ABSTRACT
Signal transduction relies largely on the activity of kinases and phosphatases that control protein phosphorylation. However, we still know very little about phosphorylation-mediated signaling networks. Plant MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASEs (MAP4Ks) have recently gained more attention, given their role in a wide range of processes, including developmental processes and stress signaling. We analyzed MAP4K expression patterns and mapped protein-MAP4K interactions in Arabidopsis (Arabidopsis thaliana), revealing extensive coexpression and heterodimerization. This heterodimerization is regulated by the C-terminal, intrinsically disordered half of the MAP4K, and specifically by the coiled coil motif. The ability to heterodimerize is required for proper activity and localization of the MAP4Ks. Taken together, our results identify MAP4K-interacting proteins and emphasize the functional importance of MAP4K heterodimerization. Furthermore, we identified MAP4K4/TARGET OF TEMPERATURE3 (TOT3) and MAP4K5/TOT3-INTERACTING PROTEIN 5 (TOI5) as key regulators of the transition from cell division to elongation zones in the primary root tip.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Protein Multimerization , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/enzymology , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , Protein Domains , Phosphorylation , Plants, Genetically ModifiedABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Phosphorylation , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Protein Processing, Post-Translational , Plants, Genetically Modified/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
Homologous recombination (HR) is a key DNA damage repair pathway that is tightly adjusted to the state of a cell. A central regulator of homologous recombination is the conserved helicase-containing Bloom syndrome complex, renowned for its crucial role in maintaining genome integrity. Here, we show that in Arabidopsis thaliana, Bloom complex activity is controlled by selective autophagy. We find that the recently identified DNA damage regulator KNO1 facilitates K63-linked ubiquitination of RMI1, a structural component of the complex, thereby triggering RMI1 autophagic degradation and resulting in increased homologous recombination. Conversely, reduced autophagic activity makes plants hypersensitive to DNA damage. KNO1 itself is also controlled at the level of proteolysis, in this case mediated by the ubiquitin-proteasome system, becoming stabilized upon DNA damage via two redundantly acting deubiquitinases, UBP12 and UBP13. These findings uncover a regulatory cascade of selective and interconnected protein degradation steps resulting in a fine-tuned HR response upon DNA damage.
Subject(s)
DNA-Binding Proteins , Homologous Recombination , Proteasome Endopeptidase Complex , Humans , Autophagy , Bloom Syndrome/metabolism , DNA-Binding Proteins/metabolism , Proteasome Endopeptidase Complex/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , trans-Golgi Network/metabolism , Golgi Apparatus/metabolism , Clathrin/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Sugar Phosphates , Sugar Phosphates/metabolism , Trehalose/metabolism , Protein Serine-Threonine Kinases/genetics , Plants/metabolism , Signal Transduction , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, PlantABSTRACT
The chromosomal passenger complex (CPC) is a heterotetrameric regulator of eukaryotic cell division, consisting of an Aurora-type kinase and a scaffold built of INCENP, Borealin, and Survivin. While most CPC components are conserved across eukaryotes, orthologs of the chromatin reader Survivin have previously only been found in animals and fungi, raising the question of how its essential role is carried out in other eukaryotes. By characterizing proteins that bind to the Arabidopsis Borealin ortholog, we identified BOREALIN RELATED INTERACTOR 1 and 2 (BORI1 and BORI2) as redundant Survivin-like proteins in the context of the CPC in plants. Loss of BORI function is lethal and a reduced expression of BORIs causes severe developmental defects. Similar to Survivin, we find that the BORIs bind to phosphorylated histone H3, relevant for correct CPC association with chromatin. However, this interaction is not mediated by a BIR domain as in previously recognized Survivin orthologs but by an FHA domain, a widely conserved phosphate-binding module. We find that the unifying criterion of Survivin-type proteins is a helix that facilitates complex formation with the other two scaffold components and that the addition of a phosphate-binding domain, necessary for concentration at the inner centromere, evolved in parallel in different eukaryotic groups. Using sensitive similarity searches, we find conservation of this helical domain between animals and plants and identify the missing CPC component in most eukaryotic supergroups. Interestingly, we also detect Survivin orthologs without a defined phosphate-binding domain, likely reflecting the situation in the last eukaryotic common ancestor.
Subject(s)
Chromosomal Proteins, Non-Histone , Histones , Animals , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Aurora Kinases/metabolism , Cell Cycle Proteins/metabolism , Centromere/metabolism , Chromatin/genetics , Chromosomal Proteins, Non-Histone/metabolism , Inhibitor of Apoptosis Proteins/metabolism , Mitosis , Phosphates/metabolism , Survivin/genetics , Survivin/metabolismABSTRACT
Nucleosomal acetyltransferase of H4 (NuA4) is an essential transcriptional coactivator in eukaryotes, but remains poorly characterized in plants. Here, we describe Arabidopsis homologs of the NuA4 scaffold proteins Enhancer of Polycomb-Like 1 (AtEPL1) and Esa1-Associated Factor 1 (AtEAF1). Loss of AtEAF1 results in inhibition of growth and chloroplast development. These effects are stronger in the Atepl1 mutant and are further enhanced by loss of Golden2-Like (GLK) transcription factors, suggesting that NuA4 activates nuclear plastid genes alongside GLK. We demonstrate that AtEPL1 is necessary for nucleosomal acetylation of histones H4 and H2A.Z by NuA4 in vitro. These chromatin marks are diminished genome-wide in Atepl1, while another active chromatin mark, H3K9 acetylation (H3K9ac), is locally enhanced. Expression of many chloroplast-related genes depends on NuA4, as they are downregulated with loss of H4ac and H2A.Zac. Finally, we demonstrate that NuA4 promotes H2A.Z deposition and by doing so prevents spurious activation of stress response genes.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Autotrophic Processes/physiology , Histones/metabolism , Nuclear Pore Complex Proteins/metabolism , Acetyltransferases , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Autotrophic Processes/genetics , Cell Nucleus/metabolism , Chloroplasts , Chromatin/metabolism , Ephrin-A1 , Gene Expression Regulation, Plant , Histones/genetics , Nuclear Pore Complex Proteins/genetics , Nucleosomes/metabolism , Stress, Physiological , Transcription Factors/metabolismABSTRACT
Plants respond to mild warm temperature conditions by increased elongation growth of organs to enhance cooling capacity, in a process called thermomorphogenesis. To this date, the regulation of thermomorphogenesis has been exclusively shown to intersect with light signalling pathways. To identify regulators of thermomorphogenesis that are conserved in flowering plants, we map changes in protein phosphorylation in both dicots and monocots exposed to warm temperature. We identify MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASE4 (MAP4K4)/TARGET OF TEMPERATURE3 (TOT3) as a regulator of thermomorphogenesis that impinges on brassinosteroid signalling in Arabidopsis thaliana. In addition, we show that TOT3 plays a role in thermal response in wheat, a monocot crop. Altogether, the conserved thermal regulation by TOT3 expands our knowledge of thermomorphogenesis beyond the well-studied pathways and can contribute to ensuring food security under a changing climate.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/growth & development , Arabidopsis/physiology , Protein Serine-Threonine Kinases/physiology , Acclimatization/genetics , Acclimatization/physiology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/physiology , Brassinosteroids/metabolism , Gene Expression Regulation, Plant , Phosphorylation , Phytochrome B/genetics , Phytochrome B/physiology , Plant Development/genetics , Plant Development/physiology , Plant Growth Regulators/physiology , Plants, Genetically Modified , Protein Serine-Threonine Kinases/genetics , Signal Transduction , TemperatureABSTRACT
Chromosome distribution at anaphase of mitosis and meiosis is triggered by separase, an evolutionarily conserved protease. Separase must be tightly regulated to prevent the untimely release of chromatid cohesion and disastrous chromosome distribution defects. Securin is the key inhibitor of separase in animals and fungi, but has not been identified in other eukaryotic lineages. Here, we identified PATRONUS1 and PATRONUS2 (PANS1 and PANS2) as the Arabidopsis homologs of securin. Disruption of PANS1 is known to lead to the premature separation of chromosomes at meiosis, and the simultaneous disruption of PANS1 and PANS2 is lethal. Here, we show that PANS1 targeting by the anaphase-promoting complex is required to trigger chromosome separation, mirroring the regulation of securin. We showed that PANS1 acts independently from Shugosins. In a genetic screen for pans1 suppressors, we identified SEPARASE mutants, showing that PANS1 and SEPARASE have antagonistic functions in vivo. Finally, we showed that the PANS1 and PANS2 proteins interact directly with SEPARASE. Altogether, our results show that PANS1 and PANS2 act as a plant securin. Remote sequence similarity was identified between the plant patronus family and animal securins, suggesting that they indeed derive from a common ancestor. Identification of patronus as the elusive plant securin illustrates the extreme sequence divergence of this central regulator of mitosis and meiosis.
Subject(s)
Arabidopsis Proteins/metabolism , Cell Cycle Proteins/metabolism , Chromosome Segregation , Chromosomes, Plant/metabolism , Securin/metabolism , Separase/metabolism , Amino Acid Sequence , Arabidopsis/cytology , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Chromatids/metabolism , Chromosomes, Plant/genetics , Conserved Sequence , Gene Expression Regulation, Plant , Meiosis , Mutation/genetics , Protein Binding , Time FactorsABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Phosphatidylinositol 3-Kinases/metabolism , 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 TransductionABSTRACT
KEY MESSAGE: Here, we used a hxk1 mutant in the Col-0 background. We demonstrated that HXK1 regulates cell proliferation and expansion early during leaf development, and that HXK1 is involved in sucrose-induced leaf growth stimulation independent of GPT2. Furthermore, we identified KINγ as a novel HXK1-interacting protein. In the last decade, extensive efforts have been made to unravel the underlying mechanisms of plant growth control through sugar availability. Signaling by the conserved glucose sensor HEXOKINASE1 (HXK1) has been shown to exert both growth-promoting and growth-inhibitory effects depending on the sugar levels, the environmental conditions and the plant species. Here, we used a hxk1 mutant in the Col-0 background to investigate the role of HXK1 during leaf growth in more detail and show that it is affected in both cell proliferation and cell expansion early during leaf development. Furthermore, the hxk1 mutant is less sensitive to sucrose-induced cell proliferation with no significant increase in final leaf growth after transfer to sucrose. Early during leaf development, transfer to sucrose stimulates expression of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSPORTER2 (GPT2) and represses chloroplast differentiation. However, in the hxk1 mutant GPT2 expression was still upregulated by transfer to sucrose although chloroplast differentiation was not affected, suggesting that GPT2 is not involved in HXK1-dependent regulation of leaf growth. Finally, using tandem affinity purification of protein complexes from cell cultures, we identified KINγ, a protein containing four cystathionine ß-synthase domains, as an interacting protein of HXK1.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Gene Expression Regulation, Plant , Hexokinase/metabolism , Monosaccharide Transport Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Chloroplasts/metabolism , Hexokinase/genetics , Monosaccharide Transport Proteins/genetics , Mutation , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/growth & development , Protein Serine-Threonine Kinases/genetics , Seedlings/enzymology , Seedlings/genetics , Seedlings/growth & development , Sucrose/metabolismABSTRACT
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 GSyellow, 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.
Subject(s)
Luminescent Agents/metabolism , Plant Proteins/analysis , Protein Interaction Mapping/methods , Zea mays/metabolism , 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/geneticsABSTRACT
Homologous recombination is central to repair DNA double-strand breaks, either accidently arising in mitotic cells or in a programed manner at meiosis. Crossovers resulting from the repair of meiotic breaks are essential for proper chromosome segregation and increase genetic diversity of the progeny. However, mechanisms regulating crossover formation remain elusive. Here, we identified through genetic and protein-protein interaction screens FIDGETIN-LIKE-1 INTERACTING PROTEIN (FLIP) as a new partner of the previously characterized anti-crossover factor FIDGETIN-LIKE-1 (FIGL1) in Arabidopsis thaliana. We showed that FLIP limits meiotic crossover together with FIGL1. Further, FLIP and FIGL1 form a protein complex conserved from Arabidopsis to human. FIGL1 interacts with the recombinases RAD51 and DMC1, the enzymes that catalyze the DNA strand exchange step of homologous recombination. Arabidopsis flip mutants recapitulate the figl1 phenotype, with enhanced meiotic recombination associated with change in counts of DMC1 and RAD51 foci. Our data thus suggests that FLIP and FIGL1 form a conserved complex that regulates the crucial step of strand invasion in homologous recombination.
Subject(s)
ATPases Associated with Diverse Cellular Activities/genetics , Arabidopsis Proteins/genetics , Homologous Recombination , Microtubule-Associated Proteins/genetics , Nuclear Proteins/genetics , ATPases Associated with Diverse Cellular Activities/classification , ATPases Associated with Diverse Cellular Activities/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/classification , Arabidopsis Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Microtubule-Associated Proteins/classification , Microtubule-Associated Proteins/metabolism , Multiprotein Complexes/metabolism , Mutation , Nuclear Proteins/classification , Nuclear Proteins/metabolism , Phylogeny , Protein Binding , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Rec A Recombinases/genetics , Rec A Recombinases/metabolism , Two-Hybrid System TechniquesABSTRACT
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.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/growth & development , Basic-Leucine Zipper Transcription Factors/physiology , Plant Leaves/growth & development , Plant Roots/growth & development , 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 ReactionABSTRACT
Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana This suggested that ubiquitylated abscisic acid (ABA) receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knockdown fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling.
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.
Subject(s)
Oryza/physiology , Plant Proteins/isolation & purification , 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/physiologyABSTRACT
The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Indoleacetic Acids/metabolism , Membrane Transport Proteins/metabolism , Protein Phosphatase 2/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , In Situ Hybridization , Membrane Transport Proteins/genetics , Microscopy, Confocal , Models, Biological , Mutation , Plant Leaves/genetics , Plant Leaves/growth & development , Plant Leaves/metabolism , Plant Roots/genetics , Plant Roots/growth & development , Plant Roots/metabolism , Plants, Genetically Modified , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Plants synthesize carbohydrate binding proteins in response to adverse environmental conditions such as drought, heat, pathogen attack, etc. The Arabidopsis EULS3 lectin (referred to as ArathEULS3, encoded by At2g39050) has recently been linked to the drought stress response. In this study, endogenous binding partners for this protein have been investigated. Tandem affinity purifications and mass spectrometry analyses allowed the identification of two putative interacting proteins, Embryo-specific protein 3A (ATS3A, At2g41475) and Embryo-specific protein 3B (ATS3B, At5g62200). Bimolecular fluorescence complementation experiments confirmed the interaction between ArathEULS3 and ATS3B in closed stomata of Nicotiana benthamiana plants. Transgenic lines with reduced ArathEULS3 expression exhibited an aberrant ABA-induced stomatal closure compared to plants overexpressing ArathEULS3 and control plants suggesting a role for ArathEULS3 in ABA-induced stomatal closure. Stomata are known as the major route for Pseudomonas syringae entry into the plant tissues. Bacterial infection of wild type Arabidopsis thaliana plants was accompanied by a 6-fold increase of transcript levels for ArathEULS3. Furthermore, infection experiments with ArathEULS3 overexpression lines resulted in a clear reduction of P. syringae disease symptoms whereas plants with reduced ArathEULS3 expression showed the highest levels of leaf damage at 3 days post infection. These data point towards the physiological importance of ArathEULS3 for stomatal movement.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Lectins/metabolism , Plant Stomata/physiology , Abscisic Acid/pharmacology , Amino Acid Sequence , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/microbiology , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Chromatography, Affinity , Fluorescence , Gene Expression Regulation, Plant/drug effects , Lectins/chemistry , Lectins/genetics , Molecular Sequence Data , Movement , Plant Development/drug effects , Plant Development/genetics , Plant Diseases/microbiology , Plant Stomata/drug effects , Protein Binding/drug effects , Protein Structure, Tertiary , Pseudomonas syringae/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
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 (â¼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; â¼5 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.
Subject(s)
Arabidopsis/chemistry , Arabidopsis/cytology , Multiprotein Complexes/isolation & purification , 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/methodsABSTRACT
The WEE1 kinase is an essential cell cycle checkpoint regulator in Arabidopsis thaliana plants experiencing replication defects. Whereas under non-stress conditions WEE1-deficient plants develop normally, they fail to adapt to replication inhibitory conditions, resulting in the accumulation of DNA damage and loss of cell division competence. We identified mutant alleles of the genes encoding subunits of the ribonuclease H2 (RNase H2) complex, known for its role in removing ribonucleotides from DNA-RNA duplexes, as suppressor mutants of WEE1 knockout plants. RNase H2 deficiency triggered an increase in homologous recombination (HR), correlated with the accumulation of γ-H2AX foci. However, as HR negatively impacts the growth of WEE1-deficient plants under replication stress, it cannot account for the rescue of the replication defects of the WEE1 knockout plants. Rather, the observed increase in ribonucleotide incorporation in DNA indicates that the substitution of deoxynucleotide with ribonucleotide abolishes the need for WEE1 under replication stress. Strikingly, increased ribonucleotide incorporation in DNA correlated with the occurrence of small base pair deletions, identifying the RNase H2 complex as an important suppressor of genome instability.