The anaphase-promoting complex/cyclosome (APC/C) is a large multiprotein E3 ubiquitin ligase involved in ubiquitin-dependent proteolysis of key cell cycle regulatory proteins, including the ...destruction of mitotic cyclins at the metaphase-to-anaphase transition. Despite its importance, the role of the APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, we describe the identification of a plant-specific negative regulator of the APC/C complex, designated SAMBA. In Arabidopsis thaliana , SAMBA is expressed during embryogenesis and early plant development and plays a key role in organ size control. Samba mutants produced larger seeds, leaves, and roots, which resulted from enlarged root and shoot apical meristems, and, additionally, they had a reduced fertility attributable to a hampered male gametogenesis. Inactivation of SAMBA stabilized A2-type cyclins during early development. Our data suggest that SAMBA regulates cell proliferation during early development by targeting CYCLIN A2 for APC/C-mediated proteolysis.
Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides ...insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.
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.
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 (Ambidopsis 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.
Defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. Elucidation of the ...protein-protein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study protein-protein interactions under near-physiological conditions. We have developed a high-throughput tandem affinity purification (TAP)/mass spectrometry (MS) platform for cell suspension cultures to analyze protein complexes in Arabidopsis thaliana. This streamlined platform follows an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and tandem matrix-assisted laser desorption ionization MS for the identification of purified proteins. Recently, we evaluated the GS tag, originally developed to study mammalian protein complexes, that combines two IgG-binding domains of protein G with a streptavidin-binding peptide, separated by two tobacco etch virus cleavage sites. We found that this GS tag outperforms the traditional TAP tag in plant cells, regarding both specificity and complex yield. Here, we provide detailed protocols of the GS-based TAP platform that allowed us to characterize transcription factor complexes involved in signaling in response to the plant phytohormone jasmonate.
Motivation: Genome-scale ‘omics’ data constitute a potentially rich source of information about biological systems and their function. There is a plethora of tools and methods available to mine omics ...data. However, the diversity and complexity of different omics data types is a stumbling block for multi-data integration, hence there is a dire need for additional methods to exploit potential synergy from integrated orthogonal data. Rough Sets provide an efficient means to use complex information in classification approaches. Here, we set out to explore the possibilities of Rough Sets to incorporate diverse information sources in a functional classification of unknown genes. Results: We explored the use of Rough Sets for a novel data integration strategy where gene expression data, protein features and Gene Ontology (GO) annotations were combined to describe general and biologically relevant patterns represented by If-Then rules. The descriptive rules were used to predict the function of unknown genes in Arabidopsis thaliana and Schizosaccharomyces pombe. The If-Then rule models showed success rates of up to 0.89 (discriminative and predictive power for both modeled organisms); whereas, models built solely of one data type (protein features or gene expression data) yielded success rates varying from 0.68 to 0.78. Our models were applied to generate classifications for many unknown genes, of which a sizeable number were confirmed either by PubMed literature reports or electronically interfered annotations. Finally, we studied cell cycle protein–protein interactions derived from both tandem affinity purification experiments and in silico experiments in the BioGRID interactome database and found strong experimental evidence for the predictions generated by our models. The results show that our approach can be used to build very robust models that create synergy from integrating gene expression data and protein features. Availability: The Rough Set-based method is implemented in the Rosetta toolkit kernel version 1.0.1 available at: http://rosetta.lcb.uu.se/ Contact: kuiper@nt.ntnu.no; krwab@psb.ugent.be Supplementary information: Supplementary data are available at Bioinformatics online.
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
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.
•Improved AP-MS protocols paved the way for protein complex analysis in plants.•Any sample can be assayed, from cell cultures to very specific isolated tissues.•Sensitive MS and computational ...filtering allow accurate detection of true interactors.•Orthogonal data integration underscores the reliability of observed interactors.•Established AP protocols allow extension to protein–RNA, protein–DNA, or protein–lipid interactions.
Rather than functioning independently, proteins tend to work in concert with each other and with other macromolecules to form macromolecular complexes. Affinity purification coupled to mass spectrometry (AP-MS) can lead to a better understanding of the cellular functions of these complexes. With the development of easy purification protocols and ultra-sensitive MS, AP-MS is currently widely used for screening co-complex membership in plants. Studying complexes in their developmental context through the isolation of specific organs and tissues has now become feasible. Besides, the tagged protein can be employed for probing other interactions like protein–DNA and protein–RNA interactions. With the tools at hand, protein-centred interaction studies will greatly improve our knowledge of how plant cells wire their functional components in relation to their function.
Nutrient sensing and signaling are essential for adjusting growth and development to available resources. Deprivation of the essential mineral phosphorus (P) inhibits root growth.1 The molecular ...processes that sense P limitation to trigger early root growth inhibition are not known yet. Target of rapamycin (TOR) kinase is a central regulatory hub in eukaryotes to adapt growth to internal and external nutritional cues.2,3 How nutritional signals are transduced to TOR to control plant growth remains unclear. Here, we identify Arabidopsis-root-specific kinase 1 (ARSK1), which attenuates initial root growth inhibition in response to P limitation. We demonstrate that ARSK1 phosphorylates and stabilizes the regulatory-associated protein of TOR 1B (RAPTOR1B), a component of the TOR complex 1, to adjust root growth to P availability. These findings uncover signaling components acting upstream of TOR to balance growth to P availability.
•ARSK1 acts as a hub of the gene regulatory network of phosphorus deficiency•ARSK1 controls root growth inhibition within hours of phosphorus deficiency•ARSK1 phosphorylates RAPTOR1B, a key component of TOR signaling•RAPTOR1B phosphorylation prevents root growth inhibition by P deficiency
Cho et al. report on how plant roots perceive phosphorus deficiency to adjust their growth. Arabidopsis studies led to the discovery of a signaling pathway that integrates the energy-sensing TOR pathway with P signaling.
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.
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