Transcription factors activate or repress target gene expression or switch between activation and repression. In animals and yeast, Groucho/Tup1 corepressor proteins are recruited by diverse transcription factors to induce context-specific transcriptional repression. Two groups of Groucho/Tup1-like corepressors have been described in plants. LEUNIG and LEUNIG_HOMOLOG constitute one group and TOPLESS (TPL) and the four TPL-related (TPR) corepressors form the other. To discover the processes in which TPL and the TPR corepressors operate, high-throughput yeast two-hybrid approaches were used to identify interacting proteins. We found that TPL/TPR corepressors predominantly interact directly with specific transcription factors, many of which were previously implicated in transcriptional repression. The interacting transcription factors reveal that the TPL/TPR family has been coopted multiple times to modulate gene expression in diverse processes, including hormone signaling, stress responses, and the control of flowering time, for which we also show biological validation. The interaction data suggest novel mechanisms for the involvement of TPL/TPR corepressors in auxin and jasmonic acid signaling. A number of short repression domain (RD) sequences have previously been identified in Arabidopsis (Arabidopsis thaliana) transcription factors. All known RD sequences were enriched among the TPL/TPR interactors, and novel TPL-RD interactions were identified. We show that the presence of RD sequences is essential for TPL/TPR recruitment. These data provide a framework for TPL/TPR-dependent transcriptional repression. They allow for predictions about new repressive transcription factors, corepressor interactions, and repression mechanisms and identify a wide range of plant processes that utilize TPL/TPR-mediated gene repression.
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Nonsense-mediated mRNA decay (NMD) is a conserved mechanism that targets aberrant mRNAs for destruction. NMD has also been found to regulate the expression of large numbers of genes in diverse organisms, although the biological role for this is unclear and few evolutionarily conserved targets have been identified. Expression analyses of three Arabidopsis thaliana lines deficient in NMD reveal that the vast majority of NMD-targeted transcripts are associated with response to pathogens. Congruently, NMD mutants, in which these transcripts are elevated, confer partial resistance to Pseudomonas syringae. These findings suggest a biological rationale for the regulation of gene expression by NMD in plants and suggest that manipulation of NMD could offer a new approach for crop protection. Amongst the few non-pathogen responsive NMD-targeted genes, one potential NMD targeted signal, the evolutionarily conserved upstream open reading frame (CuORF), was found to be hugely over-represented, raising the possibility that this feature could be used to target specific physiological mRNAs for control by NMD.
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Large-scale protein-protein interaction studies recently demonstrated that the Arabidopsis TPL/TPR family of transcriptional co-repressors is involved in a broad range of developmental processes. TPL/TPRs predominantly interact with transcription factors that contain repression domain (RD) sequences. Interestingly, RDs reported in the literature are quite diverse in sequence, yet TPL/TPRs interact with proteins containing all of the known motifs. These data lead us to conclude that the TPL/TPRs act as general repressors of gene transcription in plants. To investigate this further, we examined interactions between TPL/TPR proteins encoded by the moss Physcomitrella patens genome and components of the auxin signaling pathway. As in Arabidopsis, moss TPL proteins interact with AUX/IAA and ARF proteins, suggesting that they act in both forms of ARF-mediated transcriptional repression. These data suggest that the involvement of TPL in auxin signaling has been conserved across evolution, since mosses and angiosperms diverged approximately 450 million years ago.
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Since the first MADS-box transcription factor genes were implicated in the establishment of floral organ identity in a couple of model plants, the size and scope of this gene family has begun to be appreciated in a much wider range of species. Over the course of millions of years the number of MADS-box genes in plants has increased to the point that the Arabidopsis genome contains more than 100. The understanding gained from studying the evolution, regulation and function of multiple MADS-box genes in an increasing set of species, makes this large plant transcription factor gene family an ideal subject to study the processes that lead to an increase in gene number and the selective birth, death and repurposing of its component members. Here we will use examples taken from the MADS-box gene family to review what is known about the factors that influence the loss and retention of genes duplicated in different ways and examine the varied fates of the retained genes and their associated biological outcomes.
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In eukaryotes, nonsense-mediated mRNA decay (NMD) targets aberrant and selected non-aberrant mRNAs for destruction. A recent screen for mRNAs showing increased abundance in Arabidopsis NMD-deficient mutants revealed that most are associated with the salicylic acid (SA)-mediated defense pathway. mRNAs with conserved peptide upstream open reading frames (CpuORFs or CuORFs) are hugely overrepresented among the smaller class of NMD-regulated transcripts not associated with SA. Here we show that the common phenotypes observed in Arabidopsis NMD mutants are SA-dependent, whereas the upregulation of CpuORF-containing transcripts in NMD mutants is independent of SA. We speculate that CpuORFs could allow the conditional targeting of mRNAs for destruction using the NMD pathway.
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Large-scale protein-protein interaction studies recently demonstrated that the Arabidopsis TPL/TPR family of transcriptional co-repressors is involved in a broad range of developmental processes. TPL/TPRs predominantly interact with transcription factors that contain repression domain (RD) sequences. Interestingly, RDs reported in the literature are quite diverse in sequence, yet TPL/TPRs interact with proteins containing all of the known motifs. These data lead us to conclude that the TPL/TPRs act as general repressors of gene transcription in plants. To investigate this further, we examined interactions between TPL/TPR proteins encoded by the moss Physcomitrella patens genome and components of the auxin signaling pathway. As in Arabidopsis, moss TPL proteins interact with AUX/IAA and ARF proteins, suggesting that they act in both forms of ARF-mediated transcriptional repression. These data suggest that the involvement of TPL in auxin signaling has been conserved across evolution, since mosses and angiosperms diverged approximately 450 million years ago.
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TCP transcription factors constitute a small family of plant-specific bHLH-containing, DNA-binding proteins that have been implicated in the control of cell proliferation in plants. Despite the significant role that is likely to be played by genes that control cell division in the elaboration of plant architecture, functional analysis of this family by forward and reverse genetics has been hampered by genetic redundancy. Here we show that mutants in two related class I TCP genes display a range of growth-related phenotypes, consistent with their dynamic expression patterns; these phenotypes are enhanced in the double mutant. Together, the two genes influence plant stature by promoting cell division in young internodes. Reporter gene analysis and use of SRDX fusions suggested that TCP14 and TCP15 modulate cell proliferation in the developing leaf blade and specific floral tissues; a role that was not apparent in our phenotypic analysis of single or double mutants. However, when the relevant mutants were subjected to computer-aided morphological analysisof the leaves, the consequences of loss of either or both genes became obvious. The effects on cell proliferation of perturbing the function of TCP14 and TCP15 vary with tissue, as has been suggested for other TCP factors. These findings indicate that the precise elaboration of plant form is dependent on the cumulative influence of many TCP factors acting in a context-dependent fashion. The study highlights the need for advanced methods of phenotypic analysis in order to characterize phenotypes and to construct a dynamic model for TCP gene function.
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Gene duplication plays a fundamental role in evolution by providing the genetic material from which novel functions can arise. Newly duplicated genes can be maintained by subfunctionalization (the duplicated genes perform different aspects of the original gene's function) and/or neofunctionalization (one of the genes acquires a novel function). PLENA in Antirrhinum and AGAMOUS in Arabidopsis are the canonical C-function genes that are essential for the specification of reproductive organs. These functionally equivalent genes encode closely related homeotic MADS-box transcription factors. Using genome synteny, we confirm phylogenetic analyses showing that PLENA and AGAMOUS are nonorthologous genes derived from a duplication in a common ancestor. Their respective orthologs, SHATTERPROOF in Arabidopsis and FARINELLI in Antirrhinum, have undergone independent subfunctionalization via changes in regulation and protein function. Surprisingly, the functional divergence between PLENA and FARINELLI, is morphologically manifest in both transgenic Antirrhinum and Arabidopsis. This provides a clear illustration of a random evolutionary trajectory for gene functions after a duplication event. Different members of a duplicated gene pair have retained the primary homeotic functions in different lineages, illustrating the role of chance in evolution. The differential ability of the Antirrhinum genes to promote male or female development provides a striking example of subfunctionalization at the protein level.
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Gene duplication plays a fundamental role in evolution by providing the genetic material from which novel functions can arise. Newly duplicated genes can be maintained by subfunctionalization (the duplicated genes perform different aspects of the original gene's function) and/or neofunctionalization (one of the genes acquires a novel function). PLENA in Antirrhinum and AGAMOUS in Arabidopsis are the canonical C-function genes that are essential for the specification of reproductive organs. These functionally equivalent genes encode closely related homeotic MADS-box transcription factors. Using genome synteny, we confirm phylogenetic analyses showing that PLENA and AGAMOUS are nonorthologous genes derived from a duplication in a common ancestor. Their respective orthologs, SHATTERPROOF in Arabidopsis and FARINELLI in Antirrhinum, have undergone independent subfunctionalization via changes in regulation and protein function. Surprisingly, the functional divergence between PLENA and FARINELLI, is morphologically manifest in both transgenic Antirrhinum and Arabidopsis. This provides a clear illustration of a random evolutionary trajectory for gene functions after a duplication event. Different members of a duplicated gene pair have retained the primary homeotic functions in different lineages, illustrating the role of chance in evolution. The differential ability of the Antirrhinum genes to promote male or female development provides a striking example of subfunctionalization at the protein level.
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Cupuliformis mutants are defective in shoot apical meristem formation, but cup plants overcome this early barrier to development to reach maturity. CUP encodes a NAC-domain transcription factor, homologous to the Petunia NAM and Arabidopsis CUC proteins. The phenotype of cup mutants differs from those of nam and cuc1 cuc2 in that dramatic organ fusion is observed throughout development. In addition to cotyledon and floral organ fusions, severe lateral organ fusion is found in leaves and inflorescences, and the apical meristem becomes highly fasciated. These features reveal a role for CUP in the establishment of all above ground organ boundaries. Consistent with this function, CUP is expressed at the boundaries of all lateral organs and meristems. It is not currently known how NAC-domain genes act to establish organ boundaries. Here, we show that CUP directly interacts with a TCP-domain transcription factor. Members of the TCP-domain family have previously been shown to regulate organ outgrowth. Our results suggest a model for the establishment of organ boundaries based on the localised expression of NAC-domain and TCP-domain factors.
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The development of floral reproductive organs requires the activity of plant MADS-box transcription factors (MBFs) belonging to the C function. The C function can only operate within a floral context, specified by MBFs belonging to the SEPALLATA class of proteins. Here we describe the specific interaction between a novel protein, MIP1, and C-function and SEPALLATA (SEP)-like MBFs. MIP1 is the first member of a new class of proteins unique to plants. None of the family members have yet been assigned a function. Motif searches reveal a leucine zipper domain within a conserved N-terminal region of MIP1. The leucine zipper lies within a region sufficient for interaction with plant MBFs. MIP1 interacts with a domain of plant MBFs that is analogous to the domain of animal and yeast MBFs involved in ternary complex formation. The MIP1 protein is predicted to localise to the nucleus and activates yeast reporter genes in vivo. MIP1 is expressed in the fourth whorl of the flower, in an overlapping temporal and spatial expression pattern with the C-function and SEP-like genes. Taken together, this suggests that MIP1 acts as a ternary complex factor specifically with C-function and SEP-like MBFs.
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Plant research is moving into the post-genomic era. Proteomic-based strategies are now being developed to study functional aspects of the genes predicted from the various genome-sequencing initiatives. All biological processes depend on interactions formed between proteins and the mapping of such interactions on a global scale is providing interesting functional insights. One of the techniques that has proved itself invaluable in the mapping of protein-protein interactions is the yeast two-hybrid system. This system is a sensitive molecular genetic approach for studying protein-protein interactions in vivo. In this review we will introduce the yeast two-hybrid system, discuss modifications of the system that may be of interest to the plant science community and suggest potential applications of the technology.
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In contrast to animals, organogenesis in plants is continuous, allowing development in response to intrinsic and extrinsic signals. Organs arise from primordia formed on the flanks of meristems. The apical meristem produces primordia that acquire leaf identity, while floral meristems form primordia which develop into four organ types: sepals, petals, stamens and carpels. The production of mature organs involves two distinct processes, the initiation of organ primordia and the establishment of meristem, primordia and cell identities. Here we concentrate on floral organogenesis in Arabidopsis and examine the extent to which these processes utilize similar control mechanisms and regulatory molecules.
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Members of the MADS-box family of transcription factors are found in eukaryotes ranging from yeast to humans; In plants, MADS-box proteins regulate several developmental processes including flower, fruit and root development. We have investigated the DNA-binding mechanisms used by four such proteins in Antirrhinum majus, SQUA, PLE, DEF and GLO. SQUA differs from the characterised mammalian and yeast MADS-box proteins as it can efficiently bind two different classes of DNA-binding site. SQUA induces bending of these binding sites and the sequence of the site plays a role in determining the magnitude of these bends. Similarly, PLE and DEF/GLO induce DNA bending although the direction of the resulting bends differ. Finally we demonstrate that the MADS-box and I-domains are sufficient for homodimer formation by SQUA, However, the K-box in SQUA can also act as an oligomerisation motif and in the full-length protein, the K-box plays a different role in;mediating dimerisation in the context of SQUA homodimers or heterodimers with PLE, Together these results contribute significantly to our understanding of the function of SQUA and other plant MADS-box proteins at the molecular level.
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Floral organ identity is largely controlled by the spatially restricted expression of several MADS-box genes. In Antirrhinum majus these organ identity genes include DEF, GLO and PLE. Single and double mutant analyses indicated that the type of organ found in a particular whorl is dependent on which combination of these genes is expressed there. This paper reports the ectopic expression of Antirrhinum organ identity genes, alone and in combinations, in transgenic tobacco. Although the phenotypes are broadly in agreement with the genetic predictions, several unexpected features are observed which provide information concerning the action of the organ identity genes. The presumed tobacco homologue of DEF, NTDEF, has been isolated and used to investigate the influence of ectopic expression of the Antirrhinum organ identity genes on the endogenous tobacco genes. Analysis of the spatial and temporal expression patterns of NTDEF and NTGLO reveals that the boundaries are not coincident and that differences exist in the regulatory mechanisms of the two genes concerning both induction and maintenance of gene expression. Evidence is provided which indicates that organ development is sensitive to the relative levels of organ identity gene expression. Expression of the organ identity genes outside the flower or inflorescence produced no effects, suggesting that additional factors are required to mediate their activity. These results demonstrate that heterologous genes can be used to predictably influence floral organ identity but also reveal the existence of unsuspected control mechanisms.
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Most known floral homeotic genes belong to the MADS box family and their products act in combination to specify floral organ identity by an unknown mechanism. We have used a yeast two-hybrid system to investigate the network of interactions between the Antirrhinum organ identity gene products, Selective heterodimerization is observed between MADS box factors, Exclusive interactions are detected between two factors, DEFICIENS (DEF) and GLOBOSA (GLO), previously known to heterodimerize and control development of petals and stamens, In contrast, a third fatter, PLENA (PLE), which is required for reproductive organ development, can interact with the products of MADS box genes ex-pressed at early, intermediate and late stages. We also demonstrate that heterodimerization of DEF and GLO requires the K box, a domain not found in non-plant MADS box factors, indicating that the plant MADS box factors mag have different criteria for interaction, The association of PLENA and the temporally intermediate MADS box factors suggests that part of their function in mediating between the meristem and organ identity genes is accomplished through direct interaction, These data reveal an unexpectedly complex network of interactions between the factors controlling flower development and have implications for the determination of organ identity.
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