Rüdiger Simon

Cryo-electron tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues in situ. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-focused ion beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence light microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens, Arabidopsis thaliana seed embryos and Limonium bicolor salt glands. We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.

Spatial omics technologies provide new opportunities for plant biology by enabling molecular profiling within structurally intact tissues, revealing spatially organized cell states, developmental gradients, and regulatory interactions. While spatial transcriptomics has driven early advances, the field is rapidly expanding toward integrated spatial multi-omics by combining single-cell and spatial transcriptomic, epigenomic, proteomic, and metabolomic data. These approaches offer new opportunities to study development, physiology, and plant biotic and abiotic interactions in spatially preserved cellular contexts. However, despite rapid adoption, the field remains constrained by plant-specific challenges when applying technologies largely developed for animal systems. Compared with animal systems, plant tissues pose additional challenges due to rigid cell walls and diverse chemistries, complicating sample preparation, cell and subcellular segmentation, signal detection, and data integration. As a result, many studies rely on bespoke protocols and analysis pipelines that are often difficult to reproduce or generalize. Here, we provide a practical, solution-oriented synthesis of current bottlenecks across experimental and computational pipelines, highlight emerging strategies to overcome these limitations, and propose a roadmap for community-driven protocol sharing, benchmarking, and integration across spatial and multi-omics modalities. Addressing these challenges will be essential to establish spatial omics as a routine and scalable tool for plant biology.

Plants perceive and integrate diverse environmental signals through receptor kinase (RK) networks at the plasma membrane. Within this, Cysteine-Rich Receptor-Like Kinases (CRKs) constitute a large but not well-understood subfamily characterised by extracellular domains (ECDs) enriched in cysteine residues. CRKs have been implicated in plant responses to biotic and abiotic stress, as well as in developmental processes. Additionally, several CRKs have been proposed to act as redox sensors. Here, we investigate the homodimerization mechanism of Arabidopsis CRK18 and its regulation by redox conditions. By modulating pH and redox state, we assessed the stability and binding dynamics of the CRK18 ECD and its cysteine mutants. We also tested the plasma membrane localisation of all the cysteine mutants involved in the predicted disulfide bonds, and only CRK18C227A/C228A-ECD resembled the plasma membrane localization of wild-type CRK18. We combine co-immunoprecipitation, Forster resonance energy transfer-fluorescence lifetime imaging microscopy and microscale thermophoresis to quantify CRK18 self-association in planta and in vitro. Furthermore, we place CRK18 dimerization in a broader signalling context by identifying CRK18 interaction partners and CRK18-dependent signalling outputs. Using Arabidopsis CRK18 overexpression lines, we perform (phospho)proteomic and immunoprecipitation-mass spectrometry (IP-MS) analyses to map CRK18-centred signalling networks associated with stress-related responses. The constitutive activation of the CRK18 kinase domain and its interaction with many putative (cell wall) glycan-sensing RKs, including PERK15, suggest a regulatory role for CRK18. The absence of significant changes in the proteome and phosphoproteome of CRK18 overexpression lines in the absence of any trigger, and its restricted mobility after elicitation, suggest that CRK18 requires a stimulus for activation and possibly induces membrane microdomain reorganisation. This is in line with the infection assay with the nematode Heterodera schachtii, which causes modification and targeted damage to plant cell walls during infection, revealing that CRK18 acts as negative regulator of this process.

SUMMARY: The beginning of the reproductive phase of cereals is marked by inflorescence formation and stem elongation. In rice, florigenic proteins encoded by HEADING DATE 3a ( Hd3a ) and RICE FLOWERING LOCUS T 1 ( RFT1 ) coordinate both developmental transitions by inducing expression of floral identity genes at the apical meristem and establishing stem competence to respond to gibberellins. Stem elongation is repressed during the vegetative phase by the activity of PREMATURE INTERNODE ELONGATION 1 ( PINE1 ), and the florigens reduce its transcription, thus relieving growth repression. Here, we first resolve the relationships occurring between regulatory factors at the shoot apical meristem and underlying stem tissues in controlling reproductive phase transitions. We next show that the PINE1 protein forms higher order complexes with TOPLESS‐related and HISTONE DEACETYLASE proteins and is required to repress a set of target genes by reducing histone acetylation levels and increasing chromatin accessibility. We propose that PINE1 controls chromatin accessibility through histone deacetylation, preventing stem growth, and that florigenic proteins act upstream of growth‐promoting signals.

In plant sciences, single-cell and spatial transcriptomics generate large complex datasets which require structured metadata in order to make them interoperable and reproducible. In this work, we demonstrate how the Annotated Research Context (ARC) can be used for the management of such data using a barley single-cell RNA sequencing dataset integrated with spatial transcriptomics data. Experimental metadata was captured in ISA tables using ontology annotations to ensure machine readability and interpretability. The computational analyses were wrapped as Common Workflow Language (CWL) scripts to ensure reproducibility of the results and reusability of both the data and the analysis pipeline. The ARC links input materials unambiguously to the analysis results, allowing the research community to trace the entire investigation procedure. Our use case shows the benefits of using an ARC for structuring and annotating heterogeneous data, enabling comparative analyses across different datasets. While wrapping analysis scripts with CWL required some technical knowledge, the resulting standardized and reusable workflows outweigh this entry barrier. Parameter variations can be easily tested and results linked correctly without manual editing of the workflows themselves. Overall, this work highlights how using the ARC framework for single-cell datasets improves the FAIRness of the data and increases the reusability.

Leaves are the plant's main photosynthetic organs and drive Earth's primary production. Grasses form longitudinal leaves with parallel venation and graminoid stomata. Yet, how distinct leaf tissues coordinately develop to build functional grass leaf anatomy is not well understood. Here, we decoded the developing grass leaf from vegetative meristems to mature tissues using single-cell RNA-sequencing in the wild grass Brachypodium distachyon. In-depth analysis of epidermal clusters and multiplexed whole-mount RNA-fluorescence in situ hybridization resolved most epidermal lineages and confirmed them in planta. Gene regulatory network analysis distinguished the targetomes of the 2 co-expressed, yet functionally divergent stomatal transcription factors BdMUTE and BdFAMA. Finally, we used our dataset to identify and functionally describe the role of the transcription factor gene BdGRAS32 in inhibiting cell division and a stomata-specific role for the putative cell wall-modifying enzyme BdPME53-like. Our single-cell grass leaf atlas enables the dissection of developmental processes that make the grass leaf, which sustains global food production.

Plasmodesmata are nanoscopic channels that traverse plant cell walls, enabling direct intercellular exchange through membrane and cytosolic continuity. Although numerous plasmodesmal components have been identified, their molecular organization remains poorly defined. Here we used cryo-electron tomography to resolve the in situ architecture of plasmodesmata in Physcomitrium patens across tissues and physiological states. We show how callose-related cell wall remodelling shapes pore architecture to modulate permeability, including a previously undescribed fully sealed state, and resolve helical protein assemblies scaffolding the central, endoplasmic-reticulum-derived desmotubule. Candidate screening via proteomics and structure prediction indicates Multiple C2 Domain and Transmembrane Proteins (MCTPs) as key constituents of these assemblies. In this model, MCTPs tether the desmotubule to the plasma membrane, while their disordered linker regions with polyampholyte charge patterning may populate the cytosolic sleeve. These findings define core architectural features of plasmodesmata and provide a structural framework for understanding how membrane, protein and cell wall components coordinate intercellular connectivity in plants.

Reactive oxygen species (ROS) regulate plant development and immunity, but how extracellular ROS signals are decoded and whether the Cysteine-rich Receptor-like Kinases (CRKs) truly serve as the long-suspected ROS sensors remains uncertain. Here, we combine high-throughput interactomics, redox proteomics, structural modelling, and genetics to map a ROS-dependent CRK interaction landscape in Arabidopsis thaliana. Using a redox-dependent interactome assay (RIACRK) on 40 CRK extracellular domains (ECDs), we identified ROS-modulated dimerisation networks with enhanced inter-community connectivity and hub redistribution in the presence of ROS. Integrating this with developmental and flg22-induced expression profiles reveals spatiotemporally limited subnetworks that likely function during activated immunity and leaf senescence, both of which are associated with extensive ROS production. Differential cysteine alkylation coupled with mass spectrometry shows that cysteines in a subset of CRK ectodomains undergo ROS-dependent oxidation. Notably, solvent-exposed, vicinal cysteines C228/C229 in CRK28 emerge as prime redox-sensitive candidates. CRK28 homodimerises and heterodimerises with CRK17 in vivo, and mutation of C228/C229 retains plasma membrane localisation but abolishes CRK28 homodimerisation, indicating a redox-controlled dimerisation switch. Loss of CRK28 delays senescence, while CRK28 overaccumulation from its native promoter causes dwarfism, premature senescence, autoimmune-like phenotypes, extensive phosphoproteome rewiring, and associations with Pathogenesis Related (PR) proteins, ROS-detoxifying enzymes, receptor(-like) kinases, and vesicle trafficking components. These results indicate that CRK28 is a potential ROS-regulated hub connecting extracellular redox signals to CRK network organisation, immune response, and age-related senescence.

SUMMARY: The transcription factor WIP2/NO TRANSMITTING TRACT (WIP2/NTT) belongs to the WIP zinc finger family. Loss of WIP/NTT function in Arabidopsis thaliana causes alterations in specific tissues in the gynoecium. It also impairs root development, but only when combined with the loss of WIP4 and WIP5 function, due to redundancy. Certain mutant loss‐of‐function phenotypes can be recovered by cytokinin application, NTT interacts with cytokinin signaling components, and the phenotypes displayed by plants with increased WIP2/NTT expression also suggest a possible interaction with this pathway. Therefore, the objective of this study was to investigate the relationship between WIP2/NTT and the cytokinin pathway. To overcome the issue of genetic redundancy, we used a commonly used inducible system. We found that WIP2/NTT induction alters cytokinin levels and signaling in a tissue‐specific manner, as shown by cytokinin content measurements and TCSn::GFP reporter analysis. Transcriptome analyses revealed candidate target genes related to the cytokinin pathway. Yeast one‐hybrid and transactivation assays demonstrated direct NTT binding to regulatory regions of the cytokinin genes ISOPENTENYL TRANSFERASE 5 (IPT5) , ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6) , and CYTOKININ OXIDASE/DEHYDROGENASE 7 (CKX7) involved in cytokinin biosynthesis, signaling, and degradation, respectively. Moreover, immunolocalization assays revealed that cytokinin distribution was altered in loss of function mutants and after NTT induction. The results of this work indicate that WIP2/NTT modulates cytokinin homeostasis.