The evolution of nuclear auxin signalling
© Paponov et al; licensee BioMed Central Ltd. 2009
Received: 15 September 2008
Accepted: 03 June 2009
Published: 03 June 2009
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 (ARF) and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA), 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 their closest orthologous genes in poplar) and Ib, suggesting that neofunctionalization in important functional regions has driven the evolution of auxin signalling in flowering plants. Primary auxin responsive gene families (GH3, SAUR, LBD) 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.
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 (also known as phytohormones) 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 [3, 4]. Proteins of two related families, AUXIN RESPONSIVE FACTOR (ARF) and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA), act together to regulate this transcription [5, 6]. In flowering plants, ARF proteins possess a conserved DNA-binding domain which recognizes auxin responsive elements (AuxREs): short motifs which are found in the promoter sequences of many auxin-responsive genes [7, 8]. Most ARFs, and all Aux/IAAs also contain a conserved dimerization domain which mediates protein-protein interactions within and between both protein families [9, 10]. The middle region which joins ARF DNA-binding and dimerization domains is highly divergent and may be glutamine (Q) rich . Those ARFs which contain such Q-rich regions are thought to be activators of gene transcription [11, 12]. Conversely, those ARFs which repress gene transcription lack glutamine (or in one case methionine) -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 [13, 14]. Domain II contains a degron: a motif sufficient to signal Aux/IAAs for proteasome-mediated degradation [6, 15, 16]. 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 E3 ubiquitin ligases, directly promoting their interaction. Accordingly, at high cellular auxin concentrations, Aux/IAAs are ubiquitinated and subsequently degraded [19–21]. Degradation of Aux/IAA proteins then allows ARF-mediated, auxin-dependent gene transcription.
Physcomitrella patens (a moss), Selaginella moellendorffii (a vascular non-seed plant) and angiosperms diverged from each other at between 700 and 450 million years ago . The genomes of both P. patens and S. moellendorffii encode all the proteins necessary for this primary auxin response [23, 24]. Furthermore, P. patens has been shown to both synthesize auxin, and respond to exogenously applied auxin [25, 26]. 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 (dN) to synonymous (dS) 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 and discussion
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.
In all but one Arabidopsis Aux/IAA protein, domain I contains a sequence of amino acids reminiscent of an ERF-associated amphiphilic repression (EAR) motif. This LxLxL domain I motif interacts directly with TOPLESS (TPL), a transcriptional co-repressor. This interaction leads to a repression of the ARF-dependent transcription of a reporter gene driven by the DR5 promoter, a synthetic auxin-sensitive marker containing repeated TGTCTC auxin response elements (AuxREs) [13, 14].
PpAux/IAAs do not contain an LxLxL motif in domain I. Instead they all contain a similar LxLxPP motif (Figure 1, Additional file 1). A corresponding and overlapping LxLxLxPP motif was found in three AtAux/IAAs (IAA18, 26 and 28), forming a cluster with good bootstrap support (Figure 1, Additional file 1). 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 (Figure 1, Additional file 1). 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.
Number of ARFs and Aux/IAA with different domains and motifs.
ARF with DBD domain
No III &IV domain
ARF without DBD domain
Only III&IV domain
Q-rich, no DBD domain
No domain II
No KR domain
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 (Additional file 1) this domain I motif can be expanded to LXL [A, G] [L, P] [P, G, S, T]. This allows the detection of domain I in all sequences tested of these three species. If expanded further, an [L, I]X [L, I] [A, G] [L, P] [P, G, S, T] 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 (Additional file 2) also suggests the LxLxPP motif is able to inhibit (at least to some extent) ARF-mediated transcription. In flowering plants, however, the LxLxPP sequence appears to have been superseded by the LxLxL domain (Additional file 1). 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 (via the LxLxPP motif) and flowering plants (via the LxLxL motif) 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.
The alignment of domain II from several Aux/IAA proteins indicates that not all 13 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 (Additional file 3). Nevertheless, a central core of five residues, representing amino acids 4–8 (GWPPV), 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 (which can be represented by VGWPP [L, V, I]) is conserved in all Aux/IAAs, including those from P. patens and S. moellendorffii (Additional file 3).
Aux/IAAs are degraded after domain II binds to the TIR1 family of F-box proteins [19–21]. The presence of the core motif of domain II and four paralogs of the Aux/IAA-specific TIR1 family of F-box proteins (Additional file 4) in P. patens, suggests that PpAux/IAAs are degraded in an auxin-dependent manner. Homology modelling has shown that the auxin binding pocket of PpTIR1 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 [28, 29] is not due to an impaired ability to degrade Aux/IAAs in response to auxin.
Diversification of Aux/IAA
Sites under PDS in the A. thaliana/P. trichocarpa Aux/IAA gene family: „Site-specific analysis".
dN/dS (ω) under M0
2Δℓ M2 vs. M1 (df 2)
2Δℓ M8 vs. M7 (df 2)
Parameter estimates under M8
Positively selected sites under M2 (BEB)
Positively selected sites under M8 (BEB)
ρ0 = 1.000 (ρ1 = 0.000) (ρ = 0.285)
q = 1.705 ω = 2.40
ρ0 = 0.944 (ρ1 = 0.056) (ρ = 0.315)
q = 1.634 ω = 2.11
21 23 39 40 68
ρ0 = 0.948 (ρ1 = 0.052) (ρ = 0.318)
q = 3.848 ω = 1.00
16 19 40 42 49 75 80 81
ρ0 = 1.000 (ρ1 = 0.000) (ρ = 0.537)
q = 2.898 ω = 2.302
10 11 171
ρ0 = 0.977 (ρ1 = 0.023) (ρ = 0.522)
q = 3.630 ω = 1.000
ρ0 = 1.000 (ρ1 = 0.000) (ρ = 0.505)
q = 0.301 ω = 5.133
20 115 116
ρ0 = 0.898 (ρ1 = 0.102) (ρ = 0.617)
q = 4.304 ω = 1.307
13 43 49
Aux/IAA genes have been retained in the A. thaliana genome at a high rate. A two-way analysis of variance (ANOVA) 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 63 conditions in Additional file 7, A–J (after ). All ten sister pairs of Aux/IAA showed significant gene (G), sample (S), and gene by sample (GxS) effects (Additional file 7, A–J).
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 [27, 34, 35]. 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 [38, 39]. Studies in P. trichocarpa  also showed that genes of the expanded PtIAA3 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.
In A. thaliana, all ARFs contain a DNA binding domain, but some lack a C-terminal dimerization domain (CTD). 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
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 (MRs) of five AtARFs of sub-class IIa (AtARFs 5, 6, 7, 8 and 19) mediate this transcription . All five of these MRs (as defined by the region between the CTD and DBD) are significantly longer than those of all other ARFs, with the exception of AtARF2 . PpARFs and SmARFs of class IIa also contain an extended MR (Additional file 8 and 9). A second feature of the MRs of those AtARFs which function as transcriptional activators is a relatively high proportion of glutamine residues (except for AtARF5) (Additional file 9 and 10). The MRs of canonical PpARFs of this group contain fewer glutamine residues than their vascular plant counterparts at between 7.8 and 10% of all amino acid residues, compared to between 17.1 and 22.3 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 10 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 (Additional file 9). Given the character states of the MR length (Additional file 8) and glutamine content (Additional file 10) in the phylogenetic tree, a single gain of the domain (basal to the cluster starting with AtARF7 and 19) seems to have occurred. The MR seems to have been secondarily reduced in one SmARF (Selmo1_2_438333) and secondarily expanded in AtARF2. 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 [28, 29, 41]. In contrast, auxin-responsive transcription in A. thaliana is observed rapidly, and at relatively low auxin concentrations [4, 42]. 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
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 (Figure 5). 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 (Table 1, Figure 3, Figure 5). 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 (via ARF-ARF dimerization) and repression (via ARF-Aux/IAA dimerization) of the auxin response.
Evolution of ARF activators
Sites under PDS in the A. thaliana/P. trichocarpa ARF gene family: „Site-specific analysis".
dN/dS (ω) under M0
2Δℓ M2 vs. M1 (df 2)
2Δℓ M8 vs. M7 (df 2)
Parameter estimates under M8
Positively selected sites under M2 (BEB)
Positively selected sites under M8 (BEB)
ρ0 = 0.957 (ρ1 = 0.043) (ρ = 0.431)
q = 1.635 ω = 1.09
478 540 563 631 758
ρ0 = 0.997 (ρ1 = 0.003) (ρ = 0.355)
q = 1.994 ω = 4.49
372 464 476 479 482 485 539
ρ0 = 0.973 (ρ1 = 0.027) (ρ = 0.409)
q = 1.814 ω = 3.086
460 526 532 534 539 542 544 546 550 556 557 558 559 561 562 566 568 569 570 574 599 608 677 785
ρ0 = 0.998 (ρ1 = 0.016) (ρ = 0.413) q = 1.988 ω = 2.69
96 449 486 570
ρ0 = 0.932 (ρ1 = 0.068) (ρ = 0.528)
q = 2.885 ω = 1.00
6 334 370 372 376 383 399 412 413 478 529
ρ0 = 0.982 (ρ1 = 0.018) (ρ = 0.056)
q = 0.054 ω = 10.50
175 335 389 433 444 447 527 567 569 570 572
175 335 351 359 389 433 444 447 527 567 569 570 572
ARF7 and ARF19 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, MYB77, interacts with the CTD ARF7 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 MYB77 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 (and possible subsequent post-translational modification) influencing protein function within the ARF7 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 (six, three and five respectively), 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 (class Ia, class Ib and class IIb) indicating that evolution within flowering plants has favoured strongly the diversification of auxin-regulated repressor ARFs (Figure 2).
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 (or basal) regulation of auxin-responsive genes (Figure 5). 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 [9, 18, 46] (Figure 3, 5). Phosphorylation of ARF2 (a full length ARF) by BIN2, a kinase involved in brassinosteroid-dependent transcription decreases its ability to bind DNA . This path for crosstalk between two hormone signalling pathways (auxin and brassinosteroid) 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 ARF12 node (Class Ib), where little is known about protein function (Additional file 6, B). 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 1.
Class Ib ARFs are absent from the P. trichocarpa and O. sativa genome, raising the possibility of a specific role within the order Brassicales [27, 35]. 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, ARF3 mutants show pleiotropic effects in flower development . Plants expressing a miRNA-resistant version of a second CTD-deficient ARF, ARF17, have increased ARF17 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 (Phypa_159688 and Phypa_171197), both encoding full-length ARFs (Figure 4), 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 miR160, miR164, and miR167 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: ARF3 is the target of AtTAS3a-c and ARF17 is the target of miR160 . 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 (Figure 5). 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 ARF6 and ARF8 (involved in female and male reproductive organ development) is controlled by miR167, with miRNA160 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 [52, 54]. 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 (those genes whose expression is directly regulated by ARFs) can be grouped into three major families: Aux/IAAs, GH3s and SAURs. Recently, transcription of certain LOB domain (LBD) genes has also been shown to be rapidly and specifically up-regulated by auxin [4, 56]. 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 GH3 genes (which encode auxin conjugating enzymes) is regulated by auxin  Similarly, in O. sativa, the transcription of GH3 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 GH3 family of flowering plants. Both conjugate IAA to amino acids, with PpGH3-2 showing a far broader range of substrate specificity than PpGH3-1 . Surprisingly, the moss GH3 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 (Additional file 12). Auxin application increases transcription of specific flowering plant GH3 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 GH3 enzyme, and one protein belonging to group I (Additional file 12). The remaining 19 SmGH3 genes cannot be clearly assigned based on phylogeny. The transcriptional response to auxin of these genes has never been tested.
P. patens GH3 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 GH3 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 GH3-11 of A. thaliana, which catalyses the synthesis of jasmonic acid conjugates [58, 60]. P. patens plants lacking the GH3-2 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 GH3-2 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 18 SAUR genes (A. thaliana approximately 70), which cluster in two groups with low bootstrap support (Additional file 13). All AtSAUR genes of group A are auxin-responsive [4, 62]. This group shows relatively high similarity to nine PpSAUR genes (albeit with low bootstrap support) (Additional file 13) 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 17 members, forming five clades (Additional file 14). One clade, encoding four LBDs (Phypa_18666, 7278, 25219 and 48669), is monophyletic with important auxin-responsive regulators of lateral root formation in A. thaliana, LBD16 and 29 , 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 (i) whether these plants are capable of rapidly synthesizing specific mRNAs in response to auxin in the same manner as flowering plants, and (ii) 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.
Candidate gene family member selection and curation
To define and extract the ARF, Aux/IAA and TIR1 gene families we screened the published genomes of A. thaliana (TAIR7; ftp://ftp.arabidopsis.org/Sequences/blast_datasets/TAIR7_blastsets/TAIR7_pep_20070425), O. sativa (Osa1 version 5.0; ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_5.0/all.chrs/all.pep), P. trichocarpa (Poptr1_1; ftp://ftp.jgi-psf.org/pub/JGI_data/Poplar/annotation/v1.1/proteins.Poptr1_1.JamboreeModels.fa sta.gz), S. bicolor (Sbi1.4; ftp://ftp.jgi-psf.org/pub/JGI_data/Sorghum_bicolor/v1.0/Sbi/annotation/Sbi1.4/Sbi1.4.pep.fa.gz), V. vinifera (Vitis_vinifera_v1; http://www.genoscope.cns.fr/externe/Download/Projets/Projet_ML/data/annotation/Vitis_vinifera_peptide_v1.fa), G. max (Glyma0.1b.pep.fa.gz; ftp://ftp.jgi-psf.org/pub/JGI_data/Glycine_max/Glyma0/annotation/) and P. patens (Phypa1_1; ftp://ftp.jgi-psf.org/pub/JGI_data/Physcomitrella_patens/v1.1/proteins.Phypa1_1.FilteredModels.fasta.gz) by BLASTP against a database containing all (predicted) proteins of the respective organisms. As queries, the known members of the A. thaliana gene families were used. For ARF queries, At1g35240, At1g77850 and At5g60450 were selected; for Aux/IAA, At1g04550, At2g01200 and At4g14560; and for TIR1, At3g62980 and At5g49980. Based on the protein domain architecture of the A. thaliana proteins, BLAST results were inspected manually to determine query specific filtering criteria. For ARF sequences, we required that 30% of amino acids be identical and 50% of aligned amino acid sites be shared; for Aux/IAA sequences, we used an E-value threshold of 1E-40, and 1E-62 for TIR1. The S. moellendorfii candidate gene family members were detected using BLASTP against the filtered models 2 predicted proteins using the filtering criteria mentioned above. All candidate loci were manually inspected using the JGI genome browser http://genome.jgi-psf.org/Selmo1/ and curated to select the "optimal" gene model. Additionally, the genomic contexts (~40 kbp) of highly conserved gene model pairs were compared to exclude redundancies due to gene models representing loci from the two sequenced haplotypes. Furthermore, the P. patens genome v1.1 was screened for additional, as yet undetected, gene family members using Exonerate . All detected P. patens candidate loci were inspected manually using the cosmoss.org genome browser http://www.cosmoss.org/cgi/gbrowse/physcome. Under consideration of all available cDNA, EST and protein evidences the "optimal" predicted gene model was derived for each locus. To reduce complexity and maintain readability of the resulting phylogenetic trees, further analysis only included the candidate proteins from P. patens, S. moellendorfii and A. thaliana. ARF and Aux/IAA P. patens protein IDs: 108888, 127416, 170581, 50215, 61245, 164608, 219923, 159688, 165321, 167026, 171197, 171888, 188433, 196920, 218828, 225990, 77324 and BAB71765. ARF and Aux/IAA S. moellendorfii protein IDs: 431277, 431298, 405821, 438333, 181406, 61688, 51695, 437944, 81992, 406764, 412634, 26861, 405646, 421309, 446535, and 422125.
Domain annotation and multiple sequence alignments
Protein domain architectures of the ARF and Aux/IAA candidate hits were annotated using the Pfam  Hidden Markov Profiles (HMMs) PF02362.12 (B3, representing the DBD), PF06507.4 (Auxin_resp), PF02309.7 (AUX_IAA, representing the CTD) and the PROSITE  profile PS50962 (IAA_ARF) using the hmmpfam and the ps_scan tools and applying each domain profile's "trusted cutoff" as filtering criteria. To extract CTD domain region from both, ARFs and Aux/IAAs (CTD+), the FASTA output option of ps_scan was used. CTD+ domain sequences were aligned with MAFFT L-INSI , ProbCons, Muscle and T-coffee and subsequently combined into an optimal alignment using the combiner function of T-coffee . Full-length multiple sequence alignments (MSAs) were calculated using Dialign . Full-length MSAs including the protein domain annotation were visualized and manually inspected and curated using the Jalview  alignment editor. In order to generate data for the domain-based phylogenies, the full-length MSAs were clipped to either the N-terminal DNA-binding (DBD; extending the B3 + Aux_resp domain matches) or the C-terminal interaction domain (CTD; extending the Aux_IAA domain matches) regions, according to the domain annotation and alignment quality. Proteins missing both individual domains were discarded and the clipped MSAs were realigned using the MAFFT  L-INSI algorithm.
Bayesian inference was performed using MrBayes for the clipped Aux/IAA and the CTD+ MSA with 2 runs with a mixed model prior, a proportion of invariable sites and gamma distribution for a maximum of 2,000,000 using a temperature of 0.2 and a sampling rate of 5. Maximum Likelihood (ML) and Neighbor Joining (NJ) phylogenies were calculated for the full-length Aux/IAA MSA and the clipped MSAs for the DBD, the ARF-specific CTD and the CTD including the Aux/IAAs (CTD+). Bootstrapped (100×) NJ trees were calculated using a modified version of the quicktree software , with the Scoredist  matrix. ProtTest  was used to select the most appropriate evolutionary model for ML inference (DBD:JTT+G; CTD:JTT+G; Aux/IAA:JTT+G+F). Bootstrapped (100×) best-known likelihood topologies were calculated using the parallelized version of RAxML . Generally, phylogenetic trees were rooted by midpoint-rooting. The CTD, as the common feature of both families, was used to root the ARF and AUX/IAA trees. To infer the history of duplications and losses, the CTD+ phylogeny was reconciled with Notung , as used in [75–77] applying the species tree (Phypa, (Selmo, Arath)).
Character state analyses
The MR was defined as the region between DBD and CTD, or in case of a lack of the DBD as the region from the N-terminus to the start of the CTD. The length of the MR was transformed into a continuous character matrix comprising eight characters. Q-rich regions were represented by the amino acid frequency normalized to the length of the MR. The resulting character matrix was analyzed using the Mesquite  analysis tool "Trace Character History" on the basis of the Notung reconciled CTD+ MrBayes phylogeny. Nucleotide alignments of coding sequences were performed on the basis of protein alignments. The protein sequences were aligned with MAFFT . DAMBE 4.5.55  was used to translate protein alignments to nucleotide alignments.
Statistical tests for positive selection
We applied the codon-based substitution model of Yang et al.  to identify amino acid sites under positive selection using PAML3.14 . First, we ran a test for the existence of sites with a dN/dS ratio > 1 by using a likelihood ratio test (LRT) to compare null models M1a and M7(beta) (that do not allow for sites with dN/dS >1) with alternative models M2a (PositiveSelection) and M8(beta&ω). If the LRT difference was statistically significant we identified the sites that were under positive selection. Naïve empirical Bayes (NEB) and Bayes empirical Bayes (BEB) approaches were used  to calculate the posterior probability that each site belongs to a particular site class. Sites with high posterior probabilities from the class with ω>1 were inferred to be under positive selection.
The microarray gene expression data for paralogous pairs of Aux/IAA genes were analyzed in 63 diverse samples  (in our analysis, we included only data generated from wild type plants). gcRMA normalized data were used . Three biological replications were used to generate the data sets. To identify which components contribute to expression pattern divergence within each duplicate pair, the two-way ANOVA used by Duarte et al.  to partition the gene (G), sample (S), and gene by sample interaction (GxS) effects was extended to all 63 microarray samples. Analysis was done using Statistica 5.0.
AUXIN RESPONSIVE FACTOR
Auxin responsive elements
Bayes empirical Bayes
NDA binding domain
ERF-associated amphiphilic repression
ethylene response factor
Hidden Marsov Profiles
likelihood ratio test
maximum likelihood estimates
multiple sequence alignments
Naïve empirical Bayes
We are grateful to the Selaginella community http://selaginella.genomics.purdue.edu/ and to the JGI http://genome.jgi-psf.org/Selmo1/ for providing the S. moellendorffii genome sequence. Our work was supported by the Deutsche Forschungsgemeinschaft (SFB 592, grant Re 837/10-2), BMBF (grant 0313921, Freiburg Initiative in Systems Biology), ESA, EU, FCI, and the Landesstiftung Baden-Württemberg GmbH. D.L. is grateful for support by the GRK1305 International Graduate School.
- Davies PJ: Plant Hormones: Biosynthesis, Signal Transduction, Action!. 2004, Dordrecht: SpringerGoogle Scholar
- Teale WD, Paponov IA, Palme K: Auxin in action: signalling, transport and the control of plant growth and development. Nat Rev Mol Cell Biol. 2006, 7: 847-859. 10.1038/nrm2020.View ArticlePubMedGoogle Scholar
- Theologis A, Ray PM: Early auxin-regulated polyadenylylated messenger-RNA sequences In pea stem tissue. Proc Natl Acad Sci USA. 1982, 79: 418-421. 10.1073/pnas.79.2.418.PubMed CentralView ArticlePubMedGoogle Scholar
- Paponov I, Paponov M, Teale WD, Menges M, Shakrabortee S, Murray JA, Palme K: Comprehensive transcriptome analysis of auxin responses in Arabidopsis. Mol Plant. 2008, 1: 321-337. 10.1093/mp/ssm021.View ArticlePubMedGoogle Scholar
- Guilfoyle T, Hagen G, Ulmasov T, Murfett J: How does auxin turn on genes?. Plant Physiol. 1998, 118: 341-347. 10.1104/pp.118.2.341.PubMed CentralView ArticlePubMedGoogle Scholar
- Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M: Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Nature. 2001, 414: 271-276. 10.1038/35104500.View ArticlePubMedGoogle Scholar
- Ballas N, Wong LM, Theologis A: Identification of the auxin-responsive element, AuxRE, in the primary indoleacetic acid-inducible gene, PS-IAA4/5, of pea (Pisum Sativum). J Mol Biol. 1993, 233: 580-596. 10.1006/jmbi.1993.1537.View ArticlePubMedGoogle Scholar
- Li Y, Liu ZB, Shi XY, Hagen G, Guilfoyle TJ: An auxin-inducible element in soybean SAUR promoters. Plant Physiol. 1994, 106: 37-43. 10.1104/pp.106.1.37.PubMed CentralView ArticlePubMedGoogle Scholar
- Ulmasov T, Hagen G, Guilfoyle TJ: ARF1, a transcription factor that binds to auxin response elements. Science. 1997, 276: 1865-1868. 10.1126/science.276.5320.1865.View ArticlePubMedGoogle Scholar
- Kim J, Harter K, Theologis A: Protein-protein interactions among the Aux/IAA proteins. Proc Natl Acad Sci USA. 1997, 94: 11786-11791. 10.1073/pnas.94.22.11786.PubMed CentralView ArticlePubMedGoogle Scholar
- Ulmasov T, Hagen G, Guilfoyle TJ: Activation and repression of transription by auxin-response factors. Proc Natl Acad Sci USA. 1999, 96: 5844-5849. 10.1073/pnas.96.10.5844.PubMed CentralView ArticlePubMedGoogle Scholar
- Tiwari SB, Hagen G, Guilfoyle T: The roles of auxin response factor domains in auxin-responsive transcription. Plant Cell. 2003, 15: 533-543. 10.1105/tpc.008417.PubMed CentralView ArticlePubMedGoogle Scholar
- Szemenyei H, Hannon M, Long JA: TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science. 2008, 319: 1384-1386. 10.1126/science.1151461.View ArticlePubMedGoogle Scholar
- Tiwari SB, Hagen G, Guilfoyle TJ: Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell. 2004, 16: 533-543. 10.1105/tpc.017384.PubMed CentralView ArticlePubMedGoogle Scholar
- Worley CK, Zenser N, Ramos J, Rouse D, Leyser O, Theologis A, Callis J: Degradation of Aux/IAA proteins is essential for normal auxin signalling. Plant J. 2000, 21: 553-562. 10.1046/j.1365-313x.2000.00703.x.View ArticlePubMedGoogle Scholar
- Ramos JA, Zenser N, Leyser O, Callis J: Rapid degradation of auxin/indoleacetic acid proteins requires conserved amino acids of domain II and is proteasome dependent. Plant Cell. 2001, 13: 2349-2360. 10.1105/tpc.13.10.2349.PubMed CentralView ArticlePubMedGoogle Scholar
- Ouellet F, Overvoorde PJ, Theologis A: IAA17/AXR3: biochemical insight into an auxin mutant phenotype. Plant Cell. 2001, 13: 829-841. 10.1105/tpc.13.4.829.PubMed CentralView ArticlePubMedGoogle Scholar
- Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ: Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell. 1997, 9: 1963-1971. 10.1105/tpc.9.11.1963.PubMed CentralView ArticlePubMedGoogle Scholar
- Kepinski S, Leyser O: The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature. 2005, 435: 446-451. 10.1038/nature03542.View ArticlePubMedGoogle Scholar
- Dharmasiri N, Dharmasiri S, Estelle M: The F-box protein TIR1 is an auxin receptor. Nature. 2005, 435: 441-445. 10.1038/nature03543.View ArticlePubMedGoogle Scholar
- Tan X, Calderon-Villalobos LIA, Sharon M, Zheng CX, Robinson CV, Estelle M, Zheng N: Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature. 2007, 446: 640-645. 10.1038/nature05731.View ArticlePubMedGoogle Scholar
- Lang D, Zimmer AD, Rensing SA, Reski R: Exploring plant biodiversity: the Physcomitrella genome and beyond. Trends in Plant Science. 2008, 13: 542-549. 10.1016/j.tplants.2008.07.002.View ArticlePubMedGoogle Scholar
- Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H, Nishiyama T, Perroud PF, Lindquist EA, Kamisugi Y, Tanahashi T, Sakakibara K, Fujita T, Oishi K, Shin-I T, Kuroki Y, Toyoda A, Suzuki Y, Hashimoto S, Yamaguchi K, Sugano S, Kohara Y, Fujiyama A, Anterola A, Aoki S, Ashton N, Barbazuk WB, Barker E, Bennetzen JL, Blankenship R, Cho SH, Dutcher SK, Estelle M, Fawcett JA, Gundlach H, Hanada K, Heyl A, Hicks KA, Hughes J, Lohr M, Mayer K, Melkozernov A, Murata T, Nelson DR, Pils B, Prigge M, Reiss B, Renner T, Rombauts S, Rushton PJ, Sanderfoot A, Schween G, Shiu SH, Stueber K, Theodoulou FL, Tu H, Peer Van de Y, Verrier PJ, Waters E, Wood A, Yang LX, Cove D, Cuming AC, Hasebe M, Lucas S, Mishler BD, Reski R, Grigoriev IV, Quatrano RS, Boore JL: The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science. 2008, 319: 64-69. 10.1126/science.1150646.View ArticlePubMedGoogle Scholar
- Floyd SK, Bowman JL: The ancestral developmental tool kit of land plants. Int J Plant Sci. 2007, 168: 1-35. 10.1086/509079.View ArticleGoogle Scholar
- Cooke TJ, Poli D, Sztein AE, Cohen JD: Evolutionary patterns in auxin action. Plant Mol Biol. 2002, 49 (3-4): 319-38. 10.1023/A:1015242627321.View ArticlePubMedGoogle Scholar
- Hayashi K, Tan X, Zheng N, Hatate T, Kimura Y, Kepinski S, Nozaki H: Small-molecule agonists and antagonists of F-box protein-substrate interactions in auxin perception and signaling. Proc Natl Acad Sci USA. 2008, 105: 5632-5637. 10.1073/pnas.0711146105.PubMed CentralView ArticlePubMedGoogle Scholar
- Kalluri UC, DiFazio SP, Brunner AM, Tuskan GA: Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa. BMC Plant Biology. 2007, 7: 59-10.1186/1471-2229-7-59.PubMed CentralView ArticlePubMedGoogle Scholar
- Bierfreund NM, Reski R, Decker EL: Use of an inducible reporter gene system for the analysis of auxin distribution in the moss Physcomitrella patens. Plant Cell Rep. 2003, 21: 1143-1152. 10.1007/s00299-003-0646-1.View ArticlePubMedGoogle Scholar
- Imaizumi T, Kadota A, Hasebe M, Wada M: Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens. Plant Cell. 2002, 14: 373-386. 10.1105/tpc.010388.PubMed CentralView ArticlePubMedGoogle Scholar
- Hill RE, Hastie ND: Accelerated evolution in the reactive center regions of serine protease inhibitors. Nature. 1987, 326: 96-99. 10.1038/326096a0.View ArticlePubMedGoogle Scholar
- Duarte JM, Cui LY, Wall PK, Zhang Q, Zhang XH, Leebens-Mack J, Ma H, Altman N, dePamphilis CW: Expression pattern shifts following duplication indicative of subfunctionalization and neofunctionalization in regulatory genes of Arabidopsis. Mol Biol Evol. 2006, 23: 469-478. 10.1093/molbev/msj051.View ArticlePubMedGoogle Scholar
- Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Scholkopf B, Weigel D, Lohmann JU: A gene expression map of Arabidopsis thaliana development. Nat Genet. 2005, 37: 501-506. 10.1038/ng1543.View ArticlePubMedGoogle Scholar
- Remington DL, Vision TJ, Guilfoyle TJ, Reed JW: Contrasting modes of diversification in the Aux/IAA and ARF gene families. Plant Physiol. 2004, 135: 1738-1752. 10.1104/pp.104.039669.PubMed CentralView ArticlePubMedGoogle Scholar
- Jain M, Kaur N, Garg R, Thakur JK, Tyagi AK, Khurana JP: Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa). Funct Integr Genomics. 2006, 6: 47-59. 10.1007/s10142-005-0005-0.View ArticlePubMedGoogle Scholar
- Wang DK, Pei KM, Fu YP, Sun ZX, Li SJ, Liu HQ, Tang K, Han B, Tao YZ: Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa). Gene. 2007, 394: 13-24. 10.1016/j.gene.2007.01.006.View ArticlePubMedGoogle Scholar
- Walsh JB: How often do duplicated genes evolve new functions?. Genetics. 1995, 139: 421-428.PubMed CentralPubMedGoogle Scholar
- Ganko EW, Meyers BC, Vision TJ: Divergence in expression between duplicated genes in Arabidopsis. Mol Biol Evol. 2007, 24: 2298-2309. 10.1093/molbev/msm158.View ArticlePubMedGoogle Scholar
- Knox K, Grierson CS, Leyser O: AXR3 and SHY2 interact to regulate root hair development. Development. 2003, 130: 5769-5777. 10.1242/dev.00659.View ArticlePubMedGoogle Scholar
- Weijers D, Benkova E, Jager KE, Schlereth A, Hamann T, Kientz M, Wilmoth JC, Reed JW, Jurgens G: Developmental specificity of auxin response by pairs of ARF and Aux/IAA transcriptional regulators. EMBO J. 2005, 24: 1874-1885. 10.1038/sj.emboj.7600659.PubMed CentralView ArticlePubMedGoogle Scholar
- Guilfoyle TJ, Hagen G: Auxin response factors. Curr Opin Plant Biol. 2007, 10: 453-460. 10.1016/j.pbi.2007.08.014.View ArticlePubMedGoogle Scholar
- Fujita T, Sakaguchi H, Hiwatashi Y, Wagstaff SJ, Ito M, Deguchi H, Sato T, Hasebe M: Convergent evolution of shoots in land plants: lack of auxin polar transport in moss shoots. Evol Dev. 2008, 10: 176-186.View ArticlePubMedGoogle Scholar
- Abel S, Theologis A: Early genes and auxin action. Plant Physiol. 1996, 111: 9-17. 10.1104/pp.111.1.9.PubMed CentralView ArticlePubMedGoogle Scholar
- Salmon J, Ramos J, Callis J: Degradation of the auxin response factor ARF1. Plant J. 2008, 54: 118-128. 10.1111/j.1365-313X.2007.03396.x.View ArticlePubMedGoogle Scholar
- Okushima Y, Overvoorde PJ, Arima K, Alonso JM, Chan A, Chang C, Ecker JR, Hughes B, Lui A, Nguyen D, Onodera C, Quach H, Smith A, Yu GX, Theologis A: Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: Unique and overlapping functions of ARF7 and ARF19. Plant Cell. 2005, 17: 444-463. 10.1105/tpc.104.028316.PubMed CentralView ArticlePubMedGoogle Scholar
- Shin R, Burch AY, Huppert KA, Tiwari SB, Murphy AS, Guilfoyle TJ, Schachtman DP: The Arabidopsis transcription factor MYB77 modulates auxin signal transduction. Plant Cell. 2007, 19: 2440-2453. 10.1105/tpc.107.050963.PubMed CentralView ArticlePubMedGoogle Scholar
- Tiwari SB, Wang XJ, Hagen G, Guilfoyle TJ: AUX/IAA proteins are active repressors, and their stability and activity are modulated by auxin. Plant Cell. 2001, 13: 2809-2822. 10.1105/tpc.13.12.2809.PubMed CentralView ArticlePubMedGoogle Scholar
- Vert G, Walcher CL, Chory J, Nemhauser JL: Integration of auxin and brassinosteroid pathways by Auxin Response Factor 2. Proc Natl Acad Sci USA. 2008, 105: 9829-9834. 10.1073/pnas.0803996105.PubMed CentralView ArticlePubMedGoogle Scholar
- Sessions A, Nemhauser JL, McColl A, Roe JL, Feldmann KA, Zambryski PC: ETTIN patterns the Arabidopsis floral meristem and reproductive organs. Development. 1997, 124: 4481-4491.PubMedGoogle Scholar
- Mallory AC, Bartel DP, Bartel B: MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell. 2005, 17: 1360-1375. 10.1105/tpc.105.031716.PubMed CentralView ArticlePubMedGoogle Scholar
- Axtell MJ, Snyder JA, Bartell DP: Common functions for diverse small RNAs of land plants. Plant Cell. 2007, 19: 1750-1769. 10.1105/tpc.107.051706.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu PP, Montgomery TA, Fahlgren N, Kasschau KD, Nonogaki H, Carrington JC: Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages. Plant J. 2007, 52: 133-146. 10.1111/j.1365-313X.2007.03218.x.View ArticlePubMedGoogle Scholar
- Wu MF, Tian Q, Reed JW: Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction. Development. 2006, 133: 4211-4218. 10.1242/dev.02602.View ArticlePubMedGoogle Scholar
- Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP: MicroRNAs in plants. Genes Dev. 2002, 16: 1616-1626. 10.1101/gad.1004402.PubMed CentralView ArticlePubMedGoogle Scholar
- Axtell MJ, Bartel DP: Antiquity of microRNAs and their targets in land plants. Plant Cell. 2005, 17: 1658-1673. 10.1105/tpc.105.032185.PubMed CentralView ArticlePubMedGoogle Scholar
- Fattash I, Voss B, Reski R, Hess WR, Frank W: Evidence for the rapid expansion of microRNA-mediated regulation in early land plant evolution. BMC Plant Biology. 2007, 7: 13-10.1186/1471-2229-7-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Okushima Y, Fukaki H, Onoda M, Theologis A, Tasaka M: ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. Plant Cell. 2007, 19: 118-130. 10.1105/tpc.106.047761.PubMed CentralView ArticlePubMedGoogle Scholar
- Jain M, Kaur N, Tyagi AK, Khurana JP: The auxin-responsive GH3 gene family in rice (Oryza sativa). Funct Integr Genomics. 2006, 6: 36-46. 10.1007/s10142-005-0142-5.View ArticlePubMedGoogle Scholar
- Ludwig-Müller J, Jülke S, Bierfreund NM, Decker EL, Reski R: Moss (Physcomitrella paterns) GH3 proteins act in auxin homeostasis. New Phytol. 2009, 181 (2): 323-38. 10.1111/j.1469-8137.2008.02677.x.View ArticlePubMedGoogle Scholar
- Bierfreund NM, Tintelnot S, Reski R, Decker EL: Loss of GH3 function does not affect phytochrome- mediated development in a moss, Physcomitrella patens. J Plant Physiol. 2004, 161: 823-835. 10.1016/j.jplph.2003.12.010.View ArticlePubMedGoogle Scholar
- Staswick PE, Tiryaki I: The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis. Plant Cell. 2004, 16: 2117-2127. 10.1105/tpc.104.023549.PubMed CentralView ArticlePubMedGoogle Scholar
- Hagen G, Guilfoyle T: Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Mol Biol. 2002, 49 (3-4): 373-85. 10.1023/A:1015207114117.View ArticlePubMedGoogle Scholar
- Jain M, Tyagi AK, Khurana JP: Genome-wide analysis, evolutionary expansion, and expression of early auxin-responsive SAUR gene family in rice (Oryza sativa). Genomics. 2006, 88: 360-371. 10.1016/j.ygeno.2006.04.008.View ArticlePubMedGoogle Scholar
- Slater GS, Birney E: Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 2005, 6: 31-10.1186/1471-2105-6-31.PubMed CentralView ArticlePubMedGoogle Scholar
- Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer ELL, Bateman A: The Pfam protein families database. Nucleic Acids Res. 2008, 36: D281-D288. 10.1093/nar/gkm960.PubMed CentralView ArticlePubMedGoogle Scholar
- Hulo N, Bairoch A, Bulliard V, Cerutti L, Cuche BA, de Castro E, Lachaize C, Langendijk-Genevaux PS, Sigrist CJA: The 20 years of PROSITE. Nucleic Acids Res. 2008, 36: D245-D249. 10.1093/nar/gkm977.PubMed CentralView ArticlePubMedGoogle Scholar
- Katoh K, Kuma K, Toh H, Miyata T: MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 2005, 33: 511-518. 10.1093/nar/gki198.PubMed CentralView ArticlePubMedGoogle Scholar
- Notredame C, Higgins DG, Heringa J: T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000, 302: 205-217. 10.1006/jmbi.2000.4042.View ArticlePubMedGoogle Scholar
- Morgenstern B: Alignment of genomic sequences using DIALIGN. Methods Mol Biol. 2007, 195-203.Google Scholar
- Clamp M, Cuff J, Searle SM, Barton GJ: The Jalview Java alignment editor. Bioinformatics. 2004, 20: 426-427. 10.1093/bioinformatics/btg430.View ArticlePubMedGoogle Scholar
- Hanekamp K, Bohnebeck U, Beszteri B, Valentin K: PhyloGena – a user-friendly system for automated phylogenetic annotation of unknown sequences. Bioinformatics. 2007, 23: 793-801. 10.1093/bioinformatics/btm016.View ArticlePubMedGoogle Scholar
- Sonnhammer ELL, Hollich V: Scoredist: A simple and robust protein sequence distance estimator. BMC Bioinformatics. 2005, 6: 108-10.1186/1471-2105-6-108.PubMed CentralView ArticlePubMedGoogle Scholar
- Abascal F, Zardoya R, Posada D: ProtTest: selection of best-fit models of protein evolution. Bioinformatics. 2005, 21: 2104-2105. 10.1093/bioinformatics/bti263.View ArticlePubMedGoogle Scholar
- Stamatakis A: RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006, 22: 2688-2690. 10.1093/bioinformatics/btl446.View ArticlePubMedGoogle Scholar
- Durand D, Halldorsson BV, Vernot B: A hybrid micro-macroevolutionary approach to gene tree reconstruction. J Comput Biol. 2006, 13: 320-335. 10.1089/cmb.2006.13.320.View ArticlePubMedGoogle Scholar
- Wildman DE, Chen CY, Erez O, Grossman LI, Goodman M, Romero R: Evolution of the mammalian placenta revealed by phylogenetic analysis. Proc Natl Acad Sci USA. 2006, 103: 3203-3208. 10.1073/pnas.0511344103.PubMed CentralView ArticlePubMedGoogle Scholar
- Als TD, Vila R, Kandul NP, Nash DR, Yen SH, Hsu YF, Mignault AA, Boomsma JJ, Pierce NE: The evolution of alternative parasitic life histories in large blue butterflies. Nature. 2004, 432: 386-390. 10.1038/nature03020.View ArticlePubMedGoogle Scholar
- Whittall JB, Hodges SA: Pollinator shifts drive increasingly long nectar spurs in columbine flowers. Nature. 2007, 447: 706-U712. 10.1038/nature05857.View ArticlePubMedGoogle Scholar
- Maddison WP, Maddison DR: Mesquite: A modular system for evolutionary analysis. Version 1.12. 2006, [http://mesquiteproject.org]Google Scholar
- Xia X, Xie Z: DAMBE: Software package for data analysis in molecular biology and evolution. J Hered. 2001, 92: 371-373. 10.1093/jhered/92.4.371.View ArticlePubMedGoogle Scholar
- Yang ZH, Nielsen R, Goldman N, Pedersen AMK: Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics. 2000, 155: 431-449.PubMed CentralPubMedGoogle Scholar
- Yang ZH: PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci. 1997, 13: 555-556.PubMedGoogle Scholar
- Yang ZH, Wong WSW, Nielsen R: Bayes empirical Bayes inference of amino acid sites under positive selection. Mol Biol Evol. 2005, 22: 1107-1118. 10.1093/molbev/msi097.View ArticlePubMedGoogle Scholar
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