BACKGROUND
the evolution of signal transduction pathways since the divergence of plants and animals has been influenced by very different selection pressures.
hormone signalling, though analogous in both kingdoms, differs in the signalling molecules employed as well as in their perception and mode of action.
plants are adapted to a sessile lifestyle, being able continuously to form new organs during their postembryonic development.
this process, in addition to embryonic development, is closely associated with specific growth regulators, effective at low concentrations.
the signalling pathways of these growth regulators  are relatively well understood, but their evolution, as well as their relationship to the evolution of embryonic and post-embryonic development in the plant kingdom, is less clear  <cit> .
auxin, one such phytohormone, is a principal regulator of growth and development in flowering plants  <cit> , quickly triggering the transcription of auxin-responsive genes  <cit> .
proteins of two related families, auxin responsive factor  and auxin/indole-3-acetic acid , act together to regulate this transcription  <cit> .
in flowering plants, arf proteins possess a conserved dna-binding domain which recognizes auxin responsive elements : short motifs which are found in the promoter sequences of many auxin-responsive genes  <cit> .
most arfs, and all aux/iaas also contain a conserved dimerization domain which mediates protein-protein interactions within and between both protein families  <cit> .
the middle region which joins arf dna-binding and dimerization domains is highly divergent and may be glutamine  rich  <cit> .
those arfs which contain such q-rich regions are thought to be activators of gene transcription  <cit> .
conversely, those arfs which repress gene transcription lack glutamine  -rich regions.
the n-terminal region of aux/iaa proteins contains two other domains: domain i and ii.
domain i contains a short amphiphilic repression motif, which binds to the co-repressor topless, enabling aux/iaas to repress arf function  <cit> .
domain ii contains a degron: a motif sufficient to signal aux/iaas for proteasome-mediated degradation  <cit> .
specific point mutations in domain ii confer strong, auxin insensitive phenotypes  <cit> .
at low cellular auxin concentrations, aux/iaa proteins dimerize with arf transcriptional activators, repressing their activity  <cit> .
auxin itself can bind at the interface of aux/iaa proteins and tir1-family f-box proteins, components of specific scf e <dig> ubiquitin ligases, directly promoting their interaction.
accordingly, at high cellular auxin concentrations, aux/iaas are ubiquitinated and subsequently degraded  <cit> .
degradation of aux/iaa proteins then allows arf-mediated, auxin-dependent gene transcription.
physcomitrella patens , selaginella moellendorffii  and angiosperms diverged from each other at between  <dig> and  <dig> million years ago  <cit> .
the genomes of both p. patens and s. moellendorffii encode all the proteins necessary for this primary auxin response  <cit> .
furthermore, p. patens has been shown to both synthesize auxin, and respond to exogenously applied auxin  <cit> .
here we use the complete genomic sequences of p. patens and s. moellendorffii to address how a relatively simple signalling mechanism has evolved into, in flowering plants, a central regulator of many essential and diverse developmental processes.
a driving force of this evolution has been positive darwinian selection.
such positive selection is a measure of the adaptation of amino acid sequences following a gene duplication event.
the unambiguous indicator of positive selection, a high ratio of non-synonymous  to synonymous  nucleotide substitutions, was detected in the flowering plant arfs.
based on a comparative analysis of the fully-sequenced genomes of p. patens, s. moellendorffii, and selected flowering plants, we are able to draw conclusions about ancestral auxin target genes and signalling mechanisms, and about the pressures which have driven the radiation of auxin-signalling genes in flowering plants.
RESULTS
endogenous auxin is a widely used signalling molecule in vascular plants, but is also found in bryophytes, algae and prokaryotes  <cit> .
in the present study, we identify similarities and differences between the auxin signalling components in moss and flowering plants by comparing the fully sequenced genomes of p. patens with those of model flowering plant species.
additional support, where appropriate, is drawn from the genome of s. moellendorffii, a vascular non-seed plant.
here we present an analysis of two gene families central to auxin signalling: the arfs, encoding transcription factors, and aux/iaas, encoding their repressors.
we also analyze three families of primary auxin responsive genes, which are among the first targets of auxin-induced transcription in flowering plants.
aux/iaa
the p. patens genome encodes three aux/iaa proteins  .
these proteins, at between  <dig> and  <dig> amino acids, are significantly longer than all  <dig> a. thaliana aux/iaa  family members .
the three aux/iaas of s. moellendorffii vary in length between  <dig> and  <dig> amino acids.
aux/iaa proteins typically comprise four domains: domain i confers the proteins' transcriptional repressor function, domain ii is a degradation motif, and domains iii and iv form a protein dimerization domain, evolutionarily related to the c-terminal dimerization  domain of arf proteins.
domain i
in all but one arabidopsis aux/iaa protein, domain i contains a sequence of amino acids reminiscent of an erf-associated amphiphilic repression  motif.
this lxlxl domain i motif interacts directly with topless , a transcriptional co-repressor.
this interaction leads to a repression of the arf-dependent transcription of a reporter gene driven by the dr <dig> promoter, a synthetic auxin-sensitive marker containing repeated tgtctc auxin response elements   <cit> .
ppaux/iaas do not contain an lxlxl motif in domain i. instead they all contain a similar lxlxpp motif .
a corresponding and overlapping lxlxlxpp motif was found in three ataux/iaas , forming a cluster with good bootstrap support .
the genome of s. moellendorffii encodes three aux/iaa proteins, one of which contains an lxlxpp motif in domain i. the other two contain the lxlxl motif typical of flowering plants .
the genes containing lxlxl motifs found in domain i of s. moellendorffii and flowering plants do not form monophyletic groups.
therefore the motif is likely to have become established at least twice in each lineage.
of the  <dig> aux/iaas encoded by the populus trichocarpa genome,  <dig> are predicted to contain an lxlxl motif and six an lxlxpp motif  <cit> .
in rice, it is predicted that  <dig> out of  <dig> aux/iaas contain an lxlxl motif; of these, two contain an lxlxlxpp motif.
in rice, one aux/iaa contains lxlxpp .
at present, there is no evidence that this lxlxpp motif, in any species, represents a functional repression domain.
there are also no experimental data available which test the role of flowering plant aux/iaas which contain no apparent functional domain i motif.
these proteins may function as competitive regulators of the auxin response.
gaining empirical functional evidence on these proteins will allow hypotheses on their evolution to be tested.
.
* the number of arfs and aux/iaas from kalluri et al.
<cit>  is shown in brackets.
** in addition, one a. thaliana arf, arf <dig>  has a methionine-rich middle region.
there is one homologous position for the ear-like motif of domain i. based on the alignment of a. thaliana, s. moellendorffii and p. patens aux/iaas  this domain i motif can be expanded to lxl   .
this allows the detection of domain i in all sequences tested of these three species.
if expanded further, an x     motif can, according to our present knowledge, be used to detect domain i in all land plant aux/iaas.
this analysis does not preclude the possibility that other non-homologous domains serving a similar function are also present.
mutations in the leucine positions of the domain i motif of arabidopsis have been shown to result in significantly weaker repression of arf-mediated transcription to aux/iaa proteins  <cit> .
nevertheless, the widespread conservation of the lxlxpp sequence suggests it is a functional motif.
the predicted presence in p. patens of two tpl-like transcriptional co-repressors  also suggests the lxlxpp motif is able to inhibit  arf-mediated transcription.
in flowering plants, however, the lxlxpp sequence appears to have been superseded by the lxlxl domain .
notably, the genome of s. moellendorffii encodes proteins predicted to contain both motifs.
although the relative efficiency of domain i-dependent transcriptional repression in non-seed plants  and flowering plants  is not possible to assess with the data that are currently available, it is highly significant, as they would be expected profoundly to influence the role of auxin-dependent transcriptional activation.
domain ii
the alignment of domain ii from several aux/iaa proteins indicates that not all  <dig> amino acids of the consensus sequence, which in flowering plants mediate the specific proteasomal degradation of aux/iaas in response to auxin, are faithfully conserved .
nevertheless, a central core of five residues, representing amino acids 4– <dig> , is required for targeted protein degradation  <cit> .
though not sufficient to confer protein instability to a luciferase reporter fusion on its own, the functionally essential central motif  is conserved in all aux/iaas, including those from p. patens and s. moellendorffii .
aux/iaas are degraded after domain ii binds to the tir <dig> family of f-box proteins  <cit> .
the presence of the core motif of domain ii and four paralogs of the aux/iaa-specific tir <dig> family of f-box proteins  in p. patens, suggests that ppaux/iaas are degraded in an auxin-dependent manner.
homology modelling has shown that the auxin binding pocket of pptir <dig> is intact  <cit> .
together, these data suggest that auxin-mediated targeted protein degradation is relevant in p. patens, and that the relatively slow response of p. patens to auxin  <cit>  is not due to an impaired ability to degrade aux/iaas in response to auxin.
diversification of aux/iaa
the dramatic radiation of aux/iaa genes in land plants   underpins a corresponding increase in the complexity of auxin signalling.
after the separation of lycophytes and seed plants, the aux/iaa family in the a. thaliana lineage was expanded by  <dig> additional duplication events .
to test whether this radiation has been driven by neofunctionalization at the amino acid level , rates of positive selection were measured  <cit> .
specifically, we applied a likelihood ratio test  to selected aux/iaa sub-families of a. thaliana and p. trichocarpa, and compared data fits to two models: m <dig> vs. m <dig> and m <dig> vs. m <dig> .
a comparison of these models measures the likelihood that differences in non-synonomous/synonomous substitution ratios happened by chance.
for aux/iaa proteins, no significant differences between test and null hypotheses were found in any of the data sets tested.
each comparison has n sequences, dn/ds is average ratio over sites under a codon model with one ω. bold underline, pp ≥  <dig>  of being under positive selection; bold,  <dig> >pp ≥  <dig> ; italic,  <dig> >pp ≥  <dig> ; underline,  <dig> >pp ≥  <dig> ; normal,  <dig> >pp >  <dig>
aux/iaa genes have been retained in the a. thaliana genome at a high rate.
a two-way analysis of variance  test of microarray data has previously shown that the gene expression patterns of aux/iaa sister pairs of a. thaliana are significantly different  <cit> .
we extended this analysis by widening the conditions tested.
two-way anova results for ten pairs of aux/iaa genes are reported as graphs of expression levels at  <dig> conditions in additional file  <dig>  a–j .
all ten sister pairs of aux/iaa showed significant gene , sample , and gene by sample  effects .
aux/iaa genes have radiated through segmental duplication events  <cit> .
in p. trichocarpa and o. sativa, both arf and aux/iaa gene families have been expanded, also largely due to segmental duplication  <cit> .
after such events, the gradual appearance of deleterious mutations generally leads to the loss of one of the duplicated genes  <cit> .
if both gene copies are retained, there is a higher probability that mutations leading to a split in the expression pattern of the ancestral gene between duplicated genes, rather than mutations that lead to a new function in one copy, have occurred  <cit> .
such a split can occur through changes in transcription-factor binding sites within promoter regions that result in differential expression of the two gene copies.
we therefore conclude that changes in expression pattern have driven aux/iaa radiation.
indeed, when compared to amino acid substitution rates, changes in expression pattern contribute more to aux/iaa function  <cit> .
studies in p. trichocarpa  <cit>  also showed that genes of the expanded ptiaa <dig> subgroup, which is represented by six members, are differentially transcribed.
these data lend further support to the hypothesis that the diversification of aux/iaa family members in flowering plants has been sustained by changes in their expression patterns.
arf
in a. thaliana, all arfs contain a dna binding domain, but some lack a c-terminal dimerization domain .
the genomes of s. moellendorffii and p. patens also encode arfs with c-terminal truncations, as well as those with n-terminal truncations.
all of these variants are discussed below.
full-length and c-terminally truncated arfs
to examine evolutionary relationships among p. patens , s. moellendorffii  and a. thaliana  arf proteins, a rooted phylogenetic tree was constructed from the alignment of the predicted protein sequences of the  <dig> pparfs, the  <dig> smarfs and the  <dig> atarfs predicted to contain a dna binding domain .
all  <dig> arf genes analysed could be grouped into five major classes .
in addition to the five previously described classes of arfs  <cit> , we detected an additional cluster of four p. patens genes and one cluster of two s. moellendorffii genes, each with good bootstrap support.
six pparfs are similar to subclass iia  and two are similar to class iii .
pparfs, therefore, fell into one of three classes .
as in p. patens, s. moellendorffii has representatives of subclass iia  and class iii .
the s. moellendorffii- and p. patens-specific subclasses are not monophyletic.
in addition, p. patens and s. moellendorffii each encode one c-terminally truncated arf with no ctd .
flowering plants encode more ctd-truncated arfs.
this trend is seen in a. thaliana , o. sativa  and p. trichocarpa  .
diversification of ctd-truncated arfs in flowering plants suggests a role for auxin-independent regulation of auxin responsive genes.
full-length arf transcriptional activators
in a. thaliana, the first transcriptional response to exogenously applied auxin is a rapid up-regulation of auxin-responsive genes  <cit> .
the so-called middle regions  of five atarfs of sub-class iia  mediate this transcription  <cit> .
all five of these mrs  are significantly longer than those of all other arfs, with the exception of atarf <dig>  <cit> .
pparfs and smarfs of class iia also contain an extended mr .
a second feature of the mrs of those atarfs which function as transcriptional activators is a relatively high proportion of glutamine residues  .
the mrs of canonical pparfs of this group contain fewer glutamine residues than their vascular plant counterparts at between  <dig>  and 10% of all amino acid residues, compared to between  <dig>  and  <dig>  for the q-rich arfs of a. thaliana.
pparfs are unidentifiable as q-rich both by the normalized amino acid frequency used for additional file  <dig> and by a prosite domain search.
nevertheless, these mrs all contain a higher proportion of glutamine residues than all but two of the repressor atarfs .
given the character states of the mr length  and glutamine content  in the phylogenetic tree, a single gain of the domain  seems to have occurred.
the mr seems to have been secondarily reduced in one smarf  and secondarily expanded in atarf <dig>  the subsequent enrichment of the mr with glutamine residues apparently evolved several times independently within the genes containing the prolonged mr. s. moellendorffii contains three class iia canonical arf transcriptional activators.
these proteins all contain an extended, q-rich mr. the simultaneous appearance of an lxlxl motif in s. moellendorffii aux/iaas allows the possibility that this motif co-evolved with the appearance of canonical q-rich arfs.
the exogenous application of auxin to p. patens has been shown to have only a weak effect on the expression of transgenic flowering plant auxin-responsive markers  <cit> .
in contrast, auxin-responsive transcription in a. thaliana is observed rapidly, and at relatively low auxin concentrations  <cit> .
the slower response in p. patens could be due to a number of reasons relating either to an inability of the moss to recognize auxin-responsive flowering plant promoter elements, or to a slower auxin response in p. patens per se.
direct experimental evidence is needed if we are to state firmly that there is indeed a slower auxin response in p. patens, and that this is due to a relatively weak activation of gene transcription by arfs.
however, the observations that i) the lxlxpp motif of aux/iaa domain i has been gradually replaced by an lxlxl motif in most flowering plants aux/iaas, ii) q-rich arfs and the lxlxl ear-like domain appear together in s. moellendorffii, iii) mutations in the canonical lxlxl motif confer weaker transcriptional repression in a. thaliana  <cit> , and iv) there is a relatively slow transcriptional response of p. patens to auxin together lead us to hypothesize that the q-enriched subclass iia arfs of p. patens are moderate rather than strong transcriptional activators.
n-terminally truncated arfs are candidate trans-acting arf regulators
two proteins encoded by the p. patens genome, phypa_ <dig> and phypa_ <dig>  contain both a ctd and an extended q-rich mr  .
however, neither protein contains a recognizable dna-binding domain ; both gene models were manually checked for accuracy.
this phylogenetic analysis placed both truncated p. patens proteins in class iia with atarf transcriptional activators  <dig>   <dig>   <dig>   <dig> and  <dig>  the genome of s. moellendorffii also encodes two dbd-truncated arfs in a monophyletic group with n-terminally truncated p. patens arfs.
artificially truncated atarf <dig>   <dig>   <dig> and  <dig> proteins  have previously been shown to activate strongly  transcription of an auxin-responsive reporter gene by dimerizing with canonical arfs  <cit> .
therefore, it is possible that the activation of an auxin response in p. patens could be relayed by a ctd-dependent heterodimerization between a dbd-truncated arf and a canonical arf .
the presence of dbd-truncated q-rich arfs allows an alternative transcriptional control, alongside the evolution of a functional motif in domain i of aux/iaas.
such an n-terminal truncation enables the spatial separation of transcription-activating mrs and dbds by the competitive inhibition of arf ctds by aux/iaas .
a functional domain i-motif would not be necessary for such inhibition.
since such an inhibitory mechanism is not able to separate the dna-binding and activation domains present in a single arf, we hypothesize that a strong selection pressure on domain i of aux/iaas for the efficient recruitment of transcriptional co-repressors could have been a feature of aux/iaa evolution after the appearance of canonical q-rich arfs.
this hypothesis would predict that at least two mechanisms have evolved through which the evolution of a strong arf activation domain has been accommodated: firstly, the appearance of a strong aux/iaa repressor domain, as seen in flowering plants, and secondly, the spatial separation of the arf activation domain from the dna-binding domain, as seen in p. patens.
notably, s. moellendorffii is predicted to employ both.
a second group of proteins with an n-terminal truncation is encoded by the genomes of p. patens and s. moellendorffii .
here the truncation is larger, and the encoded proteins are predicted to have neither a dbd-domain, nor a middle region.
we propose that proteins of this group act as auxin-independent competitive inhibitors of arf dimerization, inhibiting both potentiation  and repression  of the auxin response.
evolution of arf activators
to test whether positive selection, and therefore possible neofunctionalization, has driven evolution within the extended arf mr of class iia, we compared the relative rates of synonymous and non-synonomous substitutions in full-length coding sequences from all arfs of two fully-sequenced dicotyledonous species: a. thaliana and p. trichocarpa.
a likelihood ratio test  was applied to selected arf sequences from a. thaliana and p. trichocarpa .
the maximum likelihood estimates  of parameters under model m2a and m <dig> are listed in table  <dig>  together with the sites inferred to be under positive selection by the bayes empirical bayes  approach.
for node arf <dig>  both m2a and m <dig> have significantly higher likelihood values than their corresponding null models m1a and m <dig>  suggesting the presence of sites under positive selection.
for node arf <dig>  m <dig>  had significantly higher likelihood values than its corresponding null model m <dig> .
consequently, this model identified  <dig> sites under positive selection , all within the extended mr , supporting the hypothesis that positive selection within the mr plays a role in neofunctionalization, possibly directly influencing the acquisition of a transcriptional activation function.
it is however possible that positive selection influences an unrelated function of the mr. for example, the mr may influence protein stability, as is the case for the mr of arf <dig>  <cit> .
yet, if the enrichment of glutamine is important for transcriptional activation, the fact that  <dig> of the  <dig> sites under positive selection in the mr encode a glutamine in at least one protein clearly argues for the involvement of positive selection in the acquisition of that particular function.
each comparison has n sequences, dn/ds is average ratio over sites under a codon model with one ω. bold underline, pp ≥  <dig>  of being under positive selection; bold,  <dig> >pp ≥  <dig> ; italic,  <dig> >pp ≥  <dig> ; underline,  <dig> >pp ≥  <dig> ; normal,  <dig> >pp >  <dig>
arf <dig> and arf <dig> dimerize with aux/iaas to regulate the expression of partially overlapping sets of auxin-responsive genes in the control of lateral root development and gravitropism  <cit> .
however, arfs do not only dimerize with aux/iaas.
in arabidopsis, a member of a second class of transcription factors, myb <dig>  interacts with the ctd arf <dig> to control auxin-responsive gene expression and lateral root number  <cit> .
therefore a third interaction, besides dna or aux/iaa interaction, influences arf evolution.
as the interaction between myb <dig> and arfs occurs with the arf ctd, it cannot explain positive selection within the proteins' mr. it does, however, represent a precedent for aux/iaa independent protein-protein interactions  influencing protein function within the arf <dig> node, and the presence of, as yet unconsidered, evolutionary pressures influencing arf function.
evolution of arfs which lack a q-rich middle region
in contrast to the relatively constant numbers of class iia arf transcriptional activators encoded by the genomes of p. patens, s. moellendorffii and a. thaliana , the number of arf repressors has increased from five and four in p. patens and s. moellendorffii to fourteen in a. thaliana.
there is only one p. patens-specific and one s. moellendorffii-specific arf class, whilst there are three flowering plant-specific sub-classes  indicating that evolution within flowering plants has favoured strongly the diversification of auxin-regulated repressor arfs .
two polyphyletic groups of arf lacking a q-rich region can be differentiated: those with a ctd and those without.
this suggests at least two distinct mechanisms of transcriptional regulation.
arfs which lack a ctd are responsible for the auxin-independent  regulation of auxin-responsive genes .
these arfs cannot interact with aux/iaas and therefore their transcriptional activity is independent of cellular auxin concentration.
however, identity within their dna-binding domain suggests they are able to bind to auxin responsive promoter elements.
the second type of arf has a ctd and is, at least according to the accepted paradigm, able to dimerize with aux/iaas  <cit>  .
phosphorylation of arf <dig>  by bin <dig>  a kinase involved in brassinosteroid-dependent transcription decreases its ability to bind dna  <cit> .
this path for crosstalk between two hormone signalling pathways  represents a precedent for arf repressors to perform in other signalling functions.
after analysis of all genes encoding a. thaliana arf transcriptional repressors, positive selection was only observed in the arf <dig> node , where little is known about protein function .
in this class, single knockouts do not show obvious aberrant phenotypes  <cit> , and the generation of double knockout lines has been hampered by the genes' close proximity on chromosome  <dig>
class ib arfs are absent from the p. trichocarpa and o. sativa genome, raising the possibility of a specific role within the order brassicales  <cit> .
positive selection does not prove the acquisition of novel and specific function in the class ib arfs of a. thaliana.
however, together with the subgroup's rapid and significant diversification, followed by the retention of duplicated genes, it suggests neofunctionalization.
any putative new function is also likely to be related to the amino acid residues under positive selection in the middle region of the protein, possibly facilitating new protein-protein interactions, protein stability, or post-translational modifications.
auxin-independent regulation of arf activity
arfs without a ctd cannot dimerize with aux/iaas and are therefore not expected to be regulated directly by auxin.
but is auxin-independent arf signalling relevant to flowering plants, or is arf function dependent on a functional ctd?
ctd-deficient arf mutants do indeed have aberrant phenotypes.
four arf proteins which lack a ctd are predicted to be encoded by the a. thaliana genome.
of these, arf <dig> mutants show pleiotropic effects in flower development  <cit> .
plants expressing a mirna-resistant version of a second ctd-deficient arf, arf <dig>  have increased arf <dig> mrna levels and display dramatic developmental defects.
these include embryo and emerging leaf symmetry anomalies, leaf shape defects, premature inflorescence development, altered phyllotaxy, reduced petal size, abnormal stamens, sterility, and root growth defects  <cit> .
the search for alternative mechanisms of arf regulation has centred on small rnas.
two p. patens arf transcripts , both encoding full-length arfs , have been identified as targets of small rnas  <cit> .
regulation of arfs by mirna in a. thaliana can be considered as auxin-independent because auxin treatment does not alter appreciably mir <dig>  mir <dig>  and mir <dig> accumulation, at least in seedlings  <cit> .
in a. thaliana, mrnas encoding two out of the four arfs which have no ctd have also been identified as targets of small rnas: arf <dig> is the target of attas3a-c and arf <dig> is the target of mir <dig>  <cit> .
small rnas do not only target transcripts of arfs without a ctd, but also aux/iaa-binding arfs.
the regulation of arf activity is therefore complex and involves the integration of auxin-dependent and auxin-independent mechanisms .
mirnas are also potentially important regulators of cross-talk between auxin and other signalling pathways, for example between auxin and abscisic acid  <cit> .
auxin-independent, cell-dependent regulation of auxin signalling activity has previously been identified as an important factor in plant development  <cit> .
indeed, endogenous small regulatory rnas seem to play a relatively important role in the regulation of arf gene expression  <cit> .
for example, in a. thaliana the expression pattern of both arf <dig> and arf <dig>  is controlled by mir <dig>  with mirna <dig> also involved in the control of arf expression in p. patens and a. thaliana as well as in s. moellendorffii, suggesting a conserved mechanism of arf post-transcriptional regulation  <cit> .
the p. patens genome encodes a surprisingly diverse population of mirnas.
however, in contrast to arf and aux/iaa genes, the number of mirnas conserved between p. patens and a. thaliana is relatively large  <cit> .
primary auxin response genes
primary auxin response genes  can be grouped into three major families: aux/iaas, gh3s and saurs.
recently, transcription of certain lob domain  genes has also been shown to be rapidly and specifically up-regulated by auxin  <cit> .
all four of these major gene families are represented in the genomes of p. patens and s. moellendorffii.
however, a detailed analysis of their response to auxin application is precluded by the lack of global transcriptional data from these species.
microarray analysis has showed that, in a. thaliana, only the transcription of group ii gh <dig> genes  is regulated by auxin  <cit>  similarly, in o. sativa, the transcription of gh <dig> genes which were most strongly up-regulated in response to auxin treatment also belong to group ii  <cit> .
the p. patens genome contains two genes that are homologous to the gh <dig> family of flowering plants.
both conjugate iaa to amino acids, with ppgh3- <dig> showing a far broader range of substrate specificity than ppgh3- <dig>  <cit> .
surprisingly, the moss gh <dig> genes form a common clade with the group i genes of a. thaliana, and not with those encoding the auxin conjugating enzymes of group ii.
furthermore, the clades are separated by a relatively high genetic distance, suggesting that they diverged a relatively long time ago .
auxin application increases transcription of specific flowering plant gh <dig> genes of group ii.
this increase has never been demonstrated in p. patens  <cit> .
the genome of s. moellendorffii is predicted to encode one group ii gh <dig> enzyme, and one protein belonging to group i .
the remaining  <dig> smgh <dig> genes cannot be clearly assigned based on phylogeny.
the transcriptional response to auxin of these genes has never been tested.
p. patens gh <dig> enzymes are nevertheless able to conjugate auxin.
direct measurements of auxin conjugates in moss plants have give valuable insights into the developmental role of auxin conjugation by gh3s.
p. patens plants lacking both gh <dig> enzymes, when grown on iaa, still conjugate auxin.
these results suggest other classes of enzymes may also conjugate auxin in p. patens  <cit> .
based on phylogeny p. patens gh3s are more closely related to gh3- <dig> of a. thaliana, which catalyses the synthesis of jasmonic acid conjugates  <cit> .
p. patens plants lacking the gh3- <dig> gene show an increased sensitivity to high jasmonic acid concentrations, suggesting a potential role for jasmonic acid conjugation as well for this enzyme  <cit> .
a broad substrate specificity of gh3- <dig> in p. patens could suggest that the enzyme has retained this characteristic from the common ancestor of all land plants.
in flowering plants, saur genes are a diverse family of unknown function with differing responsiveness to auxin  <cit> .
the p. patens genome contains  <dig> saur genes , which cluster in two groups with low bootstrap support .
all atsaur genes of group a are auxin-responsive  <cit> .
this group shows relatively high similarity to nine ppsaur genes   and therefore could participate in the auxin response in p. patens.
the lob domain family of transcription factors also contains important auxin-responsive signalling proteins.
in p. patens, the lbd gene family has  <dig> members, forming five clades .
one clade, encoding four lbds , is monophyletic with important auxin-responsive regulators of lateral root formation in a. thaliana, lbd <dig> and  <dig>  <cit> , and therefore represents candidates for p. patens auxin primary response, an attractive target for future research.
CONCLUSIONS
it is clear that auxin signalling is responsible for many aspects of vascular plant growth and development.
in this manuscript, we demonstrate that the genome of p. patens encodes all of the basic components necessary for an auxin response.
we also suggest that the evolution of an alternative, competitive mechanism of transcriptional control in p. patens, involving the truncation of arf transcriptional activators, substitutes for a mechanism which, in s. moellendorffii and flowering plants, confers a rapid auxin response.
however, without a systematic analysis of the auxin transcriptional response in p. patens and s. moellendorffii it remains difficult to assess  whether these plants are capable of rapidly synthesizing specific mrnas in response to auxin in the same manner as flowering plants, and  the role any such response plays in auxin homeostasis and plant development.
it is, however, clear that an expansion of the aux/iaa gene family accounts for much of the diversification of auxin signalling proteins in flowering plants.
furthermore, the smaller size of many gene families relevant to auxin signalling in p. patens is probably correlated to the lower structural complexity of this plant.
this correlation is especially pronounced in aux/iaa gene families.
auxin and its polar transport are crucial factors in flowering plant development, and have come to direct many processes which are not relevant to mosses such as apical dominance, formation and maintenance of shoot and root apical meristems and vascular differentiation.
mosses nevertheless require auxin for cell differentiation and division.
understanding the differences in the underlying mechanisms of auxin signalling, which drive these different physiological processes, and of their evolutionary relationship, will be a fascinating challenge for the future.

 the evolution of signal transduction pathways since the divergence of plants and animals has been influenced by very different selection pressures. hormone signalling, though analogous in both kingdoms, differs in the signalling molecules employed as well as in their perception and mode of action. plants are adapted to a sessile lifestyle, being able continuously to form new organs during their postembryonic development. this process, in addition to embryonic development, is closely associated with specific growth regulators, effective at low concentrations. the signalling pathways of these growth regulators are relatively well understood, but their evolution, as well as their relationship to the evolution of embryonic and post-embryonic development in the plant kingdom, is less clear. auxin, one such phytohormone, is a principal regulator of growth and development in flowering plants, quickly triggering the transcription of auxin-responsive genes. proteins of two related families, auxin responsive factor and auxin/indole-3-acetic acid, act together to regulate this transcription. in flowering plants, arf proteins possess a conserved dna-binding domain which recognizes auxin responsive elements: short motifs which are found in the promoter sequences of many auxin-responsive genes. most arfs, and all aux/iaas also contain a conserved dimerization domain which mediates protein-protein interactions within and between both protein families. the middle region which joins arf dna-binding and dimerization domains is highly divergent and may be glutamine rich. those arfs which contain such q-rich regions are thought to be activators of gene transcription. conversely, those arfs which repress gene transcription lack glutamine -rich regions. the n-terminal region of aux/iaa proteins contains two other domains: domain i and ii. domain i contains a short amphiphilic repression motif, which binds to the co-repressor topless, enabling aux/iaas to repress arf function. domain ii contains a degron: a motif sufficient to signal aux/iaas for proteasome-mediated degradation. specific point mutations in domain ii confer strong, auxin insensitive phenotypes. at low cellular auxin concentrations, aux/iaa proteins dimerize with arf transcriptional activators, repressing their activity. auxin itself can bind at the interface of aux/iaa proteins and tir1-family f-box proteins, components of specific scf e ubiquitin ligases, directly promoting their interaction. accordingly, at high cellular auxin concentrations, aux/iaas are ubiquitinated and subsequently degraded. degradation of aux/iaa proteins then allows arf-mediated, auxin-dependent gene transcription. physcomitrella patens, selaginella moellendorffii and angiosperms diverged from each other at between and million years ago. the genomes of both p. patens and s. moellendorffii encode all the proteins necessary for this primary auxin response. furthermore, p. patens has been shown to both synthesize auxin, and respond to exogenously applied auxin. here we use the complete genomic sequences of p. patens and s. moellendorffii to address how a relatively simple signalling mechanism has evolved into, in flowering plants, a central regulator of many essential and diverse developmental processes. a driving force of this evolution has been positive darwinian selection. such positive selection is a measure of the adaptation of amino acid sequences following a gene duplication event. the unambiguous indicator of positive selection, a high ratio of non-synonymous to synonymous nucleotide substitutions, was detected in the flowering plant arfs. based on a comparative analysis of the fully-sequenced genomes of p. patens, s. moellendorffii, and selected flowering plants, we are able to draw conclusions about ancestral auxin target genes and signalling mechanisms, and about the pressures which have driven the radiation of auxin-signalling genes in flowering plants.  endogenous auxin is a widely used signalling molecule in vascular plants, but is also found in bryophytes, algae and prokaryotes. in the present study, we identify similarities and differences between the auxin signalling components in moss and flowering plants by comparing the fully sequenced genomes of p. patens with those of model flowering plant species. additional support, where appropriate, is drawn from the genome of s. moellendorffii, a vascular non-seed plant. here we present an analysis of two gene families central to auxin signalling: the arfs, encoding transcription factors, and aux/iaas, encoding their repressors. we also analyze three families of primary auxin responsive genes, which are among the first targets of auxin-induced transcription in flowering plants. aux/iaa the p. patens genome encodes three aux/iaa proteins. these proteins, at between and amino acids, are significantly longer than all a. thaliana aux/iaa family members. the three aux/iaas of s. moellendorffii vary in length between and amino acids. aux/iaa proteins typically comprise four domains: domain i confers the proteins' transcriptional repressor function, domain ii is a degradation motif, and domains iii and iv form a protein dimerization domain, evolutionarily related to the c-terminal dimerization domain of arf proteins. domain i in all but one arabidopsis aux/iaa protein, domain i contains a sequence of amino acids reminiscent of an erf-associated amphiphilic repression motif. this lxlxl domain i motif interacts directly with topless, a transcriptional co-repressor. this interaction leads to a repression of the arf-dependent transcription of a reporter gene driven by the dr promoter, a synthetic auxin-sensitive marker containing repeated tgtctc auxin response elements. ppaux/iaas do not contain an lxlxl motif in domain i. instead they all contain a similar lxlxpp motif. a corresponding and overlapping lxlxlxpp motif was found in three ataux/iaas, forming a cluster with good bootstrap support. the genome of s. moellendorffii encodes three aux/iaa proteins, one of which contains an lxlxpp motif in domain i. the other two contain the lxlxl motif typical of flowering plants. the genes containing lxlxl motifs found in domain i of s. moellendorffii and flowering plants do not form monophyletic groups. therefore the motif is likely to have become established at least twice in each lineage. of the aux/iaas encoded by the populus trichocarpa genome, are predicted to contain an lxlxl motif and six an lxlxpp motif. in rice, it is predicted that out of aux/iaas contain an lxlxl motif; of these, two contain an lxlxlxpp motif. in rice, one aux/iaa contains lxlxpp. at present, there is no evidence that this lxlxpp motif, in any species, represents a functional repression domain. there are also no experimental data available which test the role of flowering plant aux/iaas which contain no apparent functional domain i motif. these proteins may function as competitive regulators of the auxin response. gaining empirical functional evidence on these proteins will allow hypotheses on their evolution to be tested.. * the number of arfs and aux/iaas from kalluri et al. is shown in brackets. ** in addition, one a. thaliana arf, arf has a methionine-rich middle region. there is one homologous position for the ear-like motif of domain i. based on the alignment of a. thaliana, s. moellendorffii and p. patens aux/iaas this domain i motif can be expanded to lxl. this allows the detection of domain i in all sequences tested of these three species. if expanded further, an x motif can, according to our present knowledge, be used to detect domain i in all land plant aux/iaas. this analysis does not preclude the possibility that other non-homologous domains serving a similar function are also present. mutations in the leucine positions of the domain i motif of arabidopsis have been shown to result in significantly weaker repression of arf-mediated transcription to aux/iaa proteins. nevertheless, the widespread conservation of the lxlxpp sequence suggests it is a functional motif. the predicted presence in p. patens of two tpl-like transcriptional co-repressors also suggests the lxlxpp motif is able to inhibit arf-mediated transcription. in flowering plants, however, the lxlxpp sequence appears to have been superseded by the lxlxl domain. notably, the genome of s. moellendorffii encodes proteins predicted to contain both motifs. although the relative efficiency of domain i-dependent transcriptional repression in non-seed plants and flowering plants is not possible to assess with the data that are currently available, it is highly significant, as they would be expected profoundly to influence the role of auxin-dependent transcriptional activation. domain ii the alignment of domain ii from several aux/iaa proteins indicates that not all amino acids of the consensus sequence, which in flowering plants mediate the specific proteasomal degradation of aux/iaas in response to auxin, are faithfully conserved. nevertheless, a central core of five residues, representing amino acids 4–, is required for targeted protein degradation. though not sufficient to confer protein instability to a luciferase reporter fusion on its own, the functionally essential central motif is conserved in all aux/iaas, including those from p. patens and s. moellendorffii. aux/iaas are degraded after domain ii binds to the tir family of f-box proteins. the presence of the core motif of domain ii and four paralogs of the aux/iaa-specific tir family of f-box proteins in p. patens, suggests that ppaux/iaas are degraded in an auxin-dependent manner. homology modelling has shown that the auxin binding pocket of pptir is intact. together, these data suggest that auxin-mediated targeted protein degradation is relevant in p. patens, and that the relatively slow response of p. patens to auxin is not due to an impaired ability to degrade aux/iaas in response to auxin. diversification of aux/iaa the dramatic radiation of aux/iaa genes in land plants underpins a corresponding increase in the complexity of auxin signalling. after the separation of lycophytes and seed plants, the aux/iaa family in the a. thaliana lineage was expanded by additional duplication events. to test whether this radiation has been driven by neofunctionalization at the amino acid level, rates of positive selection were measured. specifically, we applied a likelihood ratio test to selected aux/iaa sub-families of a. thaliana and p. trichocarpa, and compared data fits to two models: m vs. m and m vs. m. a comparison of these models measures the likelihood that differences in non-synonomous/synonomous substitution ratios happened by chance. for aux/iaa proteins, no significant differences between test and null hypotheses were found in any of the data sets tested. each comparison has n sequences, dn/ds is average ratio over sites under a codon model with one ω. bold underline, pp ≥ of being under positive selection; bold, >pp ≥; italic, >pp ≥; underline, >pp ≥; normal, >pp > aux/iaa genes have been retained in the a. thaliana genome at a high rate. a two-way analysis of variance test of microarray data has previously shown that the gene expression patterns of aux/iaa sister pairs of a. thaliana are significantly different. we extended this analysis by widening the conditions tested. two-way anova results for ten pairs of aux/iaa genes are reported as graphs of expression levels at conditions in additional file a–j. all ten sister pairs of aux/iaa showed significant gene, sample, and gene by sample effects. aux/iaa genes have radiated through segmental duplication events. in p. trichocarpa and o. sativa, both arf and aux/iaa gene families have been expanded, also largely due to segmental duplication. after such events, the gradual appearance of deleterious mutations generally leads to the loss of one of the duplicated genes. if both gene copies are retained, there is a higher probability that mutations leading to a split in the expression pattern of the ancestral gene between duplicated genes, rather than mutations that lead to a new function in one copy, have occurred. such a split can occur through changes in transcription-factor binding sites within promoter regions that result in differential expression of the two gene copies. we therefore conclude that changes in expression pattern have driven aux/iaa radiation. indeed, when compared to amino acid substitution rates, changes in expression pattern contribute more to aux/iaa function. studies in p. trichocarpa also showed that genes of the expanded ptiaa subgroup, which is represented by six members, are differentially transcribed. these data lend further support to the hypothesis that the diversification of aux/iaa family members in flowering plants has been sustained by changes in their expression patterns. arf in a. thaliana, all arfs contain a dna binding domain, but some lack a c-terminal dimerization domain. the genomes of s. moellendorffii and p. patens also encode arfs with c-terminal truncations, as well as those with n-terminal truncations. all of these variants are discussed below. full-length and c-terminally truncated arfs to examine evolutionary relationships among p. patens, s. moellendorffii and a. thaliana arf proteins, a rooted phylogenetic tree was constructed from the alignment of the predicted protein sequences of the pparfs, the smarfs and the atarfs predicted to contain a dna binding domain. all arf genes analysed could be grouped into five major classes. in addition to the five previously described classes of arfs, we detected an additional cluster of four p. patens genes and one cluster of two s. moellendorffii genes, each with good bootstrap support. six pparfs are similar to subclass iia and two are similar to class iii. pparfs, therefore, fell into one of three classes. as in p. patens, s. moellendorffii has representatives of subclass iia and class iii. the s. moellendorffii- and p. patens-specific subclasses are not monophyletic. in addition, p. patens and s. moellendorffii each encode one c-terminally truncated arf with no ctd. flowering plants encode more ctd-truncated arfs. this trend is seen in a. thaliana, o. sativa and p. trichocarpa. diversification of ctd-truncated arfs in flowering plants suggests a role for auxin-independent regulation of auxin responsive genes. full-length arf transcriptional activators in a. thaliana, the first transcriptional response to exogenously applied auxin is a rapid up-regulation of auxin-responsive genes. the so-called middle regions of five atarfs of sub-class iia mediate this transcription. all five of these mrs are significantly longer than those of all other arfs, with the exception of atarf. pparfs and smarfs of class iia also contain an extended mr. a second feature of the mrs of those atarfs which function as transcriptional activators is a relatively high proportion of glutamine residues. the mrs of canonical pparfs of this group contain fewer glutamine residues than their vascular plant counterparts at between and 10% of all amino acid residues, compared to between and for the q-rich arfs of a. thaliana. pparfs are unidentifiable as q-rich both by the normalized amino acid frequency used for additional file and by a prosite domain search. nevertheless, these mrs all contain a higher proportion of glutamine residues than all but two of the repressor atarfs. given the character states of the mr length and glutamine content in the phylogenetic tree, a single gain of the domain seems to have occurred. the mr seems to have been secondarily reduced in one smarf and secondarily expanded in atarf the subsequent enrichment of the mr with glutamine residues apparently evolved several times independently within the genes containing the prolonged mr. s. moellendorffii contains three class iia canonical arf transcriptional activators. these proteins all contain an extended, q-rich mr. the simultaneous appearance of an lxlxl motif in s. moellendorffii aux/iaas allows the possibility that this motif co-evolved with the appearance of canonical q-rich arfs. the exogenous application of auxin to p. patens has been shown to have only a weak effect on the expression of transgenic flowering plant auxin-responsive markers. in contrast, auxin-responsive transcription in a. thaliana is observed rapidly, and at relatively low auxin concentrations. the slower response in p. patens could be due to a number of reasons relating either to an inability of the moss to recognize auxin-responsive flowering plant promoter elements, or to a slower auxin response in p. patens per se. direct experimental evidence is needed if we are to state firmly that there is indeed a slower auxin response in p. patens, and that this is due to a relatively weak activation of gene transcription by arfs. however, the observations that i) the lxlxpp motif of aux/iaa domain i has been gradually replaced by an lxlxl motif in most flowering plants aux/iaas, ii) q-rich arfs and the lxlxl ear-like domain appear together in s. moellendorffii, iii) mutations in the canonical lxlxl motif confer weaker transcriptional repression in a. thaliana, and iv) there is a relatively slow transcriptional response of p. patens to auxin together lead us to hypothesize that the q-enriched subclass iia arfs of p. patens are moderate rather than strong transcriptional activators. n-terminally truncated arfs are candidate trans-acting arf regulators two proteins encoded by the p. patens genome, phypa_ and phypa_ contain both a ctd and an extended q-rich mr. however, neither protein contains a recognizable dna-binding domain; both gene models were manually checked for accuracy. this phylogenetic analysis placed both truncated p. patens proteins in class iia with atarf transcriptional activators and the genome of s. moellendorffii also encodes two dbd-truncated arfs in a monophyletic group with n-terminally truncated p. patens arfs. artificially truncated atarf and proteins have previously been shown to activate strongly transcription of an auxin-responsive reporter gene by dimerizing with canonical arfs. therefore, it is possible that the activation of an auxin response in p. patens could be relayed by a ctd-dependent heterodimerization between a dbd-truncated arf and a canonical arf. the presence of dbd-truncated q-rich arfs allows an alternative transcriptional control, alongside the evolution of a functional motif in domain i of aux/iaas. such an n-terminal truncation enables the spatial separation of transcription-activating mrs and dbds by the competitive inhibition of arf ctds by aux/iaas. a functional domain i-motif would not be necessary for such inhibition. since such an inhibitory mechanism is not able to separate the dna-binding and activation domains present in a single arf, we hypothesize that a strong selection pressure on domain i of aux/iaas for the efficient recruitment of transcriptional co-repressors could have been a feature of aux/iaa evolution after the appearance of canonical q-rich arfs. this hypothesis would predict that at least two mechanisms have evolved through which the evolution of a strong arf activation domain has been accommodated: firstly, the appearance of a strong aux/iaa repressor domain, as seen in flowering plants, and secondly, the spatial separation of the arf activation domain from the dna-binding domain, as seen in p. patens. notably, s. moellendorffii is predicted to employ both. a second group of proteins with an n-terminal truncation is encoded by the genomes of p. patens and s. moellendorffii. here the truncation is larger, and the encoded proteins are predicted to have neither a dbd-domain, nor a middle region. we propose that proteins of this group act as auxin-independent competitive inhibitors of arf dimerization, inhibiting both potentiation and repression of the auxin response. evolution of arf activators to test whether positive selection, and therefore possible neofunctionalization, has driven evolution within the extended arf mr of class iia, we compared the relative rates of synonymous and non-synonomous substitutions in full-length coding sequences from all arfs of two fully-sequenced dicotyledonous species: a. thaliana and p. trichocarpa. a likelihood ratio test was applied to selected arf sequences from a. thaliana and p. trichocarpa. the maximum likelihood estimates of parameters under model m2a and m are listed in table together with the sites inferred to be under positive selection by the bayes empirical bayes approach. for node arf both m2a and m have significantly higher likelihood values than their corresponding null models m1a and m suggesting the presence of sites under positive selection. for node arf m had significantly higher likelihood values than its corresponding null model m. consequently, this model identified sites under positive selection, all within the extended mr, supporting the hypothesis that positive selection within the mr plays a role in neofunctionalization, possibly directly influencing the acquisition of a transcriptional activation function. it is however possible that positive selection influences an unrelated function of the mr. for example, the mr may influence protein stability, as is the case for the mr of arf. yet, if the enrichment of glutamine is important for transcriptional activation, the fact that of the sites under positive selection in the mr encode a glutamine in at least one protein clearly argues for the involvement of positive selection in the acquisition of that particular function. each comparison has n sequences, dn/ds is average ratio over sites under a codon model with one ω. bold underline, pp ≥ of being under positive selection; bold, >pp ≥; italic, >pp ≥; underline, >pp ≥; normal, >pp > arf and arf dimerize with aux/iaas to regulate the expression of partially overlapping sets of auxin-responsive genes in the control of lateral root development and gravitropism. however, arfs do not only dimerize with aux/iaas. in arabidopsis, a member of a second class of transcription factors, myb interacts with the ctd arf to control auxin-responsive gene expression and lateral root number. therefore a third interaction, besides dna or aux/iaa interaction, influences arf evolution. as the interaction between myb and arfs occurs with the arf ctd, it cannot explain positive selection within the proteins' mr. it does, however, represent a precedent for aux/iaa independent protein-protein interactions influencing protein function within the arf node, and the presence of, as yet unconsidered, evolutionary pressures influencing arf function. evolution of arfs which lack a q-rich middle region in contrast to the relatively constant numbers of class iia arf transcriptional activators encoded by the genomes of p. patens, s. moellendorffii and a. thaliana, the number of arf repressors has increased from five and four in p. patens and s. moellendorffii to fourteen in a. thaliana. there is only one p. patens-specific and one s. moellendorffii-specific arf class, whilst there are three flowering plant-specific sub-classes indicating that evolution within flowering plants has favoured strongly the diversification of auxin-regulated repressor arfs. two polyphyletic groups of arf lacking a q-rich region can be differentiated: those with a ctd and those without. this suggests at least two distinct mechanisms of transcriptional regulation. arfs which lack a ctd are responsible for the auxin-independent regulation of auxin-responsive genes. these arfs cannot interact with aux/iaas and therefore their transcriptional activity is independent of cellular auxin concentration. however, identity within their dna-binding domain suggests they are able to bind to auxin responsive promoter elements. the second type of arf has a ctd and is, at least according to the accepted paradigm, able to dimerize with aux/iaas. phosphorylation of arf by bin a kinase involved in brassinosteroid-dependent transcription decreases its ability to bind dna. this path for crosstalk between two hormone signalling pathways represents a precedent for arf repressors to perform in other signalling functions. after analysis of all genes encoding a. thaliana arf transcriptional repressors, positive selection was only observed in the arf node, where little is known about protein function. in this class, single knockouts do not show obvious aberrant phenotypes, and the generation of double knockout lines has been hampered by the genes' close proximity on chromosome class ib arfs are absent from the p. trichocarpa and o. sativa genome, raising the possibility of a specific role within the order brassicales. positive selection does not prove the acquisition of novel and specific function in the class ib arfs of a. thaliana. however, together with the subgroup's rapid and significant diversification, followed by the retention of duplicated genes, it suggests neofunctionalization. any putative new function is also likely to be related to the amino acid residues under positive selection in the middle region of the protein, possibly facilitating new protein-protein interactions, protein stability, or post-translational modifications. auxin-independent regulation of arf activity arfs without a ctd cannot dimerize with aux/iaas and are therefore not expected to be regulated directly by auxin. but is auxin-independent arf signalling relevant to flowering plants, or is arf function dependent on a functional ctd? ctd-deficient arf mutants do indeed have aberrant phenotypes. four arf proteins which lack a ctd are predicted to be encoded by the a. thaliana genome. of these, arf mutants show pleiotropic effects in flower development. plants expressing a mirna-resistant version of a second ctd-deficient arf, arf have increased arf mrna levels and display dramatic developmental defects. these include embryo and emerging leaf symmetry anomalies, leaf shape defects, premature inflorescence development, altered phyllotaxy, reduced petal size, abnormal stamens, sterility, and root growth defects. the search for alternative mechanisms of arf regulation has centred on small rnas. two p. patens arf transcripts, both encoding full-length arfs, have been identified as targets of small rnas. regulation of arfs by mirna in a. thaliana can be considered as auxin-independent because auxin treatment does not alter appreciably mir mir and mir accumulation, at least in seedlings. in a. thaliana, mrnas encoding two out of the four arfs which have no ctd have also been identified as targets of small rnas: arf is the target of attas3a-c and arf is the target of mir. small rnas do not only target transcripts of arfs without a ctd, but also aux/iaa-binding arfs. the regulation of arf activity is therefore complex and involves the integration of auxin-dependent and auxin-independent mechanisms. mirnas are also potentially important regulators of cross-talk between auxin and other signalling pathways, for example between auxin and abscisic acid. auxin-independent, cell-dependent regulation of auxin signalling activity has previously been identified as an important factor in plant development. indeed, endogenous small regulatory rnas seem to play a relatively important role in the regulation of arf gene expression. for example, in a. thaliana the expression pattern of both arf and arf is controlled by mir with mirna also involved in the control of arf expression in p. patens and a. thaliana as well as in s. moellendorffii, suggesting a conserved mechanism of arf post-transcriptional regulation. the p. patens genome encodes a surprisingly diverse population of mirnas. however, in contrast to arf and aux/iaa genes, the number of mirnas conserved between p. patens and a. thaliana is relatively large. primary auxin response genes primary auxin response genes can be grouped into three major families: aux/iaas, gh3s and saurs. recently, transcription of certain lob domain genes has also been shown to be rapidly and specifically up-regulated by auxin. all four of these major gene families are represented in the genomes of p. patens and s. moellendorffii. however, a detailed analysis of their response to auxin application is precluded by the lack of global transcriptional data from these species. microarray analysis has showed that, in a. thaliana, only the transcription of group ii gh genes is regulated by auxin similarly, in o. sativa, the transcription of gh genes which were most strongly up-regulated in response to auxin treatment also belong to group ii. the p. patens genome contains two genes that are homologous to the gh family of flowering plants. both conjugate iaa to amino acids, with ppgh3- showing a far broader range of substrate specificity than ppgh3-. surprisingly, the moss gh genes form a common clade with the group i genes of a. thaliana, and not with those encoding the auxin conjugating enzymes of group ii. furthermore, the clades are separated by a relatively high genetic distance, suggesting that they diverged a relatively long time ago. auxin application increases transcription of specific flowering plant gh genes of group ii. this increase has never been demonstrated in p. patens. the genome of s. moellendorffii is predicted to encode one group ii gh enzyme, and one protein belonging to group i. the remaining smgh genes cannot be clearly assigned based on phylogeny. the transcriptional response to auxin of these genes has never been tested. p. patens gh enzymes are nevertheless able to conjugate auxin. direct measurements of auxin conjugates in moss plants have give valuable insights into the developmental role of auxin conjugation by gh3s. p. patens plants lacking both gh enzymes, when grown on iaa, still conjugate auxin. these results suggest other classes of enzymes may also conjugate auxin in p. patens. based on phylogeny p. patens gh3s are more closely related to gh3- of a. thaliana, which catalyses the synthesis of jasmonic acid conjugates. p. patens plants lacking the gh3- gene show an increased sensitivity to high jasmonic acid concentrations, suggesting a potential role for jasmonic acid conjugation as well for this enzyme. a broad substrate specificity of gh3- in p. patens could suggest that the enzyme has retained this characteristic from the common ancestor of all land plants. in flowering plants, saur genes are a diverse family of unknown function with differing responsiveness to auxin. the p. patens genome contains saur genes, which cluster in two groups with low bootstrap support. all atsaur genes of group a are auxin-responsive. this group shows relatively high similarity to nine ppsaur genes and therefore could participate in the auxin response in p. patens. the lob domain family of transcription factors also contains important auxin-responsive signalling proteins. in p. patens, the lbd gene family has members, forming five clades. one clade, encoding four lbds, is monophyletic with important auxin-responsive regulators of lateral root formation in a. thaliana, lbd and, and therefore represents candidates for p. patens auxin primary response, an attractive target for future research.  it is clear that auxin signalling is responsible for many aspects of vascular plant growth and development. in this manuscript, we demonstrate that the genome of p. patens encodes all of the basic components necessary for an auxin response. we also suggest that the evolution of an alternative, competitive mechanism of transcriptional control in p. patens, involving the truncation of arf transcriptional activators, substitutes for a mechanism which, in s. moellendorffii and flowering plants, confers a rapid auxin response. however, without a systematic analysis of the auxin transcriptional response in p. patens and s. moellendorffii it remains difficult to assess whether these plants are capable of rapidly synthesizing specific mrnas in response to auxin in the same manner as flowering plants, and the role any such response plays in auxin homeostasis and plant development. it is, however, clear that an expansion of the aux/iaa gene family accounts for much of the diversification of auxin signalling proteins in flowering plants. furthermore, the smaller size of many gene families relevant to auxin signalling in p. patens is probably correlated to the lower structural complexity of this plant. this correlation is especially pronounced in aux/iaa gene families. auxin and its polar transport are crucial factors in flowering plant development, and have come to direct many processes which are not relevant to mosses such as apical dominance, formation and maintenance of shoot and root apical meristems and vascular differentiation. mosses nevertheless require auxin for cell differentiation and division. understanding the differences in the underlying mechanisms of auxin signalling, which drive these different physiological processes, and of their evolutionary relationship, will be a fascinating challenge for the future. 
site-specific analyses of positive darwinian selection revealed relatively high rates of synonymous substitution in the a. thaliana arfs of classes iia and ib, suggesting that neofunctionalization in important functional regions has driven the evolution of auxin signalling in flowering plants. the genome of p. patens encodes all of the basic components necessary for a rapid auxin response. the spatial separation of the q-rich activator domain and dna-binding domain suggests an alternative mechanism of transcriptional control in p. patens distinct from the mechanism seen in flowering plants. comparative analysis of the p. patens, s. moellendorffii and arabidopsis thaliana genomes suggests that the well-established rapid transcriptional response to auxin of flowering plants, evolved in vascular plants after their divergence from the last common ancestor shared with mosses. an n-terminally truncated arf transcriptional activator is encoded by the genomes of p. patens and s. moellendorffii, and suggests a supplementary mechanism of nuclear auxin signalling, absent in flowering plants. primary auxin responsive gene families show different phylogenetic profiles in p. patens, s. moellendorffii and flowering plants, highlighting genes for further study. to understand the evolution of auxin signalling, we compared the genes encoding two families of crucial transcriptional regulators, auxin response factor and auxin/indole-3-acetic acid, among flowering plants and two non-seed plants, physcomitrella patens and selaginella moellendorffii. significantly, the genome of s. moellendorffii is predicted to encode proteins suitable for both methods of regulation.
 the plant hormone auxin directs many aspects of plant growth and development. to understand the evolution of auxin signalling, we compared the genes encoding two families of crucial transcriptional regulators, auxin response factor and auxin/indole-3-acetic acid, among flowering plants and two non-seed plants, physcomitrella patens and selaginella moellendorffii.  comparative analysis of the p. patens, s. moellendorffii and arabidopsis thaliana genomes suggests that the well-established rapid transcriptional response to auxin of flowering plants, evolved in vascular plants after their divergence from the last common ancestor shared with mosses. an n-terminally truncated arf transcriptional activator is encoded by the genomes of p. patens and s. moellendorffii, and suggests a supplementary mechanism of nuclear auxin signalling, absent in flowering plants. site-specific analyses of positive darwinian selection revealed relatively high rates of synonymous substitution in the a. thaliana arfs of classes iia and ib, suggesting that neofunctionalization in important functional regions has driven the evolution of auxin signalling in flowering plants. primary auxin responsive gene families show different phylogenetic profiles in p. patens, s. moellendorffii and flowering plants, highlighting genes for further study.  the genome of p. patens encodes all of the basic components necessary for a rapid auxin response. the spatial separation of the q-rich activator domain and dna-binding domain suggests an alternative mechanism of transcriptional control in p. patens distinct from the mechanism seen in flowering plants. significantly, the genome of s. moellendorffii is predicted to encode proteins suitable for both methods of regulation. 