70 results on '"Eeckhout D"'
Search Results
2. Identification of factors required for m$^{6}$A mRNA methylation in $\textit{Arabidopsis}$ reveals a role for the conserved E3 ubiquitin ligase HAKAI
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Růžička, K, Zhang, M, Campilho, A, Bodi, Z, Kashif, M, Saleh, M, Eeckhout, D, El-Showk, S, Li, H, Zhong, S, Jaeger, GD, Mongan, NP, Hejátko, J, Helariutta, Y, Fray, RG, Helariutta, Yrjo [0000-0002-7287-8459], and Apollo - University of Cambridge Repository
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mRNA methylation ,HAKAI ,Arabidopsis ,N6-adenosine methylation (m6A) ,protoxylem ,VIRILIZER - Abstract
$\textit{N}$6-adenosine methylation m$^{6}$A of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m$^{6}$A writer proteins in $\textit{Arabidopsis thaliana}$. The components required for m$^{6}$A in $\textit{Arabidopsis}$ included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m$^{6}$A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m$^{6}$A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m$^{6}$A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.
- Published
- 2017
3. A la chasse au neutrinos
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Van Eeckhout, D
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Particle Physics - Research - Published
- 1994
4. Modelling the Danube-influenced North-western continental shelf of the Black Sea. Ecosystem response to changes in nutrient delivery by the Danube River after its damming in 1972
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Lancelot, C., Staneva, J., Van Eeckhout, D., Beckers, J. M., Stanev, E., Lancelot, C., Staneva, J., Van Eeckhout, D., Beckers, J. M., and Stanev, E.
- Published
- 2002
5. Sulfite both stimulates and inhibits the yeast vacuolar H(+)-ATPase.
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Kibak, H, primary, Van Eeckhout, D, additional, Cutler, T, additional, Taiz, S.L., additional, and Taiz, L, additional
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- 1993
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6. A Nitrogen-specific Interactome Analysis Sheds Light on the Role of the SnRK1 and TOR Kinases in Plant Nitrogen Signaling.
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Persyn F, Smagghe W, Eeckhout D, Mertens T, Smorscek T, De Winne N, Persiau G, Van De Slijke E, Crepin N, Gadeyne A, Van Leene J, and De Jaeger G
- Abstract
Nitrogen (N) is of utmost importance for plant growth and development. Multiple studies have shown that N signaling is tightly coupled with carbon (C) levels, but the interplay between C/N metabolism and growth remains largely an enigma. Nonetheless, the protein kinases Sucrose Non-fermenting 1 (SNF1)-Related Kinase 1 (SnRK1) and Target Of Rapamycin (TOR), two ancient central metabolic regulators, are emerging as key integrators that link C/N status with growth. Despite their pivotal importance, the exact mechanisms behind the sensing of N status and its integration with C availability to drive metabolic decisions are largely unknown. Especially for SnRK1, it is not clear how this kinase responds to altered N levels. Therefore, we first monitored N-dependent SnRK1 kinase activity with an in vivo Separation of Phase-based Activity Reporter of Kinase (SPARK) sensor, revealing a contrasting N-dependency in Arabidopsis thaliana (Arabidopsis) shoot and root tissues. Next, using affinity purification (AP) and proximity labeling (PL) coupled to mass spectrometry (MS) experiments, we constructed a comprehensive SnRK1 and TOR interactome in Arabidopsis cell cultures during N-starved and N-repleted growth conditions. To broaden our understanding of the N-specificity of the TOR/SnRK1 signaling events, the resulting network was compared to corresponding C-related networks, identifying a large number of novel, N-specific interactors. Moreover, through integration of N-dependent transcriptome and phosphoproteome data, we were able to pinpoint additional N-dependent network components, highlighting for instance SnRK1 regulatory proteins that might function at the crosstalk of C/N signaling. Finally, confirmation of known and identification of novel SnRK1 interactors, such as Inositol-Requiring 1 (IRE1A) and the RAB GTPase RAB18, indicate that SnRK1, present at the ER, is involved in N signaling and autophagy induction., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2024
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7. Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants.
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Dragwidge JM, Wang Y, Brocard L, De Meyer A, Hudeček R, Eeckhout D, Grones P, Buridan M, Chambaud C, Pejchar P, Potocký M, Winkler J, Vandorpe M, Serre N, Fendrych M, Bernard A, De Jaeger G, Pleskot R, Fang X, and Van Damme D
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- Cell Membrane metabolism, Endocytosis, Clathrin metabolism
- Abstract
Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2024
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8. Phase separation-based visualization of protein-protein interactions and kinase activities in plants.
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Safi A, Smagghe W, Gonçalves A, Wang Q, Xu K, Fernandez AI, Cappe B, Riquet FB, Mylle E, Eeckhout D, De Winne N, Van De Slijke E, Persyn F, Persiau G, Van Damme D, Geelen D, De Jaeger G, Beeckman T, Van Leene J, and Vanneste S
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- Phosphorylation, Protein Processing, Post-Translational, Plants, Genetically Modified metabolism, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Arabidopsis genetics, Arabidopsis metabolism
- Abstract
Protein activities depend heavily on protein complex formation and dynamic posttranslational modifications, such as phosphorylation. The dynamic nature of protein complex formation and posttranslational modifications is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to assay protein-protein interactions (PPIs) (separation of phases-based protein interaction reporter) and kinase activities (separation of phases-based activity reporter of kinase) in planta, based on phase separation. This technology enabled easy detection of inducible, binary and ternary PPIs among cytoplasmic and nuclear proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SNF1-related kinase 1 activity, allowing us to visualize tissue-specific, dynamic SnRK1 activity in stable transgenic Arabidopsis (Arabidopsis thaliana) plants. The SYMPL cloning toolbox provides a means to explore PPIs, phosphorylation, and other posttranslational modifications with unprecedented ease and sensitivity., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2023
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9. Informed Consent: Research Staff's Perspectives and Practical Recommendations to Improve Research Staff-Participant Communication.
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Eeckhout D, Aelbrecht K, and Van Der Straeten C
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- Humans, Qualitative Research, Informed Consent, Communication
- Abstract
Informed consent (IC) is the process of communication between research staff and potential research participants. However, ensuring that participants clearly understand what research participation entails, raises significant challenges. The aim of this study is to provide insight into some communication barriers that research staff are confronted with and make practical recommendations to improve communication between research staff and participants. A qualitative research study using semi-structured interviews ( n = 13) with research staff from Ghent University Hospital was conducted. Data were transcribed verbatim and coded thematically. Our results indicate that communication- and process-related factors affect the IC process. Emergent recommendations include communication training, more interactive information materials and the use of digital alternatives, increasing general knowledge about research participation and patient- and public involvement.
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- 2023
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10. Adaptor protein complex interaction map in Arabidopsis identifies P34 as a common stability regulator.
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Wang P, Siao W, Zhao X, Arora D, Wang R, Eeckhout D, Van Leene J, Kumar R, Houbaert A, De Winne N, Mylle E, Vandorpe M, Korver RA, Testerink C, Gevaert K, Vanneste S, De Jaeger G, Van Damme D, and Russinova E
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- trans-Golgi Network metabolism, Golgi Apparatus metabolism, Clathrin metabolism, Arabidopsis metabolism, Arabidopsis Proteins metabolism
- Abstract
Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargoes and coated vesicle accessory proteins. As in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargoes of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2023
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11. Author Correction: The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo.
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Grones P, De Meyer A, Pleskot R, Mylle E, Kraus M, Vandorpe M, Yperman K, Eeckhout D, Dragwidge JM, Jiang Q, Nolf J, Pavie B, De Jaeger G, De Rybel B, and Van Damme D
- Published
- 2023
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12. The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo.
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Grones P, De Meyer A, Pleskot R, Mylle E, Kraus M, Vandorpe M, Yperman K, Eeckhout D, Dragwidge JM, Jiang Q, Nolf J, Pavie B, De Jaeger G, De Rybel B, and Van Damme D
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- Cell Membrane metabolism, Ubiquitin metabolism, Ubiquitination, Clathrin genetics, Clathrin metabolism, Endocytosis
- Abstract
Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2022
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13. Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins.
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Van Leene J, Eeckhout D, Gadeyne A, Matthijs C, Han C, De Winne N, Persiau G, Van De Slijke E, Persyn F, Mertens T, Smagghe W, Crepin N, Broucke E, Van Damme D, Pleskot R, Rolland F, and De Jaeger G
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- Trehalose metabolism, Protein Serine-Threonine Kinases genetics, Plants metabolism, Signal Transduction, Gene Expression Regulation, Plant, Sugar Phosphates metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism
- Abstract
The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2022
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14. Correction to: Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems.
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Arora D, Abel NB, Liu C, Van Damme P, Yperman K, Eeckhout D, Vu LD, Wang J, Tornkvist A, Impens F, Korbei B, Van Leene J, Goossens A, De Jaeger G, Ott T, Moschou PN, and Van Damme D
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- 2022
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15. Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components.
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Dahhan DA, Reynolds GD, Cárdenas JJ, Eeckhout D, Johnson A, Yperman K, Kaufmann WA, Vang N, Yan X, Hwang I, Heese A, De Jaeger G, Friml J, Van Damme D, Pan J, and Bednarek SY
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- Clathrin metabolism, Endocytosis, Proteome metabolism, Proteomics, Transcription Factor AP-1 analysis, Transcription Factor AP-1 metabolism, Arabidopsis genetics, Arabidopsis metabolism, Clathrin-Coated Vesicles chemistry, Clathrin-Coated Vesicles metabolism
- Abstract
In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
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- 2022
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16. ROPGAP-dependent interaction between brassinosteroid and ROP2-GTPase signaling controls pavement cell shape in Arabidopsis.
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Zhang C, Lauster T, Tang W, Houbaert A, Zhu S, Eeckhout D, De Smet I, De Jaeger G, Jacobs TB, Xu T, Müller S, and Russinova E
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- Brassinosteroids metabolism, Cell Shape, GTP Phosphohydrolases metabolism, Glycogen Synthase Kinase 3 metabolism, Phosphorylation, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism
- Abstract
The epidermal pavement cell shape in Arabidopsis is driven by chemical and mechanical cues that direct partitioning mechanisms required for the establishment of the lobe- and indentation-defining polar sites. Brassinosteroid (BR) hormones regulate pavement cell morphogenesis, but the underlying mechanism remains unclear. Here, we identified two PLECKSTRIN HOMOLOGY GTPase-ACTIVATING proteins (PHGAPs) as substrates of the GSK3-like kinase BR-INSENSITIVE2 (BIN2). The phgap1phgap2 mutant displayed severe epidermal cell shape phenotypes, and the PHGAPs were markedly enriched in the anticlinal face of the pavement cell indenting regions. BIN2 phosphorylation of PHGAPs was required for their stability and polarization. BIN2 inhibition activated ROP2-GTPase signaling specifically in the lobes because of PHGAP degradation, while the PHGAPs restrained ROP2 activity in the indentations. Hence, we connect BR and ROP2-GTPase signaling pathways via the regulation of PHGAPs and put forward the importance of spatiotemporal control of BR signaling for pavement cell interdigitation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)
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- 2022
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17. SAMBA controls cell division rate during maize development.
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Gong P, Bontinck M, Demuynck K, De Block J, Gevaert K, Eeckhout D, Persiau G, Aesaert S, Coussens G, Van Lijsebettens M, Pauwels L, De Jaeger G, Inzé D, and Nelissen H
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- Crops, Agricultural genetics, Crops, Agricultural growth & development, Gene Expression Regulation, Plant, Genes, Plant, Genetic Variation, Genotype, Phenotype, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cell Division genetics, Zea mays genetics, Zea mays growth & development
- Abstract
SAMBA has been identified as a plant-specific regulator of the anaphase-promoting complex/cyclosome (APC/C) that controls unidirectional cell cycle progression in Arabidopsis (Arabidopsis thaliana), but so far its role has not been studied in monocots. Here, we show the association of SAMBA with the APC/C is conserved in maize (Zea mays). Two samba genome edited mutants showed growth defects, such as reduced internode length, shortened upper leaves with erect leaf architecture, and reduced leaf size due to an altered cell division rate and cell expansion, which aggravated with plant age. The two mutants differed in the severity and developmental onset of the phenotypes, because samba-1 represented a knockout allele, while translation re-initiation in samba-3 resulted in a truncated protein that was still able to interact with the APC/C and regulate its function, albeit with altered APC/C activity and efficiency. Our data are consistent with a dosage-dependent role for SAMBA to control developmental processes for which a change in growth rate is pivotal., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
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- 2022
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18. The DREAM complex represses growth in response to DNA damage in Arabidopsis .
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Lang L, Pettkó-Szandtner A, Tunçay Elbaşı H, Takatsuka H, Nomoto Y, Zaki A, Dorokhov S, De Jaeger G, Eeckhout D, Ito M, Magyar Z, Bögre L, Heese M, and Schnittger A
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- Arabidopsis Proteins genetics, Cell Cycle Checkpoints genetics, DNA Repair genetics, E2F Transcription Factors genetics, Gene Expression Regulation, Plant, Mutant Proteins genetics, Mutation, Plant Roots genetics, Plant Roots growth & development, Plants, Genetically Modified, Trans-Activators genetics, Arabidopsis genetics, Arabidopsis growth & development, Arabidopsis Proteins metabolism, DNA Damage genetics, E2F Transcription Factors metabolism, Mutant Proteins metabolism, Signal Transduction genetics, Trans-Activators metabolism, Transcription Factors metabolism
- Abstract
The DNA of all organisms is constantly damaged by physiological processes and environmental conditions. Upon persistent damage, plant growth and cell proliferation are reduced. Based on previous findings that RBR1, the only Arabidopsis homolog of the mammalian tumor suppressor gene retinoblastoma, plays a key role in the DNA damage response in plants, we unravel here the network of RBR1 interactors under DNA stress conditions. This led to the identification of homologs of every DREAM component in Arabidopsis, including previously not recognized homologs of LIN52. Interestingly, we also discovered NAC044, a mediator of DNA damage response in plants and close homolog of the major DNA damage regulator SOG1, to directly interact with RBR1 and the DREAM component LIN37B. Consistently, not only mutants in NAC044 but also the double mutant of the two LIN37 homologs and mutants for the DREAM component E2FB showed reduced sensitivities to DNA-damaging conditions. Our work indicates the existence of multiple DREAM complexes that work in conjunction with NAC044 to mediate growth arrest after DNA damage., (© 2021 Lang et al.)
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- 2021
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19. Arabidopsis casein kinase 2 triggers stem cell exhaustion under Al toxicity and phosphate deficiency through activating the DNA damage response pathway.
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Wei P, Demulder M, David P, Eekhout T, Yoshiyama KO, Nguyen L, Vercauteren I, Eeckhout D, Galle M, De Jaeger G, Larsen P, Audenaert D, Desnos T, Nussaume L, Loris R, and De Veylder L
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- Aluminum pharmacokinetics, Arabidopsis physiology, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Ataxia Telangiectasia Mutated Proteins metabolism, Casein Kinase II genetics, Intercellular Signaling Peptides and Proteins, Phosphates pharmacology, Phosphorylation, Plant Cells drug effects, Plant Roots growth & development, Plant Roots metabolism, Plants, Genetically Modified, Transcription Factors genetics, Transcription Factors metabolism, Aluminum toxicity, Arabidopsis cytology, Arabidopsis drug effects, Casein Kinase II metabolism, Phosphates metabolism
- Abstract
Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses strongly inhibit root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche in Arabidopsis thaliana under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED (ATM). Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation., (� American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
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- 2021
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20. Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding.
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Yperman K, Papageorgiou AC, Merceron R, De Munck S, Bloch Y, Eeckhout D, Jiang Q, Tack P, Grigoryan R, Evangelidis T, Van Leene J, Vincze L, Vandenabeele P, Vanhaecke F, Potocký M, De Jaeger G, Savvides SN, Tripsianes K, Pleskot R, and Van Damme D
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- Adaptor Proteins, Signal Transducing genetics, Arabidopsis Proteins, Calcium-Binding Proteins genetics, Cell Membrane metabolism, Crystallography, X-Ray, Membrane Proteins chemistry, Molecular Dynamics Simulation, Plant Proteins genetics, Plants, Genetically Modified, Protein Domains, Protein Transport, Sequence Alignment, Nicotiana genetics, Adaptor Proteins, Signal Transducing chemistry, Calcium-Binding Proteins chemistry, Endocytosis, Plant Proteins chemistry, Protein Binding
- Abstract
Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. However, the mechanistic contribution of the individual TPC subunits to plant CME remains elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.
- Published
- 2021
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21. Conditional destabilization of the TPLATE complex impairs endocytic internalization.
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Wang J, Yperman K, Grones P, Jiang Q, Dragwidge J, Mylle E, Mor E, Nolf J, Eeckhout D, De Jaeger G, De Rybel B, Pleskot R, and Van Damme D
- Subjects
- Arabidopsis, Arabidopsis Proteins genetics, Heat-Shock Response, Mutation, Arabidopsis Proteins metabolism, Endocytosis
- Abstract
In plants, endocytosis is essential for many developmental and physiological processes, including regulation of growth and development, hormone perception, nutrient uptake, and defense against pathogens. Our toolbox to modulate this process is, however, rather limited. Here, we report a conditional tool to impair endocytosis. We generated a partially functional TPLATE allele by substituting the most conserved domain of the TPLATE subunit of the endocytic TPLATE complex (TPC). This substitution destabilizes TPC and dampens the efficiency of endocytosis. Short-term heat treatment increases TPC destabilization and reversibly delocalizes TPLATE from the plasma membrane to aggregates in the cytoplasm. This blocks FM uptake and causes accumulation of various known endocytic cargoes at the plasma membrane. Short-term heat treatment therefore transforms the partially functional TPLATE allele into an effective conditional tool to impair endocytosis. Next to their role in endocytosis, several TPC subunits are also implicated in actin-regulated autophagosomal degradation. Inactivating TPC via the WDX mutation, however, does not impair autophagy, thus enabling specific and reversible modulation of endocytosis in planta., Competing Interests: The authors declare no competing interest.
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- 2021
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22. Molecular architecture of the endocytic TPLATE complex.
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Yperman K, Wang J, Eeckhout D, Winkler J, Vu LD, Vandorpe M, Grones P, Mylle E, Kraus M, Merceron R, Nolf J, Mor E, De Bruyn P, Loris R, Potocký M, Savvides SN, De Rybel B, De Jaeger G, Van Damme D, and Pleskot R
- Abstract
Eukaryotic cells rely on endocytosis to regulate their plasma membrane proteome and lipidome. Most eukaryotic groups, except fungi and animals, have retained the evolutionary ancient TSET complex as an endocytic regulator. Unlike other coatomer complexes, structural insight into TSET is lacking. Here, we reveal the molecular architecture of plant TSET [TPLATE complex (TPC)] using an integrative structural approach. We identify crucial roles for specific TSET subunits in complex assembly and membrane interaction. Our data therefore generate fresh insight into the differences between the hexameric TSET in Dictyostelium and the octameric TPC in plants. Structural elucidation of this ancient adaptor complex represents the missing piece in the coatomer puzzle and vastly advances our functional as well as evolutionary insight into the process of endocytosis., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)
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- 2021
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23. Unraveling the MAX2 Protein Network in Arabidopsis thaliana: Identification of the Protein Phosphatase PAPP5 as a Novel MAX2 Interactor.
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Struk S, De Cuyper C, Jacobs A, Braem L, Walton A, De Keyser A, Depuydt S, Vu LD, De Smet I, Boyer FD, Eeckhout D, Persiau G, Gevaert K, De Jaeger G, and Goormachtig S
- Subjects
- Arabidopsis genetics, Arabidopsis growth & development, Arabidopsis metabolism, Arabidopsis Proteins chemistry, Arabidopsis Proteins genetics, Carrier Proteins chemistry, Carrier Proteins genetics, Germination, Nuclear Proteins genetics, Phosphoprotein Phosphatases genetics, Plants, Genetically Modified growth & development, Plants, Genetically Modified metabolism, Seedlings genetics, Seedlings growth & development, Seedlings metabolism, Nicotiana genetics, Arabidopsis Proteins metabolism, Carrier Proteins metabolism, Nuclear Proteins metabolism, Phosphoprotein Phosphatases metabolism
- Abstract
The F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke-derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/β-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. In addition, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1)-LIKE (SMXL) family control KAR/KL and SL responses. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14, and KAI2 protein network by tandem affinity purification in Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were copurified, among which were general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than in D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination under suboptimal conditions and seedling development. In addition, a phosphopeptide enrichment experiment revealed that PAPP5 might dephosphorylate MAX2 in vivo independently of the synthetic SL analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14, and KAI2 belong, we revealed a new MAX2 interactor, PAPP5, that might act through dephosphorylation of MAX2 to control mainly KAR/KL-related phenotypes and, hence, provide another link with the light pathway., Competing Interests: Conflict of interest Authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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24. Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems.
- Author
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Arora D, Abel NB, Liu C, Van Damme P, Yperman K, Eeckhout D, Vu LD, Wang J, Tornkvist A, Impens F, Korbei B, Van Leene J, Goossens A, De Jaeger G, Ott T, Moschou PN, and Van Damme D
- Subjects
- Arabidopsis cytology, Arabidopsis metabolism, Biotin metabolism, Biotinylation, Carbon-Nitrogen Ligases genetics, Carbon-Nitrogen Ligases metabolism, Cell Membrane metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Lotus genetics, Lotus metabolism, Solanum lycopersicum chemistry, Solanum lycopersicum metabolism, Plant Proteins chemistry, Plant Proteins genetics, Plants, Genetically Modified, Protein Subunits, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Repressor Proteins genetics, Repressor Proteins metabolism, Temperature, Nicotiana genetics, Nicotiana growth & development, Nicotiana metabolism, Biotin chemistry, Plant Proteins metabolism, Protein Interaction Maps
- Abstract
Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes., (© 2020 American Society of Plant Biologists. All rights reserved.)
- Published
- 2020
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25. FRS7 and FRS12 recruit NINJA to regulate expression of glucosinolate biosynthesis genes.
- Author
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Fernández-Calvo P, Iñigo S, Glauser G, Vanden Bossche R, Tang M, Li B, De Clercq R, Nagels Durand A, Eeckhout D, Gevaert K, De Jaeger G, Brady SM, Kliebenstein DJ, Pauwels L, Goossens A, and Ritter A
- Subjects
- Cyclopentanes, Gene Expression Regulation, Plant, Glucosinolates, Nuclear Proteins, Oxylipins, Repressor Proteins metabolism, Transcription Factors genetics, Transcription Factors metabolism, Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism
- Abstract
The sessile lifestyle of plants requires accurate physiology adjustments to be able to thrive in a changing environment. Plants integrate environmental timing signals to control developmental and stress responses. Here, we identified Far1 Related Sequence (FRS) 7 and FRS12, two transcriptional repressors that accumulate in short-day conditions, as regulators of Arabidopsis glucosinolate (GSL) biosynthesis. Loss of function of FRS7 and FRS12 results in plants with increased amplitudes of diurnal expression of GSL pathway genes. Protein interaction analyses revealed that FRS7 and FRS12 recruit the NOVEL INTERACTOR OF JAZ (NINJA) to assemble a transcriptional repressor complex. Genetic and molecular evidence demonstrated that FRS7, FRS12 and NINJA jointly regulate the expression of GSL biosynthetic genes, and thus constitute a molecular mechanism that modulates specialized metabolite accumulation., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)
- Published
- 2020
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26. UBP12 and UBP13 negatively regulate the activity of the ubiquitin-dependent peptidases DA1, DAR1 and DAR2.
- Author
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Vanhaeren H, Chen Y, Vermeersch M, De Milde L, De Vleeschhauwer V, Natran A, Persiau G, Eeckhout D, De Jaeger G, Gevaert K, and Inzé D
- Subjects
- Arabidopsis genetics, Peptide Hydrolases metabolism, Plant Development physiology, Plant Leaves metabolism, Plants, Genetically Modified metabolism, Arabidopsis Proteins metabolism, Endopeptidases metabolism, Gene Expression Regulation, Plant physiology, Ubiquitin metabolism, Ubiquitin-Specific Proteases metabolism
- Abstract
Protein ubiquitination is a very diverse post-translational modification leading to protein degradation or delocalization, or altering protein activity. In Arabidopsis thaliana , two E3 ligases, BIG BROTHER (BB) and DA2, activate the latent peptidases DA1, DAR1 and DAR2 by mono-ubiquitination at multiple sites. Subsequently, these activated peptidases destabilize various positive growth regulators. Here, we show that two ubiquitin-specific proteases, UBP12 and UBP13, deubiquitinate DA1, DAR1 and DAR2, hence reducing their peptidase activity. Overexpression of UBP12 or UBP13 strongly decreased leaf size and cell area, and resulted in lower ploidy levels. Mutants in which UBP12 and UBP13 were downregulated produced smaller leaves that contained fewer and smaller cells. Remarkably, neither UBP12 nor UBP13 were found to be cleavage substrates of the activated DA1. Our results therefore suggest that UBP12 and UBP13 work upstream of DA1, DAR1 and DAR2 to restrict their protease activity and hence fine-tune plant growth and development., Competing Interests: HV, YC, MV, LD, VD, AN, GP, DE, GD, KG, DI No competing interests declared, (© 2020, Vanhaeren et al.)
- Published
- 2020
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27. TPX2-LIKE PROTEIN3 Is the Primary Activator of α-Aurora Kinases and Is Essential for Embryogenesis.
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Boruc J, Deng X, Mylle E, Besbrugge N, Van Durme M, Demidov D, Tomaštíková ED, Tan TC, Vandorpe M, Eeckhout D, Beeckman T, Nowack MK, De Jaeger G, Lin H, Liu B, and Van Damme D
- Subjects
- Amino Acid Sequence, Arabidopsis embryology, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Cell Cycle Proteins metabolism, Enzyme Activation genetics, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Microscopy, Confocal, Microtubule-Associated Proteins genetics, Microtubule-Associated Proteins metabolism, Plants, Genetically Modified, Protein Binding, Protein Serine-Threonine Kinases metabolism, Proteomics methods, Seeds embryology, Seeds metabolism, Sequence Homology, Amino Acid, Arabidopsis genetics, Arabidopsis Proteins genetics, Cell Cycle Proteins genetics, Protein Serine-Threonine Kinases genetics, Seeds genetics
- Abstract
Aurora kinases are key regulators of mitosis. Multicellular eukaryotes generally possess two functionally diverged types of Aurora kinases. In plants, including Arabidopsis ( Arabidopsis thaliana ), these are termed α- and β-Auroras. As the functional specification of Aurora kinases is determined by their specific interaction partners, we initiated interactomics analyses using both Arabidopsis α-Aurora kinases (AUR1 and AUR2). Proteomics results revealed that TPX2-LIKE PROTEINS2 and 3 (TPXL2/3) prominently associated with α-Auroras, as did the conserved TPX2 to a lower degree. Like TPX2, TPXL2 and TPXL3 strongly activated the AUR1 kinase but exhibited cell-cycle-dependent localization differences on microtubule arrays. The separate functions of TPX2 and TPXL2/3 were also suggested by their different influences on AUR1 localization upon ectopic expressions. Furthermore, genetic analyses showed that TPXL3, but not TPX2 and TPXL2, acts nonredundantly to enable proper embryo development. In contrast to vertebrates, plants have an expanded TPX2 family and these family members have both redundant and unique functions. Moreover, as neither TPXL2 nor TPXL3 contains the C-terminal Kinesin-5 binding domain present in the canonical TPX2, the targeting and activity of this kinesin must be organized differently in plants., (© 2019 American Society of Plant Biologists. All Rights Reserved.)
- Published
- 2019
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28. Capturing the phosphorylation and protein interaction landscape of the plant TOR kinase.
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Van Leene J, Han C, Gadeyne A, Eeckhout D, Matthijs C, Cannoot B, De Winne N, Persiau G, Van De Slijke E, Van de Cotte B, Stes E, Van Bel M, Storme V, Impens F, Gevaert K, Vandepoele K, De Smet I, and De Jaeger G
- Subjects
- Arabidopsis growth & development, Arabidopsis Proteins genetics, Cell Culture Techniques, Mass Spectrometry methods, Phosphatidylinositol 3-Kinases genetics, Phosphoproteins metabolism, Phosphorylation, Plants, Genetically Modified, Protein Interaction Mapping, Ribosomal Protein S6 Kinases, 90-kDa metabolism, Seedlings metabolism, Signal Transduction, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Phosphatidylinositol 3-Kinases metabolism
- Abstract
The target of rapamycin (TOR) kinase is a conserved regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Despite the increased appreciation of the essential role of the TOR complex in plants, no large-scale phosphoproteomics or interactomics studies have been performed to map TOR signalling events in plants. To fill this gap, we combined a systematic phosphoproteomics screen with a targeted protein complex analysis in the model plant Arabidopsis thaliana. Integration of the phosphoproteome and protein complex data on the one hand shows that both methods reveal complementary subspaces of the plant TOR signalling network, enabling proteome-wide discovery of both upstream and downstream network components. On the other hand, the overlap between both data sets reveals a set of candidate direct TOR substrates. The integrated network embeds both evolutionarily-conserved and plant-specific TOR signalling components, uncovering an intriguing complex interplay with protein synthesis. Overall, the network provides a rich data set to start addressing fundamental questions about how TOR controls key processes in plants, such as autophagy, auxin signalling, chloroplast development, lipid metabolism, nucleotide biosynthesis, protein translation or senescence.
- Published
- 2019
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29. POLAR-guided signalling complex assembly and localization drive asymmetric cell division.
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Houbaert A, Zhang C, Tiwari M, Wang K, de Marcos Serrano A, Savatin DV, Urs MJ, Zhiponova MK, Gudesblat GE, Vanhoutte I, Eeckhout D, Boeren S, Karimi M, Betti C, Jacobs T, Fenoll C, Mena M, de Vries S, De Jaeger G, and Russinova E
- Subjects
- Arabidopsis enzymology, Basic Helix-Loop-Helix Transcription Factors metabolism, Cell Lineage, Cytosol enzymology, Cytosol metabolism, Glycogen Synthase Kinase 3 metabolism, MAP Kinase Signaling System, Multiprotein Complexes chemistry, Phenotype, Phosphorylation, Plant Stomata cytology, Protein Binding, Protein Kinases metabolism, Substrate Specificity, Arabidopsis cytology, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Asymmetric Cell Division, Cell Cycle Proteins metabolism, Cell Polarity, Multiprotein Complexes metabolism, Signal Transduction
- Abstract
Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival
1-4 . In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7 , to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.- Published
- 2018
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30. DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis.
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Nassrallah A, Rougée M, Bourbousse C, Drevensek S, Fonseca S, Iniesto E, Ait-Mohamed O, Deton-Cabanillas AF, Zabulon G, Ahmed I, Stroebel D, Masson V, Lombard B, Eeckhout D, Gevaert K, Loew D, Genovesio A, Breyton C, De Jaeger G, Bowler C, Rubio V, and Barneche F
- Subjects
- Amino Acid Sequence, Arabidopsis genetics, Genes, Plant, Intracellular Signaling Peptides and Proteins, Light, Mutation genetics, Open Reading Frames genetics, Peptides chemistry, Protein Binding, Protein Multimerization, Protein Processing, Post-Translational, Protein Subunits metabolism, Saccharomyces cerevisiae metabolism, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Histones metabolism, Homeostasis, Multiprotein Complexes metabolism, Nuclear Proteins metabolism, Proteolysis, Ubiquitination
- Abstract
DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo , linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status., Competing Interests: AN, MR, CB, SD, SF, EI, OA, AD, GZ, IA, DS, VM, BL, DE, KG, DL, AG, CB, Gd, CB, VR, FB No competing interests declared, (© 2018, Nassrallah et al.)
- Published
- 2018
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31. GS yellow , a Multifaceted Tag for Functional Protein Analysis in Monocot and Dicot Plants.
- Author
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Besbrugge N, Van Leene J, Eeckhout D, Cannoot B, Kulkarni SR, De Winne N, Persiau G, Van De Slijke E, Bontinck M, Aesaert S, Impens F, Gevaert K, Van Damme D, Van Lijsebettens M, Inzé D, Vandepoele K, Nelissen H, and De Jaeger G
- Subjects
- Arabidopsis Proteins analysis, Arabidopsis Proteins genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Chromatin Immunoprecipitation methods, Luminescent Agents analysis, Luminescent Proteins genetics, Luminescent Proteins metabolism, Plant Leaves genetics, Plant Leaves metabolism, Plant Proteins genetics, Plants, Genetically Modified, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Trans-Activators analysis, Trans-Activators genetics, Trans-Activators metabolism, Zea mays genetics, Luminescent Agents metabolism, Plant Proteins analysis, Protein Interaction Mapping methods, Zea mays metabolism
- Abstract
The ability to tag proteins has boosted the emergence of generic molecular methods for protein functional analysis. Fluorescent protein tags are used to visualize protein localization, and affinity tags enable the mapping of molecular interactions by, for example, tandem affinity purification or chromatin immunoprecipitation. To apply these widely used molecular techniques on a single transgenic plant line, we developed a multifunctional tandem affinity purification tag, named GS
yellow , which combines the streptavidin-binding peptide tag with citrine yellow fluorescent protein. We demonstrated the versatility of the GSyellow tag in the dicot Arabidopsis ( Arabidopsis thaliana ) using a set of benchmark proteins. For proof of concept in monocots, we assessed the localization and dynamic interaction profile of the leaf growth regulator ANGUSTIFOLIA3 (AN3), fused to the GSyellow tag, along the growth zone of the maize ( Zea mays ) leaf. To further explore the function of ZmAN3, we mapped its DNA-binding landscape in the growth zone of the maize leaf through chromatin immunoprecipitation sequencing. Comparison with AN3 target genes mapped in the developing maize tassel or in Arabidopsis cell cultures revealed strong conservation of AN3 target genes between different maize tissues and across monocots and dicots, respectively. In conclusion, the GSyellow tag offers a powerful molecular tool for distinct types of protein functional analyses in dicots and monocots. As this approach involves transforming a single construct, it is likely to accelerate both basic and translational plant research., (© 2018 American Society of Plant Biologists. All rights reserved.)- Published
- 2018
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32. Recent Trends in Plant Protein Complex Analysis in a Developmental Context.
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Bontinck M, Van Leene J, Gadeyne A, De Rybel B, Eeckhout D, Nelissen H, and De Jaeger G
- Abstract
Because virtually all proteins interact with other proteins, studying protein-protein interactions (PPIs) is fundamental in understanding protein function. This is especially true when studying specific developmental processes, in which proteins often make developmental stage- or tissue specific interactions. However, studying these specific PPIs in planta can be challenging. One of the most widely adopted methods to study PPIs in planta is affinity purification coupled to mass spectrometry (AP/MS). Recent developments in the field of mass spectrometry have boosted applications of AP/MS in a developmental context. This review covers two main advancements in the field of affinity purification to study plant developmental processes: increasing the developmental resolution of the harvested tissues and moving from affinity purification to affinity enrichment. Furthermore, we discuss some new affinity purification approaches that have recently emerged and could have a profound impact on the future of protein interactome analysis in plants.
- Published
- 2018
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33. Author Correction: The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis.
- Author
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Ritter A, Iñigo S, Fernández-Calvo P, Heyndrickx KS, Dhondt S, Shi H, De Milde L, Bossche RV, De Clercq R, Eeckhout D, Ron M, Somers DE, Inzé D, Gevaert K, De Jaeger G, Vandepoele K, Pauwels L, and Goossens A
- Abstract
This corrects the article DOI: 10.1038/ncomms15235.
- Published
- 2018
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34. Identification of factors required for m 6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.
- Author
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Růžička K, Zhang M, Campilho A, Bodi Z, Kashif M, Saleh M, Eeckhout D, El-Showk S, Li H, Zhong S, De Jaeger G, Mongan NP, Hejátko J, Helariutta Y, and Fray RG
- Subjects
- Adenosine metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Conserved Sequence, Methylation, Methyltransferases genetics, Methyltransferases metabolism, Plants, Genetically Modified metabolism, Sequence Alignment, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Arabidopsis metabolism, Arabidopsis Proteins physiology, Methyltransferases physiology, RNA, Messenger metabolism, Ubiquitin-Protein Ligases physiology
- Abstract
N6-adenosine methylation (m
6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m6 A writer proteins in Arabidopsis thaliana. The components required for m6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)- Published
- 2017
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35. The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis.
- Author
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Ritter A, Iñigo S, Fernández-Calvo P, Heyndrickx KS, Dhondt S, Shi H, De Milde L, Vanden Bossche R, De Clercq R, Eeckhout D, Ron M, Somers DE, Inzé D, Gevaert K, De Jaeger G, Vandepoele K, Pauwels L, and Goossens A
- Subjects
- Aldehyde Oxidoreductases metabolism, Arabidopsis growth & development, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Base Sequence, Basic Helix-Loop-Helix Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors metabolism, Circadian Rhythm physiology, Flowers growth & development, Flowers metabolism, Hypocotyl genetics, Hypocotyl growth & development, Hypocotyl metabolism, Isoenzymes genetics, Isoenzymes metabolism, Light, Photoperiod, Signal Transduction, Transcription, Genetic, Aldehyde Oxidoreductases genetics, Arabidopsis genetics, Flowers genetics, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant
- Abstract
Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes.
- Published
- 2017
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36. Isolation of protein complexes from the model legume Medicago truncatula by tandem affinity purification in hairy root cultures.
- Author
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Goossens J, De Geyter N, Walton A, Eeckhout D, Mertens J, Pollier J, Fiallos-Jurado J, De Keyser A, De Clercq R, Van Leene J, Gevaert K, De Jaeger G, Goormachtig S, and Goossens A
- Subjects
- Agrobacterium genetics, Medicago truncatula genetics, Plant Roots genetics, Plant Roots metabolism, Plants, Genetically Modified genetics, Symbiosis genetics, Symbiosis physiology, Medicago truncatula metabolism, Plants, Genetically Modified metabolism
- Abstract
Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most powerful techniques to isolate protein complexes and elucidate protein interaction networks. Here, we describe the development of a TAP-MS strategy for the model legume Medicago truncatula, which is widely studied for its ability to produce valuable natural products and to engage in endosymbiotic interactions. As biological material, transgenic hairy roots, generated through Agrobacterium rhizogenes-mediated transformation of M. truncatula seedlings, were used. As proof of concept, proteins involved in the cell cycle, transcript processing and jasmonate signalling were chosen as bait proteins, resulting in a list of putative interactors, many of which confirm the interologue concept of protein interactions, and which can contribute to biological information about the functioning of these bait proteins in planta. Subsequently, binary protein-protein interactions among baits and preys, and among preys were confirmed by a systematic yeast two-hybrid screen. Together, by establishing a M. truncatula TAP-MS platform, we extended the molecular toolbox of this model species., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)
- Published
- 2016
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37. Functional characterization of the Arabidopsis transcription factor bZIP29 reveals its role in leaf and root development.
- Author
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Van Leene J, Blomme J, Kulkarni SR, Cannoot B, De Winne N, Eeckhout D, Persiau G, Van De Slijke E, Vercruysse L, Vanden Bossche R, Heyndrickx KS, Vanneste S, Goossens A, Gevaert K, Vandepoele K, Gonzalez N, Inzé D, and De Jaeger G
- Subjects
- Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Basic-Leucine Zipper Transcription Factors genetics, Gene Expression Profiling, Genome-Wide Association Study, Meristem growth & development, Real-Time Polymerase Chain Reaction, Arabidopsis growth & development, Arabidopsis Proteins physiology, Basic-Leucine Zipper Transcription Factors physiology, Plant Leaves growth & development, Plant Roots growth & development
- Abstract
Plant bZIP group I transcription factors have been reported mainly for their role during vascular development and osmosensory responses. Interestingly, bZIP29 has been identified in a cell cycle interactome, indicating additional functions of bZIP29 in plant development. Here, bZIP29 was functionally characterized to study its role during plant development. It is not present in vascular tissue but is specifically expressed in proliferative tissues. Genome-wide mapping of bZIP29 target genes confirmed its role in stress and osmosensory responses, but also identified specific binding to several core cell cycle genes and to genes involved in cell wall organization. bZIP29 protein complex analyses validated interaction with other bZIP group I members and provided insight into regulatory mechanisms acting on bZIP dimers. In agreement with bZIP29 expression in proliferative tissues and with its binding to promoters of cell cycle regulators, dominant-negative repression of bZIP29 altered the cell number in leaves and in the root meristem. A transcriptome analysis on the root meristem, however, indicated that bZIP29 might regulate cell number through control of cell wall organization. Finally, ectopic dominant-negative repression of bZIP29 and redundant factors led to a seedling-lethal phenotype, pointing to essential roles for bZIP group I factors early in plant development., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)
- Published
- 2016
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38. The SBT6.1 subtilase processes the GOLVEN1 peptide controlling cell elongation.
- Author
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Ghorbani S, Hoogewijs K, Pečenková T, Fernandez A, Inzé A, Eeckhout D, Kawa D, De Jaeger G, Beeckman T, Madder A, Van Breusegem F, and Hilson P
- Subjects
- Arabidopsis growth & development, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Cell Differentiation genetics, Hypocotyl genetics, Hypocotyl metabolism, Peptide Hydrolases metabolism, Plant Roots genetics, Plant Roots metabolism, Serpins metabolism, Subtilisins genetics, Subtilisins metabolism, Arabidopsis genetics, Arabidopsis Proteins genetics, Gene Expression Regulation, Plant, Peptide Hydrolases genetics, Serpins genetics
- Abstract
The GOLVEN (GLV) gene family encode small secreted peptides involved in important plant developmental programs. Little is known about the factors required for the production of the mature bioactive GLV peptides. Through a genetic suppressor screen in Arabidopsis thaliana, two related subtilase genes, AtSBT6.1 and AtSBT6.2, were identified that are necessary for GLV1 activity. Root and hypocotyl GLV1 overexpression phenotypes were suppressed by mutations in either of the subtilase genes. Synthetic GLV-derived peptides were cleaved in vitro by the affinity-purified SBT6.1 catalytic enzyme, confirming that the GLV1 precursor is a direct subtilase substrate, and the elimination of the in vitro subtilase recognition sites through alanine substitution suppressed the GLV1 gain-of-function phenotype in vivo Furthermore, the protease inhibitor Serpin1 bound to SBT6.1 and inhibited the cleavage of GLV1 precursors by the protease. GLV1 and its homolog GLV2 were expressed in the outer cell layers of the hypocotyl, preferentially in regions of rapid cell elongation. In agreement with the SBT6 role in GLV precursor processing, both null mutants for sbt6.1 and sbt6.2 and the Serpin1 overexpression plants had shorter hypocotyls. The biosynthesis of the GLV signaling peptides required subtilase activity and might be regulated by specific protease inhibitors. The data fit with a model in which the GLV1 signaling pathway participates in the regulation of hypocotyl cell elongation, is controlled by SBT6 subtilases, and is modulated locally by the Serpin1 protease inhibitor., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)
- Published
- 2016
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39. Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues.
- Author
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Dedecker M, Van Leene J, De Winne N, Eeckhout D, Persiau G, Van De Slijke E, Cannoot B, Vercruysse L, Dumoulin L, Wojsznis N, Gevaert K, Vandenabeele S, and De Jaeger G
- Subjects
- Anaphase-Promoting Complex-Cyclosome isolation & purification, Anaphase-Promoting Complex-Cyclosome physiology, Cloning, Molecular, Cyclin-Dependent Kinases isolation & purification, Cyclin-Dependent Kinases physiology, Mass Spectrometry, Oryza metabolism, Plant Leaves metabolism, Plant Leaves physiology, Plant Proteins physiology, Recombinant Proteins metabolism, Seedlings metabolism, Seedlings physiology, Oryza physiology, Plant Proteins isolation & purification
- Abstract
Proteins are the cell's functional entities. Rather than operating independently, they interact with other proteins. Capturing in vivo protein complexes is therefore crucial to gain understanding of the function of a protein in a cellular context. Affinity purification coupled to mass spectrometry has proven to yield a wealth of information about protein complex constitutions for a broad range of organisms. For Oryza sativa, the technique has been initiated in callus and shoots, but has not been optimized ever since. We translated an optimized tandem affinity purification (TAP) approach from Arabidopsis thaliana toward Oryza sativa, and demonstrate its applicability in a variety of rice tissues. A list of non-specific and false positive interactors is presented, based on re-occurrence over more than 170 independent experiments, to filter bona fide interactors. We demonstrate the sensitivity of our approach by isolating the complexes for the rice ANAPHASE PROMOTING COMPLEX SUBUNIT 10 (APC10) and CYCLIN-DEPENDENT KINASE D (CDKD) proteins from the proliferation zone of the emerging fourth leaf. Next to APC10 and CDKD, we tested several additional baits in the different rice tissues and reproducibly retrieved at least one interactor for 81.4 % of the baits screened for in callus tissue and T1 seedlings. By transferring an optimized TAP tag combined with state-of-the-art mass spectrometry, our TAP protocol enables the discovery of interactors for low abundance proteins in rice and opens the possibility to capture complex dynamics by comparing tissues at different stages of a developing rice organ.
- Published
- 2016
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40. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis.
- Author
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Gonzalez N, Pauwels L, Baekelandt A, De Milde L, Van Leene J, Besbrugge N, Heyndrickx KS, Cuéllar Pérez A, Durand AN, De Clercq R, Van De Slijke E, Vanden Bossche R, Eeckhout D, Gevaert K, Vandepoele K, De Jaeger G, Goossens A, and Inzé D
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Arabidopsis growth & development, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Binding Sites genetics, Cyclin D3 genetics, Cyclin D3 metabolism, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Microscopy, Confocal, Multiprotein Complexes metabolism, Mutation, Phenotype, Plant Leaves growth & development, Plant Leaves metabolism, Plants, Genetically Modified, Protein Binding, Repressor Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Transcription Factors metabolism, Arabidopsis genetics, Arabidopsis Proteins genetics, Multiprotein Complexes genetics, Plant Leaves genetics, Repressor Proteins genetics, Transcription Factors genetics
- Abstract
Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls., (© 2015 American Society of Plant Biologists. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
41. Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf.
- Author
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Nelissen H, Eeckhout D, Demuynck K, Persiau G, Walton A, van Bel M, Vervoort M, Candaele J, De Block J, Aesaert S, Van Lijsebettens M, Goormachtig S, Vandepoele K, Van Leene J, Muszynski M, Gevaert K, Inzé D, and De Jaeger G
- Subjects
- Conserved Sequence genetics, Conserved Sequence physiology, Plant Growth Regulators genetics, Plant Growth Regulators physiology, Plant Leaves genetics, Plant Proteins genetics, Tandem Mass Spectrometry, Plant Leaves growth & development, Plant Proteins physiology, Zea mays genetics
- Abstract
Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated., (© 2015 American Society of Plant Biologists. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
42. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.
- Author
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Van Leene J, Eeckhout D, Cannoot B, De Winne N, Persiau G, Van De Slijke E, Vercruysse L, Dedecker M, Verkest A, Vandepoele K, Martens L, Witters E, Gevaert K, and De Jaeger G
- Subjects
- Affinity Labels, Arabidopsis growth & development, Carrier Proteins genetics, Carrier Proteins metabolism, Chromatography, Liquid methods, Immunoglobulin G, Multiprotein Complexes analysis, Protein Interaction Maps, Seedlings cytology, Seedlings metabolism, Tandem Mass Spectrometry methods, Arabidopsis chemistry, Arabidopsis cytology, Multiprotein Complexes isolation & purification
- Abstract
Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.
- Published
- 2015
- Full Text
- View/download PDF
43. Sulfenome mining in Arabidopsis thaliana.
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Waszczak C, Akter S, Eeckhout D, Persiau G, Wahni K, Bodra N, Van Molle I, De Smet B, Vertommen D, Gevaert K, De Jaeger G, Van Montagu M, Messens J, and Van Breusegem F
- Subjects
- Arabidopsis drug effects, Arabidopsis Proteins metabolism, Cysteine metabolism, Glutathione metabolism, Hydrogen Peroxide pharmacology, Kinetics, Models, Biological, Multiprotein Complexes metabolism, Oxidation-Reduction drug effects, Oxidative Stress drug effects, Protein Binding drug effects, Proteolysis drug effects, Recombinant Fusion Proteins metabolism, Signal Transduction drug effects, Time Factors, Arabidopsis metabolism, Metabolome drug effects, Sulfenic Acids metabolism
- Abstract
Reactive oxygen species (ROS) have been shown to be potent signaling molecules. Today, oxidation of cysteine residues is a well-recognized posttranslational protein modification, but the signaling processes steered by such oxidations are poorly understood. To gain insight into the cysteine thiol-dependent ROS signaling in Arabidopsis thaliana, we identified the hydrogen peroxide (H2O2)-dependent sulfenome: that is, proteins with at least one cysteine thiol oxidized to a sulfenic acid. By means of a genetic construct consisting of a fusion between the C-terminal domain of the yeast (Saccharomyces cerevisiae) AP-1-like (YAP1) transcription factor and a tandem affinity purification tag, we detected ∼ 100 sulfenylated proteins in Arabidopsis cell suspensions exposed to H2O2 stress. The in vivo YAP1-based trapping of sulfenylated proteins was validated by a targeted in vitro analysis of dehydroascorbate reductase2 (DHAR2). In DHAR2, the active site nucleophilic cysteine is regulated through a sulfenic acid-dependent switch, leading to S-glutathionylation, a protein modification that protects the protein against oxidative damage.
- Published
- 2014
- Full Text
- View/download PDF
44. The Cyclin-Dependent Kinase Inhibitor KRP6 Induces Mitosis and Impairs Cytokinesis in Giant Cells Induced by Plant-Parasitic Nematodes in Arabidopsis.
- Author
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Vieira P, De Clercq A, Stals H, Van Leene J, Van De Slijke E, Van Isterdael G, Eeckhout D, Persiau G, Van Damme D, Verkest A, Antonino de Souza JD, Júnior, Glab N, Abad P, Engler G, Inzé D, De Veylder L, De Jaeger G, and Engler JD
- Abstract
In Arabidopsis thaliana, seven cyclin-dependent kinase (CDK) inhibitors have been identified, designated interactors of CDKs or Kip-related proteins (KRPs). Here, the function of KRP6 was investigated during cell cycle progression in roots infected by plant-parasitic root-knot nematodes. Contrary to expectations, analysis of Meloidogyne incognita-induced galls of KRP6-overexpressing lines revealed a role for this particular KRP as an activator of the mitotic cell cycle. In accordance, KRP6-overexpressing suspension cultures displayed accelerated entry into mitosis, but delayed mitotic progression. Likewise, phenotypic analysis of cultured cells and nematode-induced giant cells revealed a failure in mitotic exit, with the appearance of multinucleated cells as a consequence. Strong KRP6 expression upon nematode infection and the phenotypic resemblance between KRP6 overexpression cell cultures and root-knot morphology point toward the involvement of KRP6 in the multinucleate and acytokinetic state of giant cells. Along these lines, the parasite might have evolved to manipulate plant KRP6 transcription to the benefit of gall establishment., (© 2014 American Society of Plant Biologists. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
45. A generic tool for transcription factor target gene discovery in Arabidopsis cell suspension cultures based on tandem chromatin affinity purification.
- Author
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Verkest A, Abeel T, Heyndrickx KS, Van Leene J, Lanz C, Van De Slijke E, De Winne N, Eeckhout D, Persiau G, Van Breusegem F, Inzé D, Vandepoele K, and De Jaeger G
- Subjects
- Binding Sites genetics, Biotinylation, Cells, Cultured, Chromatin Immunoprecipitation, Genes, Plant, Histidine metabolism, Molecular Sequence Annotation, Nucleotide Motifs genetics, Oligopeptides metabolism, Plants, Genetically Modified, Protein Binding genetics, Sequence Analysis, DNA, Arabidopsis cytology, Arabidopsis genetics, Arabidopsis Proteins metabolism, Chromatin metabolism, Chromatography, Affinity methods, E2F Transcription Factors metabolism, Genetic Association Studies methods
- Abstract
Genome-wide identification of transcription factor (TF) binding sites is pivotal to our understanding of gene expression regulation. Although much progress has been made in the determination of potential binding regions of proteins by chromatin immunoprecipitation, this method has some inherent limitations regarding DNA enrichment efficiency and antibody necessity. Here, we report an alternative strategy for assaying in vivo TF-DNA binding in Arabidopsis (Arabidopsis thaliana) cells by tandem chromatin affinity purification (TChAP). Evaluation of TChAP using the E2Fa TF and comparison with traditional chromatin immunoprecipitation and single chromatin affinity purification illustrates the suitability of TChAP and provides a resource for exploring the E2Fa transcriptional network. Integration with transcriptome, cis-regulatory element, functional enrichment, and coexpression network analyses demonstrates the quality of the E2Fa TChAP sequencing data and validates the identification of new direct E2Fa targets. TChAP enhances both TF target mapping throughput, by circumventing issues related to antibody availability, and output, by improving DNA enrichment efficiency.
- Published
- 2014
- Full Text
- View/download PDF
46. The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.
- Author
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Gadeyne A, Sánchez-Rodríguez C, Vanneste S, Di Rubbo S, Zauber H, Vanneste K, Van Leene J, De Winne N, Eeckhout D, Persiau G, Van De Slijke E, Cannoot B, Vercruysse L, Mayers JR, Adamowski M, Kania U, Ehrlich M, Schweighofer A, Ketelaar T, Maere S, Bednarek SY, Friml J, Gevaert K, Witters E, Russinova E, Persson S, De Jaeger G, and Van Damme D
- Subjects
- Adaptor Protein Complex 2 metabolism, Cell Membrane metabolism, Dynamins metabolism, Multiprotein Complexes metabolism, Arabidopsis cytology, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Clathrin metabolism, Endocytosis
- Abstract
Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
47. Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins.
- Author
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De Meyer T, Eeckhout D, De Rycke R, De Buck S, Muyldermans S, and Depicker A
- Subjects
- Amino Acid Sequence, Animals, Antibodies blood, Antibodies metabolism, Arabidopsis Proteins metabolism, Camelus blood, Molecular Sequence Data, Phylogeny, Seed Storage Proteins genetics, Antibodies immunology, Antigens, Plant immunology, Arabidopsis metabolism, Arabidopsis Proteins immunology, Seed Storage Proteins metabolism
- Abstract
Antibodies and antibody derived fragments are excellent tools for the detection and purification of proteins. However, only few antibodies targeting Arabidopsis seed proteins are currently available. Here, we evaluate the process to make antibody libraries against crude protein extracts and more particularly to generate a VHH phage library against native Arabidopsis thaliana seed proteins. After immunising a dromedary with a crude Arabidopsis seed extract, we cloned the single-domain antigen-binding fragments from their heavy-chain only antibodies in a phage display vector and selected nanobodies (VHHs) against native Arabidopsis seed proteins. For 16 VHHs, the corresponding antigens were identified by affinity purification and MS/MS analysis. They were shown to bind the major Arabidopsis seed storage proteins albumin and globulin (14 to albumin and 2 to globulin). All 16 VHHs were suitable primary reagents for the detection of the Arabidopsis seed storage proteins by ELISA. Furthermore, several of the anti-albumin VHHs were used successfully for storage protein localisation via electron microscopy. The easy cloning, selection and production, together with the demonstrated functionality and applicability, strongly suggest that the VHH antibody format will play a more prominent role in future protein research, in particular for the study of native proteins.
- Published
- 2014
- Full Text
- View/download PDF
48. ANGUSTIFOLIA3 binds to SWI/SNF chromatin remodeling complexes to regulate transcription during Arabidopsis leaf development.
- Author
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Vercruyssen L, Verkest A, Gonzalez N, Heyndrickx KS, Eeckhout D, Han SK, Jégu T, Archacki R, Van Leene J, Andriankaja M, De Bodt S, Abeel T, Coppens F, Dhondt S, De Milde L, Vermeersch M, Maleux K, Gevaert K, Jerzmanowski A, Benhamed M, Wagner D, Vandepoele K, De Jaeger G, and Inzé D
- Subjects
- Adenosine Triphosphatases metabolism, Arabidopsis cytology, Arabidopsis growth & development, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Binding Sites, Cell Differentiation, Cell Proliferation, Chromosomal Proteins, Non-Histone metabolism, Chromosomal Proteins, Non-Histone physiology, Cyclin B genetics, Cyclin B metabolism, Genome, Plant, Plant Leaves cytology, Plant Leaves genetics, Plant Leaves growth & development, Promoter Regions, Genetic, Repressor Proteins genetics, Repressor Proteins metabolism, Arabidopsis genetics, Arabidopsis Proteins physiology, Chromatin Assembly and Disassembly, Gene Expression Regulation, Plant, Repressor Proteins physiology
- Abstract
The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORs (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5, GRF6, CYTOKININ RESPONSE FACTOR2, CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5, GRF3, COL5, and ARR4, and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf.
- Published
- 2014
- Full Text
- View/download PDF
49. The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis.
- Author
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Di Rubbo S, Irani NG, Kim SY, Xu ZY, Gadeyne A, Dejonghe W, Vanhoutte I, Persiau G, Eeckhout D, Simon S, Song K, Kleine-Vehn J, Friml J, De Jaeger G, Van Damme D, Hwang I, and Russinova E
- Subjects
- Adaptor Protein Complex 2 isolation & purification, Cell Membrane metabolism, Plant Roots metabolism, Protein Binding, Protein Transport, Signal Transduction, Adaptor Protein Complex 2 metabolism, Arabidopsis cytology, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Clathrin metabolism, Endocytosis, Protein Kinases metabolism
- Abstract
Clathrin-mediated endocytosis (CME) regulates many aspects of plant development, including hormone signaling and responses to environmental stresses. Despite the importance of this process, the machinery that regulates CME in plants is largely unknown. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) is required for the formation of clathrin-coated vesicles at the plasma membrane (PM). Although the existence of AP-2 has been predicted in Arabidopsis thaliana, the biochemistry and functionality of the complex is still uncharacterized. Here, we identified all the subunits of the Arabidopsis AP-2 by tandem affinity purification and found that one of the large AP-2 subunits, AP2A1, localized at the PM and interacted with clathrin. Furthermore, endocytosis of the leucine-rich repeat receptor kinase, brassinosteroid insensitive1 (BRI1), was shown to depend on AP-2. Knockdown of the two Arabidopsis AP2A genes or overexpression of a dominant-negative version of the medium AP-2 subunit, AP2M, impaired BRI1 endocytosis and enhanced the brassinosteroid signaling. Our data reveal that the CME machinery in Arabidopsis is evolutionarily conserved and that AP-2 functions in receptor-mediated endocytosis.
- Published
- 2013
- Full Text
- View/download PDF
50. A protein phosphatase 2A complex spatially controls plant cell division.
- Author
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Spinner L, Gadeyne A, Belcram K, Goussot M, Moison M, Duroc Y, Eeckhout D, De Winne N, Schaefer E, Van De Slijke E, Persiau G, Witters E, Gevaert K, De Jaeger G, Bouchez D, Van Damme D, and Pastuglia M
- Subjects
- Alleles, Arabidopsis ultrastructure, Arabidopsis Proteins genetics, Germination, Isoenzymes metabolism, Microtubule-Associated Proteins genetics, Microtubules metabolism, Mutation genetics, Phenotype, Phosphoprotein Phosphatases metabolism, Prophase, Protein Binding, Protein Interaction Maps, Protein Phosphatase 2 genetics, Seedlings ultrastructure, Arabidopsis cytology, Arabidopsis enzymology, Arabidopsis Proteins metabolism, Cell Division, Microtubule-Associated Proteins metabolism, Multiprotein Complexes metabolism, Plant Cells enzymology, Protein Phosphatase 2 metabolism
- Abstract
In the absence of cell migration, the orientation of cell divisions is crucial for body plan determination in plants. The position of the division plane in plant cells is set up premitotically via a transient cytoskeletal array, the preprophase band, which precisely delineates the cortical plane of division. Here we describe a protein complex that targets protein phosphatase 2A activity to microtubules, regulating the transition from the interphase to the premitotic microtubule array. This complex, which comprises TONNEAU1 and a PP2A heterotrimeric holoenzyme with FASS as regulatory subunit, is recruited to the cytoskeleton via the TONNEAU1-recruiting motif family of proteins. Despite the acentrosomal nature of plant cells, all members of this complex share similarity with animal centrosomal proteins involved in ciliary and centriolar/centrosomal functions, revealing an evolutionary link between the cortical cytoskeleton of plant cells and microtubule organizers in other eukaryotes.
- Published
- 2013
- Full Text
- View/download PDF
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