Pearce, Simon and Ferguson, Alison and King, John and Wilson, Zoe A.

16 Pages·2017·2.29 MB·English

by  Christine Middleton

Pearce, Simon and Ferguson, Alison and King, John
and Wilson, Zoe A. (2015) FlowerNet: a gene
expression correlation metwork for anther and pollen
development. Plant Physiology, 167 (4). pp. 1717-1730.
ISSN 0032-0889
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FlowerNet: A Gene Expression Correlation Network for
1[OPEN]
Anther and Pollen Development
Simon Pearce2, Alison Ferguson2, John King, and Zoe A. Wilson*
Division of Plant Crop Sciences (S.P., A.F., Z.A.W.) and Centre for Plant Integrative Biology (S.P., J.K.,
Z.A.W.), School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough,
Leicstershire LE12 5RD, United Kingdom; and School of Mathematical Sciences, University of Nottingham,
Nottingham NG7 2RD, United Kingdom (S.P., J.K.)
Floralformation,inparticularantherandpollendevelopment,isacomplexbiologicalprocesswithcriticalimportanceforseed
setandfortargetedplantbreeding.Manykeytranscriptionfactorsregulatingthisprocesshavebeenidentified;however,their
direct role remains largely unknown. Using publicly available gene expression data from Arabidopsis (Arabidopsis thaliana),
focusing on those studies that analyze stamen-, pollen-, or flower-specific expression, we generated a network model of the
globaltranscriptionalinteractions(FlowerNet).FlowerNethighlightsclustersofgenesthataretranscriptionallycoregulatedand
therefore likely to have interacting roles. Focusing on four clusters, and using a number of data sets not included in the
generation of FlowerNet, we show that there is a close correlation in how the genes are expressed across a variety of
conditions, including male-sterile mutants. This highlights the important role that FlowerNet can play in identifying new
players in anther and pollen development. However, due to the use of general floral expression data in FlowerNet, it also
hasbroadapplicationinthecharacterizationofgenesassociatedwithallaspectsoffloraldevelopmentandreproduction.Toaid
the dissection of genes of interest, we have made FlowerNet available as a community resource (http://www.cpib.ac.uk/
anther). For this resource, we also have generated plots showing anther/flower expression from a variety of experiments:
Theseare normalizedtogetherwherepossibletoallowfurther dissectionoftheresource.
Anther and pollen development is a complex biolog- aspects of anther development have been discovered.
ical process that is indispensable for the generation of APETALA3 (AP3; Jack et al., 1994), SPOROCYTELESS/
male gametophytes and the production of the next gen- NOZZLE (SPL/NZZ; Liu et al., 2009), and EXCESS
eration in flowering plants. This development includes a MICROSPOROCYTES1 (Zhao et al., 2002) have impor-
series of crucial events that require interactions between tant roles in early anther development and differentia-
gametophytic and sporophytic genes in a cooperative tion, while ABORTED MICROSPORE (AMS; Sorensen
fashion (Goldberg et al., 1993; Ma, 2005). The mature et al., 2003; Xu et al., 2010), DEFECTIVE IN TAPETAL
anthercontainsfourlobes,eachofwhichcontainsmeiotic DEVELOPMENT AND FUNCTION1 (Zhu et al., 2008),
cells surrounded by four somatic cell layers, with the DYSFUNCTIONAL TAPETUM1 (DYT1; Zhang et al.,
sporophytictapetumplayinganimportantroleinpollen 2006), MALE STERILITY1 (MS1; Wilson et al., 2001;
grain development (Goldberg et al., 1993). The tapetum Alves-Ferreira et al., 2007; Ito et al., 2007; Yang et al.,
serves as a nutritive tissue, providing metabolites, nu- 2007a), MYB33/MYB65 (Millar and Gubler, 2005), and
trients, and cell wall precursors for the development of MYB80/MYB103 (Higginson et al., 2003; Zhang et al.,
pollen grains. 2007; Zhu et al., 2010) are involved in tapetum and
The use of classical genetic screens has uncovered a pollen wall development (Wilson and Zhang, 2009).
largenumberofgenesinvolvedinantherdevelopment MYB26(Steiner-Langeetal.,2003;Yangetal.,2007b)
andintheproductionofviablepollen.Forexample,in and RECEPTOR-LIKE PROTEIN KINASE2 (RPK2;
Arabidopsis (Arabidopsis thaliana), a number of genes Mizuno et al., 2007a) are involved in the later stages of
encoding putative transcription factors involved in all anther dehiscence and pollen release. Although a num-
ber of the main regulators are known and a large
number of genesdirectly involved inanther and pollen
1Thisworkwassupportedbythe BiotechnologyandBiological development have been identified, themeans by which
SciencesResearchCouncil(grantno.BB/J001295/1),theRoyalSoci- theyinteractandfunctioninthisprocessremainlargely
ety,andtheWolfsonFoundation. uncharacterized.
2Theseauthorscontributedequallytothearticle. Inthiswork,wetrytoaddressthisknowledgegapby
*[email protected]. uncovering groups of genes involved in floral develop-
Theauthorresponsiblefordistributionofmaterialsintegraltothe ment, specifically focusing on anther and pollen devel-
findingspresented in this article in accordancewith the policy de-
opment,throughthecreationofacoexpressionnetwork.
scribed in the Instructions for Authors (www.plantphysiol.org) is:
This coexpression network connects genes expressed
ZoeA.Wilson([email protected]).
[OPEN]Articlescanbeviewedwithoutasubscription. during floral development by their transcriptional reg-
www.plantphysiol.org/cgi/doi/10.1104/pp.114.253807 ulation.Coexpressedgeneshaveanincreasedlikelihood
Plant Physiology!, April 2015, Vol. 167, pp. 1717–1730, www.plantphysiol.org "2014AmericanSociety ofPlant Biologists. All RightsReserved. 1717
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Pearce et al.
of being involved in the same developmental or bio- sterile phenotypes linked to a failure of pollen forma-
chemical pathways; therefore, the correlation of gene tion or dehiscence. The Affymetrix microarrays were
expressionisapowerfulapproachtoanalyzelargedata renormalized together (see “Materials and Methods”)
sets to identify genes involved in the same functional to make the data comparable between samples.
pathway (Peng and Weselake, 2011; Wang et al., 2012). A number of additional highly relevant data sets
This approach has been successfully applied to two were also available, but since they utilize different
state-dependentsetsofinteractionsassociatedwithseed transcriptomic platforms, they cannot be integrated
dormancy or germination, with good correlation of easily with the Affymetrix array data sets. Alves-
transcriptionalregulatorsofknownseeddormancyand Ferreira et al. (2007) used Agilent two-color micro-
germination regulatory genes (Bassel et al., 2011). arrays to analyze the effect of MS1 (At5g22260) on
With the recent advances in postgenomic technolo- gene expression, comparing buds between the wild
gies, a large number of genome-wide transcriptomic type and ms1 mutants. In this experiment, the first
datasetshavebeengenerated,oftentolookatspecific sample consists of stage 13 flowers, with subsequent
changes such as a transcription factor mutant com- samples containing successively younger buds, until
pared with a wild type. Deposition of these data sets the seventh sample contains the very earliest buds
into publicly accessible online databases enables other and the inflorescence meristem. Xu et al. (2010) used
researcherstoanalyzethesecollateddataanduncover anin-house-printedtwo-colormicroarraytocompare
novelinformation,whichmaybetangentialtothegoal wild-typeandamsbudsatpollenmothercellmeiosis,
of the original experiment. In this study, we used pollen mitosis I, bicellular pollen, and pollen mitosis
publicly available gene expression data deposited in IIstages.Thissecond,developmentaltimecoursehas
the National Center for Biotechnology Information’s finertemporalandspatialdetailaroundthesekeystages
Gene Expression Omnibus (Edgar et al., 2002; Barrett and therefore may be used in conjunction with the one
et al., 2013), choosing microarray data sets that in- generated from the data of Alves-Ferreira et al. (2007).
cluded flowers, buds, anthers, or pollen materials to Data from these two-color microarray experiments
generate a coexpression network. Through generating (Alves-Ferreira et al., 2007; Xu et al., 2010) were
the network, compact clusters of coexpressed genes renormalized using a method similar to that of single-
werefound(i.e.setsofgeneshavingsimilarexpression color arrays (see “Materials and Methods”), producing
patternsacrossallthesamples).Thisclusteringenables two comparative developmental time courses of gene
the identification of novel genes that may be involved expression in wild-type, ms1, and ams buds. This en-
in specific biological processes based on known com- ables us to use the wild-type and mutant values as a
ponents within the cluster. biologically meaningful developmental time course. It
Additionally, we reanalyzed two previously con- is noted that this method seems more prone to noise
ducted two-color microarray experiments associated than the use of single-color microarrays, but it is par-
with anther and pollen development (Alves-Ferreira ticularly useful where genes are not present on the
et al., 2007; Xu et al., 2010) in order to compare gene Affymetrix microarrays, such as MS1.
expression across these time series as well as between The resulting plots (e.g. for AMS; Fig. 1) from both
the mutants. Along with the Affymetrix microarrays the Affymetrix and two-color array data give imme-
used to generate the correlation network, these three diate visual indicationof howthe genesare expressed
data sets give a clear indication of the spatial and tem- across various samples. These plots show the indi-
poralexpressionofanther-relatedgenes.Plotshavebeen vidual replicates rather than means, allowing those
createdforeachgeneinthecorrelationnetworkandare geneswhosebehaviorappearstobevariable,possibly
available online at http://www.cpib.ac.uk/anther. duetosensitivitytoconditionsortotheprecisestaging
In this work, we have generated a valuable tool for of the samples, to be easily visually detected. These
the analysis of genes involved in anther and pollen plots are freely available online at http://www.cpib.
developmentandshowhowthiscanbeusedtoidentify ac.uk/anther, allowing quick analysis of anther ex-
new players in anther and pollen development. How- pression data for genes of interest. Closed circles rep-
ever,thedatausedforthegenerationofthecorrelation resentwild-typesamples,andopensymbolsrepresent
network are from all floral organs and developmental the different mutants.
stages; therefore, FlowerNet also has broader applica-
tion in the characterization of all genes associated with
aspects of plant reproduction and flowering.
FlowerNet CorrelationNetwork
In order to investigate gene behavior across a wide
RESULTS range of samples, we generated a correlation network
using 66 Arabidopsis Affymetrix wild-type micro-
GeneExpressionPlots
arrays from plant reproduction-related experiments,
Arabidopsis anther, stamen, and bud microarray which included flowers, buds, anther/stamen sam-
data sets were selected that comprised wild-type or ples, and isolated pollen samples (see “Materials and
mutant samples associated with anther and pollen Methods”; Supplemental Files S1 and S2). These micro-
development; all mutants selected displayed male- array data sets were mostly publicly available in the
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FlowerNet: A Gene Discovery Correlation Network
Figure1. PlotfromtheWebsitetoolshowingtheexpressionofAMSinvariousantherandfloralmicroarrays(http://www.cpib.
ac.uk/anther).
National Center for Biotechnology Information’s Gene genes considered correlate well with each other. For
Expression Omnibus (Honys and Twell, 2004; Schmid example, the largest cluster (cluster 1) contains 14,433
et al., 2005; Mandaokar et al., 2006; Yang et al., 2007a) edges between 171 genes, 99.3% of the possible edges;
with some previously unpublished data (Z.A. Wilson thus, these genes have very similar expression profiles.
andJ.Song,unpublisheddata).Anumberofotheranther- Figure 3 shows a representative cluster (cluster 110),
related published studies were used for gene list overlay whichdemonstrateshowtheexpressionprofileofeach
comparison;however,thesecouldnotbeincludedinthis gene within the cluster has a highly correlated expres-
network,orintheexpressionplots,sincetherawdatafor sion pattern in the microarrays used to generate the
these analyses were not publicly available at the time of network.
construction. Overlaying expression data from different experi-
The resulting network has 605,686 edges between ments that have not been used in the creation of the
10,797 genes (Fig. 2); a visualization of the network is network (e.g. mutant data from the two-color micro-
available as a Cytoscape file (Supplemental File S3), arrays detailed above) indicate that certain clusters
withanavigablelistofgenesthatareconnectedtoeach aresignificantlyoverrepresentedforthesetranscripts.
genepresentedundereachgeneexpressionplotimage. Similarly, the majority of the clusters contain a signifi-
Within this network, compact highly intracorrelated cant overrepresentation of Gene Ontology (GO) terms,
clusterswereidentifiedusingtheTransClustalgorithm suggesting that genes within clusters are involved in
(Wittkop et al., 2010) and the correlation values as an the same developmental processes. A number of the
edge weight to generate small, well-connected clusters clusters are enriched for general plant pathways; for
basedonexpressioninthenetwork(see“Materialsand example, cluster 2 is enriched in the GO term photo-
Methods”). The resultant clusters describe groups of synthesis,cluster11inGAbiosynthesis,andcluster16in
genes that are highly positively correlated across all water transport, although these do not show significant
the samples included, with almost all the edges be- expression changes linked to anther/stamen-specific
tween the genes included (for cluster allocations, see gene expression. Other clusters show reproduction-
Supplemental File S4). These compact clusters have related enrichment; for example, cluster 67 is a set of
extremely similar expression patterns within the sam- 14 genes that all have higher expression in the carpel
ples used, avoiding the problem of chain-like clusters, (and to a lesser extent the petal) samples from the
wheregenesareseveralneighborsremovedfromthose AtGenExpress Developmental Map, which have not
at the other end of the cluster, leading to genes in the been used in the generation of the network. To focus
same cluster having very diverse expression patterns. onanther/stamen-relatedclusters,weusedanumber
The resulting clusters contain almost all of the possible ofdatasetstoselectclustersofinterest,suchasstamen-
edgesbetweenthenodesinvolved,ensuringthatallthe specific transcripts (Wellmer et al., 2004), the presence
Plant Physiol. Vol. 167, 2015 1719
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Pearce et al.
to have an important regulatory role during anther
and pollen development. We chose eight mutants:
ap3 (early stages only); spl/nzz and ems, which are
involved in anther cell specification (Zhang et al.,
2006; Alves-Ferreira et al., 2007; Wijeratne et al., 2007);
dyt1 and ams (combination of all four stages); ms1 and
myb80 (previously known as myb103), which are in-
volved in tapetum development and mature pollen
(Zhangetal.,2006;Alves-Ferreiraetal.,2007;Xuetal.,
2010; Phanet al.,2011); and rpk2 (flower stages 12–14),
which is involved in anther dehiscence (Mizuno et al.,
2007b). Wild-type data were used to initially generate
the FlowerNet network, but when the corresponding
mutant data were overlaid onto the network, a large
proportion of genes within each cluster showed corre-
lated expression in the specific mutants (Fig. 7). This
clustering of expression was also shown as a heat map
(Fig. 8), utilizing developmental staged microarray
datafromthems1mutant(Alves-Ferreiraetal.,2007).
Thissuggestsnotonlythatthesegenesarecoexpressed
but also that they may be coregulated.
To characterize the clusters further, we compared
the gene expression of these clusters in the develop-
Figure 2. Cytoscape visualization of the full correlation network. mental time courses generated from the data of Xu
Genes shown byWellmer et al. (2004)to have higherexpressionin etal.(2010)andAlves-Ferreiraetal.(2007).InFigure5,
stamenarecoloredinpurple.
each gene within the four clusters is represented by a
different color line, the thick black line being the me-
dian trend line, while Figure 8 shows a heat map of
of known anther-related genes within a cluster, and
expression levels of each gene within each of the four
clusters that are highly regulated by previously de-
chosen clusters. From both of these sets of data, the
scribedmale-sterilemutantssuchasamsandms1;these
genesintheclustersareexpressedinasimilarmanner
are shown in Figure 4.
and in most cases at a similar level of expression.
Therefore,despiteonlyusingthewild-typeAffymetrix
ClustersAssociated withPollenWallFormation
In order to validate the association between genes
identified within the same cluster, four clusters were
selected for detailed investigation. These were chosen
based on their expression patterns, GO annotations,
and behavior in other data sets. Each of these clusters
has overrepresented GO terms associated with pollen
or anther development. The selected clusters show
enrichment of gene expression at specific time points
during pollen development based upon staged bud
expression data (Xu et al., 2010); cluster 37 includes
genes that show up-regulation at pollen mother cell
meiosis, cluster 81 at pollen mitosis I, cluster 21 at the
bicellularpollenstage,andcluster116atpollenmitosis
II(forpollendevelopmentreview,seeBorgetal.,2009;
Table I). This allowed us to infer a good overview of
the developmental expression changes during anther
andpollendevelopmentthroughtheseclusters(Fig.5).
Figure 6 shows the expression patterns of individ-
ual genes within these particular clusters, looking at
pollen-specific expression stages and the whole bud;
both of these microarray data sets were used to create
the network and illustrate the expected similar be-
havior of gene expression within each cluster. To in- Figure3. Plotofthegenesincluster110,showingexpressioninthe
terrogate these clusters further, we collected publicly 66microarraysthatareusedtogenerateFlowerNet.Theorderofthe
availablemicroarraydatafromvariousmutantsknown microarraysislistedinSupplementalFileS1.
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FlowerNet: A Gene Discovery Correlation Network
Figure4. SelectionofFlowerNetclusters,
chosenforbeingbudrelatedbasedona
numberofcriteriasuchasknownanther-
relatedgenesandmale-sterilemutantex-
pression. Highlighted in pink are those
genesfoundtobestamenspecific(Wellmer
etal.,2004).
arrays to generate the network, the clusters show role, with the GO term pollen exine formation over-
similar expression in the developmental time courses represented; however, their specific function is cur-
available in two-color Agilent arrays (Alves-Ferreira rently undetermined. These genes are up-regulated in
et al., 2007) as well as the various sets of correspond- the stamen of stage 12 flowers (using the data from
ingmutantdata.Thisimpliesthatthegeneswithinthe Schmid et al. [2005]) and in pollen; the majority are
clusters have expression patterns that are closely cor- down-regulated in old ms1 buds, particularly in the
related, suggesting that they may be involved in the second and third samples of the time course from
same process and potentially may be regulated by the Alves-Ferreira et al. (2007; sample 1 equates to mature
same mechanism. Use of this network clustering to flowers and sample 7 to immature buds/inflorescence
find clusters that change in different mutant back- meristem). This corresponds with a peak in the wild-
grounds may allow new targets and processes to be typebicellularpollendevelopmentstageofbudsinthe
inferred from the results. To focus on these coex- datafromXuetal.(2010),withthemajorityofthegenes
pression similarities, we also looked at the individual within this cluster being down-regulated in the ams
genes in each cluster in more detail to compare ex- mutant from the same data set. The majority of these
pression and show that, within a cluster, the genes geneshavehigherexpressioninthetapetumcompared
show very similar expression in both wild-type and with other anther tissues at pollen mitosis II (Z.A.
mutant data (Fig. 9). Wilson and G. Vizcay-Barrena, unpublished data).
Twenty-one of these genes were found to be down-
regulated in stage 5 to 8 myb80 anthers (Phan et al.,
2011), suggesting that this cluster acts downstream
Cluster21:PollenExineFormation
of AMS and MYB80.
Cluster21contains31geneswith464edgesbetween Of the three pollen-specific genes, AtSAC1b shows
them(99.78%ofthepossibleedges),whichareinvolved specific expression in pollen, with higher levels at the
in pollen exine formation, and contains three charac- uninucleate stage (Despres et al., 2003), VHA-E2 is
terized genes that are known to be pollen specific, specific to the vegetative cell in the pollen (Strompen
Arabidopsis SUPPRESSOR OF ACTIN (AtSAC1b), et al., 2005), while APK3 has expression throughout
ADENOSINE-59-PHOSPHOSULFATE KINASE (APK3), development, with strongest expression in pollen
and VACUOLAR H+-ATPASE SUBUNIT E (VHA-E2; grains (Mugford et al., 2009). The single mutants in all
Despresetal.,2003;Dettmeretal.,2010;Mugfordetal., of these three genes have no phenotype, most likely
2010); 23 of these genes were found to be specifically because of functional redundancy of other isoforms
expressed in stamens (Wellmer et al., 2004). The ma- expressed within the pollen: only in the triple mutant
jorityofthegeneswithinthisclusterhaveanunknown of APK1 isoforms (apk1 apk3 apk4) is there a pollen
Plant Physiol. Vol. 167, 2015 1721
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Pearce et al.
TableI. Genespresentinthefourselectedclustersincludingtheirpredictedrolesandpublishedexpressionpatterns
ClusterNo. Name;TheArabidopsisInformationResource10Annotation Role PublishedExpression
Cluster21
AT1G04290 Thioesterasesuperfamily
AT1G07850 DOMAINOFUNKNOWNFUNCTION(DUF)064
AT1G20080 SYNAPTOTAGMIN2 Proteinsecretion
AT1G22150 SULTR1;1;sulfatetransporter Sulfatemembranetransport
AT1G32250 Calcium-bindingEF-handfamily
AT1G52570 PLDALPHA2;phospholipaseDa-2
AT1G54560 MYOSINXIE Golgimovement
AT1G72960 RHD3;roothairdefective3GTP-binding
AT2G25890 Oleosinfamilyprotein Lipidstorage
AT2G30290 VSR2;vacuolarsortingreceptor Vacuolarsorting
AT2G30630 P-loop-containingnucleosidetriphosphate Cellkilling
hydrolasesuperfamily
AT2G47770 TSPO;outermembraneTrp-richsensoryprotein Abscisicacidandsalt Seed
stressinduced
AT3G03900 APK3 Sulfationofsecondary Pollen
metabolites
AT3G08560 VHA-E2 Pollen
AT3G17630 CHX19;cation/H+exchanger Membranetrafficking
AT3G19090 RNA-bindingprotein RNAbinding
AT3G25160 Endoplasmicreticulumlumanprotein-retaining Endoplasmicreticulum
receptorfamily retention
AT3G51490 TMT3;tonoplastmonosaccharidetransporter Sugartransport Filament
AT3G60540 PreproteintranslocaseSec Translocase
AT4G02140
AT4G10440 S-Adenosyl-L-Met-dependentmethyltransferases
AT4G11030 AMP-dependentsynthetaseandligasefamily
AT4G18920 DUF1264
AT4G26770 Phosphatidatecytidyltransferasefamily
AT4G36600 Lateembryogenesis-abundantprotein
AT4G37840 HEXOKINASE-LIKE3 Hexokinase
AT5G10730 NAD(P)-bindingRossmannfoldsuperfamily
AT5G13350 Auxin-responsiveGH3family
AT5G15490 UDP-Glc-6-dehydrogenasefamily Cellwallcomposition Throughoutplant
AT5G66020 AtSAC1B;suppressorofactin Pollen
AT5G66150 Glycosylhydrolasefamily
Cluster37
AT1G01280 CYP703A2;cytochromeP450family703 Sporopolleninbiosynthesis Anther
AT1G02050 LAP6;lessadhesivepollen6 Sporopolleninbiosynthesis Anther,tapetumspecific
AT1G02813 DUF538
AT1G62940 ACOS5;acyl-CoAsynthetase Sporopolleninbiosynthesis Anther,tapetumspecific
AT1G69500 CYP704B1;cytochromeP450family704 Sporopolleninbiosynthesis Anther,tapetumspecific
AT1G76470 NAD(P)-bindingRossmannfoldsuperfamily Ligninbiosynthesis
AT2G42870 PAR1;Phytochromerapidlyregulated Cellelongation
AT3G11980 MS2;malesterility2 Exineformation Anther,tapetumspecific
AT3G13220 WHITE/BROWNCOMPLEXSUBFAMILY27(WBC27); Exineformation Anther,tapetumspecific
ABCtransporter-like
AT3G23770 O-Glycosylhydrolasesfamily17 Exineformation
AT3G42960 ATA1;tapetum1 Anther,tapetumspecific
AT4G14080 MEE48;maternaleffectembryoarrest Exineformation
AT4G20420 TAP35/44;tapetum-specificprotein Tapetumspecific
AT4G34850 LAP5;lessadhesiveprotein Sporopolleninbiosynthesis Anther,tapetumspecific
AT4G35420 DRL1/TKPR1;dihydroflavonol4-reductase-like Sporopolleninbiosynthesis Anther,tapetumspecific
AT5G07230 Bifunctionalinhibitor/lipidtransferprotein Lipidtransfer
AT5G47600 HSP20-likechaperonesuperfamily
AT5G59000 REALLYINTERESTINGNEWGENE/FYVE/PLANT
HOMEODOMAINGENEzincfingersuperfamily
AT5G62080 Bifunctionalinhibitor/lipidtransferprotein Lipidtransfer
AT5G65410 HB25;homeoboxprotein
Cluster81
AT1G13140 CYP86C3;cytochromeP450,family86 Exineformation
(Tablecontinuesonfollowingpage.)
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FlowerNet: A Gene Discovery Correlation Network
TableI.(Continuedfrompreviouspage.)
ClusterNo. Name;TheArabidopsisInformationResource10Annotation Role PublishedExpression
AT1G67990 TSM1;tapetum-specificmethyltransferase1 Spermidineformation Anther,tapetumspecific
AT1G71160 KCS7;3-ketoacyl-CoAsynthase Lipidbiosynthesis
AT2G19070 SHT;spermidinehydroxycinnamoyltransferase Spermidineformation Anther,tapetumspecific
AT3G52160 KCS15;3-ketoacyl-CoAsynthase Lipidbiosynthesis
AT4G28395 A7;anther7 Lipidtransfer Anther
AT5G13380 Auxin-responsiveGH3family
AT5G16920 Fasciclin-likearabinogalactanfamily Exineformation
AT5G16960 Zinc-bindingdehydrogenasefamily
AT5G48210 Exineformation
AT5G49070 KCS21;3-ketoacyl-CoAsynthase Lipidbiosynthesis
AT5G65205 NAD(P)-bindingRossmannfoldsuperfamily
Cluster116
AT1G14420 ArabidopsisLAT59ORTHOLOG(At59) Cellwallmodification Pollenspecific
AT1G23350 Plantinvertase/pectinmethylesterase Actinfilamentmovement
AT1G49290 Actinfilamentmovement
AT1G64740 TUA1;a-1tubulin Microtubulemovement Pollenspecific
AT2G17500 Pectinlyase-likesuperfamily Actinfilamentmovement
AT2G31500 CPK24;calcium-dependentproteinkinase24 Regulatesgrowth Pollen;pollentubes
AT3G17060 Pectinlyase-likesuperfamily Actinfilamentmovement
AT3G25170 RAPIDALKALINIZATIONFACTOR-LIKE26
AT5G23270 STP11;sugartransporter11 Sugartransport Pollenspecific
AT5G39400 PTEN1 Phosphatase Pollenspecific
lethality phenotype (Mugford et al., 2010). APK3 is an toregulatethePHOSPHOINOSITOL-4-phosphatepool
ADENOSINE-59-PHOSPHOSULFATE kinase involved and could control ATP transport (Despres et al., 2003).
insulfurmetabolism,whileAtSAC1bhasbeensuggested ThislinkswithVHA-E2,whichisavacuolarH+-ATPase
Figure 5. Gene expression levels in
the four selected clusters in different
pollen development stages in whole
buds,basedonwild-typedatafromXu
et al. (2010) for each gene within the
cluster. The thick black line shows
theaveragetrendofallthegenesinthe
cluster.
Plant Physiol. Vol. 167, 2015 1723
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Pearce et al.
Figure6. Geneexpressionlevelsinthefourselectedclusters,showingthedata(HonysandTwell,2004;Yangetal.,2007a)
usedtogenerateFlowerNet.UNM,Unicellularmicrospores;BCP,bicellularpollen;TCP,tricellularpollen;MPG,maturepollen
grain.YoungbudsrepresentpollenmothercellmitosisI,whileoldbudsrepresentmitosisII.
that plays an important role in maintaining the pH of up-regulatedinthems1mutant,particularlyinthethird
endomembranecompartmentsineukaryoticcells(Dettmer to fifth samples of the time course generated from the
et al., 2010). The double and triple knockouts of dataofAlves-Ferreiraetal.(2007),whilethemajorityof
AtSAC1bandVHA-E2wouldbeinterestingtostudyto these genes are down-regulated in ams mutants, par-
see if they have a similar phenotype to APK3 and dis- ticularly at the meiosis and pollen mitosis I stages (Xu
cover what role these families of genes have in pollen etal.,2010).Thiscorrespondstoapeakinexpressionin
development. Another two published genes from this wild-typedatafromthesamedatasetatthemeiosisstage,
cluster,AT5G15490(UDP-GLUCOSEDEHYDROGENASE; with the majority of the genes more highly expressed in
Reboul et al., 2011) and AT3G51490 (TONOPLAST thetapetum than in the other anther tissues at the tetrad
MONOSACCHARIDETRANSPORTER1;Wormitetal., stage (Z.A. Wilson and G. Vizcay-Barrena, unpublished
2006), are involved in cell wall composition and sugar data). Collectively, this suggests that these genes are
transport,whichalsomaybeimportantforpollenwall downstreamofAMSandareup-regulatedbyAMS,while
formation. they are negatively regulated by MS1 to give a specific
By focusing research on the other cluster members expression pattern.
withinthegroup,wemaybeabletoinferfurtherroles A large proportion of these genes are involved in
of this cluster, as many of the genes have predicted the biosynthesis of sporopollenin, which is a major
roles in protein movement and in secretion. It will be constituent of exine in the outer pollen wall. Recently
interestingtodiscovertherolestheyplayinantherand in Arabidopsis, a number of genes have been demon-
pollen development. strated to be involved in sporopollenin biosyn-
thesis: ACYL-COENZYME A SYNTHETASE (ACOS5),
CYTOCHROME P450 (CYP703A2), CYP704B1, LESS
ADHESIVE POLLEN3 (LAP3), LAP5 (POLYKETIDE
Cluster 37:SporopolleninBiosynthesis
SYNTHASE B), LAP6 (POLYKETIDE SYNTHASE A),
Cluster37isa20-nodeclique,withalledgesbetween MS2, FACELESS POLLEN1 (FLP1), and TETRAKETIDE
thempresent,andcontainsanumberofgenesknownto a-PYRONEREDUCTASE1(TKPR1[DIHYDROFLAVONOL
be tapetum specific and linked to sporopollenin bio- 4-REDUCTASE-LIKE1]; Ariizumi and Toriyama, 2011);
synthesis. Of these 20 genes, 11 were found to be sta- all of these except LAP3 and FLP1 are found within
men specific (Wellmer et al., 2004); however, two of this cluster. Lallemand et al. (2013) demonstrated
these 20 genes are not included in the array analysis. that ACOS5, LAP5, LAP6, TKPR1, CYP703A2, and
The genes in this cluster are up-regulated in flowers CYP704B1 all interact together on the endoplasmic
at stage 9 to 11 (Schmid et al., 2005) and are mostly reticulum of plant cells, and all of these interacting
1724 Plant Physiol. Vol. 167, 2015
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
FlowerNet: A Gene Discovery Correlation Network
Figure 7. Gene expressionchanges in the four selected clusters in different known antherand pollen development mutant
microarraydatasets;dataareshownforap3(flowerearlystagesonly),spl/nzz(flowerstages1–13),ms1(flowerstages1–13;
Alves-Ferreiraetal.,2007),ems(flowerstages8and9;Wijeratneetal.,2007),dyt1(flowerstages8–10;Fengetal.,2012),ams
(flowerstages9–12;Xuetal.,2010),myb80(flowerstages9and10;Phanetal.,2011),andrpk2(flowerstages12–14;Mizuno
etal.,2007a).Eachcirclerepresentsagene,withredsignifyingup-regulationandbluefordown-regulationineachmutant
comparedwiththewildtype.
genes are found in this cluster. Of the studied genes proteins, At5g07230 and At5g62080, which also may be
within this cluster, the phenotypes range from an ab- involved in sporopollenin biosynthesis. This strongly
sence of production of pollen due to compromised suggests that the remaining six unknown genes within
pollen walls (ACOS5 and TKPR1; de Azevedo Souza thisclustercouldplayaroleinsporopolleninsynthesis
et al., 2009; Tang et al., 2009; Grienenberger et al., in some manner, and studying the knockouts of these
2010) to having irregular exine layers (CYP703A2, genes could further our understanding of the sporo-
CYP704B1, ATP-BINDING CASSETTE G26 [ABCG26], pollenin biosynthesis pathway and transport to the pol-
LAP5, LAP6, and MS2; Morant et al., 2007; Dobritsa len coat wall.
et al., 2009, 2010; Kim et al., 2010; Choi et al., 2011),
with varying phenotypes from loss of fertility to re-
duced pollen viability to no effect on pollen viability.
Cluster81:PollenSpermidine Formation
The sporopollenin precursor biosynthesis gene WBC27
(ABCG26)is also within this clusterandis involved in Cluster 81 is a 12-gene clique, with all 12 of these
the transport of these sporopollenin precursors from genesfoundtobestamenspecific(Wellmeretal.,2004)
the tapetum, facilitating exine formation on the pollen and up-regulated in flower stages 9 to 11 (Schmid
surface(Choietal.,2011).Twoothergenesofunknown et al., 2005). These genes are differentially expressed
function are believed to play a role in pollen exine (mostlydown-regulated)inbothms1andamsmutants
formation, At3g23770 and At4g14080 (MATERNAL throughout pollen developmental stages (Alves-
EFFECTEMBRYOARREST48),andtwofurtherunknown Ferreira et al., 2007; Xu et al., 2010) as well as tapetum
genes are tapetum specific, At3g4290 (TAPETUM1) and specific (Z.A. Wilson and G. Vizcay-Barrena, unpub-
At4g20420, based on their description in AtEnsembl lished data) and particularly expressed in the pollen
(Flicek et al., 2013; http://atensembl.arabidopsis.info/ mitosis I stage (Xu et al., 2010). Two genes (At5g49070
index.html).Therefore,12outof20genesinthiscluster and At3g52160) were also down-regulated in myb80
have a direct role in the tapetum, with one-half of the anthers.
genes in this cluster involved in exine/sporopollenin Similar to cluster 37, the majority of these genes
formation. The rest of the genes in this cluster have an have suggestive roles in pollen exine formation (five),
unknownfunction,includingtwoputativelipidtransfer lipidbiosynthesis(three),andlipidtransfer(one),with
Plant Physiol. Vol. 167, 2015 1725
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.