Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant

19 Pages·2017·2.46 MB·English

Discovery and Characterization of the 3-Hydroxyacyl-ACP
Dehydratase Component of the Plant Mitochondrial Fatty
1[OPEN]
Acid Synthase System
Xin Guan2, Yozo Okazaki, Andrew Lithio, Ling Li3, Xuefeng Zhao4, Huanan Jin5, Dan Nettleton,
Kazuki Saito, and Basil J. Nikolau*
Department of Biochemistry, Biophysics, and Molecular Biology (X.G., H.J., B.J.N.), National Science
Foundation Engineering Research Center for Biorenewable Chemicals (X.G., B.J.N.), Department of Statistics
(A.L., D.N.), Department of Genetics, Development, and Cellular Biology (L.L.), Laurence H. Baker Center for
Bioinformatics and Biological Statistics (X.Z.), and Center for Metabolic Biology (B.J.N.), Iowa State University,
Ames, Iowa 50011; Metabolomics Research Group, RIKEN Center for Sustainable Resource Science, Yokohama
230-0045, Japan (Y.O., K.S.); and Graduate School of Pharmaceutical Sciences, Chiba University, Chiba
260-8675, Japan (K.S.)
ORCID IDs: 0000-0002-7983-2103 (X.G.); 0000-0003-2371-6215 (L.L.);0000-0003-1315-831X(X.Z.); 0000-0002-6045-1036 (D.N.);
0000-0001-6310-5342(K.S.);0000-0002-4672-7139(B.J.N.).
WereportthecharacterizationoftheArabidopsis(Arabidopsisthaliana)3-hydroxyacyl-acylcarrierproteindehydratase(mtHD)
component of the mitochondrial fatty acid synthase (mtFAS) system, encoded by AT5G60335. The mitochondrial localization
and catalyticcapability of mtHDweredemonstrated witha green fluorescent protein transgenesis experiment and byin vivo
complementation and in vitro enzymatic assays. RNA interference (RNAi) knockdown lines with reduced mtHD expression
exhibittraitstypicallyassociatedwithmtFASmutants,namelyaminiaturizedmorphologicalappearance,reducedlipoylationof
lipoylated proteins, and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These
alterations are reversed when mthd-rnai mutant plants are grown in a 1% CO atmosphere, indicating the link between
mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine2decarboxylase. In vivo biochemical feeding
experimentsillustratethatsucroseandglycolatearethemetabolicmodulatorsthatmediatethealterationsinmorphologyand
lipidaccumulation.Inaddition,bothmthd-rnaiandmtkasmutantsexhibitreducedaccumulationof3-hydroxytetradecanoicacid
(i.e.ahallmarkoflipidA-likemolecules)andabnormalchloroplasticstarchgranules;thesechangesarenotreversiblebythe1%
CO atmosphere,demonstratingtwonovelmtFASfunctionsthatareindependentofphotorespiration.Finally,RNAsequencing
2
analysisrevealedthatmthd-rnaiandmtkasmutantsarenearlyequivalenttoeachotherinalteringthetranscriptome,andthese
analyses further identified genes whose expression is affected by a functional mtFAS system but independent of
photorespiratory deficiency. These data demonstrate the nonredundant nature of the mtFAS system, which contributes
unique lipidcomponentsneededtosupportplant cellstructureand metabolism.
Plant cells utilize at least three different fatty acid- be incorporated into a variety of lipids, including
forming systems, which occur in multiple subcellular surface cuticular lipids (Samuels et al., 2008), the
compartments: plastids, the membranes of the endo- ceramide moiety of sphingolipids (Markham et al.,
plasmic reticulum, and mitochondria (Ohlrogge and 2013),andindiscretequantitiesinsomeglycerolipids
Jaworski,1997;Wadaetal.,1997;Samuelsetal.,2008). (Millaretal.,1998).
Plastidial(ptFAS)andmitochondrial(mtFAS)fattyacid The mtFAS system appears to use free malonic acid
synthasesystemsformfattyacidsdenovo,whereasthe asthesubstrate(GuanandNikolau,2016)toprimarily
endoplasmic reticulum-localized fatty acid elongase generateoctanoyl-acylcarrierprotein(ACP),whichis
(FAE) system utilizes preexisting acyl-CoA precursors the required precursor for the biosynthesis of lipoic
tosynthesize very-long-chainfattyacidsof20carbons acid (Yasuno and Wada, 1998; Gueguen et al., 2000;
andlonger(Jamesetal.,1995).TheptFASsystemgen- Wada et al., 2001). Lipoic acid is the cofactor that
erates the bulk of aplant cell’sfatty acids from acetyl- is essential for pyruvate dehydrogenase (PDH),
CoA, and these fatty acids serve as precursors for the a-ketoglutarate dehydrogenase (KGDH), branched-
assemblyofacyllipidsthatconstitutemembranelipids chain a-ketoacid dehydrogenase, and the Gly decar-
(e.g. phospholipids and glycoglycerolipids), storage boxylasecomplex(GDC;Tayloretal.,2004).Todate,
lipids (e.g. triacylglycerol [TAG]), and signaling lipids alternative functions for the mtFAS system have not
(e.g. sphingolipids, phosphatidylinositols, and oxy- been demonstrated, although its role in detoxifying
lipins;Benning,2009;Li-Beissonetal.,2013).Thevery- mitochondrial malonic acid (Guan and Nikolau,
long-chainfattyacidsgeneratedbytheFAEsystemcan 2016) and in remodeling cardiolipins (Frentzen and
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Mitochondrial Fatty Acid Synthase
Griebau,1994;GriebauandFrentzen,1994)havebeen supportingtheassemblyoflipidA-likemoleculesandan
suggested. unexpected function in maintaining chloroplastic starch
Based on the characterization of three Arabidopsis granulemorphology.
(Arabidopsis thaliana) mtFAS enzymatic components,
mitochondrial b-ketoacyl-ACP synthase (mtKAS; Olsen
etal.,2004;Yasunoetal.,2004;Ewaldetal.,2007),phos- RESULTS
phopantetheinyl transferase (mtPPT; Guan et al., 2015),
BiochemicalIdentificationofAT5G60335asthe
andmalonyl-CoAsynthetase(mtMCS;GuanandNikolau,
ArabidopsismtHDComponent
2016), it appears that the plant mtFAS system resembles
the type II FAS system that occurs in bacteria and plant Sequence-based identification of a candidate plant
plastids(OhlroggeandJaworski,1997;Whiteetal.,2005).
mtHDgeneissomewhatcomplex,becausethetwowell-
Type II FAS systems recruit ACP as the carrier of the
characterized mtHD homologs from yeast (Kastaniotis
intermediates of the process and utilize dissociated,
et al., 2004) and humans (Autio et al., 2008b) share het-
monofunctional enzymes to catalyze the iterative reac-
erogenousandlowsequencesimilaritywithmanygenes
tionsthatproducefattyacids(Hiltunenetal.,2010).This
from Plantae. For example, BLAST analysis with the
contrastswiththetypeIFASthatoccursinthecytosolof yeast HTD2 sequence failed to identify any significant
fungi, mammals, and some bacteria, where a multi- Arabidopsishomolog(e.3.9),whereasparallelanalysis
functionalproteinthatcontainsallofthecatalyticcenters with the human HsHTD2 identified a single mtHD
requiredforfatty acidbiosynthesisiterativelycatalyzes
candidateAT5G60335(e=3e-17;Fig.1).
the formation of fatty acids from acetyl-CoA and
TheAT5G60335proteincodingsequence(CDS)was
malonyl-CoA(Smithetal.,2003).
cloned with a reverse transcription (RT)-PCR strategy
Here, we report the identification and characteriza-
using an RNA template isolated from aerial organs of
tionofageneencodingthemitochondrial3-hydroxyacyl-
young Arabidopsis seedlings. This CDS encodes a
ACPdehydratase(mtHD)thatcatalyzesthethirdofthe
protein of 166 amino acids that contains an N-terminal
four iterative reactions that constitute the mtFAS cycle.
25-residue segment that is not homologous with human
Systematic investigations (i.e. biochemical, morphologi-
HsHTD. This N-terminal sequence is rich in basic amino
cal, metabolomic, and transcriptomic analyses) confirm
acids and lacks acidic residues (Fig. 1), these being char-
itsroleinmtFASandtheimportantrolethisprocesshas
acteristics typical of mitochondrial targeting presequence
insupportingphotorespiration.Moreover,thesecharac-
elements.TheAT5G60335-encodedproteinispredictedto
terizationsidentifyadditionalnovelmtFASfunctionsin
bemitochondriallylocalizedbyMitoProtII(24N-terminal
residueswithascoreof0.9957;ClarosandVincens,1996),
PSORT (with a score of 0.751; Nakai and Horton, 1999),
1This work was supported by the National Science Foundation
andTargetP(17N-terminalresidueswithascoreof0.68;
(grantnos.IOS1139489,EEC0813570,andMCB0820823toB.J.N.),the
Nielsenetal.,1997;Emanuelssonetal.,2000).
StrategicInternationalCollaborativeResearchProgramoftheJapan
TheAT5G60335CDSwasexpressedintheyeasthtd2
Science and Technology Agency (Metabolomics for a Low Carbon
SocietygranttoK.S.),theNationalInstituteofGeneralMedicalSci- mutantstrainthatlacksafunctionalmtHDenzyme.This
ences(NIGMS)oftheNationalInstitutesofHealthandthejointNa- strain cannot grow on glycerol as the sole carbon source
tional Science Foundation/NIGMS Mathematical Biology Program duetoarespiratorydeficiency(Kastaniotisetal.,2004).In
(grantno.R01GM109458toD.N.),andIowaStateUniversity’sPlant thisexperiment,theputativemitochondrialtargetingpre-
SciencesInstituteScholarsProgram(toD.N.). sequence (24 residues) of the AT5G60335 protein was
2Presentaddress:DepartmentofChemicalEngineering,Stanford replaced by the mitochondrial presequence of the yeast
University,Stanford,CA94305.
COQ3protein(Hsuetal.,1996)toensurethecorrectmi-
3Presentaddress:DepartmentofBiologicalSciences,Mississippi
tochondriallocalizationoftheAT5G60335proteininyeast.
StateUniversity,Starkville,MS39762.
4Presentaddress:InformationTechnology,CollegeofLiberalArts AsillustratedinFigure2,neithertheemptyplasmid
control nor the COQ3 mitochondrial targeting element
andSciences,IowaStateUniversity,Ames,IA50011.
5Presentaddress:CollegeofPlantScienceandTechnology,Huaz- arecapableofrescuingthegrowthdeficiencyofthehtd2
hongAgriculturalUniversity,Wuhan,Hubei430070,China. mutantstrainonglycerol,butboththenativeyeastHTD2
*[e-mail]. geneandtheAT5G60335CDSrestoredthegrowthofthe
Theauthorresponsiblefordistributionofmaterialsintegraltothe yeast strain on glycerol medium.Therefore, this experi-
findings presented in this article in accordancewith the policyde- ment establishes that the AT5G60335 gene codes for a
scribed in the Instructions for Authors (www.plantphysiol.org) is: functionthatovercomesthedeficiencyin3-hydroxyacyl-
BasilJ.Nikolau([e-mail]).
ACP dehydratase activity that is capable of generating
X.G.andB.J.N.designedtheresearch;Y.O.andK.S.analyzedthe
acyl-ACPthatisusedtosynthesizelipoicacidinyeast.
lipidome;L.L.andX.G.sequencedthetranscriptome;X.Z.assembled
thetranscriptome;A.L.andD.N.performedstatisticalanalysesofthe
transcriptomicdata; X.G. andH.J.performedtheinvitro enzymatic
assays;X.G.andB.J.N.carriedoutallotherexperiments;X.G.andB.J.N. InVitro Characterization oftheEnzymaticActivityofthe
coordinatedthepreparationofthearticle;allauthorscontributedto AT5G60335CodingProtein
theanalysisofthecollecteddataandwritingofthearticle.
[OPEN]Articlescanbeviewedwithoutasubscription. TheEscherichiacoli-producedrecombinantAT5G60335
www.plantphysiol.org/cgi/doi/10.1104/pp.16.01732 coding protein was assayed for the ability to catalyze
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Guan et al.
Figure1. ComparisonoftheaminoacidsequencesoftheArabidopsismtHD(encodedbyAT5G60335)andthehumanHsHTD2
proteins.Residuesshadedinblackareidentical,andthoseshadedingraysharesimilarityinthechemistryofthesidechains.
AlignmentwasperformedusingClustalV(AlignX,VectorNTI10),withagapopeningpenaltyof10andagapextensionpenalty
of0.1.
3-hydroxyacyl-ACP dehydratase activity. As with experiment,theDNAfragmentencodingtheN-terminal
3-hyroxyacyl-ACPdehydratasescharacterizedfroma 40residuesof theAT5G60335protein was testedforits
variety of different type II FAS systems (Kastaniotis ability to target GFP to a specific organelle. Figure 4
et al., 2004; Autio et al., 2008a, 2008b), this reaction showsconfocalmicrographsofrootsandleafmesophyll
was assayed in the reverse direction, namely the hy- cells of the resulting GFP transgenic plants. In roots,
dration of enoyl-CoA substrates. In addition, this as- MitoTracker Orange was applied as the mitochondrial
saywasusedtodeterminethesubstratespecificityof marker and it was recorded simultaneously with GFP
the enzyme in relation to the acyl chain length of the fluorescence,whileinmesophyllcells,chlorophyllauto-
substrate, and these data were compared with the fluorescence was used as a chloroplastic marker. In
specificityofthemtKASenzyme. plants carrying the p35S::AT5G60335-GFP transgene,
Two enoyl-CoA thioesters (i.e. trans-D2-10:1-CoA GFP signals were obtained from both roots and leaf
andtrans-D2-16:1-CoA)werechemicallysynthesizedas
substrates for these assays (Supplemental Fig. S1A),
and eight different acyl-ACP thioesters (with 4:0, 6:0,
8:0, 10:0, 12:0, 14:0, 16:0, and cis-D9-16:1 acyl moieties)
were synthesized as substrates for the mtKAS assays
(SupplementalFig. S1B). Both enzymes exhibitedclas-
sicalhyperbolicMichaelis-Mentenactivityresponsesto
increasing concentrations ofeachtested substrate,and
K andV valueswerecalculatedforeachsubstrate-
m max
enzyme combination (Fig. 3). The AT5G60335 coding
protein has the ability to catalyze the expected hydra-
tion of the enoyl-CoA thioesters, and the catalytic
efficiency(k /K )forthemedium-chainsubstrate(trans-
cat m
D2-10:1-CoA)issimilartothatofthelong-chainsubstrate
(trans-D2-16:1-CoA;Fig.3A).Similarly,mtKASisableto
use saturated acyl-ACP thioesters of between 4- and
16-carbon acyl chains as substrates, but its activity with
the unsaturated substrate (cis-D9-16:1-ACP) was barely
detectable (Fig. 3B). The catalytic efficiency of mtKAS
with different saturated acyl-ACP substrates (evaluated
byk /K )isrankedinthefollowingorder:16:0.14:0.
cat m
6:0.8:0.10:0.12:0.4:0.Thisrankingisaffectedby
differencesinbothK andV witheachsubstrate.These
characterizations inmdicate tmhaaxt plant mtFAS enzymes Figure2. Geneticcomplementationof the yeast htd2mutantbythe
ArabidopsismtHDgene(AT5G60335).ExpressionofmtHDwascon-
havetheabilitytosynthesizesaturatedfattyacidsofupto
trolledwiththephosphoglyceratekinasepromoter(pPGK)andtermi-
18-carbonchainlength.
nator(tPGK).Thedilutionoftheinoculaforeachstrainisindicated.A,
Yeasthtd2mutantstrainsweregrownonglycerolasthesolecarbon
source.Theyeaststrainineachrowcarriedtheindicatedconstructs:the
MitochondrialLocalization oftheAT5G60335 controlemptyplasmid(row1);theconstructtooverexpresstheHTD2
GeneProduct protein(row2);theconstructtooverexpressthemitochondrialprese-
quenceoftheyeastCOQ3protein(MP;row3);andtheconstructto
The subcellular localization of the AT5G60335 pro-
overexpress the mitochondria-targeted mtHD (MP-mtHD fusion pro-
tein was experimentally determined in Arabidopsis tein;row4).B,SamestrainsasinA,buttheyweregrownonGlcasthe
with a GFP-tagged transgenic fusion protein. In this solecarbonsource.
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Mitochondrial Fatty Acid Synthase
leafchlorophyllautofluorescence,revealingthatthe
AT5G60335-GFP fusion protein is mitochondrially
localized. In control experiments, no GFP fluores-
cencewasdetectableinrootsandmesophyllcellsof
thewild-typeplants;intransgenicplantscarryinga
nontargetedGFPconstruct(i.e.p35S::GFP),theGFP
fluorescence localizes to the cytosol and nucleus,
which is consistent with previous reports (Guan
et al., 2015).
In combination, therefore, these sets of experiments
presented in Figures 1 to 4 lead us to the conclusions
that AT5G60335 encodes the mtHD enzyme and that
this organelle-targeting process is guided by the
N-terminal 40-residue leader sequence. Furthermore,
the enzyme kinetic data reveal that, in Arabidopsis,
mtHD and mtKAS do not restrain the mtFAS system
from synthesizing long-chain saturated fatty acids of
up to 18 carbons in length. This contrasts with other
characterized eukaryotic mtFAS systems, where the
substrate specificity of either mtHD (Autio et al.,
2008a) or mtKAS (Zhang et al., 2005) constrains the
systemtoproducefattyacidsof12carbonatomsand
shorter.
ExpressionPatterns ofmtHDandmtKASinDifferent
ArabidopsisOrgans
The spatial and temporal expression pattern of the
mtHD gene was determined by quantitative RT-PCR,
and these data were compared directly with those for
the other well-characterized mtFAS gene, mtKAS
(AT2G04540). Quantitative RT-PCR analysis of RNA
preparations extracted from different organs shows
that the two mtFAS genes exhibit parallel expression
patterns. The expression of both mtHD and mtKAS
genes occurs in all organs tested (Fig. 5), with an ap-
proximately 3-fold difference between the highest (i.e.
flowers) and lowest (i.e. siliques) levels of expression.
These nearly ubiquitous expression patterns of the
mtHD and mtKAS genes are consistent with the
microarray data visualized by the Arabidopsis eFP
Browser (Schmid et al., 2005; Winter et al., 2007) and
Figure3. Substratespecificityof therecombinantmtHDandmtKAS also are comparable with the expression patterns of
enzymes.A,Substrateconcentrationdependenceofthehydratasereaction other already characterized mtFAS components, such
catalyzedbymtHDwithenoyl-CoAsubstratesof10-and16-carbonatom asthemtPPT(Guanetal.,2015)andmtMCS(Guanand
acylchainlengths.Michaelis-Mentenkineticparameterstabulatedbelow Nikolau,2016)genes.
thegraphwerecalculatedfromthreereplicatesforsubstrateconcentrations
of 200, 100, and 70 mM, four replicates for substrate concentrations of
50 and 30 mM,sixreplicatesforsubstrateconcentrationof20 mM,and
10replicatesforsubstrateconcentrationof10mM.B,Substrateconcen- MorphologicalAlterationsAssociated withmthd-rnaiand
trationdependenceofthecondensationreactioncatalyzedbymtKASwith mtkasMutations
acyl-ACPsubstratesofbetween4-and16-carbonatomacylchainlengths.
ThephysiologicalsignificanceofthemtHDgenewas
Michaelis-Menten kinetic parameters tabulated below the graph were
calculatedfromthreereplicatesforsubstrateconcentrationsof100and investigatedbystudyingsegregantsidentifiedamonga
50mM,fourreplicatesforsubstrateconcentrationsof20and10mM,and familyofplantsgeneratedfromanArabidopsisgenetic
10replicatesforsubstrateconcentrationsof5and2mM. stockthatcarriesaT-DNA-taggedmutantallele(mthd-1)
intheheterozygousstate(CS856112,whichistheonly
mesophyllcellsinadistinctpatternthatindicateslocal- T-DNA insertion line available; http://signal.salk.
ization in organelles. This GFP feature overlaps with edu). These characterizations demonstrated that the
theMitoTrackerOrangeinrootsbutisdistinctfromthe T-DNA-taggedmutantisnotanullalleleandthatthere
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Guan et al.
Figure4. SubcellularlocalizationofmtHD(encodedbyAT5G60335)determinedwithGFP-taggedtransgenes.A,Fluorescence
micrographs of roots of nontransgenic wild-type control plants (WT), transgenic plants carrying the p35S::mtHD1-120-GFP
transgene, and transgenic plants carrying the p35S::GFP control transgene. Confocal fluorescence micrographs imaged the
emissionofGFP,MitoTrackerOrange,orthemergedimagesofGFPandMitoTrackerOrange.B,Fluorescencemicrographsofleaf
mesophyllcellsofnontransgenicwild-typeplants,transgenicplantscarryingthep35S::mtHD1-120-GFPtransgene,andtransgenic
plantscarryingthep35S::GFPcontroltransgene.ConfocalfluorescencemicrographsimagedtheemissionofGFP,chlorophyll
autofluorescence,orthemergedimagesofGFPandchlorophyllautofluorescence.
was no obvious phenotypic difference between wild- TotestanyadditiveeffectofmutationsinthemtHD-
type plants and those that were homozygous for the and mtKAS-catalyzed reactions, we generated double
mthd-1allele(SupplementalFig.S2). mutant stocks by transforming the mtkas-2 mutant line
As an alternative, therefore, we generated RNA in- with the p35S::mtHD-RNAi transgene. We recovered
terference (RNAi) transgenic plants, which express a 41 independent transformants and selected two lines
suppressor ofthemtHDgene,under thecontrolofthe thatdisplaythelowestmtHDexpressionlevels(8%and
35S promoter. Seventy-eight independent RNAi lines 11% of the wild-type level, respectively) for additional
were recovered, and quantitative RT-PCR analysis de- analysis.Inambientair,thedoublemutantlinesexhibita
terminedthelevelsoftheremainingmtHDtranscriptin stronger growth defect compared with the mtkas-2 pa-
the aerial organs of young seedlings at 16 DAI. Two rentalplants.Inourexperimentalconditions,at16DAI,
RNAilinesthatexhibitthelowestmtHDexpressionlevels thegrowthofthesedoublemutantsisarrestedatstage
(3% and 4% of the wild-type levels) were designated as 1.02 (i.e. exhibiting two rosette leaves), whereas the
mthd-rnai-1andmthd-rnai-2strains,respectively,andthese mtkas-2 mutant plants develop to stage 1.05 (i.e. exhib-
were used in subsequent analyses. These two mutant iting five rosette leaves; Fig. 6B). In addition, these
strains exhibit aerial organs that are significantly re- double mutant plants do not progress any further in
ducedin size comparedwiththe wild-typeplants, and development and eventually die, which contrasts with
their developmental appearance was classified accord- thesituationwiththemtkas-2mutant,whichprogresses
ing to the systematic system developed by Boyes et al. tomaturity,althoughataslowerratethanthewild-type
(2001).Thus,at16DAI,thesemutantplantsdevelopto plants,andultimatelysetsseeds.Whengrowninthe1%
stage1.06(i.e.exhibitingsixrosetteleavesthatarelonger CO atmosphere,thegrowthofthesedoublemutantsis
2
than1mminlength),whereasthewild-typeplantsde- reversed to a nearly wild-type appearance, and these
velop to stage 1.07 (i.e. exhibiting seven rosette leaves plantsdeveloptostage1.06by16DAI(Fig.6B).
that are longer than 1 mm in length) within the same
timeperiod(Fig.6A).
When these mthd-rnai mutant plants were grown in ChloroplastAlterations Associatedwithmthd-rnai and
an atmosphere containing 1% CO2, they grew nearly mtkas Mutations
normally(Fig. 6A).The phenotypicreversal intheele-
vatedCO atmosphereistypicalofphotorespiratoryde- Moredetailedinsightsintothegrowthphenotypeof
2
ficiency (Somerville andOgren, 1979), with theelevated mthd-rnai and mtkas mutants were obtained by exam-
CO levelsinhibitingtheoxygenationreactionofRubisco ining leaves of these mutants by light microscopy and
2
and,thus,reducingthelevelsof2-phosphoglycolate,the transmission electron microscopy. These observations
starter metabolite of photorespiration. Furthermore, this illustratethedistinctdifferencesinleafcellmorphology
phenotypealsoisconferredbymutationsinothermtFAS and ultrastructure between the wild-type plants and
components,suchasmtkas(Ewaldetal.,2007),mtppt-rnai the mutants (Fig. 7A). Specifically, when these plants
(Guanetal.,2015),andaae13(GuanandNikolau,2016). aregrowninambientair,mesophyllcellsareenlarged
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Mitochondrial Fatty Acid Synthase
protein extracts does not indicate any dramatic dif-
ferences in the expressed proteomes of these mutant
plants. The most dramatic alteration in the lipoylation
statusoftheseproteinswasassociatedwiththeHsubunit
ofGDC,which is depleted to about 10% of wild-type
levels in the mthd-rnai mutants and is undetectable
in the mtkas-2 mutant and mtkas-2-mthd-rnai dou-
ble mutant strains. The dramatic depletion in the lip-
oylation status of this protein does not affect the
accumulation of the H protein itself, as indicated by
measurementsmadeusinganti-Hproteinantibodies.
Therefore,theseplantsharboralargepoolofinactive
apo-HsubunitofGDC.
These immuno-based lipoic acid analyses also iden-
tifiedthatthelipoylationstatusofothermitochondrial
lipoylated proteins also is reduced in these mutant
Figure5. ExpressionofmtHD(whitecolumns)andmtKAS(graycol- plants; these are decreased to as low as 30% of the
umns) genes in different organs of Arabidopsis. Quantitative RT-PCR wild-typelevel(i.e.E2subunitofKGDHinthemtkas-
analysiswasconductedonRNAtemplatesisolatedfromdifferentor- 2-mthd-rnai double mutants). In all of these mutants,
gansofplantsattheindicateddaysafterimbibition(DAI).Resultsare
the lipoylation status of the E2 subunit of plastidial
means of three biological replicates 6 SE and are presented as nor- PDHwasunaffected.
malizedvaluesrelativetotheexpressionoftheACTIN2gene.
inthemutants,makingtheleavesthickerthanthoseof
wild-type plants. Despite the fact that these mutants MetabolomicAlterationsAssociated withmthd-rnaiand
lack a mitochondrial biochemical function, the ultra- mtkasMutations
structure ofthisorganelleisunaffected.Themostdra-
Usingmultipleanalyticalplatforms,themetabolomes
matic ultrastructural alterations in the mutants are
of the mthd-rnai and mtkas mutants were analyzed and
associatedwithchloroplasts. Thylakoidmembrane as-
comparedwiththoseofwild-typeplants.Theseanalyses
semblyisaffectedbythemutations,withthethylakoid quantified 143 metabolites in aerial organs, and these
structuresbeingsparserandlessextensive.Furthermore,
metabolites were categorized as aqueous metabolites,
thestarchgranuleultrastructureappearstobedrastically
fatty acids, lipids, surface cuticular lipids, and starch
altered in the mutant lines, with many small granules
granulecomponents.Themetabolomesofthesemutants
present; this compares with the typical two to five disc-
and wild-type plants were determined when they were
shapedstructuresthatoccurinthewild-typeplants.
grownineitherambientair,whenthegrowthphenotype
Mostofthesealterationsintheleafcellmorphology
and ultrastructure of the mutants (i.e. cell size and
thylakoid membrane) are reversed when the mutant
plantsaregrowninthe1%CO atmosphere(Fig.7B).
2
The exception to this morphological reversal is the
ultrastructureofthestarchgranules,whichmaintain
theabnormalmorphology observedwhentheplants
weregrown inambient air.
Altered Protein Lipoylation Status Associated with
mthd-rnai and mtkas Mutations
Mitochondriallipoicacidbiosynthesisisprimedwith
octanoyl-ACPgeneratedbythemtFASsystem(Ewald
et al., 2007). Therefore, we examined the protein lip-
oylation status of different enzymes in the mtFAS
mutants(i.e.mthd-rnai,mtkas-2,andmtkas-2-mthd-rnai
mutants). Using a western-blot procedure with anti-
lipoic acid antibodies, we examined the lipoylation
status of the H subunit of GDC, E2 subunits of mito-
chondrial PDH and KGDH, and plastidial PDH
(Ewald et al., 2007; Guan et al., 2015; Fig. 8). Plants
were grown in the 1% CO2 atmosphere to eliminate Figure6. Morphologicalphenotypesofthemthd-rnaiandmtkasmu-
any bias due to the morphological appearance asso- tants.Singlemutants(A)anddoublemutants(B)weregrowninambient
ciatedwiththemutantalleles.SDS-PAGEanalysisof airorinthe1%CO atmosphere.WT,Wildtype.
2
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Guan et al.
Figure 7. Leaf ultrastructural phenotypes of the
mthd-rnaiandmtkasmutants.Lightmicrographs
ofleafcrosssections(column1)andtransmission
electron micrographs of chloroplasts (column 2)
and mitochondria (column 3) of leaf mesophyll
cellsareshownfortheindicatedgenotypes,which
were grown in ambient air (A) or the 1% CO
2
atmosphere(B).WT,Wildtype.
associated with the photorespiratory deficiency is electron microscopy, the total quantity of starch and
expressed, or in the 1% CO atmosphere, when the WSPwasessentiallyunchangedinthemutants.
2
photorespiratory growth phenotype is suppressed These alterations in the metabolome appear to be a
(Fig.9;SupplementalFig.S3). consequence of the deficiency in photorespiration, be-
In ambient air, the most dramatic metabolic altera- cause most of these are reversed when the mutant
tion in aqueous metabolites is a 70- to 150-fold hyper- plantsare growninacondition thatinhibits photores-
accumulation of Gly. Several additional aqueous piration (i.e. the 1% CO atmosphere). An exception is
2
metabolitesexhibitsignificant,butsmaller,increasesin Gly, which, although it is reduced significantly when
accumulation,between25%and4-foldofthewild-type these mutant plants are grown in the 1% CO atmos-
2
levels. In contrast, Suc is depleted to between 5% and phere,isstill15-to50-foldhigherthanthatinthewild-
12%ofthelevelpresentinthewild-typeplants. typeplants.
All of these mutant plants exhibit significantly re- We also compared the aqueous metabolite profiles
duced levels of saponifiable fatty acids (e.g. 16:1, 16:2, between the mtkas-2-mthd-rnai double mutant and its
and 16:3) that constitute membrane lipids. Consistent parental mtkas-2 mutant plants. Whereas malonate
with the reduction in the accumulation of these fatty levelsareunaffectedinthemtkas-2andmthd-rnaisingle
acids, the levels of the major leaf glycoglycerolipids (i.e. mutants (Fig. 9), it is elevated by 3-fold in the double
monogalactosyldiacylglycerol, digalactosyldiacylglycerol, mutant(SupplementalFig.S4),evenwhentheseplants
andsulfoquinovosyldiacylglycerol)arereducedmarkedly aregrowninthe1%CO atmosphere,whichsuppresses
2
tobelow80%ofthewild-typelevels.Chlorophyllcontent photorespiration.Thiselevationinmalonatelevels,and
also is reduced to below 80% of the wild-type level, as the associated growth penalty, resembles the situation
wouldbeexpectedfromtheyellowishappearanceofthe intheaae13-1mutant(GuanandNikolau,2016),which
mutantplants.Inaddition,theaccumulationofmostsur- cannot activate malonate to malonyl-CoA. Therefore,
face cuticular lipids is reduced significantly in these mu- the reduced size of the mtkas-2-mthd-rnai double mu-
tants. In contrast, however, TAG hyperaccumulates to tants may be associated with malonate toxicity (Guan
above5-foldofthewild-typelevel. and Nikolau, 2016), a known inhibitor of succinate
Alterations in the accumulation of phospholipids dehydrogenase activity (Quastel and Wooldridge,
(i.e. phosphatidylcholine, phosphatidylethanolamine, 1928;GreeneandGreenamyre,1995).
phosphatidylglycerol, and phosphatidylinositol) and
diacylglycerol are barely detectable. In addition, the
Biochemical MimicsoftheMutantEffectonthe
quantities of insoluble starch and water-soluble Glc
MorphologyandMetabolome
polysaccharide (WSP) were compared between the
wild-typeandmutantplants.Despitethedifferencein Basedontheobservationthatgrowingthemthd-rnai
starch granule morphology observed by transmission andmtkasmutantplantsinanelevatedCO atmosphere
2
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Mitochondrial Fatty Acid Synthase
Figure8. Proteinlipoylationstatusintheaerialorgansofmthd-rnaiandmtkasmutantplants.A,CoomassieBrilliantBlue-stained
SDS-PAGEanalysisofextractspreparedfromtheindicatedgenotypes.B,Western-blotanalysisoftheHsubunitofGDCdetected
withantiH-proteinantibodiesandlipoylationstatusoftheH-proteinandotherindicatedlipoylatedproteinsdetectedwithanti-
lipoicacidantibodies.WT,Wildtype.
suppresses the changes in morphology and metab- externally added Suc reverses the mtkas-induced alter-
olome, we considered that some of these genetic defi- ation in the ultrastructure of the thylakoid membrane
ciency effects are secondary to the deficiency in system (Supplemental Fig. S5B). Moreover, the Suc
photorespiration. We hypothesized, for example, that treatmenteffectivelycomplementsthealterationsinthe
thealterationsinmorphologyandlipidsarearesultof profiles of fatty acids, glycoglycerolipids, and chloro-
the alterations in the steady-state levels of soluble me- phylls(SupplementalFig.S5C).Incontrast,exogenous
tabolites that are intermediates of photorespiration. Suc does not reverse the enlarged mesophyll cells, ab-
Thispostulatewastestedbyexogenouslyfeedingthese normal starch grains, and hyperaccumulation of TAG
photorespiration intermediates to wild-type and mtkas in the mtkas-2 mutant. These results demonstrate that
mutant plants and testing whether these biochemicals Suc depletion in the mtkas-2 mutant contributes to the
complementormimicthemtkasmutation.Specifically, morphological phenotype (i.e. stunted growth, defec-
we targeted the effects of treating plants with Suc, tive thylakoid membrane system, and loss of chloro-
glycolate, and Gly, the metabolites associated with phylls)andthealterationsinglycoglycerolipids.
photorespiration, whose accumulation is either down- Incorollaryexperiments,weevaluatedtheeffectsof
orup-regulatedbythemutations. other exogenous biochemicals (specifically 10 mM gly-
Bygrowingwild-typeandmtkas-2mutantplantson colateandGly)onthewild-typeplantstotestwhether
mediumsupplementedwith3%Suc,wetestedwhether the mtkas-induced hyperaccumulation of these photo-
the mtkas-induced reduction in Suc accumulation was respiratory intermediates is the cause of the morpho-
the cause of the alterations in morphology and lipids. logical phenotype and of the altered lipidome in the
First,asindicatedby directlyassayingtheSuc content mutants. These exogenously provided biochemicals
of the tissue, we showed that these plants take up the appear to have been taken up by the plants, as evi-
exogenously supplied Suc (Supplemental Fig. S5C), dencedbythe4-foldincreaseinglycolatetissuelevels
and the Suc depletion that occurs in the mtkas mutant in the glycolate-treated plants (Supplemental Fig.
plantsisalleviatedsignificantly,reachingabout30%of S5C); these elevated levels are similar to those mea-
the level obtained in the Suc-treated wild-type plants. sured in the mthd-rnai and mtkas mutant plants. The
The Sucsupplementation markedlyrelievesthe dwarf application of exogenous glycolate leads to a dra-
phenotype and the yellowish leaf color of the mtkas matic dwarf appearance with dark-green leaf color
mutant plants (Supplemental Fig. S5A). In addition, (Supplemental Fig. S5A). In addition, this treatment
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Guan et al.
Figure9. Metabolomicalterationsinthemthd-rnaiandmtkasmutants.Themthd-rnai-1mutant(A)andthemtkas-2mutant(B)
weregrowneitherinambientair(blackcircles)orinthe1%CO atmosphere(whitecircles).Theyaxisrepresentstheindividual
2
metabolitesthatwereidentified.Thexaxisplotsthelog-transformedrelativeratiooftheabundanceofeachmetaboliteineach
2
mutantsample,normalizedtothesamemetaboliteinthewild-type(WT)controlsample.Resultsarepresentedasmeans6SE.
MGDG,Monogalactosyldiacylglycerol;DGDG,digalactosyldiacylglycerol;SQDG,sulfoquinovosyldiacylglycerol;PC,phos-
phatidylcholine;PE,phosphatidylethanolamine;PG,phosphatidylglycerol;PI,phosphatidylinositol;DAG,diacylglycerol.
increases the number of large starch granules in the metabolic changes resemble those observed in the
chloroplasts (Supplemental Fig. S5B). In parallel, mthd-rnai and mtkas mutant plants, which also hyper-
these plants also exhibit about a 6-fold increase in accumulate glycolate. In contrast, glycolate does not
TAG accumulation (Supplemental Fig. S5C). These affect the accumulation of other portions of the tested
2018 Plant Physiol. Vol. 173, 2017
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Mitochondrial Fatty Acid Synthase
plantlipidomeinapatternthatresemblesthemthd-rnai associated with two independent genetic blocksin the
andmtkasmutants. mtFASsystem,namelymthd-rnai-1andmtkas-2mutant
The contribution of Gly to the alterations in mor- strains. In these experiments, we sequenced the tran-
phology and lipidome is somewhat more difficult to scriptomesofthewildtypeandthetwomutantstrains
determine, as the exogenous application of Gly to the that were grown in either ambient air or the 1% CO
2
wild-type plants only leads to an increase of the en- atmosphere.Initially,wedirectlymeasuredchangesin
dogenous Gly levels by about 5-fold, which is consid- geneexpressionbyconductingquantitativeRT-PCRon
erably lower than the over 70-fold hyperaccumulation nine target genes and tested the validity of using the
that occurs in the mthd-rnai and mtkas mutants. More- count numbers obtained from the identical RNA sam-
over,whenthemtkasmutantphenotypeissuppressed ple preparations by RNA-seq analysis. Correlation co-
bygrowthinthe1%CO atmosphere,endogenousGly efficients of the data obtained by the two methods are
2
levelsarestill15-foldhigherthanthosethatoccurinthe above0.9(SupplementalFig.S7),indicatingthatRNA-
wild-typeplants.Itappears,therefore,thatGlyhyper- seqreadcountscanbeusedasaquantitativereadoutof
accumulation is only a small contributor to those al- global changes in gene expression in response to the
terationsinmorphologyandlipidome. twomutations.
Statisticalanalysesinvestigatedwhethermthd-rnai-1and
mtkas-2 elicit similar changes in the transcriptome. These
Depleted3-Hydroxytetradecanoic AcidAssociated with analyses identified differentially expressed genes (DEGs)
mthd-rnaiandmtkasMutations amongthethreegenotypes(i.e.wildtype,mthd-rnai-1,and
mtkas-2) when these plants were grown in the two envi-
Inadditiontothemetabolicalterationsdiscussedabove,
ronmental conditions (i.e. ambient air or the 1% CO at-
we also identified two novel fatty acids in Arabidopsis, 2
mosphere); these are comparisons 1 to 6 of Figure 10A
3-hydroxytetradecanoic acid and 3-hydroxyhexadecanoic
(Supplemental Data S1). Gene Ontology (GO) functional
acid (Supplemental Fig. S6A). These are components that
categories associated with these DEGs were analyzed at
havelongbeenknowntobeassociatedwithgram-negative
TAIR(www.arabidopsis.org;SupplementalDataS2).
bacterial lipid A (Raetz et al., 2007) and recently were de-
Comparisons 1 (7,626 DEGs; 37% of the expressed
scribed with Arabidopsis lipid A-like molecules (Li et al.,
genome) and 2 (9,052 DEGs; 44% of the expressed ge-
2011). While the accumulation of 3-hydroxyhexadecanoic nome) indicate that each mutation significantly alters
acidisunaffectedinthemthd-rnaiandmtkasmutantplants,
the expression of a large proportion of the genome
the accumulation of 3-hydroxytetradecanoic acid is de- whenplantsaregrowninambientair.Ax2testforthe
pletedtobetween20%and30%ofthewild-typelevel,
association between the two DEG lists indicates a sig-
irrespective of the atmospheric conditions that affect nificant(P,0.0001)overlapbetweengenesaffectedby
photorespiration(Fig.9;SupplementalFig. S3).
the mthd-rnai-1 and mtkas-2 mutations. Furthermore,
Toclarifythemetabolicoriginof3-hydroxytetradecanoic
Figure10Bshowstheplotoffoldchangeinexpression
acid,weassayeditsaccumulationintwomutantstrains
incomparison1versuscomparison2andindicatesthat
thataredeficienteitherinphotorespiration(i.e.theshmt1-
the directions and magnitudes of the estimated effects
2mutant,whichisdeficientinthephotorespiratoryGly-
of each mutation were strongly positively correlated
to-Serconversion;Volletal.,2006)orinthebiosynthesis (r = 0.943, P , 0.0001). However, comparison 3 dem-
oflipidA-likemolecules(i.e.theatlpxa-1mutant,whichis
onstratesthatthereareafewgenes(114DEGs;lessthan
deficient in the first reaction of the assembly of lipid
1% of the expressed genome) whose expression is dif-
A-likemolecules;Lietal.,2011;SupplementalFig.S6B).
ferentiallyaffectedbetweenthetwomutants.
3-Hydroxyhexadecanoicacidcontentisunaffectedin While the distinctions in gene expression profiles
the shmt1-2 mutant, demonstrating that its depletion
between the wild type and mutants were greatly re-
is independent of the photorespiratory deficiency. In
duced when these plants were grown in the 1% CO
the atlpxa-1 mutant, however, the accumulation of 2
atmosphere,similarrelationshipsbetweenthesethree
3-hydroxytetradecanoicacidisdepletedto15%ofthe genotypes are apparent. Specifically, comparisons
wild-typelevel,indicatingthatlipidA-likemolecules
4 (286 DEGs; 1% of the expressed genome) and
arethemajormetabolicsinkofthishydroxylatedfatty
5 (1,825 DEGs; 9% of the expressed genome) show
acid.Thelatterconclusionisfurthersupportedbythe
strong correlated changes in the transcriptome rela-
substratespecificityofacyltransferases(i.e.AtLpxA,
tive to the differences between the two mutants, as
AtLpxD1, and AtLpxD2) that are involved in the as- reflected by comparison 6 (63 DEGs; less than 1% of
semblyoflipidA-likemolecules;alloftheseenzymes the expressed genome). Again, a x2 test for associa-
recognize 3-hydroxytetradecanoyl-ACP as their opti-
tion between the DEG lists for comparisons 4 and
malsubstrate(Jooetal.,2012;SupplementalFig.S6C). 5 indicates a significant overlap between the genes
affectedbythetwomutations,andFigure10Bshows
apositivecorrelation(r=0.864,P,0.0001)between
ParallelAlterationsinGeneExpressionInducedbythe
mthd-rnai-1andmtkas-2Mutations the effects of each mutation. Collectively, these re-
sults demonstrate that the mthd-rnai-1 and mtkas-2
RNA sequencing (RNA-seq) experiments were per- mutations induce parallel changes in the tran-
formed to assess alterations in the transcriptomes scriptome,whichsupportsthehypothesisthatthese
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