Legume Transcription Factors: Global Regulators of Plant Development and Response to the Environment

Transcription factors (TFs) are DNA-binding proteins that interact with other transcriptional regulators, including chromatin remodeling/modifying proteins, to recruit or block access of RNA polymerases to the DNA template. Plant genomes devote approximately 7% of their coding sequence to TFs, which is a testament to the complexity of transcriptional regulation in these organisms. Extensive sequencing of cDNA and genomic DNA indicates that legumes encode upwards of 2,000 TFs per genome. Less than 1% of these have been characterized genetically, although TFs likely played seminal roles in legume evolution and clearly now play crucial roles in plant development and differentiation. Here we review the literature on legume TFs and describe technological developments that are paving the way for rapid and systematic characterization of TFs and the genetic regulatory networks they control. Plants are amazing organisms. Not only are they able to build complex organic superstructures from simple inorganic molecules that ensure their growth and reproductive success, but they do this while fixed in space and subject to environmental extremes of light, temperature, water, and nutrients, and to biological challenges from competitors, pests, and pathogens. Evolution has endowed plants with a flexible developmental program that enables them to elaborate new vegetative organs and attune reproduction to prevailing environmental conditions. Plant cells can also differentiate in the short term to cope with more immediate environmental challenges. Plant development and differentiation are programmed primarily at the level of gene transcription, which is controlled by TFs and other proteins that either recruit or block access of RNA polymerases to the DNA template. TFs are usually defined as sequence-specific DNA-binding proteins that are capable of activating and/or repressing transcription. Plant genomes appear to encode many more TFs than those of animals, such as Caenorhabditis elegans and Drosophila melanogaster, which indicates that transcriptional regulation in plants is at least as complex as in animals (Riechmann et al., 2000). Arabidopsis (Arabidopsis thaliana) possesses upwards of 1,800 TF genes representing more than 7% of all protein-coding genes (Riechmann et al., 2000; Guo et al., 2005; Iida et al., 2005). Surprisingly, only one-tenth of these have been characterized genetically (Qu and Zhu, 2006) despite the enormous resources that have been devoted to Arabidopsis research over the past decade. Not surprisingly, we know far less about the role of TFs in other plant species. For instance, less than 1% of TF genes in the model legumes Lotus japonicus (or simply Lotus) and Medicago truncatula (or Medicago) have been genetically characterized. This makes review of the literature on legume TFs a relatively simple task at present, although there are signs that this situation will change rapidly over the next few years. First of all, it is already apparent that TFs play crucial roles in agriculturally important processes in legumes, such as symbiotic nitrogen fixation (SNF), so there is great incentive to learn more about this important class of regulatory proteins. Second, the genome of three legume species, Medicago, Lotus, and soybean (Glycine max), will be completed or largely so in the next 2 years, which will enable the identification of most of the TFs in these species via bioinformatics approaches. Finally, numerous tools for functional genomics have been and are being developed that will facilitate rapid and systematic functional characterization of large numbers of TFs. This review summarizes our current state of knowledge about legume TFs and considers the opportunities and challenges for continued research in this area. As a backdrop to our discussion of legume TFs, it is salient that recent transcriptomic studies, using arrays of cDNA or oligonucleotides to measure transcript levels, have identified thousands of legume genes that are differentially expressed during various types of plant-microbe interactions (Colebatch et al., 2002, 2004; Liu et al., 2003; Barnett et al., 2004; El-Yahyaoui et al., 2004; Kouchi et al., 2004; Lee et al., 2004; Manthey et al., 2004; Mitra et al., 2004; Moy et al., 2004; Suganuma et al., 2004; Hohnjec et al., 2005; Zou et al., 2005; Alkharouf et al., 2006; Lohar et al., 2006; Starker et al., 2006; Zabala et al., 2006), development and differentiation (Vodkin et al., 2004; Aziz et al., 2005; Firnhaber et al., 2005; Dhaubhadel et al., 2007), and in response to abiotic stress (Ainsworth et al., 2006; Buitink et al., 2006). Invariably, TFs have been found among differentially expressed genes, implicating them in the regulation of specific developmental processes or responses to the biotic and abiotic environment. Such guilt by association is a theme that is elaborated upon below. Classification of putative TFs of Medicago into families and subfamilies IMGAG proteins were classified as putative TFs if they contained characteristic DNA-binding or other characteristic TF domains and if annotations of matching proteins obtained by BLAST searches were consistent with such a classification. TF families previously identified in plants are presented in the first part of the table, whereas potentially novel plant TF families, which were identified by the presence of domains associated with TFs and other transcriptional regulators outside the plant kingdom, are presented in the latter part of the table. Plant-specific TF families and subfamilies are indicated in bold (according to Riechmann, 2002). D, DNA-binding domain; P, protein-protein interaction domain; NA, nucleic acid (DNA and RNA) binding domain; RD, receiver domain; LBD, ligand binding; TA, transcriptional coactivator. Classification of putative TFs of Medicago into families and subfamilies IMGAG proteins were classified as putative TFs if they contained characteristic DNA-binding or other characteristic TF domains and if annotations of matching proteins obtained by BLAST searches were consistent with such a classification. TF families previously identified in plants are presented in the first part of the table, whereas potentially novel plant TF families, which were identified by the presence of domains associated with TFs and other transcriptional regulators outside the plant kingdom, are presented in the latter part of the table. Plant-specific TF families and subfamilies are indicated in bold (according to Riechmann, 2002). D, DNA-binding domain; P, protein-protein interaction domain; NA, nucleic acid (DNA and RNA) binding domain; RD, receiver domain; LBD, ligand binding; TA, transcriptional coactivator. Relationships and domain shuffling between Medicago TF families. TF families are represented by circles whose size is proportional to the number of members in the family. Domains are represented by rectangles, whose size is proportional to the domain length. DNA-binding domains appear in color. Protein-binding and other domains are hatched. Dashed lines indicate that a given domain is characteristic of the family or subfamily to which it is attached. Dotted lines indicate domains that define a potentially novel TF family or subfamily. Based on Figure 1 of Riechmann (2002). As noted above, very few legume TFs have been characterized genetically so far (Table II Genetically characterized TFs in legumes Genetically characterized TFs in legumes Many TF genes have been found to be expressed during nodule development and differentiation (see above) and several groups are now using the tools of reverse genetics to decipher the roles of such genes in SNF. Three TFs have been implicated in nodule development or function in this way (Table II). The first of these was Mszpt2-1, a Kruppel-like TF of the C2H2 (Zn) family that was found to be essential for differentiation of the nitrogen-fixing zone of alfalfa (Medicago sativa) nodules via an antisense RNA approach (Frugier et al., 2000). A similar approach implicated the Lotus ndx gene family in nodule function and maintenance (Gronlund et al., 2003). Most recently, RNA interference (RNAi) revealed a key role in nodule development for MtHAP2-1, a member of the CCAAT-binding family of TFs (Combier et al., 2006). Interestingly, MtHAP2-1 was found to be regulated by microRNA169, revealing an important role for microRNA in the regulation of legume development. Whereas research to identify TFs involved in SNF has profited little from previous work in nonlegumes such as Arabidopsis, knowledge from nonlegume models has been instrumental in identifying a number of TF genes involved in common plant processes, such as flower and leaf development (Table II). In fact, the first legume TF gene to be characterized functionally was pea (Pisum sativum) FLO, which was isolated by virtue of its sequence homology to the TFs FLO and LFY of snapdragon (Antirrhinum majus) and Arabidopsis, respectively. FLO and LFY control floral development in snapdragon and Arabidopsis, and a defect in pea FLO was subsequently found to be responsible for aberrant floral and leaf development in the pea unifoliata (uni) mutant (Hofer et al., 1997). The Lotus ortholog of FLO was later identified in the same way (Dong et al., 2005). Similar approaches were used to assign functions for PIM (a MADS family TF) in pea floral meristem determination (Taylor et al., 2002) and for PHANTASTICA (a MYB TF) in pea compound leaf development (Tattersall et al., 2005). Interestingly, Lotus has a duplicate pair of PHANTASTICA-like genes that probably have divergent functions in compound leaf development (Luo et al., 2005). The value of computational approaches in identifying genes likely to be involved in various aspects of flowering was nicely illustrated by Hecht et al. (2005), who utilized sequence information from Arabidopsis TFs to identify homologs in model legume sequence databases, which were then used to design PCR-cloning strategies to isolate homologs from pea. The majority of Arabidopsis flowering genes were represented in pea and other legume sequence databases. However, several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appeared to have undergone differential expansion, whereas other genes important in Arabidopsis, including FRIGIDA and members of the FLOWERING LOCUS C clade, were conspicuously absent from legumes. Several pea and Medicago orthologs mapped to syntenic chromosomal positions, demonstrating the benefit of parallel model systems for understanding flowering phenology in crop and model legume species. TFs of the TCP family, named after the founding members TB1, CYC, and PCF, help to establish the pattern of flower petals (Cubas, 2004), which gives legume flowers their typical bilateral symmetry. Citerne et al. (2003) sequenced a number of CYC homologs, sorted them into clades by phylogenetic analysis, and discussed the difficulties of assigning orthologs in cross-species comparisons. Thus, genetic map position, mutant phenotypes, and/or complementation of Arabidopsis mutants have been used in addition to sequence similarity to infer orthology. The squared standard mutant of Lotus is defective in the TCP gene LjCYC2, which is required to establish floral bilateral symmetry and was cloned by a candidate gene/sequence homology approach (Feng et al., 2006). Adaxial expression of two CYC genes was observed in the developing floral meristem of lupin (Lupinus albus), which also has bilaterally symmetrical flowers (Citerne et al., 2006). Interestingly, evolution of radially symmetrical flowers in Cadia, which belongs to the same subclade as Lupinus, may have resulted from an expanded domain of expression of an orthologous CYC gene in the former (Citerne et al., 2006). Cross-species complementation studies have indicated possible roles for several legume TFs. For example, Berbel et al. (2001) rescued the Arabidopsis apetala1 (ap1) floral development mutant using a PIM cDNA clone, which they called PEAM4. Later, the same group characterized PsPI, a pea MADS-box gene homologous to the petal and stamen identity genes PISTILLATA (PI), from Arabidopsis and GLOBOSA, from snapdragon. Interestingly, constitutive expression of PsPI in Arabidopsis rescued the floral defects caused by the strong pi-1 mutant allele, despite the fact that the pea protein, PsPI, lacked a particular C-terminal motif (Berbel et al., 2005). Similarly, Guo and Gan (2006) showed that overexpression of PvNAP, a kidney bean (Phaseolus vulgaris) NAC TF homologous to Arabidopsis AtNAP, successfully complemented the leaf abscission phenotype of an atnap null mutant, indicating a possible role of NAP in bean leaf abscission. Finally, five legume TFs have been implicated in abiotic stress tolerance (Table II). One of these, alfalfa Mszpt2-1, which was mentioned previously in the context of nodule development, was found to be induced in roots by salt treatment. Inhibition of Mszpt2-1 by antisense RNA resulted in increased sensitivity of transgenic plants to salinity (Merchan et al., 2003). Overexpression of CAP2 and Alfin1 TFs in transgenic plants conferred salt tolerance and increased growth (Winicov, 2000; Shukla et al., 2006). Constitutive overexpression of SCOF-1, a soybean protein, increased cold tolerance of transgenic Arabidopsis and tobacco (Nicotiana tabacum) plants (Kim et al., 2001). Another example of successful leveraging of knowledge from nonlegumes for legume research is provided by the Medicago WXP1 gene, a member of the AP2/ethylene-responsive element-binding protein (EREBP) family of TFs. Several members of this family have been implicated in drought tolerance in Arabidopsis and other nonlegumes. Overexpression of Medicago WXP1 in alfalfa resulted in enhanced tolerance to drought stress, which correlated with increased wax deposition in the leaf cuticle (Zhang et al., 2005). Whereas work on deciphering the roles of legume TFs is just beginning, considerable efforts have already been made to demonstrate the functionality of such proteins in terms of their DNA-binding and transactivation abilities and subcellular localization (Table III Biochemical and molecular characterization of legume TFs NL, Nuclear localization. All other abbreviations are defined in the text. Biochemical and molecular characterization of legume TFs NL, Nuclear localization. All other abbreviations are defined in the text. Other TFs have been identified based on their ability to interact with known gene cis-elements. The use of cDNA expression libraries has been valuable in this regard. Two HD-ZIP proteins (GmHDL56/57) were identified using a 160-bp fragment of the VspB promoter (Tang et al., 2001). Similarly, G/HBF-1, SGBF-1/2, and GmGT-2 were isolated based on their ability to bind specific promoter elements (Hong et al., 1995; Dröge-Laser et al., 1997; O'Grady et al., 2001). Yeast (Saccharomyces cerevisiae) one-hybrid screens have been effective in the isolation of legume proteins that bind specific cis-elements. Sangwan and O'Brian (2002) constructed a soybean nodule cDNA library in a vector containing a GAL4-activation domain to produce GAL4 fusion proteins. Introduction of this library into a yeast His-auxotroph engineered to have a HIS3 gene preceded by a (GA)27/(CT)27 dinucleotide repeat sequence resulted in the identification of a His prototroph containing a GBP-GAL4 fusion. Rarely have legume TF protein-protein interactions been identified, presumably because most of the research in this area has focused on et al. (2001) identified a protein, SCOF-1, which to DNA-binding with several candidate but was subsequently found to the acid response gene expression by to demonstrate DNA binding of legume TFs of DNA to TFs on and yeast one-hybrid (Table In example, et al. identified cDNA a protein with a putative by differential of alfalfa The DNA-binding of Alfin1 was by binding of protein to oligonucleotides in an by to identify the In most DNA-binding has been only on a few that have been identified by work in other plant families. Such approaches are likely to important approaches are now that this (see of TF has been provided using transactivation groups have in transactivation in and including of bean et al., et al., of Arabidopsis (Kim et al., and in in sativa) et al., 2002). One of a role as a TF is its localization to the For legume TFs, this has been with localization and 2006) or using or fusion proteins (Kim et al., et al., 2005; et al., 2005; et al., 2005; Shukla et al., 2006). In the of a of was of that is required to this protein et al., 2005). there has been a between TFs that have been a biological role based on genetic and TFs that have been characterized at the and/or molecular to the function of genetically characterized TFs, we to identify the genes and of genes that they control. the other for TFs that have been characterized in terms of their DNA-binding it is now important that biological function be via or reverse despite the knowledge that TFs work as part of a or complex of proteins to recruit or block of RNA to the DNA and is known about the proteins that interact with legume TFs to ensure their biological and development and differentiation and plant responses to specific environmental challenges the of networks of TFs, which in transcription. The of these networks in legumes. these in legume TF in an will a on the part of the The of this review a for this the next 2 or years, or genomic sequence for the of three legumes, Medicago, Lotus, and will be This will facilitate systematic approaches to TF functional approaches will rapidly identify putative TFs among the new genomic as above, which will for the functional Whereas genetics will as genomic sequencing in more and and genetic of which will facilitate of TFs involved in legume development and approaches are likely to play a more role in the functional characterization of TFs in the reverse genetics a more systematic way to all putative TF of the tools for systematic of TF function in legumes, such as plant for and overexpression et al., 1997; et al., 2003; et al., 2005; et al., and mutant for et al., are already in such as et al., and mutant et al., 2006) are being and a mutant from a has already been et al., 2006). gene is for reverse genetics that has successful in pea et al., In of the TF of Arabidopsis and we that of the three model legumes mentioned will at least 2,000 TF will be an task for group to this number of genes, at least at the genetic A help to the of TF gene function most One way to to such an be to first the developmental and environmental expression of TF in the context of the This several it Second, it environmental stress which about possible roles of by TF gene expression into the context of transcription, between TFs and groups of other genes be which help to about possible TF if correlated of genes are to be involved in or just a few biological Many of the tools required for such are now for Medicago, Lotus, and including containing for the majority of genes in these three In we are developing for all Medicago TFs for to obtained using the and and a similar is being developed for soybean by is more than by DNA et al., so the resources being developed for in Medicago and soybean will a more of TF expression in this species. et al., and et al., are two to identify genes that are which will only about the possible function of TF genes (see but also identify possible genes of specific TFs. have been few to the interaction between a genetically characterized legume TF and a gene, although possible genes have been identified by of TF mutants et al., 2005; et al., 2005). have been developed to identify TF genes in a the most of these is chromatin by DNA to identify DNA to DNA-binding proteins et al., 2006). can be by in expression of the TF of as a fusion protein with a at which enables the use of the to the fusion protein and proteins and This approach has been for example, to identify genomic DNA by the Arabidopsis TF et al., 2006). can also be used to identify associated proteins in DNA-binding et al., 2006). A for is an containing for promoter to gene such as the for Medicago, Lotus, and are for such However, which the genome and with short or between are to et al., 2006; et al., 2006). for a legume we have to a Medicago upon of the genome sequence in The may the that the is for rapid in TF function in model legumes. However, it is likely that there will be and in the For instance, TFs appear to with or more so that of function of gene may in a mutant plant (Riechmann and 2000). between TFs makes it if to isolate in these genes via However, approaches that phylogenetic and transcriptomic information to identify potentially genes to the of or mutants this et al., 2000; et al., 2003). the of mutant (see above) will be essential for this approach to functional is to mutants by TFs to known domains et al., 2002). approach is to the TF of in transgenic plants and to the of this on the expression of other genes and on the phenotype or of the TF overexpression be to the same and types as the gene and the control of an is that of TF function in plants from in that the expression domain of the gene, than from in the coding sequence that the or of TFs. Thus, TF mutant have been by expression of TFs that are expressed in the same as the TF 2002). Overexpression of TFs can also with processes to the function of the protein (Riechmann and 2000). be in and TF overexpression TFs interact with other proteins, in addition to the RNA complex to in gene and 2000). The specific of these is for the majority of plant genes, although this knowledge is a to understanding the control of such as yeast et al., and by of the protein et al., 2006; et al., 2006) will be in this Finally, the of genes regulated by a TF and its is just a part of a genetic regulatory that expression of genes involved in many processes during plant development and differentiation. these genetic networks will of many of the functional and approaches The are in the of this TFs among proteins.