Transcriptomics of plant responses to apical damage reveals no negative correlation between ...

14 Pages·2015·0.61 MB·English

by  Daniel R. Scholes

PlantEcol
DOI10.1007/s11258-015-0500-x
Transcriptomics of plant responses to apical damage reveals
no negative correlation between tolerance and defense
.
Daniel R. Scholes Ken N. Paige
Received:30April2015/Accepted:8July2015
(cid:2)SpringerScience+BusinessMediaDordrecht2015
Abstract While one may expect the loss of plant totalgeneexpressionviaRNA-sequencingofundam-
tissue by animal herbivores to be universally detri- aged and mechanically damaged plants of three
mental to a plant’s fitness, a wide range of tolerance Arabidopsis thaliana genotypes that differ in their
responsesexists,includingundercompensation(lower compensatory performances: Columbia-4, Landsberg
fitnesswhendamaged),equalcompensation,andeven erecta, and a recombinant inbred line (RIL) from a
overcompensation(increased fitnesswhendamaged). Columbia-4 9 Landsberg erecta cross. Among the
Theory predicts that these responses could be con- many genetic pathways that responded to clipping,
strained by the investment into defensive chemicals Columbia-4significantlyup-regulatedgenesinvolved
and structures produced for improving resistance in insecondary defense chemistryand equallycompen-
damaged tissues, and thus tolerance and induced sated for fitness while Landsberg erecta and the RIL
defense could be considered alternative strategies to both undercompensated and significantly down-regu-
the selection pressure imposed by herbivory. To lated secondary defense pathways. The genotypes’
determine which genetic pathways underlie differ- different compensatory performances are thus posi-
ences in compensatory (i.e., growth and fitness) tively correlated with their differential investments
responses to damage by tissue loss, and to test their intosecondarymetabolismfollowingtissueloss.This
relation with pathways involved in defense, we studyidentifiesdifferentialpost-damagegeneregula-
performed a controlled greenhouse study to measure tionofgrowth,developmentalsignaling,andenviron-
mentalresponsepathwaysinA.thaliana,andprovides
the first transcriptomic evidence counter to the
CommunicatedbySantiagoC.Gonzalez-Martinez. presumed tradeoff between tolerance and defense in
plant-herbivoreinteractions.
Electronicsupplementarymaterial Theonlineversionof
thisarticle(doi:10.1007/s11258-015-0500-x)containssupple-
mentarymaterial,whichisavailabletoauthorizedusers. Keywords Herbivory(cid:2)Resistance(cid:2)Compensation(cid:2)
Chemistry(cid:2)Glucosinolate(cid:2)RNA-seq
D.R.Scholes(&)
DepartmentofBiology,UniversityofIndianapolis,1400
E.HannaAve.,Indianapolis,IN46227,USA
e-mail:[email protected] Introduction
D.R.Scholes(cid:2)K.N.Paige A broad, consequential issue in the ecology and
SchoolofIntegrativeBiology,UniversityofIllinoisat
evolution of plant-animal interactions regards the
Urbana–Champaign,515MorrillHall,505S.Goodwin
Ave.,Urbana,IL61801,USA ways in which plants respond to herbivore-induced
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damage.Plantsexhibitawidevarietyofresponsesto toleranceordefenseduetothenecessityofallocating
damage by animal herbivores, including defense limited resources (Coley et al. 1985; Mauricio et al.
strategies that aim to prevent damage, and tolerance 1997;WiseandAbrahamson2005,2007).Forexam-
strategiesthataimtopreventthelossofreproductive ple, fitness following apical damage of Solidago
outputuponsustainingdamage(RosenthalandKota- altissima decreased nearly tenfold when plants were
nen1994;Stoweetal.2000;Agrawal2011;notethat nutrient-stressed versus fertilized, a result consistent
while we use these terms in accordance with their with the limiting resources model of tolerance (Wise
historicalusage,‘‘resistance’’and‘‘defense’’areoften andAbrahamson2008).Theproductionofsecondary
usedconverselytotheirpresentationhere(Straussand metabolites may also be resource-limited, where the
Agrawal1999)).Defensivetraitsoftenentailprevent- constitutivelevelandinducibilityofsuchdefensesare
ingdamageviastructuralimpedimentsorbydecreas- negativelyimpactedbylownutrientavailability(e.g.,
ingthepalatabilityoftheplanttissue,andmayinclude Sampedro et al. 2011). There is also theoretical and
the production of thorns, trichomes, lignin, latex, empirical evidence of a negative genetic correlation
numerous classes of secondary metabolites, or the between tolerance and defense strategies (van der
sequestrationofsilicon,asexamples(Levin1973;Ma Meijden et al. 1988; Fineblum and Rausher 1995;
2004;Mitho¨ferandBoland2012).Thesetraitsmaybe Stowe 1998). Specifically, van der Meijden
producedconstitutively, often asdefensive structures et al. (1988) argued that because defended plants
and baseline levels of secondary compounds, or they were likely to experience a lower frequency and
may be induced upon the detection of a potential intensity of herbivore attack, selection for tolerance
herbivore or after sustaining damage (Kaplan et al. ability would be reduced relative to less defended
2008). Defense traits may also be general or specific plants. Conversely, tolerant plants would not neces-
withregardtotheherbivoreagainstwhichtheyact— sarily face selective pressure to invest in defense if
for example, there is substantial evidence for coevo- theydonotincurfitnesslossesfromherbivory(vander
lutionbetweenplantsandtheirspecializedherbivores Meijden et al. 1988). The dynamics of the tolerance/
for defense compound toxicity and detoxification, defense tradeoff have been theorized from varied
respectively, inanumber ofsystems(Fraenkel 1959; perspectives(Stamp2003),butultimatelyunderthese
EhrlichandRaven1964;Berenbaum1983). circumstances tolerance and defense are considered
Tolerancereferstoaplant’sabilitytomaintainfitness redundantresistancestrategiessuchthattheywouldbe
despite sustainingdamage. Tolerancemechanismsare mutuallyexclusiveifeithertraitreceivedahighlevel
generallytheorizedtoincludethereallocationofstored ofinvestment(vanderMeijdenetal. 1988).
resourcestoreplacethetissuesremoved,increasedstem Despitethetheoreticaltradeoffbetweentoleranceand
productionand/orbranchingfromthehormonalrelease defense strategies, recent research into the genetic
of apical dominance, and increases in photosynthetic underpinnings of tolerance provides evidence that
output and growth rates following damage, among toleranceanddefensemaynotbealternativestrategies
others(StraussandAgrawal1999).Precisemechanisms after all. For example, the pentose phosphate pathway
underlying tolerance are not fully understood and are (PPP)isametabolicpathwaythatprovidesfive-carbon
likely variable among populations, species, types of sugarsandNADPHforgeneralizedbiosynthesisinthe
damage, and environments (Maschinskiand Whitham Calvin cycle (Kruger and von Schaewen 2003), but it
1989;StraussandAgrawal1999).Toleranceoutcomes alsoprovidestherawmaterialsforsecondarymetabolite
range from undercompensation (reduced fitness when production via the shikimate pathway (Maeda and
damaged), equal compensation (i.e., complete toler- Dudareva2012).Siddappajietal.(2013)demonstrated
ance), and overcompensation (increased fitness when that the A. thaliana genotype Columbia-4 had higher
damaged), with compensatory abilities varying across expressionofGLUCOSE-6-PHOSPHATEDEHYDRO-
genotypes and populations of a wide range of species GENASE 1 (G6PD1), which encodes the enzyme that
(Rosenthal and Kotanen 1994; Strauss and Agrawal catalyzesthefirstandrate-limitingstepofthePPP,when
1999;Stoweetal.2000;Tiffin2000). damaged compared to when undamaged and that it
Toleranceanddefensestrategieshavetraditionally overcompensatedforfruitproduction.Landsbergerecta,
been considered alternative forms of resistance to incontrast,hadreducedG6PD1expressionandunder-
herbivory with plants primarily exhibiting either compensated(Siddappajietal.2013).Theexperimental
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knockoutandcomplementationofthisgeneconfirmed positively corresponds with the expression of sec-
G6PD1’s, and therefore the PPP’s, influence in the ondary defense metabolic pathways in these geno-
compensatory performance of these genotypes. This types, providing transcriptomic evidence that
influence may arise in part via endoreduplication, the toleranceanddefensemetabolismarenotnecessarily
replication of the genome without mitosis such that mutuallyexclusivestrategiesandthattheymayinfact
cellular genome copy number increases with each beemployedconcurrentlyfollowingdamage.
replication. This process is assumed to play important
rolesinplantdevelopment,growth,fitness,andgeneral
stress tolerance through its effects on cell size, Materialsandmethods
metabolism, and gene expression (Sugimoto-Shirasu
andRoberts2003;Leeetal.2009;Breueretal.2014;for A.thalianagenotypes,growth,andexperimental
reviewseeScholesandPaige2015).AlongwiththePPP, clipping
Columbia-4isknowntoincreaseitsendoreduplication
during regrowth following damage while Landsberg Columbia-4(Col-4;TAIRstocknumber:CS933;The
erecta has no such increase (Scholes andPaige2011). Arabidopsis Information Resource 2014), Landsberg
Further, experimentally increasing the endopolyploidy erecta (Ler-0; TAIR stock number: CS20), and one
ofanA.thalianagenotypethattypicallyundercompen- RILfromtheircrossing(TAIRstocknumber:CS1936)
sates allows it to completely mitigate the otherwise wereselectedforthisstudybasedontheirpreviously
detrimentaleffectsofdamageonitsfitness(Scholesand demonstratedfitnesscompensatoryresponses(Scholes
Paige 2014). Since the PPP also produces DNA andPaige2011;Siddappajietal.2013).Allgenotypes
nucleotides, the ability of a genotype to increase its wereinbredaminimumofeightgenerationstoachieve
cellularploidyduringregrowthislikelyconstrainedby fullhomozygosity(ListerandDean1993).
itsabilitytoincreasethePPP(KrugerandvonSchaewen Sixty-fiveindividualsofeachgenotype(Col-4,Ler-
2003).Giventheseknownrolesingeneralizedprimary 0,CS1936)weregrown,witharandomizeddistribution,
metabolism, endoreduplication, and the production of inagreenhouseatapproximately21 (cid:3)Candona12 h
secondarydefensemetabolites,thePPPmay,therefore, light/darkcycle.Whenboltinginflorescencesreached6
notonlyunderlietheabilitiesoftheseplantstotolerate cm in height, the inflorescences of 35 plants of each
apical damage but also provide a mechanism for the genotype were clipped with scissors, leaving 1 cm of
concurrentproductionofdefensivecompounds. inflorescencetissueashasbeenperformedinprevious
Inthisstudy,weprovideatestofthehypothesisthat studieswiththesegenotypes(ScholesandPaige2011).
tolerance and defense strategies to herbivory are not This clipping regimen is comparable to natural apical
necessarily in opposition, but rather are positively damage of A. thaliana observed throughout its native
correlated via the co-regulation of their genetic range,stimulatesasimilarchangeinarchitecturaltraits
pathways. Specifically, using three genotypes of following damage, and approximately coincides with
A. thaliana that vary in their degree of tolerance, we A.thaliana’smaximumrateofinflorescenceelongation
initially ask whether a correlation exists between the (Scholes et al. unpublished). Thirty of the remaining
response in expression of genes in the PPP, the plantsofeachgenotyperemainedunclippedascontrols.
endocycle, and secondary metabolite biosynthesis Fiveoftheclippedplantsofeachgenotypewereutilized
following damage. We additionally ask whether the for transcriptomicanalysis (see ‘‘Transcriptomicanal-
expressionofgenesinthosepathwaysalsocorrelates ysis’’sectionfordetails).Rosette diameteratthetime
withmeasuresoffitnesscompensationandendopoly- theinflorescencereached6cminheightwasrecordedas
ploidyfollowingdamage.Toaddressthesequestions, ameasureofplantsizeatthisstage.
we performed quantitative transcriptome sequencing
of A. thaliana Columbia-4, Landsberg erecta, and a Phenotypicanalysis
Columbia-4 9 Landsberg erecta recombinant inbred
line(RIL)todeterminewhetherthegeneticexpression Measurementofendopolyploidy
oftoleranceanddefensecorrelatewiththeircompen-
satory performances following damage. Our results Attheinductionofsenescence,allinflorescencetissue
demonstrate that compensatory performance of 20 plants (10 unclipped, 10 clipped) of each
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genotype was analyzed for nuclear DNA content via plant’s rosette diameter at an inflorescence height of
flow cytometry. Tissue for cytometric analysis was 6 cm(tocontrolfordifferencesinplantsize)andthe
chopped with a razor blade, sheared in a nuclear number of days from planting to an inflorescence
isolation buffer (sodium citrate, 3-morpholino- heightof6 cm(tocontrolfordifferencesindevelop-
propane-1-sulfonic acid, magnesium chloride, Triton mentaltiming)wereusedascovariatestostandardize
X-100; Galbraith et al. 1983), filtered for debris between genotype 9 treatment groups. Counts of
removal, and stained with the DNA fluorophore silique yield, seed yield, and biomass were also
propidiumiodide.Isolatednucleiwerethenmeasured square-root transformed to approximate normality to
forDNAcontentviaaBDBiosciences(SanJose,CA, satisfy the assumptions of ANOVA. Linear contrasts
USA) FACScanto flow cytometer. Nuclei population were used to test for differences between unclipped
gating was performed using De Novo Software FCS andclippedplantsofeachgenotypeforeachmeasure.
Express(v.3;LosAngeles,CA,USA)tomeasurethe
number of nuclei at each ploidy level (2C, 4C, 8C, Transcriptomicanalysis
16C) for each plant sample. The cycle value, inter-
preted as the average number of endocycles that a Samplepreparation,libraryconstruction,
nucleus in the sample has undergone and thus an andsequencing
overall measure of endoreduplication (Barow and
Meister 2003), was calculated by the following Five of the clipped plants of each genotype were
equation: randomly selected for transcriptomic analysis, with
the clipped inflorescence tissue collected for RNA-
Cyclevalue¼ð0(cid:3)n þ1(cid:3)n þ2(cid:3)n
2C 4C 8C sequencing.Uponthegenerationofaxillarystems,the
þ3(cid:3)n Þ=ðn þn þn þn Þ
16C 2C 4C 8C 16C firstregrownaxillaryinflorescencetoreachaheightof
6 cm was likewise collected from each plant for
where the cycle value is the sum of the number of
transcriptomics. Initial and lateral stem tissues col-
nucleiateachploidylevelmultipliedbythenumberof
lected for transcriptomics (i.e., stems collected at the
endocycles required to achieve each corresponding
timeofandafterclipping,respectively)werecompa-
ploidy level, divided by the total number of nuclei
rable in mass, chronological age, and developmental
measured.
stage,allowingforthecomparisonofgeneexpression
beforeversusafterclippingforindividualplantswhile
Measurementoffitness
controlling for differences between individuals. Tis-
sues for transcriptomic analysis were flash-frozen in
At the completion of senescence, 40 plants of each
liquid nitrogen immediately upon clipping from the
genotype(20unclipped,20clipped)wereanalyzedfor
plant.TotalRNAwasextractedfromeachsamplevia
fitness. Inflorescence dry biomass and silique yield
an RNeasy Plant Mini Kit (Qiagen; Venlo, Nether-
were measured for each plant. Seed yield for each
lands). Qualityofextracted RNA wasassessed onan
plant was estimated by multiplying the number of
Agilent2100Bioanalyzer(AgilentTechnologies,Inc.,
siliquesbytheaveragenumberofseedspersiliquefor
SantaClara,CA,USA;softwarev.1.3)withtheassay
the respective genotype 9 treatment group (calcu-
class designated as Plant RNA Nano. RNA integrity
lated by counting the number of seeds for five
valuesaveraged9.0(range7.7–10.0),25S/18SrRNA
representativesiliquesfromeachplant).
ratios averaged 1.9 (range 1.3–2.7), and adequate
quality was confirmed upon visual inspection of
Statisticalanalysisofphenotypicmeasures electrophoretic images. cDNA libraries were con-
structed by the High-Throughput Sequencing and
Analysesforphenotypicmeasureswereconductedas GenotypingUnit,RoyJ.CarverBiotechnologyCenter
general linear models with SAS (v.9.3; Cary, NC, of the University of Illinois at Urbana–Champaign
USA).Genotypeandtreatment(clipping)weretested (Urbana, IL, USA), using a TruSeq Stranded Total
as fixed effects via ANOVA for silique yield, seed RNA Sample Prep Kit (Illumina; San Diego, CA,
yield,inflorescencedrybiomass,andcyclevalues.For USA). All libraries were quantified by qPCR and
siliqueyield, seedyield,andbiomassmeasures,each sequenced on three lanes (one for each genotype,
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comprising ten multiplexed samples per lane), dis- the false discovery rate (FDR) at 5% in edgeR based
tributed randomly within a single flow cell, for 101 on the total number of genes analyzed for the
cycleseachonaHiSeq2000sequencerusingaTruSeq respectivegenotype,generatingFDR-correctedpval-
SBS Sequencing Kit (v.2) and CASAVA software ues(i.e.,qvalues;BenjaminiandHochberg1995).
(v.1.8.2;Illumina;SanDiego,CA,USA).Onaverage,
20,717,090readsweregeneratedpersample. Genome-wideGOtermenrichment,GOterm
redundancyreduction,andtargetedGOtermanalysis
Readmapping,countnormalization,
andidentificationofdifferentiallyexpressedgenes Gene ontology (GO) analysis was performed to
determine pathway-level patterns of gene expression
Reads were trimmed to remove adapter sequences, enrichment among the genes deemed significantly
sequences with low quality scores (limit = 0.05, differentiallyexpressed(i.e.,q\0.05)relativetothe
Phred scores [20; Ewing and Green 1998), and entiresetofanalyzedgenes.GOtermsaredescriptors
sequenceswithmultipleambiguousbases(maximum of genes based on the cellular component, molecular
two ambiguous nucleotides per read) using CLC function, and biological process for which they are
Genomics Workbench (v.6.5; CLC bio; Aarhus, associated,andareoftendeterminedbyexperimental
Denmark). After these quality control measures, an demonstration or by sequence similarity to genes of
averageof19,303,545readspersampleweremapped known function. GO terms may therefore be used to
to the TAIR10 Arabidopsis thaliana annotated refer- inferthefunctionsofgenesofinterest(e.g.,differen-
ence genome of 28,642 genes (The Arabidopsis tially expressed genes) or for targeted analysis of
InformationResource2014). geneswithspecificfunctions(e.g.,todeterminewhich
Aftermapping,geneswithfewerthanfiveuniquely genes within a pathway of interest are differentially
mappedreadspertreatmentonaveragewereremoved expressed, and/or if the pathway as a whole is
from the analysis given the increased measurement differentiallyregulated).Geneidentitiesandqvalues
error typically associated with low read counts wereimportedintoGOrilla(Edenetal.2009),which
(Robinson and Smyth 2007). An average of approx- analyzesauser-supplied‘‘target’’genelistforsignif-
imately21,707genesremainedpersample.Statistical icant GO term enrichment relative to a user-supplied
analysis for differential gene expression was per- ‘‘background’’ gene list. The significantly up-regu-
formed using the edgeR (Robinson et al. 2010) latedgenesandthesignificantlydown-regulatedgenes
package in R (i386 of v.3.0.3; R Core Team 2014). were analyzed separately for each genotype. GO
The numbers of trimmed reads mapped uniquely to analyses were performed with Arabidopsis thaliana
each gene were normalized via the edgeR function selected as the experimental organism, with two
calcNormFactorstocontrolfordifferencesinsequenc- unrankedlistsofgenes(target:eitherup-regulatedor
ing depth among samples (Robinson and Oshlack down-regulated genes; background: all genes ana-
2010). Normalized read counts of each gene were lyzed for the respective genotype), for the biological
compared between treatments (before clipping, after process ontology, and with a p value threshold for
clipping) via a generalized linear model (GLM; significant GO term enrichment of 10-3. Briefly, for
McCarthy et al. 2012), with samples coming from each GO term for which A. thaliana genes are
the same individual paired to control for individual associated (2340 GO terms at the time of analysis),
variation. Model dispersion was estimated for each GOrilla uses the total number of genes in the user-
geneseparatelyviathefunctionestimateTagwiseDisp suppliedbackgroundgenelist(N),thetotalnumberof
in edgeR (Robinson and Smyth 2007, 2008). Genes genes associated with the GO term (B), the total
whose normalized read counts differed significantly number of genes in the user-supplied target gene list
before vs. after clipping were identified using GLM (n), and the number of genes in the target gene list
likelihood ratio tests via the function glmLRT in associated with the GO term (b) to calculate an
edgeR (Lund et al. 2012). Genotypes were analyzed enrichmentscore[definedas(b/n)/(B/N)];Edenetal.
independently with no direct comparisons made 2009). GOrilla then uses the hypergeometric method
among them. p values resulting from the GLM to calculate a p value from the enrichment score and
likelihood ratio tests were then corrected to control produces an FDR-corrected p value (i.e., q value) to
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adjust for the number of GO terms in the analysis due to their previously reported involvement in
(Benjamini and Hochberg 1995; Eden et al. 2009). tolerance (Scholes and Paige 2011, 2014; Siddappaji
Genes ineither user-supplied gene list notassociated et al.2013).To assess the effects ofthetreatment on
with a GO term were omitted from the analyses specificGOtermsofinterestselectedapriori,listsof
(amongthethreegenotypes,13,261genesonaverage A.thalianageneswithdirectandindirectassociations
wereanalyzedforGOenrichment).AcrossallsixGO with each a priori GO term were obtained using the
enrichment analyses (up- and down-regulated genes AmiGO 2 (http://amigo2.geneontology.org) Grebe
analyzed separately for three genotypes), an average search wizard.Foreachgeneassociatedwithagiven
of19.7GOtermsweredeemedsignificantlyenriched GO term ofinterest, the p value calculated by edgeR
(Supplementarymaterials1,2,3). wascorrectedforafalsediscoveryrateof5 %based
GO terms deemed significantly enriched (i.e., on the total number of genes associated with the GO
q\0.05) were imported with their q values into termthatremained afterthequalitycontrolmeasures
REViGOtoreducetheredundancyoftheGOtermlist describedabove(BenjaminiandHochberg1995).An
(Supeketal.2011).REViGOusesqvalues,semantic exact binomial test was then performed in R via the
similarity, and GO term parent-child relationships to binom.testfunctiontodetermineifthenumberofup-
calculatea‘‘dispensability’’score(rangingfrom0to1 regulatedgenesdifferedsignificantlyfromthenumber
with greater values indicating greater dispensability) ofdown-regulatedgenesforeachGOterm.
foreachGOtermandproducesashortenedGOterm
list based on a user-indicated threshold (Supek et al.
2011). Analyses in REViGO were performed with a Results
dispensabilitythreshold(i.e.,Cvalue)of0.5,imported
q values for each user-supplied GO term, the Ara- FitnessandnuclearDNAcontent
bidopsis thaliana database selected to provide GO
term relationships and sizes, and SimRel as the LandsbergerectaandtheCol-4 9 Ler-0RILCS1936
semanticsimilaritymeasure(Supeketal.2011).After bothsignificantly undercompensatedforsiliqueyield
redundancy reduction, an average of 5.8 GO terms [t(67) = 2.88, p\0.01, and t(67) = 2.52, p\0.05,
remained across the six analyses (Supplementary respectively;Fig. 1].CS1936alsosignificantlyunder-
materials1,2,3). compensated for seed yield [t(67) = 3.08, p\0.01),
Giventhenatureofourclippingtreatmentandour while Ler-0 nearly undercompensated for seed yield
interest in the integration of the PPP, secondary [t(67) = 1.93, p = 0.058; Fig. 1). Columbia-4
defense chemistry, and endoreduplication following equally compensated for both silique and seed yield
plantdamage,sevenGOtermswereselectedapriori [t(67) = 0.41, p = 0.682, and t(67) = 0.16,
(Table 1).Theseincludeoverallsecondarymetabolite p = 0.874, respectively; Fig. 1]. There were no dif-
biosynthesis as well as the biosynthesis of specific ferences between unclipped and clipped plants in
classes of secondary compounds of A. thaliana inflorescence dry biomass for any genotype [Col-4:
(Table 1; Kliebenstein 2004). We acknowledge that t(67) = 0.30, p = 0.762; Ler-0: t(67) = 0.95,
the classes of secondary compounds assessed may p = 0.348; CS1936: t(67) = 1.20, p = 0.234;
compriseavarietyofcompoundsub-classes(e.g.,sub- Fig. 1], though the change in their means closely
classes of phenylpropanoids include coumarins, fla- approximates the silique and seed compensatory
vonoids, lignins, etc., which may or may not be performanceforeachgenotype(Fig. 1).
specifically involved in herbivore defense; Solecka Although cycle values of unclipped and clipped
1997);however,limitationsintheavailabilityofsub- plantsdidnotdiffersignificantlyforanygenotype,the
class GO terms, the annotation of genes to these GO changes in direction of their means did correspond
terms, and ambiguity in the involvement of specific roughlytotheirfitnesscompensatoryresponseswitha
compounds in herbivore defense prevent the assess- trendtowarddecreasedendopolyploidyforLer-0and
ment of more finely defined compound sub-classes. CS1936 [t(39) = 0.66, p = 0.514, and t(39) = 0.25,
The‘‘pentosephosphateshunt’’and‘‘DNAendoredu- p = 0.807,respectively]andatrendtowardincreased
plication’’ GO terms were also selected for analysis ploidyforCol-4[t(39) = 1.67,p = 0.103;Fig. 1].
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Table1 Geneontologytermsofinterestselectedapriori
ID Term #Genes Col-4 Ler-0 CS1936
GO:0044550 Secondarymetabolitebiosyntheticprocess 310 56 11 51
GO:0016114 Terpenoidbiosyntheticprocess 261 6 1 2
GO:0019761 Glucosinolatebiosyntheticprocess 169 19 10 21
GO:0009699 Phenylpropanoidbiosyntheticprocess 135 40 1 30
GO:0052315 Phytoalexinbiosyntheticprocess 11 0 0 2
GO:0006098 Pentosephosphateshunt 182 0 2 2
GO:0042023 DNAendoreduplication 111 0 0 1
GeneontologyIDs,terms,andnumbersofArabidopsisthalianagenesannotatedtoeachGOtermwerecompiledfromtheAmiGO2
geneontologybrowser(http://www.geneontology.org).Duetodatatrimmingforquality,thenumberofgenesannotatedtoeachGO
term isnot necessarily equal to thenumber of genes that constituted the family for which thefalse discovery rate was corrected.
ValuesintheCol-4,Ler-0,andCS1936columnsarethenumbersofdifferentiallyexpressedgenesforeachrespectivegenotypeafter
falsediscoveryrate(FDR)correction(q\0.05).SeeSupplementarymaterials1,2,and3forfullresults
terms). Upon clipping, 302 genes were significantly
10 up-regulated in Col-4 while 663 were significantly
%) down-regulated. When considering Col-4 up-regu-
e ( 0 lated genes,21GOtermsweresignificantly enriched
r
u (Fig. 2;Supplementarymaterial1).Uponredundancy
s
a -10
e reduction, the three remaining GO terms were all
m
n involved in cell wall biosynthesis (Fig. 2; Supple-
ge i -20 mentary material 4). The 30 GO terms significantly
n
ha -30 # * enriched with down-regulated Col-4 genes were
C
generallyinvolvedinpollentubegrowthandcellwall
* *
-40 modification(Supplementarymaterial1).Afterredun-
Col-4 Ler-0 CS1936 dancy reduction, eight GO terms remained (Fig. 2;
Supplementarymaterial4).
Fig.1 Percent change in phenotypic measures of clipped
Only four GO terms were significantly enriched
Columbia-4, Landsberg erecta, and CS1936 plants relative to
unclipped controls. Asterisks(*) significant(a=0.05)differ- with Ler-0’s 38 up-regulated genes, with three
ences between unclipped and clipped plants for the given remainingafterredundancy reduction (Fig. 2).These
genotype and measure; #p=0.0584. Light gray shading
termswereallrelatedtotheresponsetovariousstimuli
silique yield, black shading seed yield, white shading mass,
(e.g.,chitin,nitrogen,fungi,oxygen-containingcom-
darkgrayshadingcyclevalue
pound; Supplementary materials 2, 4). Although 27
Geneexpression genesweresignificantlydown-regulated,noGOterms
were significantly enriched with Ler-0 down-regu-
Differentialgeneexpressionandgenome-wideGO latedgenes(Fig. 2;Supplementarymaterial4).
termenrichment The Col-4 9 Ler-0 RIL CS1936 exhibited 124
significantlyup-regulatedgenesand148significantly
For those genes differentially expressed between down-regulatedgenes.CS1936demonstratedavaried
samples before and after clipping, we conducted a transcriptomic response to the clipping treatment
geneontology(GO)analysisthatidentifiesbiological relative to its parents, with 25 and 38 GO terms
processes (i.e., GO terms) for which the genes are significantly enriched with up- and down-regulated
associated. Here we describe broad themes from our genes, respectively, relative to the genomic back-
gene regulation and biological process analyses (see ground (Fig. 2; Supplementary material 3). Upon
Supplementary materials 1, 2, and 3 for detailed redundancyreductionofup-regulatedGOterms,only
analyses and full lists of significant genes and GO fiveremainedandcollectivelywereassociatedwitha
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a b with no significant changes in the regulation of the
othermetabolitegroups(Fig. 3a).TheCol-4 9 Ler-0
RIL CS1936 displayed intermediate secondary
metabolite regulation relative to its parents, with
significant down-regulation of glucosinolates, signif-
icant up-regulation of phenylpropanoids, and no sig-
nificant changes in the regulation of biosynthesis of
phytoalexins, terpenoids, or secondary metabolites
overall(Fig. 3a).Nogenotypehadsignificantchanges
in the regulation of the pentose phosphate shunt or
Fig.2 The number of gene ontology terms significantly endoreduplicationuponclipping(Fig. 3b).
enriched with a up- and b down-regulated genes for
Columbia-4,Landsbergerecta,andCS1936followingclipping
inthegenome-wideanalysis.Boldvaluesindicatethenumbers
Discussion
ofsignificantlyenrichedGOtermsidentifiedbyGOrillaintotal;
normal-weight values indicate the numbers of significant GO
terms following redundancy reduction by REViGO. See Our results yield insight into the transcriptional
‘‘Materialsandmethods’’sectionfordetails.Valuesinregions responseofplants toapicaldamage, usingthe model
ofoverlapindicatethenumbersofGOtermsincommonamong
species Arabidopsis thaliana. We demonstrate that
genotypes. Values are additive for each genotype (e.g., there
were 21 GO terms significantly enriched with up-regulated Columbia-4,Landsbergerecta,andtheRILgenerated
genesforCol-4intotal,withthreeremainingafterredundancy from a cross between these two accessions differ in
reduction). See Supplementary material 4 fortheidentitiesof theirregulationofgenesassociatedwiththeresponse
thesesignificantGOterms
tostimuli,cellwallbiosynthesis,andavarietyofother
widevarietyofprocesses,includingxylanbiosynthe- processes. The most striking results, however, are
sis, transition metal ion transport, and cellular differences between the genotypes intheir regulation
responsetoironionstarvation(Fig. 2;Supplementary of secondary defensive chemistry that are positively
material 4). Of the 38 down-regulated GO terms, 16 correlated with their compensatory performances. In
remained after redundancy reduction, with many the context of these three genotypes, our results
involved in growth and developmental signaling indicatethattoleranceanddefensearenotnecessarily
(e.g., ethylene biosynthesis and phosphorelay signal alternative responses to the removal of apical domi-
transduction) and the response to the environment nance since one genotype appeared to employ both
(including ‘‘response to wounding,’’ ‘‘response to strategieswhiletheothertwoemployedneither.While
mechanical stimulus,’’ ‘‘response to external stimu- thegeneralitiesofourresultsremainunassessedacross
lus,’’etc.;Fig. 2;Supplementarymaterial4). populations and species, our methods were sufficient
to produce robust, reliable transcriptomic data that
DifferentialregulationofGOtermsofaprioriinterest provide the basis for conclusions for these three
commonly studied genotypes that could be more
GO terms selected a priori generally fall into two thoroughly investigated in future genetic and natural
groups:thebiosynthesisofsecondarymetabolites(i.e., fieldstudies.Together,thisstudycontributesthefirst
chemicaldefenses;Fig. 3a)andprocessespredictedto transcriptomic data counter to the presumed tradeoff
influence compensatory performance (the pentose between tolerance and defense, as well as transcrip-
phosphate shunt and endoreduplication; Table 1; tomic characterization of the plant regrowth process
Fig. 3b). Overall, Columbia-4 significantly up-regu- broadly,addinganewperspective tothisconsequen-
latedsecondarymetabolitebiosynthesiswhileLands- tialissueinplantdamagedynamics.
berg erecta significantly down-regulated this process The A. thaliana genotype Columbia-4 experienced
(Fig. 3a). Specifically, Columbia-4 significantly up- no significant change in attributes of fitness when
regulated the biosynthesis of glucosinolates and damaged relative to when undamaged (i.e., it equally
phenylpropanoids with no significant changes in the compensated). Col-4’s complete tolerance of damage
regulation of phytoalexins or terpenoids. Landsberg correspondedwiththesignificantup-regulationofgenes
erecta significantly down-regulated glucosinolates involvedinsecondarymetabolism,andparticularlythe
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PlantEcol
a Secondary metabolite Terpenoid biosynthetic Glucosinolate Phenylpropanoid
biosynthetic process process biosynthetic process biosynthetic process
3.0
14
20 * * *
m
er 2.5 12 * 30
t
O
G *
n 15 2.0 10
s i
ne 8 20
e 1.5
g 10 *
f 6
o
e 1.0
g
a 4 10
t
n 5 *
e
c 0.5
r 2
e
P
0 0.0 0 0
C ol-4LerC-0S 1936 C ol-4LerC-0S 1936 C ol-4LerC-0S 1936 C ol-4LerC-0S 1936
Phytoalexin b Pentose phosphate DNA
biosynthetic process shunt endoreduplication
30 1.2 1.0
m m
O ter 25 O ter 1.0 0.8
G G
n 20 n 0.8
s i s i 0.6
e e
n n
e 15 e 0.6
g g
of of 0.4
e 10 e 0.4
g g
a a
nt nt
e e 0.2
c 5 c 0.2
r r
e e
P P
0 0.0 0.0
C ol-4LerC-0S 1936 C ol-4LerC-0S 1936 C ol-4LerC-0S 1936
Fig.3 Differential gene regulation of a priori gene ontology regulated genes in the GO term differs significantly (exact
termsofinterest.Geneontologytermsincludeathebiosynthesis binomialtest,p\0.05;seeSupplementarymaterials1,2,and3
ofsecondarymetabolitesandbthepentosephosphateshuntand for full results). Black shading up-regulated, gray shading
endoreduplication. Asterisks percentage of up- and down- down-regulated
production of phenylpropanoids and glucosinolates. chemistry responses relative to its parents, including
Landsbergerecta,whichsufferedreducedfitnesswhen significant down-regulation of glucosinolates, the pri-
damaged relative to when undamaged (i.e., it under- mary anti-herbivore compounds produced by
compensated),displayedsignificantdown-regulationof A.thaliana(Kliebenstein2004;D’AuriaandGershen-
generalized secondary chemistry and glucosinolate zon2005;TzinandGalili2010).
biosynthesis. The Col-4 9 Ler-0 RIL CS1936 also Theory and historical perspective often consider
undercompensated and displayed a mix of defensive tolerance and defense to be alternative strategies to
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PlantEcol
damage based on the necessity of allocating limited non-defended tissues are preferred by their animal
resourcesandapresumednegativegeneticcorrelation herbivores(Coley1983;Berenbaumetal.1986;Steele
between the strategies. Across the literature, there is et al. 1993; Newman et al. 1996; Shroff et al. 2008;
no conclusive evidence for the generality of this Stowe 2013). This trait prevents damage to valuable
tradeoff, however, and in fact there is growing structures,andisthusaformof‘‘defense,’’yetitalso
evidencesupportingtheco-occurrenceofthesestrate- allows plants of these species to tolerate damage of
gies. For example, classical resource availability other structures since there is no evident cost to
theory predicts that limited resources would promote reproduction(Stowe2013).
theinvestmentofaplantintoanti-herbivoredefenses Becausethecorrelationbetweencompensationand
given the plant’s likely impaired growth rate, and the regulation of secondary chemistry is present at
subsequentlyitsimpairedabilitytorapidlyregrowand both extremes in this study (i.e., we observed a
maintainfitnessfollowingdamage(Coleyetal.1985). genotype that invested in both, as well as genotypes
However, mathematical models suggest that plant thatinvestedinneither,atleastintermsoftheprimary
tolerancecouldactuallybemaximizedwhenresources anti-herbivore compounds; Kliebenstein 2004;
are limiting since growth rates are reduced for D’Auria and Gershenzon 2005; Tzin and Galili
undamaged plants as well, allowing damaged plants 2010), a genetic pathway that influences both could
with multiple stems to match undamaged plants in beunderlyingtheresistancestrategiesoftheseplants.
total biomass and fitness production (Hillbert et al. Ourpriorstudieshave indicated thattheinduction of
1981;AlwardandJoern1993).Clearlytheorydoesnot the pentose phosphate pathway (PPP) and endoredu-
provideadequategeneralizationsfortheemployment plicationfollowingapicaldamageisimportantforthe
of tolerance versus defense strategies, and empirical compensatoryperformanceofA.thalianaplants,with
evidence often supports mixed-strategy hypotheses. consistent differences in the responses of these
Specifically, numerous studies have discovered that pathways between genotypes that differ in their
genotypesofmanyspeciesgenerallydisplayamixof compensation(ScholesandPaige2011,2014;Scholes
resistance strategies and that these strategies are thus et al. 2013; Siddappaji et al. 2013). Although we did
notaproductofdiversifying selection (i.e.,agenetic not observe a concurrent regulatory response of the
tradeoff between strategies; Leimu and Koricheva PPP or endoreduplication, this does not necessarily
2006;Nu´n˜ez-Farfa´netal.2007;CarmonaandFornini indicate that these processes are not integrated with
2013; Turley et al. 2013). For example, studies of secondarymetabolism,aswetheorize.Becausewedid
tolerance (measures of fitness) versus defense (tri- not observe overcompensation in Columbia-4, nor
chome and glucosinolate production) in A. thaliana significant changes in ploidy (though trends were in
revealed that natural populations exhibited mixed the expected directions) as in our previous studies
resistance strategies due to selection for the mainte- (Scholes and Paige 2011, 2014; Scholes et al. 2013;
nance of both traits, and thus no negative genetic Siddappaji et al. 2013), a primary constraint may be
correlation exists for these plants (Mauricio et al. variation in the year-to-year environment to which
1997). In fact, mixed resistance strategies may be correlations in pathway regulation might have been
expected when considering the concurrent selection weak or absent. Further, previous studies have
pressures applied by a diverse herbivore community observed a delay in the induction of the PPP and
and/or a genetically diverse plant population, as are endoreduplicationrangingfromafewtoseveraldays
commonly present in natural systems (Fornoni et al. after clipping (Scholes and Paige 2011; Siddappaji
2004;Agrawal2011;CarmonaandFornini2013).The et al. 2013), yet these pathways clearly influence
growing number of similar examples has even fitness compensation as measured at senescence.
spawned a revision of the semantics surrounding the While we used all available data to design our
discussion of plant resistance strategies, with Stowe experimental protocol to maximize our detection of
(2013)expressingthatthedistinctionbetween defen- differential gene regulation, it is also possible that
siveandtolerancetraitsisnotusefulinpracticedueto effectsofclippingongeneregulationcouldhavebeen
their often interdependent phenotypic expression and missed if the timing of sampling did not correspond
evolution.Forexample,manyspeciesallocatedefen- with differential expression. There thus remains a
sivechemicalstovaluabletissues,likeseeds,suchthat theoretical framework by which the PPP supports
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