in the last decade , the increased use of microarrays for global expression analyses made large - scale transcript analyses possible .
using publicly available microarray datasets , several studies showed that genes with similar function tended to be transcriptionally coordinated ( stuart et al .
, 2003 ; ihmels et al . , 2004 ) . based on this observation ,
co - expression approaches have been used to assign functions for genes involved in cell wall formation , isoprenoid and glucosinolate biosynthesis , and different metabolic pathways in arabidopsis ( wille et al . , 2004 ; brown et al . , 2005 ;
persson et al . , 2005 ; wei et al . , 2006 ; hirai et al . , 2007 ) .
interestingly , certain co - expression relationships appear to also be conserved across different species across the kingdoms of life ( stuart et al .
for example , orthologous genes involved in protein synthesis , cell cycle , and protein degradation formed comparable co - expressed clusters in different species ( stuart et al . , 2003 ) . in the above studies ,
the microarray analyses still had to be quality - controlled and evaluated by the investigators themselves to obtain co - expressed relationships .
however , web - based co - expression tools now enable users to easily mine publicly available microarray dataset to investigate their gene(s ) of interest . in this review
we present several co - expression tools and describe recent developments and updates for them .
we also give an overview of how these tools have been used to identify novel cell wall - related genes , and exemplify the use of comparative co - expression analyses across species to infer lignin - related genes .
several co - expression tools have been developed for plant biology , including act ( manfield et al . , 2006 ) , asidb ( rawat et al . , 2008 ) , atted - ii ( obayashi et al . , 2009 ) , cressexpress ( srinivasasainagendra et al . , 2008 ) , csb.db ( steinhauser et al . , 2004 ) , and genecat ( mutwil et al . , 2008 ) , which have been comprehensively described and compared elsewhere ( usadel et al . , 2009 ) .
in addition , several recent platforms have emerged , such as aranet , cornet , genemania , planet , and ricearraynet .
aranet aims at annotation of arabidopsis genes by integrating available large - scale experimental data , e.g. , co - expression and protein 
protein interaction data , as well as gene associations inferred from other species and literature queried data ( lee et al . , 2010 ) .
similarly , genemania is predicting gene function based on available genomics and proteomics data sets for arabidopsis , yeast , and several animal model species ( warde - farley et al . , 2010 ) .
cornet allows for user - defined selection of microarray experiments and cut - offs for assessing co - regulated genes in arabidopsis .
protein interaction , functional annotation , and localization data ( de bodt et al . ,
planet provides a platform for gene co - expression network analysis for seven plant species and includes information about significant enrichment for functional annotation using mapman ontology terms ( mutwil et al . ,
another interesting co - expression tool is ricearraynet , which calculates positive as well as negative correlation of gene expression profiles in rice ( lee et al . , 2009 ) .
this tool has later been extended to brassica and arabidopsis , referred to as plantarraynet . in principle , the platforms above calculate co - expression relationships between two genes of interest by comparing their respective expression profiles .
the pearson s correlation coefficient ( pcc ) is a commonly used measure to estimate transcriptional co - ordination . using this measure as a basis
, co - expression relationships between many genes can be determined , and can be visualized as networks in which nodes represent genes and the connection between nodes indicates the transcriptional co - ordination of the genes .
such co - expression networks can be divided into rank - based and value - based networks ( aoki et al . , 2007 ) .
previously , the most widely used approach was value - based networks , i.e. , edges were established between genes that were co - expressed above a certain pcc - value threshold ( lee et al . , 2004 ; oldham et al . , 2006 ) .
one major drawback of this method arises from the fact that some biological processes are tightly transcriptionally co - regulated , while other processes are not .
therefore , when a stringent global pcc - value cut - off is applied many genes involved in weakly transcriptionally coordinated processes that may be biologically relevant become disconnected . in contrast , a lower pcc - threshold will in many instances result in excessively large gene clusters , containing thousands of genes ( mao et al . , 2009 ) .
to avoid such problems , some tools have introduced the rank - based method , which is based on the ranks of two given genes in their mutual co - expression lists ( atted - ii : obayashi and kinoshita , 2009 ; planet : mutwil et al . , 2010,2011 ) .
although both the value- and rank - based networks are derived from pcc , rank - based networks appear to lead to a network topology that closer resembles biological networks ( ruan et al . , 2010 ) .
however , it is important to note that both approaches have their advantages and drawbacks ( usadel et al . , 2009 ) .
most of the co - expression tools have focused on the model plant arabidopsis , and have incorporated the main bulk of publicly available microarray datasets , thus representing condition - independent co - expression relationships ( usadel et al . , 2009 ) .
however , some biologically relevant transcriptional relationships may be revealed only under specific experimental conditions , or in certain tissues . to find such relationships , tools like cornet allow for user - defined selection of microarray experiments to calculate co - expression relationships .
another example of condition - dependent databases is the updated version of atted - ii , which enables the user to analyze co - expression relationships of genes under five predefined conditions : tissue and development , abiotic stress , biotic stress , hormone treatment , and different light regimes ( obayashi et al . , 2011 ) .
furthermore , seednet is a relatively new tissue - specific database , which returns co - expression relationships of genes during seed development ( bassel et al . , 2011 ) .
one possible caveat with co - expression analyses is the rate of  false positives ,
 i.e. , co - expressed genes that might be co - expressed by chance rather than being functionally related . a useful approach to minimize the rate of such  false positives
 may be to investigate whether orthologous genes are also co - expressed in related species .
as mentioned above , co - expression relationships are often conserved across species ( stuart et al . , 2003 ) .
hence , two co - expressed genes from one species often have orthologs in another species that in turn are also co - expressed . in theory
it should therefore be possible to enrich for co - occurring co - expression relationships across species , and hence minimize  false positives . 
therefore , across species co - expression analyses might improve the reliability of co - expression - based functional annotation .
consequently , several tools , such as starnet , cop , and atted - ii , allow pairwise comparison between species ( jupiter et al .
. moreover , comparison of several species at the same time was introduced by the networkcomparer - tool of planet ( mutwil et al . , 2011 ) .
this tool bins genes into gene families according to their pfam annotation ( finn et al . , 2010 ) ,
and then finds recurring pfams in co - expression networks across species ( mutwil et al . , 2011 ) .
cellulose is produced by the cellulose synthase ( cesa ) complex ( csc ) , which is comprised of cesa1 , 3 , and 6-related proteins in the primary wall and cesa4 , 7 , and 8 in the secondary wall ( gardiner et al .
interestingly , these primary and the secondary cesas display similar expression patterns , respectively ( brown et al . , 2005 ; persson et al . , 2005 ) .
in addition , these studies were able to show that many genes involved in xylan and lignin synthesis were co - expressed with the secondary wall cesas ( brown et al .
, 2005 ; persson et al . , 2005 ) . hence , co - expression analyses may be useful to identify new genes involved in secondary cell wall - related synthesis .
figure 1 shows a truncated node - vicinity network ( nvn ) of genes co - expressed with the primary wall - related arabidopsis 
 cesa1-gene , which was obtained from planet ( mutwil et al . , 2011 ) .
several primary wall cesa genes ( cesa2 , cesa3 , cesa5 , and cesa6 ) may be found in close vicinity of cesa1 .
in addition , many other genes important for cellulose synthesis , such as cobra ( cob ) , chitinase - like ( ctl)1 , cellulose synthase interacting ( csi)1/pom - pom2 , and korrigan ( kor ) , are present in this network ( nicol et al . , 1998 ;
2002 ; roudier et al . , 2005 ; gu et al . , 2010 ;
this result confirms previous findings which were based on a smaller number of microarrays ( persson et al . , 2005 ) , and is similar to results obtained from other co - expression tools .
for example , the many of the genes in this network are also present in the atcesa1-top 300 list of co - expressed genes in atted - ii ( 77 out of 190 genes ; see also the genes marked in bold in figure 1 ) .
subset of the co - expression gene vicinity network of atcesa1 ( turquoise node ) .
nodes indicate individual genes , and edges indicate whether two genes are co - expressed above a certain mutual rank threshold .
node colors indicate whether mutations in the gene cause gametophytic lethality ( yellow ) , any biological phenotype ( green ) , or if no mutant phenotype information currently is available ( gray ) according to tair .
green , orange , and red edges indicate a mutual rank relationship < 10 ( green ) , 1019 ( orange ) , and 2029 ( red ) , respectively , for each connected gene pair .
genes marked in bold are also present in the top 300 co - expression list of atcesa1 obtained from atted - ii ( http://atted.jp/ ) .
the respective gene family according to pfam annotation is given below the gene names / agi codes ( finn et al .
in addition to cellulose synthesis , co - expression approaches have been used to identify genes involved in the synthesis of hemicelluloses .
( 2007 ) found that the arabidopsis 
 cslc4 gene , which presumably is involved in glucan backbone synthesis of xyloglucan , was co - expressed with the xylosyltransferase atxt1 , which has previously been shown to attach xylose residues to a glucan backbone ( faik et al . , 2002 ) .
moreover , expression profiling was used to identify irx15 and an irx15-like gene , with corresponding single mutants showing a mild irregular xylem phenotype ( jensen et al . , 2010 ;
both genes belong to a gene family with a domain of unknown function ( duf ) 579 and only the corresponding double mutant showed decreased levels of xylan and altered cell wall morphology in stems ( jensen et al . , 2010 ;
although their exact function is unknown , these results suggest an important role of this duf579-gene family in xylan biosynthesis ( jensen et al . , 2010 ; brown et al . , 2011 ) .
interestingly , also many genes that are important for lignin synthesis are co - expressed .
( 2005 ) showed transcriptional co - ordination of many putative and bona fide genes involved in monolignol biosynthesis , transport , and polymerization .
( 2005 ) that found laccase genes , which are probably involved in polymerization of lignin , in the list of genes that are co - expressed with secondary wall - related cesas .
( 2007 ) was one of the first studies to compare transcript relationships of cell wall - related genes across monocots and dicots .
based on est data , the authors found that members of the gt43- , gt47- , and gt61-family are enriched in monocots and therefore might be involved in biosynthesis of the grass - specific glucuronoarabinoxylan ( mitchell et al . , 2007 ) .
indeed , the same group could recently show that several gt61-members are essential for arabinosylation of xylan ( anders et al . , 2012 ) .
another recent study compared the co - expression relationships of xylan - related genes from arabidopsis and rice ( oikawa et al . , 2010 ) . using the arabidopsis genes irx9 , irx10 , and irx14 , which have previously been associated with xylan backbone synthesis ( brown et al .
, 2005 ; pea et al . , 2007 ) , the tentative rice orthologs were identified based on sequence homology and expression patterns ( oikawa et al . , 2010 ) .
these genes were then utilized as baits in the atted - ii tool and revealed many re - occurring homologs in the respective co - expression lists ( oikawa et al . , 2010 ) .
a similar co - expression approach has been undertaken for cellulose - related genes by comparing the co - expression networks of primary and secondary cesas from seven species using planet ( ruprecht et al .
interestingly , many gene families are consistently co - expressed with cesa genes across species . the function of most of these gene families remains unknown ; however , their conserved transcriptional co - ordination suggests that they fulfill important functions during cellulose synthesis ( ruprecht et al . ,
2011 ) . based on the results , an arabidopsis mutant corresponding to pinoresinol reductase ( prr ) 1
was identified that displayed distorted xylem vessels ( ruprecht et al . , 2011 ) , probably due to decreased lignin or lignan production .
while comparative transcriptional analyses across species have been undertaken for cellulose and hemicelluloses - related genes , conserved co - expression relationships among lignin - related genes have been paid less attention . similar to previously published results ( ehlting et al . , 2005 )
, we found that the co - expression network of arabidopsis 
 phenylalanine ammonia lyase ( pal ) 1 , which marks the initial step for monolignol synthesis , comprises many genes involved in lignin formation figure 2b ) . using atpal1 as bait in the networkcomparer - tool of planet
, we identified similar co - expression networks for atpal1-orthologs in barley , medicago , poplar , rice , soybean , and wheat .
the resulting consensus network indicates that most of the gene families needed for monolignol synthesis are conserved across species ( figure 2 ) . surrounding the 
pal-gene family , we found gene families corresponding to the subsequent steps in the lignin pathway such as c4h ( which belongs to gene family 
p450  according to pfam annotation , finn et al . , 2010 ) , 4cl (  amp_binding  ) , hct (  transferase  ) , c3h (  p450  ) , ccoaomt (  methyltransf_3  ) , ccr (  epimerase  ) , and cad (  adh_zinc_n  ) .
in addition , genes functioning upstream of pal in the phenylalanine synthesis pathway were previously identified as transcriptionally linked with the phenylpropanoid - related genes in arabidopsis ( tohge and fernie , 2010 ) , which appeared to be also conserved in other species ( i.e. , gene families  dahp_synth_2 ,  
udpgt ,  corresponding to glycosyltransferases , was present in almost all pal1-derived co - expression networks across the seven species ( figure 2 ) .
members of this family have been shown to glucosylate various monolignols ( lim et al . , 2001 ) and we therefore hypothesize that the respective co - expressed genes of this family might have a similar function in the different species . recently , miao and liu ( 2010 ) showed that abc - transporters are likely to mediate transport of monolignols , and their glucoconjugates , across plasma and vacuolar membrane , respectively .
the atpal1-network contained three different genes from the abc - transporter family (  abc2_membrane  ) which might mediate this function in arabidopsis .
while single knock - out mutants for one of them ( atabcg33 ) did not show any obvious phenotypes ( kaneda et al .
, 2011 ) , it is plausible that the other two members may functionally compensate for the loss of atabcg33 .
interestingly , the consensus network also comprises several highly conserved gene families , whose function in lignin biosynthesis is still elusive , for example , the gene families  hydrolase ,  
abhydrolase_1 ,  and  abhydrolase_3 .  one of the co - expressed genes from the 
hydrolase-family in arabidopsis is responsive - to - antagonist ( ran ) 1 , which encodes for a copper transporter involved in ethylene signaling ( hirayama et al . , 1999 ) .
copper is a co - factor of laccases , which are important for polymerization of monolignols to lignin in the cell wall ( boerjan et al . , 2003 ) .
we therefore speculate that ran1 has an additional function in providing copper co - factors for enzymatic lignin formation .
abhydrolase_1-family ( at3g03990 ) are methylesterases , suggesting that these genes might function antagonistically to caffeic acid o - methyltransferases ( comts ) .
peroxidase-family , which one might associate to monolignol polymerization in the cell wall . however , the respective co - expressed gene in arabidopsis is ascorbate peroxidase ( apx)1 , a cytosolic enzyme that has an important role in scavenging hydrogen peroxide ( davletova et al . , 2005 ) .
apx1 might therefore function in preventing premature radical coupling of monolignols already in the cytosol .
thus , exemplified by the lignin - related analysis , we propose that comparative co - expression analyses might be useful in the future to reveal novel players for different aspects of cell wall biosynthesis , but also in high - lighting conserved and divergent elements in different biological processes .
a ) consensus co - expression network of lignin formation across seven plant species obtained from the networkcomparer - tool from planet ( http://aranet.mpimp-golm.mpg.de/aranet/networkcomparer ) .
co - expression network of following genes / probeset ids were used : arabidopsis , at2g37040 ; rice , loc_os02g41650 ; medicago , medtr1g076720 ; barley , contig1805_s_at ; poplar , ptpaffx.1672.3.a1_a_at ; wheat , taaffx.45277.1.s1_x_at .
( b ) monolignol biosynthesis pathway ( adopted from boerjan et al . , 2003 ) .
arabidopsis genes present in the co - expression network of atpal1 were mapped on the pathway .
question marks indicate that the function of the respective genes has not been shown yet .
co - expression approaches have been especially valuable for identifying new genes involved in secondary cell wall synthesis ( brown et al .
, 2005,2011 ; persson et al . , 2005 ; jensen et al . , 2010 ; ruprecht et al . ,
a comparison of the candidate genes from several different studies showed that similar results were obtained regardless of the underlying microarray datasets ( i.e. , condition - independent or stem - specific samples ) and the bait genes ( xylan or cellulose synthesis - related ) used ( oikawa et al . , 2010 ) .
this is probably due to the fact that secondary cell wall formation is a highly coordinated process that is mainly restricted to certain tissue and cell types .
however , many genes that are transcriptionally associated with secondary cell wall formation have already been investigated , and mutant analyses targeting only one gene are likely to only yield mild or no phenotypes ( jensen et al . , 2010 ; brown et al . , 2011 ; ruprecht et al . , 2011 ) .
one likely reason for this is genetic redundancy , and hence , mutant combinations or knock - down approaches that target several homologous genes might be needed in the future to generate informative phenotypes . in addition , detailed comparative transcriptional studies across and within species might be needed to obtain more reliable candidate genes related to cell wall synthesis .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 global transcript analyses based on publicly available microarray dataset have revealed that genes with similar function tend to be transcriptionally coordinated . 
 indeed , many genes involved in the formation of cellulose , hemicelluloses , and lignin have been identified using co - expression approaches in arabidopsis . to facilitate these transcript analyses , 
 several web - based tools have been developed that allow researchers to investigate co - expression relationships of their gene(s ) of interest . 
 in addition , several tools now also provide the possibility of comparative transcriptional analyses across species , which potentially increases the predictive power . in this short review 
 , we describe recent developments and updates of plant - related co - expression tools , and summarize studies that have successfully used expression profiling in cell wall research . 
 finally , we illustrate the value of comparative co - expression relationships across species using genes involved in lignin biosynthesis . 
INTRODUCTION
CO-EXPRESSION TOOLS
TRANSCRIPTIONAL CO-ORDINATION OF GENES INVOLVED IN CELLWALL BIOSYNTHESIS
COMPARATIVE EXPRESSION ANALYSES OF CELL WALL-RELATED GENES ACROSS SPECIES
USING A COMPARATIVE CO-EXPRESSION TOOL TO ANALYZE LIGNIN BIOSYNTHESIS
CONCLUSION AND PERSPECTIVE
Conflict of Interest Statement