Min-Yao Jhu

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.

SUMMARY: Legume root nodules enable symbiotic nitrogen fixation through the development of specialized cells that accommodate nitrogen-fixing bacteria intracellularly and support bacterial nitrogenase activity. Here, we present a 4D (3D space and time) spatial transcriptomic atlas of Medicago truncatula nodules and lateral roots, revealing specialized symbiotic cell types that develop alongside a conserved lateral-root-derived program that underpins vascularization. Spatial profiling of both plant and bacterial transcripts resolves distinct transcriptional states and previously unrecognized cell states. Spatial analysis of developmental regulator mutants uncovers a cascading series of cell-type-specific programs during nodule maturation. LSH1/LSH2 are central regulators of these programs, and lsh1/lsh2 mutants exhibit collapse of hormonal gradients and nodule identity. Strikingly, loss of nodule identity collapses to a primary-root identity rather than lateral-root fate. This work reveals how tissue complexity emerges through stacked developmental programs sustained in distinct cellular compartments, allowing the emergence of cell types specialized for harboring nitrogen-fixing bacteria. HIGHLIGHTS: >136,000 cells define a 4D spatial atlas of nodules and lateral roots 3D spatial and dual-species analysis resolves dynamic host and rhizobial cell states A shared meristem generates 3 spatially coordinated symbiotic, non-symbiotic, and vascular cell programs LSH1/LSH2 are critical for regulatory programs underlying nodule development and identity Nodule identity loss collapses toward a primary root-associated state, not a lateral root fate

The nitrogen-fixing root-nodule symbiosis provides a sustainable source of nitrogen for plants within the Nitrogen (N)-fixing clade (NFC). A debate has raged over whether nodulation evolved once, with many losses or multiple times following a predisposition event. Here we demonstrate that nodule-organogenesis is fully conserved between an actinorhizal nodulator Datisca glomerata and the legume Medicago truncatula , showing entirely conserved programmes for Nodule INception (NIN)- controlled development leading to nodule emergence. Convergent losses of N-fixation within the NFC is associated with loss of NIN and we show the engineering of nodule-like development into strawberry, a non-nodulating member of the NFC, through the repair of NIN- functionality. Similar NIN- engineering resulted in altered-root developmental responses in barley. Our work is consistent with the single-gain hypothesis, where repair of NIN can recapitulate nodules in species within the NFC, demonstrating that understanding the ancestral state of nodulation facilitates its engineering.

Elucidating the spatial and temporal regulation of gene expression during plant organogenesis is crucial for enabling precise crop improvement strategies that incorporate beneficial traits into crops while avoiding adverse effects. Root nodules, specialised organs formed in symbiosis with nitrogen-fixing bacteria, provide a valuable system to study cell-type-specific gene networks in a symbiosis-induced developmental context. However, capturing these dynamics at cellular resolution in intact plant tissues remains technically challenging. Spatial transcriptomics technologies developed for animal systems are often not directly transferable to plant tissues due to fundamental differences in tissue composition between plants and animals, including rigid and heterogeneous plant cell walls, high cell wall autofluorescence, and large vacuoles in plant cells that complicate probe access and signal detection. To address these challenges, we present an optimised protocol for applying the Xenium in situ sequencing platform to formalin-fixed paraffin-embedded (FFPE) sections of plant tissues, including Medicago truncatula roots and nodules. Key technical adaptations include customised tissue preparation, optimised section thickness, hybridisation conditions, post-Xenium staining, imaging, and downstream image analysis, all tailored specifically for plant samples. To mitigate autofluorescence and enhance detection sensitivity, we employed a strategic approach to codeword selection during probe design. Furthermore, we developed a modular probe design approach combining a custom 380-gene standalone panel with a 100-gene add-on panel. This design allows flexibility for addressing diverse research questions and includes orthologous gene sequences from two Medicago ecotypes, ensuring compatibility for downstream functional validation using mutant lines available in both genetic backgrounds. We validated the protocol across nodules at multiple developmental stages using both the 50-gene panel targeting mature nodule cell identity and the extended 480-gene panel, which includes markers across different cell types and developmental stages, as well as genes of interest identified from prior single-cell and bulk RNA-seq analyses. This optimised workflow provides a reproducible and scalable method for high-resolution spatial transcriptomics in plant tissues, establishing a robust foundation for adaptation to other plant species and developmental systems.