Expression of an Arabidopsis phosphoglycerate mutase homologue is localized to apical

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Plant Pathology and Microbiology Publications Plant Pathology and Microbiology
11-2003
Expression of an Arabidopsis phosphoglycerate
mutase homologue is localized to apical meristems,
regulated by hormones, and induced by sedentary
plant-parasitic nematodes
Mitra Mazarei
Iowa State University
Kristen A. Lennon
Iowa State University
David P. Puthoff
United States Department of Agriculture
Steven R. Rodermel
Iowa State University, [e-mail]
Follow this and additional works at:http://lib.dr.iastate.edu/plantpath_pubs
Thomas J. Baum
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Iowa State University, [e-mail]
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PlantMolecularBiology 53: 513–530,2003. 513
©2003KluwerAcademicPublishers. PrintedintheNetherlands.
Expression of an Arabidopsis phosphoglycerate mutase homologue is
localized to apical meristems, regulated by hormones, and induced by
sedentary plant-parasitic nematodes
Mitra Mazarei1, Kristen A. Lennon1,3, David P. Puthoff1,4, Steven R. Rodermel2 and Thomas
J. Baum1
1DepartmentofPlantPathologyand2DepartmentofBotany,IowaStateUniversity,BesseyHall,Ames,IA50011,
USA(authorforcorrespondence;[e-mail]);presentaddresses:3DepartmentofBiologicalSci-
ences,PurdueUniversity,WestLafayette,IN47907,USA;4USDA-ARSCropProduction&PestControlResearch
Unit,DepartmentofEntomology,PurdueUniversity,WestLafayette,IN47907,USA
Received9June2003;acceptedinrevisedform10October2003
Key words: apical meristem, Arabidopsis, AUX1, phosphoglycerate mutase, sedentary endoparasitic nematode,
soybean
Abstract
WepreviouslyisolatedapartialsoybeancDNAclonewhosetranscriptabundanceisincreaseduponinfectionbythesedentary,
endoparasiticsoybeancystnematodeHeteroderaglycines.Wenowisolatedthecorrespondingfull-lengthcDNAanddetermined
thatthepredictedgeneproductwassimilartothegroupofcofactor-dependentphosphoglyceratemutase/bisphosphoglycerate
mutaseenzymes(PGM/bPGM;EC5.4.2.1/5.4.2.4).WedesignatedthecorrespondingsoybeangeneGmPGM.PGMandbPGM
arekeycatalystsofglycolysisthathavebeenwellcharacterizedinanimalsbutnotplants.UsingtheGmPGMcDNAsequence,
weidentifiedahomologousArabidopsisthalianagene, whichwedesignatedAtPGM.HistochemicalGUSanalysesoftrans-
genic Arabidopsis plants containing the AtPGM promoter::GUS construct revealed that the AtPGM promoter directs GUS
expressioninuninfectedplantsonlytotheshootandrootapicalmeristems.Ininfectedplants,GUSstainingalsoisevidentin
thenematodefeedingstructuresinducedbythecystnematodeHeteroderaschachtiiandbytheroot-knotnematodeMeloido-
gyneincognita.Furthermore,wediscoveredthattheAtPGMpromoterwasdown-regulatedbyabscisicacidandhydroxyurea,
whereas it was induced by sucrose, oryzalin, and auxin, thereby revealing expression characteristics typical of genes with
rolesinmeristematiccells.Assessmentoftheauxin-inducibleAUX1genepromoter(agenecodingforapolarauxintransport
protein) similarlyrevealedfeeding cellandmeristemexpression, suggesting that auxinmay beresponsible for theobserved
tissue specificity of the AtPGM promoter. These resultsprovide first insight into the possible rolesof PGM/bPGM in plant
physiologyandinplant-pathogeninteractions.
Abbreviations: ABA, abscisic acid; bPGM, bisphosphoglycerate mutase; C, threshold cycles; dai, days after
t
inoculation; GUS, β-glucuronidase; IAA, 3-indoleacetic acid; PCR, polymerase chain reaction; PGM, phos-
phoglycerate mutase; PGM-d, phosphoglycerate mutase cofactor-dependent; PGM-i, phosphoglycerate mutase
cofactor-independent;qRT-PCR,quantitativereal-timereversetranscription-PCR
Introduction (Heterodera and Globodera spp.) and the root-knot
nematodes(Meloidogynespp.),whichareresponsible
Plant-parasitic nematodesareamongthemostserious for major crop losses worldwide (Sasser and Freck-
pests in world agriculture. Two of the most eco- man,1987).Forexample,thesoybeancystnematode,
nomically damaging groups are the cyst nematodes Heterodera glycines, is the most damaging pathogen
of soybean causing annual losses of several hundred
Nucleotide sequence data reported are available in the Gen-
million dollars in the USA alone (Wrather et al.,
Bank/EMBL/DDBJ databases under the accession number
AY004240. 2001a,b).Bothgroups(cystandroot-knotnematodes)
514
are sedentary biotrophic endoparasites of roots that
induce and maintain complexfeedingstructurescon-
taining specialized feeding cells. In this interaction,
infectivesecond-stagenematodejuveniles(J2s) enter
hostrootsandmigratetowardthevascularcylinderin
search of suitable cells that can serve as initial feed-
ing cells. J2s then initiate localized reorganizationof
the host cells’ morphologyand physiology, resulting
intheformationofspecializedfeedingcells.Depend-
ingonthenematodegenus,thesecellseitherdevelop
intoasyncytiabyfusionofneighboringcells(forcyst
nematodes) or several cells are stimulated to form a
system of discrete giant-cells that are embedded in a
gall(fortheroot-knotnematodes)(Jones,1981).Nem-
atodesfeedexclusivelyfromtheirfeedingcellsasthey
completetheirlifecycles.
Theplanthormoneauxinplaysakeyroleinawide
variety of growth and developmental processes. At
the cellular level, auxin acts as a signal for cell divi-
sion, extension, and differentiationduringthe course
of plant development (Davis, 1995). Several studies Figure 1. Southern blot of soybean genomic DNA probed with
have shown that auxin plays a role in feeding cell the soybean full-length D10.1 cDNA.Soybean genomic DNA
(10µg/lane)wasdigestedwithEcoRIandelectrophoresedthrough
induction by cyst and root-knot nematodes, suggest-
a 1% agarose gel. The DNA blot was hybridized with the soy-
ingthatauxinsignalingisessentialinbothsyncytium beanfull-lengthD10.1cDNA.Numbersatleftaremolecularlength
and giant-cellformation(reviewedby Goverseet al., markersinkb.
2000a; Bird and Kaloshian, 2003). Consistent with
thisidea, ithasbeenproposedthatnematodesinduce
We previouslyused differentialdisplayof mRNA
a local accumulation of auxin by controlling auxin
to isolate soybean cDNA clones corresponding to
distribution in the roots, likely by altering auxin ef-
mRNAspeciesthatchangeabundanceduringtheearly
flux (Hutangura et al., 1999; Goverse et al., 2000b).
stagesofthecompatibleinteractionbetweensoybean
Additionally, a recent study has suggested that after
andthesoybeancystnematode(H.glycines)(Herms-
aninitialauxinincreaseinthedevelopinggiant-cells,
meier et al., 1998). In these studies, we showed
root-knotnematodeslowerauxinbiosynthesisinfeed-
thattranscriptscorrespondingtoapartialcDNAclone
ingcellsatlaterstagesoftheinfectionprocesstoalter
(D10.1)increasedinH.glycines-infectedrootsofsus-
plantcelldevelopment(DoyleandLambert,2003).
ceptible soybean one day post inoculation. Here we
Feeding cell formation and maintenance are me-
report a further characterization of this gene, includ-
diated through nematode signaling and accompanied
ing the isolation of a full-length cDNA and detailed
by changes in plant gene expression (reviewed by
analysesofitsexpressioninsoybeanandArabidopsis
Davis et al., 2000; Hussey et al., 2002; William-
underavarietyofconditions.
son and Gleason, 2003). Several studies have shown
altered plant gene expression in nematode pathosys-
tems(reviewedbyGheysenandFenoll,2002;Favery
Materialsandmethods
et al., 2002; Green et al., 2002; Mazarei et al.,
2002; Juergensen et al., 2003; Puthoff et al., 2003;
Soybeanandnematodecultivation
Thurau et al., 2003). Identification and characteriza-
tion of host genesthat are potentially involvedin the ThesoybeancultivarCorsoy 79wasused throughout
plant-nematodeinteraction,asindicatedbynematode- thisstudy. Corsoy79 issusceptible to all Heterodera
induced changes in expression, might prove to be glycines biotypes tested (Bernard and Cremeens,
an important tool to aid in the understanding of the 1988). Sterile soybean plants were established by
molecular mechanisms involved in nematode-plant germination of surface-sterilized seeds. Seeds were
interactions. surface-sterilized by immersion in 70% ethanol for
515
Figure2. AlignmentofthesoybeanandArabidopsisthalianaphosphoglyceratemutase(PGM)-likeproteinswithPGMsfromotherorganisms.
The sources ofsequence data are as follows: 1, soybean GmPGM(this study, GenBank accession number AY004240); 2–6, A. thaliana
(ArabidopsisGenomeInitiativeAt2g17280,At5g64460,At1g58280,At5g04120,At3g50520);7,humanPGM(GenBankP15259);8,human
bPGM(bisphosphoglyceratemutase)(GenBankP07738);9,mousePGM(GenBankAAC13263);10,mousebPGM(GenBankP15327);11,
ratPGM(GenBankP16290);12,fruitfly(Drosophilamelanogaster)PGM(GenBankS50326);13,bacterium(Aquifexaeolicus)PGM(Gen-
BankAAC07594);14,yeast(Saccharomycescerevisiae)PGM(GenBankPMBYY);15,yeast(Schizosaccharomycespombe)PGM(GenBank
S43214).SequenceswerealignedwiththeCLUSTAL-Wprogram(Thompsonetal.,1994).Dashesindicategapsintheaminoacidsequences
usedtooptimizethealignment.Regionsofidentical(black)orsimilaraminoacid(shaded)presentinatleast50%ofthealignedsequencesare
indicated. ThetworegionsselectedasPGMsignaturepatterns(InterProteinIPR001345:PrositePS00175andProteinFamilyPF00300)are
∗
indicatedbysolidlines.Conservedresiduesthatconstitutetheactivesitesaremarkedbyasterisk( ).StrictlyconservedresiduesamongPGMs
areindicatedby#.
5 min and 2.1% sodium hypochlorite for 12 min, roots were examined for developmentalstages of the
and then rinsed three times in sterile distilled water nematodesbystainingwithacidfuchsinfollowingthe
for 10 min. Seeds were germinated in the dark at procedureofHussey(1990).Stainednematodeswere
◦
26 C on 1% water-agar for two days, transferred examinedwiththedissectingmicroscope.
to magenta boxes (Sigma, St. Louis, MO) contain-
ing sterile sand supplemented with Hoagland growth Soybeaninoculationandtissueharvesting
◦
solution(Sigma),andgrownat26 Cwitha16hpho-
toperiod of ca. 2400 lx provided by fluorescent light Nematodesforsoybeanrootinoculationweresurface-
bulbs. sterilizedfor12minin0.01%HgCl2andthenwashed
The H. glycinesinbredline OP50 (Dongand Op- threetimesin sterile distilledwater. Nematodeswere
perman,1997)waspropagatedingreenhousecultures pelleted and then re-suspendedin 1.5% low-melting-
withCorsoy79soybeansashosts.Inoculatedsoybean pointagarose(LifeTechnologies, Gaithersburg,MD)
516
(cid:3)
Figure4. Sequence ofthe AtPGM 5 promoter fragment. Nucle-
otide sequence ofthepromoterfragment directly upstream ofthe
AtPGM(ArabidopsisGenomeInitiativeAt2g17280)codingregion.
Thenucleotides corresponding totheprimersusedforamplifying
theAtPGMpromoterfragmentaremarkedbyboxes. Thetransla-
tion initiation codon is indicated by asterisks. Putative cis-acting
elements were determined bythe PLACE database (Hingo et al.,
1998).Auxin-responsiveelements(GmSAUR)(Xuetal.,1997)are
indicatedbysingleunderline;theabscisicacid-responsiveelement
(ABRE)(BuskandPage`s,1998)isindicatedbydoubleunderline;
thenematodebox(Escobaretal.,1999)isindicatedbytripleunder-
line;themitosis-specificactivator-like sequence(MSA)(Itoetal.,
1998)ismarkedbyanarrowedunderline; andthesugar-response
element (amy3Os) (Hwang et al., 1998) is marked by a dashed
Figure3. RNAblotanalysisofGmPGM.A.GmPGMmRNAac- underline.
cumulation in nematode-infected and uninfected plants. Blots of
total RNA (10 µg/lane) were obtained from roots and shoots of
Heteroderaglycines-infected(+)anduninfected(−)soybeanplants to continue growth as described above. At different
at1,3,and6days(d)afternematodeinoculation.Blotswerestand-
time points after inoculation, plants were taken out
ardized with a soybean actin probe (Actin) (Nagao et al., 1981),
whichwehavepreviouslyshowntobeconstitutivelyexpresseddur- of the sand, and their roots were washed by rinsing
ingtheearlyH.glycinesinfectionstages(Hermsmeieretal.,1998). withsteriledistilledwater. Rootandshoot(including
Theethidium bromidestainoftheagarosegelwasusedtoverify
cotyledons and two-true-leaf) tissues of eight plants
equalRNAloadingbeforeblotting(rRNA).Autoradiographswere
were harvested at each time point, frozen in liquid
recordedafterthreedaysofexposuretoaphosphorimagerscreen.
Representative blots are shown. Similar results were obtained in nitrogen, and stored at −80 ◦C until use. To ensure
threeotherexperiments.B.QuantificationofGmPGMexpression. aseptic conditions, the agarose-nematode suspension
Intensityofthehybridizationsignalsdetectedinthesoybeanroots
used for plant inoculation was also applied to sterile
wasmeasuredwithaphosphorimagerandquantifiedwiththeIm-
soybeanplantsestablishedonGamborg’sB5medium
ageQuant software. GmPGM hyrbidization intensity values were
adjusted relative to the constitutive actin hybridization intensity (Life Technologies). These cultures were monitored
values(relativecounts).Eachbarrepresentsthemeanoffourinde- for signs of microbialcontamination. An inoculation
pendentnorthernblotswiththestandarderrorsofthenotedmean.
seriesthatremainedclearofmicrobialcontamination
Significance oftheinduction ofGmPGMexpression ateachtime
point(infectedcomparedwithuninfected)wasdeterminedstatistic- wasusedfortheremainderofthiswork.
allybyatwo-samplet-test(P <0.05).Asterisksindicatesignificant
differences. SoybeancDNAlibraryscreening
◦ AUni-ZAPXRdirectionallycloned,oligo-dT-primed
at 37 C. Plants were inoculated at the two-true-leaf
cDNA library was constructed (Stratagene, La Jolla,
stage after about eight days of growth in sand-filled
CA) with RNA prepared from infected Corsoy 79
magentaboxes. Inoculationwasperformedbyapply-
soybean roots harvested at 1, 2, and 3 days after
ing1mlofanagarose-nematodesuspension,contain-
inoculation with the H. glycines inbred line OP50.
ingabout1000nematodes, toplantrootingzonesvia
cDNA clone D10.1 was used to screen the non-
narrowholescreatedinthesandsubstratepriortoin-
amplifiedlibraryfraction.Theprobewasradiolabeled
oculation. Control plants were treated with agarose
by PCR of gel-purified cDNA fragment with vector-
in the absence of nematodes. Plants were allowed
specificprimerspGEM-upandpGEM-down(Herms-
517
meier et al., 1998). About 350000 recombinantbac- A blot of EcoRI-digested soybean genomic DNA
teriophage from the cDNA library were screened by waspreparedasdescribed(Hermsmeieretal., 1998).
hybridizationwith radioactivelylabeledD10.1clone. DNA gel blot hybridization and analysis was per-
Positive plaques were isolated, purified, and excised formedasexplainedabove.
invivofollowingthemanufacturer’sinstructions.
IsolationofAtPGMpromoterregionfromA.thaliana
DNAsequencing
Sequencing and annotation of the BAC clone F5J6
Recombinantplasmidsisolatedfromthesoybeanroot fromchromosome2whichcontainstheAtPGM gene
cDNA library were purifiedwith the Qiagen Plasmid (GenBank accession number AC002329/F5J6.4) re-
Mini Kit (Qiagen, Chatsworth, CA) and subjected to vealed that 1155 bp upstream of the AtPGM cod-
sequencingattheIowaStateUniversityDNASequen- ing region lies another open reading frame (F5J6.3).
cingandSynthesisFacility(Ames,IA).Sequenceana- Therefore, the AtPGM promoter is likely positioned
lysiswasperformedwiththeBLAST(Altschuletal., entirelywithinthisintergenicregionandthe1155bp
1997) and DNASIS (Hitachi Software Engineering, genomic fragment directly upstream of the AtPGM
SanBruno,CA)programs. codingregioncontainsalltheregulatoryelementsre-
quiredtodirectcorrecttemporalandspatialexpression
RNAandDNAgelblotanalyses of the AtPGM gene. Due to the lack of information
concerningthe exact length of the AtPGM promoter,
For RNA blotanalyses, totalRNA was isolated from wePCR-amplifiedthe1130bpgenomicfragmentup-
frozen root and shoot tissues of eight H. glycines- streamoftheAtPGMcodingsequence(seebelow)and
infected and uninfected soybean plants each ground useditinthepromoteranalysesdescribedinthisstudy.
underliquidnitrogenasdescribedbyPawlowskietal.
(1994). RNA (10 µg) from each sample was separ-
atedon1%formaldehydeagarosegelsandtransferred Promoterconstruct
ontonylonmembranes(S&SNytranPlus, Schleicher
& Schuell, Keene, NH) by a wet blotting proced- The AtPGM promoter was isolated by PCR ampli-
ure (Sambrook et al., 1989). RNA was fixed to the fication from A. thaliana genomic DNA prepared as
membranesin a FB-UVXL-1000cross-linker(Fisher described by Rogers and Bendich (1994). Primers
Scientific, Pittsburgh, PA). Four independent blots were designed to amplify the AtPGM promoter
werepreparedfromthetotalRNA. (corresponding to 1130 bp upstream of the trans-
(cid:3)
GmPGM insert in the pBluescript SK(−) vector lation start site) and to create SalI (at the 5
(cid:3)
obtained from the cDNA library screen was ampli- end) and BamHI (at the 3 end) restriction sites.
(cid:3)
fied by PCR with vector-specific primers T3 and T7 The primers used were: AtPGM-P forward, 5-
(cid:3)
(Integrated DNA Technology, Coralville, IA). PCR ATGTCGACATCAACAATAAAACCAGGAACT-3
(cid:3)
product was gel-purified with a Qiaex II Kit (Qia- andAtPGM-Preverse,5-ATGGATCCGGCTACAAC
(cid:3)
gen), radiolabeledviaPCR, andthenusedasa probe AACAAGAAGGGAA-3. PCR amplification was
in soybean RNA blot analyses. Hybridizations were performed with Taq polymerase (Life Technologies)
carried out at 42 ◦C in a hybridization buffer com- in a three-temperature program (94 ◦C, 55 ◦C, and
◦
posed of 5× SSC (1× SSC is 0.15 M NaCl plus 72 C for 1 min each) with 30 cycles after 5 min of
◦
0.015 M sodium citrate), 50% formamide, 0.1% so- incubation at 94 C. The PCR product was digested
dium dodecyl sulfate (SDS), 5× Denhardt’s solution with SalI and BamHI, gel-purified, and ligated into
(Sambrooket al., 1989), 0.1 mg ml−1 herring-sperm binary vector pBI101.1 (Jefferson et al., 1987). The
DNA, and 3 × 106 cpm/ml of labeled gene probe. binary construct was maintained in Escherichia coli
The hybridized blots were washed three times for strain DH5α. Both strands of the promoter construct
15 min in 0.1× SSC/0.1% SDS at 65 ◦C. Bound weresequencedtoverifywild-typesequences.
radiolabeled probes were imaged with a Molecular
DynamicsStorm840PhosphorImager(MolecularDy- TransgenicArabidopsisthalianaplants,nematode
namics,Sunnyvale,CA),whichallowedquantification infection,andtreatments
of hybridized probe by using ImageQuant software
Agrobacterium tumefacienes strain C58 was trans-
(MolecularDynamics).
formedwiththeAtPGM::GUSconstructbythefreeze-
518
Figure5. HistochemicalstainingforGUSactivityintransgenicA.thalianaplantscontainingtheAtPGM::GUSconstruct.GUSexpressionin
shoot(A)andinroot(B,C)ofa12-dayoldA.thalianaplant.TheinsetinAandthearrowindicateGUSactivityintheshootapicalmeristem.
ThearrowheadinBindicatesGUSactivityintheroottip.Arootlongitudinalsection(C)showsGUSactivityintherootapicalmeristem(rm).
Morethan10independenttransformedlinesweretestedforGUSexpressionandsimilarobservationswereobtained. D–H.GUSexpression
intransgenicA.thalianaplantsinfectedwithcystnematodeHeteroderaschachtii.LocalizedGUSexpressioninsyncytium(s)at3daysafter
inoculation(dai)(D),at7dai(E),andat14dai(F).Arootlongitudinalsection(G)andacrosssection(H)revealtheanteriorendsofsedentary
nematodes(arrows)associatedwithfeedingcellsshowingstrongGUSactivityat3dai.I–K.GUSexpressionintransgenicA.thalianaplants
infected withroot-knotnematode Meloidogyne incognita. Localized GUSexpressionwithinthenematode-induced galls (g)containing the
giant-cells at3dai(I),at7dai(J),andat14dai(K).Arrowsindicatenematodes. ArrowheadsindicateGUSexpressioninroottips, which
alsoisobservedinuninfectedroots.ThedifferencesinGUSexpressionbetweennematode-infectedanduninfectedrootswereobservedin5
independenttransformedlinesforwhichatleast10seedlingseachwereassayed.
519
Figure6. PromoteractivityofAtPGM::GUSintransgenicA.thalianaplantssubjectedtovarioustreatments.Two-weekoldtransgenicplants
weregrownfor48honmediumsupplementedwitheachtreatment.Atleast10seedlingsfrom5independenttransgeniclineswereusedfor
eachassayandtypicalexamplesofGUSstainingareshown.AtypicalexampleofuntreatedcontrolplantsisshowninFigure5B.Histochemical
GUSexpressioninrootsofA.thalianaplantstreatedwith50µM3-indoleaceticacid(A),100µMabscisicacid(B),6%sucrose(C),30µM
oryzalin(DandE),or100mMhydroxyurea(F).rp,lateralrootprimordium;vs,vascularcylinder.
thaw method (An et al., 1988), and used to gen- washed three times with sterile distilled water, then
erate transgenic A. thaliana plants. The transformed planted aseptically, one seed per well, in 12-well
A.tumefacieneswasgrowninLB(Difco,Detroit,MI) cultureplatescontainingmodifiedKnopmedium(Sij-
medium containing 50 mg/ml gentamycin and kana- mons et al., 1991) solidified with 0.8% Daishin
mycin(Fisher,FairLawn,NJ).A.thaliana(wild-type agar(BrunschwigChemie,Amsterdam,Netherlands).
◦
Columbia) transformation was performed as previ- Plantsweregrownat26 Cwitha12-hphotoperiodof
ously described (Clough and Bent, 1998). At least ca.1500lxprovidedbyfluorescentlightbulbs.Trans-
15 independent transgenic A. thaliana plants were genicplants10–12daysoldwereinoculatedindividu-
generatedforthepromoterconstruct. ally with ca. 500 surface-sterilizedJ2. Four plants in
For nematode experiments, Heterodera schachtii the 12-well plate were arbitrarily positioned as non-
andMeloidogyneincognitawerepropagatedingreen- inoculatedcontrolplants.Theplantsweremaintained
house cultures with sugar beet (cv. Monohi) and in the temperature and lighting regimes mentioned
tomato (cv. Rutgers), respectively, as hosts. Surface- above. Histochemical GUS analyses were performed
sterilized J2 of the nematodes were prepared as (seebelow)toexaminetheGUSactivity.
previously described (Baum et al., 2000). Trans- Forexperiments,transgenicplantsweregrownon
genicA.thalianaseeds(T generation)weresurface- Knopmediumfortwoweeksunderthesametemperat-
2
sterilized with 2.6% sodium hypochlorite for 5 min, ureandlightingregimesmentionedabove.Wounding
520
vested from Knop plates and were then incubated in
Knop medium without or with, respectively, 50 µM
3-indoleaceticacid(3-IAA,Sigma),100µMabscisic
acid(ABA,Sigma),and6%sucrose(Fisher)for48h.
Forhydroxyureaandoryzalintreatments,plantswere
harvested from Knop plates and then transferred to
the same medium containing 100 mM hydroxyurea
(Sigma) or 30 µM oryzalin (Sigma), and were kept
for 48 h. Control plants were kept under the same
conditions in the absence of hydroxyurea or oryza-
lin treatments. The treated and untreated plants were
stained with GUS staining solution in histochemical
GUS analysis (see below), or were harvested, frozen
in liquid nitrogen, and kept at −80 ◦C until use in
fluorometricGUSanalysis(seebelow).
HistochemicalGUSanalysis
Histochemical assays for GUS expression were per-
formedwiththesubstrate5-bromo-4-chloro-3-indolyl
glucuronide (X-Gluc) (Research Products Interna-
tional, Mt. Prospect, IL) according to standard pro-
tocols(Jefferson,1987).GUSstainingwascarriedout
◦
overnightat37 C.
For the nematode experiments, GUS activity was
determinedintransgenicA.thalianaplantsbyadding
GUSstainingsolutiontoeachwell,withoutdisturbing
the roots. Nematode-infected and uninfected plants
were stained for GUS activity at varying times after
Figure 7. Quantitative real-time RT-PCR (qRT-PCR) analysis of
AtPGM mRNA steady-state levels. Total RNA was isolated from nematode inoculation. After GUS staining, tissues
respective tissues of A. thaliana plants. First-strand cDNA was wereeitherclearedbyreplacingtheGUSsolutionwith
synthesizedandsubjectedtoqRT-PCRintriplicate ontheiCycler
70%ethanol,withoutdisturbingtheplantorprepared
as described in Materials and methods. Relative AtPGM mRNA
for further cytological analyses, as described below.
quantities (Expression) weredetermined withiCycler software as
detailed in Materials and methods. Each bar represents the mean GUSactivitywasmonitoredmicroscopicallyinshoot
of three independent experiments with the standard errors of the androottissues.
noted mean. Significant AtPGM mRNAexpression changes were
Forthetreatmentexperiments,thetreatedandun-
determinedstatisticallybyatwo-samplet-test(P <0.05).Asterisks
indicate significant mRNA changes. A. AtPGM mRNA accumu- treated plants were incubated in the GUS staining
◦
lation in shoots and roots of 2-week old A. thaliana plants. B. solution overnight at 37 C. The stained plants were
AtPGMmRNAaccumulationinH.schachtii-infectedanduninfec- clearedwith70%ethanolandthenexaminedforGUS
tedrootsof12-dayoldA.thalianaplantsat1,3,and5days(d)after
activitymicroscopically.
nematodeinoculation. C.AtPGMmRNAaccumulationinrootsof
2-weekoldA.thalianaplantsintheabsence(untreated)andpres- StainedtissueswereobservedunderaZeissStemi
ence(treated)of50µM3-indoleaceticacid(IAA),100µMabscisic SV 11dissecting microscopeandimageswere recor-
acid(ABA),or6%sucrosefor48h. dedonto KodakEktachrome64Ttungstenslide film.
Slides were scanned into Adobe Photoshop 6.0, ad-
justedforbrightnessandcontrast,andassembledinto
was performed by repeatedly piercing the roots and
figures.
leaves of the plants with sterile pipette tips. At 10 h,
For cytological studies, GUS-stained A. thaliana
3 days, or 7 days, wounded plants were removed
plantswerecarefullyremovedfromtheirmediumand
from the Knop plates and stained for GUS activity
fixed in a mixture of freshlyprepared2% glutaralde-
(see below). Control plants were maintained under
hyde(TedPella,Redding,CA)and2%formaldehyde
the same conditionsin the absence of wounding. For
(Fisher) in 50 mM PIPES (1,4-piperazinediethane
IAA, ABA, and sucrose treatments, plants were har-
521
(cid:3)
sulfonicacid;Fisher)plus10%sucrosepH7.2under TGAGTTCTTTCGGGAAGG-3, and qAtPGM re-
◦ (cid:3)
vacuum overnight at 4 C. Samples were washed in verse, 5-CTCGATCCACGATAACCATTGAGCGA-
(cid:3)
50mMPIPESpH7.2,dehydratedinagradedethanol 3.Theseprimersamplifiedasingleproductof109bp,
seriesto95%ethanolandinfiltratedandembeddedin asconfirmedbythemeltingtemperatureoftheampl-
glycol methacrylate resin (Electron Microscopy Sci- iconsandgelelectrophoresis.TotalRNAwasisolated
ences,FortWashington,PA).Samplesweresectioned fromtherespectivefrozentissuesonRNeasycolumns
on a Reichert Ultracut S ultramicrotome (Leica Mi- (Qiagen, Valencia, CA) followingthe manufacturer’s
crosystems, Wetzlar, Germany) at 3–5 µm, mounted instructions. As described in Puthoff et al. (2003),
on glass slides and examined on an Olympus BH- 5 µg of total RNA was denatured in the presence
2 compound microscope equipped with differential of an oligo-d(T) primer, cooled on ice and divided
interferencecontrast(DIC)optics.Imageswererecor- evenly into three reverse transcriptionreactions. One
dedandfigureswereassembledasdescribedabove. ofthesereactionswasmonitoredforcDNAsynthesis
with 32P-dCTP (NEN, Boston, MA) incorporation, a
FluorometricGUSanalysis second reaction was used as the RT control (no re-
versetranscriptasewas added), andthe thirdreaction
Fluorescence of 4-methylumbelliferone (4-MU) pro-
was used for later qRT-PCR, which was conducted
duced by cleavage of 4-methylumbelliferyl-β-D-
in triplicate. The following additions were made to
glucuronide(MUG)(ResearchProductsInternational) eachreaction:4µl5×first-strandbuffer(Gibco-BRL,
was measured to quantify GUS activity according
Rockville,MD),2µl0.1MDTT,1µl10mMdNTP
to standard protocols described by Jefferson (1987).
mix,1µlSuperscriptIIReverseTranscriptase(Gibco-
Protein concentration was determined with a Pierce
BRL).IntheRTcontrolreactionwaterwassubstituted
BCA-200ProteinAssayKit(Pierce,Rockford,IL).
forSuperscriptII.Each20µlreactionwasincubated
at 42 ◦C for 1 h. With the 32P-monitoredreaction as
AtPGMmRNAexpressionanalyses
a guide, first-strand cDNA was diluted to ensure an
Quantitativereal-timereversetranscription-PCR(qRT- equal amount of cDNA (equivalent to 10 ng of total
PCR) (Bustin, 2002) was used to quantify AtPGM RNA/PCRreaction)wasplacedineachPCRreaction.
mRNA abundancechanges in A. thaliana shoots and PCRwasconductedontheiCycler(BioRad,Hercules,
rootsinavarietyoftreatments. CA) with a 96-well reaction block in the presence
Fornematodetreatment, A. thalianaplants(wild- of SYBR Green under the following conditions: 1×
typeColumbia)weregrownunderinvitro conditions PCRbuffer(Invitrogen,Carlsbad,CA),3mMMgCl2,
and inoculated with H. schachtii as described above. 0.4 mM dNTP, 0.1 µM each primer, 10 nM fluor-
Atvaryingtimepointsafterinoculation,rootandshoot escein (BioRad, Hercules, CA), 1:100000 dilution
tissuesof10plantspertreatment(inoculatedandnon- SYBRGreen, 10000×stock, 1U PlatinumTaq(In-
inoculated control) were harvested, frozen in liquid vitrogen, Carlsbad, CA) in a 25 µl reaction. PCR
nitrogen,andstoredat−80◦Cuntiluse. cycling was 95 ◦C for 3 min, followed by 45 cycles
◦ ◦
For hormone and sucrose treatments, A. thali- of15sat95 C,30sat60 C.Thresholdcycles(Ct)
anaplantsweregrowninsoil(SunshineProfessional were determined with iCycler (BioRad) software for
Growing Mix, Bellevue, WA) at 26 ◦C with a 12 h alltreatments. ToquantifyrelativemRNAconcentra-
photoperiod of ca. 1500 lx provided by fluorescent tions,astandardcurvewaspreparedforeach96-well
lightbulbs.Treatmentswereperformedon2-weekold platePCRreaction.Forthispurpose,athreefolddilu-
plants.Therootsystemsofwholeplantswerewashed tionseriesofatotalofsixdilutionswaspreparedfrom
gently with water to remove soil and then the plants an A. thaliana root RNA sample (which was known
were used for each treatment. Ten plants per treat- to contain detectable AtPGM mRNA amounts), and
mentwereused.Thewholeplantswereincubatedfor each dilution was subjected to qRT-PCR analyses in
48 h in either 50 µM IAA, 100 µM ABA, or 6% triplicate with the AtPGM-specific primers. Obtained
sucrose.Controlplantswereincubatedinwateralone. Ct valueswere used by the iCycler software package
Rootandshoottissueswereharvested,frozeninliquid toplotastandardcurvethatallowedquantificationof
nitrogen,andkeptat−80◦Cuntiluse. AtPGM mRNA in other RNA samples relative to the
In order to conduct qRT-PCR assays for AtPGM, RNAsampleusedtopreparethestandardcurve.These
the following gene-specific primers to the 3(cid:3) region relativemRNAquantitiesofRNAsamplesarepresen-
weredesigned:qAtPGMforward,5(cid:3)-CACGGTCTCT tedas‘Expression’valuesinFigure7andallowedthe