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Sharyn E Perry - One of the best experts on this subject based on the ideXlab platform.
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Global identification of targets of the Arabidopsis MADS Domain Protein AGAMOUS-Like15.
The Plant cell, 2009Co-Authors: Yumei Zheng, Na Ren, Huai Wang, Arnold J. Stromberg, Sharyn E PerryAbstract:AGAMOUS-Like15 (AGL15) is a MADS Domain transcriptional regulator that promotes somatic embryogenesis by binding DNA and regulating gene expression. Chromatin immunoprecipitation (ChIP) analysis previously identified DNA fragments with which AGL15 associates in vivo, and a low-throughput approach revealed a role for AGL15 in gibberellic acid catabolism that is relevant to embryogenesis. However, higher throughput methods are needed to identify targets of AGL15. Here, we mapped AGL15 in vivo binding sites using a ChIP-chip approach and the Affymetrix tiling arrays for Arabidopsis thaliana and found that ∼2000 sites represented in three biological replicates of the experiment are annotated to nearby genes. These results were combined with high-throughput measurement of gene expression in response to AGL15 accumulation to discriminate responsive direct targets from those further downstream in the network. LEAFY COTYLEDON2, FUSCA3, and ABA INSENSITIVE3, which encode B3 Domain transcription factors that are key regulators of embryogenesis, were identified and verified as direct target genes of AGL15. Genes identified as targets of the B3 genes are also targets of AGL15, and we found that INDOLEACETIC ACID-INDUCED Protein30 is involved in promotion of somatic embryo development. The data presented here and elsewhere suggest that much cross-regulation occurs in gene regulatory networks underpinning embryogenesis.
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the embryo MADS Domain Protein agamous like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism
The Plant Cell, 2004Co-Authors: Huai Wang, Leonardo V Caruso, Bruce A Downie, Sharyn E PerryAbstract:AGL15 (for AGAMOUS-Like 15) is a member of the MADS Domain family of DNA binding transcriptional regulators that accumulates to its highest amounts during embryo development. To better understand how AGL15 functions, a chromatin immunoprecipitation approach was used to identify directly regulated genes. One DNA fragment that coprecipitated with AGL15 corresponded to a portion of the regulatory region of a gene named DTA1 (for Downstream Target of AGL15-1). The expression of DTA1 was positively correlated with AGL15 abundance during embryogenesis. In this report, a cis element for response to AGL15 was identified, and the activity of DTA1 as a gibberellin (GA) 2-oxidase was confirmed. DTA1 corresponds to AtGA2ox6 and was renamed to indicate this identity. Further experiments related the function of AtGA2ox6 to regulation by AGL15. Constitutive expression of AGL15 and of AtGA2ox6 altered endogenous GA amounts and caused GA-deficient phenotypes in Arabidopsis thaliana that could be at least partially rescued by application of biologically active GA. The phenotype of plants with decreased expression of AtGA2ox6 was the converse of plants overexpressing AtGA2ox6 in terms of seed germination attributes and effects on somatic embryo production.
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binding site selection for the plant MADS Domain Protein agl15 an in vitro and in vivo study
Journal of Biological Chemistry, 2003Co-Authors: Weining Tang, Sharyn E PerryAbstract:AGL15 (for AGAMOUS-like 15) is currently the only reported member of the plant MADS Domain family of transcriptional regulators that preferentially accumulates during embryo development. Additionally, AGL15 is one of the more divergent members of the MADS Domain family, including within the DNA-binding Domain. Previous studies have shown that MADS Domain Proteins bind to DNA sequences with an overall consensus of CC(A/T)6GG (called a CArG motif). Nonetheless, different MADS Domain Proteins exhibit similar yet distinct binding site preferences that may be critical for differential gene regulation. To determine the consensus sequence preferentially bound by AGL15 in vitro, PCR-assisted binding site selection assays were performed. AGL15 was observed to prefer a CArG motif with a longer A/T-rich core and is to date the only plant MADS Domain Protein having such a preference. Next, the Arabidopsis genome data base was searched for genes containing AGL15 binding sites as candidates for direct regulation by AGL15. One gene, DTA4 (for Downstream Target of AGL15-4), was identified by this method, and then confirmed as a direct target of AGL15 in vivo.
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a chromatin immunoprecipitation chip approach to isolate genes regulated by agl15 a MADS Domain Protein that preferentially accumulates in embryos
Plant Journal, 2002Co-Authors: Huai Wang, Weining Tang, Cong Zhu, Sharyn E PerryAbstract:AGAMOUS-like-15 (AGL15) is a member of the MADS-Domain family of DNA-binding regulatory factors that accumulates preferentially in tissue developing in an embryonic mode. To better understand how AGL15 functions, we developed a chromatin immunoprecipitation (ChIP) approach to isolate genes regulated directly by AGL15. ChIP allows purification of in vivo Protein-DNA complexes. The co-purified DNA is recovered and used to isolate the putatively regulated gene. Several tests must be performed to show that the putative downstream target gene is truly regulated by the DNA-binding Protein. The DNA-binding regulatory Protein must interact with cis regulatory elements. The downstream gene expression pattern should respond to the level of the trans-acting regulatory factor. The cis element should be able to confer regulation in response to the trans-acting factor. We describe, in this report, our ChIP protocol, and discuss in detail, tests to confirm regulation by AGL15 for two targets identified by ChIP. These targets are referred to as Downstream Target of AGL15 (DTA1 and DTA2). Expression of DTA1, which encodes a Protein with high similarity to GA-2 oxidase-like Proteins, is induced by AGL15. DTA2 encodes a novel Protein and expression of this target is repressed by AGL15.
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the MADS Domain Protein agamous like 15 accumulates in embryonic tissues with diverse origins
Plant Physiology, 1999Co-Authors: Sharyn E Perry, Melissa D Lehti, Donna E FernandezAbstract:AGL15 (AGAMOUS-like 15), a member of the MADS-Domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 Protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.
Gerco C Angenent - One of the best experts on this subject based on the ideXlab platform.
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Comparative analysis of binding patterns of MADS-Domain Proteins in Arabidopsis thaliana
'Springer Science and Business Media LLC', 2018Co-Authors: Niels Aerts, Gerco C Angenent, Hilda Van Mourik, Suzanne De Bruijn, Aalt D J Van DijkAbstract:Abstract Background Correct flower formation requires highly specific temporal and spatial regulation of gene expression. In Arabidopsis thaliana the majority of the master regulators that determine flower organ identity belong to the MADS-Domain transcription factor family. The canonical DNA binding motif for this transcription factor family is the CArG-box, which has the consensus CC(A/T)6GG. However, so far, a comprehensive analysis of MADS-Domain binding patterns has not yet been performed. Results Eight publicly available ChIP-seq datasets of MADS-Domain Proteins that regulate the floral transition and flower formation were analyzed. Surprisingly, the preferred DNA binding motif of each Protein was a CArG-box with an NAA extension. Furthermore, motifs of other transcription factors were found in the vicinity of binding sites of MADS-Domain transcription factors, suggesting that interaction of MADS-Domain Proteins with other transcription factors is important for target gene regulation. Finally, conservation of CArG-boxes between Arabidopsis ecotypes was assessed to obtain information about their evolutionary importance. CArG-boxes that fully matched the consensus were more conserved than other CArG-boxes, suggesting that the perfect CArG-box is evolutionary more important than other CArG-box variants. Conclusion Our analysis provides detailed insight into MADS-Domain Protein binding patterns. The results underline the importance of an extended version of the CArG-box and provide a first view on evolutionary conservation of MADS-Domain Protein binding sites in Arabidopsis ecotypes
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differences in dna binding specificity of floral homeotic Protein complexes predict organ specific target genes
The Plant Cell, 2017Co-Authors: Cezary Smaczniak, Gerco C Angenent, Jose M Muiño, Dijun Chen, Kerstin KaufmannAbstract:Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS Domain Proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic Protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS Domain Protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily Protein interactions affect DNA binding specificity of floral MADS Domain Proteins. Differential DNA binding of MADS Domain Protein complexes plays a role in the specificity of target gene regulation.
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the abc of MADS Domain Protein behaviour and interactions
Seminars in Cell & Developmental Biology, 2010Co-Authors: Richard G H Immink, Kerstin Kaufmann, Gerco C AngenentAbstract:Development of eudicot flowers is under tight developmental control by genes belonging to the MADS box transcription factor family, as is nicely represented by the well-known ABC model of floral organ development. During the last two decades enormous progress has been made in our understanding of the molecular mechanisms underlying the combinatorial activity of the encoded MADS Domain Proteins. Here, we review how various state-of-the-art technologies were implemented in order to unravel the Protein-Protein interaction network for the plant MADS Domain transcription factor family. In addition, results from in planta studies of MADS Domain Protein behaviour and interactions will be discussed. Dimerisation and higher-order complex formation of MADS Domain Proteins appear to be instrumental and essential for floral organ identity determination and the precise regulation of specific target gene sets. According to the current molecular model, the floral MADS Proteins assemble into quaternary complexes consisting of two dimers, which is mediated by the E class Proteins. Furthermore, evidence has been provided that MADS Protein-Protein interactions specify DNA binding capacity, inter- and intracellular localisations of the Proteins and the biological function of the constituted transcription complexes.
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in planta localisation patterns of MADS Domain Proteins during floral development in arabidopsis thaliana
BMC Plant Biology, 2009Co-Authors: Susan L Urbanus, Richard G H Immink, Kerstin Kaufmann, Stefan De Folter, A V Shchennikova, Gerco C AngenentAbstract:MADS Domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS Domain Proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the Proteins. We have made GREEN FLUORESCENT Protein (GFP) translational fusions with the four MADS Domain Proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the Protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). We unravelled the Protein localisation patterns of the four MADS Domain Proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The Protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeoDomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 Proteins, supporting the hypothesis that both Proteins together suppress WUSCHEL expression in the ovule. This approach provides a highly detailed in situ map of MADS Domain Protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during development, points to mechanisms other than transcriptional control. Together this information is essential to understand the role of these Proteins in the regulatory processes that drive floral development and leads to new hypotheses.
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the MADS Domain Protein diana acts together with agamous like80 to specify the central cell in arabidopsis ovules
The Plant Cell, 2008Co-Authors: Marian Bemer, Mieke Woltersarts, Ueli Grossniklaus, Gerco C AngenentAbstract:MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent Protein-beta-glucoronidase fusion Protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.
Donna E Fernandez - One of the best experts on this subject based on the ideXlab platform.
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the MADS Domain Protein agamous like 15 accumulates in embryonic tissues with diverse origins
Plant Physiology, 1999Co-Authors: Sharyn E Perry, Melissa D Lehti, Donna E FernandezAbstract:AGL15 (AGAMOUS-like 15), a member of the MADS-Domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 Protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.
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the MADS Domain Protein agl15 localizes to the nucleus during early stages of seed development
The Plant Cell, 1996Co-Authors: Sharyn E Perry, Karl W Nichols, Donna E FernandezAbstract:Little is known about regulatory factors that act during the earliest stages of plant embryogenesis. The MADS Domain Protein AGL15 (for AGAMOUS-like) is expressed preferentially during embryogenesis and accumulates during early seed development in monocotyledonous and dicotyledonous flowering plants. AGL15-specific antibodies and immunohistochemistry were used to demonstrate that AGL15 accumulates before fertilization in the cytoplasm in the cells of the egg apparatus and moves into the nucleus during early stages of development in the suspensor, embryo, and endosperms. Relatively high levels of AGL15 are present in the nuclei during embryo morphogenesis and until the seeds start to dry in Brassica, maize, and Arabidopsis. AGL15 is associated with the chromosomes during mitosis, and gel mobility shift assays were used to demonstrate that AGL15 binds DNA in a sequence-specific manner. To assess whether AGL15 is likely to play a role in specifying the seed or embryonic phase of development, AGL15 accumulation was examined in Arabidopsis mutants that prematurely exit embryogenesis. lec1-2 mutants show an embryo-specific loss of AGL15 at the transition stage, suggesting that AGL15 interacts with regulators in the leafy cotyledons pathway.
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agl15 a MADS Domain Protein expressed in developing embryos
The Plant Cell, 1995Co-Authors: Gregory R Heck, Sharyn E Perry, Karl W Nichols, Donna E FernandezAbstract:To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS Domain family of regulatory Proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS Domain were used to probe immunoblots, and AGL15-related Proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo.
Heinz Saedler - One of the best experts on this subject based on the ideXlab platform.
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the MADS Domain Protein mpf1 of physalis floridana controls plant architecture seed development and flowering time
Planta, 2010Co-Authors: Ying Tian, Rainer Saedler, Nadia Efremova, Simone Riss, Muhammad Ramzan Khan, Alexander Yephremov, Heinz SaedlerAbstract:Floral and vegetative development of plants is dependent on the combinatorial action of MADS-Domain transcription factors. Members of the STMADS11 subclade, such as MPF1 of Physalis, are abundantly expressed in leaves as well as in floral organs, but their function is not yet clear. Our studies with transgenic Arabidopsis that over-express MPF1 suggest that MPF1 interacts with SOC1 to determine flowering time. However, MPF1 RNAi-mediated knockdown Physalis plants revealed a complex phenotype with changes in flowering time, plant architecture and seed size. Flowering of these plants was delayed by about 20% as compared to wild type. Expression of PFLFY is upregulated in the MPF1 RNAi lines, while PFFT and MPF3 genes are strongly repressed. MPF1 interacts with a subset of MADS-Domain factors, namely with PFSOC1 in planta, and with PFSEP3 and PFFUL in yeast, supporting a regulatory role for this Protein in flowering. The average size of seeds produced by the transgenic MPF1 RNAi plants is increased almost twofold. The height of these plants is also increased about twofold, but most axillary buds are stunted when compared to controls. Taken together, this suggests that members of the STMADS11 subclade act as positive regulators of flowering but have diverse functions in plant growth.
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the MADS Domain Protein ppm2 preferentially occurs in gametangia and sporophytes of the moss physcomitrella patens
Gene, 2007Co-Authors: Vanessa Quodt, Heinz Saedler, Wolfram Faigl, Thomas MunsterAbstract:To date, the function of MADS-Domain transcription factors in non-seed plants remains largely elusive, although a number of genes have been isolated and characterized from a variety of species. In our study we analyzed PPM2, a classical MIKC-type MADS-box gene from the moss Physcomitrella patens, taking advantage of the unique technical properties Physcomitrella offers in terms of efficient homologous recombination. We determined mRNA and Protein distribution and performed targeted disruption of the genomic locus for functional analysis of PPM2. Despite weak ubiquitous expression, PPM2 Protein is mostly found in male and female gametangia and basal parts of developing sporophytes. Therefore, PPM2 seems to function in both the haploid and the diploid phase of the moss life cycle. This situation reflects an evolutionary transition state of gene recruitment from an ancestral gametophytic generation into a derived sporophytic generation which became dominating in tracheophytes. However, a knock-out of the PPM2 gene did not cause visible phenotypical changes in the respective structures. The implications of our findings for the understanding of the evolutionary history of MADS-box transcription factors in plants are discussed.
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pfmago a mago nashi like factor interacts with the MADS Domain Protein mpf2 from physalis floridana
Molecular Biology and Evolution, 2007Co-Authors: Hans Sommer, Britta Grosardt, Peter Huijser, Heinz SaedlerAbstract:MADS-Domain Proteins serve as regulators of plant development and often form dimers and higher order complexes to function. Heterotopic expression of MPF2, a MADS-box gene, in reproductive tissues is a key component in the evolution of the inflated calyx syndrome in Physalis, but RNAi studies demonstrate that MPF2 has also acquired a role in male fertility in Physalis floridana. Using the yeast 2-hybrid system, we have now identified numerous MPF2-interacting MADS-Domain Proteins from Physalis, including homologs of SOC1, AP1, SEP1, SEP3, AG, and AGL6. Among the many non-MADS-Domain Proteins recovered was a homolog of MAGO NASHI, a highly conserved RNA-binding Protein known to be involved in many developmental processes including germ cell differentiation. Two MAGO genes, termed P. floridana mago nashi1 (PFMAGO1) and PFMAGO2, were isolated from P. floridana. Both copies were found to be coexpressed in leaves, fruits, and, albeit at lower level, also in roots, stems, and flowers. DNA sequence analysis revealed that, although the coding sequences of the 2 genes are highly conserved, they differ substantially in their intron and promoter sequences. Two-hybrid screening of a Physalis expression library with both PFMAGO1 and PFMAGO2 as baits yielded numerous gene products, including an Y14-like Protein. Y14 is an RNA-binding Protein that forms part of various "gene expression machines." The function of MPF2 and 2 PFMAGO Proteins in ensuring male fertility and evolution of calyx development in Physalis is discussed.
Richard G H Immink - One of the best experts on this subject based on the ideXlab platform.
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Characterization of MADS-Domain transcription factor complexes in Arabidopsis flower development.
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Cezary Smaczniak, Richard G H Immink, Jose M Muiño, Robert Blanvillain, Marco Busscher, Jacqueline Busscher-lange, Q D Peter Dinh, Shujing Liu, Adrie H Westphal, Sjef BoerenAbstract:Floral organs are specified by the combinatorial action of MADS-Domain transcription factors, yet the mechanisms by which MADS-Domain Proteins activate or repress the expression of their target genes and the nature of their cofactors are still largely unknown. Here, we show using affinity purification and mass spectrometry that five major floral homeotic MADS-Domain Proteins (AP1, AP3, PI, AG, and SEP3) interact in floral tissues as proposed in the "floral quartet" model. In vitro studies confirmed a flexible composition of MADS-Domain Protein complexes depending on relative Protein concentrations and DNA sequence. In situ bimolecular fluorescent complementation assays demonstrate that MADS-Domain Proteins interact during meristematic stages of flower development. By applying a targeted proteomics approach we were able to establish a MADS-Domain Protein interactome that strongly supports a mechanistic link between MADS-Domain Proteins and chromatin remodeling factors. Furthermore, members of other transcription factor families were identified as interaction partners of floral MADS-Domain Proteins suggesting various specific combinatorial modes of action.
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predicting the impact of alternative splicing on plant MADS Domain Protein function
PLOS ONE, 2012Co-Authors: Edouard Severing, Richard G H Immink, Aalt D J Van Dijk, Giuseppa Morabito, Jacqueline Busscherlange, Roeland C H J Van HamAbstract:Several genome-wide studies demonstrated that alternative splicing (AS) significantly increases the transcriptome complexity in plants. However, the impact of AS on the functional diversity of Proteins is difficult to assess using genome-wide approaches. The availability of detailed sequence annotations for specific genes and gene families allows for a more detailed assessment of the potential effect of AS on their function. One example is the plant MADS-Domain transcription factor family, members of which interact to form Protein complexes that function in transcription regulation. Here, we perform an in silico analysis of the potential impact of AS on the Protein-Protein interaction capabilities of MIKC-type MADS-Domain Proteins. We first confirmed the expression of transcript isoforms resulting from predicted AS events. Expressed transcript isoforms were considered functional if they were likely to be translated and if their corresponding AS events either had an effect on predicted dimerisation motifs or occurred in regions known to be involved in multimeric complex formation, or otherwise, if their effect was conserved in different species. Nine out of twelve MIKC MADS-box genes predicted to produce multiple Protein isoforms harbored putative functional AS events according to those criteria. AS events with conserved effects were only found at the borders of or within the K-box Domain. We illustrate how AS can contribute to the evolution of interaction networks through an example of selective inclusion of a recently evolved interaction motif in the MADS AFFECTING FLOWERING1-3 (MAF1–3) subclade. Furthermore, we demonstrate the potential effect of an AS event in SHORT VEGETATIVE PHASE (SVP), resulting in the deletion of a short sequence stretch including a predicted interaction motif, by overexpression of the fully spliced and the alternatively spliced SVP transcripts. For most of the AS events we were able to formulate hypotheses about the potential impact on the interaction capabilities of the encoded MIKC Proteins.
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the abc of MADS Domain Protein behaviour and interactions
Seminars in Cell & Developmental Biology, 2010Co-Authors: Richard G H Immink, Kerstin Kaufmann, Gerco C AngenentAbstract:Development of eudicot flowers is under tight developmental control by genes belonging to the MADS box transcription factor family, as is nicely represented by the well-known ABC model of floral organ development. During the last two decades enormous progress has been made in our understanding of the molecular mechanisms underlying the combinatorial activity of the encoded MADS Domain Proteins. Here, we review how various state-of-the-art technologies were implemented in order to unravel the Protein-Protein interaction network for the plant MADS Domain transcription factor family. In addition, results from in planta studies of MADS Domain Protein behaviour and interactions will be discussed. Dimerisation and higher-order complex formation of MADS Domain Proteins appear to be instrumental and essential for floral organ identity determination and the precise regulation of specific target gene sets. According to the current molecular model, the floral MADS Proteins assemble into quaternary complexes consisting of two dimers, which is mediated by the E class Proteins. Furthermore, evidence has been provided that MADS Protein-Protein interactions specify DNA binding capacity, inter- and intracellular localisations of the Proteins and the biological function of the constituted transcription complexes.
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in planta localisation patterns of MADS Domain Proteins during floral development in arabidopsis thaliana
BMC Plant Biology, 2009Co-Authors: Susan L Urbanus, Richard G H Immink, Kerstin Kaufmann, Stefan De Folter, A V Shchennikova, Gerco C AngenentAbstract:MADS Domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS Domain Proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the Proteins. We have made GREEN FLUORESCENT Protein (GFP) translational fusions with the four MADS Domain Proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the Protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). We unravelled the Protein localisation patterns of the four MADS Domain Proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The Protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeoDomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 Proteins, supporting the hypothesis that both Proteins together suppress WUSCHEL expression in the ovule. This approach provides a highly detailed in situ map of MADS Domain Protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during development, points to mechanisms other than transcriptional control. Together this information is essential to understand the role of these Proteins in the regulatory processes that drive floral development and leads to new hypotheses.
