Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Differences in DNA binding specificity of floral homeotic protein complexes predict organ-specific target genes
Smaczniak, Cezary ; Muiño, Jose M. ; Chen, Dijun ; Angenent, Gerco C. ; Kaufmann, Kerstin - \ 2017
The Plant Cell 29 (2017)8. - ISSN 1040-4651 - p. 1822 - 1835.

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.

SELEX-seq : A method to determine DNA binding specificities of plant transcription factors
Smaczniak, Cezary ; Angenent, Gerco C. ; Kaufmann, Kerstin - \ 2017
In: Plant gene regulatory networks / Kaufmann, Kerstin, Mueller-Roeber, Bernd, Humana Press Inc. (Methods in Molecular Biology - Springer Protocols ) - ISBN 9781493971244 - p. 67 - 82.
DNA binding specificity - MADS-domain proteins - Protein-DNA interactions - SELEX, EMSA - Transcription factors

Systematic evolution of ligands by exponential enrichment (SELEX) is a method that allows isolating specific nucleotide sequences that interact with a DNA binding protein of choice. By using a transcription factor (TF) and a randomized pool of double-stranded DNA, this technique can be used to characterize TF DNA binding specificities and affinities. The method is based on protein-DNA complex immunoprecipitation with protein-specific antibodies and subsequent DNA selection and amplification. Application of massively parallel sequencing (-seq) at each cycle of SELEX allows determining the relative affinities to any DNA sequence for any transcription factor or TF complex. The resulting TF DNA binding motifs can be used to predict potential DNA binding sites in genomes and thereby direct target genes of TFs.

Post-transcriptional control of GRF transcription factors by microRNA miR396 and GIF co-activator affects leaf size and longevity
Debernardi, J.M. ; Mecchia, M.A. ; Vercruyssen, L. ; Smaczniak, C. ; Kaufmann, K. ; Inze, D. ; Rodriguez, R.E. ; Palatnik, J.F. - \ 2014
The Plant Journal 79 (2014)3. - ISSN 0960-7412 - p. 413 - 426.
chromatin-remodeling complexes - tandem affinity purification - arabidopsis-thaliana - gene-expression - organ size - cell-division - protein complexes - shoot development - small rnas - growth
The growth-regulating factors (GRFs) are plant-specific transcription factors. They form complexes with GRF-interacting factors (GIFs), a small family of transcriptional co-activators. In Arabidopsis thaliana, seven out of the nine GRFs are controlled by microRNA miR396. Analysis of Arabidopsis plants carrying a GRF3 allele insensitive to miR396 revealed a strong boost in the number of cells in leaves, which was further enhanced synergistically by an additional increase of GIF1 levels. Genetic experiments revealed that GRF3 can still increase cell number in gif1 mutants, albeit to a much lesser extent. Genome-wide transcript profiling indicated that the simultaneous increase of GRF3 and GIF1 levels causes additional effects in gene expression compared to either of the transgenes alone. We observed that GIF1 interacts in vivo with GRF3, as well as with chromatin-remodeling complexes, providing a mechanistic explanation for the synergistic activities of a GRF3-GIF1 complex. Interestingly, we found that, in addition to the leaf size, the GRF system also affects the organ longevity. Genetic and molecular analysis revealed that the functions of GRFs in leaf growth and senescence can be uncoupled, demonstrating that the miR396-GRF-GIF network impinges on different stages of leaf development. Our results integrate the post-transcriptional control of the GRF transcription factors with the progression of leaf development.
Structural determinants of DNA recognition by plant MADS-domain transcription factors
Muino Acuna, J.M. ; Smaczniak, C. ; Angenent, G.C. ; Kaufmann, K. ; Dijk, A.D.J. van - \ 2014
Nucleic acids research 42 (2014)4. - ISSN 0305-1048 - p. 2138 - 2146.
in-vitro - binding properties - flower development - antirrhinum-majus - homeotic proteins - crystal-structure - ternary complex - floral organs - box proteins - chip-chip
Plant MADS-domain transcription factors act as key regulators of many developmental processes. Despite the wealth of information that exists about these factors, the mechanisms by which they recognize their cognate DNA-binding site, called CArG-box (consensus CCW6GG), and how different MADS-domain proteins achieve DNA-binding specificity, are still largely unknown. We used information from in vivo ChIP-seq experiments, in vitro DNA-binding data and evolutionary conservation to address these important questions. We found that structural characteristics of the DNA play an important role in the DNA binding of plant MADS-domain proteins. The central region of the CArG-box largely resembles a structural motif called ‘A-tract’, which is characterized by a narrow minor groove and may assist bending of the DNA by MADS-domain proteins. Periodically spaced A-tracts outside the CArG-box suggest additional roles for this structure in the process of DNA binding of these transcription factors. Structural characteristics of the CArG-box not only play an important role in DNA-binding site recognition of MADS-domain proteins, but also partly explain differences in DNA-binding specificity of different members of this transcription factor family and their heteromeric complexes.
Naturally occurring allele diversity allows potato cultivation in northern latitudes
Kloosterman, B.A. ; Abelenda, J.A. ; Carretero Gomez, M. ; Oortwijn, M.E.P. ; Boer, J.M. de; Kowitwanich, K. ; Horvath, B.M. ; Eck, H.J. van; Smaczniak, C. ; Prat, S. ; Visser, R.G.F. ; Bachem, C.W.B. - \ 2013
Nature 495 (2013)7440. - ISSN 0028-0836 - p. 246 - 250.
flowering-time - arabidopsis - constans - tuberization - gene - plants - fkf1
Potato (Solanum tuberosum L.) originates from the Andes and evolved short-day-dependent tuber formation as a vegetative propagation strategy. Here we describe the identification of a central regulator underlying a major-effect quantitative trait locus for plant maturity and initiation of tuber development. We show that this gene belongs to the family of DOF (DNA-binding with one finger) transcription factors1 and regulates tuberization and plant life cycle length, by acting as a mediator between the circadian clock and the StSP6A mobile tuberization signal2. We also show that natural allelic variants evade post-translational light regulation, allowing cultivation outside the geographical centre of origin of potato. Potato is a member of the Solanaceae family and isone of the world’s most important food crops. This annual plant originates from the Andean regions of South America3. Potato develops tubers from underground stems called stolons. Its equatorial origin makes potato essentially short-day dependent for tuberization and potato will not make tubers in the long-day conditions of spring and summer in the northern latitudes. When introduced in temperate zones, wild material will form tubers in the course of the autumnal shortening of day-length. Thus, one of the first selected traits in potato leading to a European potato type4 is likely to have been long-day acclimation for tuberization. Potato breeders can exploit the naturally occurring variation in tuberization onset and life cycle length, allowing varietal breeding for different latitudes, harvest times and markets.
MADS interactomics : towards understanding the molecular mechanisms of plant MADS-domain transcription factor function
Smaczniak, C.D. - \ 2013
Wageningen University. Promotor(en): Gerco Angenent, co-promotor(en): Kerstin Kaufmann. - S.l. : s.n. - ISBN 9789461734525 - 178
planten - moleculaire biologie - transcriptie - transcriptiefactoren - arabidopsis - mads-box eiwitten - regulatoren - dna-bindende eiwitten - plants - molecular biology - transcription - transcription factors - mads-box proteins - regulators - dna binding proteins

Protein-protein and protein-DNA interactions are essential for the molecular action of transcription factors. By combinatorial binding to target gene promoters, transcription factors are able to up- or down-regulate the expression of these genes. MADS-domain proteins comprise a large family of transcription factors present in all eukaryotes. In plants, and especially in seed plants, this family has significantly expanded. For example, more than 100 representatives are found in the Arabidopsis genome. MADS-box genes have initially been shown to play major roles in flower development, however their emerging functional characterization revealed functions in almost all developmental processes throughout the plant life cycle. How MADS-domain transcription factors acquire their functional specificity remains unresolved. The goal of this thesis was to characterize some of the molecular mechanisms by which MADS-domain proteins act in Arabidopsis.

Chapter 1 comprehensively reviews functions of MADS-domain transcription factors in flowering plants, with a main focus on Arabidopsis. Major classes of MADS-domain proteins are introduced, and their modular structures are described. Additionally, it is shown that several distinctive subfamilies of MADS-box genes can be inferred from the phylogenetic analysis of the whole gene family. By compiling recent studies on MADS-domain protein-protein and protein-DNA interactions, we present a hypothetical model of MADS-domain protein action that combines higher-order protein complex formation and active chromatin remodeling by large transcriptional machineries.

Chapter 2 describes MADS-domain protein complexes that are potentially formed during Arabidopsis flower development. By using a targeted proteomics approach we were able to characterize the protein interactome of major floral homeotic MADS-domain proteins (APETALA1, APETALA3, PISTILLATA, AGAMOUS, SEPALLATA3 and FRUITFULL) in native plant tissues, confirming interactions suggested in the ‘floral quartet’ model. Additionally, we discovered transcription factors from other families and chromatin-associated proteins as possible interaction partners of MADS-domain proteins. These interactions shed light on the combinatorial modes of action of MADS-domain transcription factors and suggest that they can act by recruiting or redirecting the chromatin remodeling machinery to control the expression of their target genes.

In Chapter 3 we review recent advances in proteomics approaches used to study cellular signaling and developmental processes in plants. We mention the emerging tools for of whole plant proteome characterization as well as sub-cellular protein localization. The major focus, though, is on the description of complete cellular signaling cascades in plants, starting from the characterization of signaling mobile molecules (e.g. peptide or protein), through identification of receptors and receptor protein complexes, ending with identification of intermediate signaling pathway members. Two examples of biochemical procedures used to identify complexes of membrane-bound receptors and transcriptional regulators from nuclei are described in Chapter 4. In our optimized method we make use of fluorophore-tagged single step affinity purification of protein complexes and label-free mass spectrometry-based protein quantification to distinguish true complex partners from non-specifically precipitated proteins.

The exact molecular mechanisms of DNA sequence recognition by MADS-domain transcription factors are still unknown. Particularly intriguing is the question whether various MADS-domain protein complexes possess different DNA-binding specificities. We address this question in Chapter 5. We used systematic evolution of ligands by exponential enrichment (SELEX) followed by high-throughput sequencing (seq) approach to discriminate DNA-binding specificities of several MADS-domain protein homo- and heterodimers.

Finally, in Chapter 6, we aimed to identify the molecular features of different DNA-binding specificities of MADS-domain transcription factors.With help of bioinformatics tools and in vitro DNA-binding assays we found that structural characteristics of the DNA play an important role in DNA-binding of MADS-domain proteins.

Taken together, research described in this thesis advances our knowledge on the molecular mechanisms of MADS-domain transcription factor action in plants. Chapter 7 concludes the thesis and describes future perspectives in MADS-domain protein research. Highlighted are the advances of high-throughput (proteomics and genomics) technologies that could be used to unravel not only the static characteristics of transcriptional regulation but also the dynamic and stoichiometric changes of complex protein and gene regulatory networks during plant development.

Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues.
Smaczniak, C. ; Li, N. ; Boeren, J.A. ; America, A.H.P. ; Dongen, W.M.A.M. van; Goerdayal, S.S. ; Vries, S.C. de; Angenent, G.C. ; Kaufmann, K. - \ 2012
Nature protocols 7 (2012). - ISSN 1754-2189 - p. 2144 - 2158.
arabidopsis-thaliana - mass-spectrometry - quantitative proteomics - gene-expression - bac transgeneomics - quantification - strategy - reveals - cells
Owing to the low abundance of signaling proteins and transcription factors, their protein complexes are not easily identified by classical proteomics. The isolation of these protein complexes from endogenous plant tissues (rather than plant cell cultures) is therefore an important technical challenge. Here, we describe a sensitive, quantitative proteomics-based procedure to determine the composition of plant protein complexes. The method makes use of fluorophore-tagged protein immunoprecipitation (IP) and label-free mass spectrometry (MS)-based quantification to correct for nonspecifically precipitated proteins. We provide procedures for the isolation of membrane-bound receptor complexes and transcriptional regulators from nuclei. The protocol consists of an IP step (~6 h) and sample preparation for liquid chromatography-tandem MS (LC-MS/MS; 2 d). We also provide a guide for data analysis. Our single-step affinity purification protocol is a good alternative to two-step tandem affinity purification (TAP), as it is shorter and relatively easy to perform. The data analysis by label-free quantification (LFQ) requires a cheaper and less challenging experimental setup compared with known labeling techniques in plants.
Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies
Smaczniak, C. ; Immink, R.G.H. ; Angenent, G.C. ; Kaufmann, K. - \ 2012
Development 139 (2012). - ISSN 0950-1991 - p. 3081 - 3098.
floral organ identity - box gene family - flowering-locus-c - genome-wide analysis - arabidopsis fruit-development - protein-protein interactions - reproductive meristem fates - abscission zone development - transcription factor family - homeotic genes
Members of the MADS-box transcription factor family play essential roles in almost every developmental process in plants. Many MADS-box genes have conserved functions across the flowering plants, but some have acquired novel functions in specific species during evolution. The analyses of MADS-domain protein interactions and target genes have provided new insights into their molecular functions. Here, we review recent findings on MADS-box gene functions in Arabidopsis and discuss the evolutionary history and functional diversification of this gene family in plants. We also discuss possible mechanisms of action of MADS-domain proteins based on their interactions with chromatin-associated factors and other transcriptional regulators.
Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development
Smaczniak, C. ; Immink, G.H. ; Muiño, J.M. ; Blanvillain, R. ; Busscher, M. ; Busscher-Lange, J. ; Dinh, Q.D. ; Liu, S. ; Westphal, A.H. ; Boeren, S. ; Parcy, F. ; Xu, L. ; Carles, C. ; Angenent, G.C. ; Kaufmann, K. - \ 2012
Proceedings of the National Academy of Sciences of the United States of America 109 (2012)5. - ISSN 0027-8424 - p. 1560 - 1565.
meristem identity - chromatin immunoprecipitation - reproductive development - homeodomain proteins - floral quartets - pound-foolish - dna-binding - time genes - in-vitro - repression
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
Strigolactone Biosynthesis in Medicago truncatula and Rice Requires the Symbiotic GRAS-Type Transcription Factors NSP1 and NSP2
Liu, W. ; Kohlen, W. ; Lillo, A. ; Camp, R. op den; Ivanov, S. ; Hartog, M. ; Limpens, E.H.M. ; Jamil, M. ; Smaczniak, C. ; Kaufmann, K. ; Yang, W.C. ; Hooiveld, G.J.E.J. ; Charnikhova, T. ; Bouwmeester, H.J. ; Bisseling, T. ; Geurts, R. - \ 2011
The Plant Cell 23 (2011)10. - ISSN 1040-4651 - p. 3853 - 3865.
arbuscular mycorrhizal fungi - affymetrix genechip data - tiller bud outgrowth - quality assessment - lotus-japonicus - germination stimulants - transduction pathway - phosphate deficiency - parasitic plants - red-clover
Legume GRAS (GAI, RGA, SCR)-type transcription factors NODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression after symbiotic signaling. However, legume NSP1 and NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved in higher plants. Here, we show that NSP1 and NSP2 are indispensable for strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice. Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is essential for conversion of orobanchol into didehydro-orobanchol, which is the main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds correlates with reduced expression of DWARF27, a gene essential for SL biosynthesis. Rice and M. truncatula represent distinct phylogenetic lineages that split approximately 150 million years ago. Therefore, we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. NSP1 and NSP2 are single-copy genes in legumes, which implies that both proteins fulfill dual regulatory functions to control downstream targets after rhizobium-induced signaling as well as SL biosynthesis in nonsymbiotic conditions.
Proteomics insights into plant signaling and development.
Kaufmann, K. ; Smaczniak, C.D. ; Vries, S.C. de; Angenent, G.C. ; Karlova, R.B. - \ 2011
Proteomics 11 (2011)4. - ISSN 1615-9853 - p. 744 - 755.
tandem affinity purification - difference gel-electrophoresis - protein identification technology - arabidopsis leaf peroxisomes - cell-wall proteome - mass-spectrometry - quantitative proteomics - phosphoproteomic analysis - ubiquitinated proteins - metabolic pat
Mass spectrometry-based proteomics is used to gain insight into the abundance and subcellular localization of cellular signaling components, the composition of molecular complexes and the regulation of signaling pathways. Multicellular organisms have evolved signaling networks and fast responses to stimuli that can be discovered and monitored by the use of advanced proteomics techniques in combination with traditional functional analysis. Plants are multicellular organisms and products of tightly regulated developmental programmes that respond to environmental conditions and internal cues. Plant development is orchestrated by inter- and intracellular signaling molecules, receptors and transcriptional regulators, which act in a temporal and spatially coordinated manner. Here we review recent advances in proteomics applications used to understand complex cellular signaling processes in plants.
Arabidopsis thaliana transcription factors bZIP19 and bZIP23 regulate the adaptation to zinc deficiency
Assuncao, A.G.L. ; Herrero, E. ; Lin, Y.F. ; Huettel, B. ; Talukdar, S. ; Smaczniak, C.D. ; Immink, R.G.H. ; Eldik, M. van; Fiers, M. ; Schat, H. ; Aarts, M.G.M. - \ 2010
Proceedings of the National Academy of Sciences of the United States of America 107 (2010). - ISSN 0027-8424 - p. 10296 - 10301.
iron-deficiency - metal homeostasis - dna recognition - binding-factors - genes - transporter - protein - expression - family - mechanisms
Zinc is an essential micronutrient for all living organisms. When facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. The molecular regulators controlling this adaptation are not known. We present the identification of two closely related members of the Arabidopsis thaliana basic-region leucine-zipper (bZIP) transcription factor gene family, bZIP19 and bZIP23, that regulate the adaptation to low zinc supply. They were identified, in a yeast-one-hybrid screening, to associate to promoter regions of the zinc deficiency-induced ZIP4 gene of the Zrt- and Irt-related protein (ZIP) family of metal transporters. Although mutation of only one of the bZIP genes hardly affects plants, we show that the bzip19 bzip23 double mutant is hypersensitive to zinc deficiency. Unlike the wild type, the bzip19 bzip23 mutant is unable to induce the expression of a small set of genes that constitutes the primary response to zinc deficiency, comprising additional ZIP metal transporter genes. This set of target genes is characterized by the presence of one or more copies of a 10-bp imperfect palindrome in their promoter region, to which both bZIP proteins can bind. The bZIP19 and bZIP23 transcription factors, their target genes, and the characteristic cis zinc deficiency response elements they can bind to are conserved in higher plants. These findings are a significant step forward to unravel the molecular mechanism of zinc homeostasis in plants, allowing the improvement of zinc bio-fortification to alleviate human nutrition problems and phytoremediation strategies to clean contaminated soils
Target genes of the MADS transcription factor SEPALLATA3, ChIP-chip
Kaufmann, K. ; Muiño, J.M. ; Jauregui, R. ; Airoldi, C.A. ; Smaczniak, C. ; Krajewski, P. ; Angenent, G.C. - \ 2009
Arabidopsis thaliana - GSE14635 - PRJNA114431
The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs, are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences, are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs.
Target genes of the MADS transcription factor SEPALLATA3, ChIP-seq
Kaufmann, K. ; Muiño, J.M. ; Jauregui, R. ; Airoldi, C.A. ; Smaczniak, C. ; Krajewski, P. ; Angenent, G.C. - \ 2009
Arabidopsis thaliana - GSE14600 - PRJNA114507
The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs, are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences, are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs.
The transcription machinery underlying flower formation
Kaufmann, K. ; Smaczniak, C.D. ; Angenent, G.C. - \ 2009
NPC Highlights 2009 (2009)9. - p. 5 - 7.
The Kluyver Centre for Genomics of Industrial Fermentations and the Netherlands Proteomics Centre collaborate in a Hotel Project to study secretion of proteins in the mycelium of the filamentous fungus Aspergillus niger. Han Wösten’s Microbiology group and Albert Heck’s Biomolecular Mass Spectrometry and Proteomics group analyze the secretome of this filamentous fungus that is used as a cell factory for industrial proteins. In this study, state of the art proteomics and genomics will be combined to obtain more insight in which parts of the colony actually contribute to secretion of (sets of) proteins. By using a defined culturing technique, secretion of enzymes at different zones of the fungus can be harvested. Using an advanced mass spectrometric technique, about seventy enzymes were identified at the periphery of the fungus. Of the proteins identified, all but one showed a predicted signal sequence for secretion. This shows that lysis of cells is minor, if present at all, in this zone of the colony. Lysis seems also absent in the colony centre, despite the fact that this part of the colony was already seven days old. Remarkably, six proteins that were not produced at the periphery of the colony were secreted in this zone. Thus, heterogeneity is reflected in the secretion of the mycelium. With this knowledge production of industrial relevant proteins can be improved
Target Genes of the MADS Transcription Factor SEPALLATA3: Integration of Developmental and Hormonal Pathways in the Arabidopsis Flower
Kaufmann, K. ; Muiño, J.M. ; Jauregui, R. ; Airoldi, C.A. ; Smaczniak, C.D. ; Krajewski, W. ; Angenent, G.C. - \ 2009
PloS Biology 7 (2009)4. - ISSN 1545-7885
auxin response factors - dna-binding - chromatin immunoprecipitation - floral organ - box gene - signal-transduction - meristem identity - domain proteins - plant-growth - in-vivo
The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals, and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs
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