BACKGROUND
transcription factors  play an important role in the regulation of gene expression.
the arabidopsis thaliana  tf databases agris  <cit> , rartf  <cit>  or datf  <cit>  contain approximately  <dig> entries, that correspond to  <dig> % of the  <dig> protein coding genes present in the tair <dig> genome release.
forward and reverse genetics, inducible expression systems and, more recently, large scale methods, such as chromatin immunoprecipitation followed by array hybridization or massive parallel sequencing, have provided a vast amount of information regarding target genes and functions of many arabidopsis tfs.
however, obtaining a complete overview of the transcriptional interactions for a given organism or developmental process is still a challenging and expensive task.
brady et al.
obtained a stele-enriched root trn containing protein-dna and protein-protein interactions identified by y1h and y2h assays using stele-enriched tfs, the promoters of these same tfs, and promoters from several mirna coding genes  <cit> .
however, the low percentage of tf promoters bound by at least one tf and the little overlap in expression enrichment between tfs and their targets suggest that several genes that might be important components of the stele trn could have been missed in this network.
gene expression microarrays allow for the rapid quantification of the expression level for a large number of genes in a given biological sample.
the most used arabidopsis gene expression microarray is the affymetrix ath1- <dig>  genechip microarray.
as of october  <dig>  there were  <dig> experiments using the ath <dig> chip listed in the ebi arrayexpress database  <cit> .
all of these experiments provide a quantitative analysis of gene expression in arabidopsis tissues under a variety of experimental conditions and are therefore a suitable data source for arabidopsis transcriptional regulatory network  inference.
although databases such as genevestigator  <cit> , atted-ii  <cit> , or bar expression angler  <cit>  have tools for the analysis of arabidopsis microarray data, they either use a limited set of microarray experiments, the atgenexpress series  <cit>  , or their quality controlled, curated, annotated and normalized data is not publicly available .
in light of these limitations we decided to create our own curated and annotated arabidopsis microarray database and use this data to infer trns.
the  <dig> microarray experiments indexed in the arrayexpress database contain over  <dig> individual chip hybridizations or cel files.
preliminary work done in our lab showed that network inference from samples obtained from different tissues, for example whole plant, roots and leaves, yields sub-optimal results and the inferred networks are difficult to interpret in a biological context.
we therefore decided to use microarray data obtained from a single organ, namely roots.
the arabidopsis root has several characteristics that make it a suitable organ for our purposes: root anatomy is relatively simple and developmental alterations can be readily observed, there is a vast amount of literature regarding root development and root-expressed tfs and, finally, there is a considerable amount of high quality ath <dig> microarray data obtained from root samples.
in order to identify the transcriptional interactions occurring in root tissues we used the microarray data as input for the aracne algorithm  <cit> .
aracne is an information-theoretical method for identifying transcriptional interactions between gene products using microarray expression profile data, which is able to recover non-linear statistical dependencies between variables and has been previously used for trn reconstruction  <cit> .
in this work we show that our database, and the trns derived from it, have been able to recover functions and target genes for previously characterized tfs.
we further show that the inferred trns can accurately predict new tf functions, as exemplified by the predicted role of the mads-box tf xal1/agl <dig>  in secondary cell wall formation and its confirmation with loss-of-function mutant root phenotypes for this gene.
RESULTS
in order to infer the trns underlying root development and physiological processes in arabidopsis, we used two carefully curated datasets obtained from  <dig> root-specific cel files from  <dig> ath <dig> microarray experiments .
the first dataset, that we call the tfs-only dataset, is a  <dig> columns by  <dig> rows table that corresponds to our list of  <dig> tf probesets.
the second dataset, that we call the complete dataset, is a  <dig> by  <dig> table that contains all  <dig> probesets present in the ath <dig> chip.
we used both datasets as input for the aracne software  <cit> .
the aracne output is a list of interacting probeset pairs ranked through a mutual information value and its associated p-value.
details for the theoretical background and practical use of aracne can be found in  <cit>  and  <cit>  but, briefly, an interaction between gene a and gene b means that the expression profile of gene a along all  <dig> experiments explains the expression profile of gene b along those same  <dig> experiments, and vice versa, as the interactions are not directed.
in a biological context, an interaction between gene a and gene b will imply that gene a and gene b participate in the same physiological process and, even further, if gene a is a tf and gene b is a non-tf, the interaction  will suggest that gene a is a transcriptional regulator of gene b.
network inference was centered on the  <dig> tf probesets present in the ath <dig> chip and was obtained at three data processing inequality  values,  <dig> ,  <dig>  and  <dig> .
dpi is a known information-theoretical property and is explained in the supplementary manual in  <cit> .
briefly, at dpi  <dig> , when a three-node clique  is present, the interaction with the lowest mutual information will be removed, as this interaction is considered to represent an indirect interaction.
at dpi values other than  <dig> , three genes loops are allowed and, at dpi  <dig> , no interactions are removed.
a dpi value of  <dig>   increases the recovery of true positive interactions while still minimizing the recovery of false positives  <cit> .
after translation of the aracne output adjacency files into cytoscape compatible tables, we obtained the corresponding tfs-only  and complete  databases.
as shown in table  <dig>  the number of edges increases dramatically from dpi  <dig>  to dpi  <dig>  to dpi  <dig> .
for clarity, all graphical representations of the networks in this paper are those obtained at dpi  <dig> .
nodes and edges present at dpi  <dig>  are part of the network obtained at dpi  <dig>  and both are part of the network obtained at dpi  <dig> .
tfs participating in inferred interactions are expressed in roots
an important question regarding our networks is to determine if the tfs participating in the inferred interactions are actually being expressed in root tissues.
the mas5calls function from the affy r package, used to flag microarray expression values as present, absent or marginal, is an unreliable tool to determine if a gene is being expressed or not  <cit> , specially when it involves arabidopsis tfs  <cit> .
therefore, in order to determine if the tfs present in our networks are expressed in root tissues, we extracted from both the tfsnet and fullnet obtained at dpi  <dig>  all tfs that participate in an interaction and we compared both lists to lists of experimentally determined root-expressed genes .
results are presented in table  <dig> and additional file  <dig>  over 92% of the recovered tfs in the two types of networks have been experimentally determined to be expressed in roots.
we are therefore confident that the tfs present in our datasets are indeed root tfs and the interactions that we have recovered represent true in planta transcriptional interactions.
tfs that participate in the same processes are grouped together in the tfsnet
the tfsnet was obtained from a tfs-only dataset that excludes all non-tf genes and constitutes an overview of arabidopsis roots tfs inferred interactions .
tfs participating in the same processes are expected to be grouped together in distinct clusters or modules.
some of these functional modules have been identified and experimentally characterized and serve as probes of the reliability of the inferred networks.
two transcriptional pathways controlling stem-cell niche patterning have been identified  <cit> .
the first pathway is composed of the gras-family short root  and scarecrow  and the c2h2-family, indeterminate domain  magpie  and jackdaw .
as shown in figure 1b, these four tfs are grouped together with idd nutcracker , the ssxt-domain transcriptional co-activator angustifolia  <dig>  and the gras-family scarecrow-like  <dig> .
nuc and scl- <dig> are proposed direct transcriptional targets of shr  <cit> .
note that, as networks obtained at dpi  <dig>  cannot contain triangles, the absence of an edge, for example between shr and nuc, does not imply a lack of interaction between these two genes but merely that both genes have other interactions with better mi scores.
also, interactions between the genes in this module have relatively low mi values, corresponding to p-values of 1e- <dig> and 1e- <dig> .
this is probably not surprising since this pathway has a complex mode of molecular interaction  <cit>  that will hinder the ability of the aracne algorithm to recover their interaction from microarray data with a higher p-value  <cit> .
additional idd genes, atidd <dig> , atidd <dig> , atidd <dig> , atidd <dig>  and atidd <dig> , are present in this module.
protein-protein interactions have been reported for scl-3-nuc, mgp-scr, mgp-shr, mgp-jkd, scr-jkd, and shr-jkd  <cit> .
on the other hand, the idd proteins jkd and mgp regulate shr and scr expression and movement across root tissues via both transcriptional and protein-protein interactions  <cit> .
finally, movement of the shr protein is abolished by the substitution of a single threonine residue in its vhiid motif, which is proposed to mediate protein-protein interactions of shr  <cit>  and its nuclear localization  <cit> .
it is therefore interesting to speculate that atidd <dig>  atidd <dig>  atidd <dig>  atidd <dig> and atidd <dig> could also be involved in root development and patterning via transcriptional regulation of, or protein-protein interactions with shr and scr.
the second pathway involves auxin signaling through the activation of as yet unidentified auxin response factors  and the plethora  tfs, of the ap2-erebp family.
the plt genes, plt <dig> , plt <dig> , plt3/ail <dig>  and baby boom , have overlapping expression profiles and act in a redundant manner  <cit> .
in the tfsnet, the four plt genes are part of the same group, that also includes the bhlh spatula , arf5/monopteros  and the erf-family cytokinin response factors crf2/tmo <dig>  and crf <dig> .
remarkably, the four plethora proteins  <cit>  and arf <dig> <cit>  are all expressed in the seedling root stele initials.
root vascular patterning has been shown to be dependent on an auxin-cytokinin cross-talk  <cit>  and the participation in this cross-talk of a few genes, such as shy <dig> <cit> , brx <cit>  or ahp <dig> <cit>  has been demonstrated.
however, a transcriptional network linking the plethora pathway and cytokinin responsive tfs is still missing.
the presence of two crf tfs in this module provides new clues in this direction.
bodenlos , a member of the aux/iaa family, is a transcriptional inhibitor of arf <dig> and its expression is controlled by arf <dig> in embryos  <cit> .
curiously, bdl, as well as two other target of monopteros  genes, ataig1/tmo <dig>  and tmo <dig> , do not group with arf <dig> in the tfsnet.
instead, they are part of a group of tfs involved in vascular development that includes genes such as iaa <dig> , iaa3/shy <dig>   <cit> , athb-14/phabulosa , athb-15/corona   <cit> , ifl/revoluta   <cit> , athb- <dig>   <cit> , athb9/phavoluta  and attcp <dig>   <cit>  .
pbdl::gfp expression has been observed in the root stele of 4–5 days-old seedlings , thus pointing to possible novel roles for these auxin-related genes in vascular development.
other tfs involved in organ development are also grouped together in the tfsnet.
for example, the closely related atham <dig> , atham <dig>  and atham <dig>  genes, belonging to the gras family, are involved in the maintenance of meristem indeterminacy, and are functionally redundant  <cit> .
these three tfs also group in the same module in the tfsnet that we inferred .
another example concerns the atgrf genes, of the grf family, which are expressed in developing tissues, such as shoot tips, flower buds and roots.
single mutants of the atgrf <dig> , atgrf <dig>  or atgrf <dig>  genes have no phenotype and double mutants have minor phenotypes  <cit> , suggesting that these three genes have redundant roles.
atgrf <dig>  atgrf <dig> and atgrf <dig> group together in the tfsnet put forward here .
interestingly, our network inference also recovers the interactions atgrf3-an <dig>  and an3-scr , suggesting a link between the atgrf module and the shr-scr module during root development.
the tfsnet also recovers transcriptional interactions between genes known to participate in root physiological processes other than development.
a first example concerns genes involved in jasmonate response .
this group includes the tify domain genes jaz <dig> , jaz <dig> , jaz <dig> , jaz <dig> , jaz <dig> , jaz <dig> , jaz <dig> , jas1/jaz <dig> , two wrky genes involved in pathogen response, wrky <dig>  and wrky <dig>   <cit> , the bhlh-family aib   <cit>  and myc <dig>   <cit>  and the ap2/erf rrtf <dig> .
interestingly, chromatin immunoprecipitation experiments have shown that wrky <dig> binds jaz <dig> and rrtf <dig> regulatory regions  <cit> , while myc <dig> was recently shown to be involved in jasmonate-dependent root development inhibition  <cit> .
a second example includes the bhlh tf bhlh <dig> , bhlh <dig> , bhlh <dig> , bhlh <dig> , popeye  and the dna-binding protein-coding brutus , which are involved in iron deficiency stress regulation  <cit> .
bhlh <dig>  bhlh <dig>  pye and bts are grouped together in the tfsnet .
a third example involves nitrate response tfs  <cit> .
the earliest tfs to be expressed in response to nitrate stimulus are hrs <dig> , lbd <dig> , ldb <dig> , lbd <dig>  and at3g <dig> .
four of these five tfs, hrs <dig>  ldb <dig>  lbd <dig> and at3g <dig> are grouped together in the tfsnet .
note that the microarray data for long et al., e-geod- <dig>  and krouk et al., e-geod- <dig> in the ebi database, were released a few days after our microarray experiments download and are not part of the data used for our analysis.
using the fullnet to integrate and analyze high-throughput functional genomics data
the fullnet was obtained from data which included all  <dig> probesets present in the ath <dig> chip, and was centered on the  <dig> tf probesets list .
in this network, tfs will be central nodes, with their interactors, either tfs or non-tfs, as neighboring nodes.
genes participating in the same processes should again be grouped together.
for example, the tf groups identified in the tfsnet are still present in the same groups in the fullnet.
one must bear in mind that, in this network, non-tf nodes are present.
when a non-tf interacts with two tfs, and these interactions have better mi scores than the tf-tf interaction, then the latter interaction will, at dpi  <dig> , be considered an indirect interaction, and thus will not appear in the network.
however, this does not mean that the tf-tf interaction does not exist, only that it is “masked” by an intermediary non-tf node.
when the tf-tf mi value is not the lowest in a triangle it is visible in the dpi  <dig>  fullnet.
this is the case for the interactions between plt <dig>  plt <dig> and plt3/ail <dig>  at p-values of 1e- <dig>  the scr-shr interaction at a p-value of 1e- <dig>  the interaction of the early nitrate-responsive tf hrs <dig> with lbd <dig>  ldb <dig> and at3g <dig> at p-values of 1e- <dig> and lower, as well as the interaction of bhlh <dig> with bhlh <dig> at a p-value of 1e- <dig> and with pye at a p-value of 1e- <dig>  interaction between agl <dig> and agl <dig>  which was present at a p-value of 1e- <dig> in the tfsnet, is now recovered with a p-value of 1e- <dig>  these two mads-box genes have recently been shown to act redundantly in apical and axillary meristems  <cit> .
in the fullnet, interactors of a tf node are potential target genes for that tf.
if this is the case, one would expect a significant number of experimentally identified target genes for that tf to be present in the corresponding lists of aracne interactors.
one example of a tf for which aracne-inferred interactions are confirmed experimentally corresponds to vnd7/anac <dig> .
vnd <dig> is a nac-family tf involved in secondary cell wall synthesis and several lists of its putative target genes are available  <cit> .
we compared these lists of experimentally identified vnd <dig> target genes with our list of vnd <dig> interactors from the complete dataset at dpi  <dig> ,  <dig>  and  <dig>  .
<dig> out of  <dig> genes at dpi  <dig> ,  <dig> out of  <dig> at dpi  <dig>  and  <dig> out of  <dig> at dpi  <dig>  from our vnd <dig> neighbor list are differentially expressed in at least one of the experimental settings.
almost all differentially expressed genes are found at high mi values, corresponding to p-values of 1e- <dig> and lower.
finally, three of the four differentially expressed tfs identified by yamaguchi et al.
<cit> , jlo , myb <dig>  and myb <dig> , are part of the vnd <dig> cluster in the tfsnet, at p-values of 1e- <dig> and lower.
curiously, a top-ranked vnd <dig> interactor in our dataset, the pinoresinol reductase atprr <dig> , is not present in any of the experimental vnd <dig> target genes lists.
atprr <dig> has, at dpi  <dig> , tf interactors with higher mi values than vnd <dig>  suggesting that it could instead be regulated by one, or more, of these higher-score tfs.
alternatively, the vnd7-atprr <dig> transcriptional interaction could be age-specific and not detectable in any of the above-mentioned experimental settings.
anac030
/
vnd <dig>
interactors in the fullnet obtained at dpi  <dig>
mi: mutual information value.
the target gene column indicates if the gene is an experimentally identified target gene in  <cit> ,  <cit> , an upregulated gene in  <cit>  or a putative direct target gene in  <cit> .
there are also examples of tfs for which there is little overlap between aracne-inferred interactors lists and experimental target gene lists.
two examples are the shr and scr tfs.
shr and scr are important genes for root development and several lists of their proposed transcriptional target genes are available  <cit> .
sozzani et al.
<cit>  obtained, through microarray data analysis, a comprehensive list of differentially expressed genes during a time-course of scr or shr induction, while cui et al.
<cit>  identified shr target genes through chromatin inmunoprecipitation .
a direct comparison of the target gene lists from sozzani et al., to which we will refer as the sozzani-scr and sozzani-shr lists, to our aracne list of inferred scr or shr interactors obtained at dpi  <dig> ,  <dig>  and  <dig> , resulted in a low overall overlap: there are  <dig> aracne-scr and  <dig> aracne-shr interactors at dpi  <dig> , of which  <dig>  and  <dig>  were found in the corresponding scr- or shr-sozzani lists.
in particular, we would expect to find in both the aracne and sozzani lists genes known to participate in the shr-scr transcriptional regulation pathway, namely jkd, mgp, nuc and cycd6; <dig> .
the first three genes are tfs and they can be found in the same module as shr and scr in the tfsnet.
cycd6; <dig>  a non-tf, is present in both the scr-sozzani and shr-sozzani lists, but is not an aracne-inferred interactor of shr, scr, jkd, mgp nor nuc.
at dpi  <dig>  its only interacting tf is agl <dig> , which is not close to the shr-scr module in either the tfsnet or fullnet.
while disappointing, this result is perhaps not surprising: cycd6; <dig> is expressed in very particular wild type root cell types, the cortex/endodermis initial stem cells and lateral root primordium endodermal cells  <cit> .
furthermore, cycd6; <dig> participates in a complex regulatory mechanism involving protein-protein interactions, protein phosphorylation and protein degradation  <cit> .
it is likely that these mechanisms are poorly translated into transcript levels of the corresponding genes in whole root samples, which is the input data for aracne.
the ability of aracne to recover experimentally identified tf target genes will most likely mirror the number and complexity of the regulatory interactions in which that tf participates.
vnd <dig> is a tf involved exclusively in secondary cell-wall synthesis   <cit> .
as such, we expect vnd <dig> to participate in a very specific transcriptional module, and aracne to accurately recover its experimentally identified target genes.
on the other hand, shr and scr are most likely involved in numerous transcriptional pathways, as mutants for these genes are strongly affected in root development  <cit> , and over  <dig> tfs can be found in the lists of differentially expressed genes for shr or scr inductions, which analyzed a specific root cell-type, i.e.
ground tissue  <cit> .
such an important number of differentially expressed tfs  further suggests that a significant number of these experimentally identified target genes are indirect targets.
additionally, regulation of root development by scr and shr involves expression in defined cell types, transport across cell-types, nucleus-cytoplasm translocation, protein-protein interactions and protein phosphorylation  <cit> .
in this case, we expect that better results could be obtained by visualizing experimentally identified target genes in the context of the networks where they participate.
we therefore decided to retrieve from the fullnet dataset, obtained at dpi  <dig>  and with a cutoff p-value of 1e- <dig>  all interactions for which both nodes are present in the list of  <dig> differentially expressed genes in the shr induction kinetic from sozzani and collaborator’s study  <cit> , to which we added shr .
the resulting dataset now contains  <dig> genes  and the corresponding network was drawn with cytoscape  <cit> .
<dig> nodes , including shr, are grouped together in a single subnetwork .
we observe that this subnetwork is clearly divided in two sections, corresponding to genes that, as time progresses in the induction kinetic, switch from an under-expressed to an over-expressed state and vice versa.
an analysis of this subnetwork can now help identify relevant nodes, which should play important roles in the shr transcriptional pathway.
for example, three of the main nodes that switch from under- to over-expression are prmt <dig> , kyp  and hd2a , which are genes coding for chromatin modification  proteins.
an analysis of all genes that switch from under- to over-expression when using david  <cit>  and enrichment map  <cit>  reveals that this module is enriched, among others, in cell-cycle, microtubule, rna-processing and putative chromatin modification protein-coding genes .
aracne-inferred networks allow for the prediction of novel genetic interactions for root-expressed tfs: a possible role for spatula in the plethora pathway
the tfsnet was obtained from data which included exclusively our list of  <dig> tf probesets .
in this network, tfs that participate in the same biological process should be grouped together.
therefore, we expect higher order mutant plants for genes in a same module to exhibit root phenotypes not observed in single mutant plants.
we set to test this hypothesis with genes that are present in the same module, but 1) belong to different tf families, 2) are not immediate neighbors in the tfsnet, and 3) whose mutants have distinct root phenotypes.
the genes baby boom  and spatula  matched these criteria.
both genes are present in the same module , and mutants of the bbm gene, an ap2-domain tf, have slightly shorter roots  <cit> , while mutants of the spt gene, a bhlh tf, have slightly longer roots than wild type plants  <cit> .
when grown on vertical plates, the bbm-2/spt- <dig> double mutant exhibited longer roots than either spt- <dig> or bbm- <dig> single mutant seedlings .
a previous report showed that pin <dig> and dr5::gus expression is altered in the root meristem of spt- <dig> mutant seedlings  <cit> .
taken together, these results point to a possible transcriptional interaction between the plethora pathway and spatula in the regulation of auxin transport and/or response in arabidopsis root meristems.
aracne-inferred networks allow for the prediction of novel functions for root-expressed tfs: the case of xal1/agl <dig>  a mads-box tf involved in secondary cell-wall synthesis
since our aracne inferred networks are able to recover known gene associations, we expect them to also be able to predict novel tf functions.
as an example of the predictive power of our database, we decided to look for new tfs that could be participating in secondary cell wall synthesis .
for this aim, our strategy consisted in selecting several genes, both tf and non-tf, known to be involved in scws, recover their interactions from the fullnet and draw the resulting network in order to identify new scws tfs.
several tfs are known to be involved in scws, among which we chose vnd6/anac <dig> , vnd7/anac <dig>   <cit> , snd2/anac <dig>   <cit> , myb <dig> <cit>  and ixr <dig>   <cit> .
as scws non-tf genes we chose the cellulose synthases cesa <dig> , cesa <dig>  and cesa <dig>   <cit> , the laccases lac <dig>  and lac <dig>   <cit> , the cysteine peptidases xcp <dig>  and xcp <dig>   <cit> , the chitinase-like atctl <dig>   <cit> , the duf <dig> domain wat <dig>   <cit> , ted <dig>   <cit> , the duf <dig> domain tbl <dig>   <cit>  and the family  <dig> glycosyl-transferase gaut12/irx <dig>   <cit> .
we then retrieved from the fullnet all interactions involving these genes at dpi  <dig>  and a p-value cutoff of 1e- <dig> and used cytoscape  <cit>  to visualize the corresponding network .
it immediately appears that these genes are indeed part of a network of scws genes that includes our input genes plus several other known, or putative, scws genes including myb <dig>   <cit> , anac007/vnd <dig>   <cit>  or atprr <dig> <cit> , but also vascular development tfs like athb- <dig> <cit> , athb- <dig>   <cit>  and jlo  a target of vnd <dig>  <cit> .
in a highly connected part of this scws network,  <dig> tfs that were not part of our input gene list are now present .
we retrieved from the fullnet all interactions involving these tfs at dpi  <dig> ,  <dig>  and  <dig>  and a p-value cutoff of 1e- <dig>  an enrichment analysis, using david, of the lists of interactors for three of the newly identified tfs, xal1/agl <dig> , bee <dig> and at1g <dig>  revealed that they are particularly enriched in scws genes ; the lists of high mi value interactors for each tf are shown in additional file  <dig>  as these three tfs are present in the highly connected part of the scws network, it is not surprising to find that they share several of their interactors.
agl12/xal <dig> is a mads-box transcription factor that is expressed in phloem tissues and is involved in the regulation of both root development and flowering time  <cit> .
bee <dig> was first identified as a brassinosteroid-responsive tf  <cit> .
brassinosteroids promote root growth  <cit> , are essential for the development of the vascular system in arabidopsis stems  <cit>  and enhance xylem vessel transdifferentiation of arabidopsis suspension cultures  <cit> .
at1g <dig> is 1) a tf that we found as part of the vascular development cluster in the tfsnet, 2) closely related to ataig1/tmo <dig>  which is also part of the tfs-only vascular development cluster and 3) a protein-protein interactor of lonesome highway, a transcriptional activator involved in vascular development  <cit> .
these results predict that xal <dig>  bee <dig> and at1g <dig> are important tfs for scws.
as mads-box tfs are not usually associated with scws, we decided to look for scw deposition in xal1- <dig> loss-of-function mutant roots  <cit> .
since xal1- <dig> presents a delay in flowering time, roots from plants of the same chronological age might reveal developmental stage-related scws differences rather than a direct scws phenotype.
therefore, both col- <dig> and xal1- <dig> roots were collected when the main stem was 29–32 cm in length, which arguably corresponds to plants at the same developmental stage.
as predicted by our inferred network, xal1- <dig> adult roots indeed have altered secondary cell-wall patterns with gaps in the secondary xylem and fiber ring , a phenotype rarely observed in wild type plants of the same size .
in an intriguing paper, sibout et al.
have shown that xylem expansion in hypocotyls and roots is linked to flowering time  <cit> .
coincidentally, xal1- <dig> plants have delayed flowering  <cit>  and altered root scws, strongly suggesting that xal <dig> could be part of a trn that connects both processes.
the confirmation of scws alterations in xal1- <dig> root tissues shows that our bioinformatics methodology to infer trns is a successful approach for the accurate prediction of novel functions for root-expressed tfs.
this result further strengthens that our networks will likely provide novel hypothesis concerning functional modules involved in root development.
as an additional example, the duf <dig> protein wat <dig>  <cit>  has, at dpi  <dig>  and a p-value cutoff of 1e- <dig>  the tf interactors athb-15/cna, at1g <dig>  at4g <dig>  sth <dig>  athb- <dig> and at2g <dig>  all of which are part of the vascular development cluster of the tfsnet .
this suggests, first, that one, or more, of these tfs is the transcriptional regulator of wat <dig> in root tissues and, second, that one, or more, of these tfs control vascular development, at least partly, through the direct transcriptional regulation of wat <dig>  finally, the duf6-domain protein-coding genes at1g <dig>  at1g <dig>  at3g <dig>  at3g <dig> and at4g <dig> are interactors of tfs known to be involved in scws, suggesting that they might have similar roles to wat <dig> in root scws.
CONCLUSIONS
in this work we show that network inference from multiple compounded, carefully selected and curated microarray datasets allows for the reconstruction of reliable root transcriptional interaction networks.
we show that such inferred networks recover both known, functionally characterized tf modules and reliably predict novel components of such modules, as well as novel modules, including unexpected roles for particular tfs.
we particularly highlight the discovery of a new module underlying secondary cell wall synthesis that involves the mads-box tf xal1/agl <dig>  our transcriptional interactions database further provides an overview of the transcriptional pathways present in arabidopsis roots and will likely yield a plethora of novel hypotheses to be tested experimentally.

 transcription factors play an important role in the regulation of gene expression. the arabidopsis thaliana tf databases agris, rartf or datf contain approximately entries, that correspond to % of the protein coding genes present in the tair genome release. forward and reverse genetics, inducible expression systems and, more recently, large scale methods, such as chromatin immunoprecipitation followed by array hybridization or massive parallel sequencing, have provided a vast amount of information regarding target genes and functions of many arabidopsis tfs. however, obtaining a complete overview of the transcriptional interactions for a given organism or developmental process is still a challenging and expensive task. brady et al. obtained a stele-enriched root trn containing protein-dna and protein-protein interactions identified by y1h and y2h assays using stele-enriched tfs, the promoters of these same tfs, and promoters from several mirna coding genes. however, the low percentage of tf promoters bound by at least one tf and the little overlap in expression enrichment between tfs and their targets suggest that several genes that might be important components of the stele trn could have been missed in this network. gene expression microarrays allow for the rapid quantification of the expression level for a large number of genes in a given biological sample. the most used arabidopsis gene expression microarray is the affymetrix ath1- genechip microarray. as of october there were experiments using the ath chip listed in the ebi arrayexpress database. all of these experiments provide a quantitative analysis of gene expression in arabidopsis tissues under a variety of experimental conditions and are therefore a suitable data source for arabidopsis transcriptional regulatory network inference. although databases such as genevestigator, atted-ii, or bar expression angler have tools for the analysis of arabidopsis microarray data, they either use a limited set of microarray experiments, the atgenexpress series, or their quality controlled, curated, annotated and normalized data is not publicly available. in light of these limitations we decided to create our own curated and annotated arabidopsis microarray database and use this data to infer trns. the microarray experiments indexed in the arrayexpress database contain over individual chip hybridizations or cel files. preliminary work done in our lab showed that network inference from samples obtained from different tissues, for example whole plant, roots and leaves, yields sub-optimal results and the inferred networks are difficult to interpret in a biological context. we therefore decided to use microarray data obtained from a single organ, namely roots. the arabidopsis root has several characteristics that make it a suitable organ for our purposes: root anatomy is relatively simple and developmental alterations can be readily observed, there is a vast amount of literature regarding root development and root-expressed tfs and, finally, there is a considerable amount of high quality ath microarray data obtained from root samples. in order to identify the transcriptional interactions occurring in root tissues we used the microarray data as input for the aracne algorithm. aracne is an information-theoretical method for identifying transcriptional interactions between gene products using microarray expression profile data, which is able to recover non-linear statistical dependencies between variables and has been previously used for trn reconstruction. in this work we show that our database, and the trns derived from it, have been able to recover functions and target genes for previously characterized tfs. we further show that the inferred trns can accurately predict new tf functions, as exemplified by the predicted role of the mads-box tf xal1/agl in secondary cell wall formation and its confirmation with loss-of-function mutant root phenotypes for this gene.  in order to infer the trns underlying root development and physiological processes in arabidopsis, we used two carefully curated datasets obtained from root-specific cel files from ath microarray experiments. the first dataset, that we call the tfs-only dataset, is a columns by rows table that corresponds to our list of tf probesets. the second dataset, that we call the complete dataset, is a by table that contains all probesets present in the ath chip. we used both datasets as input for the aracne software. the aracne output is a list of interacting probeset pairs ranked through a mutual information value and its associated p-value. details for the theoretical background and practical use of aracne can be found in and but, briefly, an interaction between gene a and gene b means that the expression profile of gene a along all experiments explains the expression profile of gene b along those same experiments, and vice versa, as the interactions are not directed. in a biological context, an interaction between gene a and gene b will imply that gene a and gene b participate in the same physiological process and, even further, if gene a is a tf and gene b is a non-tf, the interaction will suggest that gene a is a transcriptional regulator of gene b. network inference was centered on the tf probesets present in the ath chip and was obtained at three data processing inequality values,, and. dpi is a known information-theoretical property and is explained in the supplementary manual in. briefly, at dpi, when a three-node clique is present, the interaction with the lowest mutual information will be removed, as this interaction is considered to represent an indirect interaction. at dpi values other than, three genes loops are allowed and, at dpi, no interactions are removed. a dpi value of increases the recovery of true positive interactions while still minimizing the recovery of false positives. after translation of the aracne output adjacency files into cytoscape compatible tables, we obtained the corresponding tfs-only and complete databases. as shown in table  the number of edges increases dramatically from dpi to dpi to dpi. for clarity, all graphical representations of the networks in this paper are those obtained at dpi. nodes and edges present at dpi are part of the network obtained at dpi and both are part of the network obtained at dpi. tfs participating in inferred interactions are expressed in roots an important question regarding our networks is to determine if the tfs participating in the inferred interactions are actually being expressed in root tissues. the mas5calls function from the affy r package, used to flag microarray expression values as present, absent or marginal, is an unreliable tool to determine if a gene is being expressed or not, specially when it involves arabidopsis tfs. therefore, in order to determine if the tfs present in our networks are expressed in root tissues, we extracted from both the tfsnet and fullnet obtained at dpi all tfs that participate in an interaction and we compared both lists to lists of experimentally determined root-expressed genes. results are presented in table  and additional file over 92% of the recovered tfs in the two types of networks have been experimentally determined to be expressed in roots. we are therefore confident that the tfs present in our datasets are indeed root tfs and the interactions that we have recovered represent true in planta transcriptional interactions. tfs that participate in the same processes are grouped together in the tfsnet the tfsnet was obtained from a tfs-only dataset that excludes all non-tf genes and constitutes an overview of arabidopsis roots tfs inferred interactions. tfs participating in the same processes are expected to be grouped together in distinct clusters or modules. some of these functional modules have been identified and experimentally characterized and serve as probes of the reliability of the inferred networks. two transcriptional pathways controlling stem-cell niche patterning have been identified. the first pathway is composed of the gras-family short root and scarecrow and the c2h2-family, indeterminate domain magpie and jackdaw. as shown in figure 1b, these four tfs are grouped together with idd nutcracker, the ssxt-domain transcriptional co-activator angustifolia and the gras-family scarecrow-like. nuc and scl- are proposed direct transcriptional targets of shr. note that, as networks obtained at dpi cannot contain triangles, the absence of an edge, for example between shr and nuc, does not imply a lack of interaction between these two genes but merely that both genes have other interactions with better mi scores. also, interactions between the genes in this module have relatively low mi values, corresponding to p-values of 1e- and 1e-. this is probably not surprising since this pathway has a complex mode of molecular interaction that will hinder the ability of the aracne algorithm to recover their interaction from microarray data with a higher p-value. additional idd genes, atidd, atidd, atidd, atidd and atidd, are present in this module. protein-protein interactions have been reported for scl-3-nuc, mgp-scr, mgp-shr, mgp-jkd, scr-jkd, and shr-jkd. on the other hand, the idd proteins jkd and mgp regulate shr and scr expression and movement across root tissues via both transcriptional and protein-protein interactions. finally, movement of the shr protein is abolished by the substitution of a single threonine residue in its vhiid motif, which is proposed to mediate protein-protein interactions of shr and its nuclear localization. it is therefore interesting to speculate that atidd atidd atidd atidd and atidd could also be involved in root development and patterning via transcriptional regulation of, or protein-protein interactions with shr and scr. the second pathway involves auxin signaling through the activation of as yet unidentified auxin response factors and the plethora tfs, of the ap2-erebp family. the plt genes, plt, plt, plt3/ail and baby boom, have overlapping expression profiles and act in a redundant manner. in the tfsnet, the four plt genes are part of the same group, that also includes the bhlh spatula, arf5/monopteros and the erf-family cytokinin response factors crf2/tmo and crf. remarkably, the four plethora proteins and arf are all expressed in the seedling root stele initials. root vascular patterning has been shown to be dependent on an auxin-cytokinin cross-talk and the participation in this cross-talk of a few genes, such as shy, brx or ahp has been demonstrated. however, a transcriptional network linking the plethora pathway and cytokinin responsive tfs is still missing. the presence of two crf tfs in this module provides new clues in this direction. bodenlos, a member of the aux/iaa family, is a transcriptional inhibitor of arf and its expression is controlled by arf in embryos. curiously, bdl, as well as two other target of monopteros genes, ataig1/tmo and tmo, do not group with arf in the tfsnet. instead, they are part of a group of tfs involved in vascular development that includes genes such as iaa, iaa3/shy, athb-14/phabulosa, athb-15/corona, ifl/revoluta, athb-, athb9/phavoluta and attcp. pbdl::gfp expression has been observed in the root stele of 4–5 days-old seedlings, thus pointing to possible novel roles for these auxin-related genes in vascular development. other tfs involved in organ development are also grouped together in the tfsnet. for example, the closely related atham, atham and atham genes, belonging to the gras family, are involved in the maintenance of meristem indeterminacy, and are functionally redundant. these three tfs also group in the same module in the tfsnet that we inferred. another example concerns the atgrf genes, of the grf family, which are expressed in developing tissues, such as shoot tips, flower buds and roots. single mutants of the atgrf, atgrf or atgrf genes have no phenotype and double mutants have minor phenotypes, suggesting that these three genes have redundant roles. atgrf atgrf and atgrf group together in the tfsnet put forward here. interestingly, our network inference also recovers the interactions atgrf3-an and an3-scr, suggesting a link between the atgrf module and the shr-scr module during root development. the tfsnet also recovers transcriptional interactions between genes known to participate in root physiological processes other than development. a first example concerns genes involved in jasmonate response. this group includes the tify domain genes jaz, jaz, jaz, jaz, jaz, jaz, jaz, jas1/jaz, two wrky genes involved in pathogen response, wrky and wrky, the bhlh-family aib and myc and the ap2/erf rrtf. interestingly, chromatin immunoprecipitation experiments have shown that wrky binds jaz and rrtf regulatory regions, while myc was recently shown to be involved in jasmonate-dependent root development inhibition. a second example includes the bhlh tf bhlh, bhlh, bhlh, bhlh, popeye and the dna-binding protein-coding brutus, which are involved in iron deficiency stress regulation. bhlh bhlh pye and bts are grouped together in the tfsnet. a third example involves nitrate response tfs. the earliest tfs to be expressed in response to nitrate stimulus are hrs, lbd, ldb, lbd and at3g. four of these five tfs, hrs ldb lbd and at3g are grouped together in the tfsnet. note that the microarray data for long et al., e-geod- and krouk et al., e-geod- in the ebi database, were released a few days after our microarray experiments download and are not part of the data used for our analysis. using the fullnet to integrate and analyze high-throughput functional genomics data the fullnet was obtained from data which included all probesets present in the ath chip, and was centered on the tf probesets list. in this network, tfs will be central nodes, with their interactors, either tfs or non-tfs, as neighboring nodes. genes participating in the same processes should again be grouped together. for example, the tf groups identified in the tfsnet are still present in the same groups in the fullnet. one must bear in mind that, in this network, non-tf nodes are present. when a non-tf interacts with two tfs, and these interactions have better mi scores than the tf-tf interaction, then the latter interaction will, at dpi, be considered an indirect interaction, and thus will not appear in the network. however, this does not mean that the tf-tf interaction does not exist, only that it is “masked” by an intermediary non-tf node. when the tf-tf mi value is not the lowest in a triangle it is visible in the dpi fullnet. this is the case for the interactions between plt plt and plt3/ail at p-values of 1e- the scr-shr interaction at a p-value of 1e- the interaction of the early nitrate-responsive tf hrs with lbd ldb and at3g at p-values of 1e- and lower, as well as the interaction of bhlh with bhlh at a p-value of 1e- and with pye at a p-value of 1e- interaction between agl and agl which was present at a p-value of 1e- in the tfsnet, is now recovered with a p-value of 1e- these two mads-box genes have recently been shown to act redundantly in apical and axillary meristems. in the fullnet, interactors of a tf node are potential target genes for that tf. if this is the case, one would expect a significant number of experimentally identified target genes for that tf to be present in the corresponding lists of aracne interactors. one example of a tf for which aracne-inferred interactions are confirmed experimentally corresponds to vnd7/anac. vnd is a nac-family tf involved in secondary cell wall synthesis and several lists of its putative target genes are available. we compared these lists of experimentally identified vnd target genes with our list of vnd interactors from the complete dataset at dpi, and. out of genes at dpi, out of at dpi and out of at dpi from our vnd neighbor list are differentially expressed in at least one of the experimental settings. almost all differentially expressed genes are found at high mi values, corresponding to p-values of 1e- and lower. finally, three of the four differentially expressed tfs identified by yamaguchi et al., jlo, myb and myb, are part of the vnd cluster in the tfsnet, at p-values of 1e- and lower. curiously, a top-ranked vnd interactor in our dataset, the pinoresinol reductase atprr, is not present in any of the experimental vnd target genes lists. atprr has, at dpi, tf interactors with higher mi values than vnd suggesting that it could instead be regulated by one, or more, of these higher-score tfs. alternatively, the vnd7-atprr transcriptional interaction could be age-specific and not detectable in any of the above-mentioned experimental settings. anac030 / vnd interactors in the fullnet obtained at dpi mi: mutual information value. the target gene column indicates if the gene is an experimentally identified target gene in,, an upregulated gene in or a putative direct target gene in. there are also examples of tfs for which there is little overlap between aracne-inferred interactors lists and experimental target gene lists. two examples are the shr and scr tfs. shr and scr are important genes for root development and several lists of their proposed transcriptional target genes are available. sozzani et al. obtained, through microarray data analysis, a comprehensive list of differentially expressed genes during a time-course of scr or shr induction, while cui et al. identified shr target genes through chromatin inmunoprecipitation. a direct comparison of the target gene lists from sozzani et al., to which we will refer as the sozzani-scr and sozzani-shr lists, to our aracne list of inferred scr or shr interactors obtained at dpi, and, resulted in a low overall overlap: there are aracne-scr and aracne-shr interactors at dpi, of which and were found in the corresponding scr- or shr-sozzani lists. in particular, we would expect to find in both the aracne and sozzani lists genes known to participate in the shr-scr transcriptional regulation pathway, namely jkd, mgp, nuc and cycd6;. the first three genes are tfs and they can be found in the same module as shr and scr in the tfsnet. cycd6; a non-tf, is present in both the scr-sozzani and shr-sozzani lists, but is not an aracne-inferred interactor of shr, scr, jkd, mgp nor nuc. at dpi its only interacting tf is agl, which is not close to the shr-scr module in either the tfsnet or fullnet. while disappointing, this result is perhaps not surprising: cycd6; is expressed in very particular wild type root cell types, the cortex/endodermis initial stem cells and lateral root primordium endodermal cells. furthermore, cycd6; participates in a complex regulatory mechanism involving protein-protein interactions, protein phosphorylation and protein degradation. it is likely that these mechanisms are poorly translated into transcript levels of the corresponding genes in whole root samples, which is the input data for aracne. the ability of aracne to recover experimentally identified tf target genes will most likely mirror the number and complexity of the regulatory interactions in which that tf participates. vnd is a tf involved exclusively in secondary cell-wall synthesis. as such, we expect vnd to participate in a very specific transcriptional module, and aracne to accurately recover its experimentally identified target genes. on the other hand, shr and scr are most likely involved in numerous transcriptional pathways, as mutants for these genes are strongly affected in root development, and over tfs can be found in the lists of differentially expressed genes for shr or scr inductions, which analyzed a specific root cell-type, i.e. ground tissue. such an important number of differentially expressed tfs further suggests that a significant number of these experimentally identified target genes are indirect targets. additionally, regulation of root development by scr and shr involves expression in defined cell types, transport across cell-types, nucleus-cytoplasm translocation, protein-protein interactions and protein phosphorylation. in this case, we expect that better results could be obtained by visualizing experimentally identified target genes in the context of the networks where they participate. we therefore decided to retrieve from the fullnet dataset, obtained at dpi and with a cutoff p-value of 1e- all interactions for which both nodes are present in the list of differentially expressed genes in the shr induction kinetic from sozzani and collaborator’s study, to which we added shr. the resulting dataset now contains genes and the corresponding network was drawn with cytoscape. nodes, including shr, are grouped together in a single subnetwork. we observe that this subnetwork is clearly divided in two sections, corresponding to genes that, as time progresses in the induction kinetic, switch from an under-expressed to an over-expressed state and vice versa. an analysis of this subnetwork can now help identify relevant nodes, which should play important roles in the shr transcriptional pathway. for example, three of the main nodes that switch from under- to over-expression are prmt, kyp and hd2a, which are genes coding for chromatin modification proteins. an analysis of all genes that switch from under- to over-expression when using david and enrichment map reveals that this module is enriched, among others, in cell-cycle, microtubule, rna-processing and putative chromatin modification protein-coding genes. aracne-inferred networks allow for the prediction of novel genetic interactions for root-expressed tfs: a possible role for spatula in the plethora pathway the tfsnet was obtained from data which included exclusively our list of tf probesets. in this network, tfs that participate in the same biological process should be grouped together. therefore, we expect higher order mutant plants for genes in a same module to exhibit root phenotypes not observed in single mutant plants. we set to test this hypothesis with genes that are present in the same module, but 1) belong to different tf families, 2) are not immediate neighbors in the tfsnet, and 3) whose mutants have distinct root phenotypes. the genes baby boom and spatula matched these criteria. both genes are present in the same module, and mutants of the bbm gene, an ap2-domain tf, have slightly shorter roots, while mutants of the spt gene, a bhlh tf, have slightly longer roots than wild type plants. when grown on vertical plates, the bbm-2/spt- double mutant exhibited longer roots than either spt- or bbm- single mutant seedlings. a previous report showed that pin and dr5::gus expression is altered in the root meristem of spt- mutant seedlings. taken together, these results point to a possible transcriptional interaction between the plethora pathway and spatula in the regulation of auxin transport and/or response in arabidopsis root meristems. aracne-inferred networks allow for the prediction of novel functions for root-expressed tfs: the case of xal1/agl a mads-box tf involved in secondary cell-wall synthesis since our aracne inferred networks are able to recover known gene associations, we expect them to also be able to predict novel tf functions. as an example of the predictive power of our database, we decided to look for new tfs that could be participating in secondary cell wall synthesis. for this aim, our strategy consisted in selecting several genes, both tf and non-tf, known to be involved in scws, recover their interactions from the fullnet and draw the resulting network in order to identify new scws tfs. several tfs are known to be involved in scws, among which we chose vnd6/anac, vnd7/anac, snd2/anac, myb and ixr. as scws non-tf genes we chose the cellulose synthases cesa, cesa and cesa, the laccases lac and lac, the cysteine peptidases xcp and xcp, the chitinase-like atctl, the duf domain wat, ted, the duf domain tbl and the family glycosyl-transferase gaut12/irx. we then retrieved from the fullnet all interactions involving these genes at dpi and a p-value cutoff of 1e- and used cytoscape to visualize the corresponding network. it immediately appears that these genes are indeed part of a network of scws genes that includes our input genes plus several other known, or putative, scws genes including myb, anac007/vnd or atprr, but also vascular development tfs like athb-, athb- and jlo a target of vnd. in a highly connected part of this scws network, tfs that were not part of our input gene list are now present. we retrieved from the fullnet all interactions involving these tfs at dpi, and and a p-value cutoff of 1e- an enrichment analysis, using david, of the lists of interactors for three of the newly identified tfs, xal1/agl, bee and at1g revealed that they are particularly enriched in scws genes; the lists of high mi value interactors for each tf are shown in additional file as these three tfs are present in the highly connected part of the scws network, it is not surprising to find that they share several of their interactors. agl12/xal is a mads-box transcription factor that is expressed in phloem tissues and is involved in the regulation of both root development and flowering time. bee was first identified as a brassinosteroid-responsive tf. brassinosteroids promote root growth, are essential for the development of the vascular system in arabidopsis stems and enhance xylem vessel transdifferentiation of arabidopsis suspension cultures. at1g is 1) a tf that we found as part of the vascular development cluster in the tfsnet, 2) closely related to ataig1/tmo which is also part of the tfs-only vascular development cluster and 3) a protein-protein interactor of lonesome highway, a transcriptional activator involved in vascular development. these results predict that xal bee and at1g are important tfs for scws. as mads-box tfs are not usually associated with scws, we decided to look for scw deposition in xal1- loss-of-function mutant roots. since xal1- presents a delay in flowering time, roots from plants of the same chronological age might reveal developmental stage-related scws differences rather than a direct scws phenotype. therefore, both col- and xal1- roots were collected when the main stem was 29–32 cm in length, which arguably corresponds to plants at the same developmental stage. as predicted by our inferred network, xal1- adult roots indeed have altered secondary cell-wall patterns with gaps in the secondary xylem and fiber ring, a phenotype rarely observed in wild type plants of the same size. in an intriguing paper, sibout et al. have shown that xylem expansion in hypocotyls and roots is linked to flowering time. coincidentally, xal1- plants have delayed flowering and altered root scws, strongly suggesting that xal could be part of a trn that connects both processes. the confirmation of scws alterations in xal1- root tissues shows that our bioinformatics methodology to infer trns is a successful approach for the accurate prediction of novel functions for root-expressed tfs. this result further strengthens that our networks will likely provide novel hypothesis concerning functional modules involved in root development. as an additional example, the duf protein wat has, at dpi and a p-value cutoff of 1e- the tf interactors athb-15/cna, at1g at4g sth athb- and at2g all of which are part of the vascular development cluster of the tfsnet. this suggests, first, that one, or more, of these tfs is the transcriptional regulator of wat in root tissues and, second, that one, or more, of these tfs control vascular development, at least partly, through the direct transcriptional regulation of wat finally, the duf6-domain protein-coding genes at1g at1g at3g at3g and at4g are interactors of tfs known to be involved in scws, suggesting that they might have similar roles to wat in root scws.  in this work we show that network inference from multiple compounded, carefully selected and curated microarray datasets allows for the reconstruction of reliable root transcriptional interaction networks. we show that such inferred networks recover both known, functionally characterized tf modules and reliably predict novel components of such modules, as well as novel modules, including unexpected roles for particular tfs. we particularly highlight the discovery of a new module underlying secondary cell wall synthesis that involves the mads-box tf xal1/agl our transcriptional interactions database further provides an overview of the transcriptional pathways present in arabidopsis roots and will likely yield a plethora of novel hypotheses to be tested experimentally. 
in this study we present a simple bioinformatics methodology that uses public, carefully curated microarray data and the mutual information algorithm aracne in order to obtain a database of transcriptional interactions. in contrast to previous efforts to obtain root trns, that have focused on particular functional modules or tissues, our root transcriptional interactions provide an overview of the transcriptional pathways present in arabidopsis thaliana roots and will likely yield a plethora of novel hypotheses to be tested experimentally. we further show that these networks are a powerful tool to integrate and analyze high-throughput expression data, as exemplified by our analysis of a short root induction time-course microarray dataset, and are a reliable source for the prediction of novel root gene functions. this study demonstrates that network inference using carefully curated microarray data yields reliable trn architectures. in particular, we used our database to predict novel genes involved in root secondary cell-wall synthesis and identified the mads-box tf xal1/agl as an unexpected participant in this process. however, a vast amount of data from public microarray experiments is available, which can be subject to inference algorithms in order to recover reliable trn architectures.
 uncovering the complex transcriptional regulatory networks that underlie plant and animal development remains a challenge. however, a vast amount of data from public microarray experiments is available, which can be subject to inference algorithms in order to recover reliable trn architectures.  in this study we present a simple bioinformatics methodology that uses public, carefully curated microarray data and the mutual information algorithm aracne in order to obtain a database of transcriptional interactions. we used data from arabidopsis thaliana root samples to show that the transcriptional regulatory networks derived from this database successfully recover previously identified root transcriptional modules and to propose new transcription factors for the short root/scarecrow and plethora pathways. we further show that these networks are a powerful tool to integrate and analyze high-throughput expression data, as exemplified by our analysis of a short root induction time-course microarray dataset, and are a reliable source for the prediction of novel root gene functions. in particular, we used our database to predict novel genes involved in root secondary cell-wall synthesis and identified the mads-box tf xal1/agl as an unexpected participant in this process.  this study demonstrates that network inference using carefully curated microarray data yields reliable trn architectures. in contrast to previous efforts to obtain root trns, that have focused on particular functional modules or tissues, our root transcriptional interactions provide an overview of the transcriptional pathways present in arabidopsis thaliana roots and will likely yield a plethora of novel hypotheses to be tested experimentally. transcriptional regulatory networksrootarabidopsistranscription factoraracne 