Regulation of Respiration and Fermentation to Control the Plant Internal Oxygen
Concentration
Author(s): Ana Zabalza, Joost T. van Dongen, Anja Froehlich, Sandra N. Oliver, Benjamin Faix,
Kapuganti Jagadis Gupta, Elmar Schmälzlin, Maria Igal, Luis Orcaray, Mercedes Royuela, Peter
Geigenberger
Reviewed work(s):
Source: Plant Physiology, Vol. 149, No. 2 (Feb., 2009), pp. 1087-1098
Published by: American Society of Plant Biologists (ASPB)
Stable URL: http://www.jstor.org/stable/40537697 .
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Regulation of Respirationand Fermentationto Control
the Plant InternalOxygen Concentration1[OA1
Ana Zabalza2,JoostT. van Dongen2*,Anja Froehlich,Sandra N. Oliver,BenjaminFaix,
KapugantiJagadisGupta,ElmarSchmälzlin,Maria Igal, Luis Orcaray,
MercedesRoyuela,and PeterGeigenberger
D-14476 Golm-Potsdam,
of MolecularPlantPhysiology,
Max-Planck-Institute
A.F.,
Germany(A.Z., J.T.v.D.,
de
Ciências
Medio
Natural,UniversidadPública de Navarra,Campus
S.O., B.F.,K.J.G.,P.G.); Departamento
Arrosadia,E-31006Pamplona,Spain (A.Z., M.I., L.O., M.R.); Universityof Potsdam,Instituteof Chemistry,
of Vegetableand OrnamentalCrops,D-14979
D-14476Potsdam-Golm,
Germany(E.S.); and Leibniz-Institute
Grossbeeren,
Germany(P.G.)
candropwellbelowambientevenwhentheplantgrowsunderoptimalconditions.
Plantinternal
Using
oxygenconcentrations
roots,we showhow amenablerespiration
adaptstohypoxiato save oxygenwhentheoxygenavailability
pea (Pisumsativum)
ofa regulatory
as substrate
butindicatetheexistence
decreases.Thedatacannotsimplybe explainedbyoxygenbeinglimiting
at whichthe adaptiveresponseis initiatedis independentof the actual
because the oxygenconcentration
mechanism,
is followed
rate.Twophasescan be discernedduringtheadaptivereaction:an initiallineardeclineofrespiration
respiratory
In
in
rate
decreased
faster
a
inhibition
which
the
nonlinear
availability.
upon
decreasing
oxygen
progressively
respiratory
by
ofthe
theinhibition
ofthealternative
oxidasepathwayshowsonlythelinearcomponent
c pathway,
tothecytochrome
contrast
led to
rate,whichultimately
adaptiveresponse.Feedingpyruvateto therootsled to an increaseoftheoxygenconsumption
in thetissuewas further
ofbalancingthein vivo pyruvateavailability
anoxia.The importance
Usingvarious
investigated.
itwas shownthatevenunderaerobicconditions,
linesofArabidopsis(Arabidopsis
knockout
alcoholdehydrogenase
thaliana),
alcohol
role in the controlof the level of pyruvatein the tissue.Interestingly,
alcoholfermentation
plays an important
than
a
low
a
in
the
tissue
rather
induced
the
status
of
to
be
fermentation
by
oxygen
drop
energy
by
primarily
appeared
tochanges
ofoptimizing
thatsensingtheenergystatusis an important
concentration,
plantmetabolism
component
indicating
in theoxygenavailability.
consumingmetabolicpathwaysare adjustedto the
seeGeigenberger,
actualoxygenavailability
(forreview,
and Voesenek,2008). By saving
2003; Bailey-Serres
theplantdecreasesthedemandforrespiratory
energy,
thatcould help to postponeor
oxygenconsumption
evenpreventthetissuefrombecominganoxic.Indeed,
itwas observedthatthemetabolicfluxthrough
glycoloxygenconsumption
ysis slows down, respiratory
decreases,and adenylatelevels drop in responseto
etal.,2000;Bologa
low internal
oxygen(Geigenberger
ofrespiration
is noteasily
et al.,2003).Thisinhibition
c oxilimitation
of
substrate
cytochrome
explainedby
becomes
ofrespiration
dase (COX). First,thereduction
1 Thisworkwas
around20%
alreadyat oxygenconcentrations
by theDeutscheForschungsgemein- apparent
supported
whereastheKmvalue foroxygenof
ofair saturation,
of Educationand
schaft(grantno. Ge 878/1-5),by theMinistry
COX lies around0.05%ofairsaturation
felScience(Spain;grantno. AGL-2004-03784/AGR;
(whichequals
predoctoral
ofNavarre(predocunderstandard
0.14μ,Μ
conditions;
Drew,1997).Second,
lowshipto L.O.),and bythePublicUniversity
toralfellowship
toM.I.).
could be clearlydistinthe inhibitionof respiration
2 Theseauthorscontributed
equallyto thearticle.
whichdid
the
induction
of
from
fermentation,
guished
*
author;[email protected]. not occur until
Corresponding
to zero
to
levels
close
fell
oxygen
to
ofmaterials
forthedistribution
Theauthorresponsible
integral
2003).
(Geigenberger,
in thisarticlein accordancewiththepolicy
thefindings
presented
Based on thesestudies,it is reasonableto assume
is:
intheInstructions
forAuthors(www.plantphysiol.org)
described
that
a sensitivetuningmechanismmust existthat
T. vanDongen([email protected]).
Joost
[OA]
the plant to regulateoxygenconsumption
allows
a
Open access articlescan be viewedonlinewithout subwhile
simultaneously
preventinganoxia. However,
scription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.129288
hardlyanythingis knownabout the mechanismby
withoxygen
Plantsare obligateaerobicorganisms,
formitochondrial
energy
beingan essentialsubstrate
However,thepoordistribution
efficiency
production.
foroxygenthroughroot,tuber,seed, or stemtissue
ofvariousspeciesresultsin steepdropsof theinternal oxygenconcentration,
rangingfromvalues near
above zero to just below 40% of air saturation(e.g.
et al, 2000;
et al., 1994; Geigenberger
Armstrong
et al.,2002;van Dongenet al.,2003,2004;
Rolletschek
Vigeolaset al, 2003).
affect
Theselow levelsofinternaloxygenstrongly
that
studies
showed
metabolism.
Several
energyplant
© 2008 AmericanSocietyof PlantBiologists
PlantPhysiology,
February2009,Vol. 149,pp. 1087-1098,www.plantphysiol.org
1087
Zabalza et al.
which a plant induces adaptive responsesto low
and Chang,2005). Vice versa,
oxygen(Bailey-Serres
comparablylittleis knownabouthow metabolicactheplantinternaloxygenconcentration.
tivityaffects
thatfeedingof
Previousstudiesfocusedon theeffect
had
the
rateof tissue
different
on
respiration
sugars
slices(Lœfetal.,2001)orused transgenic
approaches
a moreenergymetabolism
tostimulate
byintroducing
consumingrouteof Sue degradationvia invertasein
growingtubers(Bologaet al.,2003).In thelattercase,
rateswereincreasedand internaloxygen
respiration
felltoverylow levelsthatwerecloseto
concentrations
zero.Thisshowsthatplantinternal
oxygenconcentrationsrespondverysensitively
tochangesin metabolic
activities.However,the underlyingmechanismremainsunclear.
is partofthecentral
backboneofprimary
Glycolysis
and
metabolism
respiration.
Pyruvate
carbohydrate
servesas a key metabolitelinkingglycolysisin the
Underaerobic
respiration.
cytosolwithmitochondrial
intomitochondria
conditions,
pyruvateis transported
and is oxidizedthroughthetricarboxylic
acid (TCA)
acids
and
NADH.
into
Moreover,
pyrucycle
organic
vate has regulatory
potential,as it was shown that
oxidase(AOX)becomesmoreactivein the
alternative
of
(Millaretal.,
presence a-ketoacidssuchas pyruvate
and
etal, 1995;Vanlerberghe
1993,1996;Vanlerberghe
of ATP
theefficiency
Mclntosh,1997),thusaffecting
production
per unitofoxygenbeingrespired.Under
conditions,pyruvatecan eitherbe
oxygen-limiting
convertedinto ethanolby pyruvatedecarboxylase
(ADH) ortolactate
(PDC) and alcoholdehydrogenase
by lactatedehydrogenase.
Pyruvatealso servesas a
precursorforthe synthesisof Ala via the reversible
which
reactioncatalyzedby Ala amino transferase,
was shownto play a crucialrolein therapidconversion of Ala to pyruvateduringrecoveryfromlowoxygenstress(Miyashitaet al., 2007). Furthermore,
pyruvateis the substrateof acetolactatesynthase,
to thebiosynthewhichis thefirst
enzymecommitted
sisofthebranched-chain
aminoacidsVai,Leu,and He.
Inhibitionof acetolactatesynthaseby the herbicide
in plants
inducesaerobicfermentation
imazethapyr
(Gastonet al., 2002).Despiteitscentralrolein energy
and oxygenmetabolismunder both oxygen-rich
no investigations
weremade,to
depletedconditions,
untilnow on theimpactpyruvatehas
ourknowledge,
on theplantinternal
oxygenconcentration.
The aim of thisstudywas to investigate
theregulation of respirationand its relationto the plant
internal
To investigate
this,we
oxygenconcentration.
of the nutrient
changed the oxygenconcentration
solutionofhydroponically
grownpea (Pisumsativum)
of severalsugarsand
plantsand testedtheinfluence
acids.
We
measured
root
internal
organic
oxygenconcentrations
as well as theenergystatusof thetissue
and relatedthisto therateofoxygenconsumption
by
c pathwayand the AOX. The
both the cytochrome
in relationto thefunction
resultsareinvestigated
and
offermentative
metabolism.
regulation
1088
RESULTS
underHypoxieConditions
RootOxygenConcentrations
andafterFeedingPyruvate
ofmolecularoxygen
Therootinternal
concentration
fromtheoutside
a transect
was measuredthroughout
to thecenterusinga smallneedle-type
oxygensensor
thatwas injectedintothetissue.Pea rootsappeared
hypoxicinsidewithlowestinternaloxygenconcen40% of normalair
trationsof only approximately
saturation(Fig. 1). As the actual amountof oxygen
thatcan dissolvein an aqueous solutiondependson
and solute
suchas temperature
factors,
manydifferent
of diswe describethe concentration
concentration,
thisstudyin relativeunits
solvedoxygenthroughout
of
thatindicatetheamountofoxygenas a percentage
themaximumamountofoxygenthatthesolutioncan
withnormalair.As a
containwhenitis inequilibrium
reference
value,Bensonand Krause(1980)determined
fresh
theoxygenconcentration
of 100%air-saturated
waterat 25.0°Cat 258.2μ,Μ.
when the oxygenconUnderhypoxicconditions,
centration
ofthenutrient
solutionin whichtheroots
weregrowingwas reducedto25% oftheairsaturated
devalue, the plant internaloxygenconcentration
40% to
creased 2 times only,fromapproximately
had no
20% (Fig. 1). This treatment
approximately
a
effect
on thesurvivalofthepea plants.Apparently,
mechanismexiststhatallowstheplanttissueto minimizetheinternaldecreaseof theoxygenconcentrationwhentheoxygensupplyis reduced.
In contrast,
plantssuppliedwith8 mMpyruvatein
thenutrient
solutionshoweda steeperinternal
oxygen
gradientcomparedto control,and the centerof the
rootbecamealmostanoxiceven thoughthenutrient
10011
c
■|
control
ο
D φ
V'
' '
80 - '' Cl
hypoxia
pyruvate
I! 60-'V^^x
U 40 t' ^
I
20
o I"
0.0
Y
0.1
^^5
^
ΤΤτΤτ.·., ,f
0.4
0.2
0.3
Relativediameter
0.5
Figure1. Profileof the oxygen concentrationwithinpea roots.The
surfaceof the root is indicated by 0 and the centerof the root is
indicatedby 0.5 of relativediameter.The measurementswere performedon nontreatedroots(control,whitecircles)after1 d of incubation in a hypoxicnutrientsolution(whitesquares) or after1 d of
incubationin a well-aeratednutrient
solutionsupplementedwith8 mM
pyruvate(black circles).Mean ± se (π = 7-15).
Plant Physiol.Vol. 149, 2009
and PlantInternalOxygen
Regulationof Respiration
solutionwas continuously
aeratedand the oxygen
levelofthesolutionwas wellabovethatofthehypoxic
treatment
(Figs.1 and 2A). Aftera fewdays ofpyruvatetreatment,
a reduction
ofthegrowthratecouldbe
and after3 weeksofpyruvate
the
observed,
treatment,
A
died.
similar
effect
was
observed
mM
when
8
plants
succinatewas suppliedto thenutrient
solution.However,theadditionof8 mMSue or Glc to themedium
had no apparenteffect
on theinternal
oxygenconcentration
(Fig.2A).
Apparently,
pea rootsare not able to controlthe
oftheinternaloxygenconcentration
when
regulation
theTCA cycleis directly
suppliedwithextrasubstrate
by feedingthe organicacids pyruvateor succinate,
whereastheadditionofsimilaramountsofsugarsas
substratesforglycolysisdid not affectthe internal
oxygenconcentration
(Fig.2).
ofRespiration
underHypoxic
Conditions
and
Regulation
after
FeedingPyruvate
Oxygenconsumptionrates were measuredwith
Clark-typeoxygen electrodeson root pieces that
were cut fromplantsthatwere pretreatedfor 1 d
with8 mMofSue,Glc,pyruvate,
orsuccinate(Fig.2B).
The additionof sugarshad no stimulatory
effecton
rate(B) of
(A) and respiratory
Figure2. Internaloxygenconcentration
nutrient
solutions.The hydropea rootsthatwere grownin different
solutionswere well aeratedand eitherused directlyfor
ponic nutrient
the measurements
(control)or supplementedwithSue, Glc, pyruvate,
or succinate(finalconcentration
8 mM)1 d beforemeasuring.Forthe
the nutrient
solutionwas aeratedfor1 d withgas
hypoxiatreatment,
thatconsistedof4% (v/v)O2, 0.035% CO2, and therestvolumeN2.The
values for the internaloxygen concentrationrepresentthe lowest
as measuredin the middleof a root.Mean ± se
oxygenconcentration
lettersmarkmean values thatare significantly
differ(η = 3). Different
entfromeach other(ANOVA; Ρ < 0.05).
PlantPhysiol.Vol. 149, 2009
whereas feedingpyruvateor succinate
respiration,
increasedtheoxygenconsumption
rateby2-to3-fold.
the
rateunderhypInterestingly, oxygenconsumption
oxic conditionsdecreasedsignificantly
as compared
to the controltreatment.
Hypoxic respirationwas
measuredon root pieces thatwere incubatedin a
bufferwith an oxygenconcentration
of 25% of air
saturation(Fig. 2B). The inhibitionof respiration
at
hypoxiais likelyto be associatedwithan adaptive
metabolicresponsein orderto save oxygenwhenits
becomeslimited.
availability
To evaluatethechangesin respiration
underhypoxic conditionsmoreprecisely,
theoxygenconsumption rate of rootpieces was measuredin a tightly
closed Clark-type
oxygenelectrode,startingwitha
buffer
and lastinguntilall oxygen
fullyair-saturated
was consumedfromthe solution(Fig. 3). As the
in thebuffer
decreases,therespioxygenavailability
of
the
tissue
slows
down in a biphasic
ratoryactivity
manner.Untilan oxygenconcentration
of approxiis reached,therespiratory
mately20%ofairsaturation
rateis decreasingslowlyaridtheinhibition
correlates
in a linearmannerto theoxygenconcentration
ofthe
solution.However,when oxygenconcentrations
decreasebelow thisthresholdvalue,the declineof the
curvesgetsprogressively
oxygenconsumption
steeper
untiltherespiration
ratereacheszero.Itis noteworthy
thatthissecondphase does notshowlinearbehavior.
Theinhibition
ofrespiration
as shownin Figure3 is
notdue tocelldeathoccurring
duringthecourseofthe
because identicaloxygenconsumption
experiment,
curveswereobservedrepeatedly
whentheincubation
solutionwas re-aerated
aftertheoxygenconcentration
inthemediumreachedzeroand themeasurement
was
performedagain (data not shown). It can also be
excludedthattherateofoxygendiffusion
the
through
tissuebecomeslimitingat low externaloxygenconin the medium,as similaroxygenconcentrations
sumption curves were observed for pea roots
2 mm;Fig.3A),Arabidopsis
(diameterapproximately
thaliana)roots (diameterapproximately
(Arabidopsis
200 μτη.;Fig. 3B), and Chlamydomonas
cell culture
20 μ,ΐη;data
(individualcell size of approximately
not shown).Apparently,
the diffusion
resistancefor
does
not
influence
the
oxygen
adaptiveresponseof
to hypoxia.
respiration
Feedingtheorganicacids pyruvateor succinateto
theroots(Fig.4) resultedin a strongincreaseof the
rateat all oxygenconcentrations
oxygenconsumption
measured,buttheshapeofthecurveremainedsimilar
to the controltreatment,
as describedin Figure3.
Sue
or
Glc
to
the
mediumhad no effecton
Adding
eithertheoxygenconsumption
rateortheshapeofthe
curve.
Our data show thattherateofrespiration
depends
on the externaloxygenconcentration.
The biphasic
behaviorof thishypoxicresponseindicatestheexistence of an adaptive mechanismthat changesthe
extentto whichrespiration
is inhibitedas a function
oftheoxygenavailability.
1089
Zabalza et al.
fromrootsof pea (A) and
Figure3. Rateof respiratory
oxygenconsumptionas a functionoftheexternaloxygenconcentration
was
rootswere keptin a hermetically
closed vial in which the oxygenconcentration
Arabidopsis(B). Forthe measurements,
measuredthroughtime.Due to the respiratory
activityofthetissue,oxygendepletedfromthe solution.Respirationrateswere
calculated fromthe raw data and plottedhere againstthe externaloxygen concentration.The graphsshow the values of
thatwere repeatedat leastfourtimes.
representative
experiments
ofAOXandCOXunderHypoxia
Regulation
To investigatehow theoxygenavailabilityaffects
thedifferent
terminaloxidasesofthemitochondrial
chain(AOX and COX), we deterelectrontransport
mined the adaptive response to hypoxia in the
presenceof inhibitorsforeitherCOX or AOX. The
acid (SHAM)
additionof 20 mMsalicylhydroxamic
resultedin a slightdecrease of respirationfrom
about 12% oxygenmin"1100 mg freshweight(FW)
to 8% oxygenmin"1100 mg FW (Fig. 5). Subsequently,therateof oxygenconsumptioncontinued
todeclinesteadilyuntilitreachedzero (Fig.5,A and
B). A slightdeviationbetweenthe measured data
linethatfittedto the
straight
pointsand a theoretical
of
data obtainedbetweenan oxygenconcentration
40% and 20% of air saturationcould be observed
(Fig.5, A and B). Thisis likelytobe explainedby the
same mechanismthatcauses thedeclinein respirabelow 20% of air
tion at oxygen concentrations
as observedforrootsthatwerenottreated
saturation
c pathwithSHAM. In eithercase, the cytochrome
way changes its activityin a hypoxia-dependent
manner,ultimately
reachingzeroactivitywhenoxygen depletescompletely.
c
In contrastto this,inhibition
of the cytochrome
pathwayusing200μΜKCN resultedin an immediate
oxygenconsumption
steep drop of the respiratory
ratebeforeKCN addition,
(Fig.5; oxygenconsumption
KCN ad16%O2min"1100mgFW;after
approximately
dition,3% O2min"1100mgFW).Ittookapproximately
5 minbeforecyanidereachedits maximalinhibitory
therateofoxygenconsumption
effect.
Subsequently,
in a remarkably
decreasedfurther
lineardependency
thateven at
to the oxygenavailability.
It is striking
near-anoxic
whenhardlyanyfreeoxygen
conditions,
was availableinthesolution,
theoxygenconsumption
ratestillremainedwell above zero. Clearly,hypoxia
rateby AOX
does notaffect
theoxygenconsumption
to the same extentas it does for the cytochrome
c pathway.
1090
underHypoxiaand
Induction
ofFermentative
Pathways
afterFeedingPyruvate
was investiThe inductionoffermentative
activity
gatedin pea rootsin whichtheinternaloxygenconwas reducedeitherby treatingthe roots
centration
solution(25%ofairsaturation)
witha hypoxicnutrient
of the respiratory
or via up-regulation
oxygenconsumptionafterfeedingpyruvatefor1 or2 d. Fermentative activitywas evaluated by measuringthe
extractable
enzymeactivitiesof both PDC (Fig. 6C)
theirprotein
and ADH (Fig.6D) as well as detecting
abundance using westernblottingand measuring
subsequentethanoland acetaldehyde(Fig. 6, Ε and
in theroots.Lactatemetabolism
was
F) accumulation
Figure4. Rateof respiratory
oxygenconsumptionas a functionofthe
externaloxygen concentrationof pea roots incubated in different
The concentration
nutrient
solutionsfor1 d priorto themeasurements.
or organicacids was always8 πγιμ.
ofthesupplementedcarbohydrates
D, Pyruvate;A, succinate;■,Sue; Δ, Glc. Respirationratesfromroots
wereas shownin Figure
thatwere notsupplementedwithanysubstrate
3. Also, the experimentaldetails are as describedforFigure3. The
experimentsthatwere regraphsshow the values of representative
peated at leastthreetimes.
Plant Physiol.Vol. 149, 2009
and PlantInternalOxygen
Regulationof Respiration
inhibitorsSHAM (black symbols)and
Figure5. Effectof respiratory
KCN (whitesymbols)on the rateof respiratory
oxygenconsumption
whiletheoxygenavailabilitydeclines.A, The fullprofileof respiration
fromfullsaturation(100% of air saturation)ofthe incubationsolution
to full depletion (0% of air saturation).Inhibitorswere added as
indicatedby the arrowsin the figure.The graydottedline fitsto the
thecytochromec pathway.B, The
linearpartofthegraphrepresenting
same data as in A, but scales on the horizontaland verticalaxis are
adjusted such thatthe low oxygen responsecan be observed more
precisely.The experimentalsetup is as described for Figure3. The
experimentsthatwere regraphsshow the values of representative
peated at leastthreetimes.
thatcan be detectedwithintissueis underestimating
therealamountofethanolbeingproduced.Nevertheless,theincreasein ethanolthatwas measuredproves
unequivocallythatnot onlyare the enzymesof the
fermentative
pathwayinducedbut thatfermentation
is also operatingin vivo.
on thehypoxictreatment,
Unliketheobservations
theabundanceofPDC and ADH enzymetook2 d of
pyruvatefeedingto increase(Fig. 6, C and D). This
because theroot
observation
is especiallyinteresting
internaloxygenconcentration
(Fig. 6A) was much
lowerin thepyruvate-fed
plantsthanin thehypoxic
the steadytreatment.
As in the hypoxictreatment,
statelevelsof pyruvateand acetaldehydewithinthe
rootdid notchangeafterfeedingpyruvate(Fig.6, F
and G). However,the strongincreaseof respiratory
as wellas theincreaseofthelevel
oxygenconsumption
ethanolwithintherootsafteraddingpyruvateto the
mediumare a clearindicationfortheuptakeofpyruvateby therootsand subsequentmetabolization.
thattheinductionoffermentation
The observation
roots even thoughthe
is delayed in pyruvate-fed
was muchlowerthan
internaloxygenconcentration
in rootsfromthe hypoxiatreatment
indicatesthat
feedingpyruvateattenuatessignalsthatregulatefermentativeactivityin responseto low oxygen.To
bethisfurther,
we testedthecorrelation
investigate
and the energy
tweenthe inductionof fermentation
status,expressedas the ratioof ATP to ADR In the
plants,the energystatus dropped
hypoxia-treated
roots
the
first
day,whereasin pyruvate-fed
during
theenergystatusdid notdecreasebeforethesecond
a decreasein
dayofthetreatment
(Fig.6H). Obviously,
oxygenis linkedwiththe inductionof fermentative
enzymesvia changesin theenergystatus.
HaveIncreased
adh-nulloradh~ Mutants
Pyruvate
LevelsunderNormoxia
Pyruvateis theprimarysubstrateforfermentation
inplants.As theavailability
was shownto
ofpyruvate
as well as the
affect
respiratory
oxygenconsumption
it was investiplant internaloxygenconcentration,
metabolism
is involvedin
not consideredin our work,because earlierstudies
whether
fermentative
gated
withpea concludedthatbothlactatedehydrogenase the regulationof the pyruvatecontentin plants.
we comparedpyruvateaccumulationin
Therefore,
activityas well as lactate accumulationwere not
in
roots
in different
of
fermentation
adh
two
mutants
considerable
pea
Arabidopsisecotypes
parameters
Zabalza
et
and
al.,
Rees,
1979;
2005).
(Bensheim[Be-0] and Columbia [Col-0]) with the
(Smith ap
external
As expected,
hypoxiainducedfermentative pyruvatelevelsin wild-typeplantsof the same ecois a lossandproteinabundancewithin1 d and
type.Theadh mutantintheBe-0background
enzymeactivity
ADH
mutant
without
of-function
led to sustainedlevelsafter2 d. BothPDC and ADH
(adh-null)
activity
underanoxia(Jacobs
etaL,1988),whereasthemutation
were induced more than 2 timescomparedto the
intheCol-0background
leadstoa severereduction-ofcontrol.The level of pyruvate(Fig. 6G) as well as
as itis stillable to induce
function
(adh~) phenotype,
(Fig.6F) intheroottissuedid notchange
acetaldehyde
but ethanolprobut
ethanol
mutated
in
of
the
the
of
mRNA,
treatments,
expression
significantly any
withintherootswas detectableafter4
ductionunderanoxia is reducedto verylow levels
accumulation
ethanol
d ofhypoxiaand after7 d thelevelofinternal
(Banti et al., 2008). Seedlingswere kept eitherat
increased(Fig. 6E). It should be
ambientoxygenor underhypoxicconditionsfor24
was significantly
thelevelsofpyruvateand phosphonoted thatethanolis a highlyvolatilemetabolite. h. Subsequently,
weremeasuredand comparedto
itis tobe expectedthatthelevelofethanol
Therefore,
(PEP)
enoZpyruvate
PlantPhysiol.Vol. 149, 2009
1091
with
onvarious
tobe linked
oftheeffect
ofhypoxia
orpyruvate
thataresuggested
6. Overview
parameters
Figure
supplementation
in themiddleofa root(A),oxygen
in pea roots:internal
theinduction
offermentation
as measured
oxygenconcentration
insidetheroottissue(E),
inthenutrient
concentration
ofethanol
solution
(C),ADHactivity
(D),theconcentration
(B),PDCactivity
theroottissue(F),theconcentration
ofpyruvate
theroottissue(G),andtheratioof
ofacetaldehyde
within
within
theconcentration
the
ATPtoADPas a measure
ofthetissue(H).Theinsetsingraphs
immunoblots
fortrieenergy
status
C andD represent
showing
thevarious
PDC andADH.Samplesfrom
weretaken24 h (day1) and48 h(day2)
treatments
abundance
of,respectively,
protein
toa nutrient
solution
orafter
after
ofpyruvate
toair-saturated
addition
nutrient
solution
(barsarelabeledwithpyruvate)
switching
nutrient
withan oxygen
of25% ofairsaturation
Control
concentration
(barslabeledwithhypoxia).
plantsgrewinair-saturated
were
Further
theexperimental
started.
andsamplesfrom
weretakenshortly
before
control
solution
theseplants
treatments
samples
Becausethecontrol
withthesamplestakenfrom
thevarious
treatments.
takenduring
thecourseoftheexperiment,
simultaneously
The
andgiveninthefigure.
themeanvalueofall control
variation,
sampleswascalculated
samplesdidnotshowanysignificant
weretaken4 and7 d after
orhypoxic
started.
thepyruvate
treatment
nation
(A)oracetaldehyde
(F)determi
samplesusedforethanol
± se.Different
Thebarsrepresent
mark
different
theaverage
ofatleastthree
measurements
letters
meanvaluesthataresignificantly
from
eachother(ANOVA;Ρ < 0.05).
1092
Plant Physiol.Vol. 149, 2009
and PlantInternalOxygen
Regulationof Respiration
thelevelsinwild-type
plants(Fig.7). Inbothwild-type
accessions,PEP and pyruvateaccumulatedsignifi(P < 0.05).The
cantlyduringthelowoxygentreatment
ratioPEP topyruvate
(P < 0.05),
droppedsignificantly
flux
an
increased
metabolic
of carbon
to
pointing
mediatedby theenzymepyruvatekinase(PK). In a
it was observedthat ethanol
parallel experiment,
emissionfromthe roots of wild-typeCol-0 plants
increased4.8 timesunder near anoxia
significantly
(1% [v/v]oxygenin air)as comparedto thenormoxic
control(P < 0.05;Fig. TO). Fromrootsof wild-type
Be-0,itleaked2.7timesmoreethanolintothemedium
as comparedto
duringthe near anoxic treatment
itis plausible
normoxia(P < 0.05;Fig.7D). Therefore,
of
to assumethatthePasteureffect
(i.e. theactivation
to
attributes
is
when
fermentation
induced)
glycolysis
thechangesdescribedin theratioPEP to pyruvate.
Boththe adh-nulland the adh mutantline had
increasedlevelsofpyruvateas compared
significantly
to wild-typeplantsundernormoxicconditions(P <
thePEP to pyruvateratio
0.05;Fig.7). Concomitantly,
was muchlowerthanin wild-type
plants.Upon lowno
further
increase
of thelevel of
treatment,
oxygen
in
the PEP to
or
change
pyruvate any significant
pyruvateratio was detectedin eitherof the adh
mutantslines(P < 0.05).
The increasedlevelsofpyruvatein theadh mutant
thatthe
linescomparedtowildtypecanbe interpreted
alcoholfermentation
pathwayalso playsa roleunder
aerobic conditions.This idea is supportedby the
abovethatevenunderaerobic
mentioned
observation
conditions,a small amountof ethanolcould be obnotonlyfuncserved.Thisimpliesthatfermentation
NAD fromNADH tokeepglycolysis
tionstoregenerate
Figure7. The impactof ADH activityin 3-week-old
Arabidopsisseedlingson the levels of pyruvate(A),
PEP (B), and the ratioPEP to pyruvate(B) was tested
underambientair conditions(21% [v/v]oxygenin
the air; gray bars) or after24 h of a near-anoxic
withair containing1% (v/v)oxygen(white
treatment
bars). The seedlings used for these analyses were
growingon sterilehorizontalagar-plates.The levels
of pyruvateand PEP were measured in plants of
the wild-type(WT) Arabidopsisaccessions Be-0 and
Col-0 and comparedwiththevalues measuredintwo
independentadh mutants(adh-null in the backgroundof Be-0, and adh" in the Col-0 background).
In a parallel experiment,ethanolproductionby the
two wild-typeaccessions was determined(D) by
measuringethanol leakage into liquid incubation
theseedlings,whichwas either
mediumsurrounding
aeratedwithambientair,or withair containing1%
(v/v)oxygen. Multiplecomparisonanalysis by the
Holm-Sidaktestfollowinga two-wayANOVA indicated the oxygen-induced differencesbetween the
ratio
levelsof pyruvate,
PEP,and the PEP-to-pyruvate
in wild-typeplantsas significant
(P < 0.05). Within
knockoutlines,all values measuredwere notstatisfromeach other (P < 0.05). Bars
tically different
±
the mean of at leastthreemeasurements
represent
se. Bars markedwith the same letterdo not differ
fromeach other,n.d., Not determined.
significantly
Plant Physiol.Vol. 149, 2009
1093
Zabalza et al.
but it also helpsto lowerthelevel ofpyrurunning,
vate, therebypreventingthe inductionof oxygen
consumption
by pyruvateas describedbefore.
thismechanismhelps to determinethe steady-state
withinplanttissue.The control
oxygenconcentration
each
mechanismcomprisesat leasttwo components,
actingat different
oxygenconcentrations.
DISCUSSION
Controlof the InternalOxygenConcentration
by the
Level of Respiratory
Substrates
Plantsdo nothave an efficient
distribution
system
foroxygenthroughout
theirtissues.Therefore,
oxygen
etal.,
can reachverylow internal
levels(Geigenberger
2000; Gibon et al., 2002; Rolletscheket al., 2002;
Vigeolaset al., 2003;van Dongenet al., 2004).Several
and-consuming
studiesshowedthatenergy-generating
metabolicpathwaysareadjustedto theactualoxygen
(forreview,see Geigenberger,
2003).Until
availability
now,littleis knownaboutthemechanisms
by which
inplants.In this
theseadaptiveresponsesaretriggered
study,we used hydroponically
grownpea rootsto
in relationto
theregulation
ofrespiration
investigate
theplantinternal
oxygenconcentration.
Regulationof Respirationby Low OxygenInvolvesTwo
ComponentsActingat Different
OxygenConcentrations
in Plants
Undernormalconditions,
pea rootshave internal
concentrations
that
are
about40% ofair satuoxygen
ration(Fig.1), similarto whathas been describedfor
othertissues(Geigenberger
et al., 2000; Rolletschek
et al., 2002;van Dongenet al, 2003,2004).Whenthe
externaloxygenconcentration
is reduced4-fold,the
internaloxygenconcentration
goes down only2-fold
(Fig. 1, hypoxiatreatment),
indicatingan adaptive
of
the
ratehelpingthe
regulation
oxygenconsumption
tissueto preventanoxia.Indeed,it can be observed
thattherateof oxygenconsumption
dependson the
As shownin Figure3, a reduction
oxygenavailability.
oftheoxygenavailability
resultsin a slow but linear
decrease of the respiratory
rate until a remaining
of20% ofair saturation.
Below
oxygenconcentration
thisconcentration,
a steepdeclineoftheoxygenconsumptionratecan be observed.Suchbiphasickinetic
behaviorcan only be explainedwhen at least two
mechanismsare involvedin parallel to
controlling
controlrespiratory
as a function
oftheoxygen
activity
It is not credibleto describethe kinetic
availability.
behaviorsimplyby a passivedependenceto theoxysuch as Michaelis-Menten
kinetics
gen concentration
superimposedon a lineardiffusion
component.An
forthisis thattheinternal
important
argument
oxygen
concentration
reachesvalues close to anoxia after
feedingpyruvate,whereasthe oxygenconsumption
ratebecomesmuchhigherthanundercontrolconditions.This indicatesthatthe oxygenaffinity
of the
must
be
not
to
limit
respiratory
system
highenough
eveniftheoxygenconcentration
decreases
respiration,
below25% ofairsaturation.
It is therefore
concludedthattherateofrespiration
is closelyregulatedby the oxygenavailabilityin a
manner.It is suggestedthat
concentration-dependent
1094
In contrastto the inhibitionof respiration
under
or
to
the
roots
led
succinate
hypoxia,feedingpyruvate
to a sharpincreasein the oxygenconsumption
rate
(Fig.2B).Thiscoincideswitha severedropintheplant
internaloxygenconcentration
(Figs. 1 and 2A) even
whenthenutrient
solutionwas well aeratedand the
ofthissolutionneverdecreased
oxygenconcentration
below 80% of air saturation.The firstday afterthe
thetissuein themiddleof
started,
pyruvatetreatment
therootbecamealmostanoxic(Fig. 1). Interestingly,
thebiphasiccharacteristic
shape ofthekineticcurves
is independentof the substratebeing added to the
medium(Fig. 4). Apparently,
feedingorganicacids
does notchangethemechanism
by whichrespiration
is reducedwhen theoxygenavailability
goes down,
butthestrongincreaseoftheoxygenconsumption
rate
as comparedto thecontroltreatment
(comparewith
Fig.3) does lead to a severedropin theplantinternal
forwhichtheadaptiveresponse
oxygenconcentration,
Itis notewortohypoxiacannotcompensate
anymore.
does not
thythatthe maximumrate of respiration
increasewhenextraSue ofGlcis suppliedtotheplants
withinthistimeframe.Apparently,
is able
glycolysis
to regulatethe amountof pyruvatethat becomes
availableforrespiration,
theinterthereby
regulating
nal oxygenconcentration
ofthetissue.
Fromthesedata it is concludedthatthe internal
oxygenconcentration
dependson therateofrespiraand thatthisis linkedtothe
toryoxygenconsumption
amountof substratethatis availableforrespiration.
theglycolytic
Apparently,
pathwayis capableofbufftotheTCA cycle,thereby
eringthesupplyofsubstrate
notonlytherespiratory
butalso the
affecting
activity
plantinternaloxygenconcentration.
Several controlsites are suggestedto be able to
regulatethe flux of substrateinto the TCA cycle.
couldcontroltheproduction
Firstly,
glycolytic
activity
of pyruvate(for review,see Plaxtonand Podestá,
2006).Important
regulatory
enzymesin theglycolytic
PEP directlyintopyrupathwayare PK (converting
vate)and PEP carboxylase
(whichis thefirst
stepofan
PEP intopyruvate).
alternative
pathwayconverting
Hatzfeldand Stitt(1991) showed that
Interestingly,
ofglycolysis
onlythestimulation
byPK resultedin an
increaseofoxygenconsumption.
Moreover,
analysisof
a suiteofmetabolite
and theTCA
levelsofglycolysis
from
cycleafterdecreasingtheoxygenconcentration
100%to20% airsaturation
in tubertissuerevealedPK
as one of theseveralregulatory
sitesthatlead to an
inhibitionof respiration(Geigenberger
et al., 2000).
one possiblemechanismfortheregulation
Therefore,
ofoxygenconsumption
and thusplantinternal
oxygen
Plant Physiol. Vol. 149, 2009
and PlantInternalOxygen
Regulationof Respiration
This
concentration
is via the controlof PK activity.
in
is
with
our
hypothesis
good agreement
present
observation
thattheavailability
ofpyruvate
affects
the
Severalallosteric
internal
oxygenconcentration.
reguPK activity
lationmechanisms
thataffect
are already
described(Podestaand Plaxton,1991,1992)and itwill
be ofsubstantial
interest
to investigate
theregulation
ofPK inresponsetolow oxygenand toinvestigate
the
adaptivelow oxygenresponsein plantswithaltered
PK activity.
pyruvatelevels were stronglyinInterestingly,
creased in tubersexpressinga more effective
but
lessenergy-saving
routeofSue degradation
via invertase (Trethewey
et al., 1998) or Suc phosphorylase
et
Theincreaseinpyruvate
levels
al.,
2001).
(Trethewey
in theselineswas accompaniedby a stimulation
of
and a strongdecreasein internaloxygen
respiration
in growingtubers(Bologaet al., 2003).
concentration
both
Interestingly, COX- and AOX-dependent
pathin the transwere up-regulated
ways of respiration
genictuberscomparedto wild type(Centenoet al.,
2008).
A secondcontrolmechanismofinterest
is theregulationoftheimportofpyruvate
intotheTCA cycleby
thepyruvatedehydrogenase
(PDH) complexthatcatofpyruvate
intoacetyl-CoA.
The
alyzestheconversion
PDH
of
the
is
to
a
activity
complex subject
varietyof
mechanisms.First,the PDH complexis
regulatory
feedbackregulatedby NADH (Randalland Miernyk,
1990). So, when oxygenis completelyabsent and
NADH accumulates,pyruvatewill not be shuttled
intotheTCA cycleanymore.This would resultin a
reductionof the respiratory
rate.Second,the PDH
via PDH
complexis inactivated
by phosphorylation
kinase(Tovar-Méndez
et al., 2003).Interestingly,
this
kinaseis inhibited
ofthe
by pyruvate.So, theactivity
PDH complexis stimulated
whenpyruvateis present.
A betterunderstanding
of the importanceof the
possiblecontrolsiteswill need reversedgeneticapproachesand morestudiesresolvingthe subcellular
of pyruvateand NADH between
compartmentation
cytosol,plastid, and mitochondria.Such studies
wouldbe mostinteresting
toobtaininformation
about
thefineregulation
ofthepyruvatecontrolmechanism
at low oxygen.
brane.Therefore,
the alternative
pathwayof respirationvia AOX has a lowerpotentialto produceATP
fromoxygenthantheCOX pathwayhas.
Pyruvateand otherα-ketoacidshave a significant
effect
on theactivity
ofAOX (Millaretal.,
stimulating
et al., 1995).Althoughthesignif1993;Vanlerberghe
icance of this controlmechanismin vivo has been
discussed(Vanlerberghe
and Mclntosh,1997;Pastore
et al., 2001),it could be shownin transgenic
potato
thata decreasein the level of
(Solanumtuberosum)
ofcytosolic
PK in
pyruvate
bysilencingtheexpression
tubersleads to a concomitant
decreasein theamount
ofAOX protein(Oliveret al.,2008).In linewiththese
earlierobservations,
pyruvatefeedingto pea roots
induced
the
rate of oxygenconsumption
strongly
(Figs.2 and 4). However,fromour data it cannotbe
concludedif thisis theresultof an activationof the
oxidasesor due to an increaseoftheoxidaseprotein
abundance.
Tofindouthowtheindividualrespiratory
pathways
reacton changesintheoxygenavailability,
theactivity
ofeitherAOX orCOX was inhibited
usingthespecific
chemicalinhibitors
SHAM and KCN,respectively.
The
data showthatbothpathwaysare presexperimental
entin roots,thoughthecapacityoftheAOX pathway
is much less than the capacityof the cytochrome
therewas a differential
pathway.Most interesting,
effect
of hypoxiaon thekineticbehaviorof theindividual pathways.In the presence of SHAM, the
oftheCOX pathwayis stillshowing
remaining
activity
the biphasicdeactivationat low oxygen,ultimately
leadingtoa completestopoftheoxygenconsumption.
In contrastto this, the cyanide-resistant
pathway,
which is mainlydue to the activityof AOX, does
onlyshowlinearinhibition
bylow oxygenbutremains
activeuntilall oxygenis absolutelybeingconsumed.
itwas shownforbovineheartCOX that
Previously,
of theenzymecomplexat low oxygen
preincubation
concentrations
reducedVmax
and slightly
increasedKm
foroxygen(Chandel et al., 1996). It is temptingto
speculatethatplantCOX canbe regulatedin a similar
way,whereasAOX does notpossesssucha regulatory
mechanism.
becauseoftheloweroxygenNevertheless,
of
itis reasonabletoassume
AOX,
to-energy
efficiency
thatAOX activityin vivo shouldneverexceedCOX
underhypoxicconditions.
Thiscould simply
activity
be
achieved
when the affinity
foroxygenof COX is
The Role of COX and AOX in theControlof theInternal
much higherthan thatof AOX. Also, it cannotbe
OxygenConcentration
excludedthatthe breakdownof energyparameters
An alternativeregulatorymechanismto control
afterKCN additionand further
detrimental
effects
of
is
located
within
the
mitochondrial
thispreventedtheadaptiveresponseofAOX to oxyrespiratory
activity
electron
chainitself.Thereare twodifferent gen.Directanalysesof kineticproperties
on purified
transport
COX andAOX.
COX and AOX willbe necessarytoclarify
thisfurther.
enzymesthatuse oxygenas a substrate,
In thefirst
COX (also knownas complex-IV)
pathway,
is catalyzing
thetransfer
ofelectrons
fromcytochrome Inductionof Fermentation
Is Mediatedby a Drop in the
c to molecularoxygen,therebytransporting
protons
Status
Energy
acrossthemitochondrial
innermembrane.
Thesecond
The best-known
functionof fermentative
metaborespiratory
pathwayis catalyzedbytheenzymeAOX,
whichtransfers
electronsdirectlyfromubiquinolto
lismis torecycleNADH toNAD toavoiddepletionof
the cytosolicNAD pool and inhibition
of glycolysis
oxygenwithoutcarryingprotonsacross the memPlantPhysiol.Vol. 149, 2009
1095
Zabalza et al.
whenoxidativephosphorylation
is limited.Becauseall
fermentative
prominent
pathwaysuse pyruvateas
fluxis maintainedor
initialsubstrate,
the glycolytic
via glycolyevenincreasedto secureATP production
sis. The resultsof our biochemicalapproachesshow
thatcontrolling
is veryimportant
thelevelofpyruvate
when the
forthe regulationof oxygenconsumption
we showed
becomeslow.Moreover,
oxygenavailability
by using Arabidopsisknockoutlines thatwere imthatthealcoholfermentation
pairedin ADH activity
is
for
pathway important balancingpyruvatelevels
fermentation
undernormoxicconditions.
Apparently,
NAD from
in plantsservesnot only to regenerate
the
butalso toregulate
NADH underanoxicconditions
rate.
the
level
to
control
respiration
pyruvate
underaerobicconditions
Inductionoffermentation
has beendescribedearlier(Bûcheret al., 1995;Tadege
and Kuhlemeier,
1997).In germinating
pollen,it was
fermentafoundthatatnormaloxygenconcentrations,
occursin orderto increase
tivepyruvatedegradation
via
thefluxofcarbonfrompyruvateintorespiration
that
Our
data
indicate
PDC and theglyoxylate
cycle.
underaerobic
metabolism
thefunction
offermentative
conditionsto regulaterespiratory
activityis much
assumed.
moregeneralthanpreviously
notlimitedto
is apparently
As alcoholfermentation
ofwhat
is
raised
the
anoxicconditions
only, question
ofPDC and ADH under
theinduction
factorinitiates
ofboth
InFigure6,theinduction
ofactivity
lowoxygen.
of theseenzymesis comparedto theinternaloxygen
concentration
as wellas theenergychargeofthetissue.
Plantsthatweretreatedwithpyruvatein thenutrient
as
solutionhad lowerinternaloxygenconcentrations
(Fig. 6A), but both
comparedthe hypoxictreatment
PDC andADH werenotinducedafter1 d (Fig.6,C and
well withthe
correlated
D). Inductionoffermentation
hereas theratioof
statusofthetissue,expressed
energy
ATPto ADP. In thehypoxia-treated
plants,theenergy
inpyruvatewhereas
statusdroppedduringthefirst
day,
fedroots,theenergystatusdid notdecreasebeforethe
seconddayoftreatment
(Fig.6F).Inparalleltothis,the
ofbothPDC and ADH wereinduced.Thisinactivity
theinductionofferdicatesthatpyruvateattenuates
at low oxygenbykeepingtheenergystatus
mentation
entheinductionoffermentative
constant.
Obviously,
dependentontheoxygenconzymesis notexclusively
butitis also linkedtochangesintheenergy
centration,
is oneofthemostsensitive
status.As ADH expression
tohypoxiabeingaffected
reactions
bya milddecrease
inoxygenavailability
already(vanDongenetal.,2009),
ofoxygenon
itis plausibletoassumethatdirecteffects
modlevelofthegenearesubsequently
theexpression
ifiedbytheenergystateofthetissue.
CONCLUSION
Fromthe data presentedin this study,it is conis
cludedthattheplantinternal
oxygenconcentration
ofthe
activity
regulatedvia adjustingtherespiratory
1096
Thisadaptive
tissueto theactualoxygenavailability.
to
or
is
response suggested postpone even prevent
anoxia in the tissue when the oxygenavailability
decreases.The decline in respiratory
activityas a
canbe described
oftheoxygenconcentration
function
thatmorethanone
phases,indicating
bytwodifferent
to COX,
mustexist.In contrast
mechanism
regulatory
whichshows the biphasicresponseto hypoxia,the
whentheoxygenavailability
decreaseinAOX activity
down
is
justlinearlyrelatedto theoxygencongoes
This indicatesthatthe various terminal
centration.
chain
electron
oxidasesofthemitochondrial
transport
to
mechanisms
have different
adjusttheir
regulation
concentration.
to
the
internal
oxygen
activity
to
our resultsshow thatmechanisms
Furthermore,
and thustheinternal
adjusttheoxygenconsumption
of
are linkedto theavailability
oxygenconcentration
fermentaunder
aerobic
conditions,
Also,
pyruvate.
rolein balancing
tivemetabolism
playsan important
inthecell.Evidenceis shownthat
thelevelofpyruvate
theinductionof
theenergystatusofthetissueaffects
under
low
fermentative
oxygen.
enzymes
MATERIALS AND METHODS
PlantMaterial
as
culture
Pea (Pisumsativum
'SnapSugarBoys')was growninhydroponic
solutionwas continuously
describedin Zabalza et al. (2005).The nutrient
excessivegrowthofbacteria
aeratedand was replacedevery4 d to prevent
and algaeinthesolution.
Fortheexperiments,
plantsthatwere12d oldwere
solution
weresuppliedtothenutrient
andsuccinate
used.Sue,Glc,pyruvate,
thenutrient
ata finalconcentration
of8 mM.Hypoxiawas appliedbyaerating
witha gasmixture
ofoxygenandnitrogen
solution
suppliedwith0.035%CO2
in thenutrient
solutionof25%ofair
to achievea finaloxygenconcentration
was doneinthemiddleofthelightperiod.Thepyruvate
saturation.
Sampling
in
in thenutrient
solutionwas evaluatedto studyitsstability
concentration
Theinitialconcentration
thenutrient
solution.
(8 mM)hardlychangedduring
after24 h eitherin the
thefirstday oftreatment
(7.27 ± 0.33mMpyruvate
ofplants).However,
absenceorpresence
duringthesecondday,thepyruvate
in thenutrient
solutionreducedto 5.78 ± 1.01mMwithout
concentration
in thenutrient
solution.
plantsand to 1.23± 0.37mMwithplantsgrowing
of Arabidopsis{Arabidopsis
thaliana)
ecotypeCol-0
Respiratory
activity
Forthis,seeds
onhydroponically
rootswasinvestigated
grownplantmaterial.
and
with70%ethanoland 10%sodiumhypochlorite
weresurfacesterilized
rinsedseveraltimeswithdistilledwaterbeforesowingthemon stonewool
GrodanBV) thatwas
tray(Klemplugstandard,
plugskeptin a polysterene
fromLoque et al.,2003:1 mM
modified
soakedin nutrient
solution[slightly
50
1 mMMgSO4,250μΜCaCl2,0.1mMNa-Fe-EDTA,
NH4NO3,1 mMKH2PO4,
μΜKC1,50 μΜH3BO3,5 μΜMnSO4,1 μΜZnSO4,1 μΜCuSO4,and 0.1 μΜ
Seedswereincubatedfor3 d at 4°C in darknessbeforethe
(NH4)6Mo7O24].
150
toa phytotron
(12-hday/12-h
lightintensity,
night;
traysweretransferred
activeradiation;
20°C/18°C;
μηιοίs"1 irT2photosynthetically
temperature,
relative
oftheair,70%)andplacedina 101tankfilledwithnutrient
humidity
twicea week.Rootsfrom3-week-old
solutionthatwas refreshed
plantswere
used fortherespiratory
experiments.
oxygenconsumption
old Arabidopsis
Three-week
(adhseedlingsfroma ADH loss-of-function
null) mutant(ArabidopsisecotypeBe-0,ethylmethanesulfonate
mutagenized; Jacobset al., 1988)and a reduction-of-function
(adhT)mutantin the
NASC ref. N552699;Bantiet al.,2008)
Col-0background
(T-DNAknockout,
weregrownon vertical
half-strength
Hoaglandnutriagarplatescontaining
withnormalaircontaining
with2% Sue and treated
entsolutionsupplement
21%oxygen(normoxia)
or 1% oxygen(hypoxia)and 0.035%CO2for24 h as
of ethanol
describedby van Dongenet al. (2009).For the determination
fromtheplatesto
seedlingsweretransferred
by Arabidopsis,
production
21%or1%of
withaircontaining
either
liquidmediumthatwas inequilibrium
mediumwas determined
Theethanolconcentration
oftheincubation
oxygen.
afterincubation
(Bantietal.,2008).
overnight
Plant Physiol. Vol. 149, 2009
and PlantInternalOxygen
Regulationof Respiration
OxygenMeasurements
Theoxygenconcentration
ofthenutrient
solutionand theplantinternal
contents
weremeasuredusingan opticaloxygensensor(MicroxTX2,
oxygen
PreSensPrecision
Sensing).
theplantinternal
concentrations
wereverified,
oxygen
using
Additionally
a self-made
laser-based
opticaloxygenmeasureapparatusforminiaturized
betweensamplefluorescence
ments.Thesetup,whichis abletodifferentiate
sensors
and sensorphosphorescence,
allowstheuse ofultrafine
fiber-optic
withtipdiameters
downto 10 μηι.The setupand thedata evaluationare
etal. (2005).This
etal. (2005)andbySchmälzlin
described
byLöhmannsröben
allows precisedetermination
of internaloxygen
kind of measurements
withminimal
disturbance
ofthetissue.
concentrations
RespirationMeasurements
electrodes
wasmeasured
consumption
usingClark-type
Respiratory
oxygen
H.SaurLaborbedarf)
(Hansatech
usingsmall(5-10mm)rootpieces
Oxygraph,
as thenutrient
solution
infresh
solution
withthesamecomposition
incubating
Measurements
weredoneat25°Cina finalvolumeof
therootsweretakenfrom.
was
rinsedintactroots,and thecuvette
1 mLcontaining
25 mgofthoroughly
of oxygenfromthe air.The alternative
closedto preventdiffusion
tightly
with20 mMSHAM (1 mstocksolutionin methoxypathwaywas inhibited
200μΜKCNwas added(stock
thecytochrome
andtoinhibit
ethanol)
pathway,
were
concentrations
in 20 mMHEPES,pH 8.0).Theoptimalinhibitor
solution
curveas explained
froma titration
determined
byM0lleretal. (1988).
Metaboliteand EnzymeActivityMeasurements
wereextracted
fromfrozenrootmaterial
Metabolites
byTCA extraction.
as in van Dongenet al.
PEP and pyruvateweremeasuredenzymatically
(2004).TheamountofATPand ADP was measuredbyHPLC (vanDongen
etal, 2004).
wereassayedin desalted
PDC (EC 4.1.1.1)andADH (EC 1.1.1.1)activities
as described
extract
byGastonetal. (2002).
in pea roots,500mgrootmaterial
To measureethanoland acetaldehyde
15testtube.After
in5 mLof1 ν HC1O4ina Teflon-sealed
was homogenized
theheadspaceand
minincubation
at70°C,a 1-mLgassamplewas takenfrom
intoa gasChromatograph
(6890HP; Hewlett-Packard)
equippedwith
injected
detector.
Ethanolproduction
a HP-Innowax
columnanda flameionization
by
as describedby Banti
enzymatically
seedlingswas performed
Arabidopsis
etal. (2008).
WesternBlots
Totalproteinwas isolatedfromrootsthatwerequicklyfrozenin liquid
blotswereproduced
as described
byZabalza et al. (2005).Western
nitrogen
was kindlyprovidedbyDr.
PDC antibody
tostandard
techniques.
according
FuHalle-Wittenberg,
Germany).
University,
StephanKönig(Martin-Luther
fumarase
raisedagainstArabidopsis
marasewas analyzedusingantibodies
was producedbya custompeptide
etal.,2007).ADH antibody
(Nunes-Nesi
facility
(Biogenes)and a shortconjugatedpeptideas antigen(C-KGTFYGwas raisedinrabbit
NYKPRTDL-COOH).
protocols
usingstandard
Antibody
dilutionwas 1:2,000forPDC and
fromthemanufacturer.
Primary
antibody
1:500forADH. Antirabbit
Aldrich)was used as
IgG peroxidase(Sigmaat a dilutionofdilution1:200,000.
secondary
antibody
ACKNOWLEDGMENTS
theADH mutants,
Perataforproviding
TheauthorsthankPierdomenico
and
andMarkStitt
theArabidopsis
KarinKoehlforestablishing
hydroponics,
andGustavoGarijo
TineSchmaedicke
discussions.
HansLambersforhelpful
assistance.
fortheirexperimental
areacknowledged
December
Received
15,2008;published
3,2008;acceptedDecember
September
19,2008.
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