]in thereactionmedium.Asinmost
-
2-
.
experimentscarriedoutC0 2 , H 2 C0 3 ,HC0 3 andC0 3 wereinequilibrium with each other,thetermbicarbonatewillbeusedin
thisthesistoindicatepropertiesoreffectsofasolutionto
whichbicarbonate isadded,withoutimplyingthatthebicarbonateionisthespecies responsable fortheobservedeffects.
Bicarbonate
effects
on processes
involved
in
photosynthesis
Bicarbonate is involved intheprocesofphotosynthesis in
severalways:
1.C0 2 isthe.substrate forthecarboxylationofRUBP,the C0 2 fixingreactionofphotosynthesis(7).
2. C0 2 is required to activate RUBISCO, which catalyzes the
carboxylationofRUBP(7).
3.Bicarbonate can inhibit the reduction of C0 2 in the RPP
pathway by dissipation of theproton gradient across the
chloroplastenvelope (5, 6 ) .
4.Bicarbonate is aweakuncouplerofphotosynthetic electron
flow(29).
5.Bicarbonate can stimulate photophosphorylation inbroken
chloroplasts. This appears tobe due to an effect onthe
conformationofthechloroplastATP-ase(32).
6.Bicarbonate stimulates electron flow in isolated broken
chloroplasts.Although stimulation canbeobserved innontreatedchloroplasts (31),themostdramaticeffectsofbicarbonate on electron flow are observed when thechloroplastshavebeendepletedofC0 2 inapretreatment(30).
10
.
This thesis will mainlydealwiththeeffectsofbicarbonate on electron flow as outlined in item 6, although also
items 3, 4 and 5will bebrieflydiscussed.Therefore amore
detailed descriptionoftheeffectsofbicarbonate onphotosyntheticelectron flowisappropriatehere.
Bicarbonate
effects
on photo synthetic
electron
flow
Basically two different stimulatingeffectsofbicarbonate
onphotosynthetic electron flowhavebeendescribed.
1.Bicarbonateeffects inisolatednon-treatedchloroplasts.
Addition of bicarbonate to non-C02-depleted chloroplasts
canresultinastimulationoftheHillreaction (31).This
stimulation requires relatively highconcentrations ofbicarbonate andthemagnitude ofthestimulation isrelatively small,usually less than 100%.Stimulation oftheHill
reaction is thought to be due to inhibition of a cyclic
electron flow around PSII, increasing the efficiency of
linear electron flow from PSII to PSIorartificialelectronacceptors(31).
2.Bicarbonate effects inC02-depletedchloroplasts.
Incontrastwiththebicarbonate effectselectronflowmentioned above,theeffectsdescribedhererequiremuchlower
bicarbonate concentrations and themagnitude ofthestimulationofelectron flowismuchhigher;usually theratein
thepresenceofbicarbonate ismorethan5timeshigherthan
intheabsenceofbicarbonate.
Sincethediscovery ofthestimulating effectofbicarbonateontheHillreaction inisolatedchloroplastsbyWarburg
and Krippahl in 1958 (33).considerable effortshavebeen
madetounravelthemechanismofbicarbonate actiononphotosynthetic electron flow.Theresultsoftheseeffortscan
besummarized asfollows (seealso refs.36;analternative
interpretation isgivenby ref.34).
11
- C02-depletion of isolated brokenchloroplastsresultsin
a strong inhibition of electron flow. TheC02-depletion
method isbasedondark-incubationofbroken chloroplasts
in amedium containing high anion concentrations atlow
pHintheabsenceofC0 2(35).
-Dark-incubation of C02-depleted chloroplastswithbicarbonate results inbinding ofbicarbonate tothechloroplasts and in anincreased capacitytoreduce artificial
electron acceptors. Actually two types ofbicarbonatebinding siteswere described,butonlythehigh-affinity
binding siteswere correlatedwithstimulationofelectronflow(37).
- InC02-depleted chloroplasts electronflowappearstobe
inhibited between Q, andPQ (38).Althoughminoreffects
onthe oxidizing side ofPSII havebeen reported (39),
thereisevidencethattheseeffectsmaypartlybedueto
interactions between the reducing andtheoxidizing side
ofPSII (36).Intheabsenceofbicarbonate theoxidation
of Q~ is slowed down considerably (38),but the major
site of inhibition appears to be on the protonation of
Q B and/ortheexchangeQ„ofwiththePQpool(40).
-The actual species involved inthe stimulationofelectronflowinC02-depletedchloroplasts isnotknown.Both
bicarbonate and C0 2 havebeensuggested tobetheactive
species. A special problem isthatthebicarbonatebinding site may be located in the vicinity of negative
charges atthe thylakoid surface (41),causing achange
in the local pHandthereforeashiftintheequilibruim
2_
concentrations of HC0 3 , C0 2 and C0 3 near the membrane
surface.
- Thestimulation oftheHill reactioninC02-depletedchloroplastsbybicarbonate appearstoobeykineticsthatresembleMichaelisMentenkinetics;adoublereciprocal plot
ofthe Hill reactionratevs.thebicarbonateconcentration yields straight lines (42).FromtheseplotsanapparentK ofabout~1mMwascalculated(42).
12
-Thebicarbonate effectsonelectron flowappeartointerferewith the actionofherbicidesonelectron flow (see
nextsection).
Mechanism of herbicide action on electron
side of photosi/stem
II.
flow at the
acceptor
Several classes of herbicides have been shown to inhibit
electron flow at the acceptor side of PSII; in this thesis
these herbicides will becalledPSIIherbicides (seerefs.43
and48foraclassificationofherbicides).
ThePSIIherbicidesinhibittheoxidationofQ~. Itwassuggested by Velthuys and Amesz (44)thatbinding of e.g.DCMU
(diuron)to PSII affectstheE oftheredoxcouple Q^/oZ in
lu
15 o
an aliostericway. The E was sugested to be decreasedupon
binding of DCMU, i.e. the equilibrium of the reactionQ~Q B ^ Q A ' Q R I S shifted totheleft.Reversed electron flowfrom
Q" toQ A was observed when DCMUwasaddedtochloroplastsin
thestateQA-Q~(44).
Several classes of PSIIherbicideshavebeenshowntobind
to a common binding site at thethylakoidmembrane.Thiswas
judged fromexperimentswhichshowedthatabound radio-active
labeled herbicide could bedisplaced fromthebindingsiteby
another,unlabeledherbicide (45).Usingthetechniqueofphoto-affinity labeling, itwas shown that an atrazine analog,
azido-atrazine,was specifically bound toapolypeptideof32
kDmolecularweight(46).
As both PSII herbicides and C02-depletion cause inhibiton
ofelectron flowattheacceptor sideofPSII, ithasbeensuggested that the modesofactionofherbicides andbicarbonate
mighthaveacommonbase.Itwasshownthatinthepresenceof
PSIIherbicides theaffinityofthylakoidmembranes forbicarbonatewasdecreasedbyatleastafactor2 (42).Moreover C0 2 depletion of chloroplasts caused adecrase inaffinityofthe
chloroplasts foratrazine andthiseffectofC02-depletionwas
abolished after readdition ofbicarbonate (47).Theseexperi13
meritssuggestthatthemodesofactionofbicarbonate andherbicidesmayhaveacommonbase.
Objectives of this
thesis
Thisthesisdealswithsomeaspectsofregulationofphotosyntheticelectronflowinplants.Themainobjectivesofthis
thesisistogetadeeper insightintothemechanism andphysiological significance of the regulation of electron flowby
bicarbonate,formate and,tosomeextent,herbicides.
References
1.Coombs,J., A.D.Greenwood. 1976. In: Theintactchloroplast. (J.Barber, ed.), pp.1-51,Elsevier/North-Holland
BiomedicalPress,Amsterdam.
2.Edwards,G., D.A. Walker. 1983. C 3 ,C 4:mechanisms,cellular and environmental regulation of photosynthesis.
Blackwell ScientificPublications,Oxford.
3.Heber,U.,H.W.Heldt.1981. Ann.Rev.PlantPhysiol.32:
139-168.
4.Bassham,J.A., B.B.Buchanan. 1982. In: Photosynthesis
(Govindjee,ed.), Vol II,pp.141-191,AcademicPress,New
York.
5.Enser,U., H.W.Heber. 1980. Biochim.Biophys.Acta592:
577-591.
6.Wefdan,K., H.W.Heldt, M.Milovancev. 1975. Biochim.
Biophys.Acta396:276-292.
7.Lorimer,G.H. 1981. Ann.Rev.PlantPhysiol.32:349-384.
14
8.Tolbert,N.E.1981. Ann.Rev.Biochem.50:133-157.
9.Ogren,W.L.,R.Chollet.1982. In: Photosynthesis (Govindjee, ed.), Vol.II,pp.191-230,AcademiePress,NewYork.
10. Loriraer,G.H., T.J. Andrews. 1981. In: The Biochemistry
ofPlants;Vol.8 (M.d.Hatch,N.K.Boardman, eds.),pp.
329-374,AcademiePress,NewYork.
11. Haehnel,W.1984. Ann.Rev.PlantPhysiol.35:659-693.
12. Kaplan,S., C.J.Arntzen. 1982. In: Photosynthesis (Govindjee, ed.). Vol. I, pp. 67-151,Academic Press,New
York.
13. Govindjee (ed.). 1982. Photosynthesis,Vols. IandII,
AcademicPress,NewYork.
14. Pearlstein,R.M. 1982. In: Photosynthesis (Govindjee,
ed.). Vol.I,pp.293-331,AcademicPress,NewYork.
15. Parson,W.W. 1982. In: Photosynthesis (Govindjee, ed.).
Vol. I,pp.331-386,AcademicPress,NewYork.
16. Klimov,V.V. 1984. In: Advances inPhotosynthesis Research
(C. Sybesma,ed.), Vol.I,M.Nijhoff/Dr.W.JunkPublishers, TheHague.
17. Junge,W.,J.B.Jackson.1982. In: Photosynthesis (Govindjee, ed.), Vol.I,pp.589-647,AcademicPress,NewYork.
18. Wydrzynski,T.J. 1982. In: Photosynthesis (Govindjee, ed.),
Vol I,pp.470-507,AcademicPress,NewYork.
19. Cramer,W.A.,A.R.Crofts. 1982. In: Photosynthesis (Govindjee, ed.),Vol.I,pp. 389-468,Academic Press,New
York.
20. Velthuys,B.R. 1980. Ann. Rev. Plant Physiol. 31:
545-567.
21. Wraight,C.A. 1982. In: FunctionofQuinonesinEnergyconserving systems (B.L.Trumpower, ed.), pp. 181-197,AcademicPress,NewYork.
15
22.Trumpower,B.L. (ed.)- FunctionofQuinonesinEnergyconservingsystems.AcademicPress,NewYork.
23.Vermaas,W.F.J., Govindjee. 1981. Photochem. Photobiol.
34:775-793.
24.Hall,D.O. 1976. In: The Intact Chloroplast (J.Barber,
ed.), pp.135-170,Elsevier/North-HollandBiomedicalPress,
Amsterdam.
25.Trebst,A.1972. In: MethodsinEnzymology,VolXXIV,Photosynthesis and Nitrogen Fixation,PartB (A.SanPietro,
ed.), AcademicPress,NewYork.
26.Ort,D.R., B.A.Melandri. 1982. In: Photosynthesis (Govindjee, ed.),Vol.I,pp. 539-588,Academic Press,New
York.
27.Vredenberg,W.J. 1976. In: The IntactChloroplast
(J.Barber, ed.), pp. 53-88,Elsevier/North-HollandBiomedicalPress,Amsterdam.
28.Walker,N.A.,F.A. Smith,I.R.Cathers.1980. J.Membrane
Biol.57:5158.
29.Good,N.E.1962. Arch.Biochem.Biophys.96:653-661.
30.Govindjee,J.J.S.vanRensen,1978. Biochim.Biophys.Acta505:183-213.
31.Barr,R., R.Melhem, A.L. Lezotte, F.L. Crane.1980. J.
Bioenerg.Biomembr.12:197-203.
32.Nelson,N., N.Nelson, E.Racker. 1972. J.Biol.Chem.
247:6506-6510.
33.Warburg,O., G.Krippahl. 1958. Z.Naturforsch. 15B:
509-514.
34. Stemler,A. 1982. In: Photosynthesis (Govindjee,ed.),
Vol. II,pp.513-540,AcademicPress,NewYork.
35. Stemler,A.,Govindjee,1973. PlantPhysiol.52:119-123.
16
36.Vermaas,W.F.J.,Govindjee,1982. In: Photosynthesis (Govindjee, ed.),Vol.II,pp. 541-558,Academic Press,New
York.
37. Stemler,A.1977. Biochim.Biophys.Acta545:36-45.
38.Govindjee, M.P.J.Pulles, R.Govindjee, H.J.vanGorkom,
L.N.M.Duysens.1976.Biochim.Biophys.Acta449:602-605.
39. Stemler,A.1980. Biochim.Biophys.Acta593-103-112.
40.Vermaas,W.F.J., Govindjee, 1982. Biochim.Biophys.Acta
680:202-209.
41.Vermaas,W.F.J., J.J.S.vanRensen. 1981. Biochim.Biophys.Acta636:168-174.
42.VanRensen,J.J.S., W.F.J.Vermaas. 1981. Physiol.Plant
51:106-110.
43.Moreland,D.E. 1980.Ann.Rev.PlantPhysiol.31:597-638.
44.Velthuys,B.R., J.Amesz. 1974. Biochim. Biophys.Acta
333:85-94.
45.Tischer,W., H. Strotmann. 1977. Biochim. Biophys.Acta
460:113-125.
46.Gardner,G.1981. Science211:937-940.
47.Khanna,R., K.Pfister, A.Keresztes, J.J.S.vanRensen,
Govindjee.1981. Biochim.Biophys.Acta634:105-116.
48.VanRensen,J.J.S., 1982. Physiol.Plant.54:515-521.
17
CHAPTER2
KINETICS OF THE REACTIVATION OF THE HILL REACTION IN C02DEPLETED CHLOROPLASTS BY ADDITION OF BICARBONATE IN THE
ABSENCEAND INTHEPRESENCEOFHERBICIDES
Introduction
Carbon dioxide (orbicarbonate) is required forphotosyntheticelectrontransportbetweenPhotosystem IIandPhotosystem Iinisolatedbrokenchloroplasts.Themajoreffectofbicarbonate on electron transportislocated betweenthereoxidation of the firststablePhotosystem IIacceptorQ,andthe
reduction of the PQ pool. For reviews on this matter, see
Govindjee andVanRensen (3)andVermaasandGovindjee(15).
Usually broken chloroplasts are depleted of C0 2 ina C0 2 freemedium containing formate (pH5.0).IftheHill reaction
ofthesedepleted chloroplasts ismeasured inaC02-freemedium
atahigherpHwithFeCyasanelectronacceptor,itisalmost
completely lost.The Hill reaction can bepartially restored
by incubating the chloroplasts withbicarbonate inthedark,
according to Stemler and Govindjee (11),Stemler (10)and
Vermaas andVanRensen (17).Maximal restorationoccursatpH
6.5 (16).About 60-80%oftheoriginal activity remainslost,
and this irreversible deactivation is probably locatedator
before the reoxidation of Q~according to Khanna et al. (5)
andVermaas andVanRensen(16).
Whether C0 2 , bicarbonate orcarbonate istheactive species
remainstobeestablished.Vermaas andVanRensen (16)founda
strong pH-dependence of the stimulation of theHill reaction
by 10mMbicarbonate,withasharppH-optimum nearpH 6.5,suggestingthe involvementofboth C0 2 andbicarbonate.Theyhypothezised thatbicarbonate isthebinding species,whereas C0 2
isinvolved indiffusion fromthebulkaqueousphase toabinding site below the membrance surface. Sarojini andGovindjee
18
(8)alsopointed toanactiveroleofC0 2 indiffusiontothe
bindingsite.
Stemler (10)andVermaasandVanRensen (16,17)haveshown
that, in the presenceof100mM formate,bicarbonatecanonly
reactivate electron transport when incubation takes placein
thedark;reactivationdoesnotoccurinthelight.Therestoration by dark incubationwith bicarbonate takes sometime.
So, when the incubation time isvaried,therestored initial
Hill reaction rate canbeusedasanindication forthefraction of the binding sites that are occupied bybicarbonate.
Thisprocedure enabled us to measure rate constants for the
bindingofbicarbonate tothethylakoids.
There are many electron transport inhibitors, actingbetween Q and PQ. Van Rensen andVermaas (13)haveshownthat
e.g.DCMU,simetonandDNOCinterferewithbicarbonateaction.
If these herbicides act by displacing bicarbonate from its
bindingsite,thekineticsofreactivation shouldbe influenced
by these herbicide's.Recently the idea has emerged that the
bindingsites forthephenolicherbicidesononehand,andfor
the phenylureas and the triazines on theother,arecloseto
each other although not identical (Oettmeier etal. (7),Van
Rensen (12)).Thereforeitwasinvestigated whetherthephenylurea DCMU and the dinitrophenol i-dinoseb havedifferenteffectsonthekineticsofthereactivationoftheHill reaction
bybicarbonate inC02-depletedchloroplasts.Ifso,thiscould
lead towards amoreprecise localizationoftheactionofbicarbonateonelectronflow.
Materials
and Methods
Peas (Pisum sativum L.,cv Rondo)were grown in agrowth
chamber at 20°C.Brokenchloroplastswereobtained from10to
15daysoldplantsbythefollowingprocedure.Freshlyharvestedleaves (20g)werewashedoncewithicecolddistilledwater
and ground in a Sorvall omnimixer for5sin50ml isolation
19
medium, consisting of 0.4M sorbitol, 20mMtricine-NaOH (pH
7.8), 10mMNaCl, 5mMMgCl 2 , 2mMNa-ascorbate and0.2% (w/v)
BSA. The homogenate was squeezed through eight layers of
cheesecloth,andthechloroplastswerecollected inacentrifuge step: acceleration to full speed (2500 g) in 15s, and
after 30s full speed the rotor was decelerated. The supernatantwas decanted andthechloroplastswerebrokenin50ml
Na-phospate buffer (50mM, pH 7.8). The thylakoids werecollected againby centrifugation at 1000 g for 5min. and the
pelletwasresuspended intheisolationmedium toafinalconcentration of 2mg Chl/ml. The entire procedure was carried
out at2°C.The thylakoids were stored at-80°C.Thechlorophyll (a+b) concentrationwasdetermined accordingtoBruinsma
(1).
After thawing, the chloroplast fragmentswere suspended in
C02-free standard medium, containing 50mM Na-phosphate,100
mMNaCl, 100mMNa-formate and5mMMgCl 2 (pH5.0); chloroplast
concentration was equivalent to50pgChl/ml.Thechloroplast
suspension was gently shakenonaBrunswick shaker for10min
at room temperature,whileN2-gas ([C02]<1ppm)was flushed
over the solution. The chloroplasts were pelleted andstored
oniceuntilused.Media andtubesweremadeC02-freebyflushingorbubblingwithN 2 -gas.
The chloroplasts were resuspended inC02-freestandardmedium (pH6.5).TheHillreactionwasmeasured asoxygenevolution according to VanRensenetal. (14), with0.5mMFeCyas
an electron acceptor.Normally the 02-electrodewasequipped
with a teflon membrane (YellowSprings Instruments»Co.,standard)witharesponsetimeofabout30s (to90%offinalvalue), but forthekineticexperiments athinnerteflonmembrane
(YSI Co., high sensitivity) was used, providing a response
timeof5s.
Allexperiments shownwererepeated atleastthreetimes.
20
Results
Figure2.1 showsatypicalreactivationexperiment.Thedepletedchloroplastswereincubated inC02-freestandardmedium
(pH6.5) for2mininthedark,andthentheHillreactionwas
measured during1minintheabsenceofaddedbicarbonate.Subsequentlythechloroplastswere incubated for5mininthedark,
during which bicarbonate (orherbicides)was added. Inthis
particular experiment bicarbonate was added 2min beforethe
lightwas switched on; the Hill reaction was increased from
4.4 to34.6pmol02/mgChi-hbytheadditionofbicarbonate.
5nmol02/ml
2mMNaHCOi
LIGHTON LIGHTOFF
LIGHTON
40
60
Incubationtime (s)
F i g . 2 . 1 ( l e f t ) : R e a c t i v a t i o n of the H i l l r e a c t i o n in C0 2 -depleted pea
c h l o r o p l a s t s by 2 mM NaHC03 (measured as 0 2 - e v o l u t i o n ) . Reaction medium:
100 mM Na-formate, 100 mM NaCl, 50 mM Na-phosphate (pH 6 . 5 ) , 5 mMMgCl2
and 0.5 mM FeCy. C h l o r o p l a s t s were added t o a f i n a l c o n c e n t r a t i o n e q u i v a l e n t t o 33 M8 Chi/ ml.
F i g . 2.2 ( r i g h t ) : R e a c t i v a t i o n of the H i l l r e a c t i o n i n C0 2 -depleted c h l o r o p l a s t s by 2 mMNaHC03 as a function of the dark i n c u b a t i o n time with b i c a r b o n a t e . The H i l l r e a c t i o n a t t h e s t a r t of b i c a r b o n a t e i n c u b a t i o n could
not be measured a c c u r a t e l y due t o the P e r t u b a t i o n caused by t h e i n j e c t i o n
of b i c a r b o n a t e . I n s t e a d we used the average of t h e H i l l r e a c t i o n r a t e s i n
the absence of b i c a r b o n a t e . Reaction medium as i n F i g . 2 . 1 .
The k i n e t i c s of the r e a c t i v a t i o n of the H i l l r e a c t i o n were
studied by varying t h e bicarbonate incubation period (Fig. 2 . 2 ) .
The r e a c t i v a t i o n i s almost complete a f t e r 120 s, which i s i n
agreement with e a r l i e r observations of Stemler (18). The shape
of t h i s r e a c t i v a t i o n curve suggests t h a t the binding of b i c a r bonate t o the t h y l a k o i d s i s a r e a c t i o n with (pseudo) f i r s t o r der k i n e t i c s and a h a l f time of 23 s .
21
As preliminary experiments indicated that this half-time
was influenced bythepresence ofaherbicide, a reaction
scheme isproposed, whichcanexplain these effects. Itis assumed thatthefollowing reactions occur:
ki
A+E^ EA
k,
I+ E ^ E I
k4
k2
(K =i—)
reaction1
r
kl
k4
(K.= T—)
x
K
reaction2
3
with A= activator (bicarbonate or C0„)
I = inhibitor (herbicide)
E = bicarbonate/inhibitor binding s i t e at the thylakoids membrane
K , K. = dissociation constants for the activator - resp. inhibitor - binding s i t e complex.
The r e l a x i o n of t h i s system upon a change in t h e c o n c e n t r a t i o n
of A and/or I can be d e s c r i b e d by t h e following d i f f e r e n t i a l
equations.
d
i | A J - = k 1 - [ A ] - [ E ] - k 2 - [EA]
(2.1)
d
4 | ^ - = k 3 - [ I ] - [ E ) - k 4 - [EI]
(2.2)
This setofdifferential equations hasbeen solved inAppendix 1. This solution ishowever rather complex andmaybesimplifiedbymakingthefollowing assumption.
If itisassumed that theinhibitor equilibrates much faster
with thebinding site thantheactivator,thesolutionofeqs.
fEI1
2.1 and2.2canbeobtainedbysubstituting [ E ] = K - ' , ..
When [A]and[I]areconstant,thesolution becomes:
[EA] (t)= [ E t o t ] - { K [fa-]+ C - e _ ( k i * f A ' ] + k 2 ) * t } (2.3)
22
where A'=
t
E
C
T^TTT'
K
i
= time after addition of A (or I) to the reaction of mixture,
= t o t a l number of binding s i t e s ,
= integration constant.
S t a r t i n g with a s i t u a t i o n where no a c t i v a t o r Ai s p r e s e n t
and we add At oa mixture ofE, I en EI a ttime t = 0 , t h e f o l lowing equation d e s c r i b e s t h e a s s o c i a t i o n ofE and A:
(EA](t) =[ E ^ l - j ^ J L ^ l - e - ^ - r * * !
+ k
2>-t,
(2
.4)
Itisassumed thatEisthebinding site forbicarbonateat
the thylakoids,which allows electron flowbetweenQ.andPQ
onlywhen bicarbonate isattached. IftheHillreactionrate
isproportionaltothenumberofbinding sitesoccupiedbybicarbonate,thebicarbonate supportedHillreactionmayberepresentedbyequation (2.4), withtheactualrate (V„.,->)substituted for[EA]andthemaximal rate ( V ) for[E. . ] :
v
Hill*t;
v
max ï Ç + W TS
j
K
'
Several implicationsofequation (2.5)were investigated:
1 Thetime-independent partofequation (2.5)closelyresemblestheMichaelis-Menten equationforaone-substrateenzyme catalyzed reaction. Thiscaneasilybeseenbymultiplyingnumeratoranddenominatorwith1+£—Lyielding:
K
i
V
H
i n =v m amax
v K ^[+A ][.A .
Hill
]
(2-6)
withK'=K -(1+-Ljp-)
XT
IT
|\•
23
Fig. 2.3: Double reciprocal plot of
the Hill reaction of C0 2 -depleted
chloroplasts as a function of the
bicarbonate concentration in the absence and the presence of i-dinoseb.
Reaction medium as in Fig. 2 . 1 .
O
20
1.0
[NaHCoJ-1(mMf'
Therefore, we measured the Hill reaction as a function of
the bicarbonate concentration. Figure 2.3 shows a double
reciprocal plot of a typical reactivation experiment. From
these plots the dissociation constant (K ) of the enzymeactivator complex and the maximal Hill reaction rate ( v m a x )
that can be obtained in the presence of bicarbonate can be
calculated. I t turns out that
V
max =
Kr
39
-9 * 15
Mmo1
=1.5 ±1mMNaHCO,
°2/mg
°
Chl
'
n
(n=6)
(n=6)
Figure2.3 alsoshowstheeffectof100nMi-dinoseb onthe
bicarbonate supportedHillreaction.V
isnotsignificantly influenced by i-dinoseb, but the apparentdissociation
K' is raised more than 4 times.Thus, intermsoftheMichaelis-Mentenformalism,i-dinoseb isacompetitiveinhibitorofbicarbonatebinding,resulting inaninhibitedelectron transport. From 3identical experiments the inhibitor
constant (K-)oftheinhibitor-enzyme complexwascalculatedusingequation (2.6):K.=31±6nM.
24
1
1
1
06
n
H=104
»/
0.4
• /
controL
02
/
//
/
" > Q0
>
/
X
CD
•//
E
> 2
f-o/
•
/
/
/o
/
-0.4
//
/ ^H:0.99
n
Fig.2.4: HillplotoftheHillreactionofC02-depletedchloroplasts
asafunctionofthebicarbonateconcentration.v=Hillreactionrateat
theindicatedbicarbonateconcentration;Vmaxwascalculated froma
LineweaverBurkplotofthesedata.
ReactionmediumasinFig.2.1.
/\
/
>/
-0.6
»O-lyuM
i-dinoseb
-25
.,
Log[NaHC0 3 ]
-20
Figure 2.4 shows aHillplotofthe fullyreactivatedHill
reaction as a function of the bicarbonate concentration.
The apparentHill coefficient (i^)isabout1,bothinthe
absence and in the presence of i-dinoseb, indicatingthat
onlyonetypeofactivation-siteforbicarbonate existsand
thatthereisnocooperativitybetweentheactivation-sites.
Thetime-dependentpartofequation (2.5)represents asimpleexponentaldecaywithahalf-time
t
_
0.693
,.
S "kl-lA'j+k2 ( s )
Figure2.5 showstheeffectoftheherbicides i-dinoseb and
DCMUonthereactivationoftheHillreactionby2mMNaHC03
Intheabsenceofherbicidesthehalf-timeofthereactivationisabout27s,whereas inthepresenceof100nMi-dinosebor100nMDCMUthishalf-time isincreased toapprox.
57s. From the half-time tx and thedissociationconstant
K r , the rate constants kx and k 2 can be calculated,
kj=7.3 M - ^ s " 1 ; k 2=0.011 s"1.
25
1
1
T
•/^
80
3
1
i
100
•
-
60
o
fi—
140
-
20
/
i
0'
0
•
60
i
1
i
120
180 240
Incubation time(s)
1
300
60
90
120
Incubationtime (s)
Fig. 2.5 (left): Reactivation of theHill reaction of C02-depleted chloroplasts by 2mM NaHC0 3 in the presence of herbicides,»control;O+100nM
i-dinoseb;ci+100nMDCMU.Reactionmedium as inFig.2.1.
Fig. 2.6 (right): Hill reactionofC02-depleted chloroplasts in thepresence of 1mM NaHC0 3 and 100 nM i-dinoseb asa function of the i-dinoseb
incubation time in the dark. The bicarbonate incubation timewas 300s.
Reactionmedium as inFig.2.1.
3.An inhibitor,which competeswithbicarbonate forthesame
bindingsite,isexpected toinhibitbydisplacingbicarbonate from its binding site.Therefore, thekineticsofthe
inhibition of i-dinoseb should be related tothekinetics
ofthedissociationofthebicarbonate-binding sitecomplex.
Figure2.6 shows the kinetics of the inhibitionby100nM
i-dinoseb in the presence of 1mM NaHCCU. The half-time
(t,)of the inhibition is less than 15s,which ismuch
smallerthanpredictedbytheequation (2.5).
(r - 0-693
26
c/ Ï
Discussion
Ithas now beenwidely accepted that themajor effectof
bicarbonate is located between Q. and PQ. VanRensen and
Vermaas (13)havereported thatitisadifficultprocedureto
deplete thylakoids of C0 2 . Thisisreflected inaratherhigh
variability inour values forV
andK .Thesevaluesare,
however, consistent with data givenby Khannaetal. (4)and
VanRensen and Vermaas (13), although the latterauthorsreportsomewhatlowervalues forV
max
VanRensen and Vermaas (13)firstdemonstrated aninteraction between the bicarbonate ion and the herbicides DCMU,
simeton and DNOC. Theysuggested thattheseherbicides actas
(partially)competitive inhibitors ofbicarbonate binding to
the thylakoid membrane.They did not check,however,whether
thethylakoid-herbicide-bicarbonate systemcouldbetreatedas
anenzyme-inhibitor-substrate system.
Thereactionschemeaspresented canaccount fortheobservedkineticsofbicarbonate dependentelectron flow.Figures 2.3
and2.4 showthatthereactivationoftheHillreactioncanbe
described by the time-independentpartofequation (2.5).So,
it seems that the assumption thatwas made byputtingV .,,
proportional tothenumberofbinding sitesoccupiedbybicarbonate, is justified. This gives additional support for the
conclusion that in C02-depletedchloroplasts,therate-limitingstepinlinearelectron flowislocatedatthesiteofactionofbicarbonate andnotattheoxidationofPQH 2 (cf.9 ) .
Competitive inhibition of thebicarbonate stimulation of
electron flow by DNOC was already reportedbyVanRensenand
Vermaas (13).The resultspresented inthisChapter showthat
i-dinoseb has the same effect. Itappearsthatthiscompetitiveinhibitionisageneral featureofthedinitrophenolherbicides. The dissociation constant (K- =31nM)thatwascalculated forthethylakoid-inhibitor complex,isslightly lower
than the binding constant (K. =69nM)givenbyOettmeierand
27
Masson (6).These authors used however "normal"chloroplasts
atpH 8.0, whiletheexperiments shownherewereperformed at
pH 6.5 with C02-depleted chloroplasts.Oettmeier and Masson
(6)have shown that atpH 6.0 three times morei-dinoseb is
boundthanatpH8.0 andthismightexplainthedifference.
The half-time of the reactivation of theHillreactionis
increased from27sintheabsenceofherbicides toabout58s
inthepresenceof100nMi-dinoseb or100nMDCMU. Ithasbeen
shownby VanRensen andVermaas (13)thatDCMUapparentlydecreases the affinity of the thylakoids forbicarbonate,with
an inhibition constant of about 30nM, whichisalmostequal
tothevalue shownhere fori-dinoseb.Withinexperimental limits, no difference is found in theeffectsofDCMUandi-dinoseb on the half-time of the reactivationoftheHillreactionbybicarbonate.
The kinetics of the inhibition of theHillreactionbyidinoseb cannotbe explained by equation (2.5),withthecalculated values for kj, k 2 and K..Theestimatedhalf-timeof
theinhibitionislessthan15s,whichisalmost4timeslower thanpredicted by equation (2.5). Thisdiscrepancy canbe
explainedbyassumingthatanadditional reactionoccurs:
X +E 1
EX
reaction3
k6
X isanothercompound thatcanbindtothebicarbonate/i-dinoseb binding site. This compound is most probably formate,
which is abundantly present in the reactionmedium.Anumber
of investigators,e.g. Good (2),Stemler andGovindjee(11),
Vermaas and VanRensen (16)and Sarojini and Govindjee(8),
have already suggested that formate and acetatecompetewith
bicarbonate for the samebinding site. Itisobviousthatif
reaction3occurs,thecalculatedvalues forK ,kjandk 2are
not correct. This means that in the absence of formate the
half-timeofthereactivation couldbelessthan15s.Suchan
effectofformateonthehalf-timeofthereactivationwasre28
portedbyVermaasandVanRensen (17),butinthose experiments
thereactivationoftheHill reactionwasstudiedbytheaddition ofbicarbonate inthelighttoC02-depleted chloroplasts
in reaction media containing low formate concentrations.Resultspresented in thenextChapter indicatethatformateindeedisacompetitive inhibitorofthestimulationofelectron
flowbybicarbonate.Moreover,itappearsthatthe dissociation
ofthethylakoid-formatecomplexmaybearate-limiting stepin
thebindingofbicarbonate.
So far only the case inwhich bicarbonate, i-dinoseb and
formate are competing forthesamebinding sitehasbeenconsidered. The effect ofacompetitive inhibitorisanapparent
decrease intheaffinityofthethylakoids forbicarbonate;kj
andk 2are,however,notinfluencedbytheinhibitor.Thepresentdatadonotallowtodistinguishbetween acompetitiveinhibitor and anallosteric inhibitor thatonlyaltersK ,while
Vmax
_„isnotaffected,
Thusitappearsthatthebindingofbicarbonate toitsbindingsiteatthethylakoidmembraneisretarded inthepresence
of herbicides and metabolites like formate and acetate. It
wouldbeinterestingtoseeiftheoppositeeffectalsooccurs.
Ifso, the binding sites forherbicides andbicarbonatecould
be identical (inwhichcasedisplacementofherbicidesbybicarbonate should occur)ormerelyoverlapping (allowingbindingofbicarbonate andherbicide atthesame time).
References
1.Brüinsma,J. 1963. The quantitative analysis ofchlorophylls a and b inplant extracts.Photochem. Photobiol.
2:214-249.
2.Good,N.E. 1963. Carbon dioxide and the Hill reaction.
PlantPhysiol.38:298-304.
29
3.Govindjee, J.J.S. vanRensen. 1978. Bicarbonate effects
ontheelectron flowinisolatedbrokenchloroplasts.Biochim.Biophys.Acta505:183-213.
4.Khanna,R.,GovindjeeT.Wydrzynski.1977. Siteofbicarbonate effect inHill reaction. Evidence fromtheuseof
artificial electron acceptors and donors.Biochim.Biophys.Acta462:208-214.
5.Khanna,R., K. Pfister, A. Keresztes, J.J.S. vanRensen,
Govindjee 1981. Evidence foraclosespatiallocationof
the binding sites for C0 2 and forPhotosystem IIinhibitors.Biochim.Biophys.Acta634:105-116.
6.Oettmeier,W., K. Masson. 1980. Synthesis and thylakoid
membrane binding of the radioactively labeled herbicide
dinoseb.Pest.Biochem.Physiol.14:86-97.
7.Oettmeier,W., K. Masson, U. Johanningmeier. 1982. Evidence for two differentherbicide-bindingproteins atthe
reducing site ofPhotosystem II.Biochim. Biophys.Acta
679:376-383.
8.Sarojini,G., Govindjee. 1981. Ontheactivespeciesin
bicarbonate stimulationofHillreaction inthylakoidmembranes.-Biochim.Biophys.Acta 634:340-343.
9.Siggel,U., R. Khanna,G.Renger,Govindjee.1977.Investigation of the absorption changes of the plastoquinone
system inbroken chloroplasts.The effect ofbicarbonate
depletion.Biochim.Biophys.Acta462:196-207.
10. Stemler,A.1979. Adynamic interactionbetweenthebicarbonate ligand and Photsystem IIreactioncenter complexes
inchloroplasts.Biochim.Biophys.Acta545:36-45.
11. Stémler,A.,Govindjee.1973. Bicarbonate ionasacriticalfactorinphotosynthetic oxygenevolution.PlantPhysiol.52:119-123.
30
12.VanRensen,J.J.S.1982. Molecularmechanism ofherbicide
actionnearPhotosystem II.Physiol.Plant.54:515-521.
13.VanRensen,J.J.S.,W.F.J.Vermaas.1981. Actionofbicarbonate and Photosystem II inhibiting herbicides onelectron transport inpea grana and inthylakoidsofabluegreenalga.Physiol.Plant.51:106-110.
31
CHAPTER3
REEVALUATIONOFTHEROLEOFBICARBONATEANDFORMATE INTHEREGULATIONOFPHOTOSYNTHETICELECTRONFLOW INBROKEN CHLOROPLASTS
Introduction
Although ithas been recognized for alongtimethatformate issomehowinvolved intheregulationofelectron flowby
bicarbonate, the mode of action of formate isstillunknown.
Good (2)first demonstrated thatformateandacetate increase
thedependency oftheHill reactiononbicarbonate. Ithasbeen
suggested (2,6,15,20)that formateandacetatecompetewith
bicarbonate forbindingsitesatthethylakoidmembrane.Assuming thatbicarbonate isrequired forelectron flowbetweenQ.
andPQ, itwasarguedthatdisplacementofbicarbonatebyformate would result in a lowering of the apparent affinityof
thebinding sites forbicarbonate.ThishypothesiswassupportedbyStemler (12),whodescribed twodifferenttypesofbicarbonatebinding sitesandshowed thatformatecanremovebicarbonate from these binding sites. The high-affinity binding
sites were shown tobeinvolved intheregulationofelectron
flow: removal ofbicarbonate fromthesehigh-affinitybinding
siteswas correlated with an inhibition ofelectron flow.As
bicarbonate was actually removed from the thylakoid membrane
by formate in these experiments, it is conceivable that the
inhibitionofelectron flowisnotaconsequenceoftheremoval ofbound bicarbonate,but due totheinhibitory actionof
formate. Stimulation of electron flowbybicarbonatemaythen
be explained by assuming that formate isdisplacedbybicarbonate. Inthe former explanation (6,15,20)electron flow
should be strongly inhibited in the absence of bicarbonate\
32
whether or not formate ispresent. As experimental data on
this matter are conflicting (1,2, 6, 15,20),thisChapter
describes experiments thatwere carried out toelucidatethe
mode of action offormate.Theseexperiments arebasedonthe
observation that there appears to be aMichaelis-Mententype
of relationship between the uncoupled Hill reactionrateand
thebicarbonate concentration inthereactionmedium (19).As
hasbeen pointed out inChapter 3, this means thattheHill
reaction rate canbedescribedbytwoparameters:themaximal
Hill reaction rate (v_ )a n d the apparentreactivationconstant (KM. The effectsofformateonK'andV
wereinvesJT
r
ntcix
tigated to seewhether formate is acompetitive inhibitorof
thebicarbonate stimulation of electron flow. TheHillreactioninC02-depletedchloroplastswasmeasured alsointhevirtual absence ofboth formateandbicarbonate todetermine the
modeofactionof formateandbicarbonate.
These experiments require large amounts of C02-depleted
chloroplasts with low activity in the absenceofbicarbonate
and a good stimulation of electron flowby bicarbonate.As
these requirements were notmetby thecurrentC02-depletion
method, anewC02-depletionprocedurewasdeveloped.
Anotherreason forexamingtheroleof formateintheregulation of linear electron flowinmoredetailscomes fromthe
fact that formate is ametabolite which is involved ine.g.
photorespiration (3,9 ) .If linear electron flow in vivo is
regulated by formate andbicarbonate,thenmetabolicpathways
affecting formate levels inthe chloroplast would influence
electron flow.
Materials
and Methods
Brokenchloroplasts {Pisum sativum L.cvRondo)wereisolated as described inChapter 2.Thylakoidsweredepletedof C0 2
33
using the following procedure (Snel,Vermaas andVanRensen,
unpublished data). A 0.5ml thylakoid suspension containing
2mg Chl/mlwas,washed once in4.5 ml medium consisting of
0.3 M sorbitol, 50mM sodium phosphate (pH 5.8),10mMNaCl
and 5mM MgCl 2 . The thylakoids were collectedbycentrifugation (10min 1000 g and resuspended in4.0 ml depletionmedium. This depletion medium contained 0.3 mM sorbitol,10mM
sodiumphosphate (pH5.8), 10mM NaCl, 5mM MgCl 2 and 10mM
sodium formate.The finalchlorophyll concentrationwas250|jg
Chl/ml. Depletion was accomplished by incubating thechloroplasts at 25°C indepletionmedium foratleast60mininthe
darkunderanitrogenatmosphere.TheHillreactionwasmeasured as oxygen evolutionwithFeCyanelectronacceptorat25°C
asdescribedbefore (18).Forthedeterminationofthe initial
Hill reactionrateweusedthe first20softheHillreaction.
AllmediaweremadeC02-freebybubblingwithpureN 2 -gas.
ThekineticconstantsK'andV
weredeterminedgraphicalr
max
lyfromLineweaverBurkplotsthatwereobtained frommeasurements (induplo)oftheHill reactionratesat6differentbicarbonateconcentrations.
Results
CO2-depletion
of
chloroplasts
Applicationofthenewprocedure fordepleting chloroplasts
ofC0 2 yields largeamountsofC02-depletedchloroplasts.Figure3.1 showstheresultsofatypicalC02-depletionprocedure.
At the indicated times a sample of100piofthe chloroplast
suspension was injected into 1150 \xl of C02-free reaction
medium and the Hill reaction was measured as illustrated in
Fig.3.2.After 1mindark-equilibration theHillreactionwas
recorded during 30s. Subsequently thelightwasswitched off
and NaHC03 was added to a final concentration of 5mM. The
34
+ 5mM NaHC03
150
^ , .
sz
E
5 >JH 0,
.
1
30 s
100
cf 1
o
E
3.
.
•/
JZ
uO)
J
/"
NH(C1 /
V
50
NaHC03
s
- NaHC03
v
>*
0
J
'^—
60
i — •
•
1
)
•
120
160
incubation time
(min)
^^
0 •
0
Fig. 3.1 (left): The Hill reaction rate in the absence and presence of
bicarbonate as a function of the incubation time in the depletion medium.
Reaction mixture: 0.3 M sorbitol, 10 mM NaCl, 5 M MgCl2, 20 mM sodium
phosphate (pH 6.5), 20 mM Na-formate, 5 mM NH4C1, 0.5 mM FeCy and thylakoids equivalent to 20 pg/ml. Hill reactions were determined as described
in Fig. 3.2.
Fig. 3.2 (right): Effect of NH4C1 upon the reactivation of the Hill reaction of C02-depleted chloroplasts by bicarbonate. Reaction medium as in
Fig. 3.1 with the exception that NH4C1was added at the indicated time.
c h l o r o p l a s t s were incubated i n t h e dark for 2 min and subsequently the H i l l r e a c t i o n (now i n the presence of NaHC03) was
measured. Addition of 5 mM NH4C1 causes a 4-fold s t i m u l a t i o n
of e l e c t r o n flow. Figure 3.1 shows f u r t h e r t h a t i t t a k e s about
90 min, i n t h i s p a r t i c u l a r experiment, t o reach a s t e a d y - s t a t e
s i t u a t i o n during which the H i l l r e a c t i o n r a t e i n t h e absence
of added bicarbonate i s l e s s than 20 pmol 0 2 /mg Chl-h. In t h e
presence of 5 mM added bicarbonate the uncoupled H i l l r e a c t i o n
r a t e i s about 130 pmol 0 2 /mg Chl-h. These r a t e s approach t h e
uncoupled r a t e s of non-depleted c o n t r o l c h l o r o p l a s t s suspended
i n t h e same r e a c t i o n mixture. The time needed t o reach a steady
s t a t e with minimal a c t i v i t y i n the absence of bicarbonate (Fig.
3.1) dépends on t h e composition of the d e p l e t i o n medium; a t
higher formate c o n c e n t r a t i o n s and/or a t low pH the steady s t a t e
can be reached much f a s t e r (data not shown). At low formate
c o n c e n t r a t i o n s (S 10 mM) t h i s C0 2 -depletion method i s not r e l i 35
a b l e when t h e pH i n t h e d e p l e t i o n medium i s above 5 . 9 ; 10 niM
formate and pH 5.8 were choosen t o allow for easy v a r i a t i o n s
i n t h e formate c o n c e n t r a t i o n and/or t h e pH-value of t h e r e a c t i o n medium without a d d i t i o n s or adjustments t o t h i s r e a c t i o n
medium, which might i n t r o d u c e C0 2 -contamination.
F i g . 3 . 3 : Effect of t h e pH on t h e
uncoupled H i l l r e a c t i o n i n c o n t r o l
(Q) and i n C0 2 -depleted c h l o r o p l a s t s t h a t were r e a c t i v a t e d by 20
mMNaHC03 ( • ) . Reaction medium: 0 . 3
Ms o r b i t o l , 10 mMNaCl, 5 mMMgCl 2 ,
50 mM b u f f e r , 5 mM NH4C1, 0.5 mM
FeCy and t h e c h l o r o p h y l l c o n c e n t r a t i o n was 20 (Jg Chl/ml. Buffers used
were MES, phosphate, HEPES and T r i c i n e . The r e a c t i o n medium of t h e
r e a c t i v a t e d c h l o r o p l a s t s contained
a d d i t i o n a l l y 20 mM Na-formate and
20 mMNaHC03.
Figure3.3depictstheeffectofthepHofthereactionmedium ontheHill reaction rate inreactivated C02-depleted
chloroplasts.Althoughthereactivation oftheHillreaction
by bicarbonate isalmost complete atpH6.5,thisappearsto
benotthecase athigherpHvalues.Omissionofformateand
bicarbonate fromthereactionmediumdidnotpreventthisinhibition atalkalinepHandtherefore this inhibition might
notberelatedtothe"bicarbonate-effect".
Inthefollowingexperimentsthechloroplastswereinjected
intothereactionmediumcontainingtheappropriate amountsof
formate,bicarbonate andNH.C1. After 2min. dark-incubation
theHill reactionwasmeasured.ThedeterminationoftheHill
reaction intheabsenceofaddedbicarbonatewasperformedon
separate samples atthebeginningandtheendofasetofexperiments tocheck whether thechloroplasts werestill "C0 2 depleted".Thisprocedurewasadoptedtoavoideffectsoflight
36
on the binding ofbicarbonate (cf.réf.13)andpossiblyformate (7).In thepresence of10mM formatethestimulationof
theHillreactionbysaturatingamountsofbicarbonatewasalways atleast8-fold,whenmeasured asinFig.3.2 inthepresenceofuncoupler.
-03
-
JZ
Ol
E
/.
.02
O™
O
e
3.
/./
/
•-^
^«
•-*^"^
./
.01
>*
-:'oo
-2
-1
0
1
2
3
[NaHC0 3 ]"
Interaction
4
1
F i g . 3 . 4 : Double r e c i p r o c a l p l o t
of t h e H i l l r e a c t i o n r a t e a g a i n s t
t h e b i c a r b o n a t e c o n c e n t r a t i o n added
t o the r e a c t i o n mixture i n t h e p r e s ence of 5.8 (•) and 50.8 mM (•) Naformate. C h l o r o p l a s t s were p r e v i o u s l y depleted of C02 and incubated i n
the r e a c t i o n medium for 2 min. The
r e a c t i o n medium c o n s i s t e d of: 0.3 M
s o r b i t o l , 20 mM sodium phosphate
(pH 6 . 5 ) , x mMNa-formate (50.8-x)
mM NaCl, 5 mM MgCl 2 , 0.5 mM FeCy
and 5 mM NH4C1. The c h l o r o p h y l l
c o n c e n t r a t i o n was 20 |Jg Chl/ml.
5
(mM}"
1
of formate and bicarbonate
on the Hill
reaction
In order t o e s t a b l i s h the kind of i n t e r a c t i o n of formate
and bicarbonate on e l e c t r o n flow, we have measured t h e r e a c t i v a t i o n of t h e H i l l r e a c t i o n by bicarbonate i n the presence of
5.8 and a t 50.8 mMHCOO-. Figure 3.4 shows a double r e c i p r o c a l
p l o t of the r e s u l t s . I t appears t h a t the maximal H i l l r e a c t i o n
r a t e (V ) i s not affected by formate, whereas the apparent
r e a c t i v a t i o n c o n s t a n t (K') i s increased from 0.29 mM bicarbona t e i n the presence of 5.8 mM formate t o 1.6 mMbicarbonate i n
t h e presence of 50.8 mM b i c a r b o n a t e . Formate apparently a c t s
as a competitive i n h i b i t o r of the r e a c t i v a t i o n of the H i l l r e a c t i o n by b i c a r b o n a t e .
37
Thisinhibitioncanbeinterpreted intwoways:
1.Electron flow is notpossiblewhennobicarbonate isbound
tothebindingsiteatthethylakoidmembrane.Formatemerely actsby competingwithbicarbonate forthesamebinding
site orby lowering the affinity of thebinding sitefor
bicarbonate.This view ispresented ine.g. refs.2,6,15
20.
2.Electron flow ispossible when nobicarbonate isboundto
itsbindingsite.Formate inhibitselectronflowwhenbound
to this site.Bicarbonate can relieve this inhibition of
electron flowby displacing formate fromitsbindingsite,
seerefs.16,17.
At high formate andbicarbonate concentrationsthetwomechanisms yield the same result: atthese concentrations nearly
allbinding siteswill be occupied, either by formate orby
bicarbonate andinbothmodelselectronflowshouldonlyoccur
inthosePS IIcentersthatcontainabicarbonatemoleculeattached to itsbinding site.Only atbicarbonate and formate
concentrations far below their respective dissociationconstants themajority ofthebinding siteswillbe "free",i.e.
these binding sites contain neither bicarbonate norformate.
Undertheseconditionsweshouldbeabletodistinguishbetween
thetwomechanisms.
Fig. 3.5:The apparent reactivation
constant (K') asa function of the
formate concentration in thereactionmedium. Chloroplastswerepreviously depleted of C0 2 . Reaction
medium as inFig.3.1;thechloroplasts were incubated for2min in
reaction medium + bicarbonate at
the appropriatebicarbonate and formate concentrations .
38
The d i s s o c i a t i o n c o n s t a n t s of t h e b i c a r b o n a t e - and t h e formatebinding s i t e complexes were estimated by measuring t h e r e a c t i v a t i o n c o n s t a n t K and t h e i n h i b i t i o n c o n s t a n t K.. Figure 3.5
shows t h a t K' i s l i n e a r l y dependent on t h e formate concentrat i o n i n the r e a c t i o n medium. According t o eqn. (2.6) extrapolat i o n t o [HCOO~] = 0 mMy i e l d s t h e r e a c t i v a t i o n c o n s t a n t K and
K^ can be c a l c u l a t e d from t h e slope of t h e l i n e . K appears t o
be 78 ± 31 uM bicarbonate and K. = 2.0 ± 0.7 mM formate a t pH
6.5.
5
MH ° i
ƒ
30 s
(305)
/
HC0 3 '
chloroplasts
(75! /
(177) ƒ
/
.^-""""^
0 f
J
Û
Fig. 3.6 ( l e f t ) : Reactivation of the Hill reaction by bicarbonate in C02depleted chloroplasts in the absence (B) and in the presence (A) of formate. Chloroplasts were transferred from the depletion vessel to the reaction medium in the reaction vessel and incubated for 90 s in the dark. Reaction medium as in Fig. 3.8, but in the experiments shown in trace A 10 mM
formate was added.
Fig. 3.7 ( r i g h t ) : Time-course of the reactivation of the Hill reaction in
C0 2 -depleted chloroplasts in the absence of bicarbonate. Reaction media as
in Fig. 3.6. A: +10 mMformate, B: -formate.
In the experiment shown i n Fig. 3.6 C0 2 -depleted c h l o r o p l a s t s were t r a n s f e r r e d t o C0 2 -free r e a c t i o n medium c o n t a i n i n g
e i t h e r 10 mM formate (A) or no formate (B); due t o t r a n s f e r of
formate t o g e t h e r with t h e c h l o r o p l a s t s t h e f i n a l formate conc e n t r a t i o n i n t h e sample i s about 0.8 mMh i g h e r . In the p r e s 39
ence of 10 itiM formate the Hill reaction rate i s about 14 pmol
02/mg Ch'h after 90 s incubation in the reaction medium; subsequent addition of 10 mM bicarbonate results in a nearly complete reactivation of electron flow (trace A). At low formate
concentration a relatively high rate of electron flow i s observed. Addition of bicarbonate s t i l l stimulates electron flow,
but only by a factor of 3 (trace B). I t i s noteworthy that in
the virtual absence of formate and bicarbonate there i s s t i l l
some reversible deactivation of electron flow during illumination (Fig. 3.6B, f i r s t illumination period), which i s not observed when bicarbonate has been added. Figure 3.7 shows the
dependence of the i n i t i a l Hill reaction rate in the presence
of 0.8 or 10.8 mMformate on the dark incubation time in the
reaction medium. At low formate concentration electron flow i s
100
.05
80 "
S .04
O
Dl
E
•e
3
O
-s.
°
W
.03
60 „ o
.02
40
o
e
3-
J
~
n
*
ir
20
.01
0
2
.00
4
6
8
10
1
2
iHCOO"] ImM)
F i g . 3.8 ( l e f t ) : I n h i b i t i o n of the H i l l r e a c t i o n by formate in the a b sence of b i c a r b o n a t e . Reaction medium: 0.3 M s o r b i t o l , 10 mMNaCl, 5mM
MgCl 2 , 50 mM sodium phosphate (pH 6 . 5 ) , 5 mMNH4C1, 0.5 mMFeCy and c h l o r o p l a s t s e q u i v a l e n t to 20 (Jg Chl/ml. A: H i l l r e a c t i o n r a t e as a function
of the formate c o n c e n t r a t i o n ( o ) .
B: r e c i p r o c a l H i l l r e a c t i o n r a t e as a function of the formate concentrat i o n (Dixon p l o t ) ( • ) .
F i g . 3.9 ( r i g h t ) : Double r e c i p r o c a l p l o t of the H i l l r e a c t i o n r a t e as a
function of the b i c a r b o n a t e c o n c e n t r a t i o n . Reaction mixture as in F i g . 3 . 8 ,
except t h a t 10 mM formate was added.
40
reactivated to a finalvalue of95pmol02/mgChi*hwitha
half-time ofapproximately 35s.At10.8mM formatetheHill
reaction remains much lower andeven after3minincubation
theHillreactionrateislessthan20pmol02/mgChl-h.These
experiments suggestthatintheabsenceofbicarbonateandformateelectron flowcanproceed fromQ.toPQ.
Figure3.8 showstheeffectoflowformate concentrations
on theHill reactionofC02-depletedchloroplastsinC02-free
reaction medium. Therelationship between theHill reaction
rate andtheformate concentration appears tobe hyperbolic
(curve A ) ; aplotofthereciprocalHillreactionrateagainst
the formate concentration yields astraight line (curveB ) .
The interceptwith they-axisgivestheHillreactionratein
theabsenceofformateandbicarbonate.TheHillreactionrate
is about 88(jrnol02/mgChi-h andK. inthis experimentwas
3.45mM formate,which ishowevernotsignificantly different
from thevalue forK.givenabove.Figure3.9showsthat C0 2 depleted chloroplasts from thesame batch exhibit aV
of
about 190Mmol02/mgChi-hinthepresence of10mM formate
and saturating amounts ofNaHC0 3 . Itisobvious fromFig.3.8
thatelectron flowcanproceedat[HCO3]<<K and[HCOO~]<<
K., although V
isnotreached. Thereforeweconcludethat
electron flowfromQ.toPQcanproceedwhennobicarbonateis
bound tothehigh-affinitybicarbonatebindingsites;formate
mightberegardedasaninhibitorofPSIIelectronflow.
Discussion
CO2-depletion
of
chloroplasts
Incontrasttoearliermethods (e.g.15,21,22)themodifiedC02-depletionprocedureyieldsthylakoids thatcanevolve
oxygeninexcessof150pmol02/mgChi-hatpH6.5atsaturating bicarbonate concentrations. Figure3.1 shows that these
41
high rates canonlybeobservedafterprolonged incubationof
the chloroplasts in depletion medium atpH 5.8. The rather
slowdevelopmentofthedependenceoftheHillreactiononbicarbonateprobablyreflectsthebindingofformate.Astheaction of formate ispH dependent, i.e.morepronounced atlow
pH (10,14)itcould be thattheprotonatedbase isinvolved
intherate-limitingstepoftheinhibition.
IntheC02-depletedchloroplasts obtainedbythenewprocedure electron flow inthe presence ofsaturatingbicarbonate
concentrations canbestimulated about4-foldby5mMNH4C1or
5 pMGramicidin (notshown)whenFeCyistheelectronacceptor.
Thisstronglysuggeststhatinthesechloroplasts FeCy accepts
electronsafterPQ.
InC02-depleted chloroplasts thathavebeenreactivatedby
incubation with bicarbonate, the pH dependence is different
from that innon-treated chloroplasts (Fig.3.4);atmorealkalinepHelectron flowisonlypoorlyreactivated.Thismight
be due to an irriversible ageingprocessduringonehour de^
pletion at 25°Ccausing another step inelectron flowtobe^
comerate-limiting.
Interaction
of formate and bicarbonate
on the Hill
reaction
Stemler (12)hasdemonstrated theexistenceoftwotypesof
bindingsitesforbicarbonate.Thehigh-affinitybindingsites,
with apool sizeofabout1binding siteper400Chimolecules,
were shown tobeinvolved intheregulationofelectronflow.
Thedissociationconstantofthisbindingsite/bicarbonatecomplex was reported tobe 80pM NaH 1 4 C0 3 atpH6.5 (17),which
ispractically identical tothevalueof78 MM NaHC0 3obtained here forK atpH 6.5 (seeFig. 3.5).Inmyopinionthis
close agreement indicates thattheinitialHillreactionrate
reflects the state of the regulatory high-affinity binding
sites,perhaps attheQ„-protein,withrespecttothebinding
offormateandbicarbonate.Thismeansthatintheexperiments
illustrated in Figs.3.5, 3.6B and 3.7B, alarge fractionof
42
the binding sites must have been empty,i.e.notoccupiedby
either formate orbicarbonate.Therefore Isuggest that the
high-affinity binding sites do not require thatacomplexis
formedwithbicarbonate toallowelectron flowfromQ A toPQ.
However,thequestionwhetherthebindingofbicarbonateto
the regulatory site atthe Qß-protein is arequirement for
electron flow is stillaratherhypothetical one.Theresults
do not exclude apossible involvement ofasmallpoolofbicarbonate-binding siteswithaveryhighaffinity forbicarbonate.Sofarhowever,thereareno indicationsthatsuchapool
exists. Another possibility is that binding of bicarbonate
brings the regulatory site in an "active"stateafterdissociation of the regulatory site-bicarbonate complex. Inthis
wayonlyafewbicarbonatemoleculescouldkeep allregulatory
sitesinthe "active"statewhenno formateispresent.
Ididnotsucceed inachieving atotal restorationofelectronflowintheabsenceofformateandbicarbonate (Figs.3.5
and 3.8).An explanation for this observation is notknown,
obviously further experiments areneededtoclarifytheexact
mechanism offormate/bicarbonate regulationofelectronflow.
InChapter2 the kinetics of the reactivationoftheHill
reactionbybicarbonatehavebeenanalyzed .Theretheassumptionwas made that electron flow fromQ A toPQcanonlyproceed whenbicarbonate isboundtothebindingsite.According
to the present data this assumption might notbe correct.
Those experiments were done, however, in the presence of
100mM formate, a condition atwhich the number of "empty"
binding sites must have been negligible. Therefore the only
conformationoftheregulatory sitethatallowedelectronflow
in those experiments was the one inwhichbicarbonate was
boundtothebindingsite.
Physiological
consequences
tion on electron
flow
of the formate/bicarbonate
interac-
Theresultspresented inthischapterclearly showthatformate andbicarbonatecanregulatelinearelectron flowinCO?43
depleted thylakoids. These C02-depletedchloroplastswereobtained by incubation with formate atpH5.8.This incubation
may seem a ratherunphysiological treatment,butStemler (14)
hasalreadyshownthatC02-depletedchloroplastscanalsobeobtainedbyilluminatingbrokenchloroplasts atpH8inthepresenceofhigh formateconcentrations andanartificial electron
acceptor.
Fig. 3.10: A hypothetical scheme for the interaction betweenphotosyntheticelectron flowand photorespiration.
Formateisametabolite thatisinvolved inseveralmetabolic reactions, among other things breakdown ofglyoxylateby
H 2 0 2 toformateand C0 2 inperoxisomes (3,9)and chloroplasts
(23). Glyoxylate is an intermediate inphotorespiration and
highratesofglyoxylatedecarboxylation havebeenobserved in
isolated peroxisomes (3).Figure3.10 schematically showshow
photorespiration andphotosynthetic electron flowcouldinteract in vivo viathe formateandbicarbonatepools.Underhighlyphotorespiratory conditions, e.g.high lightintensityand
low C0 2 concentration, there is a large carbon flux through
theglycolatepathway. Iftheamountof "free"H 2 0 2 (i.e.H 2 0 2
notbound tocatalase)ishighenough,highratesofglyoxylate
decarboxylation canbeexpected intheperoxisomes (3).Inorder to inhibitphotosynthetic electron flowtheproduced formatewouldhavetoenterthechloroplast.Alternatively glyoxylatecouldenterthechloroplastandreactwithH 2 0 2 ,generated in areaction of0 2 withreduced ferredoxin (11),togive
44
formate and bicarbonate.The bicarbonate formed subsequently
will be remetabolized in the Calvin Cycle and the remaining
formate will inhibit linear electron flow. So,atahigh0 2 /
C0 2 ratio there isadirectpath fromphotosynthetically produced oxygen to formate,whichinhibitslinearelectronflow.
Suchapathwaycouldberegarded asanegative feedbackmechanism that is involved inthe regulation of linear electron
flow.Asinhibitionoflinearelectronflowcanstimulatecyclic electron flow (8),regulation of linearelectronflowby
formate andbicarbonate will result in an altered ratiobetweenlinearandcyclicelectronflow.
Conclusions
Thepresentdatashowthatformate inhibitslinearelectron
flowinisolatedbrokenpeachloroplasts.Bicarbonatecanabolish this inhibition, probablybydisplacing formate fromthe
thylakoidmembrane.Thephysiological significance ofthisregulation of electron flowisstill farfrombeingunderstood.
However, asphotorespiration may be involved as a source of
formate,regulationofphotosyntheticelectron flowby formate
andbicarbonatemayprovide amechanismbywhichphotorespirationmight control photosynthetic efficiency in intactcells
orleaves.
References
Fischer,K.,H.Metzner.1981. Bicarbonateeffectsonphotosyntheticelectrontransport.I.Concentration dependence
and influenceofmanganese reincorporation.Photochem.Photobiophys.2:133-140.
45
2.Good,N.E. 1963.Carbondioxide&theHillreaction.Plant
Physiol.38:298-304.
3.Grodzinski,B. 1979. A study of formate production and
oxidation in leaf peroxisomes during photorespiration.
PlantPhysiol.63:2P9-293.
4. Izawa,S.1962. Stimulatoryeffectofcarbondioxideupon
theHillreactionasobservedwiththeadditionofcarbonic
anhydrase to reactionmixture.Plant& Cell Physiol.3:
221-227.
5.Jursinic,P., J.Warden,Govindjee.1976. Amajorsiteof
bicarbonate effect in system II reaction.Evidence from
ESR signal H v f / fast fluorescence yield changesanddelayedlightemission.Biochim.Biophys.Acta440:322-330.
6.Khanna,R.,Govindjee,T.Wydrzynski.1977. Siteofbicarbonate effect inHill reaction.Evidence fromtheuseof
artificialelectronacceptorsanddonors.Biochim.Biophys.
Acta452:208-214.
7.Lavergne,J. 1982. Mode ofactionof 3-(3,4-dichlorophenyl)-l, 1-dimethyl urea.Evidencethattheinhibitorcompetes with plastoquinone forbinding to acommonsiteon
the acceptor side of photosystem II.Biochim.Biophys.
Acta682:345-353.
8.Mills,J.D.,R.E.Slovack,G.Hind.1978. Cyclicelectron
transportinisolated intactchloroplasts.Further studies
withantimycin.Biochim.Biophys.Acta504:298-309.
9.Oliver,D.J. 1981. Mechanism andcontrolofphoto-respiratory glycolate metabolism: C0 2 lossfrom (theC-2)carbon
ofglycolate. Im Proceedingsofthe5 InternationalCongress on Photosynthesis (G.Akoyunoglou, ed.). Vol.IV.
pp. 501-508,Balaban International ScienceServices,PhiladelphiaPa.
10.Robinson, H.H., J.J. Eaton-Rye, J.J.S. van Rensen,
Govindjee.1984. Theeffectsofbicarbonate depletionand
formateincubationonthekineticsoftheoxidation-reduc46
tionreactionsofthephotosystem IIquinone acceptorcomplex.Z.Naturforsch.39c:382-385.
11. Steiger,H.,BeckE.1981. Formationofhydrogenperoxide
and oxygen dependence ofphotosynthetic C0 2 assimilation
byintactchloroplasts.Plant&CellPhysiol.22:561-576.
12. Stemler,A.1977. Thebindingofbicarbonate ionstowashedchloroplastgrana.Biochim.Biophys.Acta460:511-522.
13. Stemler,A. 1979. A dynamic interaction between thebicarbonate ligand and photosystem II reaction centercomplexes in chloroplasts. Biochim. Biophys. Acata 545:
36-45.
14. Stemler,A. 1980. Forms of dissolved carbondioxiderequired for photosystem II activity in chloroplasts membranes.PlantPhysiol.65:1160-1165.
15. Stemler,A.,Govindjee.1973. Bicarbonate ionasacritical factorinphotosynthetic oxygenevolution.PlantPhysiol.52:119-123.
16. Stemler,A., P.Jursinic. 1983. The effects of carbonic
anhydrase inhibitors formate,bicarbonate,acetazolamide,
and imidazole on photosystem II inmaize chloroplasts.
Arch.Biochem.Biophys.221:227-237.
17. Stemler,A., J.Murphy. 1983. Determinationofthebind14
ing constant ofH C0 3 to the photosystem II complex in
maizechloroplasts:effectofinhibitors andlight.Photochem.Photobiol.,38:701-707.
18.VanRensen,J.J.S., W.vanderVet,W.P.vanVliet.1977.
Inhibition anduncouplingofelectrontransportinisolated chloroplasts by the herbicide 4,6-dinitro-o-cresol.
Photochem.Photobiol.25:579-583.
19.VanRensen,J.J.S.,W.F.J.Vermaas.1981. Actionofbicarbonate andphotosystem IIinhibitingherbicides onelectron
transport inpea grana and in thylakoidsofabluegreen
alga.Physiol.Plant51:106-110.
47
20.Vermaas,W.F.J.,Govindjee.1981. Uniquerole(s)ofcarbon
dioxide and bicarbonate in the photosynthetic electron
transport chain. Proc. Indian Natl. Sei. Acad. B47:
581-214.
21.Vermaas,W.F.J., J.J.S.vanRensen. 1981. Mechanism of
bicarbonate action on photosynthetic electron transport
in broken chloroplasts. Biochim. Biophys. Acta 636:
168-174.
22.Wydrzynski,T., Govindjee. 1975. Anewsiteofbicarbonate effect inphotosystem II ofphotosynthesis:Evidence
from chlorophyll fluorescence transients inspinachchloroplasts.Biochim.Biophys.Acta387:403-408.
23. Zelitch, I.1972. Thephotooxidation ofglyoxylatebyenvelope-free spinach chloroplasts anditsrelationtophotorespiration.Arch.Biochem.Biophys.150:698-707.
48
CHAPTER 4
EFFECTS OF FORMATE ANDBICARBONATEONPHOTOSYNTHETICELECTRON
FLOW INTHEABSENCEAND INTHEPRESENCEOFUNCOUPLER
Introduction
Effectsofformateonphotosynthetic electron flowareknown
forsometime.Isolatedthylakoidsthatareincubated inmedia
containing formate are inhibited in their abilitytoproduce
oxygen in the presenceofanartificial electronacceptor(5,
10, 12,19).Theinhibitory effectofformate iscounteracted
by bicarbonate.As several other anions besides formate are
also inhibitory and asbicarbonate is theonlycompound that
hasbeenshowntorelievethis inhibition (5),ithasbeensuggested thatbinding of bicarbonate to "active sites"on the
thylakoids isarequirement fortheHill reactionin (12).The
stimulating-effect of bicarbonate on the Hill reaction the
presenceofinhibitory saltsisgenerallyknownasthe"bicarbonate-effect"onelectronflow.
The stimulation of theHillreactionbybicarbonate isdependent on the formate concentration (8); formateappearsto
be a competitive inhibitorofboththebinding ofbicarbonate
to thylakoids (13)and the reactivation oftheHill reaction
by bicarbonate in C02-depleted chloroplasts (see Chapter3 ) .
Ithasbeen suggested thatbinding ofbicarbonatemaynotbe
required for electron flow at the acceptor sideofPSII(13,
see also Chapter 3)butthatbicarbonate relieves theinhibitory action of monovalentanionssuchasformate,acetateand
Cl".
InChapter 3 a scheme has beenpresented inwhich formate
acts as anegative feedback inhibitoroflinearelectronflow
at low bicarbonate concentrations.Athigherbicarbonateconcentrations the feedback inhibitionwillbedecreased duetoa
decrease ofphotorespiratory formate production and ahigher
49
reactivation of electron flowby bicarbonate.As formate is
produced duringphotorespiration, inhibitionofelectronflow
in vivo developsduringilluminationandnotinapreviousdark
period.During illumination of isolated non-depleted chloroplasts the Hill reaction rapidly declines inthepresenceof
formate (5,9, 11)and bicarbonate is rather ineffective in
stimulating electron flow in C02-depleted chloroplasts when
added inthelightinthepresenceofformate (9,18).
Inthemodelpresented inChapter3bicarbonate and formate
are suggested to affect the relative yieldsofNADPHandATP
only by inhibition oflinearelectron flow.There arehowever
reports that anions mayuncoupleelectrontransport fromphotophosporylation (4).Someoftheseanions (e.g.citrate,phosphate) areparticularly effective while other ions are only
weak uncouplers (e.g.bicarbonate).Acetateandbutyratealso
uncouple, but electron flowrapidlydeclinesduringillumination (4).This decline is caused by inhibition of electron
flow; acetate and butyrate resemble formate inbeingableto
increase the dependence of the Hill reaction on bicarbonate
(5).Although formatewasnotinvestigatedwithrespecttouncoupling of electron flow (5,6 ) ,it is to beexpected that
formateposesses someuncouplingproperties aswell.
Ifformatewoulduncouple ataconcentration lowerthanthat
required for inhibition oflinearelectron flow,thenegative
feedback mechanism might not function in vivo since ATP is
required togenerateRUBP,thesubstrate forphotorespiration.
Therefore the inhibitory anduncouplingproperties offormate
werecompared inisolatedbrokenchloroplasts.
Materials
and methods
Isolation of pea thylakoids andmeasurementofoxygenevolutionwere performed asdescribed inChapter2.Isolationof
spinachthylakoidswasessentially thesamewiththeexception
thattheisolationmediumwasdifferent:0.4 Msorbitol,10ttiM
50
NaCl, 5 itiM MgCl2, 2 mM Na-ascorbate, 20 mM tricine/NaOH (pH
7.8) and 0.2% (w/v) BSA. Reaction medium used with pea chlorop l a s t s (PRM): 100 mM NaCl, 100 mMNa-formate, 50 mMNa-phosphate (pH 6 . 5 ) , 5 mM NH4C1 and 1 mMFeCy. For spinach t h y l a koids the following r e a c t i o n medium was used (SRM): 0.3 M sorb i t o l , 10 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 2 mMEDTA, 50mM
buffer (MES/KOH or HEPES/KOH). Bicarbonate and formate were
added t o the r e a c t i o n medium were a p p r o p r i a t e .
F i g . 4 . 1 ( l e f t ) : S t i m u l a t i o n of the i n i t i a l H i l l r e a c t i o n r a t e by formate
i n the absence of uncoupler. Reaction medium: SRM (pH 6 . 9 ) , 1 mMFeCy and
spinach c h l o r o p l a s t a t a c o n c e n t r a t i o n of 25 |Jg Chl/ml. Numbers along t h e
t r a c e s i n d i c a t e the formate c o n c e n t r a t i o n ( i n mM) p r e s e n t in the r e a c t i o n
medium.
F i g . 4.2 ( r i g h t ) : I n h i b i t i o n of the H i l l r e a c t i o n by formate i n the p r e s ence of uncoupler. Reaction c o n d i t i o n s as described in F i g . 4 . 1 with t h e
exception t h a t 5 mMNH4C1 was included i n the r e a c t i o n medium. The numbers
along the t r a c e s i n d i c a t e t h e formate c o n c e n t r a t i o n used (mM).
51
Results
Effects
of formate on basal electron
flow
Figure4.1 showstheeffectofincreasing formateconcentrations onbasal electron flow inbroken chloroplasts.At low
formate concentrations (£ 10mM)theHillreactionishardly
affected but atconcentrations of 20mM formate and abovea
significant stimulation is observed. Especially athighformateconcentrations (i 100mM)thestimulationofelectronflow
isonlymaximalduringtheinitialphaseofillumination. Initially proceeding at ahigh rate, theHillreactionprogressively slows down.Thistypeofresponsecanalsobeobserved
in thepresence ofhighacetateconcentrationsbutnotinthe
presence of citrate (datanot shown). Theeffectsofformate
ontheinitialHillreactionrate (asdetermined fromtheinitial slope)are indicated inFig.4.3A. Half-maximalstimulationofelectronflowoccursat~70mMformate.
Effects
of formate on uncoupled electron
flow.
Inthepresence ofanuncoupler formatehasno stimulatory
effectontheinitialHillreactionrate (Fig.4.3B);uncoupled electron flow isonlyinhibitedby formate.The inhibition
is not constant but increases during theilluminationperiod
(Figs.4.2 and 4.4).At ahighformateconcentrationthedecrease of theHill reaction rate appearstobemono-exponential (data not shown). The half-time of the deactivation of
theHillreactionisdependentonseveralparameters,e.g.formateconcentration (Fig.4.2),pH (Fig.4.4),surfacepotential
(15), light intensity (9)and biotype (14).InFig.4.3B the
inititialHillreactionrateinthepresenceofNH4C1 isplotted as a function of the formateconcentration.Half-maximal
inhibition is observed at approx. 20mM formate.Thus itappears that atpH6.9 formateinhibitsuncoupled electron flow
52
at concentrations atwhich hardly a stimulatory (uncoupling)
effectonbasalelectron flowisobserved.
250
c
2
(J
Ol
E
200
°
O
E
150
\
\
B
•
•
•
S 100
£
_^«—
>
_^—-—î"
50
3
A
50
100
,
150
,
200
[HC00"] (mM)
Fig.4.3 (left): EffectsofformateontheinitialHillreactionratein
spinachthylakoidsintheabsence (A)andthepresence(B)ofuncoupler(5
mMNH4C1).ReactionconditionsasdescribedinFigs.4.1 resp.4.2.
Fig.4.4 (right): EffectoftheambientpHontheHillreactioninthe
presence of20mM formate.Reaction conditions asdescribedinFig.4.2
with theexceptionthatHEPES/KOHwasused atpH8.1.Thenumbersalong
thetracesindicatethepHofthereactionmedium.
Effects of bicarbonate on the inhibition
formate during
illumination.
of electron flow by
Afterdark-incubation ofC02-depletedthylakoidswith10mM
bicarbonate the (initial)Hillreactionrateisalmostcompletelyreactivated,eveninthepresenceofhighformateconcentrations (8,18).Figure4.5A showsthatintheabsenceofbicarbonate the Hill reaction rate rapidly declines andthata
subsequent incubation inthedarkintheabsenceofbicarbonate does not result in a reactivation of theHillreaction;
only dark-incubation withbicarbonate results in areactiva-
53
10 >JH 0 ,
30 s
/
t
û
HCO,"
l
B
/
0
0
A
/ -
•
ô
•
/
0
Fig.4.5: Inhibition oftheHill reactionbyformate during prolongedillumination intheabsence (A)andthepresence (B)of10mM bicarbonate.
Reaction medium: PRM (pH6.5),1mMFeCy,5mMNH 4 C1andpeathylakoids
equivalent to25M8Chi/ml.
tion of the Hill reaction. InFig.4.5B asimilarexperiment
was carried out in thepresence of10mMbicarbonate.Itappears that also inthepresenceofbicarbonate theHillreaction becomes inhibited duringillumination.Theinhibitionis
howeverlesssevereandonlyadark-incubation sufficestoreactivate the Hill reaction.This observation isinagreement
withobservationsbyGood (5)andStemler (9)and demonstrates
oncemorethatreactivationofelectronflowbybicarbonateis
only optimal after adark-incubationperiod (seealsoChapter
2). Figure4.6 showsthatthebicarbonateconcentrationrequired forhalf-maximal reactivation of the initial rate of the
Hillreactionisabout1mM.
Thesamebicarbonateconcentrations thatreactivate theinitial Hill reaction rate inC02-depleted chloroplastsaftera
dark-incubation period (see e.g.Fig.4.6),aremuchlesseffective inpreventing theinhibitory actionofformateduring
54
.03
S
r:
u
/
Ol
E
o
-i.O
-0.5
*
/
o
E
3-
^
>?
•
/
.02
/
.01
.00
0.0
0.5
1.0
1.5
2.0
[NaHC03]-1
2.5
Fig. 4.6: Reactivation of the Hill
reaction by bicarbonate. Pea thylakoids were illuminated for 3 min.
in the presence of formate and subsequently reactivated by incubation
in the dark for 2 min. with various
bicarbonate concentrations as described in Fig. 4.5A. The i n i t i a l
Hill reaction rates were determined
from the f i r s t 20s of the Hill reaction and plotted versus the bicarbonate concentration in a double-reciprocal plot. Reaction conditions
as described in Fig. 4.5A.
(mM)" 1
i l l u m i n a t i o n (see e . g . Fig. 4.5B). Inclusion of 5 mM bicarbona t e i n the r e a c t i o n medium, a c o n c e n t r a t i o n which u s u a l l y react i v a t e s the i n i t i a l r a t e of e l e c t r o n flow for about 60-80%,
cannot prevent formate (100 mM) from i n h i b i t i n g e l e c t r o n flow
considerably i n non-depleted c h l o r o p l a s t s (Fig. 4.7C); even i n
t h e presence of 20 mM bicarbonate i n h i b i t i o n can be observed
(Fig. 4.7C). After some time the H i l l r e a c t i o n r a t e reaches a
steady s t a t e . Figure 4.7 shows t h a t the H i l l r e a c t i o n r a t e i n
steady s t a t e i s dependent on the bicarbonate c o n c e n t r a t i o n . As
i n t h e presence of 20 mM bicarbonate and 100 mM formate the
H i l l r e a c t i o n r a t e i s only ~ 13% of t h e i n i t i a l r a t e i n t h e
c o n t r o l without formate (compare F i g s . 7A en B), i t can be concluded t h a t more than 20 mMbicarbonate i s r e q u i r e d t o countera c t s the i n h i b i t o r y e f f e c t of 100 mM formate. This i n d i c a t e s
t h a t i n the l i g h t e i t h e r formate i s a more e f f e c t i v e i n h i b i t o r
of e l e c t r o n flow than i n the dark or a l t e r n a t i v e l y t h a t b i c a r bonate i s l e s s e f f e c t i v e i n s t i m u l a t i n g of e l e c t r o n flow during prolonged i l l u m i n a t i o n .
55
10/JM0,
Fig. 4.7: The kinetics of the Hill reaction inthepresence of formate
and bicarbonate. A: control,-formate,-bicarbonate;B:+ 100mM formate,
+ 20 mM bicarbonate; C:+ 100mM formate,+ 5mM bicarbonate; D:+ 100mM
formate, + 1mM bicarbonate.Reaction medium: 0.3 M sorbitol, 10mM NaCl,
5 mM MgCl 2 , 50mM MES/NaOH (pH6.5),1mM FeCy and pea chloroplasts equivalent to25 Mg'Chi/ml.
Discussion
Uncoupling of electron
flow by formate
Formate isshowntoposessuncouplingproperties (Figs.4.1
and 4.3).The half-maximal stimulationofbasalelectronflow
is observed at approximately 70mM formate atpH 6.9. This
valuemaybeslightlyunderestimated sinceathighformateconcentrationstheconcomittantinhibitoryeffectwillinterfere,
even in the first 20s of the Hill reaction (seeFig.4.3B).
This means that atahighformateconcentrationelectron flow
wouldhaveproceeded fasterintheabsenceofconcurrentinhibition.Theresultofthisinhibitionisthatthemaximalstimulation of theHillreactionrateisobserved atalowerformate concentration. Thus thetruehalf-maximal stimulationof
basal electron flow due to the uncoupling action of formate
56
may occur at formate concentrations higher than 70mM under
theconditionsused.
Thefactthatformateuncoupleselectron flowinthylakoids
indicates that formate does not enter thethylakoid lumenas
formicacid. Ifonlytheprotonated specieswouldbediffusing
into the thylakoid lumen,thiswouldleadtoanacidification
in the lumen.As acidification of the thylakoid lumen slows
down the reoxidation ofPQH 2 (6),inhibitionelectronflowby
formatewouldbeexpected.This isnotthecase (seeFigs. 4.1
and 4.3) and therefore formate must enter the chloroplasts
either as aneutral complex or asananion.Preliminarymeasurementsofthe flash-induced change inthemembranepotential
bymeansoftheP515-absorbance changes,showthatat25°CNaformateenhancesthedissipationofthetransmembranepotential
(J.Snel, unpublished data), indicating that formate moves
acrossthemembrane asanion.
Inhibition
of electron flow by formate
Theexperiments showninFigs.4.2 and4.3 demonstrate that
the initial Hill reaction rate is inhibited by formate.The
initial rate is inhibited for 50%by approx. 80mM formate
(Fig. 4.3).TheHill reaction rate decreases however during
illumination inthepresenceofformate (Fig.4.2) andafter2
min illumination the inhibition ismuchhigherthan50%at80
mM formate (not shown). Several carbonyl compounds (e.g.bicarbonate, dimethyl carbonate)have been shown to stimulate
linear electron flow from PSII toPSI (1).Itwas suggested
thatthesecompounds actbyinhibitingacyclic electron flow
aroundPSII (1).A stimulationofelectron flowby formatewas
never observed in the presence of NH 4 C1.Thisindicatesthat
formateprobablydoesnotinhibitacyclicelectron flowaround
PSII (cf.réf. 2 ) .Obviously formate eitherdoesnotbindto
the bicarbonate-binding site involved ininhibitionofcyclic
electron flowaroundPSII,oritmaybindbutthenwithamuch
57
lower affinity than theaffinity forthebindingsitethatis
involved in inhibition of linear electron flow. Results of
Barr et al. (1)and Bublaugh andGovindjee (2)indicatethat
the bicarbonate-binding site involved ininhibitionofcyclic
electronflowhasalowaffinity forbicarbonate.
The action of formate ismarkedly pHdependent.Thekinetics of the declineoftheHillreactionrate (Fig.4.4)suggestthateither formicacidoraproteinmoietywithapK a of
approx.6.5-6.8isinvolved inthemechanism offormate action
in the light. As the limited time-resolution of the oxygen
electrode prevents aprecisedeterminationofthekineticsof
the formate action at lowpH, adiscriminationbetweenthese
twomechanismshasnotbeenpossible sofar.
Interaction
prolonged
of formate and bicarbonate
illumination.
on electron
flow
during
Figures 4..5 and 4.6 show that after illumination inthe
presence of formate andanelectronacceptorchloroplastsappeartobeinastatethatisvery similartothestateobtainedafterC02-depletioninthedarkatlowpH.TheapparentreactivationconstantK'obtainedbyilluminationofchloroplasts
in thepresence of formate appears to be approx. 1mM (Fig.
4.6), which is identicaltothevalueobtained forK'in C0 2 depletedchloroplasts (8,16;seealsoChapter2)andthedissociation constant K,forbicarbonate inthepresenceofformate (13),both in the presence of 100mM formate.Themain
difference between the two C02-depletion methods is that
electron flow in the presence of saturatingbicarbonateconcentrations ismuch lowerin chloroplastsdepleted ofC0 2 as
described ine.g.Chapter2.
Ithas been reported thatbicarbonate isnotabletostimulateelectron flowwhenadded inthelightinthepresenceof
high formate concentrations (9,18).Figure4.7 showsthatin
thepresenceof100mM formatethepresenceofbicarbonatehas
58
a beneficial effect on the rate ofelectron flowduringprolonged illumination. In thepresenceof20mMbicarbonate and
100mM formate theHill reaction rateinthesteadystateis
about13%oftheinitial rateincontrol chloroplasts (compare
Figs. 4.7A and B). This means thatinthepresenceof100mM
formate more than 20mM bicarbonate is required to maintain
electron flowinsteady stateatahalf-maximal rate.Assuming
thattheeffectsofformateand electron flowarereversible,
which is indicated ine.g. Fig. 4.5B, theapparentreactivation constant K' can be calculated fromtheMichaelisMenten
equation (rewritten):
1- Ï
v
Kl= [A]- — — 5 ^ :20- 1 ~ 0 - 1 3 =134mMbicarbonate
J
0.13
max
(seeChapter2 foradescriptionofparameters)
This value of 134mM may beanoverestimationsincetheHill
reactioninthecontrolchloroplasts (Fig.4.7A) alsoseemsto
decline slighthly. But nevertheless it is clear thatK' in
steadystateinthelightismorethanamagnitudehigherthan
in the dark. StemlerandMurphy (13)havereportedeffectsof
lightonthebindingofbicarbonate. Intheabsenceofformate
only a small effectwas found butinthepresenceofformate
(20mM) a3-foldincrease inthedissociationconstantK,was
measured.Thislight-effectonK,ismuchlowerthanthatonK'
d
as estimated above,butinthebinding experiments thelightintensitywasrather low (5W/m 2 )andnoelectronacceptorwas
present (13).Illuminationwithsaturating lightinthepresenceofanelectronacceptor andhigh formateconcentrations are
conditions thatpromote the deactivation of electron flowby
formate (9;J. Snel,unpublished data).Therefore itistobe
expected thatundersuchconditions theeffectoflightonthe
bindingofbicarbonate ismuch largerthanthe factor3reported by Stemler andMurphy (13).Intheabsenceofformateonly
a small effect oflightonthebindingofbicarbonatewasde-
r
59
tected (13).Thusthemajoreffectoflightmustbeonthebindingofformate,i.e. formatebindsmuchtightertoitsbinding
site inthe light.Usingthedata fromFig.4.5 inref.13,I
havecalculated theK.offormatetobeabout5mMinthedark.
InChapter 3K. has been determined to be 2-3mM formatein
the dark. Thus K.and K. in the lightmightbesmallerthan
0.5 resp.0.2mMformate.
The results of these calculations are consistentwiththe
observationbyVermaasandVanRensen (18)thatformateconcentrations as low as 0.1 mM have a significant effect on the
stimulationofelectron flowbybicarbonate inthelight.From
these data K was estimated to beabout1pMHCO3 (18);this
estimation was based on the assumption thatK.wasabout100
mM formate in the dark. The evidence available now (13;see
also Chapter 3 andthisChapter)stronglysuggeststhatK- is
more than anorder ofmagnitude lower.Usingthenewvalues,
the same calculation yields aK of~20-150 \JMbicarbonate,
which ismore inagreementwithexperimental data (13,3;see
alsoChapter 3 ) .
Conclusions
The data presented in this chapter indicate that formate
inhibitselectron flow.Itisestimated thatinthelightformatecaninhibitlinearelectron flowatsub-mM concentrations
atpH6.5. Formatecanalsostimulatebasalelectron flow,probably by dissipating theprotongradientacrossthe thylakoid
membrane. This effect occurs at formate concentrations above
10mM.Therefore itisconcluded thatformatecaninhibitelectronflowatconcentrations thathardlyuncoupleelectronflow
fromphotophosporylation.Bicarbonatecancounteracttheinhibitoryactionofformatebutnottheuncouplingeffectsofformateasbicarbonate itselfalsohasuncouplingproperties(4).
60
References
Barr,R.,R.Melhem,A.L.Lezotte,F.L.Crane.1980.
Stimulation of electron transport from photosystem IIto
photosystem Iin spinach chloroplasts. J.Bioenerg.Biomembr.12:197-203.
Blubaugh,D.J., Govindjee. 1984. Comparison ofbicarbonate effects on thevariablechlorophyll afluorescenceof
C02-depleted and non-C02-depleted thylakoids inthepresenceofdiuron.Z.Naturforsch.39c:378-381.
Fischer,K., H.Metzner. 1981. Bicarbonate effects on
photosynthetic electron transport. I. Concentrationdependence and influence ofmanganese incorporation.Photochem.Photobiophys.2:133-140.
Good,N.E. 1962. Uncoupling of the Hill reaction from
photophosphorylation by anions. Arch. Biochem.Biophys.
96:653-661.
Good,N.E. 1963. Carbon dioxide & the Hill reaction.
PlantPhysiol.38:298-304.
Good,N.E., S. Izawa, G.Hind. 1966. Uncoupling and
energy transfer inhibition in photophosphorylation. Im
Current Topics inBioenergetics (D.R.Sanadi,ed.),Vol.
I,pp.59-76,AcademicPress,NewYork.
Huber,H.L., B.Rumberg. 1977. Determination ofpotentials and fixed charges and their light-induced changes
on the inner surface of the thylakoidmembrane. In: Photosynthesis I. Photophysical Processes -Membrane Energization (G.Akoyunoglou ed.), pp. 419-429, Balaban International Science Services,Philadelphia,Pa.
Khanna,R., Govindjee,T.Wydrzynski. 1977. Site ofbicarbonate effect inHill reaction. Evidence fromtheuse
of artifial electron acceptors and donors.Biochim.Biophys.Acta462:208-214.
61
9.Stemler,Ä. 1979. A dynamic interaction between thebicarbonate ligand andphotosystem II reaction centercomplexes in chloroplasts. Biochim. Biophys. Acta 545:
36-45.
10. Stemler,A. 1980. Inhibition ofphotosystem IIby formate. Possible evidence for a direct roleofbicarbonate
in photosynthetic oxygen evolution. Biochim. Biophys.
Acta593:103-112.
11. Stemler,A. 1980. Forms of dissolved carbondioxiderequired for photosystem II activity in chloroplasts membranes.PlantPhysiol.65:1160-1165.
12. Stemler,A., Govindjee. 1973. Bicarbonate ion as acritical factor in photosynthetic oxygen evolution. Plant
Physiol.52:119-123.
13.Stemler,A., J.Murphy. 1983. Determinationofthebinding constant ofH 14 C0a to the photosystem II complex in
maize chloroplasts:Effects ofinhibitorsand light.Photochem.Photobiol.38:701-707.
14.VanRensen,J.J.S. 1984. Interaction of photosystem II
herbicides with bicarbonate and formate in theireffects
on photosynthetic electron flow. Z. Naturforsch. 39c:
374-377.
15.VanRensen,J.J.S, J.H.Hobé, R.M.Werner, J.F.H. Snel.
1984. Reactivation of electrontransportinC02-depleted
chloroplasts by bicarbonate is influenced by thylakoid
surfacepotential. In: Advances inPhotosynthesisResearch
(C.Sybesma, ed.). Vol. I, M.Nijhoff/Dr.w.Junk Publishers,TheHague.
16.VanRensen, J.J.S., W.F.J.Vermaas. 1981. Action ofbicarbonate and photosystem II inhibiting herbicides on
electron transport in pea grana and in thylakoids ofa
blue-green alga.Physiol.Plant.51:106-110.
17.Vermaas,W.F.J., Govindjee. 1982. Bicarbonateorcarbondioxide as a reguirementforefficientelectron transport
62
on the acceptor side ofphotosystem II. In: Photosynthesis (Govindjee, ed.),Vol. II pp. 541-558, Academic
Press,NewYork.
18.Vermaas, W.F.J., J.J.S.vanRensen, 1981. Mechanism of
bicarbonate action on photosynthetic electron transport
in broken chloroplasts. Biochim. Biophys. Acta 636:
168-174.
19.Wydrzynski,T., Govindjee. 1975. Anewsiteofbicarbonate effect inphotosystem II ofphotosynthesis:Evidence
from chlorophyll fluorescence transients inspinachchloroplasts.Biochim.Biophys.Acta387:403-408.
63
CHAPTER5
STIMULATION OF THE FLASH-INDUCED TRANSMEMBRANE POTENTIAL IN
CO2-DEPLETEDCHLOROPLASTSBYADDITIONOFBICARBONATE
Introduction
InChapter3amodelhasbeenproposed thatdescribesapossible regulation of electron flowby formateandbicarbonate
in vivo. InChapter4someoftheeffectsofformateandbicarbonate on electron flow in isolated brokenchloroplastshave
been characterized inmore detail. Demonstration of formate
and bicarbonate effects on electron flow infunctionallyintact systems (e.g. intact chloroplasts, algae or leaves) in
situ requires anon-invasive technique.Twooptical techniques
useful for measuring parametersofelectron flow in situ are:
chlorophyll a fluorescence and electrochromic absorption
changes. Both have been used to studybicarbonate effectson
electron flowinisolatedbrokenchloroplasts.
P5j5~absorbance
chances
Thismethodhasbeenapplied so farinonlytwocasestomeasure the effects of bicarbonate on electron flow inchloroplasts. Jursinic and Stemler (7)have measured theeffectof
bicarbonateonthe flash-inducedP 5 1 5 response inC02-depleted
brokenchloroplasts andGarabetal. (3)havereported effects
ofC0 2 ontheflash-inducedP 5 1 5 response inboth chloroplasts
and leaves.Transmembrane charge separations inPSIandPSII
result in the formationofatransmembranepotential.Theresultingelectrical field issensedbyspecialpigments,mainly
carotenoids andchlorophylls (5).The flash-induced electrical
fieldcauses ared shiftoftheabsorptionbands;light-inducedabsorbancechanges at515nmappeartobelinearlycorrelatedwiththetransmembranepotential (1,5,6 ) .Theflash-induc-
64
ed absorbance change at 515nm (P515~response)inthylakoids
ischaracterizedbycomplexkinetics.Atleast5differentkinetic components canbeobserved inbrokenandintactchloroplasts (10).After a fast rise,which takes less than20ns
(e.g. 5, 6 ) ,a slower rise intheP 5 1 5 response isoftenobserved. This slow risehasarisetimevarying fromseveralms
(1, 5, 6)toseveral tensofms (14).Aftertheslowrisethe
p,1,.responsedecays toitsdarkvaluewithtriphasic kinetics
with life-times of~ 100ms, 1000msand>1s (10,14).The
different components have been named phase a (initial fast
rise), phase b (slow rise1) and thedecayphasesc',candd
(9). Although theinterpretationofseveralcomponents,notablyphasesb,candd, isstillamatterofdiscussion (seee.g.
reviews by Cramer and Crofts (1),Junge and Jackson (6)and
Vredenbergetal. (14)), theassumptionthatphase aisalinear indicator of the transmembrane potential hasbeenwidely
accepted.
Materials
and Methods
Thylakoids were isolated from pea leaves as described in
Chapter 2 and stored at-80°C.Beforeusethethylakoidswere
thawed andstoredoniceinthedark.Flash-induced absorbance
changeswere measured usingamodifiedAminco-Chancespectrofotometer in the double beam mode.Byremovaloftheoptical
chopper,DC-operation was possible.Themeasuring beamswere
positioned next to eachothergiving ameasuring areaof 1.75
x 1cm2; theopticalpathlengthwas1cmandtheintensityof
themeasuring lightwas7jjW/cm2at518nm.Theopticaldispersionwas14nm.Measuring lightwasprovidedby atungstenlamp
1
In this chapter phase b will be used todescribe the slow rise of the
P,.,responsewithout substraction of any other component.
65
connected to a stabilized power supply (Oltronics B32-20R).
Saturating actinic flashes were generated by twoXenon flash
tubes (General Electric FT-230)connected toa8pFcapacitor
givingaflashdurationof8psathalf-intensity;asmalltail
of less than 1% of the totalenergywashoweverextendingto
about 150 M S . The light reached the sample via aSchottRG630/ RG-645 filter combination and a glass-fiberlightguide.
The photomultiplier (power supply: Oltronics A2,5k-10HR)was
shielded fromtheactinic lightby9mmBG-39 filters (Schott).
The photomultiplier signal was amplified by aTektronix 5A22
differential amplifieraftercompensating thedarksignalwith
anoffsetvoltage.Furtherprocessingofthesignalandgenerationofthetriggers forthe flasheswascarriedoutbyaminicomputer (Minc-11/23,Digital EquipmentCorporation)equipped with the following modules:MNCAG (preamplifier),MNCAD
(AD converter), MNCAA (DA converter), 2 x MNCKW (real-time
clock)andMNCDO (triggering).A fastMNCADprocessing routine
with a maximal sampling-frequency of 6 kHz was written by
G.H.vanEck, Computer Centre,AgriculturalUniversity,Wageningen. The electrical bandwithoftheamplifierwas adjusted
to the sampling-frequency. Unless indicated otherwise the
measurementswerecarried outat3°Candsignalswereaveraged
atarepetitionrateof0.1Hz.
Thylakoids were depleted ofC0 2 bypre-illuminationofthe
sample with continuous red light for 1or 2min.informate
containing reaction medium at 0°C in the measuring cuvette.
Illumination was provided by a laboratory-built lamphouse
equippedwitha250W tungsten lamp.Red lightwasobtainedby
including a3mmRG-630 filterinthelight-path;the incident
lightintensitywasapprox.150W/m2.
66
Results
P
515
res onse
*D control
P
chloroplasts
-tin
>L
.o«L
150 ns
A: c o n t r o l
vW.V
—
B: +GHCD
T
t
T
t
B: C0 B -depleted
-AI/I
5x1CT*
30 DS
• ""-'^V:::^,:.:,,^/^ , ^ y .
C: +GHC0
_|v^_N—k—N~
t
T
t
f
't
Figure5.1(left): Flash-induced absorbance changes (AI/I)at518nmin
pea thylakoids.Reactionmedium:0.3Msorbitol,10mMNaCl,5mMMgCl 2 ,
20mMNa-formate,25mMHEPES/K0H(pH7.2).Furtheradditionsare:0.25mM
BQandthechlorophyllconcentrationwas25jJgChl/ml.A:control;B:same
sampleafteradditionof1|JMgramicidine;C:samesamplemeasuredwith
higher time resolution.IntracesAandB16signalswereaveragedata
repetition rateof0.1HzandintraceC36.Thearrowsindicatethe time
atwhichtheactinic flashesweregiven.IntraceCtheelectricalbandwith(-3dB)wasincreasedfrom100Hzto1kHz.
Figure5.2(right): EffectsofC02-depletionandadditionofbicarbonate
ontheflash-inducedM518atpH6.3.Thereactionmediumwasessentially
thesameasinFig.5.1but
25mMMES/KOH(pH6.3)wasusedasabuffer.
Furtheradditions are:5mMNH4C1,1mMFeCyand[Chi]=40|jgChl/ml.A:
chloroplastsnotdepletedofC0 2 ;B:sampleApre-illuminatedfor2minat
0°C;C:sampleBafter2mindark-incubationwith10
mMNaHC03.Forthese
experiments16measurementswereaveragedatarepetitionrateof0.1Hz;
theelectricalbandwithwas1kHz.
67
Figure 5.IA shows the flash-induced P 5 1 5 response inpea
thylakoids that have not been depleted of C0 2 . The signal
clearly contains the components a and c'.Inthepresenceof
gramicidin (Fig.5.IB)acomponentremainsvisiblewithanunresolved risetimeoflessthan7.5msandabiphasicdecaywith
anoverallhalf-time of~460ms.Theslowestcomponentisprobably identical to phase d in intactchloroplasts (7,9, 13)
as it is also observed at\>535nmandasthehalf-lifeof
this slower component islargerthan1s. Inpreviousexperimentswithbrokenchloroplastsphasedhasbeenreported tobe
negligably small (8,10)butthisappearstobeonlythecase
inmedia of relatively high ionic strength intheabsenceof
anelectron acceptor (datanotshown). InthepresenceofMV,
FeCy or BQ theamplitudeofthegramicidin insensitive signal
wasabout8-25%ofthetotal signal,dependingonexperimental
conditions. Figure 1C showsthegramicidin insensitive signal
athigher time resolution;therisetimeislessthan1.5ms.
Thehalf-lifeofthe fastdecaycomponentis~50ms.
Pcjß response in COz-depleted.
chloroplasts
Figure 5.2 shows the effects of C02-depletionontheP 5 1 5
response inbrokenchloroplastsbeforeandafterreadditionof
bicarbonate.After2minpreillumination inthepresenceof20
mM formate theP 5 1 5 response is drastically altered (B).The
magnitude ofphaseaisdiminishedby~ 50%inthe firstflash
compared to the control (A)and even to a greaterextentin
subsequent flashes.This ispartly due toacombinationofa
faster decay of thesignal andtheuseofanelectricalbandwith of 1kHz;at 3 kHz the magnitude ofphase awas ~10%
higher (data not shown). After incubation ofthesamplewith
10mMbicarbonate astimulationoftheP 5 1 5 response isobservedinthesecond and following flashes.Qualitatively thesame
results were obtained atpH 6.1 (data not shown) andpH 5.7
(Fig.5.3).AtpH5.7phasedisclearlypresent inthesignal
(Fig.5.3).
68
•L
•L
^~. -~
A: CO z -depleted
B: CO-depleted
_f- k k~-J—
C: + HCO,"
_Jv__k—J- k_
D: C0 2 -depleted
~J
f
t
T
f~-~J*~
t
r
t
T
t
T
Figure 5.3 ( l e f t ) : Effect of C 0 2 - d e p l e t i o n and a d d i t i o n of b i c a r b o n a t e
and gramicidin on AA518 a t pH 5 . 7 . Reaction medium as i n F i g . 5.2 with
omission of NH4C1. A: non-depleted c h l o r o p l a s t s ; B: sample A a f t e r 1 min
i l l u m i n a t i o n a t 0°C; C: sample B a f t e r 2 min d a r k - i n c u b a t i o n with 10mM
NaHC03 D: sample C a f t e r a d d i t i o n of 2.5 |JM g r a m i c i d i n . Other c o n d i t i o n s
as i n F i g . 5 . 2 .
F i g u r e 5.4 ( r i g h t ) : Effect of b i c a r b o n a t e on t h e gramicidin i n s e n s i t i v e
absorbance changes a t 518 nm (A, B and C) and 535 nm (D, E and F ) . Reaction
medium as in F i g . 5 . 3 . The c h l o r o p h y l l c o n c e n t r a t i o n was 25 Ug Chl/ml and
0.5 mMBQ was used as an e l e c t r o n a c c e p t o r . A(D): c h l o r o p l a s t s i l l u m i n a t e d
for 2 min a t 0°C; B(E) : Sample A(D) a f t e r a d d i t i o n of 1 uM g r a m i c i d i n e ; C:
Sample B(E) a f t e r about 3 min d a r k - i n c u b a t i o n with 10 mMNaHC03. The e l e c t r i c a l bandwith was 100 Hz in t h e s e experiments; 16 measurements were
averaged a t a r e p e t i t i o n r a t e of 0.1 Hz.
The e f f e c t s of C0 2 -depletion and subsequent r e a d d i t i o n of
bicarbonate on P 5 1 5 response are dependent on the formate conc e n t r a t i o n , t h e ambient pH and t h e p r e i l l u m i n a t i o n time. F i g ure 5.4A shows the Pcjc response in thylakoids depleted of C02
by 2 min p r e i l l u m i n a t i o n a t pH 5 . 7 . In t h e presence of 2 uM
gramicidin a s i g n a l remains (B) which i s influenced by t h e
a d d i t i o n of bicarbonate (C).
69
Thesameeffectswereobserved at535nm, althoughthegramicidinsensitivepartoftheresponseisofcoursemuchsmaller at this wavelength (Figs.5.4A, Een F ) . Thismeansthat
the gramicidin insensitive signal has a spectrum thatbears
moreresemblencetothespectrum oflightscattering (14)than
to the spectrum of theP 5 1 5 response (1,5,6 ) .Thedecayof
thegramicidin insensitive signal atpH5.7 inthepresenceof
formate and bicarbonate contains2componentswithhalf-lives
of~ 50ms and > Is. Figure 5.4 clearly showsthatthefast
componentofthegramicidin insensitive signal (tv$~ 50-60ms)
in C02-depleted chloroplasts appears tobe suppressed inthe
second and subsequent flashes in the absenceofbicarbonate.
It seems that this fast component is saturated by thefirst
flashandreverseswithadecaywhichisslowerintheabsence
than inthepresence ofbicarbonate. Thiscauses anapparent
inhibitioninthesecond andsubsequentflashesintheabsence
ofbicarbonate.After incubationwithbicarbonate thekinetics
of the gramicidin insensitive signal appeartobethesameas
innon-depleted chloroplasts (seee.g. Fig. 5.IB).
Discussion
Prj5 response in CO2-depleted
chloroplasts
The hypothesis that in C02-depleted chloroplasts electron
flowisinhibitedbetweenQ B andPQimplicatesthatinC02-depleteddark-adapted chloroplastsPSIIcanmake3turnovers(4).
Thishasbeenobserved inseveralcases (2,4 ) .SincebothPSI
and PSII contribute to the transmembrane potential,thefast
riseofthePc, 5 response iscomposed ofthemoreorlessequal
contributions of PSI and PSII (5).Ifthe flow ofelectrons
outofPSII isinhibited andtheintersystem electrontransport
components are in afullyoxidized state,PSIcanmakeonly1
turnover,leavingtheprimarydonorP 7 0 0 intheoxidizedstate:
P-QQ. Incase of amore reducedelectrontransportchainthe
70
contributionofPSItotheP 5 1 5 responseislessclear.ThereforethecontributionofPSItotheP 5 1 5 response inFigs. 5.2B
and5.3B isratheruncertain,especially inthesecond andsubsequent flashes. In Figs. 5.2B and 5.3B the amplitude after
thesecond flashisalready inhibitedwithrespecttothefirst
flash indicating that eitherP ? 0 0 isnot fullyreducedinthe
dark time between the flashes (250ms)and/oraconsiderable
amount of PSII centers are in one of the states [PCQO'^Ä^'
[P 680 'Q^] or [P6 80 -Q A ]- Since the reduction ofP 6 g 0 by the
watersplittingsystem isnotstrongly inhibitedbyC02-depletion (12)andsincetheequilibrium Qä'Q B*Qa'^Bm a ^^ e s h -i f t _
ed slightly to the left in theintheabsenceofbicarbonate
(12), thereduced amplitudecausedbythesecond flashisprobably aconsequence of thepresence of the state
(^(.on'Ol]
and perhaps an incomplete reduction ofP 700 - From Figs. 5.4B
andEitisclearthatthereducedamplitude inthesecondand
following flashesispartlycausedbysuppressionofthegramicidin insensitive signal.Thehalf-timeofelectron flowfrom
PSIItoPSI isslowed downto100-200ms inC02-depletedchloroplasts, asjudged fromP 7 0 0 reductionkinetics (11).Theprogressive inhibitionoftheP 5 1 5 response inC02-depletedchloroplasts in successive flashes given at astateof4Hzalso
points to an overallhalf-timeofelectron flowof200-250ms
(Figs.5.2B and5.3B, seealsoref.7 ) .
Inthe presence ofbicarbonate the amplitude of the fast
rise of theP 5 1 5 responseremains approximately constantata
flash rate of4Hz (Figs.5.2C and 5.3C), althoughtheamplitude of the 1st flash is always somewhathigherthanthatin
subsequent flashes.But even inthe presence of bicarbonate
theamplitude isonly~ 50%ofthevalueinnon-depleted chloroplasts (Figs.5.2 and5.3).Thisisinagreementwithresults
ofJursinic andStemler (7).Intheirexperiments onlythecontribution ofPSII tothePcic responsewasmeasured;theP 51 c
responsewasirreversibly reducedby~50%afterC02-depletion
ofthethylakoids.Therearebasically twoexplanations;either
theelectrical fieldisdiminishedortheP,.,cpigmentpoolis
71
less sensitive to fieldchanges.Thisreducedsensitivitymay
be caused by areduced sensitivity ofallthepigmentsoralternatively anumber ofP 5 1 5 pigments havebeen inactivated.
Themagnitude of the flash-induced transmembranepotential is
determinedbythenumberof"open"reactioncentersandbythe
membrane capacitance (4,5 ) .Several factors might influence
thenumberofreactioncentersthatparticipate inthegeneration of a transmembrane charge separation, e.g. absorption
crosssection,conversionofPSIIcenters toanon-electrogenic
form and destruction of reaction centers byphotoinhibition.
The electrical field isaffectedbychanges inphysicalparametersofthemembrane suchasthedielectricconstantandthe
thicknessofdielectriccore.TheresponseoftheP 5 1 5pigment
to an applied electrical field isnotlinearbutpseudolinear
(5,6 ) .A loweringofthelocalstaticelectrical fieldby e.g.
a lowering of asurfacepotential differencemightreducethe
magnitude of the P5-i5response.The ioniccompositionofthe
reaction medium appears tobe important with respect to the
amplitudeoftheP 5 1 5 response;astimulationofthisamplitude in low-salt conditions has been observed (R.L.A.Peters,
O.vanKooten, unpublished results). Thismightindicatethat
C02-depletion lowers the surface potential difference across
the thylakoid membrane.Another explanation may be found by
the report that a low pH in the thylakoid lumen appears to
cause oxidation of some carotenoids (6).It is however not
clear whether these carotenoids areinvolved intheP 5 1 5 response.
Gramicidin insensitive
absorbance
changes
Theabsorbance changesobserved inthepresenceofgramicidin have the sameamplitudeandkinetics at518nmand535nm
(see Fig. 5.4).This suggests that these absorbance changes
may be identical totheabsorbance changesthathavebeenreferred toasreaction III (14).Thegramicidin insensitivesig72
nalobservedherehowevercontains2kineticcomponents incontrastwith reaction III.Bothcomponents arecharacterizedby
a (unresolved)risetime of < 1.5 ms.A fastcomponentdecays
with ahalf-time of~ 50-60mswhiletheslowdecay component
hasat, >I s . Accordingtopublished data (14 )thefastcomponentapparently isabsentoralternativelythedecayisslower inintact chloroplasts and leaves.The fastcomponentappearstobeinhibitedbybicarbonatedepletion (Figs.5.4B and
E)andcanbereactivatedbyincubationwithbicarbonate (Figs.
5.4C and F ) .Vredenberg et al. (14)haveshownthatreaction
IIIcanonlybeobserved inthe firsttwo flashes inthepresence ofDCMU;moreoverreaction IIIshowsanoscillatorypattern; at least one component has aperiodicity of 2 (14,U.
Schreiber,unpublished data).Theseobservations indicatethat
reaction IIIcanprobablybeassociatedwithPSIIactivity(23).
The gramicidin insensitive signal observed here probablyhas
the same spectrum asreaction IIIandismoreoveraffectedby
formate and bicarbonate. Therefore these absorbance changes
probably arealsoassociatedwithPSII.Comparisonofthekineticsofthe fastcomponentofthegramicidin insensitive signal
(t*s~50-60ms)withpublished data forotherreactions reveals
thatthefastcomponentmightbecorrelatedwithprotonuptake
attheacceptorsideofPSII,whichoccurswithahalf-timeof
approx. 60ms (9).Obviously furtherexperiments arerequired
to see whether this fastcomponent indeed can be correlated
withprotonuptake attheacceptor sideofPSII.
Conclusions
C02-depletion of thylakoids appearstodiminishtheflashinduced absorbancechangesat518nm,theP 5 1 5 response,using
multiple flashesgivenatahighrate,e.g.4Hz.Partofthis
diminution is reversible, i.e.canberelievedbybicarbonate
addition.However ~ 50%of theP 5 1 5 response is irreversibly
diminished, i.e.cannotberestoredbyadditionofbicarbonate.
73
C02-depletionalsoaffectsgramicidin insensitive absorbance
changesinthe500-550nmregion. Intheabsenceofbicarbonate
theseflash-induced changesarepartlyinhibited.Thispartis
restoredbyadditionofbicarbonate.These flash-inducedabsorbance changes show a spectrum that differs fromthespectrum
ofthe flash-inducedP515-response.
References
1.Cramer, W.A., A.R. Crofts. 1982. Electron and proton
transport. In: Photosynthesis (Govindjee, ed.),vol. I,
pp.387-467,AcademicPress Inc.,NewYork.
2.Farineau, J., P. Mathis. 1983. Effect ofbicarbonate on
electron transfer between quinones inphotosystem II. In:
The Oxygen-evolving system of Plant Photosynthesis
(Y. Inoue,ed.), AcademicPress,NewYork.
3.Garab, G., A.A. Sanchez Burgos, L. Zimànyi, A. FaludiDàniell.1983. EffectofC0 2 ontheenergizationofthylakoidsinleavesofhigherplants.FEBSlett.154:323-327.
4.Govindjee, M.P.J. Pulles, R. Govindjee, H.J. van Gorkom,
L.M.N. Duysens. 1976. Inhibition of the reoxidation of
thesecondaryelectronacceptorofphotosystem IIbybicarbonatedepletion.Biochim.Biophys.Acta449:602-605.
5.Junge, W. 1977. Membrane potentials in photosynthesis.
Ann.Rev.PlantPhysiol.28:503-536.
6.Junge,W.,J.B.Jackson.1982.Thedevelopmentofelectrochemical potential gradients across photosynthetic membranes. In: Photosynthesis (Govindjee, ed.),Vol. I,pp.
589-646,AcademicPress Inc.,NewYork.
7.Jursinic,P. A. Stemler. 1982. A secondsrangecomponent
of the reoxidation of the photosystem IIacceptor,Q.Effects of bicarbonate depletion in chloroplasts. Biochim.
Biophys.Acta681:491-428.
74
8.Peters,R.L.A.,M.Bossen,O.vanKooten,W.J.Vredenberg.
1983. On the correlation between the activityofATP-hydrolase andthekineticsofthe flash-inducedP 5 1 5electrochromic bandshift in spinach chloroplasts. J. Bioenerg.
Biomembr.15:335-346.
9.Polie,A.,W.Junge.1984. Diffusionbarriers forprotons
at the external surface of thylakoids. In: Advances in
Photosynthesis Research (C. Sybesma, ed.), Vol. II,
M.Nijhoff/Dr.W.JunkPublishers,TheHague.
10. Schapendonk,A.H.CM.,W.J.Vredenberg,W.J.M.Tonk.1979.
Studiesonthekineticsofthe515nmabsorbance changesin
chloroplasts. Evidence for the induction of a fast and a
slow P 5 1 5 response upon saturation light flashes. FEBS
lett.100:325-330.
11. Siggel,U.,R.Khanna,G.Renger,Govindjee.1977. Investigation of the absorption changes of the plastoquinone
system in broken chloroplasts. The effect of bicarbonate
depletion.Biochim.Biophys.Acta462:196-207.
12.Vermaas, W.F.J. 1984. The interaction of quinones,herbicides and bicarbonate with theirbinding environmentat
the acceptor side of photosystem II in photosynthesis.
Dissertation, Agricultural University, Wageningen, The
Netherlands.
13.Vermaas,W.F.J., Govindjee. 1982. Bicarbonate effects on
chlorophyll fluorescence transients in the presence and
theabsenceofdiuron.Biochim.Biophys.Acta680:202-209.
14.Vredenberg, W.J., O. van Kooten, R.L.A. Peters. 1984.
Electrical eventsandP 5 1 5 response inthylakoidmembranes.
In: Advances inPhotosynthesisResearch (C.Sybesma, ed.).
Vol II,M.Nijhoff/dr.W.Junk,Publishers,TheHague.
75
CHAPTER6
EFFECTS OF BICARBONATE ANDFORMATEONPHOTOSYNTHETICELECTRON
FLOW INISOLATED INTACTCHLOROPLASTS
Introduction
Severalproposalshavebeenmadewithrespecttoapossible
roleofbicarbonate and formate intheregulationofphotosynthetic electron flow in vivo. Radmer and Ollinger (24)have
suggested thatregulationoflinearelectron flowbybicarbonatewill also affectcyclicelectronflow,becauselinearand
cyclic electron flow share apart of the electron transport
chain (16).This meansthattheNADPH/ATP ratiowillbelower
at sub-saturating bicarbonate concentrations.Vermaas andVan
Rensen (36)suggested thatinhibitionoflinearelectronflow
at lowbicarbonate concentrations mightprevent overreduction
of the electron transportchainandthebuild-up ofasurplus
ofreducingequivalents (e.g.NADPH,NADH)whichmightdisturb
cellmetabolism. InChapter3evidence isgiventhatbicarbonatemay notbe required for electron flow from Q A toPQbut
thatbicarbonate onlycounteractstheinhibitory actionofformate. As formate canbe generated duringphotorespiration,a
model was proposed that links regulation ofelectron flowto
photorespiration via the bicarbonate and formatepools inthe
chloroplasts.
The models mentioned above were however entirelybasedon
experimentscarriedoutwith isolatedbrokenchloroplasts.Demonstration of the existenceofabicarbonate-effectonelectron flow in vivo isacomplicatedmatter (seethediscussion
section). Therefore demonstration of abicarbonate-effect on
electron flowinisolated intactchloroplasts seemed tobethe
mostlogicalstep.
There arebasically twomethods to inducea"bicarbonateeffect"inbrokenchloroplasts.Thesetwomethods are (i)prolongeddark-incubationofthechloroplastswith formate atlow
76
pH (35,41andChapter3)and (ii)illuminationofchloroplasts
for several minutes inthepresenceofformateandanartificial electron acceptor (34,seealsoChapter 4 ) . Bothmethods
have their advantages andtheirdrawbackswhenappliedtointactchloroplasts aswillbeshownbelow.
A special problem in demonstrating a "bicarbonate-effect"
inisolated intactchloroplasts isthatusually C0 2 (orbicarbonate) is the ultimate electron acceptor. TheuseofC0 2 as
electron acceptor would make measurement of electronflowin
the absence ofbicarbonatenearly impossible.FortunatelyPGA
isreadilytakenupbyintactchloroplasts (11)andPGAcanbe
reduced to GAL3P atthe expense of1NADPHand1ATP (15).In
contrastto C0 2 reduction,PGAreductionbyintactchloroplasts
isnearly independent ofthepHofthestroma (40),whichimpliesthatacidificationofthestromabyanionsofweakacids
(5)willnotinterferewithPGAreduction.
Inthe experiments described in this Chapterbothmethods
of C02-depletion have beenappliedtoobtainC02-depletedintact chloroplasts.Dark-incubation with formate atlowpHappearstobeamethodthatcombinesafairdegreeofC02-depletionandagoodreactivation ofelectronflowbybicarbonate.
Material
and
methods
Spinach plants (Spinacia
oleracea) were grown inagreenhouse under high-pressure mercury lamps (PhilipsMGR 102-400)
at a light-intensity ofapproximately80W/m2. Severalcultivars were used: Kir,Bergola andAmsterdamsbreedblad.During
thedailyilluminationperiodof8hthetemperature oftheleaf
and the soil surface was keptbetween 18-20°C.The relative
humiditywasminimal70%.
Intact chloroplasts were isolated from freshly harvested
leaves of4-5 weeks old plants.Themethod was basedonthe
procedure described by Enser and Heber (5).The leaves (ap77
prox.20g)werederibbed,cutinsmallpiecesandhomogenized
in50mlisolationmedium inaSorvallOmnimixerin2-3 bursts
of3s.After filtrationofthebrei throughtwolayersofperlonnet (pore diameter40jjm)the filtratewascentrifuged for
60sat1500 g intwocentrifuge tubesinaMSEChillSpincentrifuge.Thepelletwasgently suspended in25ml resuspension
medium and centrifuged foranother60sat1500 g. Thesupernatantwasdiscarded andthe loosepellet (brokenchloroplasts)
was removed by gentlywashingthepelletwith0.5-1 mlresuspensionmedium.Theremainingpelletwasresuspended inasmall
volumeofresuspensionmedium andkeptonice.Theentireprocedurewascarriedoutat0-4°C. Isolationmedium: 0.3 Msorbitol, 10mM NaCl, 1mM MgCl 2 , 1mM MnCl 2 , 2mM EDTA, 0.5mM
KH 2 P0 4 , 2mM ascorbate, 4mM cystein and 50mM MES/KOH (pH
6.2). Resuspension medium: 0.3 M sorbitol, 1mM MgCl 2 , 1mM
MnCl 2 , 2mM EDTA, 0.2 mMKH 2 P0 4 , 1mgBSA/mland50mMHEPES/
KOH (pH6.7).
The percentage intact chloroplasts was usually between80
and95%,asdeterminedbytheFeCymethod (12).Intheexperiments shown, the percentage intact chloroplasts was minimal
75%.Thechlorophyll concentrationwasdetermined asdescribed
inChapter 2.
Intactchloroplastsweredepleted ofC0 2 byincubationwith
Na-formate atlowpH.Formatewasaddedtoaconcentrated suspension of chloroplasts inresuspensionmedium (0.5-2mg Chi/
ml)andthepHwasadjusted tothedesiredvaluewithHCl.The
incubation took place on ice in darkness;seetheresultsof
thisChapter formoredetails.
Oxygenevolutionwasmeasuredwith aClarkelectrode at20°C
as described in Chapter2. Standard reaction medium: 0.33 M
sorbitol, 1mMMgCl 2 , 1mMMnCl 2 , 2mMEDTA,0.2 mMKH 2 P0 4 and
50mM buffer (MES orHEPES, adjusted with KOH). Formeasurementswithbrokenchloroplastsphosphatewasomitted and10mM
NaClwasadded.Brokenchloroplastswereobtainedby suspending
intact chloroplasts in 2mM MgCl 2 and 5mM MES/Tris (pH7 ) ;
after 1min incubation anequalvolumeofdouble strengthreactionmediumwasadded.
78
P
515 m e a s u r e m e n t s were carriedoutasdescribed inChapter
5. Reaction medium: 0.3 M sorbitol, 10mM NaCl, 5mM MgCl 2 ,
50mMbuffer (MES orHEPES,adjustedwithKOH)andNa-formate
whenappropriate.
Results
Effects
of formate on PGA reduction
C0 2 -depletion of broken c h l o r o p l a s t s by i l l u m i n a t i o n i n the
presence of an e l e c t r o n acceptor and formate can properly be
achieved by using a high l i g h t - i n t e n s i t y ; a low pH and a high
formate c o n c e n t r a t i o n speed up the d e p l e t i o n process (see Chapt e r 4 for more d e t a i l s ) . On t h e other hand a high pH and a low
formate c o n c e n t r a t i o n are p r e r e q u i s i t e s for optimal carbon r e duction i n i n t a c t c h l o r o p l a s t s ( 5 ) . These two requirements are
10 / J M 0 a
/
HC0,"
60 S
HCOO" GMCD BQ
V
1
V
1
1
/
0
*
HC00"
•
0
0
F i g . 6 . 1 : Effect of formate (12.5 mM) on PGA dependent 0 2 e v o l u t i o n i n
i s o l a t e d i n t a c t spinach c h l o r o p l a s t s . The c h l o r o p l a s t s were suspended i n
standard r e a c t i o n medium (19 |Jg Chl/ml) t o which 3 mM PGA was added; the
pH was 7. Gramicidin (GMCD) and 1,4-benzoquinone (BQ) were added to a f i n a l
c o n c e n t r a t i o n of 2 pM r e s p . 0.4 mM. At the end of the i l l u m i n a t i o n p e r i o d
the formate c o n c e n t r a t i o n was increased t o 50 mM.
79
c o n f l i c t i n g and t h e r e f o r e a compromise was chosen by using pH 7
and 10-50 mM formate. Figure 6.1 shows t h a t PGA dependent oxygen evolution a t pH 7 i s s i g n i f i c a n t l y i n h i b i t e d by 12.5 mM
formate, with maximal i n h i b i t i o n a t t a i n e d a f t e r about 3 min.
Subsequent a d d i t i o n of an uncoupler and an e l e c t r o n acceptor
provides a means of assaying e l e c t r o n flow without i n t e r f e r ence of carbon metabolism. Figure 6.1 shows t h a t e l e c t r o n flow
i n t h e presence of BQ and gramicidin i s much f a s t e r than i n
the presence of PGA a l o n e . Moreover bicarbonate does not stimu l a t e e l e c t r o n flow i n the presence of BQ and gramicidin. So
i t seems t h a t the i n h i b i t i o n of PGA reduction by formate i s
not caused by i n h i b i t i o n of e l e c t r o n flow.
Experiments s i m i l a r t o the one described i n Figure 6.1 have
been c a r r i e d out a t higher formate c o n c e n t r a t i o n s and/or longer i l l u m i n a t i o n times. The r e s u l t of these experiments was t h a t
PGA r e d u c t i o n g e n e r a l l y was i n h i b i t e d t o a g r e a t e r e x t e n t . Also e l e c t r o n flow was more i n h i b i t e d in the absence of bicarbon-
100.0
3
c
o
75.0
CT
E
O™
O
E
50.0
3,
c
•
o
>
O
•
25.0
•
O™
5 5
6.0
6 5
7.0
7.5
B.O
PH
F i g . 6.2 ( l e f t ) : Effect of ambient pH on the i n h i b i t i o n of PGA dependent
0 2 e v o l u t i o n i n i n t a c t c h l o r o p l a s t s by formate. A: standard r e a c t i o n medium a t pH 7 . 0 . B: standard r e a c t i o n medium a t pH 7 . 5 . The PGA concentrat i o n was 3 mM; where i n d i c a t e d 20 mMNa-formate was added.
F i g . 6.3 ( r i g h t ) : Effect of ambient pH on PGA dependent 0 2 evolution i n
i n t a c t c h l o r o p l a s t s . Standard r e a c t i o n medium with MES as a buffer a t pH
6.0 and pH 6 . 5 ; HEPES was used a t the o t h e r pH v a l u e s . The PGA concentrat i o n was 3 mM.
80
B.5
ate, but addition ofbicarbonate mostly did not result ina
significantstimulationofelectronflow.
Figure6.2 shows that 20mM formate strongly inhibitsPGA
reduction atpH7while only a slightinhibitionisobserved
atpH 7.5. These observations do suggest that formate might
affectPGAreductioninaway similartotheinhibitionof C0 2
reduction by formate (5).This, however, implies that there
must exist apHdependentstep inthereductionofPGAbyintact chloroplasts. Therefore the effect of theambientpHon
PGA reduction by intactchloroplastswasmeasured.Figure 6.3
shows the pH dependence ofPGA reduction.OptimalPGAreduction is observed atpH 7.9, avaluepHwhichisalmostidentical to the optimum pH for C0 2 dependent oxygen evolution
(40).
The results shown inthissectionindicatethatinhibition
ofPGAreductionby intactchloroplasts inthepresenceofformate isprobably not due to inhibition of electron flowbut
due to an effect on carbon metabolism;perhaps acidification
of the stroma inducedby formatecouldberesponsible forthe
observed inhibition.
CO2-depletion
by dark-incubation
with formate at low pH
Prolonged treatment of isolatedintactchloroplasts atlow
pH (<6)was foundtosuppressPGAdependent0 2 evolutioncompletely; increasing the pHofthereactionmedium didnotrestore PGA dependent oxygen evolution (D.Naber and J. Snel,
unpublished results). Therefore anartificial electronacceptor like e.g. BQ had to beused forourpurposes.Theuseof
anartificialelectronacceptorhasacleardisadvantage:both
intact and broken chloroplasts canreducetheelectronacceptor.Thereforeonlybicarbonate and formateeffects largerthan
5-25% (thepercentagebrokenchloroplasts)canbeascribedwith
certaintytotheintactchloroplasts.
Table 6.1 showstheresultsofanexperimentinwhich intact
chloroplastsweredark-incubatedwith100mM formate at4dif81
ELECTRON FLOW (Mmol0 2 /mgChi'h)
INCUBATION
time (min
1
)
PH
-HCO;
+HCO3
+HCO3/-HCO3
15
6.4
140
169
1.2
46
6.4
53
158
3.0
90
6.4
24
88*
3.7
15
6.1
110
180
1.6
45
6.1
46
176
3.8
90
6.1
26
110
4.2
15
5.5
44
174
4.0
47
5.5
24
143
6.0
90
5.5
18
79
4.4
15
5.2
30
153
5.1
45
5.2
24
143
6.0
90
5.2
-
-
-
This valuemaybe an underestimation; the samplewas twice
illuminated for 30 sin the absence ofbicarbonate.
Table 6.1. Effect ofbicarbonate on theHill reaction in isolated
intact chloroplasts after dark-incubation with formate at theindicated pH. A suspension of intact chloroplasts was supplemented
with 100mM formate and thepHwas adjusted withHCl to thedesired value. Subsequently the chloroplasts were incubated on icein
the dark. At the indicated times a small sample of C02-depleted
chloroplasts was drawn and injected into the reactionvessel containing the standard reaction medium (pH 6.8) supplemented with
10 mM NH4CI, 2 mM BQ and 25mM Na-formate. Bicarbonate was added
toa final concentration of20mM.The control activity ofbroken
chloroplasts in the presence of FeCy (0.5 mM) and NH 4 C1 (10mM)
was 193pmol/mg Chi.h.
82
ferentpHvalues.Atvarioustimessamplesweredrawnandoxygen evolution wasmeasuredintheabsence andinthepresence
ofbicarbonate using theprotocol described inFig.6.4.The
resultsclearlydemonstrate thatatallpHvaluestested abicarbonate-effect on electron flowisinducedduringtheincubationperiod;howeveratpH5.2 thisinductionismuchfaster
thanatpH6.4,butnevertheless aclearinhibitionofelectron
flow inthe absence ofbicarbonate is observed after 90min
incubationatpH6.4.Theinitialrateofelectron flowinthe
presenceofbicarbonate ishigh aslongastheincubationtime
is not exceeding 45min. Figure6.4 shows that although the
initial rate of electron flowishighinthepresenceofformate and bicarbonate, electron flow is decliningduringprolonged illumination.A similarphenomenonhasalsobeenobservedinbrokenchloroplasts (seeChapter4 formore details).
10 MH %
30 s
NaHCO,
\
0
•
Effects of bicarbonate
response
f
F i g . 6 . 4 : Effect of b i c a r b o n a t e on
02 evolution in C02-depletion i n t a c t
c h l o r o p l a s t s in the presence of a
H i l l o x i d a n t . The c h l o r o p l a s t s were
depleted of C02 by d a r k - i n c u b a t i o n
with 100 mM formate a t pH 5.8 as
described i n m a t e r i a l s and methods.
The standard r e a c t i o n medium a d d i t i o n a l l y contained 25 mM Na-formate,
10 mMNH4C1 and 2 mMBQ.
0
and formate on the flahs-induced
P
515
Theflash-inducedP 5 1 5 responseofC02-depletedintactchloroplasts isshowninFig.6.5B. Six flashesweregivenandthe
amplitude of the absorbancechangesat518nmafterthefirst
flash is about equaltotheamplitudeofthe fastrise (phase
83
a)of theP 5 1 5 response in controlchloroplasts (Fig.6.5A).
Inthefollowing flashesthemagnitude ofphase agraduallydecreasestoavalueof-\>30%oftheamplitude inthe firstflash.
The flash-induced AA 5 1 Q measured afterdark-incubation ofthe
samesamplewithbicarbonate isshowninFig.6.5C. Thegradual
decreaseofphase a,whichisapparentinC02-depletedchloroplasts, is almost absent inthepresenceofbicarbonate.The
magnitude ofphase aafterthe6thflashisabout70%ofthat
after the first flash.Theseresults indicatethatintheabsence ofbicarbonate onlypartofthereactioncentersarein
an "open" state (i.e.canperform astable charge-separation)
withinthedark-period of250msbetweentheflashes.Itshould
bekeptinmindthatbothPS IandPS IIcontribute tothegenerationofatransmembranepotential (37)andthattotal inhibition of e.g. PS IIresults inonly 50%reductionoftheamplitudeoftheP 5 1 5 response (providedPS Iis "open").
-AI/I
.ooaj
200 ns
"*.\
A: c o n t r o l
T
B: COj-depleted
',, \ . '''H V ~- ^ ~
-.'-A
,W.
-.,. \, \, VN "-'•-
C: + HC0,"
Fig. 6.5: Effect ofbicarbonate on
the flash-induced P515 response in
COg-depleted chloroplasts atpH 6.4.
A: control (non-depleted);B: C0 2 depleted chloroplasts; C: C0 2 -depleted chloroplasts in the presence
of 10mM NaHC0 3 . The chloroplasts
were depleted of C 0 2 as described
inFig. 6.4, the formate concentration was 20mM and the chlorophyll
concentration was 27jigChl/ml. The
number of averages was:A:11,B:3,
C:7; the flash-trains were given at
a rateof0.05Hz.
• > , ,
V"*
T
t
t
t
t
t
Another aspectoftheP 5 1 5 response inC02-depletedchloroplastsisthatthekineticsofthedark-relaxation aredrasticallyaltered incomparisonwiththeresponse incontrolchlo84
roplasts. The slow (phaseb)riseapparentincontrolchloroplasts is absent in C02-depleted chloroplasts andphasebis
notrestored inthepresenceofbicarbonate. Inbothcasesthe
half-time of the decay of AA g is about 120ms,which is
slightlyhigherthanusually found forthedecayofreactionI
(38).Thedecayofreaction1isknownhowevertobe dependent
ontheioniccompositionofthereactionmedium(21,
F i g . 6 . 6 : Effect of formate on t h e
flash-induced P515 response i n i n t a c t c h l o r o p l a s t s (not depleted of
C 0 2 ) . A: c o n t r o l ; B: +50 mM Ka-formate; C: sample B a f t e r 90 min darki n c u b a t i o n on i c e . The c h l o r o p h y l l
c o n c e n t r a t i o n was 33 pg Chl/ml. The
number of average was: A:6, B : 8 ,
C:13.
2 6 ) . Under t h e c o n d i t i o n s used here, a d d i t i o n of sodium f o r mate was found not t o a l t e r t h e r a t e of decay of AA5 „ s i g n i f i c a n t l y b u t formate does appear t o i n h i b i t t h e slow r i s e
s l i g h t l y (Fig. 6 . 6 ) . Only a f t e r prolonged incubation the amp l i t u d e of t h e f a s t r i s e (phase a) appears t o be decreased as
well ( F i g . 6.6C). On t h e o t h e r hand t h e ambient pH has dramatic
e f f e c t s on the k i n e t i c s of t h e flash-induced AA 5 1 ß . Figure 6.7
shows t h a t e s p e c i a l l y a t low pH and a f t e r long dark-incubation
p e r i o d s the r a t e of decay i s g r e a t l y increased and phase b i s
no longer observable. These r e s u l t s i n d i c a t e t h a t t h e absence
of t h e slow r i s e observed in C0 2 -depleted i n t a c t c h l o r o p l a s t s
i s more l i k e l y t o be due t o by the incubation a t low pH than
by an e f f e c t of formate on t h e k i n e t i c s of AA 5 1 g .
85
-tl/l
A
.003
100 RS
-al/I
B
.003
100 RS
pH 6.4
'r""""-^^~<^^.
pH 6.4
~~'
• ' • • • -
;V
"~''"" ' ~-*~~.«~w
pH 5.9
'''*'~~-''^^^-^^,^,;^._^_M^^
pH 5.9
~.w
'~"~^—"^^^..,
pH 5.5
pH 5.5
..>,,.
"""^":'-"'^^^:-<„:^,^^
^"~~-«-~*
pH 5.2
PH 5.2
' V ''-'" S ,V. ...
.:.. •
Î
t
Fig. 6.7: Effect ofambientpHon the flash-induced P515 response inintact chloroplasts. A: measurements started after approx. 20min incubation; B: measurement started after 60min incubation. Incubation inreaction medium at the indicated pHvaluewas carried out inthedark onice.
The chlorophyll concentration was 27 pg Chl/ml.The number ofaverages was
9; the repetition rate 0.1Hz.
Discussion
Effects
of formate on PGA reduction
Figure 6.1 clearly demonstrates that formate inhibitsPGA
dependent 0 2 evolution at another step than photosynthetic
electron flow. This inhibition of PGA reductionismorepronounced atlowpHthanathighpH,aphenomenonthatresembles
theinhibitionofC02-dependent0 2 evolutionbyanionsofweak
acids (5,40).PGAdependent0 2 evolutionappearedtobedependentontheambientpH (Fig.6.3).Themaximal rateofPGAdependent0 2 evolutionwasobserved atpH7.9.ThispHdependen-
86
cemightexplaintheinhibitionofPGAdependentoxygenevolution by formate at pH 7 as formate appears to inhibit the
light-induced alkalinizationofthestroma(5).
There is however strong evidence that reductionofPGAto
GAL3P invitro (18)and in isolated intactchloroplasts (40)
isnot dependent on the ambientpH atpH valuesbetween 6.7
and 9.2. The experimental conditions in ourexperimentswere
however slightly different fromtheconditionsusedbyWerdan
etal. (40)withrespecttotheP. concentration;inthoseexperiments 3mM P. was used. At ahighratioofPGA/P. starch
formation is stimulated (13)byactivationofADPglucosepyrophosphorylase (23).As starchsynthesis requiresphosphorylation ofG1P (23),a lowered energy chargemight resultin
inhibition of PGA reduction (15,18).Theshapeofthecurve
inFig.6.3 mightthenbeexplainedbythe factthatphotophosphorylationundercontinuous illumination ispHdependent.The
optimumpH forphotophosphorylation hasbeenreported tobepH
8.4(1).
Ifphotophosphorylation is the rate-limiting step inPGAdependent0 2 evolutionundertheconditionsused,itisdifficult to see howinhibitionofFBP-aseactivityby formate (5)
could inhibit PGA reduction. Inhibition of FBP-ase activity
should increase the energycharge,wichinturnwouldstimulatePGA-kinase activity (18).There arehowevertwoeffectsof
formate that could inhibitPGAreduction.The firsteffectis
that formate can uncoupleelectron flow (seeChapter4)which
will lower the energycharge.Thesecond effectisthedissipationoftheApHacrosstheenvelope (5).PGAtransportacross
thechloroplastenvelopeismediatedbythephosphatetranslocator (7,11).Thekineticparameters ofthisenzyme aredependentontheambientpHandonthemagnitude anddirectionofa
transmembrane protongradient (7,11).Inthepresenceofthe
light-inducedprotongradientacrosstheenvelope (insidealkaline), PGA uptake willbe faster (17).As formate isknownto
dissipate the proton gradient, PGA reduction to DHAP may be
inhibitedbysubstratedepletion.ThereductionofPGAtoDHAP
87
is known to behighlyreversible (18).Obviouslymoreexperiments are required to elucidate themechanism oftheinhibitionofPGAreductionbyformate.
The irreversible inhibition of electron flow in isolated
intactchloroplasts thathavebeenpreilluminatedwithsaturatinglightinthepresenceofformate indicatesthatphotoinhibition may have occurred. Inchloroplasts orleavesthatare
illuminated with strong actinic light in the absence of an
electron acceptor or in the presence ofDCMU photobleaching
occurs andelectron flowisimpaired (17).As formate alsoinhibits electron flow attheacceptor sideofPSII,theeffect
offormatewithrespecttophotoinhibitionmightbesimilarto
theeffectofDCMU. InhibitionofC0 2 andPGA reductionbyformatemightresultindepletionofelectronacceptorswhichwill
enhancephotoinhibition(17).
C02-depletion
of intact
formate at low pH
chloroplasts
bu dark-incubation
ThedatagiveninTable 6.1 clearlydemonstrate thata"bicarbonate-effect" on electron flowcanbeinduced inisolated
intactchloroplastsbydark-incubationwith formate atlowpH.
This method has twoseriousdrawbacks.The firstisthatPGAdependent 02-evolution is inhibited after C02-depletion and
thisinhibitionisnotrelieved inthepresenceofbicarbonate.
The second drawback isthataftermorethan45-90minincubation (depending on pH)electron flow is inhibited in sucha
way that it cannotbe fully reactivated by bicarbonate (see
Table 6.1).TheresultsofTable 6.1 alsoprovideevidencethat
the irreversible inhibition of PGA reduction isprobablynot
caused by inhibition of electron flow.Thisdepletionmethod
was optimized however to demonstrate abicarbonate-effect on
electronflow.Themethodcanprobablybe improved,especially
withrespecttocarbonmetabolism.
88
with
Effects
of bicarbonate
and formate on the P51c
response
Incontrastwiththeexperiments showninChapter 5theamplitude ofphase a (the fast rise)oftheflash-inducedP 5 1 5
response after the first flash is aboutequal incontroland
C02-depleted chloroplasts (Fig.6.5).This is in agreement
withresultsofQ7decayexperimentswhichshowthatQ~isreoxidized within 20s (6,14,25),thetimebetweentheflashtrains (Fig. 5 ) .Prolonged dark-incubationof intactchloroplasts at lowpHwas foundtodecreasetheamplitudeofphase
a,both in theabsence (Fig.6.7) andinthepresenceofformate (datanotshown).
Inthe absence ofbicarbonate themagnitudeofphaseadecreases in subsequent flashes.Figure 6.5 shows that in the
presenceofbicarbonatethemagnitudeofphaseaafterthe6th
flashisafactor2higherthanintheabsenceofbicarbonate.
Thismeansthatittakesabout250mstoregenerate 50%ofthe
reaction centers in an"open"state,i.e.reactioncentersin
whichtheprimarydonorisreduced andtheacceptorisoxidized.Thishalf-timeofabout250msisinthesameorderofmagnitude as the half-times for the reductionofPQandP 7 0 0 in
C02-depleted broken chloroplasts (33).Fromtheoxygenevolutionmeasurement shown inFigure6.4 ahalf-timeofabout165
mswascalculated fortheturnoverofPQintheabsenceofbicarbonate.Inthepresenceofbicarbonate thehalf-timeofPQturnover mustbe much smaller than 250ms.From Fig. 6.4 a
half-time of about 30 ms is estimated, avalue which is in
agreement with data givenby Siggel et al. (33)for broken
chloroplasts.
In C02-depleted intact chloroplasts phase b is absent in
theP 5 1 5 response (Fig.6.5).Thisobservation isinagreement
withexperimentsofGarabetal. (8),butinthose experiments
phase b reappeared afteradditionofC0 2 . Thequestionarises
whether in those "C02-depleted" leaves or chloroplastselectron flow really wasinhibited attheacceptor sideofPSII.
ThemethodofC02-depletionused in (8)isquitedifferentfrom
89
themethods used in this thesis.Thedepletiontimesusedby
Garab and co-workers arevery short (5-30min)whencompared
with the incubation times shown inTable 6.1, especially if
one considers that no formateoracetatewasusedbyGarabet
al. (8).Moreoverphaseb showssomecharacteristics thatmake
the use ofphase b as anindicatorofbicarbonateeffectson
electron flowratherhazardous.Thesecharacteristicsare:
1.phaseb ispresent inthylakoidvesiclesthatcontainnoPS
II,whichindicatesthatphasebmightberelatedtocyclic
electron flow (19,20).
2.phaseb isdependentontheambientredoxpotential (9,10).
Atredoxpotentialshigherthan150mVphaseb isverysmall
(10)andphaseb appearswithanE ofapprox.+200mV,at
pH7.5,avaluewhichispHdependent(10).
3.phase b is dependent on the pHofthereactionmedium;at
lowpHphaseb isabsent (39,seeFig.6.7).
4.phase b is dependent on the state of energization ofthe
thylakoid membrane. In actively ATP-hydrolyzing thylakoid
membranepreparationsphaseb isabsent (30,31,32).
With respect to theeffectofpre-energization ofthethylakoidmembrane itisnoteworthy tomentionthat,inthepresence ofDTE andATP,thechloroplastATP-asecanbe activated
by only a fewsaturating light-flashes andtheATP-hydrolysis
continues for several minutes (30;R.L.A.Peters,unpublished
data). According to Garabetal. (8)theeffectofC02-depletioncould onlybeobserved "afterafewexciting flashes"and
thedisappearanceofphasebmighthavebeencausedbyenergization of themembrane by ATP-hydrolysis. Reversed electron
flow from PQH 2 to Q. caused by ATP-hydrolysis can partially
reduceQ.toQ~ (2,29)andacidificationofthethylakoidlumen lowers Chi a fluorescence yield (3,4 ) .This meansthat
ATP-hydrolysis, induced by the C02-depletion procedure,may
havecaused theinhibitionofphaseb andthecomplex kinetics
of Chia fluorescence inductionobservedbyGarabetal.(8).
Addition of C0 2 totheseleavesmightperhaps lowertheAG- T p
byactingasasubstrate incarbonmetabolism, thereby slowing
90
downATP-hydrolysis.Thisalternative interpretation israther
speculative and needs further investigations. Nervertheless
this alternative interpretation showsthatthe interpretation
ofGarabetal. (8)needs furtherevidence aswell.
Theabsenceofanacceleratingeffectofformateonthedecay of theP 5 1 5 response (Fig. 6.6) indicates that, at3°C,
the formateaniondoesnotreadilymoveacrosseitherthethylakoid membrane or thechloroplastenvelope.Experimentswith
broken chloroplasts show that formatedoesacceleratethedecay at 25°C,whichisinagreementwiththefactthatformate
uncouples electron flow at 25°C (see Chapter 3 ) ,but at3°C
formate has no significant effect on theP 5 , 5response (data
not shown).
TheeffectoftheambientpHontheP 5 1 5 response (Fig. 6.7)
isinagreementwithdatagivenbySchapendonk (26)forbroken
chloroplasts. Between pH 5 and pH 6phase b appears to be
strongly dependent on theambientpH;atthemomentitisnot
knownwhether the disappearance ofphase b is reversible or
whether phaseb is irreversibly suppressed by incubation at
lowpH.
Conclusions
ItisshowninthisChapterthatdark-incubation ofisolated intactchloroplasts,thatareabletoevolve0 2 inthepresenceofPGA,with formateatlowpHrendersthesechloroplasts
"C02-depleted". These C02-depleted chloroplastshavelostthe
ability to use PGA as electron acceptorandelectron flowto
an artificial electron acceptor is inhibited. The inhibition
of electron flow inC02-depletedchloroplasts isrelievedafter dark-incubation with bicarbonate.PGA-dependent0 2evolutionhowever isnotrestored inthepresenceofbicarbonate.
91
Inhibition of electron flowinC02-depletedintactchloroplasts was also inferred fromthe flash-inducedP 5 1 5 response
AsthePc-icresponsecanbemeasured inintactleavesofwhole
plants, measurement of theP 5 1 5 responsecanbeausefultool
in the search for a "bicarbonate-effect" onelectron flow in
vivo.
References
Avron,M. 1972. The relation of light inducedreactions
of isolated chloroplasts to proton concentrations. In:
Proceedings of the U n d International Congress onPhotosyntheisis Research (G.Fort et al., eds.). Vol.2:p.p.
861-871,Dr.W.JunkPublishers,TheHague.
Avron,M., U.Schreiber. 1979. PropertiesofATP-induced
chlorophyll luminescence inchloroplasts.Biochim.Biophys.
Acta546:448-454.
Briantais,J.M.,C.Vernotte,M.Picaud,G.H.Krause.1979.
A quantitative study oftheslowdeclineofchlorophyll a
fluorescence in isolated chloroplasts. Biochim. Biophys.
Acta548:128-138.
Briantais,J.M.,C.Vernotte,M.Picaud,G.H.Krause.1980.
Chlorophyll fluorescence as aprobe forthe determination
of the photo-induced proton gradient in isolatedchloroplasts.Biochim.Biophys.Acta 591:198-202.
Enser,U., U.Heber. 1980. Metabolic regulation by pH
gradients. Inhibitionofphotosynthesisbyindirectproton
transfer acrossthechloroplastenvelope.Biochim.Biophys.
Acta592:577-591.
Farineau,J., P.Mathis. 1983. Effect of bicarbonate on
electron transfer between plastoquinones in photosystem
92
II. In: TheOxygen-evolving systemofPlantPhotosynthesis
(Y. Inoue,ed.).AcademicPress,NewYork.
7.Flügge,U.I.,H.W.Heldt.1984. Influenceofaprotongradient on the activity of the reconstituded chloroplast
phosphatetranslocator. In: Advances inPhotosynthesisResearch, (C.Sybesma,ed.),Vol.II,M.Nijhoff/dr.W.Junk
Publishers,TheHague.
8.Garab,G.,A.A.SanchezBurgos,L.Zimànyi,A.Fàludi-Daniel
1983. Effect of C0 2 ontheenergizationofthylakoids in
leavesofhigherplants.FEBSLett.154:323-327.
9.Giorgi,L.,N.Packham,J.Barber.1984. Redox titrations
of the fast and slowphase of the 515 nm electrochromic
absorption change inchloroplasts. In: Advances inPhotosynthesisResearch (C.Sybesma,ed.),Vol.II,M.Nijhoff/
dr.W.JunkPublishers,TheHague.
10.Girvin,M.,W.A.Cramer.1984. Redoxtitrationoftheslow
electrochromicphaseinchloroplasts. In: AdvancesinPhotosynthesisResearch (C.Sybesma, ed.). Vol.II,M.Nijhoff/
dr.W.JunkPublishers,TheHague.
11.Heber,U., H.W.Heldt. 1981. The chloroplast envelope:
structure,function androleinleafmetabolism.Annu.Rev.
Plant.Physiol.32:139-169.
12.Heber,U., K.A. Santarius. 1970. Direct and indirect
transfer ofATP andADP across thechloroplastenvelope.
Z.Naturforsch.25b:718-827.
13.Heldt,H.W., C.J. Chon,D.Maronde,A.Herold, Z.S.Stankovic,D.A. Walker,A.Kraminer,M.R.Kirk,U.Heber.1977.
Roleoforthophosphate andother factorsinthe regulation
of starch formation in leaves and isolatedchloroplasts.
PlantPhysiol.59:1146-1155.
14. Jursinic,P.,A. Stemler.1982. A secondsrangecomponent
ofthereoxidationoftheprimaryphotosystem IIacceptor,
Q.Biochim.Biophys.Acta681:419-428.
93
15. Latzko,E., G.J.Kelly. 1979. Enzymes of the reductive
pentosephospatecycle. In: Encyclopedia ofPlantPhysiology, Photosynthesis II (A.Pirson, ed.). New SeriesVol.
6,p.p.239-250,Springer-Verlag,Berlin.
16.Mills,J.D., R.E.Slovacek,G.Hind. 1978. Cyclicelectron
transportinisolated intactchloroplasts.Further studies
withantimycin.Biochim.Biophys.Acta682:345-353.
17.Osmond,C.B. 1981. Photorespiration andphotoinhibition.
Some implications for the energetics ofphotosynthesis.
Biochim.Biophys.Acta639:77-98.
18.Pacold, I., L.E.Anderson. 1975. Chloroplast and cytoplasmatic enzymes.VIPealeaf3-phosphoglyceratekinases.
PlantPhysiol.55:168-171.
19.Peters,F.A.L.J.,R.H.M. vanderPal,R.L.A.Peters,
W.J.Vredenberg, R.Kraayenhof. 1984. Studies onwellcoupled photosystem Ienriched subchloroplast vesicles.
Discrimination offlash-induced fastandslowelectricpotential components.Biochim.Biophys.Acta766:169-178.
20. Peters,F.A.L.J., G.A.B. Smit, R.Kraayenhof. 1984. On
the origin of theslowelectricpotential inducedbyferredoxin-mediatedcyclicelectron transfer inphotosystem-Ienriched subchloroplastvesicles. In: Advances inPhotosynthesis Research (C.Sybesma, ed.),Vol II,M.Nijhoff/
dr.W.JunkPublishers,TheHague.
21.Peters,R.L.A., O.vanKooten,W.J.Vredenberg. 1984. The
kineticsofthe flash-induced P515electrochromicbandshift
independenceoftheenergetic stateofthethylakoidmembrane andtheionconcentration intheouteraqueousphase.
In: Advances inPhotosynthesis Research (C.Sybesma, ed.),
Vol. II,M.Nijhoff/Dr.W.JunkPublishers,TheHague.
22.Peters,R.L.A., M.Bossen,O.vanKooten,W.J.Vredenberg.
1983. On the correlationbetween theactivityofATP-hydrolase andthekineticsofthe flash-inducedP515electrochromic bandshift in spinach chloroplasts. J.Bioenerg.
94
Biomembr.15:335-346.
23. Preiss,J., C.Levi.1979. Metabolism ofprimaryproducts
ofphotosynthesis.Metabolismofstarchinleaves. In: EncyclopediaofPlantPhysiology,Photosynthesis II (M.Gibbs,
E.Latzko,eds.).NewSeriesVol.6,pp.282-312,Springer
Verlag,Berlin.
24.Radmer,R., 0.Ollinger. 1980. Isotopic composition of
photosynthetic 0 2 flash yields in the presence ofH 2 1 8 0
andHC 18 0ä. FEBSlett.110:57-61.
25.Robinson,H.H.,J.J. Eaton-Rye,J.J.S.vanRensen,
Govindjee. 1984. The effects bicarbonate depletion and
formate incubation on thekineticsofoxidation-reduction
reactions of thephotosystem IIquinoneacceptorcomplex.
Z.Naturforsch.39c:382-385.
26.Schapendonk,A.H.CM. 1980. Electrical events associated
with primary photosynthetic reactions inchloroplastmembranes.Agric.Res.Rep.905,ISBN9022007561 (doctoral
thesis).
27. Schapendonk,A.H.CM., W.J.Vredenberg. 1979. Activation
of the reaction II component of P515 in chloroplasts by
pigmentsystem I.FEBSlett.106:257-261.
28. Schapendonk,A.H.CM.,W.J.Vredenberg,W.J.M.Tonk.1979.
Studiesonthekineticsofthe515nmasorbancechangesin
chloroplasts. Evidence for the induction of afastanda
slow P515 response upon saturating light flashes.FEBS
lett.100:325-330.
29. Schreiber,U., M.Avron, 1979. Properties of ATP-driven
reversed electron flow inchloroplasts.Biochim.Biophys.
Acta 546:436-447.
30. Schreiber,U., S.DelValle-Tascon. 1982. ATP-synthesis
with single turnover flashes in spinach chloroplasts.In
situ monitoring with the firefly luciferase method.FEBS
lett. 150:32-37.
95
31. Schreiber,U., K.G.Rienits. 1982. Complementarity of
ATP-induced and light-induced absorbance changes around
515nm.Biochim.Biophys.Acta682:115-123.
32. Schuurmans,J.J., A.L.H.Peters,F.J. Leeuwerik,
J.Kraayenhof. 1981. On the association of electrical
events with the synthesisandhydrolysis ofATP inphotosynthetic membranes. In: Vectorial Reactions in Electron
and IonTransport inMitochondria andBacteria (F.Palmieri
et al., eds.), pp. 359-369,Elseviers/North-HollandBiochemicalPress,Amsterdam.
33. Siggel,U.,R.Khanna,G.Renger,Govindjee.1977. Investigation of the absorption changes of the plastoquinone
system inbroken chloroplasts.Theeffectofbicarbonatedepletion.Biochim.Biophys.Acta462:196-207.
34. Stemler,A. 1979. A dynamic interaction between thebicarbonate ligand and photosystem II reaction centercomplexes inchloroplasts.Biochim.Biophys.Acta545:36-45.
35. Stemler,À.,Govindjee.1973. Bicarbonate ionasacritical factorinphotosynthetic oxygenevolution.PlantPhysiol.52:119-123.
36.Vermaas,W.F.J.,J.J.S.vanRensen.1981. Bicarbonateactiononphotosynthetic electrontransport anditspossible
physiological role. In: Photosynthesis II.Electrontransport and photophosphorylation (G.Akoyunoglou, ed.)pp.
157-165,Balaban InternationScience Service,Philadelphia,
Pa.
37.Vredenberg, W.J. 1981. P515:amonitorofphotosynthetic
energization inchloroplastmembranes.Physiol.Plant.53:
598-602.
38.Vredenberg,W.J.,O.vanKooten,R.L.A.Peters 1984.Electrical events and P515 response in thylakoid membranes.
In: Advances inPhotosynthesis Research (C.Sybesma, ed.),
Vol. II,M.Nijhoff/Dr.W.JunkPublishers.
96
39.Vredenberg, W.J., A.H.C.M.Schapendonk. 1981. Reaction
kinetics ofP515 inchloroplasts:transmembrane (Reaction
I)and inner membrane (Reaction II)electric fields. In:
Photosynthetisis I. Photophysical Processes - Membrane
Energization (G.Akoyunoglou, ed.), pp.489-498,Balaban
International ScienceServices,Philadelphia,Pa.
40.WerdanK.,H.W.Heldt, M.Milovancev. 1975. The role of
pHintheregulationofcarbon fixationinthechloroplast
stroma.StudiesonC02-fixationinthelightanddark.Biochim.Biophys.Acta396:276-292.
41.Wydrzynski,T.,Govindjee.1975. Anewsiteofbicarbonate
effect inphotosystem IIofphotosynthesis:evidencefrom
chlorophyl fluorescence transients inspinachchloroplasts.
Biochim.Biophys.Acta387:403-408.
97
CHAPTER7
GENERALDISCUSSION
This thesis presents results of experiments, designed toobtainmore insight intotheregulationofphotosyntheticelectronflowinisolatedchloroplastsbybicarbonate andformate.
Moreovertheinfluenceofherbicidesontheregulationofelectronflowbybicarbonate and formatewas investigated.
An allosteric
relation between the binding of the
i-dinoseb and the binding of
bicarbonate/formate
herbicides
The results presented in Chapter 2 could be explained in
two ways, i).Bicarbonate and i-dinoseb compete for a common
siteattheacceptor sideofPSIIwhichintheirabsenceregulatory is occupied by another competitive inhibitor. Itwas
suggested that formate mightbe this competitive inhibitor,
ii)Bicarbonate and i-dinoseb do notbind to a commonsite,
buti-dinoseb affectsthereactivation oftheHill reactionby
bicarbonate in an allostericmanner.TheresultsofChapter3
show that formate indeed acts as a competitive inhibitor of
the stimulation of the Hill reaction by bicarbonate. Inthe
presenceoftwocompetitive inhibitors thereactivationofthe
Hillreactionbybicarbonate canbedescribedbyequation7.1.
V
Hill=vmax
1
V
TT-Tl
K7+ K;>+A
+
with Ij, I 2 : concentration of formate resp. i-dinoseb
K 1 } K 2 : dissociation constant of the formate- resp.
i-dinoseb-binding site complex.
v
„ . - , , , V ,K ,A: as defined inChapter 2. r
Hill' max' r'
98
(1 X)
'
Recalculationofthedatapresented inFig.2.3 yieldsavalue
forK 2 of1.6 nMi-dinoseb (Ij=100niMformate, Kt =5mMformate). This lowvalue is inconsistentwith measured binding
constants, which have been found in the range between 30nM
and 105nM (5,10).Therefore it isprobable that i-dinoseb
and formate/bicarbonate do not compete for acommon binding
site at the acceptor side of PSII.Thisleavesanallosteric
interaction between i-dinoseb binding andbicarbonate/formate
binding site as the most likely mechanism.Thebindingsites
ofbicarbonate/formate andi-dinoseb arebothlocatedinthe
vicinity ofQ A , since both i-dinoseb and formate affect the
EPR signal at g=1.82.ThisEPRsignal isattributed toaQ~
2+
Fe complex (11).Interactionofotherherbicideswithbicarbonate/formateattheacceptor sideofPSIIhavebeendiscussedindetailelsewhere(10).
Quantitative
parameters for the interaction
formate with photosynthetic
electron
flow
of bicarbonate
and
Theresultsoftheexperimentsdescribed inChapter3indicatethatformateandbicarbonatecompete foracommonregulatorysite,probablylocated atthePSIIproteincomplexinthe
vicinityof QH/QO- Thissite,thatcanbind formateandbicarbonate, is involved in electron flow attheacceptor sideof
PSII. The datapresented inChapter3canbeexplainedbyassuming that i)electron flow from Q.toP Q isinhibitedwhen
formate isbound to this site, ii)electron flowcanproceed
when bicarbonate isboundtothissiteandiii)electron flow
is allowed when neither formate nor bicarbonate isbound to
this site.These assumptions implythatthemodel forthereactivationoftheHill reactionbybicarbonate inthepresence
of a competitive inhibitor, asproposed inChapter2,should
be adapted. According to assumptions i, iiandiii,theHill
reactionrateislinearlydependenton [EA]+ [A](seeChapter
2 andAppendix 1foradescriptionoftheparameters).TheHill
reaction rate in the presence ofbicarbonate and formatecan
99
bedescribedbythefollowing equation:
V
Hill *V max ^
+
E
-r^-5
E
tot
tot
(7.2)
Thescaling factor,a,hasbeen introducedineqn.7.2toindicatethattherateofelectrontransportwhenthebinding site
is inthestateEisonlyafraction (a)oftheratewhenit
isinstateEA.Equation7.2canbereducedto:
V
=V
Hill max *K . ( 1+
a-Kr+A
I,+ A
r
Ki
(7
'3)
Undertheexperimental conditions usedinChapter3thevalues
oftheparametersineqn.7.3werethefollowing:
Vmax=150 -200umolOp/mgChi.h
£
a= 0.4- 0.5
K = 50 -110MM bicarbonate
K.= 1.3- 2.7mM formate
These values give asatisfactory description oftheinitial
Hill reaction rate asafunctionofthebicarbonateandformateconcentration.InChapter4evidenceisgiventhatK.may
be much lower during prolonged illumination. InthatcaseK.
inthelightmaybeaslowas0.2mM formate atpH6.5.At
higherpHformatemaybelessinhibitory (seee.g.Fig.6.4in
Chapter 4 ) .
Effects
of formate and bicarbonate
on carbon
metabolism
TheC02-depletionmethod appliedinChapter6yields intact
chloroplastsbothelectron flowandthereductionofexogenous
100
PGAareinhibited. Incontrasttoelectronflowinthepresence of BQ, PGA dependent oxygen evolutionwas foundnottobe
stimulatedbyadditionofbicarbonate.This irreversible inhibition ofPGA reductionmayhavebeencausedbythehighformate concentration orby the lowpHofthedepletionmedium.
TheC02-depletionprocedure howeverwasdevelopedprimarilyto
demonstratetheexistenceofalargeandreproduciblebicarbonateeffectonelectron flow. In vivo onlysmalleffectsofbicarbonateand formate areexpected.Therefore,whenonlysmall
effects on electron flowarerequired,amilderC02-depletion
procedure issufficienttoevoketheseeffects.
Formate and other anions ofweak acidshavebeenreported
to inhibit C02-dependentoxygen evolution in isolated intact
chloroplasts (1,12)andprotoplasts (3).C02-dependentoxygen
evolution in intact chloroplasts isinhibited for50%by5mM
formate atpH 6.9; the degree of inhibition ispHdependent
andinhibitionisonlyobserved atsub-optimalpH (1,10).The
inhibition of C0 2 dependent oxygen evolution by formate has
been ascribed to a dissipation of theprotongradientacross
the envelope in thepresenceofformate andnoeffectofformateontheprotongradientacrossthethylakoidmembranecould
bedetected (1).Theresultspresented inChapter4alsoindicatethatformatedoesnotsignificantlyuncoupleelectron flow
at a concentration of 5mM. Formateapparently also inhibits
FBP-ase atrelatively high concentrations (1). Thisinhibitionoccurshowever atmuchhigher formateconcentrations than
thoserequired forinhibitionofelectron flow (seeChapter 4)
orinhibitionofC02-dependent02-evolution.
In Chapter 4 the inhibitor constant K. for inhibition of
linear electron flow by formate has been estimated to bein
thesub-mMrangeinthelight.Itseemsthereforethatformate
affects!electron flow atlowerconcentrations thancarbonmetabolism.
101
Interaction
of photo synthetic
tion in higher
plants
electron
flow and
photorespira-
Figure 7.1 shows aschemeinwhichthelinkbetweenphotorespiration photosynthetic electron flowvia the formateand
the bicarbonate pools in the cytoplasm isvizualized. This
scheme is anextension of the scheme presented in Chapter3
andisbasedontheobservations thati)formateandbicarbonate can regulate electron flow at the acceptor side ofPSII
and ii)formatecanbeproduced duringphotorespiration.
CHL.OROPLAST
PEROXISOME
glycolate •
•glycolate-*
glyoxylate •
•glyoxylate
HJOJ-J
H20-«-4-W»
formate •
•formate
Figure 7 . 1 . Hypothetical scheme of reactions that are involved in the
coupling of photosynthetic electron flow to photorespiration in higher
plants. Heavy arrows indicate reactions that are proposed to be included
in the negative feedback loop from oxygen to formate.
At a high 0 2 /C0 2 r a t i o p h o t o r e s p i r a t i o n proceeds a t high
r a t e s (4, 6 ) . P h o t o r e s p i r a t i o n s t a r t s with the oxygenation of
RUBP, a r e a c t i o n which y i e l d s PGA and P - g l y c o l a t e (4, 6, 9 ) .
In subsequent r e a c t i o n s (not shown) P - g l y c o l a t e i s converted
back t o PGA; t h i s s e r i e s of r e a c t i o n s i s a l s o known as the C 2 cycle ( 4 ) . Glyoxylate i s an i n t e r m e d i a t e i n t h e C 2 -cycle and
i s generated by o x i d a t i o n of g l y c o l a t e i n t h e peroxisome (4,
6, 9 ) . Glyoxylate can e i t h e r be decarboxylated i n a r e a c t i o n
with H 2 0 2 (2, 6, 7, 8, 9) or converted t o glycine by a t r a h s 102
amination reaction (4,6, 8, 9, 13).Although high ratesof
glyoxylatedecarboxylationhavebeenobserved inisolatedbrokenperoxisomes (2),recentdatasuggestthat in vivo glyoxylatedecarboxylation doesnotsignificantlycontributetophotorespiratory C0 2 evolution (8).Itappeared thatonlyinthe
absence of amino donors,i.e. when transaminationofglyoxylatewasinhibited, glyoxylate decarboxylation couldbeobserved (8).Thismeansthatregulationofelectron flowby formate
produced duringphotorespiration isprobably limitedtoconditionsinwhichtransaminationofglyoxylate istoslowtocompensatethesynthesisofglyoxylate,i.e.glyoxylatecanaccumulateandreactwithH 2 0 2 yielding formate and C0 2 .
References
Enser,U., U.Heber. 1980. Metabolic regulation by pH
gradients. Inhibition ofphotosynthesis by direct proton
transfer across the chloroplast envelope.Biochim.Biophys.Acta592:577-591.
Grodzinski,B. 1979. A study of formate production and
oxidation inleafperoxisomes duringphotorespiration.Biochim.Biophys.Acta63:289-293.
Kaiser,G.,U.Heber. 1983. Photosynthesis of leaf cell
protoplasts and permeability of the plasmalemma to some
solutes.Planta 157:462-470.
Lorimer,G.H.,T.J.Andrews.1981. In: TheBiochemstry of
Plants, Vol. 8 (M.D.Hatch, N.K.Boardman, eds.), pp.
329-374,AcademicPress,NewYork.
Oettmeier,W., K.Masson. 1980. Synthesis and thylakoid
membrane binding of the radioactively labeled herbicide
dinoseb.Pest.Biochem.Physiol.14:86-97.
103
6.Ogren,W.L. 1984. Photorespiration: Pathways,Regulation
andmodification.Ann.Rev.PlantPhysiol.35:415-442.
7.Oliver,D.J. 1981. Mechanism andcontrolofphotorespiratory glycolate metabolism: C0 2 loss fromthe (C-2)carbon
ofglycolate. In: Proceedings ofthe5th InternationalCongressonPhotosynthesis (G.Akounoglou,ed.), Vol.IV,pp.
501-508,BalabanInternational ScienceServices,Philadelphia,Pa.
8.Somerville,C.R., W.L.Ogren. 1981. Photorespiration-deficientmutants ofArabidopsisthaliana lackingmitochondrial serine transhydroxymethylase activity. Plant Physiol.67:666-671.
9.Tolbert,N.E.1981. Metabolicpathways inperoxisomes and
glyoxysomes.Ann.Rev.Biochem. 50:133-157.
10.Vermaas,W.F.J. 1984. Theinteractionofquinones,herbicides and bicarbonate with theirbinding environment at
theacceptor sideofphotosystem IIinphotosynthesis.Dissertation,AgriculturalUniversity,Wageningen,TheNetherlands.
11.Vermaas,W.F.J., A.W.Rutherford. 1984. EPR-measurements
on the effects ofbicarbonate and triazineresistanceon
the acceptor side of photosystem II. FEBS lett. 175:
243-248.
12.Werdan,K., H.W.Heldt, M.Milovancev. 1975. Theroleof
pHintheregulationofcarbon fixationinthe chloroplast
stroma. Studies on C02-fixation inthe light and dark.
Biochim.Biophys.Acta396:276-292.
13.YuC , Z.Liang,A.H.C.Huang.1984. Glyoxylatetransamination in intact leaf peroxisomes. Plant Physiol. 75:
7-12.
104
APPENDIX1
KINETICS OF THE BINDING OF TWO LIGANDS TO A COMMON BINDING
SITEATAMACROMOLECULE
ThebindingoftwoligandsAand Itoamacromolecule Ecanbe
describedbythefollowingreactions:
A+E Z£- EA
[1]
and
*3
I+E^
EI
k4
[2]
Theequilibrium constants forthesereactionscanbeexpressed
asfollows:
kl
[E]-[A]
Ka k 2
[EA]
= r.— = rj.»i—•*-
*• ,
c
forreaction1
and
E
1
K, =_i4= t ]'t ]
*
"
f o r reaction2
[EI]
Strictly speakingtheseequilibrium constantsarethedissociationconstantsandareusuallyexpressed inmM.
In the following considerations it is assumedthatligandsA
and Icompete forthecommonbinding siteE;A and Ionlybind
to the "free"binding site, i.e. bindingtothestatesEAor
EI does not occur.Under these conditions therateofformation of the twobinarycomplexescanbedescribedbythefollowingdifferentialequations:
105
ât5*l=kl.[E].[A]-k2.[EA]
[3]
and
^ f 1 1 =k3-[E]-[I]-k4-[EI]
[4]
It is assumed that the number of binding sites remains
constant:
[E]+[EI]+[EA]=constant=Efct
[5]
(For the sake of simplicity the brackets will be omittedin
the following calculations.)
The amount of free binding sites (E)canbe eliminated from
equations [3]and [4]withtheaidofeqn.[5]:
^ J ! ^= k i ' A ' E t o t -kj-A-EI - (k1-A+k2)-EA
[6]
d
[7]
and
1 | i=k 3 -I-E t o t-k3-I-EA- (k3-I+k4)-EI
Generally thissetofdifferential equations ishard tosolve.
IfhoweverA and Iareconstants,i.e.whenA>> Efc.and I>>
E. .,equations [6] and [7] form asetoflinear independent
differential equations forwhichananalytical solutionexists.
Equations [6]and [7]canbesimplifiedbydifferentatiation.
Firstdefinex = ..
and ys
andafterdifferentia-
tionthefollowing resultisobtained:
106
g|= -k1-h-y-(k1-h+k2)-x
[8]
g =-k3-I-x-(k3-I-k4)-y
[9]
Next it is assumed thatx =X-e z t andy=Y-e z twithXandY
beingconstants.Furthersimplification isobtainedbysubstituting a=k!-A+k2;b=k3-I+k4;c=ka-Aandd=k 3 -I.
Substitution inequations [8]and [9]yields:
z-X-ezt=-c -Y-e zt -a-X-e zt
[10]
and
z-Y-ezt=-d -X-e xt -b-Y-e zt
[11]
This set of equations has only asolution fort>0whenthe
coefficientsofX andYhavethefollowingrelation:
T*=bfi
I"]
orwritteninadifferentform:
z 2 + (a+b)-z+a-b-c-d=0
[13]
yielding
Zj
= - (a+b)±V(a-b)
2
-4(a-b-c-d)
2
[14]
Now X and Y can be calculated fromequation [12]forthetwo
distinctvaluesofz:
a+zx
a)
z - z,:letX=A,then Y=-A
—
[15]
107
forthesecondvalueofz,z 2 ,thecalculationisidentical:
b)
z=z 2 :putting X=p,yieldsY= -JJ
a+z 2
[16]
—
Thegeneralsolutioniscomposed ofthetwopartialsolutions:
x =\ - e Z l , t +(j-eZ2"t
[17]
and
a+Zj 1 u ^
e^ •e-z M
y =-x
a+z:
e^2 «-
[18]
dEA __... dEI
Reminding thatx = d t andy= ..,equations [17]and [18]
canbewrittenintheirfinal form:
^
=X - e 2 ! ^ +M -e Z 2 , t :
[19]
and
dEI_ .a + Z l Zi-t
"dt"
K
S
a+Zg
e
~ »'S
.z,-t
r - n1
e
[20]
These differential equations canbesimply integrated simply,
becauseA,a,z 1 # z 2 andcaretime-independent.Thereforethe
generalsolutionbecomes:
[EA] (t)=C'+* - • e 2 * * 1 +U — e 2 2' t
z
l
z
[21]
2
and
[EI] (t)=C» - X - —
a+z
i r, +e 2 l - t -M - —
a+z 2
e 2 2' t
withC'andC"asintegrationconstants.
108
^
[22]
Equations [21] and [22]describethetime-dependentchangein
the concentration of thebinary complexes EA and EI upon a
change inone of theparameters,e.g. [A]or [I].Thevalues
fortheconstantsC',C",A.andparedeterminedbytheboundaryconditions.
For a few casesitmaybeillustrative tocalculatetheexact
solution.
Example1
At time t=0 an amount of ligand A is addedtoasolution
containing macromolecules E, competitive inhibitor Iand
their binary complex EI. This case simulates theexperimentsdescribed inFigs.2and5inChapter2.
Theboundaryconditionsinthiscaseare:
a ) a t t < 0:
[EI] = E t o t - — t j
[I] = I
[A] = 0
b ) a t t = 0:
[A]
=A
[EA] = 0
[EI]
and
c ) a t t < a> : [EA] = E
and
[EI]
"totK ^ I
F
A'
'-totKr+A'
"totK + I ,
, with
A'
,with
I1
A
1+I
K
i
I
K
r
109
From these boundary conditions the values for C', \ andpcan
be calculated:
A=—
E^ .-{kx-A'+z 2 - - ^ — }
zx-z2 tot
Kr+A'
M=
E...{k^A*zi--^-5
zrz2 t0t
K r +A'
[23]
(241
and
C'=E
L
A'
•——
H o t Kr+A'
r251
lZJ
Substitutionofthesevaluesinequation[21]yields:
EA(t)=E ^ . C ^ j r +ir èi I -(k 1 -A' +Z2 - R A ^ 1 -).e z ^ t-
+z
) eZ2 t}
"Ï ii-z.2
T W ^ - * ' i-iTTA^ - *
r
[26]
with
{ k!-A+k 2 + k 3 -I+k 4 ,+
Z, = —1
and
V(k x -A+k 2 -k 3 'I-k 4 ) 2 +4kj-A-k 3 -I
i
k x -A+k 2 +k 3 -I+k 4 j _V(k t •A+k 2 -k 3 -I-k 4 ) 2 +4k!-A-k 3 -I
Equation [26] describes theformation oftheEAcomplex asa
function oftheincubation time t after addition ofligandA
to a mixture ofEandI.Equation [26] canbesplitupinto
three parts: a time-independent part andtwotime-dependent
parts with relaxation constants z x andz 2 :
EA (t)=Cj -C 2 - e Z l t -C 3 - e Z 2 * t
110
[27]
Theconstants Clt C 2 ,C 3 ,z t andz 2havebeencalculated fora
number ofparameter sets. The results areshowninTableAl.
Line 3 shows that, at [I]= K. and [A]=K r , thecondition
thatk_ = 10-kjandk4=10-k2 sufficestomakeC 3muchsmallerthanC 2 .Thismeansthatequation [27]reducestothe following form:
EA (t):C^-U-e 2 ' 1 )
[28]
with
C
i = E tot'ÏÇÎV
a n d Z = Zl
Line 3 of TableAl furthershowsthat Zx =-0.0146s"1,which
is only slightly different from the value for an infinitely
fast inhibitor: -0.0150 s"1.TableAl furthershowsthat increasingk 3 andk 4 furtherreducestheerrormadeby omitting
thesecondterm fromequation[39].
Example2
Attimet=0anamountofcompetitive inhibitor Iisaddedtoa
solution containing macromolecules E,ligandA andtheirbinarycomplexEA.Thisexample simulatestheexperimentdescribed inFig.6ofChapter2.
Theboundaryconditionsare:
a ) a t t < 0: [EA] = [E,. . ] J
tot
[A]
JM.
K r +[A]
=A
[EI] = 0
[I]
b ) a t t=0
[A]
=0
=A
[I]
= I
5
U =
41
k3-I-{Etot-EA}
111
and c)att•»»: [EA]=E
(A'=-=-y)
tot Kr+A'
[EI]=E
(I' =
tot K i +I'
-^-K)
1 +K—
r
Calculationoftheconstants asinexample 1yields:
EA ( t ) = E _ . • [ { - ^
K r +A'
•eZl,t +
{k3
^— • - ° — • ( k 3 - I ' + z 2
zj-z2
a+z x
+ z.
Ki+I,J
^-)}
K.+I'
[29]
J
The c o n s t a n t s used i n equation [29] have been described e a r l i e r in t h i s appendix. Comparing equations [26] and [29] i t
appears t h a t the time-indepent p a r t s are i d e n t i c a l , which i s
of course not s u r p r i s i n g for r e v e r s i b l e r e a c t i o n s . When i n h i b i t o r I e q u i l i b r a t e s much f a s t e r with E than A e q u i l i b r a t e s
with E, equation [29] can be s i m p l i f i e d by omitting t h e t h i r d
term. Although I binds r a p i d l y t o E, f u l l equilibrium i s only
reached a f t e r ligand A i s i n equilibrium with the macromolecule E, i . e . d i s s o c i a t i o n of the EA-complex i s the r a t e - d e t e r mining s t e p i n a t t a i n i n g e q u i l i b r i u m .
Table Al. Effect of the magnitude of the r a t e c o n s t a n t s k 3 and k on t h e
k i n e t i c s of the binding of ligand A to the macromolecule E i n the presence
of a competitive ligand I . The parameters have been described in example 1
and the values of C 1 ; C 2 , C 3 , Zj and Z2 were c a l c u l a t e d according t o equat i o n [ 2 6 ] . The following values were used for the p a r a m e t e r s : [A] = 1 mM,
0.01 s _ 1 , [ I ] = 1 mM.
10 M"
(M-i-s
1
10
100
1000
112
-1
)
(S
-1
c2
C3
0.3333
0.3333
0.3333
0.3333
0
0
0
0
0968
2500
3326
3333
-0.00146
-0.01000
-0.01461
-0.01496
-0.02054
-0.03000
-0.20540
-2.00500
1
)
0.001
0.010
0.100
1.000
(s" )
Z2
(s"1)
Zi
-0 2366
-0 0833
-0 .0007
-0 0000
SUMMARY
Thisthesisdescribessomeeffortsthatweremadetogaina
better understanding of theprocesses involved intheregulation ofphotosynthetic electron flowby bicarbonate,formate
and herbicides in chloroplasts. Inthepast decade a large
amount of research has been devoted to get insight intothe
mechanism ofherbicide actiononelectronflowattheacceptor
sideofphotosystem II.Thisthesiswilldealmainlywithstudies on the regulation ofelectron flowattheacceptorside
ofphotosystem II bybicarbonate and formate.Somedetailsof
themechanism ofthisregulation aswell asitsintegrationin
theoverallprocessofphotosynthesiswere investigated.
InChapter 2 experiments are described thatwereaimedto
provideamorequantitativedescriptionofthemutualinteractionbetweenherbicides,bicarbonate andtheirbindingenvironment at the acceptorsideofphotosystem II.This interaction
wasstudiedbymeasuringtheeffectsofbicarbonate andherbicidesonelectron flowinC02-depletedchloroplasts.Thekinetics of the reactivation of the inhibited Hill reaction in
C02-depleted chloroplasts by dark-incubationwithbicarbonate
suggest that thebinding ofbicarbonate involves a reaction
with (pseudo) first order kinetics. It is shown that inthe
presenceoftheherbicides i-dinoseb andDCMUthereactivation
oftheHillreactioninC02-depletedchloroplastsbybicarbonateisretarded. Itisshownthatanycompetitive inhibitorof
thebinding ofbicarbonate canbeexpectedtoeffectivelyretard thebinding ofbicarbonate (atheoretical treatment is
giveninAppendix 1). Although i-dinoseb appeared tobeanapparently competitive inhibitor ofthestimulation oftheHill
reactionbybicarbonateunderequilibrium conditions, i-dinoseb
andbicarbonate probably do not compete foracommonbinding
site. This was inferred from the kinetics of thebindingof
i-dinoseb. These kineticswere shown to beto fasttobeex-
113
plained by simple competition.Therefore itisconcludedthat
thebindingofi-dinoseb affectsthebindingofbicarbonate in
anallostericway.
InChapter 3 anew procedure forC02-depletionofchloroplasts ispresented. This method yields C02-depletedchloroplasts inwhichtheHillreactioncanbealmostcompletelyreactivated by bicarbonate atpH 6.5. Itisfurthershownthat
formate is a competitive inhibitorofthereactivationofthe
Hill reaction by bicarbonate. The true reactivationconstant
K wascalculatedtobe78pMbicarbonate atpH6.5.Underthe
sameconditions theinhibitorconstantK.wascalculated tobe
2mM formate.Experimentsperformed atbicarbonate and formate
concentrations lower than K resp.K. show that under these
conditions electron flowproceeds athighrates.Theseobservationssuggestthat,contrarytohithertopresentedconcepts,
binding ofbicarbonate totheregulatory siteatthe acceptor
sideofPSII isnotarequirement forelectron flowattheacceptor side of PS II.Theresultspresented inChapter3further suggest that formate is apotent inhibitor of electron
flow atthe acceptor side of PSII. The inhibitory actionof
formateiscounteracted inacompetitivewaybybicarbonate.
It isproposed in Chapter 3 thatphotorespirationmayaffect photosynthetic electron flow.Under certain conditions
formate isproduced duringphotorespiration.Asphotorespiratory formate production isdependentontheoxygenconcentration,anegative feedback loop fromphotosyntheticallyproduced oxygen to formate may exist under these conditions.This
negative feedback loop canbe powerful mechanism fortheregulationofelectron flow in
vivo.
InChapter 4 theeffectsofformate andbicarbonate onthe
Hillreactionare furtherstudied andcharacterized,especiallyduringprolonged illumination.Formate isshowntostimulate
theHillreactioninnon-depleted chloroplasts.Itistherefore
proposed thatformate actsasanuncouplingagent.Inthepresence of anuncoupler formatewas foundnottostimulateelec-
114
tron flow. Instead onlyinhibitonofelectron flowby formate
was observed. The extent of this inhibitionwasnotconstant
intimebutappeared toincreaseduringprolonged illumination.
The inhibition of electron flowby formatecouldbepartially
preventedbyincludingbicarbonate inthereactionmedium,but
inthelightbicarbonate appeared tobealessefficientcounteracting agentthaninthedark. Itisestimated thattheinhibitor constant K. of formate may be more than amagnitude
lowerinthelightthaninthedark,implyingthatformateaffectssteady stateelectron flowatsubmMconcentrations.
InChapter 5 an alternative method ispresentedwhichcan
effectivelybeusedtostudytheeffectsofformate andbicarbonateonelectron flowinintactchloroplasts.Thismethodis
based on measurement of the magnitude of the flash-induced
transmembrane potential using theP 5 1 5 response as a linear
indicator of the transmembrane potential difference. In C0 2 depleted chloroplasts the overallrate-limiting stepofelectron flowwas foundtoposess ahalf-time ofabout200-250ms
intheabsenceofbicarbonate.Thishalf-timewasmuchsmaller
in the presence ofbicarbonate.Moreovergramicidininsensitive absorbance changes inthe500-550nmregionwere foundto
be affected by C02-depletion and readdition ofbicarbonate.
These absorbance changes arespeculated tobeassociatedwith
protolytic reactions attheacceptor sideofPSII.
Chapter 6 describes effects of formate andbicarbonate on
electron flow in isolated intact chloroplasts. Formate was
found to inhibit PGAdependentoxygenevolution,butthisinhibition could not be correlated with inhibitionofelectron
flowattheacceptor sideofPSII. Inhibitionofelectronflow
was observed when isolated intactchloroplastswere incubated
with formate at lowpH.This inhibitionofelectron flowwas
abolished by addition ofbicarbonate.Thetreatmentwithformate at low pH however appeared to be detrimental tocarbon
metabolism; PGA and C02-dependentoxygenevolutionwere found
tobeirreversibly inhibited.
115
Ä summarizing discussion is given in Chapter 7, inwhich
the physiological significance ofregulationofelectronflow
by formate and bicarbonate isdiscussed inrelationtocarbon
metabolism.
116
SAMENVATTING
Ditproefschrift beschrijft eenaantal experimentendieer
op gericht zijn om tot een beterbegrip tekomenvandeprocessen die betrokken zijn bij de reguleringvanhetfotosynthetisch elektronentransport in chloroplasten doorbicarbonaat, formiaaten herbiciden. Inde laatste10jaariseral
veel onderzoek verricht naar hetwerkingsmechanisme vanherbiciden die het elektronentransport remmen aan de acceptor
zijde van fotosysteem II.Het in ditproefschriftbeschreven
onderzoek zal voornamelijk betrekkinghebbenopderegulering
van het fotosynthetisch elektronentransport door bicarbonaat
en formiaat.Het onderzoek omvatte zowelsommige aspectenvan
het mechanisme van dezeregulering alsdeintegratieervanin
het fotosyntheseproces.
In hoofdstuk 2worden experimenten beschreven die zouden
moeten leiden tot een meer kwantitatieve beschrijvingvande
effecten van de interacties van herbiciden enenbicarbonaat
met hun bindingsomgeving aandeacceptorzijdevan fotosysteem
II en de effecten daarvan op hetelektronentransport. Ingeisoleerde chloroplasten waaraan C0 2 onttrokken is, is het
elektronentransport sterk geremd. Door toevoeging vanbicarbonaat (ofC0 2 )wordt het elektronentransport gereactiveerd.
Dekinetiekvandezereactivatie doetvermoedendatdebinding
vanbicarbonaataanchloroplastenverlooptvolgenseenreactie
met een (pseudo)eerste ordekinetiek. Indeaanwezigheidvan
het herbicide i-dinoseb blijkt de affiniteit van dechloroplasten voor bicarbonaat verlaagd te zijn; erismeerbicarbonaat nodig om tot een maximale reactivering van het elektronentransport te komen. Op grond van theoretische overwegingen wordt aangetoond dat een competitieve remmer van de
binding van bicarbonaat deze binding kanvertragen (zieook
Appendix 1), hetgeen experimenteel ook voor i-dinoseb wordt
aangetoond. Desondanks moet echter aangenomen worden datbicarbonaat en i-dinoseb niet aangrijpen op dezelfdebindings117
plaats.Deze conclusie komtvoortuithetfeitdatderemming
vanhetelektronentransport indeaanwezigheidvanbicarbonaat
door i-dinoseb sneller optreedt danverwacht magworden van
een competitieve remmer.Demogelijkheid wordt geopperd dat
de interactie tussen debindingsplaatsenvani-dinoseb enbicarbonaatallosterischvanaard is.
Inhoofdstuk 3wordt een nieuwe methodebeschrevenom C0 2
aan geïsoleerde chloroplasten teonttrekken.Dezemethodeleverde chloroplasten op waarin het elektronentransport beter
gereactiveerd kanworden door bicarbonaat danbijvorigemethoden het geval was. Indit hoofdstukwordtderolvanformiaat in de regulering van het elektronentransportnaderbestudeerd. Formiaatblijkt eencompetitieve remmertezijnvan
de reactivering van het elektronentransport inC02-vrijgemaakte chloroplasten door bicarbonaat. De reactiveringsconstante (Kr)blijkt 78 uMbicarbonaat tebedragen bij eenpH
van 6.5; onder dezelfde omstandigheden werd eenwaardevan2
mM gevondenvoor deremmingsconstantevanformiaat.Uitexperimenten uitgevoerd met chloroplasten waaraan C0 2 onttrokken
is, blijkt dat het elektronentransport goed verlooptwanneer
debicarbonaat en formiaat concentraties veel lagerzijndan
K r resp.K..Dezewaarnemingdoetvermoedendat,integenstelling tot hetgeen totdantoeaangenomenwerd,bindingvanbicarbonaataanchloroplastennietvereistisvoorhetdoenverlopenvan elektronentransport aan de acceptorzijde vanfotosysteem II.Dit houdt in dat de remming van hetelektronentransportinafwezigheidvanbicarbonaatveroorzaaktmoetzijn
door formiaat.Deze,somssterke,remmingvanformiaatkandoor
bicarbonaatopeencompetitievewijzeongedaangemaaktworden.
Inhoofdstuk 3wordtverdernogeenmechanismevoorgesteld
waarbijfotorespiratie het fotosynthetisch elektronentransport
beïnvloedt.Onderbepaaldeomstandighedenkanindefotorespiratie formiaat gevormd worden.Erwordtbeschrevenhoedefotorespiratie eennegatieve terugkoppeling op het elektronentransportteweegkanbrengen.Eendergelijkmechanismezoukun-
118
nen dienen om door middel van fotorespiratiehetelektronentransporttereguleren.
Inhoofdstuk 4 wordendeeffectenvan formiaatenbicarbonaat op het elektronentransport inonbehandelde chloroplasten
nader bestudeerd, met nametijdens langerebelichting.Formiaatblijkt in onbehandelde chloroplastenhetelektronentransport te kunnen stimuleren.Deze stimuleringwordttoegeschreven aan een ontkoppelende werking van formiaat. Involledig
ontkoppeldechloroplastenhad formiaatalleeneenremmendewerking op het elektronentransport. Demate van de remming nam
toe tijdens de belichting. De aanwezigheid van bicarbonaat
bleek de remming van het elektronentransport door formiaat
slechts gedeeltelijk te kunnen tegengaan. Volgens een ruwe
schatting is de remmende werking van formiaat in het licht
meer dan een orde van grootte sterkerdaninhetdonker.Dit
betekentdatformiaathetelektronentransport alkanbeïnvloedenbijeenconcentratievanminderdan1mM.
Inhoofdstuk 5wordteenmethodebeschrevenwaarmeeopeen
andere wijze de effecten van bicarbonaat en formiaat op het
fotosynthetisch elektronentransport bestudeerd kunnenworden.
Deze methode is gebaseerd ophetmetenvanflits-geïnduceerde
potentiaalveranderingen overhetthylakoidmembraanmetbehulp
van absorptieveranderingen van het pigmentcomplex P,.,. In
chloroplastenwaaraan C0 2 onttrokkenwaskonookopdezewijze
aangetoond worden dat het elektronentransport geremd was.Na
toevoeging van bicarbonaat bleekhetelektronentransportversneld teverlopen. Het onttrekken van C0 2 aan chloroplasten
hadookinvloed opabsorptieveranderingen inhetgebied tussen
500 en 550 nm.Deze absorptieveranderingen,welkehoogstwaarschijnlijknietgerelateerd zijnaande flits-geïnduceerdemembraanpotentiaalveranderingen, worden verondersteld te zijn
gecorreleerd metprotonopname aan de acceptorzijdevan fotosysteem 11.
Hoofdstuk 6beschrijfteffectenvan formiaatenbicarbonaat
119
op het elektronentransport ingeïsoleerde intactechloroplasten. Er werd gevondendat formiaateenremmendewerking heeft
op de zuurstofproduktie in aanwezigheid van een natuurlijke
electronacceptor, PGA. Deze remmingbleeknietveroorzaakt te
worden door remming vanhetelektronentransport aandeacceptorzijde van fotosysteem II.Door incubatievanintactechloroplastenmet formiaatbijlagepHkonhet elektronentransport
wel geremd worden.Na toevoeging van bicarbonaat bleek deze
remming te zijn opgeheven, d.w.z. het "bicarbonaat-effect"
blijkt ook in intacte chloroplasten op te kunnen treden.De
incubatie met formiaat bij lage pH had echter wel eenschadelijkeffectophetkoolstofmetabolisme.
Inhoofdstuk 7 tenslottewordenenkeleresultatennogeens
besprokenenwordtgespeculeerd overdebetekenisvandereguleringvanhet fotosynthetisch elektronentransport doorbicarbonaatenformiaat.
120
PUBLICATIONS
Several topics of this thesis are covered in the following
papers:
Snel,J.F.H., J.J.S.vanRensen,1982. Influenceofphotosynthetic inhibitors on thereactivationoftheHill reaction
inC02-depletedchloroplastsbybicarbonate.PlantPhysiology69:114.
Snel,J.F.H., J.J.S.vanRensen,1983. KineticsofthereactivationoftheHillreactioninC02-depletedchloroplastsby
addition ofbicarbonate intheabsence andinthepresence
ofherbicides.PhysiologiaPlantarum57:422-427.
VanRensen,J.J.S.,J.H.Hobé,R.M.Werner,J.F.H. Snel,1983.
Reactivation of electron transportinC02-depletedchloroplasts by bicarbonate is influenced by thylakoid surface
potential.' In: Advances in Photosynthesis Research
(Sybesma,C , ed.),Vol. I,Martinus Nijhoff/Dr. W. Junk
Publishers,TheHague.
Snel,J.F.H., A.Groote-Schaarsberg, J.J.S.vanRensen,1983.
Mechanism and physiological role ofbicarbonate actionon
electron flow;apossible linkbetweenphotorespiration and
photosynthetic electron flow? In: Advances inPhotosynthesis Research (Sybesma,E.,ed.), Vol.I,MartinusNijhoff/
Dr.W.JunkPublishers,TheHague.
Snel,J.F.H., J.J.S.vanRensen, 1984. Réévaluation of the
roleofbicarbonate and formateintheregulationofphotosynthetic electron flowinbrokenchloroplasts.PlantPhysiology 75:146-150.
Snel,J.F.H., D.Naber, J.J.S.vanRensen, 1984. Formate as
an inhibitor ofphotosynthetic electron flow. Zeitschrift
fürNaturforschung 39c:386-388.
121
VanRensen, J.J.S., F.deKoning, J.F.H. Snel, (in press).
Effectsofbicarbonate,formate andherbicides onphotosynthetic electron transport inisolatedbrokenchloroplagts.
Proceedingsofthe3rdEBECconference, ICSUPress,Miami.
Snel, J.F.H., D.Naber, J.J.S.vanRensen, (inpress). Regulation ofphotosynthetic electron flowby formate andbicarbonate in isolated intact chloroplasts. Proceedings of
the3rdEBECconference, ICSUPress,Miami.
VanRensen, J.J.S., J.F.H. Snel, 1985. Regulation ofphotosynthetic electron transport by bicarbonate, formate and
herbicides inisolatedbrokenandintactchloroplasts.PhotosynthesisResearch (inpress).
122
CURRICULUMVITAE
Ikben geboren op 31juli 1952 teLaag-Soerenindegemeente
Rheden (Gld). De middelbare school heb ikgevolgd inZutphen
ophetBaudartiusCollege,alwaar ikhetdiplomaHBS-Bbehaalde in 1970.Daarnaheb ik2jaargestudeerd aandeonderafdelingVliegtuigbouwkunde vandeTechnischeHogeschool inDelft.
In 1974ben ikaandeRijksuniversiteitteGroningenbegonnen
aandestudieBiologie.HetkandidaatsexamenB3,d.w.z.metde
hoofdvakken Biologie enNatuurkunde, heb ikbehaald in1978.
Inmijn doctoraalfase hebikeerstbijdevakgroepPlantenfysiologieonderzoekgedaannaarhetgebruikvanbicarbonaatals
koolstofbrondoorwaterplanten;debegeleidingdoorDr.Hidde
PrinsenProf.RienderHelderheeftmijerggestimuleerd.Vervolgens heb ikbij devakgroep Fysischechemieonderleiding
van Dr.Klaas Nicolay enProf.Rob Kaptein onderzoek gedaan
naar mogelijke toepassing van 31P-NMR op het gebied van de
energieconversie in eukaryote fotosynthetische organismen en
organellen. Daar heb ikmijneerstechloroplastengeisoleerd.
In1981heb ikhetdoctoraalexamen Biologie behaald met als
specialisatiePlantenfysiologie enalsbijvakFysischechemie.
Van 1981 tot 1984ben ik alswetenschappelijk assistent in
dienst geweestbij devakgroep Plantenfysiologisch Onderzoek
van de Landbouwhogeschool teWageningen,alwaarikonder leidingvanDr.ir.JacquesvanRensenenProf.WimVredenberghet
onderzoek gedaan heb dat in ditproefschrift beschreven is.
Vanaf december 1984ben ik als universitair docentintijdelijkedienstbijdevakgroepPlantenfysiologisch onderzoek.
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