Root microbiota dynamics of perennial Arabis alpina are dependent on soil residence time but

13 Pages·2016·2.55 MB·English

by  Nina Dombrowski

OPEN TheISMEJournal(2016),1–13
©2016InternationalSocietyforMicrobialEcology Allrightsreserved 1751-7362/16
www.nature.com/ismej
ORIGINAL ARTICLE
Arabis alpina
Root microbiota dynamics of perennial
are dependent on soil residence time but
independent of flowering time
Nina Dombrowski1, Klaus Schlaeppi2, Matthew T Agler1, Stéphane Hacquard1,
Eric Kemen1,3, Ruben Garrido-Oter1,3,4, Jörg Wunder5, George Coupland5 and
Paul Schulze-Lefert1,3
1Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne,
Germany; 2Plant–Soil-Interactions, Institute for Sustainability Sciences, Agroscope, Reckenholzstrasse 191,
Zurich, Switzerland; 3Cluster of Excellence on Plant Sciences (CEPLAS), Max Planck Institute for Plant
BreedingResearch,Cologne,Germany;4DepartmentofAlgorithmicBioinformatics,HeinrichHeineUniversity
Düsseldorf, Düsseldorf, Germany and 5Department of Plant Developmental Biology, Max Planck Institute for
Plant Breeding Research, Cologne, Germany
Recent field and laboratory experiments with perennial Boechera stricta and annual Arabidopsis
thaliana suggest that the root microbiota influences flowering time. Here we examined in long-term
time-course experiments the bacterial root microbiotaofthe arctic-alpine perennial Arabis alpina in
natural and controlled environments by 16S rRNA gene profiling. We identified soil type and
residence time of plants in soil as major determinants explaining up to 15% of root microbiota
variation,whereasenvironmentalconditionsandhostgenotypeexplainmaximally11%ofvariation.
Whengrowninthesamesoil,therootmicrobiotacompositionofperennialA.alpinaislargelysimilar
tothoseofits annualrelativesA.thalianaandCardaminehirsuta.Non-floweringwild-type A.alpina
andfloweringpep1mutantplantsassembleanessentiallyindistinguishablerootmicrobiota,thereby
uncouplingfloweringtimefromplantresidencetime-dependentmicrobiotachanges.Thisrevealsthe
robustness of the root microbiota against the onset and perpetual flowering of A. alpina. Together
withpreviousstudies,thisimpliesamodelinwhichpartsoftherootmicrobiotamodulateflowering
time,whereas,aftermicrobiotaacquisitionduringvegetativegrowth,theestablishedroot-associated
bacterialassemblageisstructurallyrobusttoperturbationscausedbyfloweringanddrasticchanges
inplant stature.
TheISME Journal advance onlinepublication, 2August 2016; doi:10.1038/ismej.2016.109
Introduction seed germination (Edwards et al., 2015). Soil
represents the most diverse ecosystem on earth
Plants host taxonomically structured bacterial con-
with an exceptionally high bacterial species diver-
sortia on andinside roots and leaves, designated the
sity that varies greatly between different soil types
root and leaf microbiota (Vorholt, 2012; Bulgarelli
(Fierer and Jackson, 2006; Lauber et al., 2009).
et al., 2013; Hacquard et al., 2015). The start
Numerous studies employing next generation
inoculumoftheleaf-associatedmicrobiotaisderived
sequencing technologies have shown that soil type
from multiple sources, likely involving bacteria
is amajor determinant of rootmicrobiotacomposi-
transmitted by aerosols, insects or soil particles
tion, most likely reflecting the different bacterial
(Vorholt, 2012; Bodenhausen et al., 2013; Maignien
start inocula present in each soil type (Bulgarelli
et al., 2014; Bai et al., 2015). Conversely, root-
et al., 2012; Lundberg et al., 2012; Peiffer et al.,
associated bacterial consortia are mostly derived
2013; Schlaeppi et al., 2014; Edwards et al., 2015).
from the bacterial soil biome surrounding roots and
Despite significant variation in microbiota compo-
establish rapidly. For example, a stable taxonomic
sition at low taxonomic ranks, for example, genus-
structureinricerootsdevelopedwithin14daysafter
or species-level, a recent direct comparison of
the root microbiota of eight flowering plant
Correspondence: Dr P Schulze-Lefert, Department of Plant species, including monocots and dicots, revealed
Microbe Interactions, Max Planck Institute for Plant Breeding a co-occurrence of three main bacterial phyla
Research,Cologne50829,Germany. comprising Actinobacteria, Bacteroidetes and
E-mail:[email protected]
Proteobacteria(Hacquardetal.,2015).Thisfinding
Received4February2016;revised27June2016;accepted3July
2016 suggests that the root microbiota and its overall
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taxonomic structure is a conserved plant trait conditions in native or non-native soils (‘soil type
across flowering plants. and environment’ experiment). We then investigated
An unresolved question in microbiota research potential changes of the A. alpina root microbiota at
is whether plant-associated bacterial assemblages two developmental stages (flowering vs vegetative)
contribute to the plasticity of complex plant traits. during an extended residence time of WT and pep1
For example, field experiments with perennial mutant plants in soil (‘time-course’ experiment).
Boechera stricta and laboratory experiments with Finally, we examined the diversification of the A.
annual A. thaliana have indicated that the bacterial alpina bacterial microbiota by comparison with root
root microbiota modulates flowering time (Wagner microbial communities collected from two other
et al., 2014; Panke-Buisse et al., 2015). Conversely, Brassicaceae species, A. thaliana and Cardamine
root-associated bacterial consortia of A. thaliana hirsuta (‘diversification’ experiment).
appear to be affected by plant development stages
and metatranscriptome studies revealed bacterial
Materials and methods
transcripts induced at bolting and flowering stages
(Chaparro et al., 2014).
Soil types used and plant material
Plant growth in the arctic-alpine environment French soil was harvested in fall 2012 (‘FS Fall-12’)
requires an adaptation to a range of abiotic stresses,
at the Col du Galibier, France (45.061 N/6.402 E).
including water limitation, extreme temperature
Similar to earlier work (Bulgarelli et al., 2012),
shifts and low nutrient availability (Billings and
Cologne soil batches were collected in fall 2010, in
Mooney, 1968; Chapin, 1983; Chapin and Shaver, spring2013andinfall2013(termed‘CSFall-10’,‘CS
1989; Margesin and Miteva, 2011). Root-associated Spring-13’ and ‘CS Fall-13’) at the Max Planck
bacterial members from three arctic-alpine plant
Institute for Plant Breeding Research in Cologne,
species (Oxyria digyna, Diapensia lapponica and
Germany (50.958 N/6.856 E). Geochemical charac-
Juncus trifidus) appear to be enriched for potent
terization of soil types was carried out by the 'Labor
microbial solubilizers of mineral phosphorus, a
fürBoden-undUmweltanalytik'(EricSchweizerAG,
common but plant-inaccessible source of phos-
Thun, Switzerland).
phorus in arctic-alpine environments (Nissinen
ThreeBrassicaceaeplantspecieswereinvestigated
et al., 2012). This suggests the potential importance
during this study. Experiments with A. alpina were
of microbiota members for plant growth and health
conducted using the Spanish reference ecotype
in arctic-alpine environments (Richardson et al.,
Pajares (Paj), the two French ecotypes F1-Gal5 and
2009). Perennial Arabis alpina is an arctic-alpine
Gal60, as well as the mutant line pep1 (Paj back-
plant closely related to the annual Arabidopsis
ground; Wang et al., 2009). A. alpina Gal60 was
thaliana, which allows comparative studies of
harvested during the course of this study at the
inter-species trait diversification such as flowering
Col du Galibier, France. In addition, A. thaliana
time (Beilstein et al., 2010; Wang et al., 2009). For
(ecotype Columbia, Col-0) and C. hirsuta (ecotype
example, the orthologue of the A. thaliana gene
Oxford, Ox) were investigated.
FLOWERING LOCUS C (FLC) that inhibits flowering
until A. thaliana is exposed to winter temperature,
was shown to be A. alpina PEP1 (PERPETUAL Plant growth
FLOWERING 1), which limits flowering duration We characterized A. alpina root-associated bacterial
(Wangetal.,2009).Perpetualfloweringischaracter- communities using three sets of experiments
istic for A. alpina pep1 mutant plants, resulting in a (Table 1, Supplementary Figure S1). First, for the
drasticdifferenceinplantstatureduetothepresence ‘soil type and environment’ experiment, individual
of reproductive shoots compared with wild-type flowering A. alpina plants of unknown age were
(WT) plants (Wang et al., 2009). Extensive allelic excavated (designated Gal60) from their native
variation at PEP1 also exists in natural populations, habitat at the Col du Galibier (France) in fall 2012
includinglossofPEP1functionalleles(Albanietal., to evaluate their natural root microbial communities
2012). The adaptation of A. alpina to arctic-alpine (Figure 1a). To evaluate the effect of environmental
environments,itsperennialnature,theavailabilityof factors on community assembly, we also collected
genetic lines, as well as the close evolutionary soil from this site and transported it to Cologne
relationship to A. thaliana make this plant species to compare microbial communities from A. alpina
a suitable model to investigate the interplay of Gal60 grown its native French soil in the natural
determinants for root microbiota composition and environment with controlled environmental condi-
diversification. tions in the greenhouse. To investigate possible host
Here we characterized A. alpina root-associated genotype-dependent effects on microbiota composi-
bacterial communities using several experimental tion, we grew Gal60 alongside with a French
approaches. We employed culture-independent com- genotype from a nearby location (Gal5) and a
munity profiling by 16S rRNA gene sequencing to Spanish reference genotype (Paj) in the French soil
assess bacterial community composition of A. alpina in the greenhouse for 3 months. During harvest,
plants grown in their natural habitat compared with Gal60andPajresidedinthevegetativegrowthstage,
plants grown under controlled environmental whileGal5plantsstartedtoflower(Figure1b).Apart
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Table1ExperimentaldesignandnumbersofreplicatesperDNAsampleacrossexperimentalsetups CompartmentHostplantSoiltypeandenvironmentTimecourseDiversification454vsIllumina CS_CFS_CFS_N6weeks12weeks28weeks aarep3rep2rep1rep2rep1rep2rep1rep2rep1rep2 —Soil4433333333333 —————RhizosphereA.alpina(Paj)45334444———————A.alpina(pep1)334444————————————A.alpina(Gal5)5———————————A.alpina(Gal60)55 —————————RootA.thaliana(Col-0)3333—————————C.hirsuta(Ox)3332—A.alpina(Paj)453344443333———————A.alpina(pep1)334444————————————A.alpina(Gal5)5———————————A.alpina(Gal60)55 SoiltypeCologneFranceCologneCologneCologneResidencetime12weeks12weeksND6weeks6weeks12weeks12weeks28weeks28weeks6weeks6weeks6weeks6weeksHarvestdatesoilSpring13Fall12Spring13Fall13Spring13Fall13Spring13Fall13Fall10Spring13Fall13Spring13EnvironmentControlledNativeControlledControlledControlledSeq.technologyIlluminaIllumina454Illumina Abbreviations:CS,Colognesoil;FS,Frenchsoil;ND,notdetermined.‘’‘’Experimentalsetupcomparingbacterialcommunitycompositionundernaturalgrowthconditions(soiltypeandenvironmentexperiments),prolongedresidencetimeofplantsinsoil(time-course‘’experiment)onthreedifferentplantspecies(diversificationexperiment).Thefirsttwocolumnscharacterizethetestedcompartment,hostplantandplantgenotype.Thenumbersindicatethe‘’numberofsequencedDNAsamples.Soiltypeandenvironmentexperiment:plantsweregrowninCSorFSundercontrolledenvironmentalconditions(C)orintheirnativehabitatinFrance(N).‘’‘’Timecourseexperiment:A.alpinaplantsweregrownundercontrolledenvironmentalconditionsinCSfor6,12and28weeks.Diversificationexperiment:A.alpina,C.hirsutaandA.thaliana–weregrownundercontrolledenvironmentalconditionsinCSfor6weeks.Indicatedarethenumberofindependentbiologicalreplicates(rep1rep3),thesoiltypeusedforgrowingplants(seealsoSupplementaryTablesS3andS4),theharvestdateofthesoil,theenvironmentalconditionsusedforplantgrowthandtheemployedsequencingtechnology.ND,notdetermined.aRepresentsthesameDNAsamplesthatwereprocessedwithboth454andIlluminasequencingtechnologies.
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Figure1 GrowthphenotypeofA.alpinainanaturalFrenchhabitatandundercontrolledenvironmentalconditions.(a)Naturalhabitat
ofA.alpinaintheFrenchAlps.(b)Growthmorphologyof differentA.alpinaaccessionsinaFrenchsoil(FS)inits nativehabitatin
the French Alps, in FS under controlled environmental conditions and Cologne soil (CS) under controlled environmental conditions.
Gal60,FrenchA.alpinaaccessioncollectedinitsnaturalhabitatintheFrenchAlps,ColduGalibier;Gal5,FrenchA.alpinaaccession;
Paj,SpanishA.alpinaaccession.
from their geographic origin, the three genotypes 16S rRNA gene sample preparation, sequencing and
differ from each other in that the genotype Paj analysis
requires a vernalization treatment to flower, while We collected soil, rhizosphere and root compart-
Gal5 and Gal60 do not. In an additional subset of ments and prepared a DNA template for further
this experimental setup, we planted the A. alpina processing using established protocols (Schlaeppi
genotype Paj not only in French but also Cologne et al., 2014). Briefly, amplicon libraries were pre-
soil to study the effect of soil type on community pared using the primers 799F (Chelius and Triplett,
composition. For the ‘soil type and environment’ 2001) and 1193R (Bodenhausen et al., 2013).
experimentconductedinthegreenhouse,A.alpina Sequencing of samples with 454 pyrosequencing
seeds were surface-sterilized, sown onto the (Branford, CT, USA; ‘diversification’ experiment)
respective soil type, stratified for 4 days and a was conducted as previously described (Schlaeppi
singleplantperpot(atleastfourtofiveindividuals et al., 2014), while the majority of the samples (‘soil
per genotype) was grown for 3 months until typeandenvironment’and‘timecourse’experiment)
harvest. In a second experimental setup, denoted were sequenced using Illumina (MiSeq) paired-end
‘time course’ experiment, individual A. alpina Paj sequencing (San Diego, CA, USA; using a slightly
plants were grown in two batches of Cologne soil, modified PCR-amplification protocol, but targeting
thatis,twofullfactorialbiologicalreplicates,under the same 16S rRNA gene region, see Supplementary
controlledenvironmentalconditionsandharvested Notes for details). We prepared a total of 201 DNA
after 6, 12 and 28 weeks. Finally, in the ‘diversifi- samples for sequencing: 59, 106 and 36 samples
cation’ experiment, A. alpina (Paj), C. hirsuta belonging to the ‘soil type and environment’, ‘time
(Oxford) and A. thaliana (Col-0) plants (four plants course’ and ‘diversification’ experiment, respec-
per pot) were grown in three batches of Cologne tively.Inaddition,11samplesofthe‘diversification’
soil,thatis,threefullfactorialbiologicalreplicates, experiment sequenced by the 454 technology, were
under controlled environmental conditions for re-sequenced using the Illumina protocol to investi-
6 weeks until harvest. gate potential methodological biases (Table 1,
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Supplementary Figure S1). The bioinformatic analy- pattern between compartments was consistent
sis of sequence reads included de-multiplexing of between 454 and Illumina methodologies
samples using the QIIME software package (Supplementary Figure S2) and we did not detect
(Caporaso et al., 2010) and determination of opera- marked differences in taxonomic assignments
tional taxonomic units (OTUs) on the three con- (Supplementary Figure S3, see Supplementary
catenated datasets using the UPARSE pipeline Notes for details). This shows that the two sequen-
(Edgar, 2013). Afterwards, OTU tables were sepa- cing technologies produce comparable results.
rated and investigated independently based on the For the 201 samples included in the main analysis
three experimental setups (if not stated otherwise) of this study, we defined 8969 OTUs (⩾97%
and closer investigated by employing Principal sequence similarity of 16S rRNA gene sequences),
Coordinate Analysis (PCoA) analyses and estab- all belonging to the kingdom Bacteria. Across
lished linear model statistics using analysis of experiments, the tested compartment (soil, rhizo-
variance (ANOVA) and Bayesian model-based mod- sphere or root) explained 18–36% of community
erated t-tests to calculate differentially enriched variation among samples (Figure 2a, Table 2).
OTUs (Bulgarelli et al., 2012, 2015). At phylum rank, unplanted soil communities were
dominated with decreasing relative abundance by
Proteobacteria, Actinobacteria and Gemmatimona-
Data deposition detes(SupplementaryFiguresS4–S7).Incomparison
Raw sequencing data were deposited in the
to unplanted soil, root microbial communities
NCBI Short Read Archive (SRA), BioProjectID
displayed a significantly reduced alpha-diversity
PRJNA317760, accession number SRP073035. The (Supplementary Figure S8, Supplementary Table S1)
reference numbers for the original raw sequencing
and were mainly inhabited by Proteobacteria, Acti-
data are SRR3350817 (L1151), SRR3351958 (L1264),
nobacteria and Bacteroidetes in all soil types tested.
SRR3353796 (L1291), L35 (SRR3355061), L905 This co-occurrence of three bacterial phyla corre-
(SRR3355062) and L1118 (SRR3355063). Custom R
sponds well with a recent assessment of the root
scripts can be found at http://www.mpipz.mpg.de/
microbiota of eight flowering plant species
R_scripts. This provides a downloadable folder (Hacquard et al., 2015). Taken together, the tested
including the used R script, input data files and compartments had a pronounced effect on commu-
custom R-packages that were separated for the
nity structure, with the rhizosphere community
individual parts of the analysis.
displaying an alpha-diversity that is comparable
to unplanted soil, whereas its taxonomic composi-
tion shared features with the soil and the highly
Results
distinctive root microbiota (Figure 2a, Supplementary
Marked bacterial community shifts in both A. alpina Figures S4–S8).
rhizosphere and root compartments
Plants were grown in microbe-containing soils and
root, rhizosphere and soil compartments sampled as The A. alpina rootmicrobiotaisdependentonsoiltype
described previously (Figure 1; Bulgarelli et al., but exhibits a converging taxonomic structure
2012; Schlaeppi et al., 2014). Briefly, the ‘soil We analyzed the 59 samples from the ‘soil type and
compartment’referstosoilcollectedfromunplanted environment’ experiments separately, enabling us to
pots and the ‘rhizosphere compartment’ defines soil dissect the relative contribution of the factors soil
particlestightlyadheringtorootsthatwerecollected type, environment and host genotype on bacterial
by two washing steps. Washed roots were addition- communitycomposition(Table1).Wecomparedthe
ally sonicated to deplete bacterial epiphytes and taxonomic composition of bacterial assemblages in
enrich for root-inhabiting bacteria, designated ‘root root and rhizosphere compartments with the soil
compartment’.Bacterialcommunityprofilesforeach biome of the A. alpina accession Gal60 in its native
compartment were generated by PCR amplification arctic-alpine environment at the Col du Galibier
of the 16S rRNA gene by targeting region V5-V7 (France, Figure 1a). In addition, the Gal60 accession
usingPCRprimers799F(CheliusandTriplett,2001) was grown in the same native soil, but under
and 1193R (Bodenhausen et al., 2013) followed controlled environmental conditions, together with
by 454 or Illumina sequencing (Table 1 and the accessions Gal5 and Paj that are derived from a
Supplementary Information). We generated a total differentsiteattheColduGalibierandtheCordillera
of 10138758 high-quality sequences from 201 Cantábrica in Spain, respectively. Ina further subset
samples (average of 50342 and 2804-155004 of this experiment, the accession Paj was grown
sequences per sample, individual read counts under controlled environmental conditions in
supplied in Supplementary Data S1). In addition, Cologne soil (Figure 1b, Supplementary Tables S3
11 samples of the ‘diversification’ experiment that and S4).
were sequenced with 454 were re-sequenced using To quantify the contribution of individual factors
theIlluminamethodologyandprotocol,toestimatea to the observed variation in community profiles,
potential methodological bias (533801 total reads, we used Bray–Curtis (accounting for OTU abun-
Table 1, Supplementary Figure S1). The separation dances but not phylogenetic distances), unweighted
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Figure2 BacterialcommunityshiftsinA.alpinarhizosphereandrootcompartments.(a)Unconstrainedordinationrevealingthatmost
ofthevariationamongall201samplesfromthe3experimentalsetupsisexplainedbythefactorcompartment(firstprincipalcoordinate
axis)andsoiltype(secondprincipalcoordinateaxis)basedontheBray–Curtisdistancemetric.(b)Ternaryplotdepictingthenumberof
OTUsenrichedinthesoil,rhizosphereandrootcompartmentsofthe59samplesofthe‘soiltypeandenvironment’experiment(SoilOTUs
(black),RhizoOTUs(brown),RootOTUs(green),respectively).EachcircledepictsoneindividualOTU.Thesizeofthecirclereflectsthe
relativeabundance(RA).ThepositionofeachcircleisdeterminedbythecontributionoftheindicatedcompartmentstotheRA.Number
inbrackets:EnrichedOTUsbasedonaBayesmoderatedt-test;Po0.05(FDR-corrected).(c)Constrainedprincipalcoordinatesanalysison
thegenotypesPaj,Gal60andGal5grownintheFrenchSoilinthegreenhouseusingtheBray–Curtisdissimilarityandconstrainingbyhost
genotype.Ineachcase,thepercentageofvariationexplainedbyeachaxisreferstothefractionofthetotalvarianceofthedataexplained
bytheconstrainedfactor.CS-C,Colognesoilgrownundercontrolledenvironmentalconditions'FS-C,Frenchsoilgrownundercontrolled
environmentalconditions;FS-N,Frenchsoilgrownundernativeenvironmentalconditions.
(accounting for phylogenetic distances but not Supplementary Figure S9) revealed that the factor
abundances) and weighted (accounting for OTU soil type explained 11–15% of the observed varia-
abundances and phylogenetic distances) UniFrac tion, followed by environment (8–11%) and host
distance metrics as β-diversity (between-sample genotype (5–12%). Both soil type and environment
diversity) estimates (Lozupone and Knight, 2005). significantly influenced community composi-
A constrained Canonical Analysis of Principal tion, and we found no significant interaction
coordinates (CAP), followed by a permutation- between the soil type and environment variables
based ANOVA (PERMANOVA, Table 2, Figure 2c, (Supplementary Figure S9a). Notably, host genotype
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Table2 DeterminingdriversofbacterialcommunityassemblyusingCAP
Constrainedfactor Bray–Curtis WeightedUnifrac UnweightedUnifrac
% P-value CI % P-value CI % P-value CI
Soiltype+environment Compartmenta 31 *** 21,48 33 *** 20,52 36 *** 23,64
Environmenta 11 *** 8,16 8.2 *** 5,13 9.8 *** 7,14
Compartmentb 25 *** 15,47 24 *** 13,46 37 *** 19,78
Genotypeb 9.1 *** 7.3,11 12 *** 8.5,16 4.8 * 4.2,5.5
Compartmentc 31 *** 20,50 39 *** 22,67 33 *** 21,53
Soiltypec 15 *** 9,26 11 *** 6,20 15 *** 10,25
Timecourse Compartment 19 *** 14,27 25 *** 17,39 18 *** 14,26
Timepoint 9.9 *** 7.8,13 12 *** 8.5,18 6.6 *** 5.7,7.8
Soilbatch 6.6 *** 4.9,9.1 5.3 *** 3.6,8 8.4 *** 6.6,11
Floweringstage 1.5 NS 1.2,1.9 1.3 NS 1.0,1.9 1.8 * 1.3,2.5
Mutantbackground 0.6 NS 0.4,0.8 0.8 NS 0.5,1.2 0.7 NS 0.5,1.1
Diversification Compartment 21 *** 12,37 26 *** 12,51 18 *** 12,31
Soilbatch 19 *** 14,28 20 *** 12,32 18 *** 14,24
Plantspecies 10 *** 8,14 10 *** 7,15 7.3 *** 6,9
Abbreviations:CAP,constrainedanalysisofprincipalcoordinates;CI,confidenceinterval;NS,non-significant;PERMANOVA,permutation-based
analysisofvariance.
Variation(in%)betweensamplesinthe‘soiltypeandenvironment’,‘timecourse’and‘diversification’experimentsbasedonBray–Curtis,
weightedandunweightedUniFracdistances,constrainingfortheindicatedfactors.P-valuebasedonPERMANOVA(999permutations).*Po0.05,
**Po0.01,***Po0.001.SampleswereanalyzedseparatelyaccordingtothethreedifferentexperimentalsetupsasstatedinTable1.Inaddition,
the‘soiltypeandenvironment’sampleswereseparatedasfollows:
aGal60grownatthenaturalsiteandthegreenhouse.
bGal60,Gal5andPajgrowninFrenchsoilinthegreenhouse.
cPajgrownintheFrenchandColognesoilinthegreenhouse.
affected community composition with a higher significantly enriched in soil, rhizosphere and root
significance using weighted compared with compartments as SoilOTUs, RhizoOTUs and RootO-
unweighted UniFrac, suggesting that host genotype TUs, respectively. Of a total of 1676 OTUs, the
preferentially acts on abundant community mem- compartment-enriched OTUs gradually declined
bers. At the time of harvest, the accession Gal5 was from 713 SoilOTUs to 365 RhizoOTUs and 135
flowering while Gal60 and Paj resided in the RootOTUs, with the remaining 463 OTUs being
vegetative stage (Figure 1b). Because only Paj shared among compartments (Figure 2b). Of the
requires a vernalization treatment to flower (Gal5 RootOTUs, ~30% were shared across all tested soil
and Gal60 do not), the differences in developmental typesandenvironmentalconditions(Supplementary
stages could potentially confound the observed host Figure S10). Taken together, this analysis shows
genotype-dependent effects (see below). In addition, that A. alpina roots accommodate a taxonomically
the three genotypes were only grown in one soil congruent bacterial community in contrasting
type; therefore, we cannot make conclusions on environments and produce a marked rhizosphere
potential environment-specific adaptations of indi- effect, as evidenced by the identification of 365
vidual genotypes. The impact of soil type on RhizoOTUs.
community profiles was apparent at the highest
taxonomic rank. For example, differential enrich-
ments of the phyla Actinobacteria and Firmicutes
were found in French and Cologne soils The A. alpina root microbiota is dependent on soil
(Supplementary Table S5 and Supplementary residence time and independent of flowering time
Figure S4). In addition, soil type-dependent differ- To evaluate potential changes of A. alpina root- and
ences of the bacterial soil biome at phylum rank rhizosphere-associated microbiota composition over
converged progressively towards more soil type- time, A. alpina samples were collected at 6, 12 and
independent communityprofiles inrhizosphereand 28 weeks after sowing in Cologne soil under
root compartments (Supplementary Table S5), indi- controlled environmental conditions (Table 1,
catingthatrootsprovideanichefortheassemblyofa Supplementary Information). In the context of this
stable bacterial consortium despite contrasting soil ‘time-course’ experiment we also investigated
types and environments. whether host developmental stage (non-flowering
Next,wequantifiedthenumberofOTUsthatwere andflowering)affectsbacterialcommunitystructure.
enriched in a compartment across soil types We planted the WT A. alpina accession Paj together
and environments (Supplementary Information; with the perpetually flowering mutant pep1 (Wang
Bulgarelli et al., 2012). We refer to OTUs et al., 2009). This mutant started to flower after
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a b
c d
Figure3 A.alpinaroot-associatedbacterialcommunitiesdependonsoilresidencetime.(a)Principalcoordinateanalysisonsamples
fromthe‘timecourse’experiment(106samples)constrainedforthefactortimepointforsoil,rhizosphereandrootcompartmentsat6,12
and 28 weeks, respectively. The percentage of variation explained by each axis refers to the fraction of the total variance of the data
explainedbytheconstrainedfactor.(b–d)Soil-,rhizosphere-androot-enrichedOTUs(RootOTUs,RhizoOTUs,SoilOTUs)includingOTUs
observedacrossalltestedtimepointsandOTUsspecificforindividualtimepoints.Timepoint-specificSoilOTUs(b),RhizoOTUs(c)and
RootOTUs(d).Themeanofsoil(b),rhizosphere(c)orroot(d)samplesateachtimepointisplotted.Numberinbrackets:totalnumberof
OTUs enriched in the respective compartment or time point. Enriched OTUs are based on a Bayes moderated t-test; Po0.05 (FDR-
corrected).
12 weeks, whereas WT Paj plants remained in the abundance across all tested time points. The
vegetative stage until the end of the experiment. observed strong impact of residence time of plants
Within this experimental setup, the factor time in soil on bacterial root and rhizosphere commu-
point explained 7–12% of the observed variation nities was apparent at high taxonomic rank, for
(Table 2, Figure 3a). Considering the interaction example, by an increase in the relative abundance
between time point and compartment, we found a of Proteobacteria and decrease of Bacteroidetes in
strong interaction between those two factors, roots over time (Supplementary Figure S5 and
explaining 11% of the variance of the data Supplementary Table S6). To identify OTUs that
(Supplementary Figure S9b). Interestingly, only root explainthesetimepoint-dependentchanges,wefirst
and rhizosphere community profiles were affected determined all SoilOTUs (745), RhizoOTUs (271)
over time, while the initial bacterial community and RootOTUs (200). Around 62% of RhizoOTUs
present in unplanted soil remained largely stable and54%RootOTUssignificantlychangedinrelative
overthetested timeframefrom6to28weeksacross abundanceovertime,whereasthiswasonlythecase
the used soil batches (Figure 3, Supplementary for ~10% of SoilOTUs (Figures 3b–d). Closer
Figure S5). The stability of soil samples over time inspection showed that changes in rhizosphere and
was apparent since these samples did not separate root microbiota profiles were characterized by
withinthePCoAandmostSoilOTUsremainedinthe a continuous increase in the total number of Rhizo-
center of the ternary plot, reporting their equal and RootOTUs over time (Figures 3c and d).
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RootmicrobiotadynamicsofperennialArabisalpina
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Figure4 TheA.alpinarootmicrobiotaisindependentoffloweringtime.(a–c)GrowthmorphologyofA.alpinawild-type(Paj)andpep1
mutantplantsacrossthe‘timecourse’experiment.(d–f)TernaryplotsofOTUsenrichedinthe‘timecourse’experimentacrossthesoiland
rootcompartmentsoftheA.alpinawild-type(Paj)andpep1mutantplantsafter6weeks(a,d),12weeks(b,e)and28weeks(c,f).Each
circledepictsoneindividualOTU.Thesizeofthecirclereflectstherelativeabundance(RA).Thepositionofeachcircleisdeterminedby
thecontributionoftheindicatedcompartmentstotheRA.Numberinbrackets:totalnumberofOTUsenrichedintherespectiveplantline.
OTUsenrichedinthetwoplantlinesarebasedonaBayesmoderatedt-test;Po0.05(FDR-corrected).
Remarkably, despite drastic differences in plant profiles in which the factor host species explained
stature, we did not detect significant changes 7–10% of the observed variation (Table 2). PCoA,
between the root and rhizosphere microbiota of WT constrained by host species, revealed that samples
and pep1 mutant plants (Table 2, Figure 4). None of from A. alpina roots clustered closely together
the Root- or RhizoOTUs differentially accumulated with those of A. thaliana although these plant
in 6- or 12-week-old WT and pep1 plants, when 12- species diverged ~45 Myr ago (Figure 5,
week-oldpep1plantswereflowering(Figures4dand Supplementary Figure S11a; Beilstein et al.,
e). A single low-abundant OTU (OTU927, order 2010). Root samples of C. hirsuta clustered sepa-
Myxococcales) was enriched in 28-week-old WT ratelyfromtheformertwohostsalthoughA.alpina
plants when pep1 plants were flowering and WT diverged from C. hirsuta only ~10 Myr ago
plantsremainedinthevegetativephase(Figure4f). (Figure 5a). Thus, for the tested plant species
In addition, we detected stable nutrient contents host divergence time appears to be incongruent
and soil parameters in unplanted soil compared with root microbiota diversification. The impact of
with soil samples recovered from pots containing host species on bacterial root communities was
28-week-old plants (Supplementary Tables S3 and detectable at phylum rank by a significant reduc-
S4). In summary, our findings reveal marked tion in the relative abundance of Bacteroidetes
residence-time-dependent changes of A. alpina in root samples of A. alpina (Supplementary
root- and rhizosphere-associated bacterial consor- Figure S7a). In addition, significant differ-
tia, but a stability of the root microbiota against the ences were detected for one, three and five
onset and perpetual flowering of A. alpina and bacterial families in A. thaliana, C. hirsuta and A.
concomitant drastic changes in plant stature. alpina, respectively (Supplementary Figure S7b,
Supplementary Table S7). Each tested host
species was also enriched for a few species-
The compositionof the root microbiota of perennial A. characteristic OTUs that displayed quantitative
alpina issimilarcomparedwiththeannualsA. thaliana differences in relative abundance (designated
and C. hirsuta AtOTUs, ChOTUs and AaOTUs; Figure 5b). How-
Next, we planted perennial A. alpina side-by-side ever, the majority of RootOTUs (62) were similarly
with the annuals C. hirsuta and A. thaliana detected in roots of all three host species
in Cologne soil for 6 weeks to examine potential (Figure 5b). In addition, a total of 18 RootOTUs
diversification of the corresponding root micro- were significantly enriched in roots of all 3 plant
biota (Table 1, Supplementary Figures S11a and species across the tested soil batches (Figure 5c,
b and Supplementary Information). We found Supplementary Figure S12a). These shared OTUs
evidence for host species-specific root microbiota were barely detectable in unplanted soil and
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RootmicrobiotadynamicsofperennialArabisalpina
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together represented almost 50% of the root- 12 OTUs assigned to Proteobacteria and 3 each
associated consortia (Supplementary Figure S12b). to Actinobacteria and Bacteroidetes (Figure 5c). In
The shared OTUs belonged to 3 phyla, with summary, we found few host species-characteristic
OTUs, while the majority of the root microbiota
was shared among the tested host species.
Discussion
Here, we quantified the relative contributions of
factors explaining variation in A. alpina root-
associated bacterial consortia by analyzing high-
quality 16S rRNA reads from 4200 samples and
multiple environmental factors. This revealed the
variable compartment as strongest determinant of
communityvariation(18–36%).Additionalvariables
influencing community variation were soil type
(11–15%), residence time of plants in soil (7–12%),
environmental conditions (8–11%), host species
(7–10%) and host genotype (5–12%). The relative
contributions of the factors compartment, soil type
and host species in this study are comparable to an
earlier report on root microbiota diversification
between A. thaliana relatives (Schlaeppi et al.,
2014). Despite the observed host species-dependent
differences in root microbiota profiles, we identified
a largely shared bacterial assemblage whose taxo-
nomic structure is similar to the microbiota inter-
sectionbetweenA.thaliana,A.halleri,A.lyrataand
C. hirsuta (Schlaeppi et al., 2014). This points to
potentially common services provided by these
bacterialtaxaforplantgrowthandhealthacrossfive
tested Brassicaceae host species. However, in con-
trast to previous work showing only weakly differ-
entiated bacterial rhizosphere assemblages
compared with unplanted soil biomes in annual
A. thaliana and C. hirsuta plants that were partially
performed in the same soil type and growth condi-
tions (Bulgarelli et al., 2012; Lundberg et al., 2012;
Schlaeppi et al., 2014), we detected a pronounced
rhizosphere effect for A. alpina at all tested
time points and across soil types and environments.
This suggests that within the Brassicaceae, evolu-
tionary diversification has apparently produced
host species-specific differences in bacterial com-
munity differentiation in the rhizosphere compart-
ment despite an overall convergent community
composition inside roots (Bulgarelli et al., 2012;
Lundberg et al., 2012; Schlaeppi et al., 2014). This
striking feature of A. alpina may be due to its
phylogenetic distance to A. thaliana and C. hirsuta,
Figure 5 The bacterial root microbiota of A. alpina is similar adaptation to the arctic-alpine habitat or its peren-
compared with A. thaliana and Cardamine hirsuta. (a) Con- nial lifestyle.
strainedprincipalcoordinatesanalysis(PCoA)basedontheBray– Based on similar CAP analyses, the variation
Curtis distances on the 36 samples of the ‘diversification’
of root microbiota profiles explained by intra-
experimentconstrainedbysamplegroups.(b)OTUsenrichedon
rootsofA.alpina,C.hirsutaandA.thaliana.Color-codedinblack species genetic diversity in A. alpina (7–10%)
areroot-enrichedOTUs(RootOTUs).AllenrichedOTUsarebased is remarkably similar to those reported for corn
onaBayesmoderatedt-test,Po0.05(FDR-corrected).Numberin (5–8%; Zea mays), barley (~6%; Hordeum vulgare)
brackets: total number of OTUs enriched in the respective plant and rice (3–5%; Oryza sativa), suggesting that host
species. (c) Pie chart reporting family distribution of 18 shared
OTUs based on parametric Tukey’s honest significant difference genotype determines a small, but significant
andBayesianandnon-parametricMann-Whitneystatistics. amount of microbiota variation across all tested
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