Arabidopsis MDA1, a Nuclear-Encoded Protein, Functions in Chloroplast Development and Abiotic ...

14 Pages·2012·1.93 MB·English

Arabidopsis MDA1, a Nuclear-Encoded Protein,
Functions in Chloroplast Development and Abiotic Stress
Responses
Pedro Robles, Jose´ Luis Micol, Vı´ctor Quesada*
InstitutodeBioingenier´ıa,UniversidadMiguelHerna´ndez,CampusdeElche,Elche,Spain
Abstract
Most chloroplast and mitochondrial proteins are encoded by nuclear genes, whose functions remain largely unknown
because mutant alleles are lacking. A reverse genetics screen for mutations affecting the mitochondrial transcription
terminationfactor(mTERF)familyinArabidopsisthalianaallowedustoidentify75linescarryingT-DNAinsertions.Twoof
themwerehomozygousforinsertionsintheAt4g14605gene,whichwedubbedMDA1(MTERFDEFECTIVEINArabidopsis1).
Themda1mutantsexhibitedalteredchloroplastmorphologyandplantgrowth,andreducedpigmentationofcotyledons,
leaves, stems and sepals. The mda1 mutations enhanced salt and osmotic stress tolerance and altered sugar responses
duringseedlingestablishment,possiblyasaresultofreducedABAsensitivity.LossofMDA1functioncausedup-regulation
of the RpoTp/SCA3 nuclear gene encoding a plastid RNA polymerase and modified the steady-state levels of chloroplast
genetranscripts.DoublemutantanalysesindicatedthatMDA1andthepreviouslydescribedmTERFgenesSOLDAT10and
RUG2actindifferentpathways.OurfindingsrevealanewroleformTERFproteinsintheresponsetoabioticstress,probably
through perturbed ABAretrograde signalling resulting from adisruption inchloroplast homeostasis.
Citation: Robles P, Micol JL, Quesada V (2012) Arabidopsis MDA1, a Nuclear-Encoded Protein, Functions in Chloroplast Development and Abiotic Stress
Responses.PLoSONE7(8):e42924.doi:10.1371/journal.pone.0042924
Editor:MiguelA.Blazquez,InstitutodeBiolog´ıaMolecularyCelulardePlantas,Spain
ReceivedApril25,2012;AcceptedJuly13,2012;PublishedAugust8,2012
Copyright: (cid:1)2012 Robles et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkhasbeensupportedbygrantsfromtheConselleriad’Educacio´ oftheGeneralitatValenciana(GV/2009/058)andBancaja-UMHtoV.Q.The
fundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction polymerase from the termination to the initiation site, thus
accountingforthehighmitochondrialrRNAsynthesisrateinthis
Chloroplast andmitochondrialgenomesareassumedtoderive
organelle[4].MTERF1mightalsomodulatemitochondrialDNA
from ancestral prokaryotes that established an endosymbiotic replication since the replication pause was sensitive to MTERF1
relationshipwithaprimitiveeukaryoticcell.Sincemanygenesof
over-expression in human cultured cells [5]. Inactivation of the
those ancestral endosymbionts have been transferred to the mouse MTERF2 gene leads to myopathies and memory deficits,
nucleus, contemporary plant organelle genomes harbour only
which are associated with decreased levels of mitochondrial
100–200genes, which encode components of photosynthetic,
transcripts and proteins of respiratory chain complexes, thus
transcriptional and translational apparatuses. Recent estimations
impairing the respiratory function [6]. In addition, loss of the
indicate, however, that 3,000 and 2,000 proteins localise inplant
mouse MTERF3 or the MTERF4 function very early in
chloroplasts and mitochondria, respectively [1]. Most of these development is lethal [7,8]. MTERF3 functions invivo as a re-
proteins are encoded by nuclear genes, synthesised in the
pressor of mitochondrial transcription [7], while MTERF4
cytoplasm and subsequently transported to their target organelle
regulates mitochondrial translation by targeting the methyltrans-
[1]. Therefore, the expression of the nuclear and organellar
ferase NSUN4toribosomes [8].
genomes has tobeveryprecisely coordinatedintheplant cell.
Information on the function of mTERF genes in plants is still
The mitochondrial transcription termination factor (mTERF)
scarce [9]. An Arabidopsis mTERF protein (PTAC15) has been
familywasfirstidentified andcharacterised inhumansandother describedasamemberoftheTAC(transcriptionallyactivechromosome)
metazoans. Members of this family have been found in mono-
multi-proteincomplexinvolvedinthetranscriptionofchloroplast
cotyledonous and dicotyledonous plants, and also in the moss
genes [10]. A proteomic study of plastid-enriched nucleoid
Physcomitrellapatens,butnotinfungiandprokaryotes[2].Metazoan
fractions of maize leaves identified 10 mTERFs among 750
mTERFs are required for the termination and initiation of
nucleoid-associated proteins [11], indicating a function for
transcription in mitochondria. Four vertebrate MTERF subfami- mTERFs in the expression of plastid genomes. Three mTERF
lies have been described [2,3], which suggests that mitochondrial
nuclear genes from photosynthetic organisms have been cloned
transcriptionregulationismorecomplexthaninitiallyanticipated. and functionally characterised: MOC1 (mterf-like gene of Chlamydo-
Human MTERF1 simultaneously binds to the mitochondrial monas1) in the unicellular alga Chlamydomonas reinhardtti [12], and
transcriptioninitiationandterminationsites.ThiscreatesaDNA SOLDAT10 (SINGLET OXYGEN-LINKED DEATH ACTIVA-
loop that promotes the direct delivery of mitochondrial RNA TOR10; [13] and BELAYA SMERT (BSM)/RUGOSA2 (RUG2)
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[14,15]inArabidopsis.PerturbationoftheMOC1mitochondrial Isolation of Arabidopsis T-DNA Mutants in mTERF Genes
protein alters the expression profiles of those genes encoding We followed a reverse genetics approach in Arabidopsis to
componentsofmitochondrialrespiratorycomplexes.SOLDAT10 identify mutations in the mTERF genes with a morphological
is a chloroplast-localized protein. The soldat10 mutation alters phenotype. With this purpose in mind, we screened 75 T-DNA
chloroplastfunctionandsuppressesthecelldeathcausedbysinglet insertion lines from different publicly available collections [Table
oxygen in the Arabidopsis flu (fluorescent) mutant [13]. Of these S3;SIGnAL[19];SAIL(SyngentaArabidopsisInsertionLibrary;
proteins, only BSM/RUG2 is dually targeted to chloroplasts and [20]) and WiscDsLox (https://mywebspace.wisc.edu/groups/
mitochondria. The bsm and rug2-1 mutations alter the expression Krysan/Web/2010/default.html)] by presumably tagging 29 of
ofchloroplastgenesattheRNAandproteinlevels,causingstunted the 35 mTERF genes of Arabidopsis. Regardless of their
plant growth and paleness [14,15]. The bsm mutant also exhibits phenotype, eight T plants from each line were transferred to
3
arrested embryo development. Besides, impaired RUG2 function soil andgenotyped by PCR forthe presence of the annotated T-
results in decreased levels of mitochondrial transcripts, including DNAinsertion(TableS4),andtheT plantsdisplayingamutant
4
those encoding subunitsof therespiratory chain [15]. phenotype were selected for further studies, thus confirming the
Weidentified T-DNAallelesofArabidopsis mTERFgenesand mutantphenotype intheT3,T4andT5generations.
characterised a member of this family, which we named MDA1 TheN597243andN819625lines,putativelycarryingaT-DNA
(forMTERFDEFECTIVEINArabidopsis1).MDA1lossoffunction insertion in the At4g14605 gene, exhibited a mutant phenotype
altersplantdevelopment,leadingtodefectivechloroplastfunction that was inherited with complete penetrance and constant
and gene expression, early flowering, reduced plant growth and expressivity. They were backcrossed to the Col-0 wild type and
pale pigmentation, together with increased tolerance to salt and theirF2progeniesshoweda3:1wild-type:mutantsegregationratio
osmotic stress,andaltered responsestosugarsandABA. (x2=0.81, P=0.37 and x2=1.93, P=0.16, for N597243 and
N819625, respectively), indicating that the mutant phenotypes
Results were monogenic and recessive. The presence of insertions in the
At4g14605 gene was confirmed by PCR (see Materials and
Identification of mTERF Genes in the Arabidopsis and Methods), affecting the fifth exon of the gene (Figure S1A) as
Rice Genomes annotated on the SIGnAL website (http://signal.salk.edu). For
each line, all the F mutant plants and their F progenies were
To initiate a functional characterisation of the mTERF family 2 3
homozygousfortheT-DNAinsertions.Themutantphenotypeof
ofArabidopsisgenes,weperformedsequencesimilaritysearchesin
the F plants from an N597243 6 N819625 cross further
genomedatabasesandfoundmTERFgenesintheArabidopsisand 1
confirmedtheirallelism.Wenamedthesemutantsmda1-1(mTERF
rice genomes. Rice was chosen to study the conservation of this
defectivein Arabidopsis1;N597243) andmda1-2 (N819625).
gene family between dicotyledonous and monocotyledonous
plants. We used the amino acid sequence of the Arabidopsis
Bioinformatics Analysis of the MDA1 Protein
RUG2 protein [15] as a query. In thisway, wefound 35and 29
mTERF annotated genes in the Arabidopsis and rice genomes, TheMDA1geneencodesapredictedproteinof493aminoacids
respectively,aboutasmanygenesaspreviouslyreportedbyother withamolecularmassof55.9kDa(http://www.arabidopsis.org/
authors [2,14]. Most of these genes lack introns: 20 of the 35 index.jsp). The number of mTERF motifs predicted by SMART
Arabidopsisgenesand17ofthe29ricegenes.Weconfirmedthe was eight (Figure 1A). Database searches allowed us to identify
proteins similar to MDA1 in metazoans and plants, but not in
expressionofthemTERFgenesbysearchingfortheircorrespond-
archea, eubacteria or fungi. An alignment of the amino acid
ing cDNAs, ESTs [in the Knowledge-based Oryza Molecular Biological
sequences of the mTERF motifs in MDA1 with those of other
Encyclopedia (KOME; http://cdna01.dna.affrc.go.jp/cDNA/) from
plant and metazoan mTERFs (see below) revealed the conserva-
rice and The Arabidopsis Information Resource (TAIR; http://www.
tion of a proline residue at position 8 (Figure 1B). The iPSORT,
arabidopsis.org/)], or MPSS (Massively Parallel Signature Se-
ProteinProwler and TargetP programs yielded a high probability
quencing)data(http://mpss.udel.edu/at/).Wefoundevidencefor
forchloroplastlocalisationforMDA1(TableS1),asalsoreported
thetranscriptionofallthegenesidentifiedinoursearches,except
in theSubCellular ProteomicDatabase (http://suba.plantenergy.
for one Arabidopsis gene (Table S1 and S2). Our analysis of the
uwa.edu.au/flatfile.php?id).Theseresultsareconsistentwiththose
MPSSdatarevealedthatArabidopsismTERFgenesareexpressed
obtained byBabiychuk etal.[14] usingGFP fusions.
ingerminatingseedlings(16)andinsimilarnumbersinroots(23),
The closest identity with MDA1 was found for the ARALY-
vegetativeleaves(26),inflorescences(27)andsiliques(27).Inrice,
DRAFT_915404proteinfromArabidopsislyrata:95.6%aminoacid
the highest and lowest numbers of mTERF genes were found in
identityand97.8%similarity(Figure1C).ThericeOs02g39040.1
young and mature leaves (23) and in mature pollen (4),
gene product displayed 54.5% amino acid identity and 82.3%
respectively. Regarding the mTERF proteins, our analysis with
amino acid similarity. The amino acid sequence of MDA1
SMART (http://smart.embl-heidelberg.de/) showedthat they all
exhibited 31.3% identity and 70.4% similarity to SOLDAT10
containtheconservedmTERFmotifs,whicharecharacteristicof
[13], and 25.5% identity and 57.8% similarity to BSM/RUG2
this family [3]. The average number of mTERF motifs for the
[14,15](Figure1C).OtherArabidopsismTERFscloselyrelatedto
Arabidopsis and rice proteins was 6 and 5.4, respectively.
MDA1 were the products of the At4g38160 (27.8% identity and
TargetP1.1 (http://www.cbs.dtu.dk/services/TargetP/; [16]),
65.0% similarity) and At2g44020 (24.6% identity and 59.6%
PREDOTAR V1.03 (http://urgi.versailles.inra.fr/predotar/
similarity) genes. Furthermore, MDA1 displayed 25.7%, 25.5%,
predotar.html; [17]), IPSORT (http://hc.ims.u-tokyo.ac.jp/
23.9% and 22.9% overall sequence identity with the human
iPSORT/; [18]) and ProteinProwler (http://pprowler.itee.uq.
MTERF3, MTERF4, MTERF1 and MTERF2 proteins, re-
edu.au/pprowler_webapp_1-2/) predicted most of the Arabidop-
spectively, showing a closer similarity to the MTERF3 subfamily
sis and rice mTERFs to be mitochondrial (Table S1 and S2).
members from rat, mouse, sea urchin (Paracentrotus lividus) and
Thus, TargetP1.1 and ProteinProwler predicted a mitochondrial Drosophila(from26.6%to21.7%aminoacididentity)thantothe
localisation for 63.3% and 70.0% of the rice and 54.3% and remaining subfamily members (Figure 1C). Our results indicate
57.1%of theArabidopsis mTERFfactors,respectively. that MDA1 shows closer identities to mTERFs from plants than
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Figure1.mTERFmotifsinmembersofthemTERFfamily.(A)ModulararchitectureoftheArabidopsisthaliana(At)mTERFproteinsMDA1,
BSM/RUG2andSOLDAT10,human(Hs)MTERF3,Drosophilamelanogaster(Dm)D-MTERF3andParacentrotuslividus(Pl)mtDBP.Thediagramwas
drawnusingSMART.mTERFmotifsareshownasellipses.Thenumberofaminoacidsofeachproteinisindicated.(B)SequencelogoforthemTERF
motifsoftheabovementionedproteins.NumbersontheabscissarepresentpositionsinmTERFmotifsandtheordinatesrepresenttheinformation
contentmeasuredinbits.ThesequencelogowasderivedusingWebLogo(http://weblogo.berkeley.edu/).(C)Multiplealignmentoftheaminoacid
sequenceofpartoftheproteinsencodedbytheAtMDA1,SOLDAT10andBSM/RUG2,ArabidopsislyrataAl915404,OryzasativaOs02g39040.1,mouse
[Musmusculus(Mm)]Mterf3,rat[Rattusnorvegicus(Rn)Mterf3],HsMTERF3,DmD-MTERF3andPlmtDBPgenes.Residuesconservedacrossfiveor
moresequencesareshadedinblack,andsimilarresiduesareshadedingrey.Numbersindicateaminoacidpositions.Thealignmentwasobtained
usingClustalXv1.5b.AcontinuouslineindicatesanmTERFmotifinMDA1.
doi:10.1371/journal.pone.0042924.g001
from metazoans, and hint at a functional conservation of this FigureS1A).Asinglebandoftheexpectedsizewasobtainedfrom
protein inArabidopsis and rice. Col-0cDNA,butnotfrommda1-1ormda1-2cDNAs,byusingas
primers the oligonucleotides RP and R2a flanking the insertions
MDA1 Expression Analyses (Figure S1B). In addition, chimeric transcripts were detected in
We examined the expression of the MDA1 gene in mda1 mda1 plants when using a primer (F1) which hybridises upstream
mutants.Forthispurpose,totalRNAwasextractedfrommutant theT-DNAinsertionsthatdisruptAt4g14605andanLB-specific
plantson14das(daysafterstratification),reverse-transcribedand primer(LBb1.3orLB1formda1-1ormda1-2,respectively;Figure
PCR-amplifiedusingdifferentprimercombinations(TableS4and S1C).Thetranslationofthesechimerictranscriptsispredictedto
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yield truncated proteins lacking 110 and 238 C-terminal amino analysis revealed changes in the MDA1 transcript levels in those
acidsinthemda1-1andmda1-2mutants(deleting2and5mTERF Col-0 plants grown in vitro in response to different stress stimuli
motifs), respectively. In addition,primers annealing upstream the overtime,themostsignificantbeingasubstantialdown-regulation
T-DNA insertions revealed by quantitative RT-PCR (qRT-PCR) after abscisic acid (ABA) or salt treatments. Besides, we analysed
down-regulation of MDA1 expression in mda1-1 and mda1-2 all the Arabidopsis mTERF genes using the Arabidopsis whole-
comparedwithCol-0(2.0-and1.75-fold,respectively;FigureS2A). genome tiling array express (At-TAX) data (http://www.
We followed insilicoandexperimentalapproachestostudy the weigelworld.org/resources/microarray/at-tax)[23]togetawider
spatial expression of the MDA1 gene in Arabidopsis. In the first perspectiveontheroleofmTERFfamilymembersinabioticstress
case, we examined the results from different publicly available responses. Most mTERF genes (including RUG2 and SOLDAT10)
microarray databases [(Genevestigator (https://www. werelargelydown-regulatedinresponsetoABA,saltormannitol
genevestigator.com/gv/) and the BIO-array resource (BAR; (Table S5), being these differences usually higher when the
http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi)]. We found exposure of the seedlings to the stress was more prolonged [12 h
MDA1 to be ubiquitously expressed, reaching its highest level of vs. 1h; (TableS5)].
expression in the aerial parts of seedlings, and in young and These results prompted us to study the response of the mda1
senescent leaves, and its lowest level in roots and old flowers, mutants to different agents causing abiotic stress. We first
according to the tiling array data available at BAR [21,22]. examinedtheabilityofourmutantstoformfullyexpandedgreen
Similar results were obtained when using Genevestigator. We cotyledons at different NaCl concentrations (seedling establish-
confirmed the ubiquitous expression of MDA1 by qRT-PCR. ment;Figure4A,B).Wefoundthatmda1mutantsshowedthistrait
MDA1 transcripts were detected in all organs analyzed (Figure at200mM NaCl,aconcentrationthatalmostabolisheditinCol-
S2B),withthelowestexpressionfoundinrootsandthehighestin 0 (53%, 58% and 6% of the mda1-1, mda1-2 and Col-0 seeds,
stemsandvegetativeleaves(2.9-and2.5-foldhigherthaninroots, respectively; Figure 4C). Interestingly, we found that 52% of
respectively). soldat10 (in a Ler genetic background) and 13% of Ler seeds
developedexpandedcotyledonsat150mM NaCl,indicatingthat
External Morphology and Histology of mda1 Mutants animpairedSOLDAT10functionalsocausesreducedsensitivityto
The most noticeable phenotype of mda1 plants is the pale this salt (neither Ler nor soldat10 seeds expanded cotyledons at
pigmentationoftheirrosetteandcaulineleaves,stems,sepalsand 200mMNaCl). To determine whether mda1 mutants were less
siliques,whichwasvisibleearlyindevelopment.Thecotyledonsof sensitive to specific ions or osmotic stress than Col-0, we grew
themda1seedlingsexhibitedlessgreenpigmentationthanthewild them on media supplemented with KCl or mannitol. mda1 seeds
type(Figure2A–C).Consistently,wenotedasignificantreduction yieldedhigherratesofseedlingestablishmentthanCol-0onmedia
in chlorophyll a, b and carotenoid levels in mda1 mutants containing high KCl or mannitol concentrations (Figure 4D, E),
comparedwithCol-0(Figure2I).mda1leaveswererounded,their indicatingthatmda1mutantsarelesssensitivethanthewildtypeto
margins lacked indentations and the lamina was uniformly pale K+andCl2ionsandtoosmoticstressproducedbytheosmoticum
(Figure 2D–F). mda1 plants were small in size if compared with mannitol.
Col-0plants,asconfirmedbymeasuringseveralbodyparameters, It is well-known that plant responses to environmental stresses
which revealed a significant decrease in mda1 fresh and dry are regulated by ABA, a hormone that can inhibit seed
weights, root, hypocotyl and main stem lengths (Figure 2G, H; germination in response to high ionic and/or osmotic stress
Table1).Furthermore,wefoundthatthemda1mutationsdidnot producedbysalt,coldordrought,andthatthemutationsaffecting
affect flower development, although they significantly accelerated ABA synthesis or signalling enhance germination under these
flowering,whilethemda1-1mutationdiminishedfertility(Table1). stress conditions [24–26]. Hence, weinvestigated the behavior of
The internal anatomy of mda1 leaves was studied by confocal mda1 and Col-0 seeds on media containing different ABA
microscopyusingchlorophyllautofluorescenceofmesophyllcells. concentrations. As depicted in Figure 5A, mda1 seeds clearly
Wedidnotfindanynoticeabledifferencesinthemda1-1andmda1- showed higher levels of seedling establishment than Col-0 on
2 mesophyll cells when we compared them with Col-0, although medium supplemented with either 2 mM ABA (97%, 98% and
a slight decrease in chloroplast autofluorescence was observed 65% of the mda1-1, mda1-2 and Col-0 seeds yielded green
(Figure 3A, D and G). We examined the leaf chloroplast expandedcotyledons,respectively)or3 mMABA(51%,38%and
ultrastructure in both mda1 and wild-type plants by transmission 6% of the mda1-1, mda1-2 and Col-0 seeds lead to seedlings with
electron microscopy, which allowed us to identify defects in the green expanded cotyledons, respectively). Nevertheless, ABA
mda1 chloroplast structure (Figure 3B, C, E, F, H and I). sensitivity was greater in mda1 than in the null ABA insensitive
Accordingly,chloroplastsinmda1mesophyllcellshadalowstarch san˜5(alsonamedabi4-2)mutant[24]usedasacontrol(Figure5A).
grain number, suggesting low photosynthetic activity, as well as Takentogether,thesedataindicatethattheincreasedtoleranceto
enlarged thylakoid lamellas, probably due to a breakdown of saltandosmoticstressshownbymda1mutantsmightbeexplained
thylakoidmembranes.Consistently,mda1chloroplastsrevealedan by theirreduced sensitivity toABA.
accumulation of plastoglobuli, storage sites for membrane To characterise the response of mda1mutants tosalt andABA
degradation material (Figure 3C, F and I). Vacuoles were laterindevelopment,Col-0andmutantplantsweretransferred9
occasionally found in mda1-2 (Figure 3C and I). Our microscopy das from a non-supplemented agar medium to media containing
studies did not reveal any significant differences in either the 100mMNaClor4mMABA.Theirrootlengthandfreshweight
number of chloroplasts or the morphology of the mitochondria were determined after a 12-day growth period and were referred
between mda1mutants andCol-0. tothoseoftheCol-0ormda1plantstransferredatthesametimeto
non-supplemented media. Compared with Col-0, mda1-1 and
Abiotic Stress Responses of the mda1 Mutants mda1-2 plants subjected to salt stress had a significantly reduced
Dozens of genes associated with abiotic stress are up-regulated fresh weight (20%, 34% and 41% respectively; Figure 5B) and
in the Arabidopsis soldat10 mutant, which displays a very similar mda1-2 also displayed shortened roots (31% and 43% length
morphological phenotype to that of the mda1 mutants [13]. By reduction for Col-0 and mda1-2, respectively; Figure 5C). When
employingtheBIO-arrayresourcewebsite[21,22],ourexpression grownonNaCl,thesoldat10plantsexhibitedsignificantlylessfresh
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Figure2.Some morphologicaltraits ofthemda1mutants.(A–C)Seven-day-oldseedlingsand (D–F)21-day-oldrosettesof mda1-1(B,E),
mda1-2(C,F)andCol-0(A,D).(G)Forty-five-day-oldplantsgrowninsoil.(H)Fourteen-day-oldplantsgrownonverticallyorientatedagarplates.Bars
=1mm (A–H). (I) Concentration (mg/g of fresh weight) of chlorophyll a (Ca) and b (Cb), and carotenoids in the mda1 mutants and Col-0. Data
representmeanof10samplesof15-day-oldplantspergenotype6standarddeviation(SD).Twoasteriscsindicatethatthevalueissignificantly
differentfromthewildtypeatP,0.01,respectively,usingStudent’st-test.
doi:10.1371/journal.pone.0042924.g002
weight when compared withLer (63.6% and53.9%, respectively; 25uC (we normally grow our plants at 20uC; Figure S3D-I).
P=0.015).OntheABA-supplementedmedia,mda1-1andmda1-2 Compared with 20uC, at 15uC (Figure S3A-F) the mutant plants
exhibitedconsiderablyshortenedrootscomparedwithCol-0(28%, showed reductions in fresh weight significantly higher than Col-
20% and 9%, respectively; Figure 5C), while mda1-1 individuals 0 (65.5%)formda1-1(73.8%),butnotsignificantones formda1-2
displayed significantly lower fresh weight values than Col-0(63% (71.9%;FigureS3J).Takentogether,ourresultsindicatethatmda1
and 80%ofnon-stressed plants, respectively;Figure 5B). mutations diminish salt, mild cold and ABA tolerance during
We evaluated theeffectof temperature stress onmda1 mutants vegetative growth.
becausewepreviouslyreportedthatthephenotypeofthemTERF-
defectiverug2-1mutantwastemperature-dependent[15].Growth
of mda1 mutants and Col-0 was similarly affected by culture at
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(PEP; class I), PEP and the nuclear-encoded polymerase (NEP;
Table1. Morphometricanalysis ofthe mda1mutants.
classII),andonlyNEP(classIII)[29].Accordingtothesecriteria,
theexpressionofthepsbA[(classI)encodingacoresubunitofthe
photosystem II], matk [(class II) encoding a maturase], clpP [(class
Bodyparameters Genotype
II) encoding the proteolitic subunit of the Clp ATP-dependent
Col-0 mda1-1 mda1-2 protease], rpoB, rps18 and accD [(class III) encoding the core
Freshweighta 26.167.1 9.063.0* 9.862.9* bsubunitofPEP,aribosomalproteinandasubunitoftheacetyl-
Coa carboxylase for lipid biosynthesis, respectively] genes was
Dryweightb 2.760.8 0.860.5* 0.660.4*
studied. In comparison to Col-0, in mda1-1, we found significant
Rootlengthc 75.060.3 59.266.2* 49.364.6*
differencesintheexpressionofpsbA,accD,rps18[2.2-fold(2.260.6;
Hypocotyllengthd,i 15.361.6 13.061.2* 12.861.3* P=0.01), 1.7-fold (1.760.6; P=0.04) and 1.6-fold (1.660.3;
Primarystemlengthe 77.063.2 36.663.8* 27.263.9* P=0.01) up-regulated, respectively] and of clpP [1.7-fold down-
Siliquelengthf 13.660.9 13.161.2 N.D. regulated (0.660.2; P=0.008)], whereas the transcript levels of
Numberofseedspersiliqueg 50.767.3 40.967.7* N.D. matk[1.3-fold(1.360.5;P=0.2)]andrpoB(0.960.1;P=0.3)were
onlyslightly affected.
Numberofvegetativeleavesat 13.462.2 12.061.1* 9.161.1*
boltingh Defective chloroplast development may modify the expression
ofnucleargenesthroughretrogradesignalling[30].Consequently,
Numberofdaysforbolting 27.462.5 N.D. 24.264.3*
the transcript levels of the RpoTp/SCA3 nuclear gene, encoding
Valuesshownarethemeanofatleast15measurements6standarddeviation a plastid RNA polymerase [31], change in Arabidopsis mutants
(SD).Lengthsareindicatedinmmandweightsinmg.Measurementswere which are impaired in plastid development, such as rug2-1 [15].
obtainedfromplantmaterialcollecteda,b16,c14,d11,e60,f,g42andh31days Forthisreason,weinvestigatedwhethertheexpressionofRpoTp/
afterstratification(das).iSeedlingsgrowninthedark.*Valuesweresignificantly
SCA3 was affected in mda1 mutants: RpoTp/SCA3 was found
different(Student’st-test,P,0.01)fromthoseofthewildtype(Col-0).N.D.:not
determined. significantlyup-regulatedinmda1-1(1.760.1;P=0.01)andmda1-
doi:10.1371/journal.pone.0042924.t001 2 (1.660.3;P=0.008) whencomparedwith Col-0.
Sugar Sensitivity of the mda1 Mutants Genetic Interactions among mTERF-defective Mutants
It is known that ABA-deficient or insensitive mutants display To identify the genetic interactions among the mTERF genes
altered responses to sugars upon germination (reviewed in [27]). whose perturbation led to a mutant phenotype, we crossed mda1
Besides, theArabidopsismutant shs1-1(salthypersensitive1-1)shows mutants with rug2-2 [15] and soldat10 [13] in the Col-0 and Ler
altered sensitivity to NaCl, ABA and sugars, together with genetic backgrounds, respectively. In all cases, we confirmed the
inhibition of chlorophyll synthesis, very early in development phenotypesofthedoublemutantsidentifiedinF2bystudyingthe
[28]. Therefore, given the paleness of mda1 mutants and their F3 progenies derived from selfed F2 plants displaying single or
reduced sensitivity to salts and ABA during early seedling double mutant phenotypes, whose genotypes were verified by
development, we decided to examine their responses to sugars. PCR (seeMaterials andMethods).
Seeds weresown onmediacontaining different concentrationsof Therug2-26mda1-1andrug2-26mda1-2crossesallowedusto
sucrose (0, 30, 90, 175 and 290mM) or glucose (330 and identify additive double mutant phenotypes in their F2 progenies
390mM). We normally grow our mutants on 30mM sucrose. sincetheyexhibitedacombinationofphenotypictraitsfromtheir
The mda1-1 mutant was clearly less sensitive to sugars: on the corresponding parentals (Figure 6A). Since MDA1 and RUG2 are
mediacontaininghighconcentrationsofglucose(330or390mM) linked, the four phenotypic classes found did not fit the expected
or sucrose (290mM), mutant seedlings developed fully expanded 9:3:3:1 ratio. As regards the mda1-1 6soldat10 and mda1-2 6
greencotyledonsathigherratesthanCol-0(Table2).Consistent- soldat10 crosses, we identified double mutants with a clearly
ly,18.3%(on290mMsucrose)and51.5%(on330mMglucose) additivephenotypeinalltheF2progeniesstudied:theyhadmuch
of the mda1-1 germinated seeds, and 5.8% (on 290mM sucrose) smallerleavesandrosettesthanmda1orsoldat10(Figure6A).Only
and 4.1% (on 330mM glucose) of those of Col-0 produced true threephenotypicclasseswerefoundintheF2progenies(wild-type,
leaves(observed14das).Laterindevelopment,palenessofmda1-1 single or double mutant), probably because of the similarity
between the phenotypes of soldat10 and mda1. Additivity of the
plants was almost completely suppressed when grown in the
phenotypes caused by soldat10 and mda1 was confirmed by
presenceof175mMsucrose(FigureS4A-D).Accordingly,mda1-1
analysing the F progenies. We also crossed rug2-1 with soldat10,
and Col-0 plants had higher chlorophyll concentrations on 175 3
both in the Ler genetic background, and the F progeny showed
than on 30mM sucrose, and this increase was more marked in 2
a9:3:3:1segregationratio(x2=0.97;P=0.82),andtheF andF
mda1-1 (1.5-fold) than inCol-0 (1.2-fold;Figure S4E). 2 3
double mutants were smaller and paler than their single mutant
siblings(Figure6B),aphenotypewhichweinterpretedasadditive.
Effects of mda1-1 Mutation on the Expression of Plastid
We evaluated the response of the double mutants to ABA by
and Nuclear Genes
measuring root growth and seedling establishment. Root growth
ConsideringthatMDA1isanmTERF-plastidproteinwhichis assays revealed that the rug2-1 and soldat10 mutations increase
putatively involved in transcriptional control and that the ABA sensitivity whereas no differences were found for rug2-2
mutations in the previously characterized mTERF genes SOL- comparedwithCol-0(Table3).OnABA,rootlengthofthemda1-
DAT10 and BSM/RUG2 modify the levels of chloroplast 2 rug2-2 plants was similar to that of mda1-2 whereas it was
transcripts, we decided to study the expression of several plastid shortened in rug2-1 soldat10 and mda1-1 soldat10 over that of the
genes by qRT-PCR using the RNA extracted from seedlings 14 single mutants, which suggests additivity (Table 3). Regarding
das.Weselectedgeneswhoseexpressionisknowntobeaffectedin seedling establishment on ABA, rug2-1, soldat10 and the rug2-1
the soldat10, rug2-1 and/or bsm mutants. Besides, we included soldat10 double mutant were as sensitive as Ler. On the contrary,
representativesofthethreeclassesofgenesthataretranscribedby therug2-2,mda1-2rug2-2andmda1-2soldat10seedlingsweremore
different RNA polymerases: only the plastid-encoded polymerase tolerant than the corresponding wild type (Table 3). The rate of
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Figure3.Chloroplaststructureinthemda1mutants.(A,D,G)Confocalmicrographsshowingchlorophyllautofluorescenceinmesophyllcells
ofCol-0(A),mda1-1(D)andmda1-2(G)third-nodeleaves.(B,C,E,F,HandI).TransmissionelectronmicrographsofchloroplastsofaCol-0(B),mda1-1
(E) and mda1-2 (H) mesophyll cell. Close-up views of Col-0 (C), mda1-1 (F) and mda1-2 (I) chloroplasts. Photographs were taken 21days after
stratification(das).Bars =20mm(A,D,G),5mm(B,E,H)and2mm(C,F,I).
doi:10.1371/journal.pone.0042924.g003
seedling establishment on ABA for mda1-2 rug2-2 was similar to Discussion
that of the single mutants, whereas mda1-2 soldat10 exhibited
higher and lower rates than soldat10 and mda1-2, respectively, Although chloroplasts and mitochondria are essential for life,
likelydue toitsmixedgenetic backgrounds (Table 3). and despite experimental and in silico studies estimating the
number of proteins which are located in these organelles to run
WestudiedbyqRT-PCRtheexpressionofthenuclearRpoTp/
intothousands,knowledgeoftheirroleinplantbiologyislimited.
SCA3 and plastid psbA genes, in the mda1-1 rug2-2 and mda1-1
To help elucidate the function of the nuclear-encoded proteins
soldat10 double mutants. In mda1-1 rug2-2 compared with Col-0,
RpoTp/SCA3 (2.360.2; P=0.008) and psbA (2.860.6; P=0.05) localised to chloroplasts and/or mitochondria, we studied the
Arabidopsis mTERF family of transcriptional regulators. To this
wereup-regulatedtolevelsslightlyhigherthanthoseofmda1-1(see
end, we first searched for members of this gene family in the
above). psbA was up-regulated in mda1-1 soldat10 compared with
Col-0 (2.560.8; P=0.008) or Ler (2.560.8; P=0.01) at a similar genomes of the dicotyledonous Arabidopsis thaliana and the mono-
cotyledonous Oryza sativa. Our bioinformatics analyses revealed
extent than mda1-1 (see above). RpoTp/SCA3 expression was
that the numbers of annotated mTERF genes in the Arabidopsis
clearlydependentonthegeneticbackground;itwasup-regulated
in Col-0 (2.860.2; P=0.006) compared with Ler. This would and rice genomes are similar and substantially higher than in
metazoan genomes (four in vertebrates, three in Drosophila
explain the differences found in RpoTp/SCA3 expression between
melanogaster or one in Caenorhabditis elegans [2,14]). We found
mda1-1soldat10andeitherLer(1.9-foldup-regulated)orCol-0(1.3-
experimental evidence for the expression of these genes, thus
fold down-regulated).
validating the in silico identification. Furthermore, our results
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ArabidopsisMDA1Gene
independentexperimentswithtwotofourreplicatesof50–100seeds
each.Germinationwasscoredat10das.
doi:10.1371/journal.pone.0042924.g004
suggest that Arabidopsis and rice mTERFs are targeted to
chloroplasts or mitochondria, most of which are potentially
locatedinmitochondria.Thishasbeenexperimentallyconfirmed
inArabidopsisbyGFPfusions[14].Inanimals,mTERFproteins
are mitochondrial, and their molecular characterisation has
revealed their participation in mitochondrial transcription initia-
tion, termination, translation and mtDNA replication. We
speculate that the large number of mTERF genes in plants might
beexplainedbythemrequiringtheaccurateexpressionofnotonly
mitochondrial, but also plastid genes. Besides, mTERFs might
differentially contribute to regulate the organelle gene expression
in distinct developmental stages and tissues, or in response to
environmental demandsor stress.
Ourreversegeneticsapproachunderpinstheimportanceofthe
mTERF gene family in Arabidopsis, as previously shown
throughout the characterisation of the mTERF-related genes
SOLDAT10[13]andBSM/RUG2[14,15].mda1insertionalalleles
curtail MDA1 expression. Besides, they would encode truncated
proteinslacking110(mda1-1)or238(mda1-2)residuesfromtheC-
terminus, which would likely include divergent amino acids
translatedfromT-DNAbecausewedetectedchimerictranscripts.
Thissuggeststhatmda1mutantsarenotnull.Despiteallthis,the
mda1-1andmda1-2phenotypesareindistinguishable,whichmight
be explained by the existence of a redundant function supplying
MDA1 deficiency regardless of the extent of mutational damage.
Our morphological, physiological and molecular analyses of the
mda1 soldat10 and mda1 rug2-2 double mutants suggest additivity
rather than synergy which would rule out RUG2 or SOLDA10 as
that redundant function. This indicates that the mTERF genes so
farcharacterisedinArabidopsisparticipateindifferentpathways.
Alternatively, the mTERF domains remaining in mda1-2 may be
sufficienttoaccomplishalevelofactivitysimilartothatofmda1-1.
ConsistentwiththechloroplasttargetingoftheMDA1protein,
loss-of-function mda1 alleles alter chloroplast morphology and
causedisorganisedthylakoidmembranesandareductioninstarch
grains, indicating diminished photosynthetic activity. Impaired
chloroplast activity results in reduced chlorophyll levels in mda1
plants,leadingtopalegreenorgansandgeneralstuntedgrowth,as
shownbythelowerweightandreducedheightofmda1plants.In
line with the pleiotropic effects of mda1, the MDA1 gene was
broadly expressed. Leaf morphology was almost normal in mda1
mutants, except for their roundness. Stunted plant growth,
chlorophyll levels and green pigmentation are phenotypic traits
sharedbyotherpreviouslycharacterisedmTERFmutants,suchas
rug2 [15] and soldat10 [13]. Like mda1, rug2-1 also showed
abnormal chloroplasts. Interestingly, the short hypocotyls of
mda1 plants suggest that MDA1 is required to complete the
etiolated developmental program in the dark and also suggests
a role for this gene in etioplasts. The most severe phenotype
describedsofarforanmTERF-defectivemutantwasdisplayedby
Figure 4. Effects of NaCl, KCl and mannitol on seedling bsm,whichislikelyanullalleleoftheBSM/RUG2gene,exhibiting
establishment in themda1mutants. (A, B) Col-0 (A) and mda1-1 albino cells and severe alterations in organogenesis. In addition,
(B) seedlings germinating on growth medium supplemented with bsmcellsrequirehormonesupplementationtoproliferateaswellas
200mMNaCl. The inset images correspond to magnifications of the to grow invitro [14]. Like bsm, rug2-1 and soldat10, mda1-1 plants
seedlings indicated by arrows. The mutants display green expanded
exhibitanalteredplastidgeneexpression.Thefactthatthemda1-1
cotyledons, a phenotypic trait barely observed in Col-0. Scale bars:
1mm.(C–E)Seedlingestablishmentofthemda1mutantsandCol-0in mutation modifies the transcript levels of those genes transcribed
variousNaCl(C),KCl(D)andmannitol(E)concentrations.Weconsidered by NEP, PEP or both, suggests that MDA1 might be directly or
onlythoseseedlingsdisplayinggreenexpandedcotyledons.Errorbars indirectlyrequiredbydifferentplastidtranscriptionalmachineries
represent SD. Each value corresponds to the average of two forappropriate geneexpression.
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The proper activity of the different plant cell genomes entails
tight coordination. Thus, retrograde pathways transmit the
developmental, metabolic or physiological chloroplast status to
the nucleus by modifying the expression of those nuclear genes
whoseproductsactonchloroplasts,suchastheRpoTp/SCA3gene,
which encodes a plastid RNA polymerase [31]. Accordingly, we
found that RpoTp/SCA3 up-regulates in mda1 plants as in
Arabidopsis rug2-1 [15] and rpoT;2 (affected in the chloroplast
and mitochondria-targeted RNA polymerase; [32]) mutants and
the plastid-ribosome deficient albostrians mutant from barley [33].
This up-regulation may attempt to compensate for the defective
plastid function caused by the altered plastome expression in
mutants.
OurfindingsrevealthatdisruptedMDA1activitycausesaltered
responses to abiotic stresses, according to the in silico microarray-
based results, and they support previous observations suggesting
a role for mTERFs in plant stress. Thus, soldat10 is constitutively
adaptedtolightstress[13],whereasaBrassicanapusmTERFgene
and its Arabidopsis orthologue are up-regulated under abiotic
stress conditions [34]. Consistent with this, our analysis using the
At-TAX tiling array data showed differences in the expression of
most Arabidopsis mTERF genes after salt, mannitol or ABA
treatments, suggesting a role for mTERFs in abiotic stress
responses. mda1 mutants exhibit reduced seed sensitivity to the
inhibitionofseedlingestablishmentcausedbyhighNaCl,KClor
mannitol concentrations, yielding higher rates than those of Col-
0 under stress conditions. As ABA plays a central role in plant
adaptive responses to environmental stresses, and since the
perturbation of ABA signalling in Arabidopsis abi (ABA insensitive)
mutants results in increased salt tolerance [24,35], we evaluated
the mda1 response to ABA. We found mda1 early seedling
development to be less sensitive to this hormone, indicating that
theMDA1functionisrequiredforaproperABAresponseinthis
stage.Theexperimentalresultssupportthatthereisaninteraction
between sugar and ABA signalling during early seedling de-
velopmentinArabidopsis,totheextentthatABAbiosynthesisand
signallingpathwaysareatleastpartiallymodulatedbyglucose(for
reviews, see [36–38]). Consequently, the mutants identified in
screens for an altered sugar response were actually allelic to aba2
(ABA deficient2) or abi4 mutants [27]. Therefore, we decided to
investigateifmda1mutants,whicharepartiallyinsensitivetoABA,
alsoshowanalteredsugarresponse.Wefoundthistobethecase
because,inthepresenceofhighglucoseorsucroseconcentrations,
mda1-1seedsyieldedhigherseedlingestablishmentratesthanCol-
0.
Altered sugar, salt and ABA responses during early seedling
developmentwerealsoobservedintheArabidopsisshs1-1mutant
Figure5.TolerancetoABAandNaClofthemda1mutants.(A) [28].SHS1belongstothemitochondrialcarrierfamilyofproteins
EffectsofdifferentABAconcentrationsonseedlingestablishmentinthe involved in the energy transfer being located in plants in plastids
mda1mutants.Dataaremeansoftwoindependentexperimentswith [39] or the endoplasmic reticulum [28]. We considered reduced
three replicates of 50–100 seeds each scored 10 das. Error bars
sensitivity to ABA to be a likely explanation for the altered
representSD.Thesan˜5(abi4-2)ABAinsensitiveandsalt-tolerantmutant
responses to salts, sugar and osmotic stress exhibited by mda1
wasusedasapositivecontrol[24].(B,C)SensitivitytoABAandNaClof
mda1 plants. The individuals were transplanted 9 das from non- mutants because, as previously mentioned, any perturbation in
supplemented growth media to media supplemented with 0, ABA-mediated perception mechanisms might alter stress re-
100mMNaCl or 4mM ABA. 12days after the transfer, tolerance was sponses.Consistentlywiththephenotypesthatwefound,arecent
estimated by determining the fresh weight and root length of the studyhasreportedthatthemTERFgenesplayanimportantrolein
plants transferred to NaCl or ABA supplemented media and referring
germination: an in-depth transcriptomic profiling at 10time
themtothoseofthesamegenotypestransferredtonon-supplemented
points during Arabidopsis germination identified a total of
media.Thesevaluesarerepresentedaspercentagesof(B)freshweight
and(C)rootlengthofplantstransferredtonon-supplementedmedia. 15,789 genes to be expressed, including 23 mTERFs [40].
Eachvaluecorrespondstothemean6SDofthefreshweightorroot Moreover, 15 of the 23 mTERF genes identified, including
lengthof15plantsofeachgenotype.Oneandtwoasterisksindicate MDA1, were present in a subgroup of 775 genes, which were
thatthevalueissignificantlydifferentfromthewild-typeatP,0.05or transiently expressed during germination [40]. Furthermore, we
P,0.01,respectively,usingStudent’st-test.
found that mda1 mutations enhanced NaCl, mild cold, ABA or
doi:10.1371/journal.pone.0042924.g005
sugarsensitivityduringvegetativegrowth,andthattheyextended
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Table2. Effect ofsugars onthe germinationofthe mda1mutants.
Genotype Seedlingsdisplayinggreenexpandedcotyledons(%)
Nonsupplemented Sucrose(mM) Glucose(mM)
30a 90a 175a 290b 330b 390b
Col-0 98.660.4 89.463.3 76.762.0 83.363.4 11.762.0 29.864.7 2.663.2
mda1-1 95.462.4 93.765.5 65.060.0 76.9616.2 31.860.7 68.3612.3 11.261.4
mda1-2 96.060.2 73.0611.6 71.4624.0 71.7616.5 N.D. 31.5615.8 9.864.3
Valuesshownarethemean6SDofthepercentagesofgreenexpandedcotyledonsseedlingsreferredtogerminatedseedsandobtainedintwodifferentexperiments
withatleast100seedspergenotypeineachexperiment.Measurementswereperformeda4andb10das.N.D.:notdetermined.
doi:10.1371/journal.pone.0042924.t002
MDA1 requirement for adaptation to abiotic stresses later in influencedbymTERFgenes.Accordingly,mda1,soldat10(datanot
development. shown) and rug2 [15] mutants are early flowering, which is in
Apart from germination and seedling establishment, flowering agreement with the finding that germination and flowering share
time, the other fundamental developmental transition, is also genetic controls [41]. mTERFs might affect flowering by their
Figure6.GeneticinteractionsbetweenmTERFmutants.(A)Additivephenotypesofmda1rug2-2andmda1soldat10doublemutants.Rosettes
fromawild-type(Col-0)andthesinglemutantsrug2-2,mda1-1andmda1-2(inaCol-0geneticbackground),soldat10(inaLergeneticbackground)
andthemda1-1rug2-2,mda1-1soldat10,mda1-2rug2-2andmda1-2soldat10doublemutants.(B)Geneticinteractionbetweenrug2-1andsoldat10.
Rosettesfromtherug2-1andsoldat10singlemutants,bothinaLergeneticbackground,andtherug2-1soldat10doublemutant.Picturesweretaken
21das.Scalebars:1mm.
doi:10.1371/journal.pone.0042924.g006
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