Structure and Expression Profile of the Phosphate Pht1 Transporter Gene Family in Mycorrhizal ...

14 Pages·2011·1.59 MB·English

Structure and Expression Profile of the Phosphate
Pht1 Transporter Gene Family in Mycorrhizal
Populus trichocarpa1[W]
Vero´nica Loth-Pereda2, Elena Orsini2,3, Pierre-Emmanuel Courty2,*, Fre´de´ric Lota, Annegret Kohler,
Loic Diss, Damien Blaudez, Michel Chalot, Uwe Nehls4, Marcel Bucher5, and Francis Martin
Unite´ Mixte de Recherche (UMR) 1136 Institut National de la Recherche Agronomique/Nancy Universite´
Interactions Arbres/Micro-organismes, Institut National de la Recherche Agronomique Nancy, 54280
Champenoux, France (V.L.-P., A.K., L.D., D.B., M.C., U.N., F.M.); Federal Institute of Technology, Zurich,
Institute of Plant Sciences, Experimental Station Eschikon 33, CH–8315 Lindau, Switzerland (E.O., M.B.);
Botanical Institute, University of Basel, 4056 Basel, Switzerland (P.-E.C.); and Institute of Botany, University of
Cologne, 50674 Cologne, Germany (F.L.)
Gene networks involved in inorganic phosphate (Pi) acquisition and homeostasis in woody perennial species able to form
mycorrhizalsymbiosesarepoorlyknown.Here,wedescribethefeaturesofthe12genescodingforPitransportersofthePht1
familyinpoplar(Populustrichocarpa).IndividualPht1transportersplaydistinctrolesinacquiringandtranslocatingPiindifferent
tissues ofmycorrhizaland nonmycorrhizal poplarduringdifferent growthconditionsand developmental stages.Pistarvation
triggered the up-regulation of most members of the Pht1 family, especially PtPT9 and PtPT11. PtPT9 and PtPT12 showed a
striking up-regulation in ectomycorrhizas and endomycorrhizas, whereas PtPT1 and PtPT11 were strongly down-regulated.
PtPT10 transcripts were highly abundant in arbuscular mycorrhiza (AM) roots only. PtPT8 and PtPT10 are phylogenetically
associatedtotheAM-induciblePht1subfamilyI.Theanalysisofpromotersequencesrevealedconservedmotifssimilartoother
AM-inducibleorthologsinPtPT10only.TogainmoreinsightintogeneregulatorymechanismsgoverningtheAMsymbiosisin
woody plant species, the activation of the poplar PtPT10 promoter was investigated and detected in AM of potato (Solanum
tuberosum)roots.TheseresultsindicatedthattheregulationofAM-induciblePitransportergenesisconservedbetweenperennial
woodyandherbaceousplantspecies.Moreover,poplarhasdevelopedanalternativePiuptakepathwaydistinctfromAMplants,
allowingectomycorrhizalpoplartorecruitPtPT9andPtPT12tocopewithlimitingPiconcentrationsinforestsoils.
Phosphorus(P)isrequiredformostplantprocesses, primary source of P for plants isinorganicphosphate
being essentialfordevelopment and metabolism. The (Pi). Since Pi is often present in limited amounts in
soils, it represents the second most frequently limit-
1ThisworkwassupportedbytheNationalCouncilofScienceand ingfactorforplantgrowthinavarietyofnaturaleco-
TechnologyNationalForestryCommission(Ph.D.fellowshiptoV.L.-P.), systems (Marschner, 1995; Poirier and Bucher, 2002;
bytheSwissNationalScienceFoundation(toP.-E.C.),bytheFederal Misson et al., 2004). Pi concentration in the soil solu-
InstituteofTechnology,Zurich,bytheSwissFederalOfficeforEduca- tionhardlyreaches1mMinforestsoilsandmaydrop
tion and Science COST Action GENOSILVA (E28), by the German to micromolar or even submicromolar levels at the
ScienceFoundation(grantno.BU2250/1,toM.B.),bytheEuropean root-soilinterface,whereadepletionzoneisgenerated
CommissionwithintheprojectsPOPYOMICSandENERGYPOPLAR by rapid Pi uptake of plants and rhizospheric micro-
(grant no. FP7–211917), by the U.S. Department of Energy Plant-
organisms (Poirier and Bucher, 2002; Plassard and
MicrobeInterfacesproject,bytheNetworkofExcellenceEVOLTREE
Dell, 2010).
(grantno.FP6–016322),andbytheRe´gionLorraine(toF.M.).
2Theseauthorscontributedequallytothearticle. Higher plants use a series of adaptive morpholog-
3Presentaddress:UniversityofHohenheim,350InstituteofPlant ical and biochemical strategies to increase the acqui-
Breeding, Seed Science, and Population Genetics, 70593 Stuttgart, sitionofpoorlyavailablePi.Theseadaptivestrategies
Germany. areoftenbasedonrootcell-specificactivationofgenes,
4Presentaddress:Biology/ChemistryBotany,UniversityofBre- the regulation of which can be conserved between
men,Fachbereich2,28359Bremen,Germany. distantly related species. In vascular plants, the root
5Present address: Institute of Botany, University of Cologne, epidermis including root hair cells participates in
50674Cologne,Germany. direct nutrient uptake from the soil solution at the
*Correspondingauthor;[email protected].
root-soilinterface(Marschner,1995).Theformationof
Theauthorresponsiblefordistributionofmaterialsintegraltothe
thearbuscular mycorrhizal (AM)symbiosisallows the
findings presented in this article in accordance with the policy
establishment of a second interface involved in plant
describedintheInstructionsforAuthors(www.plantphysiol.org)is:
nutrientacquisition(i.e.attheintraradicalroot-fungus
FrancisMartin([email protected]).
[W]TheonlineversionofthisarticlecontainsWeb-onlydata. interface in the inner cortex). Thus, the AM symbiosis
www.plantphysiol.org/cgi/doi/10.1104/pp.111.180646 evolved to improve the nutrient acquisition efficiency
Plant Physiology(cid:1), August 2011, Vol. 156, pp. 2141–2154, www.plantphysiol.org (cid:3) 2011 American Society of Plant Biologists 2141
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Loth-Pereda et al.
of especially Pi, by forming far-reaching extraradical specifically in cortex cells harboring arbuscules and
mycelia, which operate as functional extensions of the coiled hyphae in transgenic roots of several herba-
plantrootsystem(Paszkowskietal.,2002;Karandashov ceous plant species, including potato, Petunia hybrida,
andBucher,2005). Daucus carota, Medicago truncatula, and Lotus japonicus
The ectomycorrhizal (EM) fungi are a type of root (Rausch et al., 2001; Karandashov et al., 2004), indi-
symbionts that have been shown to improve nutrient cating evolutionary conservation of gene regulatory
availability mainly in tree species (Martin and Nehls, mechanisms in symbiotic Pi transport of distantly
2009). A typical ectomycorrhiza is characterized by a relatedplantspecies.
dense sheath of fungal hypha formed mainly around The major aim of this study was to establish a
young lateral roots.EM hyphae extend intercellularly comprehensive genome-wide inventory of Pi trans-
into the outer root cortex, forming the Hartig net porters of the Pht1 family (TC 2.A.1.9) in poplar
(Martin and Nehls, 2009). Extraradical EM fungal (Populus trichocarpa ‘Nisqually’) and to study their
hyphaesecreteenzymesabletohydrolyzesoilorganic expression. Pht1 transporters are homologs of the
P compounds that are normally unavailable to non- yeast PHO84 Pi transporter, and they belong to the
EM roots(Plassardand Dell, 2010). major facilitator superfamily of proteins (Pao et al.,
Specific transport systems are essential for the up- 1998). The release of the genome sequence of this
takeofPiandforitspartitioningwithinplants(Poirier perennialspecies(Tuskanetal.,2006)andthepresence
and Bucher, 2002). Kinetic studies have shown the of an extensive EST database enabled genome-wide
coexistenceoftwotransportsystemswithloworhigh screeningandtheidentificationof12Pht1transporter
affinityfor Pi(Dunlopet al., 1997;Missonet al., 2004; genes and corresponding transcripts. The genome
Miller et al., 2009). Transport of Pi through plant organization and evolutionary history of the poplar
membranes is mediated by a number of transporter Pht1 genes are described along with their expression
proteins,whichbelongtoatleastthreedistinctprotein patternsinvarioustissuesduringPistarvation,senes-
families, and serves Pi partitioning and recycling of cence, and EM and AM symbioses. In addition, the
Pi from, for example, senescing leaves throughout regulation of the AM-specific poplar PtPT10 gene is
a plant’s life cycle. The low-affinity transporters are revealed in transgenic potato plants harboring the
activeintheremobilizationofacquiredP(Smithetal., PtPT10 promoter/GUS chimeric gene. Reciprocally,
2001),whereasthehigh-affinityPitransportersplayan the conservation of solanaceous root hair-specific
important role in the acquisition of Pi. In annual LeExt1 and AM-specific StPT3 promoter regulation is
plants, Himelblau and Amasino (2001) have found investigated in transgenicpoplarroots.
that about 80% of P is removed from senescing
Arabidopsis (Arabidopsis thaliana) leaves. In barley
(Hordeum vulgare), expression of the Pi transporter
HvPT6, likely involved in Pi remobilization (Preuss RESULTS
et al., 2010), was increased as leaves aged (Rae et al., Annotation and Distribution of Poplar Pht1 Genes
2003).
The Pht1 family, comprising nine members in the TwelvegenemodelscodingforputativePht1trans-
Arabidopsis genome, includes proteins involved in porters were identified in the predicted gene catalog
the uptake of Pi from the soil solution and the redis- resultingfromtheautomatedannotationoftheP.tricho-
tributionofPiwithintheplant.Membersofthisfamily carpa genome assembly (version 1.1; http://genome.
areH+/Pisymporters(Bucher,2007;Javotetal.,2007). jgi-psf.org/Poptr1_1/Poptr1_1.home.html). The curated
MostofthegenesofthePht1familythatareexpressed setofPht1genes(forP.trichocarpa Pitransporter fam-
inrootsareup-regulatedinP-deprivedplants(Mudge ily 1) were named according to the Commission on
et al., 2002). The Pht2 family proteins share high Plant Gene Nomenclature as POPtr;Pht1;1 through
similarity with mammalian Na+/Pi cotransporters POPtr;Pht1;12; for simplification, they will be called
but function as H+/Pi cotransporters in plastids of PtPT1 through PtPT12 in this article. ESTs have been
plants (Versaw and Harrison, 2002; Bucher, 2007). identified for these 12 Pht1 transporters, confirming
Expression patterns, Pi transport activities, and sub- thattheywereexpressed.IntheP.trichocarpagenome
cellular localization suggest roles for Pht4 proteins in assembly (version 1.1), these 12 putative Pht1 genes
thetransportofPiinplastidsandtheGolgiapparatus were located on scaffolds 125, 197, and 273 and
(Guo et al., 2008). The expression pattern of 11 PHO1 mapped to linkage groups (LG) I, II, V, and X. In the
homologs in the Arabidopsis genome indicated a P. trichocarpa genome assembly (version 2.0; http://
likely role of the PHO1 proteins in the transfer of Pi www.phytozome.net), the Pht1 transporters were in-
tothevascularcylinderofvarioustissuesbutalsoinPi tegrated into LG XIX, XV, and X (Supplemental Table
acquisitionintocells,suchaspollenorrootepidermal S1; Supplemental Fig. S1). In genome assembly ver-
cells and cells ofthe cortex (Wanget al., 2004). sion 2.0, three more Pht1 transporters were predicted
The first described AM-inducible Pi transporter (Supplemental Table S2). Examination of these gene
gene,StPT3frompotato(Solanumtuberosum),encodes models revealed one truncated gene model (POPTR_
ahigh-affinityPitransporter(Rauschetal.,2001).The 0005s24510.1), another (POPTR_0005s15860.1) sharing
1.7-kb-long StPT3 promoter directed GUS expression 100%sequencesimilaritywithPtPT6, and genemodel
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Pht1 Family in Populus trichocarpa
POPTR_0005s24510.1 having 97% sequence similarity proteins. Whereas the wild-type yeast strain could
with PtPT1. These similarities concerned not only the normally grow on both sufficient and low-Pi media,
coding sequences but also the upstream and down- a Dpho84 strain exhibited a growth defect at low-Pi
streamnoncodingpartsofthegenes,thusmakingthese conditions.YeastcellsexpressingPtPT1,PtPT2,PtPT4,
genemodelsquestionable.Therefore,theywerenotin- PtPT9 , andPtPT10 were not able to grow on low-Pi
cludedinthisstudy. medium.Onthecontrary,yeastcellstransformedwith
PtPT4 and PtPT7, sharing 96% sequence similarity, the pFL61 vectorharboring PtPT6 cDNA showed par-
were closely located on LG V (Supplemental Table tiallyrestoredgrowth.PHO84Pwasshowntofunction
S3). These two genes belonged to tandem repeat se- also as a high-affinity manganese transporter (Jensen
quences,thatis,multiplecopiesofasimilarnucleotide etal.,2003).However,allthePht1-expressingcellsdid
sequencewithinalessthan10-kbregionofthegenome not present any growth defect on toxic manganese
(here a 5.94-kb region). PtPT4, PtPT6, PtPT7, and medium (data not shown). Therefore, yeast comple-
PtPT11 and PtPT1, PtPT2, and PtPT3, respectively, mentation revealed that PtPT6 has high-affinity Pi
located on LG V and LG X are not tandem repeat transporter activity.
sequences.PtPT4/PtPT7andPtPT9/PtPT11have96%
and 85% homology, respectively, suggesting that they
Gene Expression of Pht1 Transporters in Poplar Tissues
areduplicated genes (Supplemental TableS3).
In poplar adventitious roots, PtPT1, PtPT2, PtPT3,
Primary and Secondary Structures of Pht1 Proteins PtPT4/PtPT7, PtPT9, PtPT11, and PtPT12 showed the
highest expression levels, whereas PtPT5 and PtPT6
All putative poplar Pht1 proteins contained 12 pre- were expressed at 5-fold lower levels in the same
dicted transmembrane domains (TM) separated into tissue (Fig. 2A). In central cylinder, petiole, and ma-
two blocks of six TM spanners by a long hydrophilic ture leaves, the relative expression of PtPT1, PtPT2,
loop and hydrophilic N and C termini (Supplemental PtPT3, PtPT5, PtPT6, PtPT9, andPtPT12 was similar,
TableS1;Paoetal.,1998).ThePht1signatureGGDYPL- but PtPT4/PtPT7 were expressed at higher levels
SATIxSE (Karandashov and Bucher, 2005), conserved (from 5- to 100-fold). In inflorescences, PtPT3, PtPT9,
among all Pht1 proteins, was identified. In three of PtPT11, and PtPT12 were not detectable. PtPT2,
them, the signature was slightly modified: in PtPT6, a PtPT4/PtPT7, andPtPT5 were expressed both in fe-
Ser(S)wasexchangedwithanAla(A);inPtPT8,aThr male and male inflorescences. PtPT6 and PtPT1 were
(T)wasexchangedwithaVal(V);andinPtPT10,aPro expressed only in male and female inflorescences, re-
(P) was exchanged with a Ser (S). TM10 of PtPT10 spectively(Fig.2A).Inthreebuddevelopmentalstages,
shared a high similarity with the WW domain signa- PtPT4/PtPT7 were the most highly expressed genes
ture,alsoknownasrsp5orWWP,betweenaminoacids (Fig. 2B). PtPT2 and PtPT6 were lowly, but similarly,
400 and 425, as reported for LePT4 and StPT4 (Nagy expressed in these three stages. PtPT1, PtPT3, PtPT5,
etal.,2005).Eightoutof12Pht1genesareintronlessin PtPT9, PtPT11 , andPtPT12 were expressed at higher
poplar (Supplemental Table S1). The percentage iden- levels in stage 2 than in stages 3 and 4. Finally, PtPT8
tity between poplar and Arabidopsis Pht1 proteins andPtPT10expressionwasnotdetectedinthesetissues.
reached values from 83.5% (PtPT4 against AtPT2) to Young and mature poplar leaves showed similar
50.2%(PtPT9againstAtPT2;SupplementalTableS1). transcript levels in PtPT3, PtPT4/PtPT7, PtPT5, and
PtPT11 (Fig. 3). On the other hand, five Pht1 gene
Phylogenetic and Functional Analysis of Pht1 Proteins transcripts (PtPT1, PtPT5, PtPT6, PtPT9, and PtPT12)
weresignificantlyup-regulated(3-foldorgreater)dur-
A neighbor-joining tree was constructed using a ing autumnal leaf senescence. The highest increases
multiple sequence alignment of poplar Pht1 proteins (7.1- and 9.5-fold) corresponded to PtPT5 and PtPT6,
and sequences from fungal and other plant Pi trans- respectively.
porters. As in other dicot plant species, Pht1 proteins
clusteredintothreesubfamilies(Fig.1).Poplarharbors Pht1 Expression Depends on Pi Availability and on
twononorthologousPitransporters,PtPT8andPtPT10,
Poplar Genotype
which belong to subfamily I. PtPT10 appeared to be
closelyrelatedtoAM-inducibleStPT4andLePT4from P. trichocarpa, Populus deltoides, and genotypes of the
two solanaceous species (potato and tomato [Solanum hybridcloneP.deltoides3P.trichocarpaweretestedfor
lycopersicum]; Nagy et al., 2005). Two Pht1 proteins theirdifferentresponsivenesstoPidepletion(Fig.4).As
belongedtosubfamilyII(PtPT9andPtPT11),andeight rooted cuttings were used, the root systems relied on
Pht1 proteins belonged to subfamily III (PtPT1–PtPT7 bothPiuptakeandPiremobilizedfromperennialstem
and PtPT12). Growth of the Dpho84 yeast mutant tissues. It was speculated, however, that the growth
expressing poplar Pht1 (PtPT1, PtPT2, PtPT4, PtPT6, medium depletion would havea dramatic effectonPi
PtPT9,orPtPT10)wasassessed(SupplementalFig.S2). contentofrapidlygrowingroots.P.deltoideswashighly
As in other plants like tomato (Nagy et al., 2005) and affected by Pi starvation, showing a low growth rate
maize (Zea mays; Nagy et al., 2006), we were not able andahighnumberofnecroticspotsonleaves,whereas
to complement the yeast mutants for all poplar Pht1 the hybrid exhibited a high growth rate during Pi
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Loth-Pereda et al.
Figure1. Neighbor-joiningtreeforPht1transportersbasedonrealignedsequences(745aminoacids).Bootstrapvaluesarefrom
500replications.Sequencenamesconsistofspeciescode(firstletterofgenusandfirstletterofspeciesname)andgenename.
AccessionnumbersofthepredictedproteinsaregiveninSupplementalMaterialsandMethodsS1.Romannumeralsindicate
fourdifferentPht1subfamiliesthoughttodifferinevolutionaryage(accordingtoNagyetal.,2005).Concerningchloroplastic
Pht2transporters,numberscorrespondasfollows:1,POPtr;Pht2;2(poplar);2,LYCes;Pht2;1(tomato);3,POPtr;Pht2;1(poplar);
4,ARAth;Pht2;1(Arabidopsis);5,TRIae;Pht2;1(Triticumaestivum);6,ORYsa;Pht2;1(rice);7,PHYpa;Pht2;1(Physcomitrella
patens);and8,MEDtr;Pht2;1(M.truncatula).
starvation and no indication of Pi deficiency in leaves Finally, expression of PtPT8 and PtPT10 was not de-
(data not shown). Poplar exhibited an intermediate tectable in control and Pi-depleted roots in any of the
responsiveness to Pi deprivation. Plants growing in genotypes. Thus, there was an extensive variation in
full-Pi nutrient solution were compared with plants Pht1 gene expression levels in response to Pi depriva-
growing in Pi-free nutrient solution. Removal of Pi tionbetweenthetwoparentalspeciesandtheirhybrid.
enhanced the expression ofmost Pht1 genesinadven-
titiousrootsofthethreedifferentpoplargenotypes(Fig. Coregulation and Interspecific Conversation in Pht1
4). In P. trichocarpa, PtPT9 and PtPT11 expression was
Gene Expression in EM and AM Roots
up-regulatedmorethan10,000-fold,PtPT12expression
wasabout1,000-foldhigher,andPtPT1andPtPT3were The expression level of each Pht1 gene was quanti-
up-regulated about 100-fold. In contrast, PtPT5 and fied in Populus tremula 3 Populus alba roots colonized
PtPT6 were down-regulated about 2-fold at low-Pi either by EM fungi (Laccaria bicolor and Paxillus invo-
conditions. In P. deltoides, the homologs of PtPT3, lutus) or AM fungi (Glomus intraradices and Glomus
PtPT5, and PtPT6 were found to be up-regulated by mosseae). Expression of PtPT1 and PtPT11 was re-
more than 100-fold, whereas other homologs were pressedinbothEMandAMroots,whereasexpression
moderately up-regulated by 10- to 40-fold. In the of PtPT9 and PtPT12 was strongly enhanced in both
hybrid, the homologs of PtPT9 and PtPT11 exhibited symbiotic associations (Fig. 5). PtPT8 and PtPT10
transcript levels that were more than 100,000-fold transcripts were not detectable in both nonmycorrhi-
higher than in control plants. Other Pht1 gene homo- zalandEMroottips.PtPT3,PtPT4/PtPT7,andPtPT5
logs were moderately up-regulated by 10- to 20-fold. expression was enhanced in AM roots only. In AM
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Pht1 Family in Populus trichocarpa
Figure2. QuantificationbyqRT-PCRof
the transcript levels of the 12 poplar
Pht1 genes in different developmental
stages.Ubiquitinwasusedastherefer-
encetranscript.Thevaluesaremeansof
fivereplicates,andeachreplicaterep-
resented a pool from at least three
plants.ErrorbarsrepresentSD.A,Tissues
fromplantletsgrowninthegreenhouse.
B,Threestagesduringbuddevelopment
accordingtoCouturieretal.(2007).
roots, transcripts coding for PtPT8 were not detected, CTTC motif, exhibiting some sequence variation, was
whereas PtPT10 was only found to be expressed in presentincombinationwiththeP1BSelementinboth
these symbiotic tissues (Fig. 5). the PtPT10 and PtPT12 promoters in a subregion
Pht1 gene regions of up to 2.4 kb upstream of their between 400 and 600 bp upstream of the start ATG,
respective initiator ATG were analyzed and phyloge- similar to the StPT3 promoter (Karandashov et al.,
netically comparedwithorthologousPht1genesfrom 2004).BoththeCTTCandP1BSmotifswerepresentin
other plant species (Fig. 6A). The P1BS cis-acting the PtPT10 promoter sequences of both poplar Nis-
element (PHR1-binding site [GNATATNC]; Rubio qually 1 and poplar clone INRA 101-74 in the same
et al., 2001; Schu¨nmann et al., 2004) was found in the subregion. Moreover, the PtPT10 promoter sequence
promoter region of PtPT1, PtPT4, PtPT7, PtPT10, has three motifs out of 13 (TGTATTGATTTT, GTATT-
PtPT11, and PtPT12. The P1BS element and the GATTTTT,andATTTTAAAAAAA)similarwiththose
CTTC motif (CTTCTTGTTCTA; Karandashov et al., from MtPT4 and StPT4 as well as a motif (CAAT-
2004) were found in close proximity to each other TATTTat21,049)verysimilartothemotifreportedin
withinthe200bpupstreamoftheATGstartcodonof StPT3 (CAATTATTG at21,227; Rauschet al., 2001).
PtPT10 and PtPT12 and a set of other AM-specific Pi The other promoters of poplar Pht1 genes, and
transporter genes (Fig. 6A). A second copy of the especially PtPT8 from Pht1 subfamily I (AM induc-
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Loth-Pereda et al.
suggestedthatrootcelltype-specificcis-regulationofa
solanaceouspromoter isconserved in poplar.
To study the evolutionary conservation of AM-
specific Pi transporter gene regulation (Karandashov
et al., 2004), the activities of the 1.2-kb-long PtPT10
promoter and of the 1.7-kb-long AM-inducible StPT3
promoter were studied in transgenic hairy roots of
potatoandP.tremula3P.tremuloides,respectively.This
cannot be assessed in P. trichocarpa, as this species is
not yet amenable to routine genetic transformation.
Mycorrhizal symbiosis was established between two
potato hairy root genotypes (one wild type and one
transgenic containing the 1.2-kb pPtPT10-GUS con-
struct) and G. intraradices after 4 to 6 weeks (Fig. 7).
There was no difference in colonization pattern be-
tween the two potato hairy root genotypes. GUS
activity was colocalized with intracellular arbuscules
and coiled hyphae in transgenic potato (Fig. 7, B–D),
whileGUSactivitywasabsentinwild-typemycorrhi-
Figure3. QuantificationbyqRT-PCRofthetranscriptlevelsofthe12 zal potato (Fig. 7A) and in nonmycorrhizal wild-type
poplarPht1genesuponautumnalleafsenescence.Transcriptlevelsin and transgenic hairy roots from potato (data not
youngleaveswereusedasthecontrolvalues.Ubiquitinwasusedasthe
shown). As in hairy roots of potato, there was no
referencetranscript.Thevaluesaremeansoffivereplicates,andeach
difference in colonization pattern between the two
replicate represented a pool from at least three plants. Error bars
poplar hairy root genotypes (one wild type and one
represent SD. Genes were considered to be regulated for expression
whengreaterthanorlessthanthree.Student’sttestwasperformedfor transgenic containing the 1.7-kb pSTPT3-GUS con-
statisticalanalysis.AsterisksindicateP,0.05. struct). However, mycorrhizal (Fig. 8, A–D) and non-
mycorrhizal (data not shown) poplar hairy roots did
ible), did not contain these motifs or variations of notrevealGUSactivity,indicatinganabsenceofStPT3
them, suggesting that they might not be regulated promoteractivity.Incontrast,GUSactivitycolocalized
upon mycorrhization. with arbuscules and branched hyphae in transgenic
Phylogenetic footprinting also identified longer potato root or hairy root controls both in vitro and in
blocks of sequence homologies in closely related pro- pot cultures (Fig. 8, E and F). Together, these results
moters (Fig. 6A). No similarity was detected in the
promoter sequences of mycorrhiza-inducible Pht1
subfamily I genes PtPT8 and PtPT10 and in most of
thepoplarPht1genepromoters.Inaddition,thePtPT9
andPtPT11promoterssharedhighlysimilarsequences
in two different blocks (74% and 44%, respectively;
Fig. 6B). About 300 bp separated the two blocks in
the PtPT9 promoter, while there were no gaps in the
PtPT11 promoter. A P1BS element was identified in
the second block of the PtPT11 promoter but not in
the PtPT9 promoter. High similarity was also found
betweenthePtPT7andPtPT4promotersandthePtPT1
and PtPT2 promoters (Fig. 6B). Thus, computational
promoter analysis suggested evolutionary conserva-
tionofAM-specificgeneregulationinmycorrhizaltree
andherbaceousplantspecies.
In view of reciprocal examination of AM-specific
promoter activation in potato and poplar, the LeExt1
promoter mediating root hair-specific expression in
solanaceous species (Bucher et al., 2002) was used to
driveGUSexpressionintransgenicpoplar.GUSstain-
ing was detected exclusively in root hair-bearing
rhizodermal cells of P. tremula 3 Populus tremuloides Figure4. QuantificationbyqRT-PCRofthetranscriptlevelsofthe12
poplar Pht1 genes in response to Pi starvation. Analyses were per-
roots grown in in vitro and aeroponic systems. GUS
formedinP.trichocarpa,P.deltoides,andthehybridP.trichocarpa3
stainingwasabsentincellsattheroottipandtheroot P.deltoides.TranscriptlevelsinrootswithPiwereusedasthecontrol
cap(SupplementalFig.S3),whichwasconsistentwith
values.Ubiquitinwasusedasthereferencetranscript.Thevaluesare
the GUS expression pattern described in herbaceous meansoffivereplicates,andeachreplicaterepresentedapoolfromat
species (Bucher et al., 2002). Overall, these results leastthreeplants.ErrorbarsrepresentSD.
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Pht1 Family in Populus trichocarpa
nificantly higher in thick poplar roots compared with
that of potato and thin poplar roots, while the per-
centageofrootsectorscontainingvesicles/sporesand
hyphaeinpotatoandinthinpoplarrootsissimilar.In
vitro, thick roots of poplar never developed, while in
potato,allcolonizedrootsstainedpositiveirrespective
oftheirthicknessandage.Takentogether,theseresults
suggested that the mycorrhizal pattern of G. intra-
radices is dependent on the host plant species, and in
the case of poplar grown in pot culture, also by the
age/diameteroftheroots.Thus,theabsenceofStPT3
promoter activation in poplar could be explained by
the absence of arbuscules and a reduced number of
coiled hyphae, which might have become inactive at
thetimeofharvest.
Figure5. QuantificationbyqRT-PCRofthetranscriptlevelsofthe12
Pht1genesinP.tremula3P.albarootscolonizedeitherbyEM(P. Localization of PtPT10, an AM-Inducible
involutusandL.bicolor)orAM(G.intraradicesandG.mosseae)fungi. Phosphate Transporter
Transcript levels in nonmycorrhizal roots were used as the control
values.Thevaluesaremeansoffourreplicates,anderrorbarsrepresent Two peptides were selected as antigens for the
SD. The ubiquitin gene mRNA was used as the reference transcript. production of anti-PtPT10 antibodies. The selected
Genes were considered to be regulated for expression when greater peptides were not found in the deduced protein se-
than or less than three. Student’s t test was performed for statistical quences of other poplar gene models. PtPT10 locali-
analysis. Asterisks indicate P , 0.05. 2, PtPT8 transcripts were not zation in G.intraradices-P.trichocarpa mycorrhizal root
detectableundertheseconditions;+,PtPT10expressionwasinduced tips by indirect immunofluorescence is illustrated in
specificallybyAMfungiinmycorrhizalpoplarroots.
Figure 10. Control images in which the mycorrhizal
rootsectionswereobtainedbyreplacingprimaryanti-
suggestedthatthemechanisminvolvedintheregula- PtPT10 antibodies with preimmune immunoglobulin
tion of the PtPT10 promoter is conserved between showed no signal. For cases in which AM roots were
poplar and potato. treated with anti-PtPT10 antibody followed by fluo-
Next, we examined whether the AM colonization rescence-labeled secondary antibody, fluorescence
pattern in P. tremula 3 P. tremuloides revealed differ- was localized along the mycelial branches. The indi-
ences compared with that of potato. An intermediate vidual branches of the G. intraradices coiled hyphae
Arum-Paris-type pattern of mycorrhizal colonization were clearly visible (Fig. 10) into poplar cortical cells,
was observed both in poplar and potato. However, andthePtPT10-relatedfluorescencesignalcolocalized
insteadofdichotomousbranchingofhyphaethatform withthesymbioticstructuresfillingthecells.Nosignal
the arbuscules, as in potato, poplar developed arbus- wasdetected in noncolonized cortical cells.
culatecoils(Fig.8,A,C,andD)andcoiledhyphaethat
occasionally ramified in finger-like structures (Fig.
8A). In vitro, the total percentage of colonization of
DISCUSSION
hairyrootswasabout32%inpotatoand22%inpoplar
atbothharvestingtimes(2and8months;Supplemen- Little is known about the molecular basis of Pi
tal Fig. S4). In pot culture, it was about 90% in potato transportintreespeciesandwhichcomponentsplaya
and 44% in poplar. The detailed analysis of the my- role in symbiosis-mediated Pi uptake. In this study, a
corrhizal structures revealed a difference in the per- comprehensive genome-wide inventory of the Pht1
centage of colonization (Fig. 9) between older-thick family of Pi transporters in poplar cv Nisqually has
(Poplar )andyounger-thin(Poplar )poplar been established, including expression regulation in
R.1mm R,1mm
roots. Overall colonization was almost 70% in thick different plant tissues and AM and EMsymbioses.
rootsandabout30%inthinroots.Inpotato,thickroots
did not develop. The total percentage of root sectors
Evolutionary History of Pht1 Subfamilies in Poplar
containing arbuscules was significantly higher in po-
tato(19%)thaninpoplar(1.7%;Fig.9).Thepercentage Thephylogeneticanalysisrevealedtheclusteringof
ofrootsectorscontainingvesicles/sporesandhyphae the12poplartransporters(Fig.1)withmembersofthe
in thick poplar roots (56%; Fig. 9) was significantly previously described Pht1 subfamilies (Karandashov
higher than in potato (16%) and in thin poplar roots and Bucher, 2005; Nagy et al., 2005). Subfamily I
(14%;Fig.9).Incontrast,thepercentageofrootsectors clustered the AM-inducible Pi transporters from both
containing vesicles/sporesandhyphaein thin poplar monocotanddicotspecies,suggestingtheirevolution
rootswasnotstatisticallydifferentfromthatofpotato before the separation of the two plant groups (dicots
(Fig. 9). Thus, in pot culture, the percentage of root versusmonocots).ProteinsencodedbythepoplarEM-
sectors containing vesicles/spores and hyphae is sig- and AM-up-regulated Pht1 genes (PtPT9 and PtPT11)
Plant Physiol. Vol. 156, 2011 2147
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Loth-Pereda et al.
Figure6. PhylogeneticfootprintingofthePht1promotersfrompoplarandofmycorrhiza-induciblePht1transportersfromother
plantspecies(A),andsimilarityofthepromotersubregionsunderlinedinthephylogeneticfootprinting(B).A,Thedistance
betweentwo tick marks represents 100 bp of promotersequenceupstream of the start ATG. The phylogram represents the
phylogeny of the corresponding genes. The red lines represent the truncated CTTC motif TCTTGTTC, and the green lines
representtheP1BSmotif.Blackshapesrepresenta100%matchtotheextendedmotif.Grayshapesrepresentfewerchangesin
themotif.Blocksofsimilarpromotersequences(44%identityorgreater)areunderlinedwiththesamecolor.B,Thelengthofthe
alignmentblocksinsimilarpromotersubregionsisindicatedtogetherwiththepercentageidentity.
fallintothehighlydivergentsubfamilyII.Genesfrom regulationofparalogouspairs.Moreover,thisduplica-
dicotandmonocotplantswerefoundtobepresentin tion is strongly supported by an analysis of promoter
subfamilies III and IV, indicating their evolutionary regionsrevealingthatPtPT1/PtPT2,PtPT4/PtPT7,and
divergence after the separation of these two primary PtPT9/PtPT11 have longer blocks of sequence simi-
groupsofflowering plantsfromtheircommon ances- larities in promoter subregions.
tor. In addition, the fact that all poplar members of
subfamily III are intronless except PtPT3 suggested Transcriptional Control of Poplar Pht1 Gene Expression
that they were generated by duplication from a com-
monancestralsequence.Whateverthesubfamily,tan- Homologous Pht1 genes exhibited substantial dif-
demduplicationsareclearlyanimportantmechanism ferences in their regulation in the various organs
generating new gene copies in poplar (Tuskan et al., examined.PtPT4/PtPT7showedhightranscriptlevels
2006).PtPT1/PtPT2,PtPT4/PtPT7,andPtPT9/PtPT11 in leaf and root tissues compared with other Pht1
are likely to be recent duplications, subsequently genes (Fig. 2A). The transcripts coding for PtPT1,
followed by divergence in gene expressionlevels and PtPT5, PtPT6, PtPT9, and PtPT12 showed strong up-
2148 Plant Physiol. Vol. 156, 2011
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Pht1 Family in Populus trichocarpa
Figure7. HistochemicalstainingforGUSactivityin
mycorrhizalpotatorootsexpressingtheGUSreporter
gene under the control of the PtPT10 promoter. A,
Negativecontrol showing mycorrhizal structuresin
nontransgenic (wild-type) roots after trypan blue
staining (specific for fungal structures). No GUS
stainingofthehairyrootscompositeplantsisvisible,
meaningthatthefungusoranycontaminatingbac-
teria exhibit GUS staining in the absence of GUS
expression. B, Mycorrhizal transgenic roots with
Magenta-GUS staining (DsRED fluorescence) indi-
catingtransgenesis.C,Magenta-GUSstainingcolo-
calizeswith trypanblue staining.D,Enlargedview
ofthewhitesquareinC.Bars=0.1mmexceptfor
0.5mminB.
regulation in the oldest leaves undergoing autumnal Upon Pi starvation, PtPT9 and PtPT11 showed
senescence(Fig.3),suggestingthattheyplayarolein dramatic up-regulation in poplar (Fig. 4), as did their
Pi remobilization from source to sink tissues, which orthologs in the hybrid P. trichocarpa 3 P. deltoides.
may be specific for the perennial lifestyle of poplar. PtPT9 and PtPT11 are highly homologous to AtPT8
During bud development, PtPT1 and PtPT2 wereup- andAtPT9,twogenesfromthePht1subfamilyII(Fig.
regulated in a similar way as their homolog AtPT1 1),whichwerealsoshowntobestronglyup-regulated
from Arabidopsis Pht1 subfamily III (Figs. 1 and 2B; during Pi starvation (Mudge et al., 2002). Therefore,
Mudge et al., 2002). these Pht1 transporters likely play a role in Pi scav-
Figure8. HistochemicalstainingforGUSactivityinmycorrhizaltransgenic(AandB)andwild-type(CandD)P.tremula3P.
tremuloideshairyrootsandinmycorrhizaltransgenicpotatohairyroots(EandF)expressingtheGUSreportergeneunderthe
controloftheStPT3promoter.Mycorrhizalstructuresarevisibleinrootsafterinkbluestaining(specificforfungalstructures):
finger-likecoiledhyphae(A),vesicles(intracellular)andspores(extracellular;B),andarbusculatecoils(arrows;CandD)in
transgenicpoplarplantsandbranchedhyphae(E)andarbuscules(F)intransgenicpotatoplants.AandDshowthatMagenta-
GUSstainingdoesnotcolocalizewithinkbluestainingintransgenicandwild-typepoplarhairyroots.EandFshowapositive
control,whereMagenta-GUSstainingcolocalizeswithinkbluestainingintransgenicpotatohairyroots.Bars=200mm.
Plant Physiol. Vol. 156, 2011 2149
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Loth-Pereda et al.
(Karandashov et al., 2004; Chen et al., 2011) but was
neverdescribedintreesandespeciallyinMalphigiales,
towhichpoplarbelongs.
Interestingly, AM and EM development led to the
up-regulation of two other Pht1 transporters, PtPT9
and PtPT12 of subfamily II and III, respectively,
suggesting that they may have evolved with both
symbiotic lifestyles and that different signaling path-
ways control Pi uptake in these mycorrhizal symbio-
ses. Although the drastic accumulation of PtPT9,
PtPT10, and PtPT12 transcripts upon mycorrhizal
colonization could be symbiosis specific, it may also
be the result of a secondary effect of enhanced Pi
supply (i.e. Pi provided by the fungus). In poplar
rooted cuttings grown on Pi-depleted medium,
PtPT10 was not responsive to Pi availability whereas
PtPT9 and PtPT12 were strongly up-regulated. Thus,
Figure9. Colonizationpatternsofmycorrhizalhairyrootsofpotatoand
poplar harvested after 3 months of cocultivation with G. intraradices. PtPT10isregulatedduringAMbutnotEMsymbiosis,
Percentagesofrootsegmentscontaininghyphae(H),vesicles/sporesand indicating that the regulation of AM-inducible Pht1
hyphae(V+H),arbusculesandhyphae(A+H),andarbuscules,vesicles/ genes is conserved in woody and herbaceous spe-
spores, and hyphae (A+V+H) are indicated and compared. Note that cies.Moreover,ourdatasuggested thatanalternative
both in PoplarR.1mm and PoplarR,1mm, we observed 1.4% containing regulatory pathway distinct from the AM regulatory
vesicles/spores and hyphae and 0.3% containing arbuscules, vesicles/ pathway developed during evolution, which allowed
spores,andhyphae.However,asdataarelow,itisnotpossibletoshow EMplantstorecruitPtPT9andPtPT12forsymbioticPi
themclearlyhere.Differentlettersrepresentsignificantlydifferentmeans
uptakeinEMroottips.Alternatively,duringbothAM
betweenplantmaterial(ANOVALSD,P,0.05).
and EM development, PtPT9 and PtPT12 expression
maybetriggeredbylow-Piconditionsatthesymbiotic
enging within the Pht1 family during conditions of interfaces. This latter scenario could explain the high
low soil Pi availability. P1BS-like elements were iden- levelexpressionoftheAMfungalPitransportergene
tifiedinsix(PtPT1,PtPT4,PtPT7,PtPT11,PtPT10,and GmosPTAfromG.mosseaeinarbusculesofmycorrhizal
PtPT12) of the 12 Pht1 promoter sequences, as in a
range of Pi-regulated genes in dicots and monocots
(Fig.6A).InArabidopsis,theMYBtranscriptionfactor
PHR1(At4g28610)bindstotheP1BSelementandisa
key regulator in the Pi starvation response (Rubio
etal.,2001).AnorthologofthisMYBtranscriptionfac-
tor (Joint Genome Institute protein identifer 711538),
sharing57%similaritywiththePHR1protein,waslo-
cated on LG II of the poplar draft genome, suggest-
ingPHR1-dependentregulationofasetofPht1genes
in poplar.
Two nonorthologous mycorrhiza-inducible Pht1
genes were identified in solanaceous plant species
(i.e.LePT3/LePT4intomatoandStPT3/StPT4inpotato,
as well as their orthologs from other solanaceous
species;Nagyetal.,2005;Chenetal.,2007)belonging
to Pht1 subfamily III and I, respectively, and a single
geneortwogenesinleguminous(MtPT4andLjPT3in
M. truncatula and L. japonicus, respectively) and poa-
ceous (OsPT11 and OsPT13 in rice [Oryza sativa] and
ZmPT6inmaize)specieswerealsoidentified(Harrison
et al., 2002; Gu¨imil et al., 2005; Maeda et al., 2006;
Nagy et al., 2006). In poplar, PtPT8 and PtPT10, phy-
logenetically related to the AM-inducible Pht1 genes
from subfamily I, are functionally different; only
PtPT10 was specifically induced in poplar roots colo-
nized by AM fungi. Analysis of the promoter region
Figure 10. Immunolocalization of PtPT10 in symbiotic structures in
revealedthepresenceoftheCTTCmotifinPtPT10but rootcorticalcellsofpoplarcolonizedbytheAMfungusG.intraradices.
not in PtPT8 (Fig. 6A). This motif was shown to be CW, Cell walls of cortical cells; Sym, G. intraradices symbiotic
highlyconservedinAM-induciblePht1genesindicots structures.
2150 Plant Physiol. Vol. 156, 2011
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