Suppression of Chloroplastic Alkenal/One Oxidoreductase Represses the Carbon Catabolic

16 Pages·2016·0.93 MB·English

Suppression of Chloroplastic Alkenal/One
Oxidoreductase Represses the Carbon Catabolic
1[OPEN]
Pathway in Arabidopsis Leaves during Night
Daisuke Takagi2, Kentaro Ifuku2, Ken-ichi Ikeda, Kanako Ikeda Inoue, Pyoyun Park, Masahiro Tamoi,
Hironori Inoue, Katsuhiko Sakamoto, Ryota Saito, and Chikahiro Miyake*
Department of Biological and Environmental Science, Faculty of Agriculture, Graduate School of Agricultural
Science (D.T., K.-i.I., K.I.I., P.P., H.I., K.S., R.S., C.M.), and Center for Support to Research and Education
Activities (P.P.), Kobe University, Nada, Kobe 657–8501, Japan; Division of Integrated Life Science, Graduate
School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606–8502, Japan (K.I.); and Faculty of Agriculture,
Kinki University, Nakamachi, Nara 631–8505, Japan (M.T.)
ORCID IDs: 0000-0003-0880-5877 (D.T.); 0000-0002-2426-2377 (C.M.).
Lipid-derivedreactivecarbonylspecies(RCS)possesselectrophilicmoietiesandcauseoxidativestressbyreactingwithcellular
components. Arabidopsis (Arabidopsis thaliana) has a chloroplast-localized alkenal/one oxidoreductase (AtAOR) for the
detoxification of lipid-derived RCS, especially a,b-unsaturated carbonyls. In this study, we aimed to evaluate the
physiological importance of AtAOR and analyzed AtAOR (aor) mutants, including a transfer DNA knockout, aor (T-DNA),
andRNAinterferenceknockdown,aor(RNAi),lines.Wefoundthatbothaormutantsshowedsmallerplantsizesthanwild-type
plants when they were grown under day/night cycle conditions. To elucidate the cause of the aor mutant phenotype, we
analyzed the photosynthetic rate and the respiration rate by gas-exchange analysis. Subsequently, we found that both wild-
typeandaor(RNAi)plantsshowedsimilarCO assimilationrates;however,therespirationratewaslowerinaor(RNAi)thanin
2
wild-type plants. Furthermore, we revealed that phosphoenolpyruvate carboxylase activity decreased and starch degradation
duringthenightwassuppressedinaor(RNAi).Incontrast,thephenotypeofaor(RNAi)wasrescuedwhenaor(RNAi)plants
weregrownunderconstantlightconditions.Theseresultsindicatethatthesmallerplantsizesobservedinaormutantsgrown
underday/nightcycleconditionswereattributabletothedecreaseincarbonutilizationduringthenight.Here,weproposethat
thedetoxificationoflipid-derivedRCSbyAtAORinchloroplastscontributestotheprotectionofdarkrespirationandsupports
plantgrowth duringthenight.
Among biomolecules, lipids, especially polyunsatu- 2013).Subsequently,thelipidradicalreactswithmolec-
rated fatty acids (PUFAs), are easily oxidized by reac- ular oxygen and forms lipid peroxyl radical and lipid
tive oxygen species (ROS). When PUFAs react with alkoxylradical(Vistolietal.,2013).Lipidperoxylradical
ROS, sequential lipid peroxidation reactions start to and lipid alkoxyl radical cause radical chain oxidation
occur.Anallylic/bis-allylichydrogenatominPUFAsis tolipidperoxidationbyreactingwithneighboringlipid
highly reactive against ROS, especially the hydroxyl molecules, as a result of which lipid peroxide and
radical (Møller et al., 2007; Poon, 2009). The allylic lipidradicalaccumulate(Catalá,2010).Thissequential
hydrogen atom in PUFAs is extracted by these radical lipid peroxidation cycle disturbs membrane structures
species, and then lipid radical is formed (Vistoli et al., and their fluidity; thus, the physiological functions of
membranesareinactivated(Pamplona,2011).Further-
more, lipid peroxide produces lipid-derived reactive
1ThisworkwassupportedbytheJapanSocietyforthePromotion carbonyl species (RCS) such as a,b-unsaturated car-
ofScience(grantno.21570041toC.M.).
2Theseauthorscontributedequallytothearticle. bonyls, dicarbonyls, and keto-aldehydes as intermedi-
*[email protected]. ate products through their breakdown (Vistoli et al.,
Theauthorresponsiblefordistributionofmaterialsintegraltothe 2013). The electrophilic moieties in lipid-derived RCS
findings presented in this article in accordancewith the policy de- are capable of reacting with DNA and proteins that
scribed in the Instructions for Authors (www.plantphysiol.org) is: containnucleophilicaminoacids(suchasCys,His,Lys,
ChikahiroMiyake([email protected]). andArg)andformadvancedlipoxidationendproducts
D.T., K.I., and C.M. designed the experiments; D.T. performed through the formation of Michael adducts and Schiff
most of the work; K.I. generated RNAi mutants (aor-1 and aor-4);
bases(Esterbaueretal.,1991;Miyataetal.,2000;Aldini
K.-i.I., K.I.I., and P.P. performed transmission electron microscopy
et al., 2007). The formation of advanced lipoxidation
analysis; M.T., H.I., K.S., and R.S. supported the experiments and
end products from DNA and proteins is accompanied
theinterpretationofdata;D.T.andC.M.wroteandcompletedthe
bytheirstructuralchangesorcross-linkages,andDNA
articlewiththevaluablesuggestionofallauthors.
[OPEN]Articlescanbeviewedwithoutasubscription. andproteinslosetheirintrinsicphysiologicalfunctions
www.plantphysiol.org/cgi/doi/10.1104/pp.15.01572 (Pamplona,2011).
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Alkenal/One Oxidoreductase Supports Plant Growth
Chloroplasts in higher plants are PUFA-rich organ- plantcells,suitableamountsoflipid-derivedRCScould
elles. Monogalactosyldiacylglycerol and digalacto- be beneficial for acclimation to environmental stress
syldiacylglycerolaremajorconstituentsofchloroplasts, conditions. Therefore, the production of lipid-derived
and fatty acid moieties, which constitute these RCSshouldbestrictlyregulatedinplantscells.
lipid molecules, are highly unsaturated (monogalacto- To avoid the risk of accumulating excess lipid-
syldiacylglycerol, 18:3 approximately 60%, 16:3 ap- derived RCS in plant cells and the consequent
proximately 30%; digalactosyldiacylglycerol, 18:3 oxidative modification of biomolecules, plants have
approximately 70%, 16:3 approximately 2%; Douce detoxificationenzymeslikealdo-ketoreductase(AKR),
et al., 1973; Kelly et al., 2003; Block et al., 2007). Chlo- aldehyde dehydrogenase (ALDH), aldose/aldehyde
roplasts are major ROS-producing organelles, and reductase (ALR), alkenal reductase (AER), and
2
superoxide(O ),hydroxylradical,andsingletoxygen alkenal/one reductase (AOR; Oberschall et al., 2000;
2
(1O ) are inevitably produced by photosynthetic elec- Mano et al., 2002; Kirch et al., 2004; Yamauchi et al.,
2
tron transport reactions (Asada and Takahashi, 1987). 2011;Saitoetal.,2013).BothAKRandALRreduceal-
These facts suggest that chloroplasts are exposed to a dehydegroupstoalcoholgroupsusingNAD(P)H,and
high risk of lipid peroxidation. Indeed, Mano et al. ALDH oxidizes aldehyde groups to carboxylic acid
(2014a) reported that the Arabidopsis (Arabidopsis groups using NAD(P)+ (Oberschall et al., 2000; Kirch
thaliana) fad7fad8 double mutant contained less lipid- et al., 2004; Saito et al., 2013). Both AER and AOR de-
derived RCS (acrolein, crotonaldehyde, and malon- toxifya,b-unsaturatedcarbonylsthroughthereduction
dialdehyde) in its leaves compared with wild-type of a highly electrophilic a,b-unsaturated bond using
leaves. Both Arabidopsis FAD7 and FAD8 encode NAD(P)H (Mano et al., 2002; Yamauchi et al., 2011).
chloroplastic v-3 fatty acid desaturases that convert Overexpression of these detoxification enzymes in
16:2 and 18:2 fatty acids into 16:3 and 18:3 fatty acids plantsreducesthecontentoflipid-derivedRCSincells
(Iba et al., 1993; McConn et al., 1994). Hence, the fad7- compared with the wild type. Moreover, these over-
fad8 double mutant suppressed the production of 16:3 expressing plants acquire tolerance to oxidative dam-
and 18:3 fatty acids, and these lipid molecules that age under drought and salt stress conditions, where
constitute chloroplasts are less unsaturated (McConn ROS production in chloroplasts is stimulated (Sunkar
etal.,1994).Thesereportsindicatethatchloroplastsare et al., 2003; Mano et al., 2005; Rodrigues et al., 2006;
animportantsourceoflipid-derivedRCSinplants. Turóczy et al., 2011). On the other hand, transgenic
Lipid-derivedRCSmodifyvariousenzymesinplant plants that have suppressed activities of these detoxi-
cells and inhibit their catalytic activities. For example, fication enzymes show higher sensitivity to oxidative
theadditionofacrolein,crotonaldehyde,or4-hydroxyl- stressthanwild-typeplants(Kotchonietal.,2006;Shin
2-nonenaltoisolatedchloroplastsinhibitstheactivities et al., 2009; Stiti et al., 2011; Yamauchi et al., 2012).
oftheCalvincycleenzymes,especially thiol-regulated These reports indicate that the accumulation of lipid-
phosphoribulokinase (PRK) and glyceraldehyde-3- derived RCS indeed stimulates oxidative stress in
phosphate dehydrogenase (GAPDH; Mano et al., plants. However, in spite of these studies on lipid-
2009). The inhibition of enzyme activities reduces the derived RCS detoxification enzymes in plants, the
CO assimilationrateinchloroplasts(Manoetal.,2009). mechanism of how lipid-derived RCS affect plant
2
In addition, Rubisco, the 33-kD oxygen-evolving com- physiological reactions such as photosynthesis or res-
plex,andthelight-harvestingcomplexaremodifiedby piration is less clear in vivo. As a result, the physio-
malondialdehyde under heat stress conditions in vivo logicalimportanceofthedetoxificationoflipid-derived
(Yamauchietal.,2008;YamauchiandSugimoto,2010). RCSinplantsisstillelusive.
Furthermore, recent studies have revealed that lipid- In this study, we aimed to elucidate the physiolog-
derived RCS modify not only chloroplastic enzymes ical importance of the detoxification of lipid-derived
butalso cytosolicandmitochondrialenzymes inplant RCSinchloroplaststhatisthesourceofROSandlipid-
leaves (Fujita and Hossain, 2003; Hoque et al., 2012; derivedRCSinplants.First,wegrewtheArabidopsis
Mano et al., 2014b). These facts indicate that the pro- transfer DNA-tagged knockout mutant aor (T-DNA)
duction and the accumulation of lipid-derived RCS and analyzed plant growth. We found that aor
should be dangerous for plants. On the other hand, (T-DNA)showedgrowthretardation,comparedwith
recent studieshave alsoshown thatlipid-derivedRCS thewildtype,underday/nightcycleconditions.This
play an important role in the signaling process and result conflicted with previous results (Yamauchi
modifythegeneexpressionofseveralimportantcellu- et al., 2012). To confirm whether AOR is related to
lar processes, including detoxification, heat stress, cell growthinArabidopsis,weconstructedRNAinterfer-
division, auxin signaling, and programmed cell death encemutantsofAORinArabidopsis,aor(RNAi),and
(Farmer and Mueller, 2013; Biswas and Mano, 2015). checked their growth. We observed that aor (RNAi)
Indeed, Yamauchi et al. (2015) reported that exposure plants also showed growth retardation, compared
to lipid-derived RCS modifies the expression of tran- withthewildtype,underday/nightcycleconditions.
scriptionalfactorsinplantcells,andthemodificationof Here,wediscussthephysiologicalimportanceoflipid-
gene expression networks provides tolerance to the derived RCS detoxification through the investigation
heat stress. On the basis of these reports, although ex- ofthecauseofthegrowthretardationobservedinaor
cess amounts of lipid-derived RCS would be toxic for mutants.
Plant Physiol. Vol. 170, 2016 2025
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Takagi et al.
conflicted with the previous report that the growth of
aor (T-DNA) was the same as that of the wild type
(Yamauchietal.,2012).
To confirm whether the growth retardation in aor
(T-DNA) observed in our analysis depends on the
malfunctionofAtAOR,wegeneratedRNAinterference
(RNAi) mutants in Arabidopsis, which specifically
suppressedthemRNAlevelofAtAOR.Weisolatedtwo
RNAilines,aor-1andaor-4.ThemRNAlevelofAtAOR
decreased to approximately 2% and 50% in aor-1 and
aor-4,respectively,ascomparedwiththewildtype(Fig.
2A).Subsequently,theaccumulationofAtAORprotein
inaor-1andaor-4leaveswascomparedwiththatofthe
wild type by western-blot analysis. AtAOR protein in
thewild-typeleaveswasdetectedatamolecularmass
ofapproximately38kD(Fig.2B).AccordingtoExPASy
(http://www.expasy.org/),themolecularmassoffull-
Figure 1. Phenotype analysis of aor (T-DNA) grown under day/night length AtAOR protein was estimated as 41 kD. How-
cyclegrowthconditions.A,ConfirmationoftheexpressionofAtAORin ever, AtAOR protein has been reported to possess a
wild-type(WT)andaor(T-DNA)leavesbyRT-PCR.Asapositivecon- transit peptide sequence to chloroplasts (Yamauchi
trol,theexpressionof40SRIBOSOMALPROTEINS15A(Rps15aA)is
et al., 2011). Thus, it was speculated that the true mo-
shown.B,Phenotypesofwild-typeandaor(T-DNA)plants.Theseplants
lecular mass of AtAOR protein is smaller than its full-
are 3 weeks old after germination. Representative plants are shown.
length protein. To clarify whether AtAOR protein is
Bar=1cm.C,Plantgrowthevaluatedasanincreaseinmaximumro-
settediameter.Dataareexpressedasmeans6SE(n=4).Blacksquares localized in the chloroplasts of Arabidopsis cells, we
indicatethe wild type,andred circles indicateaor (T-DNA). D, Dry expressed a translational fusion protein between
weightofplantscomparedbetweenthewildtypeandaor(T-DNA).The AtAOR protein and GFP in protoplasts isolated from
blackbarindicatesthewildtype,andtheredbarindicatesaor(T-DNA). Arabidopsis. Green fluorescence from GFP was ob-
Dataareexpressedasmeans6SE(n=4).Asterisksindicateasignificant served in the protoplasts, and the location of emitting
differencebetweenthewildtypeandaor(T-DNA)(Student’sttest:**, green fluorescence overlapped with red fluorescence
P,0.01).
RESULTS
aorMutantsShowGrowthRetardationComparedwiththe
WildTypeunderDay/NightCycle Conditions
Tostudytheimportanceofthedetoxificationoflipid-
derivedRCSinhigherplants,wegrewthetransferDNA-
tagged knockout mutant of AtAOR, aor (T-DNA)
(Yamauchi et al., 2012), under day/night cycle condi-
tions (16 h of light and 8 h of dark) and analyzed the
phenotype.First,wecheckedthemRNAlevelofAtAOR
in aor (T-DNA) by reverse transcription (RT)-PCR. The
expression of AtAOR was successfully inhibited in aor
(T-DNA) (Fig. 1A). Subsequently, we evaluated the
acrolein-reducing activity in a crude leaf extract of aor
(T-DNA).Theactivitywasdecreasedtoabout58%inaor
(T-DNA) (2.3 6 0.2 mmol NADPH mg21 protein h21
[n = 3]) compared with the wild type (4 6 0.4 mmol
NADPHmg21proteinh21[n=3]).Becausehigherplants
have a cytosolic acrolein detoxification enzyme, aor
(T-DNA) would show the residualactivity, ashas been Figure2. ExpressionanalysisofAtAORandthedetectionofAtAOR
mentionedinpreviousreports(Manoetal.,2005;Papdi proteininwild-type(WT)andaor(RNAi)leaves.A,Relativeexpression
etal.,2008;Yamauchietal.,2012). levelsofAtAORmRNAinwild-typeandaor(RNAi)leaves.Expression
Under day/night cycle conditions, aor (T-DNA) levelwasquantifiedbyreal-timePCR.Dataareexpressedasmeans6SE
(n = 3). B, AtAOR protein extracted from wild-type and aor (RNAi)
showed smaller growth compared with the wild type
leaves was detected by western-blot analysis. M indicates a protein
(Fig. 1B). The growth rate, as evaluated by maximum
weightmolecularmarker.Proteincorrespondingto5mgwasloadedin
rosettediameter,wasslowerinaor(T-DNA)thaninthe
each lane. C, Protein profile by SDS-PAGE that correlates with the
wildtype(Fig.1C).Furthermore,thedryweightofaor
western-blot analysis shown in B. M indicates a protein weight mo-
(T-DNA) 3 weeks after germination was significantly lecularmarker.Proteincorrespondingto5mgextractedfromwild-type
lowerthanthatofthewildtype(Fig.1D).Theseresults andaor(RNAi)leaveswasloadedineachlane.
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Alkenal/One Oxidoreductase Supports Plant Growth
quantum yield of PSII (F /F ) and the incident quan-
TableI. Reductionactivityofacroleinincrudeleafextract v m
tumyieldofPSII[Y(II)]undergrowthlightconditions.
The acrolein reduction activities in crude protein extracts from
ThevaluesofF /F andY(II)weresimilaramongwild-
leavesinwild-typeandaor(RNAi)mutantplantsweredetermined.In v m
type, aor-1, and aor-4 plants (Table III). These results
thisexperiment,3-to4-week-oldplantswereused.Dataareexpressed
asmeans6SE(n=3).Valuesinparenthesesindicatetherelativeac- indicated that aor-1 and aor-4 have similar photosyn-
tivityforthewildtype. theticactivitiescomparedwiththewildtype.
In contrast to the photosynthetic activity, the dark
Line Activity
respiration rates evaluated from CO gas-exchange
mmolmg21proteinh21 2
analysis were lower in aor-1 and aor-4 compared with
Wildtype 4.560.6(100)
the wild type (Table III). We also evaluated the dark
aor-1 2.760.1(60)
aor-4 3.060.3(75) respiration rate using an oxygen electrode; aor-1 and
aor-4 again showed lower respiration rates compared
with the wild type (Supplemental Fig. S2). Further-
more, we also evaluated the photosynthetic activities
emitted from chloroplasts (Supplemental Fig. S1).
and the dark respiration rate in aor (T-DNA) grown
These results indicated that AtAOR protein was local-
under day/night cycle conditions. Both Y(II) and g
ized in chloroplasts. From these results, we concluded s
tendedtobelowerthaninthewildtype;however,CO
that AtAOR protein exists in the chloroplast as the 2
assimilation rate and C were the same between wild-
38-kDprotein.Inaor-1,noAtAORproteinwasdetected, i
type andaor(T-DNA)plants (Supplemental Table S2).
whereasasmallamountofAtAORproteinwasdetected
Incontrast,thedarkrespirationrateinaor(T-DNA)was
inaor-4(Fig.2,BandC).Next,weevaluatedtheacrolein-
lowercomparedwiththewildtype, butitwassimilar
reducing activity in crude leaf extracts of aor (RNAi)
tobothaor-1andaor-4(SupplementalTableS2).These
mutants.Theactivitydecreasedtoabout60%and75%in
results suggested that the growth retardation in aor
aor-1 and aor-4, respectively, compared with the wild
mutantswascausedbyadecreaseinthedarkrespira-
type (Table I). Like aor (T-DNA), aor-1 showed residual
tionrate.
acrolein-reducing activity even though AtAOR protein
wasnotdetectedinwestern-blotanalysis.
We evaluated the growth rates in aor-1 and aor-4
ExpressionAnalysis ofRespiration-Related Genes by
under day/night cycle conditions. Like aor (T-DNA),
Real-TimePCR
aor-1 and aor-4 showed growth retardation, and dry
weightsofaor-1andaor-4at3weeksaftergermination
In the previous section, we observed that the aor
decreased to about 49% and 54%, respectively, com-
mutantsshowedlowerrespirationratesinthedarkbut
paredwiththewildtype(Fig.3,AandB).Fromthese
not lower CO assimilation rates in the light. These
facts, we concluded that AtAOR protein function is 2
requiredforoptimalplantgrowth.
SuppressionofAtAORDecreasesDarkRespirationRate,
ButNotthePhotosynthesisRate,underDay/Night
CycleConditions
To elucidate the cause of growth retardation in aor
mutants, we analyzed the photosynthesis activity and
dark respiration rate in aor-1 and aor-4 under growth
conditions. First, we quantified the nitrogen andchloro-
phyllcontentsinaor-1andaor-4.Comparedwiththewild
type, the chlorophyll content in aor-1 and aor-4 mutants
decreasedby approximately 14%and20%, respectively,
andthenitrogencontentalsodecreasedbyapproximately
18%inbothaormutants(TableII).Thesedecreasesinthe
chlorophyllandnitrogencontentsalsowereobservedin Figure 3. Characterization of aor (RNAi) mutants grown under day/
aor(T-DNA)(SupplementalTableS1).Next,weevaluated nightcycleconditions.A,Phenotypesofwild-type(WT),aor-1,andaor-4
thephotosyntheticactivityandthedarkrespirationratein plants.Theseplantswere2weeksoldaftergermination.Representative
wild-typeandaor(RNAi)mutantplants.CO assimilation plantsareshown.Bar=1cm.B,Plantgrowthevaluatedasanincreasein
ratesinaor-1andaor-4showedsimilarvalue2stothewild maximumrosettediameter.Dataareexpressedasmeans6SE(n=6).
Blacksquaresindicatethewildtype,redcirclesindicateaor-1,andblue
type under atmospheric conditions, where actinic light
intensitywasthesameasgrowthlightintensity(150mE trianglesindicateaor-4.C,Dryweightofplantscomparedbetweenthe
m22 s21; Table III). Furthermore, internal partial wildtypeandaor(RNAi)mutants.Theblackbarindicatesthewildtype,
theredbarindicatesaor-1,andthebluebarindicatesaor-4.Dataare
pressure of CO2 (Ci) and stomatal conductance (gs) expressedasmeans6SE(n=16).Differentlettersindicateasignificant
alsoweresimilaramongthewildtype,aor-1,andaor-4 differencebetweenthewildtypeandaor(RNAi)mutants(Tukey-Kramer
(TableIII).Simultaneously,weevaluatedthemaximum honestlysignificantdifferencetest:P,0.05).
Plant Physiol. Vol. 170, 2016 2027
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Takagi et al.
subsequently decreased to the end of the night in the
Table II. Chlorophyll and nitrogen contents in wild-type and aor
wild type (Fig. 4D). However, the mRNA level of
(RNAi)mutantleavesgrownunderday/nightcyclegrowthconditions
PEPC1 in aor-1 decreased linearly from the end of the
Wild-typeandaor(RNAi)mutantplantsweregrownunderday/night
daytotheendofthenight(Fig.4D).
cyclegrowthconditions.Chlorophyllandnitrogencontentswerede-
Next, we analyzed the mRNA levels of TCA cycle
termined on leaves in to 4-week-old plants. Data are expressed as
means6SE(n=6).Asterisksindicatesignificantdifferencesbetween enzymes and respiratory electron transport chain
thewildtypeandaor(RNAi)mutants(Student’sttest:*,P,0.05and components. The mRNA levels of ACO3 (encoding
**,P,0.01). ACONITASE3) and CSY4 (encoding mitochondrion-
targeted CITRATE SYNTHASE4) showed similar
Line Chlorophyll Nitrogen
mmolm22 mmolm22 changestothatofPEPC1fromtheendofthedaytothe
Wildtype 219.067.4 75.663.0 end of the night, and the mRNA level of ACO3 was
aor-1 189.966.4** 63.064.1* significantly lower in aor-1 at midnight, as compared
aor-4 177.366.6** 62.464.2* with the wild type (Fig. 4, E and F). In contrast, the
mRNA levels of CI76 (encoding COMPLEX I 76-kD
subunit), COX6a (encoding CYTOCHROME C OXI-
observations indicatedthattheutilizationefficiencyof DASE SUBUNIT 6A) and AOX1a (encoding ALTER-
carbon acquired during photosynthesis was lower in NATIVE OXIDASE1a) showed similar changes in the
theaormutantsduringthenight.Thus,mRNAlevelsof wildtypeandaor-1(Fig.4,G–I).
genesrelatedtorespiratorymetabolismenzymes,such In addition, we analyzed the mRNA levels of en-
asthoseinvolvedinstarchdegradation,glycolysis,the zymes involved in starch degradation: GWD1 (encod-
TCAcycle,andrespiratoryelectrontransportreactions, ing GLUCAN WATER DIKINASE1), SEX4 (encoding
were analyzed. In the following experiments,we used PHOSPHOGLUCAN PHOSPHATASE), BAM1 (encod-
aor-1 as the aor mutant. For this analysis, mRNA was ingb-AMYLASE1),andBAM3(encodingb-AMYLASE3).
extracted from the leaves of both wild-type and aor-1 Inthesestarchdegradation-relatedgenes,nodifferences
plantsatthreedifferenttimes:attheendoftheday(1h in mRNA levels were observed between the wild type
before the night period started), at midnight (4 h after and aor-1 (Fig. 4, J–M).The expression patterns of these
thenightperiodstarted),andattheendofthenight(7h genesduringthenightperiodwereconsistentwithpre-
afterthenightperiodstarted).Asaninternalstandard, viousreports(Smithetal.,2004;Santeliaetal.,2011).To
we used Rps15aA (Watanabe et al., 2014), and the confirm that the mRNA levels of each gene were inde-
mRNAlevelinthewildtypeattheendofthedaywas pendent from the expression levels of an internal stan-
set to 1 (Fig. 4). First, we analyzed the mRNA levels dard, we also analyzed ACT2 (encoding ACTIN2)
involved in glycolysis. The mRNA level of FBA1 expression each time. In both the wild type and aor-1,
(encoding FRUCTOSE-1,6-BISPHOSPHATE ALDOL- ACT2hardlyshowedanychangeinmRNAlevelduring
ASE1) decreased from the end of the day to midnight the night, and no difference was observed in ACT2 ex-
andsubsequentlyincreaseduntiltheendofthenightin pression between the wild type and aor-1 (Fig. 4N).
both the wild type and aor-1 (Fig. 4A). For the mRNA Therefore,theseresultsindicatedthatthemRNAlevelsof
level of PK (encoding PYRUVATE KINASE), a signifi- each gene normalized by Rps15Aa showed unique
cantdifferencebetweenthewildtypeandaor-1wasnot changes during the night. These observations indicated
observed;however,thechangeintheamountofmRNA thatthefunctionsofglycolysisandtheTCAcycleduring
was different, in that the mRNA level tended to de- thenightperiodweremodifiedinaor-1.
crease in aor-1 at midnight, compared with the wild
type(Fig.4B).ThechangeinmRNAlevelofPDH(en-
EnzymeActivitiesofPDH,PEPC,ACO,andCSYinWild-
coding PYRUVATEDEHYDROGENASEE1 a-subunit) Typeandaor-1Leaves
increasedatmidnightandsubsequentlydecreasedatthe
endofthenightinthewildtype(Fig.4C).Incontrast,the Based on the gene expression analysis related to
mRNAlevelofPDHdidnotincreaseatmidnightinaor-1 respiration,weevaluatedtheenzymeactivitiesofPDH,
(Fig. 4C). The mRNA level of PEPC1 (encoding PEPC, ACO, and CSY in wild-type and aor-1 leaves,
PHOSPHOENOLPYRUVATE CARBOXYLASE1) also which were sampled during the night period. PDH
increased from the end of the day to midnight and activities in the wild type and aor-1 were similar (Fig.
TableIII. Darkrespirationrateandphotosyntheticparametersinwild-typeandaor(RNAi)plantsgrownunderday/nightcycleconditions
Photosynthesisactivitiesweredeterminedundergrowthlightconditions(lightintensityof150mEm22s21),andrespirationactivitywasdetermined
in the dark. Data are expressed as means 6 SE (n = 3). Dagger indicate significant differences between the wild type and aor (RNAi) mutants
(Student’sttest:†,P,0.1).
Line F/F Respiration CO Assimilation Y(II) g C
v m 2 s i
mmolCO m22s21 mmolCO m22s21 mmolmol21
2 2
Wildtype 0.82760.002 1.4160.34 4.7260.20 0.63160.004 0.07660.008 386.1612.4
aor-1 0.82160.003 0.5460.14† 4.8760.22 0.62560.016 0.07060.014 374.169.3
aor-4 0.81760.003 0.5160.15† 4.4960.10 0.62760.025 0.08660.003 384.964.4
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Alkenal/One Oxidoreductase Supports Plant Growth
Figure4. Geneexpressionanalysisrelatedto
respiration and starch degradation in leaves.
mRNAwasextractedfromwild-type(WT)and
aor-1leavesatendofday(1hbeforethenight
period started), midnight (4 h after the night
periodstarted),andendofnight(7hafterthe
nightperiodstarted).Ineachgene,themRNA
levelinthewildtypeattheendofdaywasset
to1,andrelativeexpressionchangeisshown.
Blacksquaresindicatethewildtype,andred
circlesindicateaor-1.Dataareexpressedas
means6SE(n=3–6).Asterisksindicatesig-
nificantdifferencesbetweenthewildtypeand
aor-1(Student’sttest:*,P,0.05).
5A).Incontrast,PEPCactivityinaor-1decreasedsignif- type (Fig. 6B). In contrast, starch remained in chloro-
icantlytoabout75%comparedwiththewildtype(Fig. plasts of aor-1 at the end of the night, although the
5B). The activities of ACO and CSY also were similar starchcontentdecreasedsignificantlyattheendofthe
betweenthewildtypeandaor-1(Fig.5,CandD). night, compared with the end of the day (Fig. 6). This
resultshowedthatabout62%ofthestarchaccumulated
StarchDegradationinWild-Typeandaor-1Leavesduring in the day was consumed in aor-1 chloroplasts during
thenightperiod.Comparedwiththewildtype,starch
theNight Period
content in aor-1 was significantly higher at the end of
During the night period, plants degrade starch both day and night (Fig. 6B). These results indicated
through respiratory metabolism (Zeeman et al., 2010). thatstarchdegradationwassuppressedinaor-1during
Accordingly, we quantified the amount of starch in the night, resulting in the accumulation of starch in
chloroplasts in the wild type and aor-1. For this mea- chloroplasts.Wealsoquantifiedtheamountofstarchin
surement,leavesfromwild-typeandaor-1plantswere wild-typeand aor-1 leavesbiochemically, asdescribed
harvestedattheendofthedayandnight.Thestarchin by Sawicki et al. (2012). In the wild type, starch accu-
chloroplasts was observed by transmission electron mulated during the day was degraded significantly
microscopy, and the amount of starch in chloroplasts during the night (Supplemental Fig. S3). In contrast,
was quantified by a point-counting method (Weibel, starchdegradationwassuppressedinaor-1duringthe
1979).Inwild-typeleaves,starchaccumulatedinchlo- night, and the starch content was not decreased sig-
roplastsattheendofthedaywashardlydetectedatthe nificantlyattheendthe night, comparedwiththeend
endofthenight(Fig.6A).Thisresultshowedthatabout oftheday(SupplementalFig.S3).Thisresultsupports
98% of the starch accumulated during the day was the idea that starch degradation is suppressed in aor-1
consumed during the night in chloroplasts of the wild duringthenight.
Plant Physiol. Vol. 170, 2016 2029
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Takagi et al.
aor(T-DNA)grewsimilartothewildtype(Supplemental
Fig.S4).Chlorophyllandnitrogencontentsweresimilar
betweenthewildtypeandaor(T-DNA);furthermore,the
photosynthetic activities estimated by CO assimilation
2
rate and Y(II) were not different between the wild type
and aor (T-DNA) (Supplemental Tables S3 and S4).
However, the dark respiration rate was lower in aor
(T-DNA) than in the wild type, as well as in aor-1
(SupplementalTableS4).
Figure5. EvaluationofenzymeactivitiesofPDH(A),PEPC(B),ACO
(C),andCSY(D)inwild-type(WT)andaor-1leaves.Leaveswerehar-
vested from wild-typeand aor-1plantsgrown underday/nightcycle
growth conditions, and these enzyme activities were evaluated in
leavesharvestedatmidnight.Blackbarsindicatethewildtype,andred
barsindicateaor-1.Dataareexpressedasmeans6SE(n=4).Theas-
terisk indicates a significant difference between the wild type and
aor-1(Student’sttest:*,P,0.05).
Growth ofWild-Typeand aor-1PlantsunderConstant
LightConditions
We grew both wild-type and aor-1 plants under
constant light conditions and analyzed their growth.
We found that wild-type and aor-1 plants grew simi-
larly(Fig.7A).Dryweightat3weeksaftergermination
alsowassimilarbetweenthewildtypeandaor-1under
continuouslightconditions(Fig.7B).Furthermore,the
nitrogen and chlorophyll contents also were at similar
valuesforthewildtypeandtheaor-1mutant(TableIV).
These results indicate that constant light conditions
rescuedthegrowthinaor-1.
Next, we analyzed the photosynthetic activities in
wild-typeandaor-1plants grownunderconstantlight
growthconditions.CO assimilationrateandY(II)were Figure 6. Evaluation of starch metabolism in wild-type (WT) and
2
notdifferentbetweenthewildtypeandaor-1(TableV). aor-1 leaves during the night. A, Ultrastructural observation of
The values of F /F , C, and g also were not different chloroplastsinwild-typeandaor-1leaves.Leaveswereharvestedatend
v m i s
betweenthewildtypeandaor-1(TableV).Theseresults ofday(1hbeforethenightperiodstarted)andendofnight(7hafterthe
nightperiodstarted)forthisanalysis.Representativeimagesareshown.
showed that wild-type and aor-1 plants have similar
Bars=2mm.B,Starchoccupancyratioinchloroplastsinwild-typeand
photosyntheticactivities,similartotheday/nightcycle
aor-1leavesevaluatedbyapoint-countingmethod(see“Materialsand
growthconditions.Incontrast,thedarkrespirationrate Methods”).Dataareexpressedasmeans6SE.Morethan16chloro-
inaor-1waslowercomparedwiththewildtype(Table
plastswereobservedineachtreatment.Differentlettersindicatesig-
V). We also checked the phenotype of aor (T-DNA) nificant differences between the wild type and aor-1 (Tukey-Kramer
grown under continuous light conditions. Like aor-1, honestlysignificantdifferencetest:P,0.05).
2030 Plant Physiol. Vol. 170, 2016
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Alkenal/One Oxidoreductase Supports Plant Growth
degradation during the night, leading to a lowering of
dark respiration activity and suppressed plant growth
(Figs.3and6;TableIII;SupplementalFig.S3).Thesere-
sultsconflictwithapreviousreportinwhichaor(T-DNA)
wasanalyzed(Yamauchietal.,2012).However,wefound
that the same aor (T-DNA) also showed smaller growth
size than the wild type when they were grown under
day/night cycle conditions (Fig. 1). These results corre-
sponded to those of aor (RNAi). Therefore, our different
resultsforthegrowthofaor(T-DNA)wouldbeduetothe
difference in the growth conditions, including nutrition,
growingmedium,lightintensity,orgrowthstage.Infact,
thesizesoftheaor(T-DNA)plantsreportedbyYamauchi
etal.(2012)weresmallerthanthosereportedhere(Fig.1).
We revealed that the growth retardation in aor mu-
tantsunderday/nightcycleconditionswasnotcaused
by carbon acquisition capacity during the day. Under
thegrowthlightintensity,CO assimilationandY(II)in
2
aormutantsweresimilartowild-typevalues(TableIII;
Figure7. Characterizationofaor-1plantsgrownunderconstantlight
Supplemental Table S2). Based on these results, the
growthconditions.A,Phenotypesofwild-type(WT)andaor-1plants.
growth retardation in aor mutants was not accounted
Theseplantsare3weeksoldaftergermination.Representativeplants
areshown.B,Plantgrowthevaluatedasanincreaseinmaximumrosette for by their photsynthetic ability. However, these re-
diameter.Blacksquaresindicatethewildtype,andredcirclesindicate sults did not mean that the suppression of AtAOR
aor-1.Dataareexpressedasmeans6SE(n=9–10).C,Dryweightof hardly affectsphotosynthetic ability. Inaor-1,theCalvin
plantscomparedbetweenthewildtypeandaor-1.Theblackbarin- cycleenzymesFBPaseandPRKhadsignificantlylowered
dicates the wild type, and the red bar indicates aor-1. Data are activitiescomparedwiththewildtype(SupplementalFig.
expressedasmeans6SE(n=9–10). S8). FBPase and PRK have thioredoxin-regulated Cys
in their structures, and the reduction of the disulfide
bridgebetweentwoCysresiduesactivatestheiractivities
Next,weanalyzedthemRNAlevelsofPDH,PEPC1,
(Martin et al., 2000). These enzymes are susceptible to
ACO3, and CSY4 in both wild-type and aor-1 plants
oxidationbyROS;furthermore,theseenzymesaremod-
grown under constant light growth conditions. The
ified by lipid-derived RCS, which inhibit their functions
mRNAlevelsofthesegeneswerenotdifferentbetween
(Asada and Takahashi, 1987; Tamoi et al., 1996a; Mano
thewildtypeandaor-1(SupplementalFig.S5).
et al., 2009, 2014b). Based on these previous reports, the
We also evaluated the enzyme activities of PDH,
decrease in FBPase and PRK activities in aor-1 could be
PEPC, ACO, and CSY in both wild-type and aor-1
due to oxidative stress induced by the suppression of
plants grown under constant light conditions. We
AtAOR in chloroplasts. In fact, we observed that oxida-
found that PEPC activity in aor-1 decreased compared
tivestressevalutedbythe3,3-diaminobenzidinestaining
withthewildtype(SupplementalFig.S6B).Incontrast,
methodwasstimulatedinaor-1,comparedwiththewild
the activities of PDH, ACO, and CSY were similar be-
type, under their growth conditions (Supplemental Fig.
tweenthewildtypeandaor-1(SupplementalFig.S6,A,
S9).FBPaseandPRKactivitieswerelowerinaor-1;nev-
C, and D). These results indicated that PEPC activity
ertheless,theCO assimilationrateandY(II)weresimilar
was lower in aor-1, although the growth in aor-1 was 2
between wild-type and aor-1 plants under their growth
similartothatofthewildtype.
lightconditions(TableIII).
Finally, the amount of starch in chloroplasts was
These results showed that the lack of AtAOR in
observed in both wild-type and aor-1 plants grown
chloroplastsinducesoxidativestress,whichaffectsthe
underconstantlightconditions(SupplementalFig.S7).
Although the amount of starch tended to be higher in
aor-1thaninthewildtype,nosignificantdifferencewas
Table IV. Chlorophyll and nitrogen contents in wild-type and aor
observedbetweenthewildtypeandaor-1.
(RNAi)mutantleavesgrownundercontinuouslightconditions
Wild-typeandaor(RNAi)mutantplantsweregrownunderconstant
light growth conditions. Chlorophyll and nitrogencontentswere de-
DISCUSSION terminedonleavesin3-to4-week-oldplants.Dataareexpressedas
In this study, we analyzed the physiological impor- means6SE(n=6).Thedaggerindicatesasignificantdifferencebe-
tance of the detoxification of lipid-derived RCS in tweenthewildtypeandaor-1(Student’sttest:†,P,0.1).
chloroplasts using Arabidopsis mutants that have a Line Chlorophyll Nitrogen
loweramountofAtAOR[aor(RNAi);aor-1andaor-4]or mgm22 mmolm22
donotexpressafunctionalAtAOR[aor(T-DNA)].We Wildtype 261.369.7 75.164.8
revealed that the suppression of AtAOR function aor-1 288.368.4† 83.566.5
inhibited carbon metabolism initiated by starch aor-4 290.2620.0 78.863.5
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Takagi et al.
TableV. Darkrespirationrateandphotosyntheticparametersinwild-typeandaor-1plantsgrownunderconstantlightconditions
Photosynthesis activities were determined under growth light conditions (light intensity of 150 mE m22 s21), and the respiration activity was
determinedinthedark.Dataareexpressedasmeans6SE(n=3).
Line F/F Respiration CO Assimilation Y(II) g C
v m 2 s i
mmolCO m22s21 mmolCO m22s21 mmolmol21
2 2
Wildtype 0.82260.003 0.4660.18 4.6960.44 0.68560.016 0.09760.008 378.869.1
aor-1 0.81860.003 0.2560.07 4.4060.19 0.68260.020 0.09260.008 369.767.4
Calvincycleenzymes,althoughitdidnotcausethesup- fluorescence in the dark-adapted state (Fm) levels in
pressionofphotosynthesisunderourgrowthconditions. dark-adapted wild-type and aor-1 leaves. If oxygen
Thiswouldbebecausephotosynthesiswasnotlimitedby consumption by PTOX was suppressed in the aor-1
theregenerationofribulose1,5-bisphosphate(RuBP)un- mutant,PQH wouldaccumulateinthedarkasaresult
2
derourgrowthconditions(Farquharetal.,1980).Under ofNDHactivityandF wouldincrease(Häusleretal.,
o
ourgrowthconditions,thelightintensitydidnotsaturate 2009).However,wefoundthattheF levelsweresim-
o
against photosynthesis; that is, photosynthesis was lim- ilarbetweenwild-typeandaor-1plants(Supplemental
itedbythesupplyofphotonenergytothephotosystems Fig. S11A). Second, we analyzed NDH-dependent PQ
inthethylakoidmembranes.Wild-typeandaor-1plants reduction activity in the dark. After illumination (150
showed the same F /F values; therefore, there was no mE m22 s21) for 10 min, we monitored the kinetics of
v m
difference in photosynthetic activity between wild-type chlorophyll fluorescence, which represents NDH-
and aor-1 plants grown under the growth light condi- dependentPQreduction,inthedark(Hashimotoetal.,
tions(TableIII).Ontheotherhand,underhigh-lightand 2003;Yamorietal.,2015).Weobservedsimilarkinetics
high-CO conditions,wherephotosynthesisislimitedby in both wild-type and aor-1 plants (Supplemental Fig.
2
theregenerationofRuBP,weobservedthataor-1showed S11B). These results indicate that chlororespiration ac-
lower CO assimilation rate and lower Y(II) compared tivity, which consisted of PQ reduction by NDH and
2
withthewildtype;otherwise,C wassimilarbetweenthe PQH oxidation by PTOX, was not impaired in aor-1.
i 2
wildtypeandaor-1(SupplementalFig.S10).Incontrast, Furthermore,weanalyzedthetimecourseofchangein
under high-light and ambient CO conditions, ranging thePQredoxstate(qLandFs/Fm)afterthestartofil-
2
from20to30PaC,wherephotosynthesisislimitedbythe luminationindark-adaptedwild-typeandaor-1leaves,
i
carboxylationreactionofRuBPbyRubisco,wild-typeand because the function of chlororespiration could be em-
aor-1plantsshowedthesameCO assimilationrateandY phasized in dark/light transient conditions (Casano
2
(II)(SupplementalFig. S10).Therefore,aormutantsmay etal.,2000;Joëtetal., 2002;Krameretal.,2004; Miyake
showamoreseverephenotypeunderhigh-CO andhigh- et al., 2009; Suorsa et al., 2012). After illumination
2
lightconditions. atgrowthlightintensity,qLandFs/Fmshowedthesame
Carbon utilization during the night period is sup- kineticsinbothwild-typeandaor-1plants(Supplemental
pressed in aor mutants. Although aor mutants grown Fig. S11, C and D). On the basis of these results, we
undertheday/nightcycleshowedgrowthretardation, suggest that aor-1 maintains chlororespiration activity
the growth retardation was alleviated under constant similarto the wildtype. Therefore, the decrease in oxy-
lightconditions(Figs.1,3,and7;SupplementalFig.S4). genconsumptionrateintheaormutantsinthedarkcould
These results indicated that the growth retardation be attributed to the suppression of mitochondrialrespi-
observed in aor mutants grown under the day/night ratoryreactions.
cycle might be due to the dark respiration during the Photosynthesis assimilates CO to accumulate carbon
2
night. In fact, aor mutants showed a decreased rate of asastarchinchloroplastsduringthedayperiod;incon-
darkrespirationcomparedwiththewildtype(TableIII; trast, respiration degrades starch to acquire energy to
Supplemental Table S2; Supplemental Fig. S2). In the supply carbon to the catabolic metabolism. Under day/
dark, chlororespiration is active, as is mitochondrial nightcyclegrowthconditions,relativegrowthrateshows
respiration (Nawrocki et al., 2015). Chlororespiration a linear correlation with starch degradation rate during
consists of two reactions: (1)first, the reduction of plas- thenightperiod,ratherthanstarchsynthesisrateduring
toquinone (PQ) by the NAD(P)H dehydrogenase-like the day (Gibon et al., 2009). Furthermore, the starch
complex (NDH) embedded in the thylakoid mem- degradation rate during the night period also shows a
branes; and (2) the oxidation of reduced plastoquinone linearcorrelationwiththeutilizationefficiencyoforganic
(PQH2) by PLASTID TERMINAL OXIDASE (PTOX). acid metabolism (Gibon et al., 2009). This means that
TheoxygenconsumptionbyPTOXaffectstotheoxygen carbonflowfromstarchinchloroplaststoglycolysisinthe
absorptionrateinleavesinthedark(Häusleretal.,2009). cytosol andthe TCA cycle in mitochondria is important
Thus,thereisapossibilitythatchlororespirationactivity for plant growth. Indeed, mutant analysis showed that
was lowered in the aor mutants. To determine whether the suppression of starch degradation caused a lower
thiswasthecase,weconductedthreeexperiments. respiration rate and severe growth retardation (Zeeman
First, we analyzed initial (minimum) PSII fluores- andRees,1999;Yuetal.,2001;Chiaetal.,2004;Niittylä
cenceinthedark-adaptedstate(F )andmaximumPSII etal.,2004;Köttingetal.,2005;Fultonetal.,2008).
o
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Alkenal/One Oxidoreductase Supports Plant Growth
In this study, we found that aor mutants showed a components. Moreover, lipid-derived RCS have been
lowerdarkrespirationrate(TableIII;SupplementalFig. reported to be involved in signaling processes such as
S2),where PEPC expressionlevel during the night pe- auxinsignalingandcelldivision(FarmerandMueller,
riod and its enzyme activity decreased significantly, 2013; Biswas and Mano, 2015; Yamauchi et al., 2015).
compared with the wild type (Figs. 4 and 5). PEPC Based on these reports, lipid-derived RCS diffused in
2
converts phosphoenolpyruvate and HCO to oxalo- plantcellsmightmodifythegeneexpressionnetwork,
3
acetic acid (OAA) and inorganic phosphate (Pi) by which is not analyzed in this study, and cause the de-
b-carboxylation of phosphoenolpyruvate in the pres- crease of carbon utilization during the night and the
enceofMg2+,andthisreactioncontributestothesupply growth retardation in aor mutants. Combining the re-
of carbon skeletons to the TCA cycle and amino acid sults of Mano et al. (2014a) and our study, we con-
synthesis by the production of OAA (Lepiniec et al., cluded thatchloroplastsareamajorproductionsiteof
1994; Izui et al., 2004). PEPC activity greatly affects lipid-derived RCS and might be an initiator for lipid-
carbon and nitrogen metabolism. For example, an derived RCS-dependent signaling cascades in plant
ArabidopsisknockoutmutantofPEPCshowedgrowth cells. Accordingly, further intensive studies are re-
retardation(Shietal.,2015).Furthermore,thedecrease quired to elucidate the molecular targets of lipid-
of PEPC activity in plants caused a decrease in starch derived RCS, especially the receptors of lipid-derived
degradation during the night period (Häusler et al., RCStriggeringthesignalcascadeinplantcells(Farmer
1999;Shietal.,2015).Inaddition,nitrogenassimilation andMueller,2013;BiswasandMano,2015).
alsowassuppressedandaminoacidcontentproduced PEPC is one of the targeted molecules that is oxida-
fromOAA(AspandAsn)wasloweredcomparedwith tivelymodifiedbylipid-derivedRCSinvivo.Therefore,
the wild type (Häusler et al., 1999; Shi et al., 2015). In thedecreaseinPEPCactivitymightnotbelimitedtothe
contrast, plants overexpressing PEPC showed higher suppressionofPEPCgeneexpressionintheaormutant.
nitrogen content compared with wild-type plants as Manoetal.(2014b)reportedthattheproductionoflipid-
well as the stimulation of starch degradation (Häusler derived RCS is stimulated in plants under severe salt
etal.,1999;Agarieetal.,2002;Rademacheretal.,2002; stressconditions,andtheyshowedthatseveralcandidate
Chen et al., 2004). Furthermore, the change in PEPC proteinsare carbonylatedbylipid-derivedRCSandox-
activityisrelatedtothatinthedarkrespirationratein idativestress.Amongthecandidateproteins,PEPCwas
leaves.ThePEPCsuppressionmutantshowedalower detectedasatargetforproteincarbonylation(Manoetal.,
darkrespirationrate,andplantsoverexpressingPEPC 2014b). PEPC hasnucleophilic aminoacids asa prereq-
showed a higher respiration rate, compared with the uisiteforitsactivity.Forexample,aHisresidueislocated
wild type (Häusler et al., 1999; Agarie et al., 2002; in the hydrophobic pocket of PEPC; this His residue
Rademacher et al., 2002). These changes accompanied stabilizesanintermediateofcarboxylationproductsand
thechangeofcarbonsupplyfromstarchtorespiratory extracts H+ from the carboxyl group in phosphoenol-
metabolism. The phenotype of the PEPC suppression pyruvate (Lepiniec et al., 1994; Izui et al., 2004). Fur-
mutant reported inthese previousstudies issimilar to thermore,PEPChasaLysresidueinitsactivesiteforthe
the phenotype of aor-1 observed in this study (Figs. 3 carboxylation reaction (Podesta et al., 1986; Jiao et al.,
and6;TableIII;SupplementalFig.S3). 1990). Interestingly, this Lys residue reacts with an al-
Basedontheseobservations,oneofthereasonswhy dehydegroupinpyridoxal59-phosphate,wherebyPEPC
aormutantsshowedasuppressionofthedarkrespira- activity is reduced by the formation of a Schiff base be-
tion rate, a lower nitrogen content, and growth retar- tweenLysandthealdehydegroup(Podestaetal.,1986;
dation would be the lower activity of PEPC (Table II; Jiao etal., 1990). Inaddition,plantPEPCisozymescon-
SupplementalTableS1;Reichetal.,1998;Agarieetal., servesevenCysresiduesintheiraminoacidalignments,
2002;Foyeretal.,2011).However,thedecreaseinPEPC andtheiroxidationofthiolgroupssuppressesPEPCac-
activity cannot be wholly responsible for the growth tivity (Chardot and Wedding, 1992). Based on these re-
retardationobservedintheaormutants.Thisisbecause ports,PEPCisstructurallyhighlysusceptibletooxidative
Shi et al. (2015) reported that a moderate decrease in modificationbyreactivealdehydegroups.Therefore,we
PEPCactivitydidnotresultingrowthinhibitioncom- suggestthatoxidativemodificationofthesenucleophilic
pared with the wild type, although the growth was aminoacidresidues in PEPCmighthave occurred, and
evaluatedattheearlygrowthphaseinthatstudy.Here, thismightbeoneofthereasonsforadecreaseinPEPC
wefoundthatthedecreaseinthedetoxificationactivity activity in aor-1, because the detoxification activity of
oflipid-derivedRCSinchloroplastsaffectsthecytosolic lipid-derived RCS is suppressed and oxidative stress is
enzyme and nucleus-encoded gene expression (Figs. 4 stimulatedinaormutants(SupplementalFig.S9).
and 5). That is, our results suggest that lipid-derived Underconstantlightconditions,thechangeofcarbon
RCS produced in chloroplasts spread widely through- supply from starch degradation to glycolysis and the
outplantcells.Therefore,lipid-derivedRCSalsocould TCA cyle does not affect plant growth (Izumi et al.,
affect various molecular targets, which were not ex- 2013).Furthermore,thedarkrespirationrateislargely
amined in this study in plant cells because of their re- suppressed (about 80%) under illumination compared
activities,andaormutantsmightcausethedecreaseof with under darkness, because TCA cycle activity and
carbon utilization duringthe night and the growth re- the supply of hexose molecules like Glc and Suc to
tardation as a result of the modification of plant cell glycolysisareinhibitedunderillumination(Brooksand
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