Phase separation-based visualization of protein-protein interactions and kinase activities in plants.

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.

Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins.

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.

SAMBA controls cell division rate during maize development.

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.

Unraveling the MAX2 Protein Network in Arabidopsis thaliana: Identification of the Protein Phosphatase PAPP5 as a Novel MAX2 Interactor.

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/ß-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.

The CEP5 Peptide Promotes Abiotic Stress Tolerance, As Revealed by Quantitative Proteomics, and Attenuates the AUX/IAA Equilibrium in Arabidopsis.

Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.

SYNERGISTIC ON AUXIN AND CYTOKININ 1 positively regulates growth and attenuates soil pathogen resistance.

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.

UBP12 and UBP13 negatively regulate the activity of the ubiquitin-dependent peptidases DA1, DAR1 and DAR2.

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.

Capturing the phosphorylation and protein interaction landscape of the plant TOR kinase.

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.

Quantitative Tandem Affinity Purification, an Effective Tool to Investigate Protein Complex Composition in Plant Hormone Signaling: Strigolactones in the Spotlight.

Phytohormones tightly regulate plant growth by integrating changing environmental and developmental cues. Although the key players have been identified in many plant hormonal pathways, the molecular mechanisms and mode of action of perception and signaling remain incompletely resolved. Characterization of protein partners of known signaling components provides insight into the formed protein complexes, but, unless quantification is involved, does not deliver much, if any, information about the dynamics of the induced or disrupted protein complexes. Therefore, in proteomics research, the discovery of what actually triggers, regulates or interrupts the composition of protein complexes is gaining importance. Here, tandem affinity purification coupled to mass spectrometry (TAP-MS) is combined with label-free quantification (LFQ) to a highly valuable tool to detect physiologically relevant, dynamic protein-protein interactions in Arabidopsis thaliana cell cultures. To demonstrate its potential, we focus on the signaling pathway of one of the most recently discovered phytohormones, strigolactones.

GSyellow, a Multifaceted Tag for Functional Protein Analysis in Monocot and Dicot Plants.

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.

The heterodimeric transcription factor complex ERF115-PAT1 grants regeneration competence.

Regeneration of a tissue damaged by injury represents a physiological response for organ recovery1-3. Although this regeneration process is conserved across multicellular taxa, plants appear to display extremely high regenerative capacities, a feature widely used in tissue culture for clonal propagation and grafting4,5. Regenerated cells arise predominantly from pre-existing populations of division-competent cells6,7; however, the mechanisms by which these cells are triggered to divide in response to injury remain largely elusive8. Here, we demonstrate that the heterodimeric transcription factor complex ETHYLENE RESPONSE FACTOR115 (ERF115)-PHYTOCHROME A SIGNAL TRANSDUCTION1 (PAT1) sustains meristem function by promoting cell renewal after stem cell loss. High-resolution time-lapse imaging revealed that cell death promotes ERF115 activity in cells that are in direct contact with damaged cells, triggering divisions that replenish the collapsed stem cells. Correspondingly, the ERF115-PAT1 complex plays an important role in full stem cell niche recovery upon root tip excision, whereas its ectopic expression triggers neoplastic growth, correlated with activation of the putative target gene WOUND INDUCED DEDIFFERENTIATION1 (WIND1)9. We conclude that the ERF115-PAT1 complex accounts for the high regenerative potential of plants, granting them the ability to efficiently replace damaged cells with new ones.

Functional characterization of the Arabidopsis transcription factor bZIP29 reveals its role in leaf and root development.

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.

Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues.

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.

Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf.

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.

An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

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.

Sulfenome mining in Arabidopsis thaliana.

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.

The Cyclin-Dependent Kinase Inhibitor KRP6 Induces Mitosis and Impairs Cytokinesis in Giant Cells Induced by Plant-Parasitic Nematodes in Arabidopsis.

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.

The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.

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.

A generic tool for transcription factor target gene discovery in Arabidopsis cell suspension cultures based on tandem chromatin affinity purification.

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.

The non-JAZ TIFY protein TIFY8 from Arabidopsis thaliana is a transcriptional repressor.

Jasmonate (JA) signalling is mediated by the JASMONATE-ZIM DOMAIN (JAZ) repressor proteins, which are degraded upon JA perception to release downstream responses. The ZIM protein domain is characteristic of the larger TIFY protein family. It is currently unknown if the atypical member TIFY8 is involved in JA signalling. Here we show that the TIFY8 ZIM domain is functional and mediated interaction with PEAPOD proteins and NINJA. TIFY8 interacted with TOPLESS through NINJA and accordingly acted as a transcriptional repressor. TIFY8 expression was inversely correlated with JAZ expression during development and after infection with Pseudomonas syringae. Nevertheless, transgenic lines with altered TIFY8 expression did not show changes in JA sensitivity. Despite the functional ZIM domain, no interaction with JAZ proteins could be found. In contrast, TIFY8 was found in protein complexes involved in regulation of dephosphorylation, deubiquitination and O-linked N-acetylglucosamine modification suggesting an important role in nuclear signal transduction.