Flowers
and
mycorrhizal
roots
–
closer
than
we
think?
Eva
Nouri
and
Didier
Reinhardt
DepartmentofBiology,UniversityofFribourg,Fribourg,Switzerland
Roots and flowers are formed at the extreme ends of
plants and they differ in almost every aspect of their
developmentandfunction;evenso,theyexhibit
surpris-ingmolecularcommonalities.Forexample,thecalcium
and calmodulin-dependent protein kinase (CCaMK)
plays a central role in root symbioses with fungi and
bacteria, but is also highly expressed in developing
anthers. Moreover, independent evidence from
tran-scriptomics,phylogenomics,andgeneticsreveals
com-mon developmental elements in root symbioses and
reproductivedevelopment.Wediscussthesignificance
of these overlaps, and we argue that an integrated
comparativeviewofthetwophenomenawillstimulate
researchandprovide newinsight,notonlyintoshared
components,butalsointothespecificaspectsofanther
developmentandrootsymbioses.
Roots andflowers:closerthanapplesandoranges?
Most plants engageinarbuscular mycorrhiza (AM),
mu-tualistic associationswith fungifrom theorder
Glomero-mycota [1–3]. The wide distribution of this interaction
among land plants, and its predominance in almost all
ecological niches, suggests that this symbiosis provides
diverseimportantservicestothehost[4].Establishment
ofAMrequiresasignalingcascadeinthehost,referredto
as the common symbiosis signaling pathway (CSSP)
be-causeitissharedwiththenitrogen-fixingnodulesymbiosis
betweenlegumesandrhizobia[2,5].
One of the central elements of the CSSP is CCaMK,
whichisrequiredforinfection byAM fungiandrhizobia
[6].ConstitutivelyactiveformsofCCaMKaresufficientto
triggertheformationofnodulesintheabsenceofrhizobia
or of any exogenous stimulus, hence demonstrating the
centralrole ofCCaMK innodulation[7,8].Interestingly,
CCaMKwasoriginallyidentifiedandcharacterizednotin
roots,butintheanthersofflowers,hencetheprovocative
question: does pollen development involve mechanisms
shared withrootsymbiosis?Whatmightthetwo havein
common? Based on several lines of evidence, it may be
relatedtosignalingandcellwallbiosynthesis.
Pollengrainsrepresenthighly-resistantcapsuleswhich
protect the sensitive sperm cells (or their progenitor
cell) against harsh conditions such as desiccation and
UVirradiation.Thewallofthecapsuleconsistsofasturdy
multi-layered cell wall[9]that is coatedwith oneofthe
most resistant biological materials, sporopollenin, that
consistsofahydrophobicpolymeroffattyacidsandtheir
derivatives[10].Owingtoitsextremeresistanceandwide
distribution in nature, pollen is one of the best fossil
indicatorsforevolutionaryandclimaticstudiesoverlarge
spatialandtemporalscales[11].
At the other end of the spectrum regarding longevity
residetheephemeralarbuscules,thefeedingstructuresof
AMfungi thathave alifetimeofonly afew days[12,13].
Nevertheless,surprisingly,thedevelopmentofarbuscular
mycorrhizarequiresasimilarlipid-relatedpathwayinthe
planthostasthatinvolvedinpollenwallsynthesis.
Howev-er, in the case ofsymbiosis, there is noevidence forthe
formationofanextracellularpolymerbytheplant.Instead,
thenon-polymerizedlipidicintermediatesofthepathway
arethoughttofunctionas signalsbetweentheplanthost
andthefungal endosymbiont[14–16].Moreover,genomic
and genetic analysesreveal further unexpected parallels
betweenrootsymbiosisandpollendevelopment.
Wediscusshererecenthighlightsinthesesofar
unre-latedresearch domains, explorethe significanceof
over-lapsbetween theinvolved pathways,andsuggest that a
scientific crosstalkbetween thetwo researchfieldscould
potentiallycontributetoadvancingbothfieldsinthe
com-ingyears.
Theprecedent:thecaseofCCaMK
CCaMKwas firstisolatedfrom lilly(Liliumlongiflorum)
anthers [17], and subsequently biochemically
character-ized in lilly, tobacco (Nicotiana tabacum), maize (Zea
mays),andpea(Pisumsativum)[18–25].Thecombination
of a calmodulin-binding domain and three EF hands in
CCaMK is unique (Box 1) and sets it apart from the
calcium-dependent protein kinases (CDPKs) [6]. In lilly
andtobaccoanthers,CCaMKisexpressedathighestlevels
during the meiotic phase of pollen development, in the
meiocytes as well as in the tapetum, the cell layer that
feedsthedevelopingpollen[18,26].
ThefunctionofCCaMKhaslongremainedelusive,and
whenthefirstknockoutmutantsofccamkwereidentified
inMedicagotruncatulatheirphenotypewasnotrelatedto
flowersbut,surprisingly,totheroots,whichweredefective
inthedevelopmentofbothAMandnitrogen-fixingnodules
[27,28]. Subsequent work showed that the AM-related
functionofCCaMKanditsexpressioninflowersare
con-served in rice (Oryza sativa) [29,30]. The function of
CCaMK in symbiosis is thought to bethe decoding of a
Correspondingauthor:Reinhardt,D. (didier.reinhardt@unifr.ch).
Keywords:symbiosis;arbuscularmycorrhiza;pollen;glycerol-3-phosphate acyltrans-ferase;GPAT.
Published in 7UHQGVLQ3ODQW6FLHQFH ± which should be cited to refer to this work.
calcium signal (calcium spiking) in root cells that have
perceivedAMfungalorrhizobialsignals(reviewedin[6]).
Interestingly,mutantsdefectiveintwoothercomponents
oftheCSSPthatactupstreamofcalciumspiking,
nucleo-porin85(NUP85) andNUP133,alsopointtoan overlap
between root symbiosis and reproduction because they
exhibitfertilitydefects[31–33].
Alipid-relatedpathwaysharedbetweenAMandflower
development
Toexplorepotentiallysharedregulatorypathwaysin
flow-erdevelopmentandrootsymbiosisinasystematicfashion,
acomparisonoftherespectivetranscriptomescanbeused
asanindicatorforcommondevelopmentalandregulatory
pathways.Because the well-characterized model species
Arabidopsisthalianadoesnotengageinrootsymbioses,we
chosethestandardsymbiosismodelspeciesM.truncatula
which offers excellent bioinformatics tools with the
M. truncatula Gene Expression Atlas (MtGEA; http://
mtgea.noble.org/v3).
Bycomparinggene expressionin flowerswith
vegeta-tivebuds,andapplyingathreefoldinductioncutoff,atotal
of151flower-induced geneswere identified. In a second
step,theseflower-relatedgeneswereassessedinrelation
tothree recentlyestablishedAM-related criteria [34]: (i)
threefoldinduction inmycorrhizalroots,(ii)asignificant
AM-relatedpatternofsequenceconservationinthecoding
region,and(iii)predictedAM-relatedregulatorysequences
in their promoters. Surprisingly, 81 of the 151
flower-inducedgenes(53.6%)werefoundinatleastoneofthese
three AM-related categories. Considering the 20 genes
with the highest induction levels in AM (Table 1), 10
(50%)exhibitedasignificant(P<0.05)AM-related
conser-vationpatternasdefinedin[34]orwereentirelymissing
fromA.thaliana(indicatedwithasterisksinTable1).This
isconsiderably morethan the 28.5%ofgenes(n =1334)
withanAM-relatedconservationpatternwhen4684
ubiq-uitoushousekeepinggeneswereconsidered(seeTableS5
in[34]).Thissuggeststhatthese10flower-inducedgenes
have beenunderselectionfor AM-related functions,and
thereforemayhaveoverlappingfunctionsinflowersandin
AMsymbiosis.
The gene with the highest induction ratioin AM is a
glycerol-3-phosphate acyltransferase (GPAT) (Table 1),
knownasREQUIREDFORARBUSCULAR
MYCORRHI-ZA2(RAM2),basedonitsAM-defectivemutantphenotype
[16].GPATcatalyzesthetransferoffattyacidsonto
glyc-erol-3-phosphate[35,36].Thisisthefirstcommittedstepin
the biosynthesisofphospholipids (membrane lipids)and
triacylglycerols (storagelipids), andofextracellularlipid
polyesterssuchascutinandsuberin(Box2).Animportant
distinction in these pathways is that the precursors of
Box1.Calciumandcalmodulin-dependentproteinkinase(CCaMK)
CCaMKisaproteinkinasethatcanbindthreecalciumionswiththeEF hands(redblocks)initsC-terminaldomain(Figure I).Thisdomain, whichisevolutionarilyrelatedtocalmodulin(althoughoneEFhandis nonfunctional,depictedinpink),issharedwiththecalcium-binding domainofCDPKs[70].However,incontrasttoCDPKs,CCaMKhasin additionacalmodulin-bindingdomain(CBD) whichisessentialfor properregulation[6,71].CCaMK,whichisencodedbyasingle-copy geneinmostplants,ishighlyconservedinspeciesthatcanundergo
AMand/orrootnodulesymbiosis[34,72];however,Arabidopsisthaliana andothernon-symbioticspecieshavelostit.Forphylogeneticanalysis, MtCCaMKwasusedasabaittoidentifythethreeclosesthomologsfrom Medicagotruncatula(Mt),A.thaliana(At),Solanumlycopersicum(Sl), Cucumissativus(Cs),Oryzasativa(Os),Zeamays(Zm),andSorghum bicolor(Sb)byPBLASTatNCBI.Phylogramswereproducedasdescribed using the software package at www.phylogeny.fr [73]. Support for branchseparationisindicatedwithbootstrapvaluesfor100replicates.
sb_2 Os_3 Os_2 Sb_3 ZmCDPK3MtCDPK2 Cs_2 Cs_3 SlCDPK3 ZmCDPK24 SlCDPK17 AtCDPK29 MtCDPKb AtCDPK2 AtCDPK12 CsCCaMK SbCCaMK OsCCaMK ZmCCaMK MtCCaMK SlCCaMK CCaMK Calmodulin Kinase domain CBD EF EF EF EF EF EF EF 0.99 0.99 0.88 0.8 0.39 1 0.85 0.6 1 1 0.75 0.69 0.78 0.42
TRENDS in Plant Science
FigureI.PhylogramofplantCCaMKproteins.
membrane and storage lipids are first acylated at the
sn1-position, whereas theprecursors ofthe extracellular
polyestersareacylatedatthesn2-position[35,36](Box2).
Plantshavetypically8–10GPATsdistributedoversix
subfamilies(Figure1).Basedonitsrelativeproximityto
GPAT4toGPAT8(Figure1), RAM2ispredictedtohave
sn2-acylation activity. Furthermore, RAM2 shares
con-served amino acid residues with GPAT4, GPAT6, and
GPAT8 that are required for phosphatase activity
[16,37].This activity releases the phosphate groupfrom
the sn3-position,therebygenerating precursorsfor cutin
biosynthesis (Box 2). Based on this collective evidence,
RAM2 in pollen may be involved in the synthesis ofan
extracellularlipidicpolymer,perhapssporopollenin.
What is the role of RAM2 in AM symbiosis? Lipidic
polyesters function primarily as barriers for water and
solublemolecules[38],afunctionthatisnotimmediately
obvious in mycorrhizal roots where facilitated nutrient
transfer is the central symbiotic function. Based on the
factthatram2mutantsshowextremelylowlevelsofAM
fungalcontactpoints, ithasbeenproposed thatthe
pro-ducts of the GPAT activity of RAM2 may function as
signalsfromthehosttostimulatefungalinfection[14–16].
The secondgene inthelistofsharedAM- and
flower-induced genes (Table 1)encodesa predictedCASP1-like
protein. CASPs, which are encoded by a gene family in
Arabidopsis, are membraneproteins that localize to the
Casparian strip, and are required for its formation
[39].ThethirdgeneinthelistofAM-andflower-induced
genes is a cytochrome P450 belonging to the subfamily
CYP71,andaferulate5-hydroxylase(FAH)istheclosest
homologinArabidopsis(Table1).FAH isinvolvedinthe
biosynthesis of lignin by oxidizing one of its aromatic
constituents [40,41].Becausebothfunctions,CASP1-like
andCYP71,appeartoberelatedtocellwallmodifications,
it is possible that the apoplastic symbiotic interface of
mycorrhizalrootsandthepollenwallsharesomeunknown
features.However,itshouldbeborneinmindthat,despite
theirtentativeannotationbasedonArabidopsis,the
func-tionsofCASP1-like andofCYP71 in rootsymbiosis and
pollendevelopmentareelusiveandwillrequirefunctional
analysisbyreversegeneticsapproaches.
RecognitioninAMandinpollinationmayinvolvea
commonmechanism
Atasuperficiallevel, theinteraction betweenfungal
hy-phaeandroots,andbetweenpollentubesandthestigma,
exhibitsimilaraspects.Inbothcases,atubularstructure
(fungal hyphae and pollen tubes, respectively) penetrate
planttissues(therootsurfaceandthestigma,respectively).
Aparallelatthemolecularlevelinvolvesthespecific
recog-nitionmechanismsinvolvedinthetwointeractionswhich,
however, lead to opposite downstream consequences. In
AM, the recognitionof symbiosis-specificsignals and the
activationoftheCSSPareprerequisitesforacompatible
interaction[2,3,42].Bycontrast,plantsusetherecognition
ofpollen-specific signals toidentify theirown pollen and
toreject it, a phenomenonknown as self-incompatibility
(SI)[43].
TherearetwodifferentmechanismsofSI,gametophytic
and sporophytic SI, depending on whether recognition
involvesfactorsencoded by thehaploidpollengrain(the
gametophyte) or factors encoded by the diploid anther
tissue of the sporophyte (in particular the tapetum) of
theplantthatproducedthepollen.Inthe
well-character-ized sporophytic SI of the Brassicaceae (including the
genus Arabidopsis),the diploidanthertissues produce a
protein, the S-locus cysteine-rich (SCR) protein, that is
deposited on the pollen coat during pollen development
[44].Thisextracellularproteinisrecognizedinthestigma
Table1.GenescommonlyinducedinAMsymbiosisandinflowersofMedicagotruncatulaa
GeneID Probeset LCM AM Flo/Bud Annotation Athomolog
1b MTR_1g040500.1 Mtr.36944.1.S1_at 321.9 45.1 75.2 ERglycerol-phosphateacyltransferase(RAM2) At2g38110 2b MTR_6g073040.1 Mtr.41728.1.S1_at 199.3 241.4 22.8 Putativeuncharacterizedprotein(CASP1-like) At2g39530 3b MTR_8g091690.1 Mtr.12170.1.S1_at 58.1 93.7 30.7 CytochromeP450(CYP71A1;ferulate5-hydroxylase) At4g36220
4 MTR_8g087810.1 Mtr.46057.1.S1_at 47.7 31.3 3.2 Nitrate/chloratetransporter At1g59740
5 MTR_8g038210.1 Mtr.14183.1.S1_at 27.5 2.5 11.1 Annexin At5g12380
6 MTR_8g074530.1 Mtr.43640.1.S1_at 20.5 1.6 3.9 Putativeuncharacterizedprotein At4g35690
7 MTR_4g122750.1 Mtr.38447.1.S1_at 14.0 1.1 19.5 Pectinesteraseinhibitor At1g47960
8 MTR_3g020970.1 Mtr.30156.1.S1_at 11.8 6.0 7.5 Subtilisininhibitor At2g38900
9 MTR_5g017550.1 Mtr.44890.1.S1_at 11.1 0.9 26.2 Calcium-bindingproteinCML24 At2g15680
10b MTR_5g018610.1 Mtr.29593.1.S1_at 8.4 29.6 4.0 Putativeuncharacterizedprotein nohomolog 11b MTR_2g100350.1 Mtr.8968.1.S1_at 7.8 0.4 27.9 PyridoxalphosphatephosphatasePHOSPHO2 At1g17710
12b MTR_7g083570.1 Mtr.22351.1.S1_at 7.0 0.5 3.5 GEM-likeprotein At5g13200
13 MTR_7g082570.1 Mtr.12958.1.S1_at 6.7 1.3 5.2 ClassIglutamineamidotransferase At5g38200
14b MTR_6g009260.1 Mtr.17352.1.S1_at 6.4 10.7 3.9 Synaptojanin-1 At3g63240
15 MTR_3g084200.1 Mtr.20624.1.S1_at 6.1 0.8 3.5 Auxin-inducedprotein-likeprotein At4g38840
16 MTR_2g101560.1 Mtr.12373.1.S1_at 5.1 0.9 14.4 Nitrate/peptidetransporter At1g52190
17 MTR_8g094740.1 Mtr.44450.1.S1_at 4.7 1.8 4.6 Glutamyl-tRNA(Gln)amidotransferasesubunitA At5g64440
18b MTR_3g061170.1 Mtr.43887.1.S1_at 4.5 0.8 3.8 ChaperoneproteindnaJ At3g13310
19b MTR_7g012330.1 Mtr.43426.1.S1_at 4.1 0.6 52.6 CytochromeP450(CYP71B37) At4g31500
20b MTR_3g088460.1 Mtr.17288.1.S1_at 4.0 0.7 7.4 Metaltransporter At1g15960
aAbbreviations:AM,mycorrhizalrootsvscontrolroots;ER,endoplasmicreticulum;Flo/Bud:flowersvsvegetativebuds;LCM,lasercapture-microdissectedmycorrhizal cortexcellsvsnon-colonizedcells.
bGenesthatexhibitasignificant(P<0.05)AM-relatedconservationpatternasdefinedin[34],orthatwerelostinArabidopsis.
byS-locusreceptorkinase(SRK)whichactivatesthe
abor-tionofself-pollenbyamechanismthatiscurrentlyunder
intensedebate [45–48].TheextracellulardomainofSRK
containsahighly conservedcysteine-richstretch
consist-ingof an EGF-like and a PAN_APPLE motive that are
requiredforreceptor dimerization[49],andcomprises
N-glycosylationsitesthatareessentialforlocalizationtothe
plasmamembrane[50].ThegenesencodingSCRprotein
andSRK are tightly linked in the S-locus, and occur in
many allelic variants in natural plant populations [51],
thusensuringefficientcross-pollination.
Interestingly, maize roots express a closely related
homologofSRK,referredtoasZmPK1[52].Recent
bioin-formaticsanalysisatthegenomiclevelrevealed thatthe
genomesofAM-competentspecieshaveaconserved
SRK-relatedgroupofreceptorkinasesthatareabsentfromthe
non-mycorrhizalBrassicaceae [34]. The extracellular
do-mainofthesepredictedAM-related receptorsexhibitthe
canonicalcysteineresiduesrequiredfordimerizationand
severalpredictedN-glycosylationsites(Figure2).
Consis-tent with a role in root symbioses, the three Medicago
homologs of this specific SRK-related gene family are
expressed exclusively or primarily in roots (see http://
mtgea.noble.org/v3).A potentialroleoftheseAM-related
receptor kinases, in addition to the established roles of
leucine-richrepeat(LRR)andlysine-motive(LysM)
recep-tor-like kinases (RLKs) [53], remains to be elucidated;
however, it is intriguing to note that AM and SI may
involve recognitionsystems that share a common
evolu-tionaryorigin.
Cellwall-relatedcommonalitiesbetweensymbiosisand
pollendevelopment
Fascinatingparallelshave emergedfrom comparisonsat
the molecularandmorphologicallevelsbetweengrowing
pollentubesandtheinfectionthreadinrootnodule
sym-biosis.Infectionthreadsareinvaginationsofroothaircells
thatallowrhizobiatoenter,andthatguidethemtowards
thecortex[5].Severalsimilaritieshaveledtothe
hypoth-esis that the infection thread could be regarded as an
inversepollentubethat extendsbyatip-growing
mecha-nism[54].Indeed,pollentubesandinfectionthreadsshare
severalcloselyrelatedproteinssuchaspecticenzymes[55]
andarabinogalactanproteins[56].Thecloserelationship
betweennodule-relatedandpollen-relatedpecticenzymes
extendsfromtheconservedcodingregionintothe
promo-ters. Strikingly, some promoters contain separate
ele-ments that mediate expression in flowers and nodules,
respectively.Takentogether,thesefindingsstronglyargue
for a common evolutionary origin of cell wall-related
Box2.Activitiesofglycerol-3-phosphateacyltransferase(GPAT)
GPATs mediate the first dedicated step in the biosynthesis of membranelipids,storagelipids,andextracellularlipidpolyesters (cutinandsuberin)(FigureI).Ingeneral,GPATsuseacyl-CoAasa substrate,from which they transfer theacyl chainonto glycerol-3-phosphate. In the case of membrane and storage lipids, the acylgroupistransferredtothesn1-position,resultingin lysopho-sphatidicacid(1-acyl-LPA),followedbythetransferofasecondacyl
chaintothesn2-position.Cutinandsuberinprecursorsareformed bythetransferoftheacylchaintothesn2-position(2-acyl-LPA).In the caseofcutinbiosynthesis,the productof theacyltransferase reaction is simultaneously dephosphorylated, resulting in mono-acylglycerol (MAG). Nomenclature and numbering are based on theGPATsofArabidopsisthaliana.Reproduced,withpermission, from[74]. O O OH CH3 n OH (GPAT5) (GPAT4,6) O OH OH O O O n R R O n P O P sn-1 GPATs sn-2 GPATs Fay acyl-CoA + Glycerol-3-phosphate Membrane and storage lipids Suberin Cun
R = CH3, OH, COOH, OCOR′, COOR′
(GPAT55 sn-1 GPATs 4,6) sn-2 GPAT (GPAT55) (GPAT55
TRENDS in Plant Science
FigureI.GPATsandtheirroleinthebiosynthesisofmembraneandstoragelipidsandoflipidpolyesters.
mechanisms involved in the formation of pollen tubes
andinfectionthreads[55].Furthermoleculargenetic
dis-section of the pathways involved in these two different
cellulargrowthphenomenawillprofitfromsuch
compara-tivestudies.
Evidenceforoverlapsfromgenetics
Geneticoverlapsbetweendevelopmentalpathwayscanbe
discovered by rescreening mutants for new phenotypes.
For example, the finding that approximately half the
nodulation-defectivemutantsinpeaarealsoAM-defective
0.95 0.88 1 1 0.86 0.91 0.87 0.92 0.62 0.94 1 0.96 0.39 1 0.82 0.44 0.93 0.96 0.96 0.97 0.97 0.64 0.45 0.79 0.77 0.95 0.82 0.92 1 0.55 0.96 SIGPAT5/7a SIGPAT5/7b MtGP AT5/7 OsGP AT5/7 OsGPAT4/8 SIGPAT4/8 MtGP AT4/8 AtGP AT4 AtG PAT8 OsGP AT6b OsGP AT6a OsRAM2a OsRAM2b AtG PA T6 OsGP A T-X SIGP A T1 MtGP A T1a MtGP AT1b MtGP AT2/3a
MtGPAT2/3bAtGPAT3 AtGPAT2 AtG PA T1 OsGP AT 1 OsGP AT2/3a OsGP AT2/3b AtGPAT7 At GPAT 5 MtGP A6a MtGP A T6b MtRAM2 SIGP A T 6 SlRAM2a SlRAM2b SlRAM2-lik e
TRENDS in Plant Science
Figure1.Phylogenyofglycerol-3-phosphateacyltransferases(GPATs).PhylogramoftheentireGPATfamiliesoftheAMcompetentspeciesMedicagotruncatula,Solanum lycopersicum,Oryzasativa(rice),andofthenon-mycorrhizalspeciesArabidopsisthaliana.NotethatA.thalianalacksahomologintheRAM2branch(yellow).Phylogenetic analysiswascarriedoutasdescribedusingthesoftwarepackageatwww.phylogeny.fr[73].Supportforbranchseparationisindicatedwithbootstrapvaluesfor 100replicates.
EGF-like mof PAN-APPLE mof
TRENDS in Plant Science
Figure2.ConserveddimerizationdomaininAM-relatedSRK-likeproteins.ConservedC-richextracellulardomainofArabidopsislyrataSRKb[75]andBrassicaoleraceaSRK6
[76],togetherwiththethreepredictedMedicagotruncatulaAM-relatedSRKhomologs(Mt238,Mt354,Mt4055)[34],theclosesthomologinOryzasativa(Os354),andZeamays PK1(ZmPK1)[52].Conservedcysteineresiduesarehighlightedinred,predictedN-glycosylationsitesinblue.GlycosylationsitesinAlSRKbwereexperimentallyconfirmed[50].
ledtothediscoveryoftheCSSP[57].Applyingthisapproach
to reveal overlaps between pollen development and root
symbioses appears at firstsight less pertinent, and thus
systematic rescreening of pollen mutants for symbiosis
defects has not been carried out. Furthermore, this
ap-proach has been precluded in the cases where pollen
mutants had been isolated in the non-symbiotic model
speciesA.thaliana.Conversely, AM mutantswith pollen
defectsshould immediatelybeidentifiedby theirfertility
defects.Indeed, somesymbiosismutantsexhibit
segrega-tiondistortions[32,33,58]and, inthecaseofNUP85and
crinkle,thishasbeenattributedtopollendefects[33,58].
GiventhetranscriptomicoverlapbetweenAMandflower
development, ram2 and ccamk mutants could also be
expected to exhibit fertility defects in addition to their
symbiosisdefects;however,thisisnotthecase.Thismay
befor two reasons: pollen is generally produced in large
excess,andthereforequantitativedefectsinpollenfitness
maybe overlooked,in particular in self-fertile species. A
secondreasonforthelackofpollen-relatedmutant
pheno-types may be functional redundancy. For example, the
secretionofcomponentsforpollenwallformationin
Arabi-dopsisinvolvestwoABCtransportersoftheG-type
subfam-ily, ABCG1 and ABCG16, which act redundantly [59].
Similarly, RAM2 may function redundantly with other
GPATsinpollen, whereasits role inmycorrhizalrootsis
non-redundant.Indeed,membersfromotherbranchesofthe
GPATtreewereshowntobeinducedduringpollen
develop-ment[60]ortoberequiredforpollendevelopment[61–63].
Concludingremarksandoutlook
Thecombinedevidencefromtranscriptomics,genomics,and
geneticsrevealssignificantoverlapsbetweenroot
symbio-ses and pollen development. Components of the CSSP,
includingNUP85,NUP133,andCCaMK,aswellasashared
mechanism involvingthe glycerol-3-phosphate
acyltrans-feraseRAM2,appeartofunctioninbothmycorrhizalroots
andanthers.Althoughthesharedelementsmayultimately
serve different downstream functions in AM and pollen
development, comparison of the common elements could
beinformative.Animportant aspectofsuchcomparative
workshouldinvolvetheprecisetranscriptomicanalysisof
thedifferentcelltypesinanthersandmycorrhizalrootsin
an AM-competent species such as M. truncatula or rice.
Geneexpressiondatawithhighspatialandtemporal
reso-lutioncouldbeobtainedusingtechniquessuchascell
sort-ing[64,65]orlaser-assistedmicro-dissectioninmycorrhizal
roots[66,67]andinpollendevelopment[68,69].Usingsuch
asystematiccomparativeapproach,andexchanging
infor-mation and ideas from the two research domains, could
helptoexplaincommonanddifferentialelementsinthese
seeminglyverydifferentrealmsofplantbiology.
Acknowledgments
WethankPatrickFavre forassistancewith geneexpressionanalysis. ThisworkwassupportedbySystemsXandtheSwissNationalScience Foundation.
References
1 Bucher,M.etal.(2014)Throughthedoorsofperceptiontofunctionin arbuscularmycorrhizalsymbioses.NewPhytol.204,833–840
2Gutjahr,C.andParniske,M.(2013)Cellanddevelopmentalbiologyof arbuscularmycorrhizasymbiosis.Annu.Rev.CellDev.Biol.29,593–617
3 Harrison,M.J.(2012)Cellularprogramsforarbuscularmycorrhizal symbiosis.Curr.Opin.PlantBiol.15,691–698
4 Smith,S.E.andRead,D.J.(2008)MycorrhizalSymbiosis,Academic Press
5 Oldroyd,G.E.D.etal.(2011)Therulesofengagementinthelegume– rhizobialsymbiosis.Annu.Rev.Genet.45,119–144
6Singh,S.andParniske,M.(2012)Activationofcalcium-and calmodulin-dependentproteinkinase(CCaMK),thecentralregulatorofplantroot endosymbiosis.Curr.Opin.PlantBiol.15,444–453
7 Gleason,C.etal.(2006)Nodulationindependentofrhizobiainduced by a calcium-activated kinase lacking autoinhibition. Nature 441, 1149–1152
8 Tirichine,L.etal.(2006)DeregulationofaCa2+/calmodulin-dependent
kinase leads to spontaneous nodule development. Nature 441, 1153–1156
9 Quilichini,T.D.etal.(2014)Newviewsoftapetumultrastructureand pollen exine development in Arabidopsis thaliana. Ann. Bot. 114, 1189–1201
10 Ariizumi, T. and Toriyama, K. (2011) Genetic regulation of sporopollenin synthesis and pollen exine development. Annu. Rev. PlantBiol.62,437–460
11 Mitchell,F.J.G.(2011)Exploringvegetationinthefourthdimension. TrendsEcol.Evol.26,45–52
12 Alexander,T.etal.(1988)Dynamicsofarbusculedevelopmentand degenerationinmycorrhizasofTriticumaestivumLandAvenasativa LwithreferencetoZeamaysL.NewPhytol.110,363–370
13 Alexander,T.etal.(1989)Dynamicsofarbusculedevelopmentand degenerationinonion,bean,andtomatowithreferencetovesicular– arbuscularmycorrhizaeingrasses.Can. J.Bot.Rev.Can. Bot.67, 2505–2513
14 Bonfante,P.andGenre,A.(2015)Arbuscularmycorrhizaldialogues: doyouspeak‘plantish’or‘fungish’?TrendsPlantSci.20,150–154
15 Murray,J.D.etal.(2013)Signalingattherootsurface:theroleofcutin monomersinmycorrhization.Mol.Plant6,1381–1383
16 Wang,E.T.etal.(2012)Acommonsignalingprocessthatpromotes mycorrhizal and oomycete colonization of plants. Curr. Biol. 22, 2242–2246
17 Patil,S.etal.(1995)Chimericplantcalcium/calmodulin-dependent proteinkinasegenewithaneuralvisinin-likecalcium-bindingdomain. Proc.Natl.Acad.Sci.U.S.A.92,4897–4901
18 Liu,Z.H.etal.(1998)Chimericcalcium/calmodulin-dependentprotein kinaseintobacco:differentialregulationbycalmodulinisoforms.Plant Mol.Biol.38,889–897
19 Pandey, S. and Sopory, S.K. (1998) Biochemical evidence for a calmodulin-stimulated calcium-dependent protein kinase in maize. Eur.J.Biochem.255,718–726
20 Ramachandiran,S.etal.(1997)Functionaldomainsofplantchimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitoryandvisinin-likedomains.J.Biochem.121,984–990
21 Sathyanarayanan,P.V.etal.(2000)PlantchimericCa2+ /calmodulin-dependentproteinkinase–roleoftheneuralvisinin-likedomainin regulatingautophosphorylationandcalmodulinaffinity.J.Biol.Chem. 275,30417–30422
22 Sathyanarayanan, P.V. et al. (2001) Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependentproteinkinase.J.Biol.Chem.276,32940–32947
23 Sathyanarayanan,P.V.andPoovaiah,B.W.(2002) Autophosphorylation-dependentinactivationofplantchimericcalcium/calmodulin-dependent proteinkinase.Eur.J.Biochem.269,2457–2463
24 Pandey, S. and Sopory, S.K. (2001) Zea mays CCaMK:
autophosphorylation-dependent substrate phosphorylation and down-regulationbyredlight.J.Exp.Bot.52,691–700
25 Pandey, S.etal.(2002)ACa2+/CaM-dependent kinasefrompeais
stressregulatedandinvitrophosphorylatesaproteinthatbindsto AtCaM5promoter.Eur.J.Biochem.269,3193–3204
26 Poovaiah,B.W.etal.(1999)Developmentalregulationofthegenefor chimeric calcium/calmodulin-dependent protein kinase in anthers. Planta209,161–171
27 Le´vy, J. et al. (2004) A putative Ca2+ and calmodulin-dependent
proteinkinaserequiredforbacterialandfungalsymbioses.Science 303,1361–1364
28 Mitra,R.M.etal.(2004)ACa2+/calmodulin-dependentproteinkinase required for symbiotic nodule development: gene identification by transcript-basedcloning.Proc.Natl.Acad.Sci.U.S.A.101,4701–4705
29 Chen,C.Y.etal.(2007)Fungalsymbiosisinricerequiresanorthologof a legume common symbiosis gene encoding a Ca2+
/calmodulin-dependentproteinkinase.PlantPhysiol.145,1619–1628
30 Gutjahr,C.etal.(2008)Arbuscularmycorrhiza-specificsignalingin ricetranscendsthecommonsymbiosissignalingpathway.PlantCell 20,2989–3005
31 Binder,A.andParniske,M.(2014)AnalysisoftheLotusjaponicus nuclearporeNUP107-160subcomplexrevealspronouncedstructural plasticityandfunctionalredundancy.Front.PlantSci.4,552
32 Kanamori,N.etal.(2006)Anucleoporinisrequiredforinductionof Ca2+spikinginlegumenoduledevelopmentandessentialforrhizobial
andfungalsymbiosis.Proc.Natl.Acad.Sci.U.S.A.103,359–364
33 Saito, K. et al. (2007) NUCLEOPORIN85 is required for calcium spiking, fungal and bacterial symbioses, and seed production in Lotusjaponicus.PlantCell19,610–624
34 Favre,P.etal.(2014)Anovelbioinformaticspipelinetodiscovergenes relatedtoarbuscularmycorrhizalsymbiosisbasedontheirevolutionary conservationpatternamonghigherplants.BMCPlantBiol.14,333
35 Chen, X. et al. (2011) sn-Glycerol-3-phosphate acyltransferases in plants.PlantSignal.Behav.6,1695–1699
36 Li-Beisson,Y.etal.(2013)Acyl-lipidmetabolism.ArabidopsisBook11, e0161
37 Yang, W.L. et al. (2010) A distinct type of glycerol-3-phosphate acyltransferasewithsn-2preferenceandphosphataseactivityproducing 2-monoacylglycerol.Proc.Natl.Acad.Sci.U.S.A.107,12040–12045
38 Schreiber, L.(2010)Transportbarriers madeofcutin,suberinand associatedwaxes.TrendsPlantSci.15,546–553
39 Geldner,N.(2013)Theendodermis.Annu.Rev.PlantBiol.64,531–558
40 Franke,R.etal.(2000)Modifiedligninintobaccoandpoplarplants over-expressingtheArabidopsisgeneencodingferulate5-hydroxylase. PlantJ.22,223–234
41 Marita, J.M. et al. (1999) NMR characterization of lignins in Arabidopsis alteredin theactivity offerulate 5-hydroxylase. Proc. Natl.Acad.Sci.U.S.A.96,12328–12332
42 Oldroyd,G.E.D. (2013)Speak,friend,andenter:signallingsystems thatpromote beneficial symbiotic associationsin plants. Nat. Rev. Microbiol.11,252–263
43 Takayama, S. and Isogai, A. (2005) Self-incompatibility in plants. Annu.Rev.PlantBiol.56,467–489
44 Nasrallah, J.B.and Nasrallah,M.E.(2014)S-locusreceptorkinase signalling.Biochem.Soc.Trans.42,313–319
45 Goring,D.R.etal.(2014)TheARC1E3ligasepromotesastrongand stableself-incompatibilityresponseinArabidopsisspecies:responseto theNasrallahandNasrallahcommentary.PlantCell26,3842–3846
46 Indriolo,E.etal.(2012)TheARC1E3ligasegeneIsfrequentlydeleted inself-compatibleBrassicaceaespecies andhasaconservedrolein Arabidopsislyrataself-pollenrejection.PlantCell24,4607–4620
47 Indriolo,E.etal.(2014)TheARC1E3ligasepromotestwodifferent self-pollenavoidancetraitsinArabidopsis.PlantCell26,1525–1543
48 Nasrallah,J.B.andNasrallah,M.E.(2014)Robustself-incompatibility intheabsenceofafunctionalARC1geneinArabidopsisthaliana.Plant Cell26,3838–3841
49 Naithani,S.etal.(2007)Structuralmodulesforreceptordimerization intheS-locusreceptorkinaseextracellulardomain.Proc.Natl.Acad. Sci.U.S.A.104,12211–12216
50 Yamamoto,M.etal.(2014)Site-specificN-glycosylationoftheS-locus receptorkinaseanditsroleintheself-incompatibilityresponseofthe Brassicaceae.PlantCell26,4749–4762
51 Vekemans,X.etal.(2014)Theevolutionofselfingfromoutcrossing ancestorsinBrassicaceae:whathavewelearnedfromvariationatthe S-locus?J.Evol.Biol.27,1372–1385
52 Walker,J.C.andZhang,R.(1990)Relationshipofaputativereceptor proteinkinasefrommaizetotheS-locusglycoproteinsof Brassica. Nature345,743–746
53 Antolin-Llovera,M.etal.(2014)Knowingyourfriendsandfoes–plant receptor-likekinasesasinitiatorsofsymbiosisordefence.NewPhytol. 204,791–802
54 Szczyglowski,K.andAmyot,L. (2003)Symbiosis,inventivenessby recruitment?PlantPhysiol.131,935–940
55 Rodriguez-Llorente,I.D.etal.(2004)Frompollentubestoinfection threads: recruitmentofMedicago floralpecticgenes forsymbiosis. PlantJ.39,587–598
56 Nguema-Ona,E.etal.(2012)Arabinogalactanproteinsin rootand pollen-tubecells:distributionandfunctionalaspects.Ann.Bot.110, 383–404
57 Duc,G.etal.(1989)Firstreportofnon-mycorrhizalplantmutants (myc-)obtainedinpea(PisumsativumL.)andfababean(ViciafabaL.). PlantSci.60,215–222
58 Tansengco,M.L.etal.(2004)Pollendevelopmentandtubegrowthare affectedinthesymbioticmutantofLotusjaponicus,crinkle.PlantCell Physiol.45,511–520
59 Yadav,V.etal.(2014)ABCGtransporters arerequiredforsuberin andpollenwallextracellularbarriersinArabidopsis.PlantCell26, 3569–3588
60 Cnudde, F. et al.(2006) Changes in geneexpression during male meiosisinPetuniahybrida.ChromosomeRes.14,919–932
61 Chen, X. et al. (2014) Glycerol-3-phosphate acyltransferase 4 is essentialforthenormaldevelopmentofreproductiveorgansandthe embryoinBrassicanapus.J.Exp.Bot.65,4201–4215
62 Li,X-C.etal.(2012)Glycerol-3-phosphateacyltransferase6(GPAT6)is importantfortapetumdevelopmentinArabidopsisandplaysmultiple rolesinplantfertility.Mol.Plant5,131–142
63 Zheng, Z.F. et al. (2003) Arabidopsis AtGPAT1, a member of the membrane-boundglycerol-3-phosphateacyltransferase gene family, isessentialfortapetumdifferentiationandmalefertility.PlantCell 15,1872–1887
64 Birnbaum,K.etal.(2003)AgeneexpressionmapoftheArabidopsis root.Science302,1956–1960
65 Birnbaum,K.etal.(2005)Celltype-specificexpressionprofitingin plantsviacellsortingofprotoplastsfromfluorescentreporterlines. Nat.Methods2,615–619
66 Berruti,A.etal.(2013)Applicationoflasermicrodissectiontoidentify themycorrhizalfungithatestablisharbusculesinsiderootcells.Front. PlantSci.4,135
67 Gomez,S.K.andHarrison,M.J.(2009)Lasermicrodissectionandits application to analyze gene expression in arbuscular mycorrhizal symbiosis.PestManag.Sci.65,504–511
68 Aya, K. et al. (2011) Comprehensive network analysis of anther-expressedgenesinricebythecombinationof33lasermicrodissection and143spatiotemporalmicroarrays.PLoSONE6,e26162
69 Suwabe, K. et al. (2008) Separated transcriptomes of male gametophyteandtapetuminrice:validityofalasermicrodissection (LM)microarray.PlantCellPhysiol.49,1407–1416
70 Harper,J.F.andHarmon,A.(2005)Plants,symbiosisandparasites:A calciumsignallingconnection.Nat.Rev.Mol.CellBiol.6,555–566
71 Poovaiah,B.W.etal.(2013)Recentadvancesin calcium/calmodulin-mediatedsignalingwithanemphasisonplant-microbeinteractions. PlantPhysiol.163,531–542
72 Delaux,P-M.etal.(2014)Comparativephylogenomicsuncoversthe impact of symbiotic associations on host genome evolution. PLoS Genet.10,e1004487
73 Dereeper,A.etal.(2008)Phylogeny.fr:robustphylogeneticanalysisfor thenon-specialist.NucleicAcidsRes.36,W465–W469
74 Beisson,F. etal. (2012) Solving the puzzles of cutin and suberin polymerbiosynthesis.Curr.Opin.PlantBiol.15,329–337
75 Kusaba,M.etal.(2001)Self-incompatibilityinthegenusArabidopsis: characterizationoftheS-locusin theoutcrossingA.lyrataandits autogamousrelativeA.thaliana.PlantCell13,627–643
76 Stein,J.C.etal.(1991)Molecularcloningofaputativereceptorprotein kinase gene encoded at the self-incompatibility locus of Brassica oleracea.Proc.Natl.Acad.Sci.U.S.A.88,8816–8820
