* Max-Planck-Institute for Plant Breeding Research, Cologne, Germany
Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich, United Kingdom
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Abstract |
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Key Words: bHLH Arabidopsis thaliana transcription control genomics
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Introduction |
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Typically, a bHLH domain comprises a stretch of about 18 hydrophilic and basic amino acids at the N-terminal end of the domain, followed by two regions of hydrophobic residues predicted to form amphipathic -helices separated by an intervening loop (Murre et al. 1994). Studies with mammalian bHLH proteins have shown that the conserved HLH structure is required for dimerization between two bHLH proteins (Ferré-D'Amaré et al. 1993, 1994; Ellenberger et al. 1994). The specificity for a particular partner protein resides in the
-helices, and for a sequence-specific interaction with DNA, the basic regions of both polypeptides are required. Although the bHLH domain is conserved between animals and plants, the dimeric structure of the DNA binding complex in plants has only been inferred from the conserved amino acid sequence. Some, although not all, plant bHLH proteins which have been studied so far have been shown to modulate gene expression by binding DNA.
Generally, eukaryotic transcription factors (TFs) consist of at least two discrete domainsa DNA binding domain and an activation or repression domainthat operate together to modulate the rate of transcriptional initiation from target gene promoters (Ptashne 1988). Domains other than the bHLH DNA binding domain are known to be important for the regulation of gene expression by bHLH proteins, and these regions may be conserved at the amino acid level in related proteins from different species. This has been shown for the E12 family of bHLH proteins in animals, which are a ubiquitously expressed class of animal TFs involved in the development of many cell lineages. E12 factors act via heterodimerization with other, tissue-specific bHLH proteins (Quong et al. 1993).
To date, few plant bHLH genes have been studied in detail, but those that have, have provided insights into the central roles of TFs in biology, and into their biochemical function. Genetic analysis of the anthocyanin biosynthetic pathway in Z. mays has identified a group of bHLH genes required for production of the purple anthocyanin pigments: R(R-s and R-p), B, Lc, Sn (Neuffer, Coe, and Wessler 1997, pp. 367369) and R-ch Hopi (Petroni et al. 2000). The encoded bHLH proteins interact with members of another group of TFs in Z. maysCl or P, both R2R3-MYB proteinsand together they control pigmentation in tissues wherever the two proteins are expressed together. Other members of the R gene family in Z. mays and other species are also believed to interact with MYB proteins to regulate anthocyanin production in a tissue-specific manner. The bHLH/MYB partnership has been shown to be important in the differentiation of A. thaliana trichomes through the interaction of a bHLH protein encoded by GLABRA3 (GL3) and the R2R3-MYB protein GLABROUS1 (GL1; Payne, Zhang, and Lloyd 2000). Phytochrome interacting factor 3 (PIF3) is a bHLH protein necessary for light signaling mediated by the photoreceptor phytochrome B (PhyB) in A. thaliana (Ni, Tepperman, and Quail 1998; Halliday et al. 1999). Recently it has been shown that PhyB in its active form interacts with DNA-bound PIF3, suggesting a mechanism for direct activation of photoresponsive genes by a light signal (Martinez-Garcia, Huq, and Quail 2000).
Within the A. thaliana genome (The Arabidopsis Genome Initiative 2000), 32 families of genes have been identified as encoding TFs that contain three or more members (Riechmann and Ratcliffe 2000). Each family is characterized by a unique region of highly conserved amino acid sequence, which usually comprises the DNA binding domain. From comparative analysis of these conserved domains, it is apparent that within each gene family there are subgroups of genes that are structurally more closely related to each other than to other members of the family. Regions of TFs outside the DNA binding domain often contain short amino acid sequence motifs or domains that are also conserved. The prediction is that proteins within a subgroup that share these smaller motifs will share similar (but not necessarily identical) functions. Where information on function is available, different members of a subgroup of genes tend to share very similar or related functions, even though the biological functions of the particular TF family as a whole may be very broad (for example the AP2 family [Riechmann and Meyerowitz 1998], the R2R3-MYB family [Stracke, Werber, and Weisshaar 2001], or the MADS-box family [Becker et al. 2000]). Therefore an assessment of the structural relationships between all A. thaliana bHLH genes should provide a guide for prediction of gene function and for elucidation of the range of activities carried out by members of the bHLH gene family. At least for related groups of animal genes, the amino acid sequence of the bHLH motif has retained sufficient information to identify evolutionary relationships (Atchley and Fitch 1997). In this article, structural analysis is presented for all A. thaliana genes encoding proteins with a bHLH motif. Gene and protein sequence prediction is based on sequence information from newly isolated cDNAs. The results of the structural relationships between the proteins are discussed in the context of the functional diversity of this family of TFs in plants.
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Materials and Methods |
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The exon-intron structure prediction for each gene was improved by using a combination of gene-modeling programs (GeneMark: opal.biology.gatech.edu/GeneMark; GeneScan: genome.dkfz-heidelberg.de/cgi-bin/GENSCAN/genscan.cgi) and comparison with available ESTs. Where ESTs were not available, the predicted protein and the genes most closely related to it were aligned and the gene model was altered manually if there was evidence for greater similarity in gene structure than predicted. In some cases, small exons in the basic region were missing in the existing predictions (e.g., AtbHLH16).
Isolation and Sequencing of cDNAs for the A. thaliana bHLH TFs
To verify the ORFs for each bHLH gene, and to confirm expression, cDNAs corresponding to the majority of genes were isolated using the Rapid Amplification of cDNA Ends (RACE) protocol (Frohman, Dush, and Martin 1988). If available, cDNAs encoding bHLH proteins identified in EST collections were sequenced. All sequencing was on both strands. DNA sequences were determined by the MPIZ DNA core facility on Applied Biosystems (Weiterstadt, Germany) Abi Prism 377 and 3100 sequencers using BigDye-terminator chemistry. Premixed reagents were from Applied Biosystems. Oligonucleotide primers were purchased from Metabion (Martinsried, Germany) or Invitrogen (Carlsbad, Calif.).
Expression Analysis
Total RNA was isolated from cultured A. thaliana cells (At7; Trezzini, Horrichs, and Somssich 1993) that were submitted to eight different treatments: untreated, incubation for 6 h at 4°C in the dark, incubation at 37°C in the dark, incubation under UV light at 28°C, and incubation with 10 µM 1-aminocyclopropan-1-carboxylic acid at 28°C. To simulate a stress response to pathogen attack, cell cultures were also incubated with 1 µM Flagellin22 at 28°C, with 10 µM methyl-jasmonate for 40 min at 28°C or 10 µM salicylic acid for 40 min at 28°C. In addition, RNA was isolated from roots, leaves, stems, and flowers and siliques of 6- to 8-week-old plants grown in the greenhouse under long day conditions. Using CDSIII-NotI primer (ATTCTAGAGGCCGAGGCGGCCGCCATG(T30)VN), 5 µg of each RNA was reverse transcribed in a 20 µl reaction with Superscript II RT polymerase (Invitrogen). After addition of 100 µl water, 1 µl of the diluted reaction was used for a first round of polymerase chain reaction (PCR) with a gene-specific primer, and CDSIII-NotI. PCR was carried out in a 96-well format, where 8 different genes could be probed against the array of 12 cDNA samples. After a first denaturation step of 2 min at 94°C and 30 cycles of 20 s at 94°C, 20 s at 60°C, and 2 min at 72°C, an aliquot of 1 µl of each reaction was transferred to a new 96-well plate and subjected to a second round of PCR with the same conditions as in round one, except that a second, nested, gene-specific primer was included. Resulting PCR fragments were analyzed on gels, and bands of the expected size were extracted and subsequently inserted into pTOPO (Invitrogen). Two to six clones were sequenced for each gene.
Clustering of bHLH Genes into Groups and Subgroups
For each gene, only the amino acid sequence from the bHLH domain was used for creating a manually edited alignment for sequence comparison with the ClustalW (Thompson, Higgins, and Gibson 1994) program. A similarity tree was constructed (Neighbor-Joining algorithm) followed by bootstrap analysis using PHYLIP (Felsenstein 1993). Based on the sequence information, 500 trees were calculated and all branches that appeared in more than 250 trees were used to calculate the final clustering. In addition, we used the MEME analysis tool (Bailey and Elkan 1994) to search for motifs shared by the AtbHLH proteins.
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Results and Discussion |
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The predicted gene structure of 87 bHLH genes was verified by performing 3' RACE-PCR on 12 different cDNA sources. The results highlighted a number of incorrectly annotated ORFs (37% of all annotated bHLH genes) that had been predicted by the A. thaliana genome annotation. Forty-seven entries were corrected using our RACE-PCR results, including AtbHLH125 (At1g62975), which was newly annotated; this genome region was originally described as a noncoding repeat-rich region. These new cDNA accessions have been deposited at GenBank with the assigned AtbHLHxyz gene name and the new ORF structure has been submitted to MAtDB at the Munich Institute for Protein Sequences (MIPS). The results of the RT-PCR experiment also provided a preliminary impression of the expression profile of these genes (fig. 3). In total, at least 113 bHLH genes can be confirmed as being expressed in A. thaliana, including genes previously characterized. Most of the genes tested on all 12 cDNA sources have a very broad expression spectrum; very few are specific for particular tissues (AtbHLH039, 040, 050, and 101) or treatments (AtbHLH085). Because this PCR-based approach was designed for the maximum amplification of cDNAs, it does not provide any insight into the relative expression levels of the genes. However, our results provide the basis for the creation of cDNA (and other) arrays with a high coverage of bHLH genes.
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The Structural Relationships Between Plant bHLH Genes
Based on the amino acid alignments of 133 bHLH domains, a comparative tree was constructed. The clustering showed that there are 12 major groups (subfamilies) of related sequences which were strongly supported by bootstrap analysis. The conclusions from this analysis are presented in figure 4, with closely related genes within subfamilies grouped together. Comparing all aspects of gene structure, genes within each subfamily contain a similar number of introns with conserved positions, and show similar predicted lengths for the encoded proteins and similar positions for the bHLH domain within the protein. In many subfamilies, the proteins also share related amino acid sequences outside the DNA binding domain (fig. 4). These motifs give added support to the clustering obtained using the DNA binding domain alone and in several cases help to separate the subfamilies further into subgroups. Some of these motifs probably act as activation domains and may also be important for interaction with other modules of the transcription complex, or they may be targets of signal transduction chains. For example, proteins belonging to sub-groups IIId, IIIe, and IIIf have a conserved stretch of amino acids positioned toward the N-termini of the proteins, a region that is found only in plant bHLH proteins (fig. 4). Amino acids within this region are predicted to form
-helixes, some of which are highly amphipathic (Goff, Cone, and Chandler 1992). A conserved aspartate residue in this region (within motif 11) of the AtbHLH005/ATR2 protein appears to be functionally important for correct expression of several downstream target genes that belong to the tryptophan biosynthesis pathway (Smolen et al. 2002). Between the bHLH domain and the conserved N-terminal domain is a sequence rich in acidic amino acids with significant negative charge (Lc: Ludwig et al. 1989; B: Radicella, Turks, and Chandler 1991; DEL: Goodrich, Carpenter, and Coen 1992), a feature of activating domains identified in known transcriptional activators (Ptashne 1988). Using trans-activation assays Goff, Cone, and Chandler (1992) demonstrated that a region of the anthocyanin regulator B, comprising the N-terminal domain and part of the acidic region, was important for activation of a Bz1 promoter-reporter construct. However, activation only occurred when the C1 MYB protein was also present in the assay, indicating that an interaction of B with C1 was necessary to obtain an active transcription complex. The interacting region within C1 was mapped to the MYB DNA binding domain (Goff, Cone, and Chandler 1992), and more recently, particular amino acids within the MYB domain of C1 have been shown to be important for an interaction with the R bHLH protein (Grotewold et al. 2000). It is not clear whether R-like bHLH proteins (group III) bind DNA in the bHLH-MYB complex or whether DNA binding occurs only via the MYB partner; to date only C1 has been shown to bind cis-acting elements in the promoter of the Z. mays A1 (DFR) gene (Sainz, Grotewold, and Chandler 1997). Within bHLH group III, B is most similar to A. thaliana AtbHLH012/MYC1, a bHLH protein from A. thaliana identified by Urao et al. (1996).
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Specific functions for other genes belonging to group III are known, and in contrast to the R-like proteins, the corresponding gene products have been shown to bind DNA. The Phaseolus vulgaris bHLH protein PG1 binds to a G-box motif in the ß-phaseolin gene promoter that is important for positive transcriptional regulation (Kawagoe and Murai 1996), and the A. thaliana rd22BP1 protein (AtbHLH006/rd22BP1/RAP-1) binds to a region of the rd22 gene promoter that is sufficient for dehydration- and ABA-induced expression of the rd22 gene (Abe et al. 1997; de Pater et al. 1997). The tomato Fer genestructurally closely related to A. thaliana group III bHLH geneshas been shown recently to have a role in iron uptake in roots (Ling et al. 2002), and so the diverse functional nature of this group of bHLH proteins in A. thaliana is emerging.
Members of group IV have a highly conserved leucine zipper (ZIP) motif adjacent to the second helix of the bHLH motif. The ZIP domain is predicted to adopt a coiled-coil structure that permits dimerization between proteins (Lupas 1996). Some animal proteins also contain a ZIP domain in the same position as the plant bHLH-ZIP proteins, and a few members of group III also have a partially conserved ZIP motif. In animal bHLH-ZIP proteins, there is evidence that the ZIP motif stabilizes protein dimers (Brandt-Rauf et al. 1989; Bresnick and Felsenfeld 1994) and that particular residues within the ZIP domain determine dimerization specificity (Marchetti et al. 1995). The functions of the plant group IV bHLH-ZIP proteins are unknown.
Recently, three members of group XII, AtbHLH044/BEE1, AtbHLH058/BEE2, and AtbHLH050/BEE3 (BR Enhanced Expression) from A. thaliana have been linked to multiple pathways regulating plant growth and development (Friedrichsen et al. 2002). These closely related bHLHs act redundantly as positive regulators in the early Brassinosteroid (BR) signaling pathway and they also affect signaling by abscisic acid (ABA), a known antagonist of BR.
One might anticipate that proteins clustered on the tree have similar biological functions. However the genes that make up subgroup IIIf are involved in very different processes; flavonoid/anthocyanin biosynthesis (AtbHLH0012/MYC1and AtbHLH042/TT8) and trichome initiation (AtbHLH001/GL3). The maize gene, Lc, encodes a member of the R/B family and is orthologous to the A. thaliana gene AtbHLH012/MYC1. Overexpression of Lc in wild-type A. thaliana results in additional trichomes and an elevated level of anthocyanins compared to control plants (Lloyd, Walbot, and Davis 1992). However, in maize, members of the R gene family have no influence on trichome formation. The bHLH protein bHLH001/GL3, closely related to Lc (Payne, Zhang, and Lloyd 2000), belongs to a cascade of transcriptional regulators that control the differentiation of trichome progenitor cells in shoot epidermis. Although the maize Lc gene was overexpressed in a heterologous context, the observation that it can induce trichome formation in A. thaliana suggests that closely related genes retain the potential to cross over and regulate parallel processes (requiring similar transcriptional activators) when expressed at artificially high levels. This may reflect the importance that patterns of expression and strength of expression have on the specific function(s) carried out by transcription factors in wild-type tissues.
Evolution of bHLH Protein Structure and Function
In their phylogenetic classification of animal bHLH protein sequences, Atchley and Fitch (1997) identified more than 24 protein lineages containing proteins with related function. They found that these lineages could be organized further into five groups (A to E) that relate to the preferred DNA binding sequence of the proteins within each lineage. Within each group proteins bound to a variation of the palindromic hexanucleotide sequence CANNTG known as the E-box motif. Patterns of amino acids were found at three positions within the basic region of the bHLH motif (the 5913 configuration) that defined the five different subgroups very accurately and suggested that the amino acid sequence motif of group B, with an H-E-R configuration, was the ancestral sequence (Atchley and Fitch 1997).
In A. thaliana, three proteins, namely PvPG1, AtbHLH006/rd22BP1/RAP-1 (both group III), and more recently AtbHLH008/PIF3 (group VII; Martinez-Garcia, Huq, and Quail 2000) have been shown to bind a sequence identical or very similar to the B variant of the animal E-box motif, which is also identical to the G-box core motif (CACGTG), a ubiquitous regulatory DNA element found in plants that is also bound by some bZIP TFs (Menkens, Schindler, and Cashmore 1995). Most (87 of 133) A. thaliana genes have the H-E-R configuration within the bHLH domain that is found in members of group B from animals (fig. 4), suggesting that plant bHLH proteins evolved from one or several members of group B that were present in early eukaryotic lineages. In this context it is also interesting that seven plant bHLH proteins in group IV contain their ZIP motif in the identical position to animal bHLH-ZIP proteins, which allwith one exceptionbelong to the class B proteins. Some plant proteins, notably members of groups VIII and X, have a variation of the H-E-R configuration and contain helix-breaking prolines in the basic region (fig. 4), characteristics that may interfere with affinity for DNA. These variations may enable these proteins to act as negative regulators, retaining the ability to dimerize with other bHLH proteins but lacking the ability to bind DNA. This would make group X members candidates for reclassification into group D according to Atchley and Fitch (1997).
From comparative studies it is apparent that the bHLH motif was present in early eukaryotes and that the subfamilies of genes distinguishable today have emerged following the separation of plants, animals, and fungi (Ledent and Vervoort 2001). A. thaliana possesses more bHLH genes than are found in Caenorhabditis elegans or D. melanogaster (39 and 59 genes, respectively), but is comparable to the numbers obtained for human and mouse, 125 and 102, respectively (Ledent, Paquet, and Vervoort 2002). Although no striking similarities in gene structure were detected between plants and these other lineages, except for similarities to animal group B proteins, it is still possible that some plant proteins have retained functions possessed by the ancestral eukaryotic proteins such as the developmental control of cell lineages. For example, MyoD and related proteins are involved in the determination of muscle cell lineages, and expression is confined to these cells. They interact with the ubiquitously expressed E12 family of bHLH proteins to produce active heterodimers (French et al. 1991). Furthermore, E12 and related proteins are involved in the determination and maintenance of other cell types through their ability to dimerize with other proteins that are expressed in specific cell lineages (Littlewood and Evan 1998, pp. 2735). Recently, two members of the plant group VII have been shown to be important in the development of floral structures. Mutants in the gene AtbHLH024/SPATULA are defective specifically in the development of the style and stigma and, with strong mutant alleles, cells comprising the pollen-transmitting tract tissue do not form (Alvarez and Smyth 1999; Heisler et al. 2001). However, expression is not confined to these tissues, indicating that SPATULA may have additional roles in other aspects of growth and development. The other member of group VII, AtbHLH073, is the most closely related A. thaliana protein to SPATULA and is responsible for the differentiation of a nonlignified cell layer necessary for cell separation during dehiscence (Rajani and Sundaresan 2001).
Mediating responses to the environment may be another ancient function retained by plant bHLH proteins, for example, controlling responses to light and interacting with components of the circadian clock. The PIF-like 1 protein (AtbHLH124/PIL1; group VII) has been shown to interact with APRR1, a factor that has been implicated in circadian rhythms (Makino et al. 2002) and that belongs to a class of pseudo-response regulators that are components of signal transduction systems. In addition, AtbHLH008/PIF3 (group VII) can regulate expression of the circadian clock genes CCA1 and LHY, suggesting that this protein provides an entry point to phytochrome regulation of the circadian clock (Martinez-Garcia, Huq, and Quail 2000). Studies indicate that light regulation and control of biorhythms are functionally interrelated and possibly have a common evolutionary origin (Kay 1997). Photoreceptor proteins and protein components of the circadian clock from species belonging to several kingdoms of life contain PAS (PER-ARNT-SIM) domains (Nambu et al. 1991) that have been shown to mediate proteinprotein interactions. These include the D. melanogaster PER protein that interacts via its PAS domain with Timeless to regulate the circadian clock (Zeng et al. 1996), and the White Collar 2 protein (WC2) from Neurospora crassa that is involved in the blue light regulation of gene expression (Linden and Macino 1997) and has also been shown to be a clock component (Crosthwaite, Dunlap, and Loros 1997). A PAS-like domain has been identified in PIF3 and a related protein, PIF4 (Huq and Quail 2002), but in both cases the domain shows only modest similarity to animal PAS domains, although the region encompassing the PAS domain in PIF3 is important for the interaction of PhyB (Ni et al. 1996).
ProteinProtein Interactions and Their Importance in Gene Regulation
In mammals and D. melanogaster there are many instances in which bHLH proteins have been shown to homodimerize or heterodimerize as a means of controlling transcription (Littlewood and Evan 1998). The ability to heterodimerize allows a large number of potential DNA binding complexes that may have different biochemical propertiesincluding variable DNA binding characteristics and activation or repression potentialsthereby creating a lot of opportunities for regulating different genes or sets of genes. In plants, dimerization has been shown for two related members of group VII, AtbHLH008/PIF3, and AtbHLH026/HFR1/REP1, with proposed dominant roles in phytochrome B and phytochrome A signaling, respectively (Halliday et al. 1999; Fairchild, Schumaker, and Quail 2000; Soh et al. 2000). In yeast-two-hybrid experiments, PIF3 binds preferentially to PhyB rather than PhyA, as well as being able to interact with itself (Zhu et al. 2000); AtbHLH026/HFR1/REP1 binds neither to phyA nor to phyB, but it does bind to PIF3 in vitro, suggesting that it is likely to operate as a heterodimer in regulating transcription from target promoters (Fairchild, Schumaker, and Quail 2000). Recently the AtbHLH009/PIF4 protein, which is closely related to PIF3, has been shown to have a role in phyB-regulated de-etiolation as a negative regulator (Huq and Quail 2002). PIF4 and PIF3 have similar biochemical properties, but unlike PIF3, PIF4 is unable to bind to the active form of PhyB when bound to the G-box element. In addition, PIF4 cannot regulate G-boxcontaining gene promoters including those of the circadian clock genes CCA1 and LHY, which are targets of PIF3 (Martinez-Garcia, Huq, and Quail 2000). Structurally, the most similar gene to PIF4 in A. thaliana, AtbHLH065 (fig. 4) appears on a duplicated section of the A. thaliana genome, so it may have functions similar if not identical to those of PIF4. Subgroup VIIa contains several more genes encoding factors which are related to PIF3, PIL1, and PIF4 that have not yet been studied (AtbHLH015, AtbHLH023, AtbHLH056, AtbHLH119, and AtbHLH127) but that have (due to their structural similarity) the potential to be involved in aspects of phytochrome signaling. These five genes may have redundant functions, as two are found in duplicated sections of the genome and one is part of a cluster of four structurally similar genes, indicating that there have been recent duplications of these bHLH genes in A. thaliana (fig. 4).
Interactions can also occur between different classes of TFs. For example, members of bHLH group III associate with R2R3-MYB proteins. In a similar way to the Z. mays bHLH protein B, which interacts with the R2R3-type MYB protein C1 to create an active transcription complex (Goff, Cone, and Chandler 1992), bHLH TFs related to B from other species also behave as co-activators of MYB proteins to induce anthocyanin biosynthetic genes (PhJAF13; Quattrocchio et al. 1998). AtMYC1 is the most closely related protein in A. thaliana to these R-like proteins, but it is phylogenetically distinct from two other regulators of anthocyanin biosynthesis, ANTHOCYANIN1 (AN1) from Petunia (Spelt et al. 2000) and TT8 from A. thaliana (Nesi et al. 2000). Together with a MYB protein (AN2 in P. hybrida, TT2 in A. thaliana), these petunia bHLH proteins can regulate a specific part of the anthocyanin pathway, downstream of dihydroflavonol biosynthesis. In transfection assays, co-expression of PhAN1 or PhJAF13 with PhAN2 (a P. hybrida R2R3-MYB) resulted in much stronger activation of the DFR target gene than if either PhAN1 or PhJAF13 were expressed alone (Spelt et al. 2000). Interestingly, when PhAN1 was co-expressed with ZmP, a R2R3-MYB protein that activates a branch of flavonoid biosynthesis in Z. mays maternal seed tissues, expression was enhanced 10-fold more than if ZmP was expressed alone; this did not happen if PhJAF13 was co-expressed with ZmP (Spelt et al. 2000). This experiment suggests that, at least under certain conditions, specific bHLH factors differ in their affinities for particular MYB proteins, and this finding is a reflection of their structural differences.
The AtbHLH001/GL3 protein that controls trichome development in A. thaliana interacts with the R2R3-MYB protein AtMYB0/GLABROUS1 (GL1) in yeast-2-hybrid experiments (Payne, Zhang, and Lloyd 2000). GL1 and GL3 positively control expression of the homeodomain transcription factor GLABRA2 (GL2; Rerie, Feldmann, and Marks 1994) but it is not known how GL2 then induces the cellular requirements for the directional shaping of a developing trichome cell. Interestingly, GL1 and WEREWOLF (WER) belong to the same sub-group of R2R3-MYB proteins (subgroup 15; Stracke, Werber, and Weisshaar 2001) but control opposite epidermal cell fates: WER is expressed specifically in non-hair cells of root epidermis to prevent hair formation, whereas GL1 promotes trichome formation that is expressed only in shoot epidermis. The biochemical function of the WER protein is likely to be identical to that of GL1, because each gene can complement the mutant phenotype of the other when expressed from the reciprocal promoter (Lee and Schiefelbein 2001). In this case function is dependent on cell-specific expression and the other (co-localized) TFs with which each protein can interact. Some specificity might come through the differential expression of bHLH genes with which WER might interact, as GL3 does not affect root hair formation.
There is also evidence that another member of group III, AtbHLH006/rd22BP1/RAP-1, interacts with the R2R3-MYB protein AtMYB2 to activate gene expression from a dehydration and ABA-responsive promoter element (Abe et al. 1997). In conclusion, bHLH/MYB interactions are probably conserved for group III bHLH proteins and for members of MYB subgroups 5, 6, and 15 (Stracke, Werber, and Weisshaar 2001), but it remains to be clarified whether interactions are confined to the protein products of these particular gene clades or are more widespread. The N-terminal region conserved in group III bHLH proteins may be involved in bHLH/MYB interactions and could provide the specificity for particular proteinprotein interactions (Goff, Cone, and Chandler 1992).
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Future Perspectives |
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Major segmental duplication events appear to have occurred at different times during angiosperm evolution, possibly including a polyploidization event (Blanc et al. 2000; Vision, Brown, and Tanksley 2000), and the more recent duplication events may have contributed significantly to the phenomenon of gene redundancy (Pickett and Meeks-Wagner 1995). Combining the data on inferred gene duplications with clustering analysis of genes from one species (fig. 4) provides a framework for visualizing the likely functional diversity within groups of related genes such as those encoding bHLH proteins. From these data, educated guesses can be made concerning gene redundancy, which will be important information for estimating the number of knockout mutations required for observing altered phenotypes for closely related genes. From structural similarities we estimate that the bHLH family includes at least 33 discernible functions in transcriptional regulation. An automated interrogatory gene tree founded on the Arabidopsis genes would be a useful aid for providing clues to the functions for bHLH proteins in other model and crop species and would allow subsequent experiments on these genes to be designed more effectively. Knowledge about the structure of the AtbHLH gene family, and the option to integrate new bHLH genes from other plant species into this frame, will further facilitate the identification of the functions of bHLH genes in plants.
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Acknowledgements |
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Footnotes |
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William Taylor, Associate Editor
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Literature Cited |
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