Table Of ContentTranscriptional programming during cell wall
maturation in the expanding Arabidopsis stem
Hall and Ellis
HallandEllisBMCPlantBiology2013,13:14
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HallandEllisBMCPlantBiology2013,13:14
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RESEARCH ARTICLE Open Access
Transcriptional programming during cell wall
maturation in the expanding Arabidopsis stem
Hardy Hall1,2 and Brian Ellis1*
Abstract
Background: Plant cell walls are complex dynamic structures that play a vital role incoordinatingthe directional
growth ofplant tissues. The rapid elongation of theinflorescence stem inthemodel plant Arabidopsis thaliana is
accompanied by radical changes in cell wall structure and chemistry, but analysis of the underlying mechanisms
and identification ofthegenes that are involved has been hampered by difficulties inaccuratelysampling discrete
developmental states along thedeveloping stem.
Results: Bycreating stem growth kinematic profiles for individual expanding Arabidopsis stems wehave been able
to harvest and pool developmentally-matched tissue samples, and to use thesefor comparative analysis of global
transcript profiles at four distinct phases of stemgrowth: theperiodof elongation rate increase, thepoint of
maximum growth rate, the point ofstem growth cessation and thefully matured stem. The resulting profiles
identify numerous genes whose expression is affected as the stemtissues pass through these defined growth
transitions, including both novel lociand genes identified in earlier studies. Of particular note is the preponderance
ofhighlyactive genes associated with secondarycell wall deposition inthe region ofstem growth cessation, and
ofgenes associated with defence and stress responses in the fully mature stem.
Conclusions: The use of growth kinematic profiling to create tissue samples that are accuratelypositioned along
theexpansiongrowthcontinuum of Arabidopsis inflorescence stems establishes a new standard for transcript
profiling analysesof such tissues. The resulting expression profiles identify a substantial number of genes whose
expression is correlated for thefirst time with rapid cell wall extension and subsequentfortification, and thus
provide animportant new resource for plant biologists interested in gene discovery related to plant biomass
accumulation.
Keywords: Cellwall,Anisotropy,Growthkinematicprofiling,Transcriptome,Microarray,Arabidopsis,Inflorescencestem
Background coordinationofcellwall building,reconstructionandforti-
Directional (anisotropic) cell wall expansion is an integral fication in order to harness the underlying driving force of
part of most plant developmental processes, where it turgor pressure in a spatially-defined manner (reviewed in
facilitates the structural changes necessary for proper cell [1,2]). Consistent with such developmental complexity, at
and organ morphogenesis. The initial expansive growth least one thousand genes in Arabidopsis have been shown
phase, which requires both addition of new extracellular to have some association with cell wall synthesis and
polymers and remodeling of existing components in the remodeling[3].
primarycellwalls,isoftensucceededbycellwallthickening The gene expression patterns associated with cell wall
and rigidification processes to create secondary cell walls expansion and/or secondary cell wall formation in
that enhance the structural integrity of the organ, but also Arabidopsishavebeenanalyzedinseveralstudiesinefforts
curtail further wall extension. These sequential processes toidentifyparticipatinggenesandunderstandthebiological
require a high degree of dynamic, context-specific roles of their products [4-8]. To specifically address cell
expansion processes, for example, transcript profiles have
*Correspondence:[email protected] been compared with protein accumulation profiles in
1MichaelSmithLaboratories,UniversityofBritishColumbia,VancouverBC Arabidopsisseedlinghypocotylsthatwereundergoingrapid
V6T1Z4,Canada
cell elongation without significant cell division [9].
Fulllistofauthorinformationisavailableattheendofthearticle
©2013HallandEllis;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Transcript profiling has also been conducted with in vitro stems as described in Hall and Ellis (2012) [15], and used
cultured Zinnia mesophyll cells [10,11] and Arabidopsis the resulting growth rate profiles to generate pooled
subcultured cells [12] that were induced to trans- samples of stem tissue that accurately represent four
differentiateintotrachearyelement-likecells,aprocessthat discrete growth stages along the cell wall expansion devel-
is accompanied by deposition of distinctive secondary cell opmentalcontinuum.OuruseofGKP-basedsamplingwas
wallthickeningsontopoftheoriginalprimarycellwalls.In expected to reduce the biological noise associated with
another approach, large-scale correlation analysis of public indirect sampling strategies used in previous studies, and
microarraydataenabledtheinsilicoidentificationofgenes thus increase the sensitivity (power to detect actual differ-
whoseexpressionisstronglyalignedwithexpressionofspe- ential expression) and accuracy of transcript profiling.
cific members of the Arabidopsis cellulose synthase (CesA) Microarray-based assessment of global transcript abun-
gene family that are believed to be predominantly involved dance in these GKP-matched stem samples then enabled
ineitherprimaryorsecondarycellwallbiogenesis[13]. us to generate transcriptomic datasets that can be
TheArabidopsisinflorescencestemrepresentsanattract- positioned withconfidencewithin avalidateddevelopmen-
ive experimental system for such gene discovery studies tal context of cell wall expansion performance (relative
since it provides more substantial amounts of tissue for elemental growth rates). The resulting gene expression
analyses, and its tissue architecture is largely established profiles demonstrate the participation of many genes that
prior to bolting, which means that stem expansion is pri- had earlier been linked to primary or secondary cell wall
marily the product of cell elongation, rather than division. synthesis, but they also highlight expression changes in a
However, the growing stem also presents a continuum of rangeofuniquegeneswhoserole(s)incellwallmaturation
developmentalstatesalongtheorganasitscomponentcells orstemexpansionhaveyettobeassessed.
transition from early anisotropic expansion growth to
growth cessation, and finally to cell wall fortification.
Integrated withthese changes in cell expansion activityare Results
additional changes associated with the differentiation and Stage-specifictranscriptomeanalysis
maturationofthediscretetissuesthatcomprisethestem. In order to position transcript profiles accurately within
In order to accurately monitor the gene expression the cell wall expansion continuum, we employed growth
changes, or transcriptionalprogramming, thataccompany kinematic profiling to establish relative elemental growth
these various stem growth transitions, it is essential to rates (REGRs) for contiguous stem segments harvested
sampleandpoolstemtissuesthatare verifiablyassociated from a series of individual plants [15]. These profiles
withspecificstagesofdevelopment.Anumberofinflores- allowed us to identify three developmental stages for
cence stem profiling studies have attempted to compare each plant being sampled: 1) an apical region where
the global transcriptional changes occurring between tissues begin to differentiate, and directional cell growth
specific developmental stages [4,5,7,14], but the experi- is initializing (termed ‘young’, or YNG), 2) a region
mental strategies employed have typically compared where directional growth of the stem is most rapid
tissues from visually selected regions of multiple plants, (termed 'maximum growth-rate’, or MGR), and 3) a
and have operated under two untested assumptions: 1) region where elongative growth is finishing (termed
that the pooled plants whose stems are being sampled all 'cessation', or CSS). These stages were therefore each
havesimilardevelopmentalproportioning, and2)thatthe represented by samples consisting of multiple pooled
samplingguidelinesfortheharvestedplants,derivedfrom segments, each of which had been harvested from a
destructive analysis of a different set of plants, accurately stem location centered upon a specific GKP-identified
associate features such as appearance of lignification in growth phase. The tissue selection protocol is outlined
the interfascicular fibres of the stem [4] with specific in Figure 1A-C, while the growth kinematic profiles for
developmental growth stages. Contrary to these assum- all the plants used in this study are provided in
ptions, growth kinematic profiling of expanding inflores- Additional file 1: Figure S1. To facilitate comparison of
cence stems of individual Arabidopsis plants has recently our data with the results of other transcriptome studies
demonstrated that stem growth profiles actually vary in inflorescence stems, we also harvested a segment
widely from plant to plant, even within genetically homo- (termed 'mature', or OLD) from the base of each of the
geneous populations [15]. As a consequence, data stems and pooled these for inclusion in our transcript
obtained from indirectly selected and harvested stem profilinganalysis.
regions are likely to be relatively poorly correlated with To maximize both statistical power and flexibility in
onsetofprocessesinvolvedincellwallextensionormodi- analysis,ourexperimentdirectlyco-hybridizedallpair-wise
ficationeventsassociatedwithspecificgrowthstages. combinations of developmental stage samples (‘complete
Inthisstudy,wehaveappliedgrowthkinematicprofiling factorial’ experimental design) and utilized a ‘mixed effects
(GKP) to a series of individual Arabidopsis inflorescence model’ analysis [16] to compare the four developmental
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A B B62R2 Plant 2 C
5
1
m) Apex
Growth rate (%change55443322.11.0.0.5.0.5..0.5.0.5.05050 1917503001000after tagging minutes) Distance from stem base (c510 11111112345678949123450rebmunttnemgeSSS YCMNSGGSR
/hr) Se2gme4nt n6umb8er 1fr0om1 2ap1e4x115300Time ( R0ela (t%iv0ec hGa2rnogw4et/hh r6R)a16te OLD
Figure1Representativegrowthprofilingandharvesting.A)Representativesurfaceplotofrelativeelongationgrowthrates(%changeper
hour,verticalaxis)plottedagainstthenumberofsegments(definedbyopticalmarkertags)fromtheapexdownwards,overthedurationofthe
imagingperiodin10minutesintervals.Thedarkergrey-shaded,nearestprofiledenotesthelast10-minuteintervalbeforeharvest,depictedinthe
greaterdetailintheright-handscatterplot.B)Correspondingscatterplotofgrowthrates(%changeinlengthperhour)againstdistancefrom
thestembaseforspecificsegments.Segmentsarenumberedfromthetopoftheplantdownwardsintheright-handmargin.TheLOWESS
regressioncurvefollowsthebestfitthroughthegrowthratedataforthisplantoveragiven10'interval.Greendottedlinesrepresented65%
confidenceintervalsfortheLOWESSregressioncurve.Closed-box/arrowindicatesthestempositionthatmatchesthemaximumgrowthrateof
theregressioncurve(segment5),plottedastheright-mostverticaldottedline,whiletheopen-box/arrowindicatesthefirstpositionbelowthe
topofthestemwherethegrowthratefallstozero(segment10).C)Harvestingzonesforyoung(YNG),maximumgrowth-rate(MGR),cessation
(CSS),andstembase(OLD)zonesbaseduponLOWESScurve.Seemethodsfordescriptionofzoneestablishment.SeeAdditionalfile1:FigureS1
forcompletesetof34growthkinematicprofiles.
stages on the basis of six biological replicates, each pooled predominantly populated by genes whose transcript
from a common set of thirty-four randomly-assigned abundancesarebeingmodulatedinaradicalfashionduring
plants. each associated growth transition. To look for potential
functional relationships among these short-listed, up- and
Examinationofdifferentialexpressionbetweenstages down-regulated genes, their annotations, gene ontology
The goal of this study was to identify genes whose expres- (GO)assignmentsandpossiblepromotermotifenrichment
sionintheinflorescencestemdiffersmoststronglybetween were examined. GO term enrichment was determined
different growth stages, since these are expected to repre- relative to whole-genome averages using the ‘AtCoeCis’
sent the loci most actively involved in the accompanying web-tool [17], which reports enrichment (fold-change),
transcriptional reprogramming. The mixed effects model- statistical significance (p-value), and the proportion of the
based analysis of this experiment generates six possible genes in each short-list that have been assigned that
pair-wise comparisons between the four stages, for which specific GO term ('score') (Additional file 5: Table S2,
the complete statistical analysis is presented in Additional Additionalfile6:TableS3,Additionalfile7:TableS4).
files2,3and4:TableS1. Toestablishthestatusofthestemtranscriptomepriorto
Fordetailedanalysis,wefocushereonthreegrowthstage the period at which the maximal elongation rate has been
comparisons (YNG-MGR, MGR-CSS, and CSS-OLD), and achieved, gene expression profiling was conducted on the
examinethearithmeticdifferencesbetweenthemean(log ) top1cmsegmentofthestem(withflowersremoved),and
2
signal intensities for each gene. Although as many as 4635 this YNG transcript profile was then compared to the
genes are differentially expressed (q-value<0.05) between profile generated from segments representing the GKP-
stages in these comparisons, we have restricted discussion identified maximum growth rate (MGR) phase. The forty
to the forty largest gene expression differences, which are genes exhibiting either high transcript copy number in the
presentedasconventionalfold-changeratios,togetherwith YNGstagesamplerelativetotheMGRstage(positivefold-
thelog ratiosfromwhichtheywerederived,andmeasures change values), or in theMGR stagesamplerelative to the
2
of false-discovery-corrected statistical significance of two- YNG stage (negative fold-change values) are listed in
samplet-testscores(q-values),fortheYNG/MGR(Tables1 Tables 1 and 2, respectively Among the most up-regulated
and2),MGR/CSS(Tables3and4),andCSS/OLD(Tables5 genes in the YNG-dominant set are two members of the
and 6) comparisons. Since we have applied stringent four-member ELONGATION FACTOR 1-ALPHA gene
filtering criteria (see Table legends), these lists should be family [18] (EF-1-α A2 (At1g07930) and EF-1-α A3
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Table1TwentymostdifferentiallyexpressedgeneswithhigherexpressioninYNGstagerelativetoMGRstage
Accession Geneannotation1 YNG/MGRFold-change2 q-value3
AT1G07930 elongationfactor1-alpha/EF-1-alpha 22.5 4.1E-02
AT5G25754 unknownprotein 19.2 4.7E-02
AT1G11520 spliceosomeassociatedprotein-related 18.9 3.4E-02
AT1G07940 elongationfactor1-alpha/EF-1-alpha 18.9 4.1E-02
AT5G22430 unknownprotein 17.6 2.2E-02
AT1G75240 ARABIDOPSISTHALIANAHOMEOBOXPROTEIN33(AtHB33) 16.2 1.4E-02
AT4G14080 MATERNALEFFECTEMBRYOARREST48(MEE48) 15.4 2.2E-02
AT1G01300 aspartylproteasefamilyproteinlocatedinmembrane,plant-typecellwall 14.6 4.0E-02
AT2G07739 unknownprotein 14.4 4.1E-02
AT3G13470 chaperonin,putativewithdomainCpn60/TCP-1(InterPro:IPR002423) 13.1 2.3E-02
AT4G34850 chalconeandstilbenesynthasefamilyproteininvolvedinphenylpropanoidbiosyntheticprocess 13.0 2.5E-02
AT3G17840 RECEPTOR-LIKEKINASE902(RLK902) 12.6 3.0E-02
AT1G52030 myrosinasebindingprotein,putative 12.2 2.2E-02
AT5G15720 GDSL-MOTIFLIPASE7(GLIP7) 11.6 3.7E-02
AT2G18020 EMBRYODEFECTIVE2296(EMB2296) 11.5 3.6E-02
AT5G62080 proteaseinhibitor/seedstorage/lipidtransferprotein(LTP)familyprotein 11.4 2.9E-02
AT2G01505 CLAVATA3/ESR-RELATED16(CLE16) 10.8 4.3E-02
AT3G45140 LIPOXYGENASE2(LOX2) 10.6 1.5E-02
AT1G62950 leucine-richrepeattransmembraneproteinkinase 10.4 2.7E-02
AT3G55210 ARABIDOPSISNACDOMAINCONTAININGPROTEIN63(anac063) 10.0 3.0E-02
AT4G04720 CPK21 9.9 4.2E-02
1GenedescriptionsareabbreviatedfromTAIR10genomerelease.Putativefunctionsarestatedinlowercase.
2Genesrankedaccordingtofold-changevaluesderivedfromlog2ratiosofYNG(numerator)andMGR(denominator).
3Derivedfromfalsediscovery-ratecorrectionofp-values.
(At1g07940)).Thisassociationwithactiveproteinsynthesis regulated in stems in response to mechanical load [24].
isalsoreflectedintheGOtermenrichmentanalysisofthis TheperoxidasegenefamilyinArabidopsisislarge,andits
set (Additional file 5: Table S2), which indicates >80-fold membersplayanumberofrolesincellularmetabolism,
enrichment in GO terms containing ‘translation’. Also including modulation of reactive oxygen species accu-
found within this ‘YNG-up-regulated’ list are genes related mulation [25] and the oxidative coupling of aromatic
to signaling (RLK902 [19]; CLE16(CLAVATA3 homologue) metabolites such as the monolignols that serve as
[20]; LOX2 [21]; At1g62950, a LRR protein kinase), as well precursors for the lignin polymer [26,27]. The expres-
as transcription factors (ZF-HD class AtHB33; NAC063). sion of PER64 in Arabidopsis has been shown to be
Cell-cell communication mediated by peptides derived concentrated in the protoxylem [28], where lignifica-
from CLE gene products, acting together with cognate tion of patterned secondary cell wall thickenings
receptor kinases, represents part of the elaborate signaling contributes to cell wall stabilization during vascular
network that helps guide plant development [22]. While elongation, a spatial specificity that is consistent with
known cell wall-associated genes are not notably thestrongPER64expressioninMGRtissues.
over-represented within the ‘YNG-up-regulated’ list, The MGR up-regulated list also contains several genes
one gene encoding a putative glucan endo-β(1→3)- more directly related to primary cell wall formation and
glucosidase (At4g14080) [23] is up-regulated 15-fold re-modeling,includingaxyloglucanendotransglycosylase/
overtheMGRstage. hydrolase MERISTEM-5 (MERI5B/XTH24), a putative
Although cell wall expansion is expected to be taking pectinase (At1g80170), two arabinogalactan proteins
place in both theYNG and the MGR stage tissues, genes (AGP12, AGP13) and a MYB transcription factor
whoseproductsareuniquelyrequiredforrapidexpansion (MYB61) that has recently been shown to contribute
should be relatively more highly expressed in the latter. to both cell wall synthesis and regulation of plant
The most strongly differentially up-regulated (~40-fold) carbon allocation [29-32]. In addition, several genes
gene in the MGR tissues relative to YNG is a peroxidase encoding proteins associated with phytohormone sig-
(PER64) that has been previously reported to be up- nalling are more highly expressed in the MGR tissues,
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Table2TwentymostdifferentiallyexpressedgeneswithhigherexpressioninMGRstagerelativetoYNGstage
Accession Geneannotation1 YNG/MGRFold-change2 q-value3
AT3G13520 ARABINOGALACTANPROTEIN12(AGP12) −6.8 3.1E-02
AT1G80170 putativepolygalacturonase(pectinase) −7.3 1.4E-02
AT1G09540 MYBDOMAINPROTEIN61(MYB61) −7.4 2.6E-02
AT3G05880 RARE-COLD-INDUCIBLE2A(RCI2A) −7.5 1.3E-02
AT1G77330 similarto1-aminocyclopropane-1-carboxylateoxidase −7.5 1.9E-02
AT1G72430 AuxinresponsiveSAURprotein −8.0 3.2E-02
AT4G23496 SPIRAL1-LIKE5(SP1L5) −8.1 4.7E-02
AT4G03205 SOULheme-bindingfamilyprotein −8.1 1.9E-02
AT1G67865 unknownprotein −8.2 3.4E-02
AT4G26320 ARABINOGALACTANPROTEIN13(AGP13) −8.8 1.9E-02
AT3G19710 BRANCHED-CHAINAMINOTRANSFERASE4(BCAT4) −9.6 1.5E-02
AT5G48560 basichelix-loop-helix(bHLH)familyprotein −9.9 4.5E-02
AT3G55240 OverexpressionleadstoPEL(Pseudo-EtiolationinLight)phenotype −10.0 1.5E-02
AT1G74670 putativegibberellin-responsiveprotein(GASA6) −10.2 3.3E-02
AT4G30270 MERISTEM-5(MERI5B) −11.5 1.6E-02
AT1G74660 MINIZINCFINGER1(MIF1) −12.0 4.9E-03
AT4G29905 unknownprotein −12.9 4.3E-02
AT3G45160 unknownprotein −15.3 1.1E-02
AT5G05960 proteaseinhibitor/seedstorage/lipidtransferprotein(LTP)familyprotein −24.2 1.5E-02
AT5G42180 peroxidase64(PER64)locatedinplant-typecellwall −40.0 9.4E-04
1GenedescriptionsareabbreviatedfromTAIR10genomerelease.Putativefunctionsarestatedinlowercase.
2Genesrankedaccordingtofold-changevaluesderivedfromlog2ratiosofYNG(numerator)andMGR(denominator).
3Derivedfromfalsediscovery-ratecorrectionofp-values.
including a putative ACC oxidase, the GA-responsive The ‘Higher in CSS relative to MGR’gene list (Table 4),
MINI ZINC FINGER 1 (MIF1) [33] and another gene on the other hand, would be expected to capture
GASA6(At1g74670)reportedtobeGA-responsive[34]. those genes that make a major contribution to the
Comparison of the MGR stage gene expression patterns re-programming associated with transition to a phase of
to those observed at the more mature CSS stage provides cellwallstabilizationandrigidification.Consistentwiththis
anotherviewofthosegenesthataremostrelevanttoactive prediction, this list is dominated by genes associated with
stem expansion, by contrasting their performance in the formationofnon-expandingwalls,includingallthreeofthe
rapidly expanding MGR tissues with that seen in the CSS cellulosesynthasegenesbelievedtobeinvolvedincellulose
tissues where cell wall expansion has ceased. Interestingly, microfibril deposition during secondary cell wall biosyn-
the list of twenty genes whose expression is ‘Higher in thesis (CESA4/IRX5, CESA7/IRX3, and CESA8/IRX1)
MGR relative to CSS’ (Table 3) is led, notby genes known [14,35-37],andCHITINASE-LIKEPROTEIN2(CTL2)[38]
to be associated with cell wall synthesis or modification, whose loss-of-function mutant displays cellulose biosyn-
butbyMAJORLATEXPROTEIN423(MLP423),amember thesis defects [39]. Also strongly represented are genes
of the BET V1 class of allergens that exhibits sequence required for xylan biosynthesis/modification, including a
homology to ABA- and stress-responsive proteins from UDP-GLUCURONIC ACID DECARBOXYLASE 3/UXS3
various plant species (EMBL-EBI database information). [40] that provides UDP-xylose for xylan backbone synthe-
MLP423 is accompanied by two members of the large sis, IRREGULAR XYLEM 9 (IRX9) [41] and FRAGILE
(108-member) GDSL-type lipase homologue gene family, FIBER8(FRA8)[42]whoseencodedproteinsbuildandex-
and by other genes associated with lipid metabolism/ tendtheglucuronosylxylanpolymer,andtwoxylanmodifi-
transport, but few, if any, genes known to be directly cation genes: a xyloglucan-specific endotransglycosylase/
involved in cell wall synthesis are included. This profile hydrolase 19 (XTH19) [43], and REDUCED WALL
implies that the genes populating the ‘Higher in MGR ACETYLATION 1 (RWA1) [44]. Other cell wall modifica-
relativeto CSS’ listareprimarilythosewhoseexpressionis tion genes are present, including two pectinesterases
relativelystronglyreducedasthecellsmaketheirtransition (At2g43050, At2g45220), one of which is the most strongly
fromrapidanisotropicexpansiontomaturation. differentially-expressed gene in the list. The prominence of
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Table3TwentymostdifferentiallyexpressedgeneswithhigherexpressioninMGRstagerelativetoCSSstage
Accession Geneannotation1 MGR/CSSFold-change2 q-value3
AT1G24020 MLP-LIKEPROTEIN423(MLP423) 16.7 2.9E-03
AT5G33370 GDSL-likelipase 5.7 2.3E-02
AT2G02320 PHLOEMPROTEIN2-B7(AtPP2-B7) 5.2 6.6E-03
AT2G38540 LIPIDTRANSFERPROTEIN1(LP1) 5.2 5.0E-05
AT5G24780 VEGETATIVESTORAGEPROTEIN1(VSP1) 4.8 3.6E-03
AT2G02850 PLANTACYANIN(ARPN) 3.9 2.4E-03
AT2G33810 SQUAMOSAPROMOTERBINDINGPROTEIN-LIKE3(SPL3) 3.8 4.2E-02
AT3G04290 LI-TOLERANTLIPASE1(LTL1) 3.7 2.4E-03
AT1G55490 chloroplast60kDachaperoninbetasubunit 3.6 9.1E-04
AT5G20630 GERMIN3(GER3) 3.5 3.8E-04
AT2G39670 radicalSAMdomain-containingprotein 3.5 2.1E-02
AT3G47650 bundle-sheathdefectiveprotein2family/bsd2family 3.4 3.2E-02
AT5G15230 GAST1PROTEINHOMOLOG4(GASA4) 3.4 3.1E-04
AT3G47340 GLUTAMINE-DEPENDENTASPARAGINESYNTHETASE(ASN1) 3.2 1.3E-02
AT5G20720 CHAPERONIN20(CPN20) 3.2 7.6E-03
AT5G55450 proteaseinhibitor/lipidtransferprotein(LTP)familyprotein 3.2 3.6E-03
AT5G61170 40SribosomalproteinS19(RPS19C) 3.1 4.3E-02
AT3G08740 elongationfactorP(EF-P)familyprotein 3.1 2.0E-02
AT3G21410 F-boxfamilyprotein(FBW1) 3.1 2.6E-02
AT2G02130 LOW-MWCYSTEINE-RICH68(LCR68)(PDF2.3) 3.0 3.5E-02
1GenedescriptionsareabbreviatedfromTAIR10genomerelease.Putativefunctionsarestatedinlowercase.
2Genesrankedaccordingtofold-changevaluesderivedfromlog2ratiosofMGR(numerator)andCSS(denominator).
3Derivedfromfalsediscovery-ratecorrectionofp-values.
these pectin de-methylating enzymes in the MGR→CSS (growth kinematic profiling can only distinguish stem
transition list is consistent with a current model for plant regions on the basis of their rates of expansion), it is clear
cell wall rigidification in which a reduction in the levels of from previous microscopic analysis [4,51] that the OLD
pectin methylesterification leads to enhanced calcium ion stage tissue displays an advanced phase of organ growth
cross-linkingandwallstiffening[45-48]. and cell wall maturation in the 10-15 cm tall Columbia
In addition to genes whose encoded products affect plants examined in this study. Based on our present
cell wall polysaccharide biosynthesis, the list includes understandingofthestemmaturationprocess,theCSSand
IRREGULAR XYLEM 12 (IRX12/LAC4). Laccases are OLD samples are expected to contain tissues actively
thought to contribute to polymerization of lignin in engaged in earlier and later stages of secondary cell wall
secondarywalls,andLAC4expressionhaspreviouslybeen formation and reinforcement, respectively. Tables 5 and 6
shown tobe specific toxylaryandinterfascicularfibres in present the twenty genes whose expression is ‘Higher in
the Arabidopsis stem. Lignin deposition is largely CSS relative to OLD’ and the twenty genes whose
unaffected in the lac4 loss-of-function mutant, but is expressionis‘HigherinOLDrelativetoCSS,’respectively.
stronglyreducedinthelac4/lac17doubleloss-of-function Displaying high expression in the CSS relative to OLD
mutant [49]. It is noteworthy that we observed no signifi- samples are GERMIN-LIKE PROTEIN 3 (GER3/GLP3)
cant difference in expression of LAC17 between the CSS (At5g20630) and GERMIN-LIKE PROTEIN 1 (GER1/
andMGRstages(1.3-folddifferential,CSS/MGR).Overall, GLP1) (At1g72610). GER3 also appeared in the list of
nine of the twenty genes featured in this list also occur genes more highly expressed in MGR tissues than in CSS
among a set of ‘xylem-specific’Arabidopsis genes identi- (Table3),indicatingthatexpressionofthismemberofthe
fied through analysis of public datasets [50], consistent GER gene family follows a steeply declining trajectory
with a metabolic commitment in CSS tissues to cell wall during the stem maturation process. While specific
rigidification in xylem fibres and tracheary elements once developmental roles for GLP1 and 3 have yet to be iden-
stemexpansionceases. tified, GER proteins are apoplastic glycoproteins that
While growth kinematic data cannot precisely position have been widely associated with plant disease resistance
the base of the stem along the developmental continuum and ROS modulation, particularly in the cereals [52].
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Table4TwentymostdifferentiallyexpressedgeneswithhigherexpressioninCSSstagerelativetoMGRstage
Accession Geneannotation1 MGR/CSSFold-change2 q-value3
AT5G25110 CBL-INTERACTINGPROTEINKINASE25(CIPK25)(SnRK3.25) −5.1 1.8E-02
AT2G43050 pectinmethylesterase −5.5 2.8E-03
AT4G30290 XYLOGLUCANENDOTRANSGLUCOSYLASE(XTH19) −5.5 2.8E-04
AT5G59290 UDP-glucuronicaciddecarboxylase3(UXS3) −5.6 5.8E-04
AT2G38080 LACCASE4(IRX12) −6.0 4.1E-04
AT2G37090 IRREGULARXYLEM9(IRX9) −6.1 4.3E-04
AT5G46340 REDUCEDWALLACETYLATION1(RWA1) −6.1 3.0E-03
AT1G03740 S/Tproteinkinase −6.1 7.8E-06
AT5G01360 TRICHOMEBIREFRINGENCE-LIKE3(TBL3) −6.9 2.3E-03
AT2G28315 Nucleotide/sugartransporterfamilyprotein −7.3 4.0E-03
AT1G22480 plastocyanin-likedomain-containingprotein −7.8 3.6E-04
AT5G17420 CESA7(IRX3) −8.8 1.6E-04
AT3G18660 glucuronicacidsubstitutionofxylan1(GUX1) −9.0 5.7E-04
AT4G18780 CESA8(IRX1) −9.5 9.4E-04
AT3G16920 CHITINASE-LIKEPROTEIN2(CTL2) −10.1 1.3E-05
AT2G28110 FRAGILEFIBER8(FRA8) −10.3 4.8E-04
AT2G03200 aspartylproteasefamilyprotein −10.4 9.6E-05
AT1G63910 AtMYB103 −11.5 2.3E-03
AT5G44030 CESA4(IRX5) −11.5 5.6E-05
AT2G45220 pectinmethylesterase −38.4 1.8E-05
1GenedescriptionsareabbreviatedfromTAIR10genomerelease.Putativefunctionsarestatedinlowercase.
2Genesrankedaccordingtofold-changevaluesderivedfromlog2ratiosofMGR(numerator)andCSS(denominator).
3Derivedfromfalsediscovery-ratecorrectionofp-values.
Interestingly, another Arabidopsis GER homologue distributed within the eukaryotes [56]. Since both CSS and
(GLP10, At3G62020) whose expression was previously OLD tissues were harvested only seconds apart, an
found to be highly correlated with secondary cell wall- artifactual pattern of wounding-induced gene induction is
associated CESAs (CesA4, 7 and 8) in regression analysis not likely. Instead, it appears that accumulation of the
of public microarray datasets [13], also displayed elevated products of such classical “defense” genes may form an
expression at both the CSS and OLD stages in our study integral part of the normal maturation of the inflorescence
(Additionalfiles2,3and4:TableS1). stem, perhaps reflecting a commitment to protection of
Also more highly expressed at this earlier stage of these tissues until fertilization and seed dispersal are
cell wall maturation are two pectate lyases (polyga- successfullycompleted.
lacturonases), At3g07010 and At3g15720, previously Relatively few cell wall-specific genes appear in the
associated with cell separation [53], and ALPHA- ‘higherinOLDthaninCSS’shortlist,withtheexceptionof
XYLOSIDASE 1/AXY3 (At1g68560), an exoglycosylase EXTENSIN 3/RSH and another proline-rich extensin-like
that acts specifically on non-fucosylated xyloglucans [54] family protein. EXT3/RSH plays an essential role in cell
and is essential for apoplastic xyloglucan modification wall deposition through formation of EXTENSIN protein
[55]. Several other up-regulated genes are less clearly scaffoldsthatcross-linkothercellwallconstituents,thereby
linked to cell wall processes, but the functions of contributing to cell wall rigidification [57,58]. The most
their encoded proteins may be related to the over- up-regulated of all the genes at the OLD stage relative to
representation of ‘turgor pressure’ in the GO term the CSS stage is the chloroplast-localized FATTY ACID
enrichment analysis for this gene set (Additional file 7: REDUCTASE6(FAR6).AsimilarpatternofelevatedFAR6
TableS4). expression was earlier observed in microarray analysis of
The list of genes most highly expressed in OLD tissues epidermal peelsfrom thestem base[59] aswell asinstem
relative to CSS tissues (Table 6) is particularly striking: six sections harvested from the base of mature Arabidopsis
oftheeightmosthighlyup-regulatedgenesencodePLANT Col-0 plants [6]. Accompanying FAR6 in this list of most
DEFENSIN (PDF) proteins, small cysteine-rich peptides highly expressed genes is a wax synthase homologue
homologous to anti-microbial peptides that are widely (At5g22490), a co-occurrence pattern consistent with
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Table5TwentymostdifferentiallyexpressedgeneswithhigherexpressioninCSSstagerelativetoOLDstage
Accession Geneannotation1 CSS/OLDFold-change2 q-value3
AT1G12845 unknownprotein 10.1 4.1E-02
AT5G20630 GERMIN3(GER3) 6.5 1.6E-02
AT3G07010 pectatelyasefamilyprotein 6.3 7.8E-03
AT1G72610 GERMIN-LIKEPROTEIN1(GER1) 6.2 4.0E-02
AT1G64660 ARABIDOPSISTHALIANAMETHIONINEGAMMA-LYASE(ATMGL) 5.7 9.6E-03
AT3G15720 glycosidehydrolasefamily28protein/polygalacturonase(pectinase)familyprotein 5.3 3.5E-02
AT1G80280 hydrolase,alpha/beta-foldfamilyprotein 5.3 3.4E-02
AT1G68600 unknownprotein 5.0 4.3E-02
AT5G38430 ribulosebisphosphatecarboxylasesmallchain1B/RuBisCOsmallsubunit1B(RBCS-1B)(ATS1B) 4.8 4.3E-02
AT2G39010 PLASMAMEMBRANEINTRINSICPROTEIN2E(PIP2E) 4.8 2.2E-02
AT2G38540 LIPIDTRANSFERPROTEIN1(LTP1) 4.7 2.8E-02
AT3G16240 DELTATONOPLASTINTEGRALPROTEIN(DELTA-TIP) 4.7 2.2E-02
AT4G03205 coproporphyrinogenoxidaseactivityinporphyrinbiosyntheticprocesswithinchloroplast 4.4 3.9E-02
AT3G48970 copper-bindingfamilyproteininmetaliontransport 4.3 4.9E-02
AT1G75900 familyIIextracellularlipase3(EXL3),carboxylesteraseactivity,acyltransferaseactivity 4.3 1.5E-02
AT2G05790 glycosylhydrolasefamily17protein 4.2 7.8E-03
AT5G38420 ribulosebisphosphatecarboxylasesmallchain2B/RuBisCOsmallsubunit2B(RBCS-2B)(ATS2B) 4.2 4.1E-02
AT1G68560 ALPHA-XYLOSIDASE1(XYL1) 4.2 4.1E-02
AT5G22580 unknownprotein 4.2 2.8E-02
AT3G12610 DNA-DAMAGEREPAIR/TOLERATION100(DRT100) 4.1 2.2E-02
1GenedescriptionsareabbreviatedfromTAIR10genomerelease.Putativefunctionsarestatedinlowercase.
2Genesrankedaccordingtofold-changevaluesderivedfromlog2ratiosofCSS(numerator)andOLD(denominator).
3Derivedfromfalsediscovery-ratecorrectionofp-values.
epidermal cells in fully mature stems actively synthesizing abundance at one stage relative to a hypothetical mean
both their cuticle polyester network and the associated wax value of zero across the entire experiment. It should be
matrix. The modest representation of explicitly cell wall- notedthatthese'estimates'canbecomputedwiththesame
associated genes in this CSS-to-OLD transition list implies statisticalpowerasappliestothelog differentialexpression
2
thattheCSSandOLDstagetissuessharequitesimilartran- ratios reported in Tables 1, 2, 3, 4, 5, 6. This treatment
scriptionalprofilesintermsofsecondarycellwallformation providesamoreintuitivemeansof visualizinggeneexpres-
processes, and that the metabolic commitment to cell wall sion trajectories, and provides the basis for formal
fortification in stem tissues does not change dramatically co-expression analysis. The genes associated with each co-
aftercessationofactiveelongation. expression set (cluster) are identified in a filterable column
within the full-genome dataset (Additional files 2, 3 and 4:
Stage-specific,whole-genomeco-expressionanalysis Table S1), and their AGI codes are also listed separately in
While differential gene expression datasets contrasting Additionalfile8:TableS5foreasieraccess.
discrete growth stages provide initial insights into the Hierarchical divisive clustering was performed on the
biology underlying specific developmental transitions, 4635 genes whose means were most significantly different
potential functional relationships between gene products from the gene-wise mean of all stages (q-value <0.05)
can also be revealed by considering transcript abundances (Figure 2), thereby filtering out the great majority (22 294)
across all the sampled developmental stages. The under- of genes whose expression displayed little perceptible
lying rationale is that genes co-expressed at one stage and change during the course of stem elongation. The 4635
exhibiting similar association patterns across a broader genes fall within eight major co-expression clusters that
developmental range may represent a subset of genes exhibitdistinctdevelopmentaltrajectories.Withtheexcep-
involvedinspecificbiologicalprocesses. tionofcluster 2(Figure2),which exhibitselevated expres-
The ‘mixed effects model’ approach used in this study sion at only the YNG and OLD stages, genes within the
allowedustogeneratedevelopmentalstage'estimates'from majorclustersexhibitasinglepeakintranscriptabundance
two-channel arrays, which can be expressed as mean fold- associated with a discrete developmental stage, accompan-
change values (biological replicates = 6) of transcript ied by lower expression in all the sets before and/or after
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Table6TwentymostdifferentiallyexpressedgeneswithhigherexpressioninOLDstagerelativetoCSSstage
Accession Geneannotation1 CSS/OLDFold-change2 q-value3
AT1G21310 EXTENSIN3(ATEXT3) −3.9 4.8E-02
AT5G09840 unknownprotein −4.1 3.7E-02
AT3G54580 proline-richextensin-likefamilyprotein,structuralconstituentofcellwall −5.0 4.8E-02
AT5G54230 MYBDOMAINPROTEIN49(MYB49) −5.0 4.1E-02
AT2G28780 unknownprotein −5.0 2.9E-02
AT1G70830 BetvIallergenfamily −5.2 3.9E-02
AT2G02930 GLUTATHIONES-TRANSFERASEF3(ATGSTF3) −5.4 3.0E-02
AT4G15390 acyl-transferasefamilyprotein −6.1 1.6E-02
AT4G08780 peroxidase,putative −8.2 4.5E-02
AT1G02930 GLUTATHIONES-TRANSFERASE6(GSTF6) −8.6 4.3E-02
AT2G36120 DEFECTIVELYORGANIZEDTRIBUTARIES1(DOT1) −8.8 3.5E-02
AT1G19530 unknownprotein −9.7 3.1E-02
AT1G75830 LOW-MOLECULAR-WEIGHTCYSTEINE-RICH67(LCR67) −9.9 1.5E-02
AT2G26020 PLANTDEFENSIN1.2B(PDF1.2b) −10.7 4.3E-02
AT5G22490 Waxestersynthasehomologue −11.5 4.5E-02
AT5G44420 PLANTDEFENSIN1.2(PDF1.2) −13.1 3.9E-02
AT2G26010 PLANTDEFENSIN1.3(PDF1.3) −16.2 4.4E-02
AT4G16260 O-glycosylhydrolase −17.9 4.9E-02
AT5G44430 PLANTDEFENSIN1.2C(PDF1.2c) −19.9 3.6E-02
AT3G56700 FATTYACIDREDUCTASE6(FAR6) −55.4 7.8E-03
1GenedescriptionsareabbreviatedfromTAIR10genomerelease.Putativefunctionsarestatedinlowercase.
2Genesrankedaccordingtofold-changevaluesderivedfromlog2ratiosofCSS(numerator)andOLD(denominator).
3Derivedfromfalsediscovery-ratecorrectionofp-values.
this peak. Each co-expression set (cluster) thus appears Discussion
to have a uniquely defined developmental window in Whilegeneexpressionprofilinghasbeenappliedpreviously
which the associated genes act, and the clusters are to the expanding inflorescence stem in Arabidopsis, those
engaged sequentially during elongation and maturation studies have all suffered from various limitations that
of the inflorescence stem. It is interesting that while impede our ability to accurately align the resulting expres-
most clusters (1–7) contain at least 400+ genes, Cluster sion profiles with the developmental state of the tissue
8 (out-group of Clusters 5–7), which contains genes being sampled. To address this issue, we have made use of
that are up-regulated only at the base of the stem growth kinematic profiling (GKP) to establish, for each
(OLD),islimitedto16genes. plant being sampled, the precise state of growth extension
To test for functional relatedness of the genes popu- that each stem section represents. Collectively, these
lating these clusters, we examined gene ontology en- sectionsspanthegrowthandmaturationstatesofthestem,
richment within several sub-clusters that exhibited andtheyalsorepresentacellwalldevelopmentcontinuum.
marked patterns of coordinated up- or down-regulation Thus, GKP-guided pooling of sections associated with
specific to single developmental stages (Figure 2; sub- discrete zones along that continuum (e.g. the point
clusters 1.1, 2.1, 5.1, 5.2, and Cluster 8). Although box- at which extension growth ceases) makes it possible to
plotting of the mean 'estimates' for these sub-clusters generatetissuesampleswhosegeneexpressionprofilescan
clearly demonstrates the extent to which these co- beconfidentlyalignedwithspecificdevelopmentalstates.
expression sets are synchronously up- or down-regulated Whilethegrowingstemisacomplexorganconsistingof
with respect to the other stages (Additional file 9: Figure multiple tissues, the common denominator across all of
S2),geneontologyanalysisrevealedonlymodestGOterm these cell types is the coordinatedandinitiallyrapidaniso-
enrichment within these subsets of co-expressed genes tropicexpansionoftheircellwallsalongtheaxisofgrowth.
(Additionalfile10:FigureS3,Additionalfile11:TableS6), This expansion ultimately comes to a halt as the walls of
indicating that, despite their shared expression pattern, some tissues, most notably the vascular tissues and
thegenesineachsub-clusterdonotdisplayobviousfunc- supporting fibres, are reinforced with non-extensible
tionalrelatedness. secondary wall layers. Thus, while the biological processes
