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Plant Physiol. Jul 2010; 153(3): 1398–1412.
Published online May 14, 2010. doi:  10.1104/pp.110.153593
PMCID: PMC2899937

Genome-Wide Classification and Evolutionary Analysis of the bHLH Family of Transcription Factors in Arabidopsis, Poplar, Rice, Moss, and Algae1,[W]

Abstract

Basic helix-loop-helix proteins (bHLHs) are found throughout the three eukaryotic kingdoms and constitute one of the largest families of transcription factors. A growing number of bHLH proteins have been functionally characterized in plants. However, some of these have not been previously classified. We present here an updated and comprehensive classification of the bHLHs encoded by the whole sequenced genomes of Arabidopsis (Arabidopsis thaliana), Populus trichocarpa, Oryza sativa, Physcomitrella patens, and five algae species. We define a plant bHLH consensus motif, which allowed the identification of novel highly diverged atypical bHLHs. Using yeast two-hybrid assays, we confirm that (1) a highly diverged bHLH has retained protein interaction activity and (2) the two most conserved positions in the consensus play an essential role in dimerization. Phylogenetic analysis permitted classification of the 638 bHLH genes identified into 32 subfamilies. Evolutionary and functional relationships within subfamilies are supported by intron patterns, predicted DNA-binding motifs, and the architecture of conserved protein motifs. Our analyses reveal the origin and evolutionary diversification of plant bHLHs through differential expansions, domain shuffling, and extensive sequence divergence. At the functional level, this would translate into different subfamilies evolving specific DNA-binding and protein interaction activities as well as differential transcriptional regulatory roles. Our results suggest a role for bHLH proteins in generating plant phenotypic diversity and provide a solid framework for further investigations into the role carried out in the transcriptional regulation of key growth and developmental processes.

Most biological processes in a eukaryotic cell or organism are finely controlled at the transcriptional level by transcription factors. Transcription factors usually contain two different functional domains involved in DNA binding and protein dimerization, activities that may be regulated by several mechanisms, including differential dimer formation (Riechmann et al., 2000; Amoutzias et al., 2007). In addition, transcription factors are usually encoded by multigene families, multiplying the number and complexity of possible transcriptional regulatory roles (Riechmann et al., 2000).

Basic helix-loop-helix proteins (bHLHs) are widely distributed in all three eukaryotic kingdoms and constitute one of the largest families of transcription factors (Riechmann et al., 2000; Ledent and Vervoort, 2001). bHLHs represent key regulatory components in transcriptional networks controlling a number of biological processes. In unicellular eukaryotes, such as yeast, bHLH proteins are involved in chromosome segregation, general transcriptional enhancement, and metabolism regulation (Robinson and Lopes, 2000). In animals, bHLHs have been involved in sensing environmental signals, in regulating the cell cycle and circadian rhythms, as well as in the regulation of diverse essential developmental processes, including neurogenesis, myogenesis, sex and cell lineage determination, proliferation, and differentiation (Atchley and Fitch, 1997; Ledent and Vervoort, 2001; Amoutzias et al., 2004; Stevens et al., 2008). The R gene product Lc was the first plant protein reported to possess a bHLH domain and is involved in the control of flavonoid/anthocyanin biosynthesis in maize (Zea mays; Ludwig et al., 1989). The R gene belongs to a small subfamily comprising three additional genes (R, B, and Sn) for which the corresponding orthologs have been reported in Arabidopsis (Arabidopsis thaliana; AtTT8) and rice (Oryza sativa; OsRa-c; Hu et al., 1996; Nesi et al., 2000).

The number of characterized plant bHLHs has increased in recent years, revealing the wide and diverse array of biological processes in which they are involved. They have been reported to function in light signaling (Ni et al., 1998; Halliday et al., 1999; Fairchild et al., 2000; Huq and Quail, 2002; Khanna et al., 2004; Oh et al., 2004; Hyun and Lee, 2006; Roig-Villanova et al., 2007; Leivar et al., 2008), hormone signaling (Abe et al., 1997; Friedrichsen et al., 2002; Yin et al., 2005; Lee et al., 2006), wound and drought stress responses (de Pater et al., 1997; Smolen et al., 2002; Chinnusamy et al., 2003; Kiribuchi et al., 2004), symbiotic ammonium transport (Kaiser et al., 1998), shoot branching (Komatsu et al., 2001), fruit and flower development (Rajani and Sundaresan, 2001; Liljegren et al., 2004; Szecsi et al., 2006; Zhang et al., 2006; Gremski et al., 2007), and microspore (Sorensen et al., 2003), trichome (Payne et al., 2000; Morohashi et al., 2007), stomata (Pillitteri et al., 2007; Kanaoka et al., 2008), and root (Menand et al., 2007; Ohashi-Ito and Bergmann, 2007) development.

These proteins are defined by the bHLH signature domain (Ferre-D'Amare et al., 1993), which is composed of approximately 60 amino acids arranged according to the typical bifunctional structure. The basic region, an N-terminal stretch of approximately 15 to 20 residues typically rich in basic amino acids, is involved in DNA binding. Certain conserved amino acids in the basic region determine recognition to the so-called core E-box hexanucleotide consensus sequence 5′-CANNTG-3′, whereas other residues would provide specificity for a given type of E-box (e.g. the G-box [5′-CACGTG-3′]). In addition, flanking nucleotides outside the core have also been shown to play a role in binding specificity (Shimizu et al., 1997; Atchley et al., 1999; Martinez-Garcia et al., 2000; Massari and Murre, 2000). The HLH region is composed of two amphipathic α-helices mainly consisting of hydrophobic residues linked by a more diverged (both in length and primary sequence) loop region. The HLH domain promotes protein-protein interaction, allowing the formation of homodimeric or heterodimeric complexes (Massari and Murre, 2000). Cocrystal structural analysis has shown the interaction between the HLH regions of two bHLH proteins and that each partner binds to half of the DNA recognition sequence (Ma et al., 1994; Shimizu et al., 1997).

Outside the bHLH domain, bHLH proteins usually exhibit low, if any, sequence conservation. However, groups of evolutionary and/or functionally related bHLH proteins may share additional motifs. Some of these have been characterized in animals to determine specificity in DNA-binding sequence recognition and dimerization activities, as responsible for the activation or repression of target genes or for the binding to small molecules (e.g. dioxin; Ledent and Vervoort, 2001). One example is provided by the highly conserved Leu zipper (ZIP) motif characterized by heptad repeats of Leu residues adjacent to the second helix of the bHLH domain and predicted to adopt a coiled-coil structure that permits dimerization between proteins (Lupas, 1996). Other domains commonly found in animal bHLH proteins are the PAS domain, the Orange domain, the WRPW motif, and the COE domain (Ledent and Vervoort, 2001; Stevens et al., 2008).

Previous classifications of animal bHLHs have led to the definition of six major functional and evolutionary lineages (groups A–F; Atchley and Fitch, 1997; Ledent and Vervoort, 2001) that can be further subdivided into smaller orthologous subfamilies (Simionato et al., 2007). Most bHLH proteins are classified as group A or B and are expected to bind the core E-box consensus sequences. Group B includes members specifically displaying a G-box-binding motif configuration and proteins that share a ZIP domain at the COOH-terminal end of the protein or that contain the Orange domain. Group C bHLH proteins share a pair of PAS domains and bind non E-box sequences. Group E includes bHLH proteins that contain a conserved Pro or Gly residue at a key position within the basic region, preferentially bind to sequences referred to as N-boxes, and share an additional WRPW motif. Groups D and F represent proteins particularly diverged at the basic region. Some group D proteins, described as unable to bind DNA, might form heterodimers that function as dominant-negative regulators of DNA-binding activity of otherwise DNA-binding bHLHs (Fairman et al., 1993). Group F includes the so-called COE proteins, which share the COE domain. It has been suggested that the ancestral bHLH sequence was a group B protein present in early eukaryote evolution, from which bHLHs from different lineages evolved independently (Ledent and Vervoort, 2001; Heim et al., 2003).

Previous classifications of the family of bHLH proteins encoded by the Arabidopsis and rice genomes (Heim et al., 2003; Toledo-Ortiz et al., 2003; Li et al., 2006b) are essentially based on a bHLH consensus motif constructed from the alignment of 392 sequences mostly from groups A and B of animal DNA-binding bHLHs (Atchley et al., 1999). The consensus was expected to identify bHLH domain-containing proteins with a high degree of accuracy. However, highly diverged bHLH proteins are poorly predicted from the consensus (Atchley et al., 1999), and recent studies have identified and characterized novel atypical bHLHs in Arabidopsis (Fairchild et al., 2000; Hyun and Lee, 2006; Lee et al., 2006; Roig-Villanova et al., 2007). They were particularly diverged at the basic region and usually lacked sequence features characterized as critical for proper DNA binding (Massari and Murre, 2000).

Functional diversification in gene families encoding transcription factors is emerging as a major source of morphological and physiological diversity underlying evolution (Doebley and Lukens, 1998; Riechmann et al., 2000; Tsiantis and Hay, 2003; Kellogg, 2004). We present here a comprehensive classification together with a structural and evolutionary analysis of the plant bHLH gene family. This analysis was performed at a genome-wide level across distantly related land plant evolutionary lineages, including three angiosperms, Arabidopsis (eudicot-eurosids II), Populus trichocarpa (poplar; eudicot-eurosids I), and rice (monocot), as well as Physcomitrella patens (moss; bryophyte; Arabidopsis Genome Initiative, 2000; International Rice Genome Sequencing Project, 2005; Tuskan et al., 2006; Rensing et al., 2008). In terms of evolution, moss can be considered as a basal species for land plants and, therefore, might enable inference of the ancestral state of the land plant bHLH family (Kenrick and Crane, 1997; Karol et al., 2001). Furthermore, to have a broader perspective into the early evolutionary history of the plant bHLH family, we also searched for bHLH genes in five algal species, including four green algae species (Volvox carteri, Chlamydomonas reinhardtii, Ostreococcus tauri, Ostreococcus lucimarinus), which diverged from the land plants over 1 billion years ago, and the primitive red alga Cyanidioschyzon merolae (Matsuzaki et al., 2004; Merchant et al., 2007; Palenik et al., 2007). This is a first step toward further investigations into the biological and molecular functions of novel bHLH transcription factors as well as into their role in plant evolutionary diversification.

RESULTS

Identification and Classification of Arabidopsis, Poplar, Rice, Moss, and Algae bHLH Gene Families

Previous surveys of Arabidopsis and rice bHLH gene families had identified 162 and 167 members, respectively (Bailey et al., 2003; Li et al., 2006b). All but seven of these sequences encoded for proteins annotated as matching the INTERPRO 001092 domain, corresponding to the dimerization region of the bHLH domain. To define the bHLH gene families from poplar, moss, V. carteri, C. reinhardtii, O. tauri, O. lucimarinus, and C. merolae, we searched through the corresponding whole sequenced genomes for genes encoding proteins containing the INTERPRO 001092 domain. The resulting sequences were named following the generic system proposed for Arabidopsis (Heim et al., 2003), discarding the “bHLH.” Names are composed of a number, corresponding to the relative position resulting from searches for the bHLH domain, followed by the most common name as retrieved in the literature. Correspondences of sequence names with gene and protein identifiers from the corresponding genome browsers are shown in Table I and Supplemental Table S2.

Table I.
Summary of novel atypical sequences accepted and discarded as bHLHs

In recent years, novel atypical Arabidopsis bHLH proteins, most of them not identified as such in previous surveys, have been reported: At163KDR, At164PRE5, At165PAR1, and At166PAR2 (Hyun and Lee, 2006; Lee et al., 2006; Roig-Villanova et al., 2007). Another group of putative novel bHLH sequences were identified in microarray analysis as down-regulated in At165PAR1 constitutively overexpressing plants, designated in this work as P1R1 (for PAR1-RESPONSIVE1), P1R2, and P1R3 (corresponding to At167P1R1, At159P1R2, and At168P1R3, respectively). From the latter, only At159P1R2 had been previously classified as a member of the bHLH family. With the aim of identifying additional putative homologs to these novel bHLH proteins in different plant species, we implemented a BLAST-HMM (for hidden Markov model)-based combined search strategy.

BLAST searches were performed using the novel atypical Arabidopsis bHLHs as queries. In each case, a large number of hits were obtained, mostly corresponding to proteins annotated as containing the bHLH domain. However, among the best-scoring matches, 19 Arabidopsis, poplar, and rice sequences not previously annotated as bHLHs were also retrieved. These sequences were subsequently aligned, and the resulting alignments were used as a seed to generate HMM profiles. The HMM profiles were in turn used as queries in searches against selected plant proteome databases, resulting in the identification of eight additional matches (Table I).

The 27 putative novel bHLH sequences were combined with the previous estimates of bHLH families, resulting in a primary data set of 650 amino acid sequences putatively corresponding to bHLH domains. On the basis of the corresponding alignment, a consensus motif composed of the 25 most conserved positions was obtained, 11 of them corresponding to key functional residues also conserved in a consensus previously defined from animal bHLHs (Atchley et al., 1999; Fig. 1). Some positions specific to the plant bHLH consensus were occupied by highly conserved amino acids, including R16 at the basic region and P32 at the end of the helix 1 region. Furthermore, amino acid frequencies at some of the positions common to both plant and animal bHLH consensus were sharply different (Supplemental Table S1). These differences underlie the early divergence between animal and plant bHLHs.

Figure 1.
Plant and animal bHLH consensus. Alignment of the plant and animal bHLH consensus used as predictive motifs. The plant consensus is based on an alignment of plant bHLHs and contains positions conserved in more than 50% of the sequences. In such positions, ...

To confirm our data set of amino acid sequences as bHLHs, we examined the fit of every sequence to both consensus motifs by counting the number of matches at each region of the predicted bHLH domain (Supplemental Table S2). In previous works, sequences with more than eight to 10 mismatches from the animal bHLH consensus motif were discarded (Buck and Atchley, 2003; Heim et al., 2003; Toledo-Ortiz et al., 2003; Li et al., 2006b). To ensure that atypical bHLH domains were not eliminated by lack of correspondence to the consensus, we used a low stringent criterion by allowing 10 and 13 mismatches from the animal and plant bHLH consensus, respectively.

From the whole data set of putative bHLH sequences, only 13 sequences did not match any bHLH consensus and were eliminated from further analysis (Table I). This criterion was relaxed for At168P1R3, identified in our phylogenetic analysis as a recent paralog of At169. The remaining 638 sequences, representing an updated classification of bHLH families in the species examined in this study, are shown in Supplemental Table S2.

Dimerization Activities of Atypical bHLH Proteins

As a way to evaluate the accuracy of our searches for atypical bHLHs, we tested dimerization activity of AtPAR1 by performing yeast two-hybrid assays. As shown in Figure 2A, the GAL4 activation domain (AD) fused to AtPAR1 interacts strongly with the GAL4 binding domain (BD) fused to AtPAR1, revealing the ability of AtPAR1 to specifically interact with itself. Therefore, we conclude that AtPAR1 has retained protein interaction activity. Together with previous results demonstrating that nuclear localization is required for AtPAR1 function as a direct transcriptional repressor of specific targets (Roig-Villanova et al., 2007), it supports our analyses including it as an actual bHLH.

Figure 2.
Yeast two-hybrid analysis of AtPAR1 protein interaction activities. A, Homodimerization activity of wild-type AtPAR1. B, Homodimerization activity of two mutated versions of AtPAR1, L1mut (Leu-27Glu) and L2mut (Leu-73Lys). SD-LT refers to the selective ...

Conserved hydrophobic residues in the HLH region of the animal bHLH domain presumably define protein interaction activities (Massari and Murre, 2000). Leu-27 and Leu-73 of helix 1 and 2, respectively, have been identified as the most highly conserved residues across plant bHLHs (Fig. 1; Supplemental Table S1). Furthermore, most of the amino acid changes in these positions were conservative (Supplemental Fig. S1). To test whether these residues played a role in dimerization activities of plant atypical bHLHs, two mutated versions of AtPAR1, PAR1-L1mut (Leu-27Glu) and PAR1-L2mut (Leu-73Lys), were generated. When PAR1-L1mut and PAR1-L2mut were fused to the AD and tested against wild-type BD-PAR1, yeast growth was clearly affected, indicating that the interaction was greatly reduced (PAR1-L2mut) or completely abolished (PAR1-L1mut; Fig. 2B).

Phylogenetic Analysis of Plant bHLHs

To examine the evolutionary relationships among plant bHLH proteins, a maximum likelihood (ML) phylogenetic analysis based on the alignment of the corresponding bHLH domains (Supplemental Fig. S1) was carried out. The 638 plant bHLH proteins could be classified into 32 subfamilies identified as clades with high support values (Fig. 3; Supplemental Fig. S2). A summary of information of bHLH proteins grouped into their respective subfamilies is shown in Supplemental Table S3. Our analysis was robust to the alignment method employed, as almost every sequence clustered similarly in MUSCLE and MAFFT-based analysis (data not shown). Furthermore, tree topology resulting from neighbor joining (NJ) and maximum parsimony (MP) analyses was essentially the same, most of the subfamilies being retrieved (Supplemental Fig. S2). Most plant bHLH subfamilies identified in a recent survey (Pires and Dolan, 2010) were also detected in our analysis. Newly identified atypical bHLHs either formed new subfamilies (subfamilies 18–22) or grouped within previously defined subfamilies (subfamily 16).

Figure 3.
Phylogenetic relationships, intron pattern, DNA-binding motifs, and architecture of conserved protein motifs in 32 plant bHLH subfamilies. A, ML tree of 638 plant bHLH proteins (for the full representation of the tree, see Supplemental Fig. S2). The tree ...

We found 18 sequences that were not members of any of the identified subfamilies or showed ambiguous clustering between different phylogenetic trees. In an attempt to solve their evolutionary relationships with defined plant bHLH subfamilies, a Bayesian analysis (BA) was performed on a restricted data set of the original alignment, which also included representatives from the 32 subfamilies. From the resulting tree, three additional sequences were classified within many other subfamilies (Supplemental Fig. S3). The remaining 15 sequences were considered as orphans, most likely representing highly diverged lineage-specific bHLH sequences or our phylogenetic analysis could not resolve their evolutionary relationships. As typically observed in bHLH protein phylogenies, deep nodes, those determining interclade relationships, commonly showed low statistical support and varied between different phylogenetic methods, likely reflecting the large number of sequences being examined, the high divergence of the motif combined with its short length, and the occurrence of many ancient paralogs (Atchley and Fitch, 1997). Although beyond the scope of this work, the BA tree also provided some preliminary insights into the deep evolutionary history of the plant bHLH domain.

Our phylogenetic analysis permitted the estimation of the number of ancestral bHLH genes in the most recent common ancestors (MRCA) of plants (Nam et al., 2004). For instance, assuming that shared clades, composed of ortholog sequences from the four land plant species examined, are descendants of an ancestral bHLH gene, we obtained a minimum estimate of 14 bHLH genes in the hypothetical MRCA of land plants (Fig. 4). However, this number could represent an underestimate if we assume that the four additional subfamilies including moss representatives, as well as the 13 orphan genes found in land plants, represent divergent members of additional ancestral families lost in specific plant lineages. Assuming the latter, we obtained a maximum estimate of 31 bHLH genes in the MRCA of land plants (Fig. 4). The actual number of bHLH genes will range between these two values and will be dependent on the prevalence of gene duplication or loss in specific evolutionary lineages. A similar approach was performed to get estimates of the number of genes in the MRCA of eudicots and monocots as well as of eurosids I and eurosids II (Fig. 4). Interestingly, we found chlorophyte representatives in subfamilies 4 and 14, likely representing the descendants of ancestral green plant bHLH genes. Cr7 also tends to cluster at the base of subfamily 4 (Supplemental Figs. S2 and S3), although with lower support. The rest of chlorophyte bHLHs clustered in subfamily 32 at the base of the tree. The single representative from C. merolae did not group into any of the subfamilies, suggesting that plant bHLH subfamilies evolved after divergence of red algae from other photosynthetic eukaryotes 1.5 billion years ago (Yoon et al., 2004).

Figure 4.
Evolution of bHLH gene family size in plants. Estimates of bHLH gene family size in the MRCA of examined plant species are represented at the corresponding nodes of a tree depicting their evolutionary relationships. Numbers correspond to minimum and maximum ...

Sequence and Structural Analysis Provide Further Support to Plant bHLH Subfamilies Definition

Intron/Exon Structure within the bHLH Domains

We analyzed the intron pattern, including intron distribution, positions, and phases over genomic regions encoding for the bHLH domains. Approximately 20% of bHLH genes had no introns at the bHLH coding region (Fig. 5, pattern k). The rest of the genes had up to three introns that, according to relative positions and phases, could be arranged into 21 different splicing patterns. Patterns a to g, composed of one to three introns distributed at three highly conserved specific positions, accounted for approximately 72% of bHLH genes. As previously observed in Arabidopsis and rice bHLH genes (Toledo-Ortiz et al., 2003; Li et al., 2006b), patterns a and f were found to be the most common ones also in poplar and moss bHLH genes but were not found in algae (Fig. 5). The remaining bHLH genes have introns at positions different from the rest of the family, forming patterns h to l as well as nine additional patterns exclusive of single bHLH genes. It is interesting that intron pattern distribution was almost absolutely conserved within most subfamilies, providing an independent criterion for testing the reliability of our phylogenetic analysis (Fig. 3B). An interesting exception is provided by the green plant ancestral subfamily 4, which clustered representatives of intron patterns i and j.

Figure 5.
Intron patterns within the bHLH domains. Alignment of bHLH domains representative of 11 intron patterns, named from a to l. The ? indicates nine additional gene-specific intron patterns. Locations of introns are indicated by triangles, and the number ...

Figure 5 also shows, in each case, the position of splicing with respect to codon (i.e. the intron phase). An intron was designated as occurring in one of three phases, phase 0, 1, or 2, depending on whether the splicing occurred between codons, after the first nucleotide, or after the second nucleotide of the codon, respectively. Among the 887 introns analyzed here, a great majority (840) had phase 0, whereas only 15 and 32 had phases 1 and 2, respectively. Among phase 0 introns, we found all introns from patterns a to g.

Predicted DNA-Binding Properties

By examining the amino acid sequence at the basic region of the bHLH domain, plant bHLH proteins could be classified into different DNA-binding groups. The distribution of the different predicted DNA-binding categories was represented across the bHLH phylogenetic tree, revealing that most subfamilies share predicted DNA-binding properties (Fig. 3B).

bHLH domains with at least five basic amino acids at the basic region are expected to bind DNA (Massari and Murre, 2000). A larger group composed of 471 plant bHLH proteins was found to fit this criterion (Table II). DNA-binder bHLHs can be further subdivided into additional DNA-binding categories. According to three-dimensional structural analysis of bHLH proteins, Glu-13 and Arg-16 have been reported to be essential in E-box-binding recognition (Fig. 1; Ferre-D'Amare et al., 1994; Shimizu et al., 1997). This E-box-binding recognition motif has been identified in 359 plant bHLH proteins. Seven more sequences have the conservative amino acid change Arg-16Lys, which has been shown not to interfere with E-box binding (Hua et al., 1993). Moreover, three additional residues at the basic region, His/Lys-9, Glu-13, and Arg-17, provide DNA-binding specificity for a specific type of E-box, the so-called G-box (Ferre-D'Amare et al., 1994; Shimizu et al., 1997). Eighty-six of the 366 E-box DNA-binder bHLHs lacked the G-box recognition motif, the rest (280) being classified as G-box DNA binders (Table II). The remaining 105 bHLHs, lacking residues defining E-box-binding recognition specificities but having more than five basic amino acids at the basic region, were classified as non E-box DNA binders.

Table II.
Classification of plant bHLHs according to the presence of DNA-binding motifs in the basic region of the bHLH domain

A total of 167 out of 638 plant bHLH proteins lacked a basic region and were tentatively predicted to be non DNA binders. However, a subset of these sequences displayed the E-box-binding (seven) and G-box-binding (66) recognition motifs and, in some cases, grouped within subfamilies mostly composed of DNA-binder bHLHs (Fig. 3B). It remains to be determined whether these sequences have retained DNA-binding activity in spite of their low basic region.

Some bHLH sequences displayed a significantly higher frequency of specific amino acids. For instance, subfamily 23 grouped several non E-binder bHLH sequences displaying up to four Pro residues at the basic region (Supplemental Table S3). The presence of Pro residues in the basic region has been claimed to indicate a differential positioning with respect to the DNA as a result of modified folding (Toledo-Ortiz et al., 2003). Moreover, in most non DNA-binder bHLHs, basic residues at the basic region have been replaced by specific amino acids such as Ser (e.g. subfamilies 16 and 17), Gly (e.g. subfamily 22), or even acidic amino acids (e.g. subfamily 21). The functional significance of such specific amino acid replacements at the basic region is yet unknown.

Architecture of Conserved Protein Motifs

A search for conserved motifs in plant bHLH proteins identified 50 motifs of variable length (8–80 amino acids; Supplemental Table S4). In most cases, protein architecture is remarkably conserved within specific subfamilies, giving further support to the phylogenetic analysis based on bHLH domains (Fig. 3C).

Motifs 1 and 2 were identified as the helix 2 and helix 1 regions of the bHLH domain, respectively, in almost every bHLH protein sequence analyzed. The basic and loop regions of the bHLH domain appear to be less conserved; consequently, no single motif was detected matching these regions across plant bHLHs. By contrast, some specific motifs were identified as matching the basic and loop regions of specific subfamilies (Fig. 3C).

Outside the bHLH domain, some subfamily-specific motifs had been previously characterized as defining additional functional properties. For instance, motif 9, observed in all members of subfamily 4, was unambiguously identified as a ZIP dimerization domain. Motif 6, shared by members of subfamily 23, has been characterized in AtLHW as necessary for homodimerization (Ohashi-Ito and Bergmann, 2007). Motif 14, identified in subfamilies 2, 5, and 23, corresponds to a motif previously identified in AtMYC3/ATR2. A conserved Asp residue in this region has been reported to be functionally important for correct expression of several downstream genes acting in the Trp biosynthesis pathway (Smolen et al., 2002). Motif 44, conserved among phytochrome-interacting members of subfamily 24, has been characterized as providing a phytochrome B-specific recognition module (Khanna et al., 2004). Finally, motifs 7, 4, and 19 have been reported to form the highly conserved C-terminal domain of AtSPCH, AtMUTE, and AtFMA, grouped within subfamily 10. Although the biological role of this domain is still uncertain, its overexpression leads to a weak partial reversion of the fama mutant stomata phenotype (Pillitteri et al., 2007).

To gain further insights into the origin and mode of evolution of bHLH motifs, we examined their distribution across species as well as their spatial locations across bHLH proteins. Most conserved motifs were already present in the ancestor of land plants, as all but motif 6 were identified in moss bHLH proteins. Apart from motifs 1 and 2, only four conserved motifs were also found in Cm1, while a total of 19 out of 50 motifs were detected in chlorophyte bHLH proteins. The ZIP motif (9) was the only one to have been found outside plants. However, no similarities were found between subfamily 30 of plant bHLH-ZIP proteins and animal bHLH-ZIP proteins, and previous works supported the independent acquisition of the motif multiple times during plant and animal evolution (Atchley and Fitch, 1997; Morgenstern and Atchley, 1999; Pires and Dolan, 2010). The bHLH domain itself provides an interesting example of variation in the relative spatial location. In specific subfamilies, the bHLH domain is located at the NH2-terminal, middle, or COOH-terminal region of the protein (Fig. 3C). In addition, motifs 12, 22, 26, 35, 39, 45, and 48 also showed spatial variation relative to the bHLH domain.

DISCUSSION

Comparative studies of bHLH gene numbers in different plant species show a gradual increase in the number of bHLHs from algae to flowering plants (Fig. 4), which correlates with increasing organism complexity (Richardt et al., 2007). Although the loss of ancestral bHLH genes in specific lineages cannot be ruled out, it is unlikely that gene loss explains the observed pattern. Our results, more likely, support evolutionary diversification of the bHLH family through extensive expansion at key milestones during plant evolution, a pattern similar to that observed in animal bHLHs (Amoutzias et al., 2004; Simionato et al., 2007).

According to our analysis, two subfamilies (4 and 14) might configure the set of bHLH transcriptional regulatory networks ancestral to the green plant lineage. However, the most important expansion in the bHLH family occurred after the split between green algae and land plant species. This led to the establishment of most of the diversity of DNA-binding motifs, intron patterns, and protein motifs of plant bHLH proteins and probably reflects the transition from aquatic to terrestrial habitats. A similar evolutionary scenario has also been postulated in a recent analysis (Pires and Dolan, 2010). Other studies conclude that a first evolutionary expansion of the bHLH complement in metazoans and plants might have been related to the acquisition of multicellularity (Ledent and Vervoort, 2001) or even earlier (Simionato et al., 2007). At least in certain green algae lineages, evolutionary expansion may have preceded multicellularity, as revealed by the seven bHLH genes found in the single-celled C. reinhardtii.

A second significant expansion was observed after the split between moss and vascular plants, as reflected in the 12 angiosperm-specific subfamilies and the greater size of 12 land plant ancestral bHLH subfamilies in angiosperms (Fig. 3B). This expansion might reflect the more complex body plan and specialization of vascular and flowering plants (Richardt et al., 2007).

Our results support birth-and-death evolution through repeated gene duplication and eventual loss driving plant bHLH evolutionary expansion and diversification (Nei and Rooney, 2005; Zhang et al., 2008). Signatures of birth-and-death evolution are observed at both the sequence and genomic levels. At the sequence level, this would translate into bHLH sequences showing similar or higher between-species divergence. To examine whether this was the case for the plant bHLH family, we estimated sequence divergence at the amino acid level for the four land plant species and C. reinhardtii. As expected, differences in sequence divergence appear not to be significant in any comparison at the within-species level and are slightly increased in between-species comparisons with C. reinhardtii (Supplemental Table S5). Previous studies on the genome distribution of Arabidopsis and rice bHLH genes supported a prominent role for genome segments and tandem duplication in the expansion of this gene family (Heim et al., 2003; Toledo-Ortiz et al., 2003; Li et al., 2006b). Similarly, recurrent events of single-gene duplication have been inferred to drive animal bHLH diversification (Amoutzias et al., 2004). Some duplicated genes will accumulate mutations as a pseudogene and gradually lose their function. We have identified several truncated and apparently nonexpressed bHLH genes in poplar and moss genomes, likely corresponding to pseudogenes (data not shown), which had been identified in a previous survey also in Arabidopsis and rice (Li et al., 2006b). More interestingly, some other duplicated genes remain in the genomes as differentiated functionally specialized genes, providing a source to generate evolutionary novelty in the form of new regulatory functions (Nam et al., 2004; Nei and Rooney, 2005).

Regulatory roles of bHLHs are essentially based on the recognition of a specific hexanucleotide sequence core at the promoter of target genes (Martinez-Garcia et al., 2000; Massari and Murre, 2000). A prominent role has been attributed to key residues at the basic region in discriminating between variants of this hexanucleotide core motif, allowing the classification of plant bHLHs into DNA-binding categories. None of these DNA-binding categories formed monophyletic groups, supporting the independent acquisition of specific DNA-binding properties at different times during plant bHLH gene family evolution (Fig. 3). Moreover, a role for specific amino acids outside the basic region in conferring additional DNA-binding specificity through elements that lie outside of the hexanucleotide core recognition motif cannot be ruled out. Studies of the Drosophila melanogaster bHLH transcription factor Deadpan have led to the identification of a single Lys residue at the loop region whose replacement severely reduces DNA-binding affinity (Nair and Burley, 2000; Winston et al., 2000). A similar role might be inferred in plant bHLHs, as this residue has been identified as highly conserved in 77.4% of the sequences (position 46; Fig. 1). A second position of the loop (position 56; Fig. 1) has also been found to be particularly conserved, being occupied by an Asp residue in approximately 65.5% of plant bHLHs.

Most of the novel bHLH genes and subfamilies identified, and classified by our analysis, correspond to atypical bHLHs, in which basic residues at the basic region are commonly replaced by nonbasic amino acids and are consequently predicted to lack DNA-binding activity (Supplemental Table S3). AtKDR (subfamily 16) and AtPAR1 and AtPAR2 (subfamily 21) constitute the first characterized plant bHLH proteins predicted to be non DNA binders. AtKDR has been reported to negatively regulate AtHFR1 (Hyun and Lee, 2006), a bHLH protein that had been previously reported to function as a branching point of phytochrome-dependent signaling responses (Fairchild et al., 2000). Later molecular and overexpression studies suggested that AtKDR, together with a set of five additional, closely related homologs (AtPRE1–AtPRE5), could play a role in GA-dependent responses (Lee et al., 2006). AtPAR1 and AtPAR2 act as direct transcriptional repressors of specific targets during shade-avoidance responses, including atypical bHLHs (AtP1R1–AtP1R3) and specific auxin-responsive genes (Roig-Villanova et al., 2007). Plant atypical bHLHs would act as negative regulators of DNA-binding bHLHs by forming heterodimers, as reported for ID proteins from group D of animal bHLHs. Consistently, AtKDR has been shown to heterodimerize with AtHFR1 (Hyun and Lee, 2006), and the AtPAR1 HLH domain has retained protein interaction function (Fig. 2A). However, no sequence similarity has been found between this group of plant bHLHs and group D of animal bHLHs. Plant atypical bHLHs emerge as a group of transcriptional regulators playing regulatory roles in plant-specific biological processes, notably, by integrating phytochrome- and hormone-dependent signaling pathways.

Plant bHLH proteins have been reported to dimerize with a wide and diverse range of transcriptional regulators, including members of the bHLH family (Toledo-Ortiz et al., 2003), other transcription factors, such as R2R3-MYBs (Goff et al., 1992; Dubos et al., 2008), BZR1-BES1 (Yin et al., 2005), or AP2s (Chandler et al., 2009), signal transduction proteins, such as WD40 repeat proteins (Ramsay and Glover, 2005), and epigenetic regulators of gene expression (Thorstensen et al., 2008). Dimerization activities of bHLH proteins allow expanding regulatory roles of bHLH proteins by defining additional protein interaction and DNA-binding specificities (Massari and Murre, 2000; Toledo-Ortiz et al., 2003). The HLH region of the bHLH domain is responsible for the dimerization activities of bHLH proteins. However, little is known about how the specificity of this interaction is defined. Three-dimensional structural analysis of the mammalian Max protein together with site-directed mutagenesis experiments on human E47 and E12 characterized two conserved Leu residues at the helix 1 and 2 regions, respectively, as essential for dimerization (Voronova and Baltimore, 1990; Ferre-D'Amare et al., 1993). Both Leu residues have been identified as the most conserved residues across plant bHLHs (positions 27 and 73; Fig. 1). Such an essential role in dimerization activity would also be conserved in plant bHLHs, as revealed by yeast two-hybrid protein interaction assays using two mutated versions at these positions of the highly diverged AtPAR1 protein (Fig. 2B).

We observed an excess of phase 0 introns and of symmetric exons within the bHLH domain (Fig. 5). This provides an interesting mechanism to explain the exchange of protein motifs, facilitating exon shuffling by avoiding interruptions of the open reading frame. Introns would be inserted (or eventually excised) from the bHLH coding region in a subfamily-specific manner, in accordance with previous results showing that numerous introns have been specifically inserted into plants and retained in the genome (Rogozin et al., 2003). The scattered distribution through the bHLH phylogeny of pattern k, lacking introns, together with its occurrence in bHLH sequences from algae species might be indicative of its ancestral nature, consistent with this model.

Most plant bHLH proteins are multidomain proteins composed of a set of conserved motifs already present in the MRCA of land plants. Many motifs consist of short conserved sequences arranged following a mosaic pattern (Fig. 3C). This arrangement might be mostly explained by modular evolution with domain shuffling, as suggested in animal bHLHs (Morgenstern and Atchley, 1999). Shuffling of functional domains among bHLH proteins, including specific regions of the bHLH domain, would promote further functional diversification in specific lineages.

One might anticipate that ortholog bHLH proteins closely clustering in a subfamily and sharing similar intron/exon organization, the architecture of protein motifs, predicted DNA-binding motifs, and additional sequence features should have recent common evolutionary origins and consequently related molecular and biological functions. However, the extent of functional diversification within specific subfamilies is variable, ranging from functional redundancy to members displaying highly diverged specialized functions (Table III).

Table III.
Summary of functionally characterized bHLHs from plant species examined in this study classified by bHLH subfamilies

Such apparent functional redundancy is observed in subfamilies 14 and 25, clustering AtBIM and AtBEE genes, respectively, involved in brassinosteroid signaling (Friedrichsen et al., 2002; Yin et al., 2005). Functional specialization may be observed in other plant bHLH subfamilies. AtSPCH, AtMUTE, and AtFMA, members of subfamily 10, have been characterized to control stomatal development at three consecutive steps: initiation, meristemoid differentiation, and guard cell morphogenesis, respectively (Pillitteri et al., 2007). The corresponding rice orthologs of subfamily 10 also provide an interesting example of functional conservation (Liu et al., 2009). An outstanding example of functional diversification is encountered in subfamily 1, which clusters nine plant bHLH genes involved in very diverse biological roles (Table III; Chinnusamy et al., 2003; Sorensen et al., 2003; Jakoby et al., 2004; Kiribuchi et al., 2004; Li et al., 2006a; Zhang et al., 2006; Kanaoka et al., 2008).

We found moss orthologs of bHLHs related to biological processes specific to vascular and flowering plants. The above-mentioned AtBIM and AtBEE genes provide a first example. Brassinosteroids play a key role in the differentiation of vascular tissues (xylem and phloem; Cano-Delgado et al., 2004). Consistently, nonvascular moss is devoid of brassinosteroid biosynthetic and signaling pathway genes (Rensing et al., 2008). Interestingly, subfamily 14 also grouped consistently chlorophyte representatives Cr1 and Vc3. Several moss bHLH orthologs also showed clustering in subfamily 24, whose members have been reported for their role in phytochrome-dependent photomorphogenic responses that appeared later in the evolutionary lineage of vascular plants, such as shade avoidance and seed germination (Ni et al., 1998; Huq and Quail, 2002; Yamashino et al., 2003; Oh et al., 2004). Arabidopsis AtHEC genes, also grouping within subfamily 24, have been shown to work in concert with AtSPT to coordinately regulate development of the female reproductive tract, probably in an auxin-dependent manner (Gremski et al., 2007). An interesting question for future research will be to investigate whether moss (and algae) bHLH orthologs, within these subfamilies, have retained the ancestral function or have evolved new functions. Studies on AtRHD6 and AtRSL1 (subfamily 28), which control root hair development, provide a first insight into this question. Interestingly, the corresponding orthologs PpRSL1 and PpRSL2 in moss also control the development of nonhomologous organs with a rooting function (Menand et al., 2007).

Some other bHLHs functionally characterized belong to angiosperm-specific subfamilies subjected to lineage-specific expansions, which may reflect species-specific adaptations. Subfamilies 12 and 15 provide examples of dicot- and monocot-specific expansion, respectively. Subfamily 12 clusters AtORG2 and AtORG3, regulated by iron ion deficiency-mediated stress and the phytohormones salicylic acid and jasmonic acid (Kang et al., 2003). Subfamily 15 includes AtERP, which is involved in GA signaling acting downstream of DELLA proteins (Zentella et al., 2007), conserved growth repressors that modulate GA responses. Furthermore, subfamily 23 clusters together six poplar sequences but only three Arabidopsis and rice homologs. Subfamily 23 is represented by AtLHW, which is involved in the regulation of the Arabidopsis root vascular initial population (Ohashi-Ito and Bergmann, 2007). Similar poplar-specific significant expansion has been found in the MADS box subfamily clustering AtANR1, which is also known to be involved in root development (Zhang and Forde, 1998; Leseberg et al., 2006), and in the R2R3-MYB C1 subfamily, whose members showed particularly abundant expression in roots (Wilkins et al., 2009).

Twelve out of the 32 bHLH subfamilies defined here lack any functionally characterized member. Some subfamilies might regulate biological roles essential for land plant development, as they conform to big subfamilies, including representatives from the four land plant species (e.g. subfamilies 9 and 27), or are specific to angiosperms (e.g. subfamilies 7, 18, 19, and 20). We expect the comprehensive classification and evolutionary analysis of plant bHLHs presented here to provide a useful framework to ortholog identification. This is a first step to infer the role of newly identified plant bHLH proteins in the transcriptional regulation of growth and development processes as well as toward further investigations into the role of the bHLH family in plant phenotypic diversification.

MATERIALS AND METHODS

Plant bHLH Sequence Identification and Analysis

Putative novel bHLH sequences were identified using BLAST (Altschul et al., 1997) and profile HMMs (Durbin et al., 1988), generated and calibrated with HMMER software version 2.3.3. Local searches were performed through the proteomes of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), poplar (Populus trichocarpa), and moss (Physcomitrella patens), downloaded from The Arabidopsis Information Resource, The Institute for Genomic Research Rice Genome Annotation, Joint Genome Institute (JGI) Ptri version 1.1, and JGI Ppatens version 2.0 browsers, respectively. Similar searches were performed on the whole sequenced genomes of Volvox carteri, Chlamydomonas reinhardtii, Ostreococcus tauri, and Ostreococcus lucimarinus (JGI Volca version 1.0, JGI Chlre version 4.0, JGI Ostta version 2.0, and JGI Ostlu version 2.0, respectively), as well as Cyanidioschyzon merolae (http://merolae.biol.s.u-tokyo.ac.jp/). Only hits returning E-values of less than 0.001 were considered for further analysis. Redundant sequences were identified through BLASTCLUST from the BLAST stand-alone package and subsequently discarded.

The bHLH sequences were aligned using the ClustalW, MUSCLE version 5.0, and MAFFT 6.0 (FFT-NS-2 algorithm) programs (Thompson et al., 1997; Katoh et al., 2002; Edgar, 2004), and the resulting alignments were subsequently manually edited using GENEDOC 2.6.002. Limits of the bHLH domains were taken according to the proposed predictive consensus motif (Atchley et al., 1999), constructed referring to the structure of the human MAX bHLH protein (Ferre-D'Amare et al., 1993), and further corrected for predicted plant-specific bHLH domain boundaries (Toledo-Ortiz et al., 2003; Roig-Villanova et al., 2007).

The MEME version 3.5.7 tool was used to identify conserved motifs shared among bHLH proteins (Bailey and Elkan, 1994; Bailey et al., 2006). The following parameter settings were used: maximum number of different motifs to find, 50; optimum motif width, 8 to 100. Subsequently, the MAST program was used to search detected motifs in protein databases (Bailey and Gribskov, 1998). The motifs were further scanned against different domain databases, including the National Center for Biotechnology Information's Conserved Domain Database, INTERPRO, and PROSITE (Apweiler et al., 2001).

Exon/intron location, distribution, and phases at the genomic sequences encoding for the bHLH domain were examined through comparisons with the predicted encoded protein using GENEWISE (Birney et al., 2004).

Phylogenetic Analysis

Reconstruction of evolutionary relationships was performed on the basis of amino acid sequences of bHLH proteins. Only the bHLH domain was used, because the flanking sequences of bHLH proteins from independent subfamilies are either nonhomologous or too divergent to be reliably aligned. bHLH sequences from the different species examined were added sequentially to the analysis, and the resulting trees were compared with previous classifications (Bailey et al., 2003; Buck and Atchley, 2003; Heim et al., 2003; Toledo-Ortiz et al., 2003; Li et al., 2006b; Pires and Dolan, 2010).

The Jones, Taylor, and Thorton (JTT) with an estimated proportion of the invariable sites (I) and an estimated γ-distribution parameter (G) was selected as the best-fitting amino acid substitution model with the Akaike information criterion implemented in ProtTest version 1.4 (Jones et al., 1992; Abascal et al., 2005). The ML analyses were performed using PHYML version 2.4.5 (Guindon and Gascuel, 2003), using the JTT+I+G model. Heterogeneity of amino acid substitution rates was corrected using a γ-distribution with eight categories. Tree topology searching was optimized using the subtree pruning and regrafting option. The statistical support of the retrieved topology was assessed using the Shimodaira-Hasegawa-like approximate likelihood ratio test and a bootstrap analysis with 100 replicates. NJ and MP analyses were implemented with MEGA 4.0 (Tamura et al., 2007). In NJ, distances were calculated using the JTT amino acid substitution model, the γ-distributed rate among sites, and the γ-parameter set as retrieved in ProtTest analysis. To deal with short insertions/deletions (commonly occurring throughout the loop region), “pairwise deletion” and “all sites” settings were used in NJ and MP analyses, respectively. A bootstrap analysis with 1,000 replicates was performed in each case.

BA was implemented in MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Searches were run with four Markov chains for 1 million generations and sampling every 100th tree. After stationary phase was reached (determined by independent runs sampling similar likelihood values after plotting against the number of generations), the first 100,000 trees were discarded as burn-in and a consensus tree was then constructed to evaluate clades with Bayesian posterior probabilities greater than 50%. The JTT model with rate heterogeneity across sites modeled as γ-distributed with eight categories and invariant sites was used.

Yeast Two-Hybrid Interaction Assays

The Matchmaker two-hybrid system (Clontech) was used to perform yeast two-hybrid assays. The full-length open reading frame of AtPAR1 was inserted in frame with the DNA BD and transcription AD fusion construct using the pGBKT7 and pGADT7 vectors, respectively. The NcoI-BamHI fragment of pACV9 (containing the entire coding sequence of AtPAR1; Roig-Villanova et al., 2007) was subcloned into the same sites of pGBKT7 and pGADT7, resulting in pCL3 (BD-PAR1) and pCL1 (AD-PAR1), respectively. L1mut was generated by PCR-based site-directed mutagenesis using the primers RO47 (5′-GATTGAGGCGGAGCAGAGGATTATCCCCGGAGGAG-3′) and RO48 (5′-GATAATCCTCTGCTCCGCCTCAATCTTTTCCTTGAC-3′). L2mut was similarly generated using the primers RO49 (5′-CATTCTGTCTAAACAATGTCAGATCAAAACCATTA-3′) and RO50 (5′-GATCTGACATTGTTTAGACAGAATGTAACCAGCTG-3′). In both cases, AtPAR1 was amplified from the binary vector pBF1 (P35S:AtPAR1-GFP, a pCAMBIA-1302-based binary vector containing full-length AtPAR1 flanked by NcoI and SpeI) using specific primers from the P35S and GFP coding sequences. Mutated L1mut and L2mut sequences were subcloned into pCRII-TOPO (Invitrogen) to generate pIR44 and pMR5, respectively. Site-directed mutations in these inserts were verified by sequencing. The NcoI-SpeI fragments of pIR44 and pMR5 were subcloned into the same sites of pGADT7, resulting in pCM7 (AD-PAR1-L1mut) and pCM8 (AD-PAR1-L2mut), respectively.

Yeast (AH109 strain) transformation was performed according to the manufacturer's instructions. Yeast cells were cotransformed with the different pairs of BD-AD constructs. Independent transformants were selected on minimal synthetic dropout medium (SD)-Leu-Trp (SD-LT). At least 10 independent colonies were transferred to SD-Ade-His-Leu-Trp to test for protein-protein positive interaction (SD-AHLT).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure S1. ClustalW amino acid sequence alignment of 638 Arabidopsis, poplar, rice, moss, and algae bHLH domains.
  • Supplemental Figure S2. ML phylogenetic tree of 638 plant bHLH proteins.
  • Supplemental Figure S3. BA phylogenetic tree of 50 plant bHLH proteins.
  • Supplemental Table S1. Plant and animal bHLH predictive consensus motifs.
  • Supplemental Table S2. Species classification of 638 plant bHLH sequences examined in this study.
  • Supplemental Table S3. Subfamily classification of 638 Arabidopsis, poplar, rice, moss, and algae bHLH sequences examined in this study and additional information.
  • Supplemental Table S4. Summary of conserved motifs identified by MEME in plant bHLHs.
  • Supplemental Table S5. Rates of sequence divergence at the amino acid level in Arabidopsis, poplar, rice, moss, and C. reinhardtii bHLH sequences.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank F. Paulet-Dubois for critical reading of the manuscript and all our laboratory members for stimulating discussions and suggestions. We also thank two anonymous referees for their insightful comments. This work has been carried out within the University of Almería, the University of Manchester, and the Centre CONSOLIDER for Research in Agricultural Genomics. Thanks also to the Apple Research and Technology Support scheme for support.

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