The generation of reactive oxygen species (ROS) and their regulation by antioxidant enzymes, such as IRON SUPEROXIDE DISMUTASE 1 (FSD1), are critical for managing plant responses to salt stress. However, the protein networks modulating ROS levels during salt stress remain incompletely understood. Our co-immunoprecipitation analysis identified FSD1 as an interaction partner of the scaffolding protein RECEPTOR FOR ACTIVATED C KINASE 1A (RACK1A). Bimolecular fluorescence complementation analysis revealed that RACK1A interacts with FSD1 predominantly in the cytoplasm. Despite elevated FSD1 activity in rack1a mutants, the abundance of FSD1 protein remained unchanged. Additionally, we found that the RACK1A-FSD1 module was involved in root hair tip growth, highlighting the developmental significance of this interaction. While rack1a mutants exhibited salt resilience, the fsd1-1 rack1a-1 double mutant displayed reduced salt stress resistance, which was substantiated by reduced ROS levels. Upon salt stress, a distinct pool of RACK1A and FSD1 proteins accumulated in cycloheximide-sensitive structural condensates in the cytoplasm that colocalized with the stress granule (SG) marker protein RNA-BINDING PROTEIN 47. FSD1 activity was lower in SGs compared to the soluble extract. RACK1A also interacted with TUDOR STAPHYLOCOCCAL NUCLEASE 2, which participates in SG formation. However, RACK1A knock-out completely abolished salt-stress-dependent accumulation of FSD1 in structural condensates, suggesting that RACK1A likely mediates the recruitment of FSD1 to SGs. Thus, this study uncovers a mechanism for the regulation of RACK1/FSD1-dependent antioxidant defense in response to salt stress in Arabidopsis thaliana.

Mitogen-activated protein kinases (MAPKs) play important roles in immune signalling pathways following pathogen attack or elicitor treatment. A key plant defence strategy is a localised cell death (CD) mechanism that restricts pathogen spread. This study investigated the potential role of alfalfa (Medicago sativa) MAPK kinases (MAPKKs) and MAPK pairs in regulating CD. In vitro kinase assays confirmed that STRESS-INDUCED MAPKK (SIMKK) and PATHOGEN-RESPONSIVE MAPKK (PRKK) could activate downstream targets, including STRESSINDUCED MAPK (SIMK), STRESS-ACTIVATED MAPK (SAMK), MEDICAGO MAPK 2 (MMK2), and MEDICAGO MAPK 3 (MMK3). SIMKK was more active than PRKK, with the highest activation observed in SIMKK combinations with SIMK and SAMK, perhaps associated with CD. To study this, SIMKK and PRKK were co-transformed pairwise with SIMK, SAMK, MMK2, and MMK3 in Nicotiana benthamiana, representing a reliable in planta expression system. Microscopy revealed co-localisation of fluorescently-tagged MAPKKs with MAPKs in nuclei and cytoplasm. In the case of the SIMKK-SAMK combination, stress granule (SG)-like structures appeared in the cytoplasm, and phenotypic observations showed CD symptoms including increased level of reactive oxygen species (ROS) two days and death tissues six days post-transformation. Further phenotype analysis indicated that SIMKK pairwise overexpression with SAMK or SIMK induced ROS production and CD, suggesting SIMKK promotes immune signalling that may restrict pathogen invasion. Conversely, PRKK overexpression with the same MAPKs suppressed ROS production and CD, suggesting a protective role against excessive CD. These findings highlight a differential function between SIMKK and PRKK, offering a basis for enhancing alfalfa disease resistance through genetic approaches.

Cytokinin ribosides are major mobile and precursor forms of cytokinins, plant hormones whose transport and subcellular distribution shape developmental and stress responses. Here, we identify Arabidopsis thaliana EQUILIBRATIVE NUCLEOSIDE TRANSPORTER1 (ENT1) as a tonoplast-localized cytokinin riboside transporter. Tissue-specific subcellular analysis under native regulatory elements localized ENT1 predominantly to the tonoplast of root epidermal and lateral root cap cells, where it gates intracellular cytokinin riboside availability. Accordingly, ENT1 overexpression enhanced cytokinin riboside sensitivity and signalling, whereas loss of ENT1 altered adenosine metabolism and disrupted cytokinin homeostasis, leading to the accumulation of multiple zeatin-type cytokinins. ENT1-dependent cytokinin riboside compartmentalization was required for beneficial microbe-induced protection, as ent1 mutants failed to acquire protection against the fungal pathogen Botrytis cinerea and the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. These findings reveal a vacuolar gatekeeping mechanism that controls intracellular cytokinin riboside availability and links hormone compartmentalization to beneficial microbe-dependent plant defence.

Mitogen-activated protein kinases (MAPKs) modulate the organization of the plant cytoskeleton. The spatial organization of microtubules is critical for cell division, polarity, shape control, and elongation during plant growth and morphogenesis. Here, we analyzed the impact of the stress-induced MAPK (SIMK) abundance on the tubulin cytoskeleton in different plant organs and tissues of an important legume crop species, alfalfa (Medicago sativa L.). For this purpose, we have established unique transgenic double lines with genetically manipulated SIMK, possessing microtubular molecular fluorescent marker, the tag RED FLUORESCENT PROTEIN-TUBULIN ALPHA 6 (tagRFP-TUA6). We show that lower or higher SIMK abundance can change cell division planes (CDPs) and phragmoplast orientations in roots. In addition, the transgenic line with downregulated SIMK shows disordered and disorganized microtubules, and a reduced degree of microtubule bundling mainly in leaves and stems. This may be linked to smaller habitus, shorter stems, and smaller leaves. Obtained results indicate that the genetic manipulation of SIMK abundance has effect on microtubule organization and plant development in alfalfa. This study also paves the way for testing anti-microtubular drugs on alfalfa and for biotechnological use of the newly-developed lines.

The generation of reactive oxygen species (ROS) and their regulation by antioxidant enzymes such as IRON SUPEROXIDE DISMUTASE 1 (FSD1) are critical for managing plant responses to salt stress. However, the protein networks modulating ROS levels during salt stress remain incompletely understood. Our co-immunoprecipitation analysis identified the FSD1 as an interaction partner of the scaffolding protein RECEPTOR FOR ACTIVATED C KINASE 1A (RACK1A). Bimolecular fluorescence complementation analyses revealed that RACK1A interacts with FSD1 predominantly in the cytoplasm. Despite elevated FSD1 activity in rack1a mutants, the abundance of FSD1 protein remained unchanged. Computational predictions of interaction interfaces suggested that RACK1A may interfere with the catalytic site of FSD1. Advanced fluorescence microscopy and genetic studies further confirmed localization and relocation patterns of RACK1A and FSD1 during salt stress responses. Additionally, we found that the RACK1A-FSD1 module was involved in root hair tip growth, highlighting the developmental significance of this interaction. While rack1a mutants exhibited salt resilience, fsd1-1 rack1a-1 double mutant displayed reduced salt stress resistance compared to rack1a single mutant, which was substantiated by deregulation of ROS levels. RACK1A and FSD1 accumulated in cycloheximide-sensitive structural condensates in the cytoplasm that colocalized with the stress granule marker protein TSN in roots. However, RACK1A knock-out completely abolished salt-stress-dependent relocation of FSD1 to structural condensates, suggesting that RACK1A recruits FSD1 to these stress granules. Thus, this study uncovers an entirely novel mechanism for the regulation of RACK1/FSD1-dependent antioxidant defense in response to salt stress in Arabidopsis. ### Competing Interest Statement The authors have declared no competing interest.

Actin cytoskeleton and reactive oxygen species are principal determinants of root hair polarity and tip growth. Loss of function in RESPIRATORY BURST OXIDASE HOMOLOG C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2), an NADPH oxidase emitting superoxide to the apoplast, and in ACTIN 2, a vegetative actin isovariant, in rhd2-1 and der1-3 mutants, respectively, lead to similar defects in root hair formation and elongation Since early endosome-mediated polar localization of AtRBOHC/RHD2 depends on actin cytoskeleton, comparing the proteome-wide consequences of both mutations might be of eminent interest. Therefore, we employed a differential proteomic analysis of Arabidopsis rhd2-1 and der1-3 mutants. Both mutants exhibited substantial alterations in abundances of stress-related proteins. Notably, plasma membrane (PM)-localized PIP aquaporins showed contrasting abundance patterns in the mutants compared to wild-types. Drought-responsive proteins were mostly downregulated in rhd2-1 but upregulated in der1-3. Proteomic data suggest that opposite to der1-3, altered vesicular transport in rhd2-1 mutant likely contributes to the deregulation of PM-localized proteins, including PIPs. Moreover, lattice light sheet microscopy revealed reduced actin dynamics in rhd2-1 roots, a finding contrasting with previous reports on der1-3 mutant. Phenotypic experiments demonstrated a drought stress susceptibility in rhd2-1 and resistance in der1-3. Thus, mutations in AtRBOHC/RHD2 and ACTIN2 cause similar root hair defects, but they differently affect the actin cytoskeleton and vesicular transport. Reduced actin dynamics in rhd2-1 mutant is accompanied by alteration of vesicular transport proteins abundance, likely leading to altered protein delivery to PM, including aquaporins, thereby significantly affecting drought stress responses.

Abstract The photosynthesis-induced accumulation of reactive oxygen species in chloroplasts can lead to oxidative stress, triggering changes in protein synthesis, degradation, and the assembly/disassembly of protein complexes. Using shot-gun proteomics, we identified methyl viologen-induced changes in protein abundance in wild-type Arabidopsis and oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants, which are deficient in IRON SUPEROXIDE DISMUTASE 1 (FSD1). The levels of proteins that are localized in chloroplasts and the cytoplasm were modified in all lines treated with methyl viologen. Compared with the wild-type, fsd1 mutants showed significant changes in metabolic protein and chloroplast chaperone levels, together with increased ratio of cytoplasmic, peroxisomal, and mitochondrial proteins. Different responses in proteins involved in the disassembly of photosystem II–light harvesting chlorophyll a/b binding proteins were observed. Moreover, the abundance of PATELLIN 4, a phospholipid-binding protein enriched in stomatal lineage, was decreased in response to methyl viologen. Reverse genetic studies using patl4 knockout mutants and a PATELLIN 4 complemented line indicate that PATELLIN 4 affects plant responses to oxidative stress by effects on stomatal closure.

Protein phosphorylation, the most common and essential post-translational modification, belongs to crucial regulatory mechanisms in plants, affecting their metabolism, intracellular transport, cytoarchitecture, cell division, growth, development, and interactions with the environment. Protein kinases and phosphatases, two important families of enzymes optimally regulating phosphorylation, have now become important targets for gene editing in crops. We review progress on gene-edited protein kinases and phosphatases in crops using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). We also provide guidance for computational prediction of alterations and/or changes in function, activity, and binding of protein kinases and phosphatases as consequences of CRISPR/Cas9-based gene editing with its possible application in modern crop molecular breeding towards sustainable agriculture.

Mitogen-activated protein kinases (MAPKs) transduce stress and developmental signals related to the production of reactive oxygen species (ROS). Alfalfa (Medicago sativa L.) is a valuable forage and human nutrition crop, however, the involvement of MAPKs in plant resistance to oxidative stress is poorly understood in this species. Therefore, we elucidated the role of STRESS-INDUCED MAPK (SIMK) in alfalfa response to menadione, a compound inducing ROS generation, exploiting transgenic alfalfa lines with contrasting SIMK abundance. SIMK was activated by short-term menadione treatment and relocated from the nucleus to the cytoplasm. Proteomic analysis revealed that menadione caused changes in the abundance of proteins involved in metabolism, oxidative stress, biotic stress response, detoxification of carbonyl species, glutathione homeostasis, chloroplast protein turnover, photosynthesis, and membrane trafficking. Genetic manipulations of SIMK altered the abundance of proteins involved in mitochondrial and chloroplast protein import and processing, as well as GLUTATHIONE S-TRANSFERASES (GSTs). Increased GST abundance and activity in roots, and modifications in mitochondrial and chloroplast protein turnover might be responsible for the elevated oxidative stress resistance of alfalfa line overexpressing SIMK. This was supported by the reduced ROS levels in this line. These results reveal a complex nature of plant stress response and suggest a new role of SIMK in the alfalfa resistance to menadione-induced oxidative stress.

Summary Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred‐fold their original size. Their tip growth is determined both by intrinsic and environmental signals. Although nutrient availability and temperature are key factors for a sustained plant growth, the molecular mechanisms underlying their sensing and downstream signaling pathways remain unclear. We use genetics to address the roles of the cell surface receptor kinase FERONIA (FER) and the nutrient sensing TOR Complex 1 (TORC) in RH growth. We identified that low temperature (10°C) triggers a strong RH elongation response in Arabidopsis thaliana involving FER and TORC. We found that FER is required to perceive limited nutrient availability caused by low temperature. FERONIA interacts with and activates TORC‐downstream components to trigger RH growth. In addition, the small GTPase Rho of plants 2 (ROP2) is also involved in this RH growth response linking FER and TOR. We also found that limited nitrogen nutrient availability can mimic the RH growth response at 10°C in a NRT1.1‐dependent manner. These results uncover a molecular mechanism by which a central hub composed by FER‐ROP2‐TORC is involved in the control of RH elongation under low temperature and nitrogen deficiency.

Changes in environmental conditions cause rapid accumulation of reactive oxygen species, which can generate oxidative stress in plants. Modulation of protein synthesis, degradation and assembly or disassembly of protein complexes represent primary mechanisms of plant responses to environmental stress. Our objective was to delineate early changes in protein abundances associated with the response of Arabidopsis to methyl viologen (MV)-induced oxidative stress using shot-gun proteomic analysis. Wild type (WT) plants have been compared with oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants of IRON SUREOXIDE DISMUTASE ( FSD1). Proteins localized to chloroplast were the most affected in all lines treated with MV. The mutants showed remodeling of abundances of metabolic proteins and chloroplast chaperones as well as increased ratio of cytoplasmic, peroxisomal and mitochondrial proteins compared to WT plants. These mutants also showed different responses of proteins involved in PSII-LHC disassembly compared to WT plants. The abundance of PATELLIN 4, a phospholipid-binding protein, rapidly decreased in response to MV in WT plants, as shown by proteomic and microscopic analyses. Reverse genetic studies using patl4-1 and patl4-2 knockout mutants as well as patl4-1 complemented line showed that PATELLIN 4 affects response of Arabidopsis to MV-induced oxidative stress, likely by contributing to chloroplast positioning.

Abstract Fluorescent selective probes for reactive oxygen species (ROS) detection in living cells are versatile tools for the documentation of ROS production in plant developmental or stress reactions. We employed high-resolution live-cell imaging and semiquantitative analysis of Arabidopsis (Arabidopsis thaliana) stained with CM-H2DCFDA, CellROX Deep Red, and Amplex Red for functional characterization of the spatiotemporal mode of ROS production, delivery, and utilization during root hair formation. Cell viability marker fluorescein diacetate served as a positive control for dye loading and undisturbed root hair tip growth after staining. Using a colocalization analysis with subcellular molecular markers and two root hair mutants with similar phenotypes of nonelongating root hairs, but with contrasting reasons for this impairment, we found that: (i) CM-H2DCFDA is a sensitive probe for ROS generation in the cytoplasm, (ii) CellROX Deep Red labels ROS in mitochondria, (iii) Amplex Red labels apoplastic ROS and mitochondria and shows high selectivity to root hairs, (iv) the root hair defective 2-1 (rhd2-1) mutant with nonfunctional NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2) has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks Amplex Red-reactive ROS in apoplast, and (v) the ACTIN2-deficient deformed root hairs1-3 (der1-3) mutant is not altered in these aspects. The sensitivity of CellROX Deep Red was documented by discrimination between larger ROS-containing mitochondria and small, yet ROS-free premature mitochondria in the growing tip of root hairs. We characterized spatial changes in ROS production and compartmentalization induced by external ROS modulators, ethylene precursor 1-aminocyclopropane-1-carboxylic acid, and ionophore valinomycin. This dynamic and high-resolution study of ROS production and utilization opens opportunities for precise speciation of particular ROS involved in root hair formation.

Fluorescent selective probes for reactive oxygen species (ROS) detection in living cells are versatile tools for the documentation of ROS production in plant developmental or stress reactions. We employed high-resolution live-cell imaging and semi-quantitative analysis of Arabidopsis thaliana stained with CM-H 2 DCFDA, CellROX TM Deep Red and Amplex TM Red for functional characterization of spatiotemporal mode of ROS production, delivery and utilization during root hair formation. Cell viability marker fluorescein diacetate served as a positive control for dye-loading and undisturbed tip growth after staining. Colocalization analysis with subcellular molecular markers and utilization of two root hair mutants with similar phenotype of non-elongating root hairs, but with contrast reasons for this impairment, we found that: i) CM-H 2 DCFDA is a sensitive probe for ROS generation in cytoplasm, ii) CellROX TM Deep Red labels ROS in mitochondria, iii) Amplex TM Red labels apoplastic ROS and mitochondria, and shows high selectivity to root hairs, iv) rhd2-1 mutant with nonfunctional AtRBOHC/RHD2 has a low level of CM-H 2 DCFDA-reactive ROS in cytoplasm and lacks Amplex TM Red-reactive ROS in apoplast, v) ACTIN2 -deficient der1-3 mutant is not altered in these aspects. The sensitivity of CellROX TM Deep Red was documented by discrimination between larger ROS-containing mitochondria and small, yet ROS-free pre-mature mitochondria in the growing tip of root hairs. We characterized spatial changes in ROS production and compartmentalization induced by external ROS modulators, ethylene precursor 1-aminocyclopropane-1-carboxylic acid and ionophore valinomycin. This dynamic and high-resolution study of ROS production and utilization opens new opportunities for precise speciation of particular ROS involved in the root hair formation. One sentence summary: High-resolution live-cell imaging of ROS production and subcellular localization in bulges and growing root hairs of Arabidopsis using CM-H 2 DCFDA, CellROX TM Deep Red and Amplex TM Red selective probes.

Leguminous plants have established mutualistic endosymbiotic interactions with nitrogen-fixing rhizobia to secure nitrogen sources in root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule, and shoot formation, raising the question of how SIMK modulates these processes. In particular, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during early stages of alfalfa nodulation remain unclear. Here, we characterized SIMK distribution in Ensifer meliloti-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing down-regulation of SIMKK and SIMK, we found considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads. However, this was strongly increased in the GFP-SIMK line, constitutively overexpressing green fluorescent protein (GFP)-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. Advanced light-sheet fluorescence microscopy on intact plants allowed non-invasive imaging of spatiotemporal interactions between root hairs and symbiotic E. meliloti, while immunofluorescence detection confirmed that SIMK was activated in these locations. Our results shed new light on SIMK spatiotemporal participation in early interactions between alfalfa and E. meliloti, and its internalization into root hairs, showing that local accumulation of active SIMK modulates early nodulation in alfalfa.

The forest canopy, as a biodiversity hotspot with many wildlife habitats, remains a difficult site to access for researchers and forest managers. Here, we present a novel, small, unmanned aerial vehicle, called the Druid Drone (DD), equipped with a multifunctional manipulator designed for tree crown management and biodiversity research. Its use is demonstrated by studies and the control of the widespread, obligatory hemiparasitic European mistletoe ( Viscum album L.). The DD provides integrated pest management by canopy surveying, spraying and sampling of leaves/branches for further studies. The pilot and operator, using first person view goggles, handle the lightweight backpack‐sized drone (DJI Mavic 2 Pro, Air 2, Phantom 3 or custom Rotorama quadcopter) equipped with a manipulator and five replaceable 3D‐printed functional modules. These include the peripheral high‐resolution Observer camera for close, non‐invasive inspection of tree crowns, the entomological Collector unit for arthropod trapping, the forceps arm Sampler for soft plant tissue collection, the terminal Secator saw for cutting harder tree tissues such as twigs and the Green Doctor precision sprayer, which disperses growth regulators or other compounds for targeted tree and mistletoe management. This method offers a potential solution for safe, precise and minimally invasive research on forest canopy biodiversity in different forest types across the globe, tree health care and mistletoe management as compared to the manual work of arborists or the use of canopy cranes, forest helicopters and firearms.

NADPH oxidases are enzymes localised in the plasma membrane and emitting superoxide to the extracellular space. By production of superoxide as one type of reactive oxygen species (ROS), they exert pleiotropic functions in plant development and various stress responses. Arabidopsis thaliana RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2) is an NADPH oxidase with preferential gene expression in roots. Polar localisation and ROS production by this enzyme are essential for root hair elongation. However, the proteome-wide and physiological consequences of RBOHC/RHD2 mutations are unknown. To find out potential new functions of AtRBOHC/RHD2, we employed a differential proteomic analysis of Arabidopsis rhd2-1 mutant, carrying a loss-of-function mutation in RBOHC/RHD2 . Proteomic analyses that were validated with independent biochemical, phenotypical and advanced microscopy methods, showed quantitative deregulation of proteins involved in abiotic and biotic stress response, metabolism, vesicular transport and cell wall modification. Considerable differences in the differential proteomes between roots and above-ground parts were found in the mutant. The altered abundance of aquaporins and homeostasis of transmembrane pumps and transporters most likely determine the higher sensitivity of Arabidopsis rhd2-1 mutant to drought. Highlight Proteomics and advanced microscopy reveal that the drought sensitivity of Arabidopsis mutant in ROOT HAIR DEFECTIVE 2 is linked to altered homeostasis of plasma membrane proteins and cytoskeleton remodelling.

SUMMARYMitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the organization and dynamics of mitotic chromosomes, nucleoli, and microtubules in living cells of barley root primary meristems using a series of newly developed stable fluorescent protein translational fusion lines and time‐lapse confocal microscopy. The median duration of mitosis from prophase until the end of telophase was 65.2 and 78.2 min until the end of cytokinesis. We showed that barley chromosomes frequently start condensation before mitotic pre‐prophase as defined by the organization of microtubules and maintain it even after entering into the new interphase. Furthermore, we found that the process of chromosome condensation does not finish at metaphase, but gradually continues until the end of mitosis. In summary, our study features resources for in vivo analysis of barley nuclei and chromosomes and their dynamics during mitotic cell cycle.

Legumes enter into symbiotic associations with soil nitrogen-fixing rhizobia, culminating in the creation of new organs, root nodules. This complex process relies on chemical and physical interaction between legumes and rhizobia, including early signalling events informing the host legume plant of a potentially beneficial microbe and triggering the nodulation program. The great significance of this plant-microbe interaction rests upon conversion of atmospheric dinitrogen not accessible to plants into a biologically active form of ammonia available to plants. The plant cytoskeleton consists in a highly dynamic network and undergoes rapid remodelling upon sensing various developmental and environmental cues, including response to attachment, internalization, and accommodation of rhizobia in plant root and nodule cells. This dynamic nature is governed by cytoskeleton-associated proteins that modulate cytoskeletal behaviour depending on signal perception and transduction. Precisely localized cytoskeletal rearrangements are therefore essential for the uptake of rhizobia, their targeted delivery, and establishing beneficial root nodule symbiosis. This review summarizes current knowledge about rhizobia-dependent rearrangements and functions of the cytoskeleton in legume roots and nodules. General patterns and nodule type-, nodule stage-, and species-specific aspects of actin filaments and microtubules remodelling are discussed. Moreover, emerging evidence is provided about fine-tuning the root nodulation process through cytoskeleton-associated proteins. We also consider future perspectives on dynamic localization studies of the cytoskeleton during early symbiosis utilizing state of the art molecular and advanced microscopy approaches. Based on acquired detailed knowledge of the mutualistic interactions with microbes, these approaches could contribute to broader biotechnological crop improvement.

SummaryIn plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system‐wide dissection of the vesicular proteome, the characterization of ligand‐receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.