Miguel Angel Moreno-Risueno

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.

Nitrogen (N) is an essential macronutrient and its bioavailability plays a major role in tuning plant development to the environmental nutrient status. To find novel factors in early root system architecture responses to N conditions, we performed Arabidopsis thaliana root transcriptome profiling of a short-term time course in limiting and sufficient N conditions. Using this data, we inferred transcriptional regulatory networks in each condition, which revealed the N-condition specific jasmonate responses. We found that the transcription factor (TF) ERF107 plays a generalized role in lateral root development while LBD13 is specific to N-limiting conditions. Further, we used a lateral root single-cell-identity specific transcriptome dataset to model the roles of TFs in early stages of lateral root development. We linked the N time course transcriptomics, lateral root mutant phenotypes and cell type-specific profiling approaches to find and test the roles of TFs that are involved in early root patterning responses to N conditions.

Summary: Phosphate (Pi) is an essential nutrient for plant growth, and understanding how Arabidopsis thaliana root cells respond to Pi deficiency is crucial to decipher whole plant responses. We perform high‐resolution transcriptomic profiling across five distinct root cell types, identifying differentially expressed genes (DEGs) and differential alternative splicing (DAS) events that define cell‐type‐specific Pi‐responsive regulatory layers. We identify over 7000 DEGs and 733 DAS that act largely independently and are cell‐type‐specific, affecting distinct loci and biological pathways. Transcription factors such as LHY , REV7 and MYB88 , linked to circadian rhythms, stomatal regulation and ABA perception, acquire novel functions under Pi starvation. We show that the splicing factor SR45 , previously associated with iron homeostasis, regulates Pi allocation. The sr45‐1 mutant displays increased root‐to‐shoot Pi ratio and reduced biomass. A substantial number of Pi‐responsive DAS transcripts are SR45 bound, indicating that SR45 operates as a regulatory hub for Pi transport and homeostasis. This cell‐type‐resolved dataset provides a detailed molecular map of Pi deficiency responses and highlights the importance of post‐transcriptional regulation in shaping root adaptive strategies. Transcriptional regulation and alternative splicing emerge as coordinated mechanisms orchestrating plant adaptation to Pi starvation.

This study characterizes Penicillium melinii, an endophytic fungus isolated from Arabidopsis thaliana roots, as a plant growth-promoting fungus with potential use as a model to study root development and as a biostimulant for sustainable agriculture. Although endophytes are known to promote plant growth, the underlying molecular mechanisms often remain poorly understood. Here, we aimed to elucidate how P. melinii enhances root system development and to assess its applicability across different crops. Phenotypic assays were conducted in Arabidopsis, quinoa and tomato under in vitro, greenhouse and field conditions. Root architecture and biomass were quantified using image-based phenotyping. Transcriptomic and phytohormone profiling assessed plant responses, and fungal genome sequencing coupled with secretome analysis was used to identify candidate effectors and metabolic traits. P. melinii consistently promoted root growth and increased plant biomass across species and environments, both in vitro and in the greenhouse. In tomato field trials, this translated into a significant increase in yield. The fungus colonized root surfaces without vascular penetration and triggered a mild transcriptomic response: early activation of stress-response genes followed by their attenuation and sustained upregulation of auxin-related pathways. Notably, the interaction modulates the SLR-ARF-LBD pathway and the number of pre-branch sites probably through increased auxin signalling in the oscillation zone. Additional hormonal changes were limited and mainly associated with the attenuation of the plant response to microorganisms. P. melinii enhances lateral root formation through a subtle molecular and metabolic dialogue with the host plant, underscoring its relevance as a model for studying root developmental plasticity. Its strong and reproducible growth-promoting effect, demonstrated with different fungal strains and under controlled and field conditions, supports its potential as a biostimulant for sustainable crop production.

Regeneration is a unique feature of postembryonic development extensively observed in plants. The capacity to induce regeneration exogenously is limited and usually confined to meristematic-like tissues. We show that bacterial cellulose (BC), but not other structurally similar matrixes, induces postwounding regeneration in nonmeristematic plant tissues via a distinctive route to callus-mediated regenerative programs. The BC-specific program involves cytokinin operating concurrently with strongly activated plant biotic response genes to induce plant regeneration. A reactive oxygen species (ROS) burst, normally associated with defense responses, is sustained upon BC application, involving a network of tightly interconnected transcription factors, where WRKY8, known for regulating stress responses, shows a clustering and hierarchical prevalence. WRKY8 regulates BC-mediated plant regeneration and ROS homeostasis, including superoxide anion accumulation, to potentially promote cell proliferation after wounding. Collectively, our results demonstrate that the cytokinin- and ROS-associated defense responses can be targeted by BC application to promote plant wound regeneration through alternative regenerative programs.

Understanding root development is critical for enhancing plant growth and health, and advanced technologies are essential for unraveling the complexities of these processes. In this review, we highlight select technological innovations in the study of root development, with a focus on the transformative impact of single-cell gene expression analysis. We provide a high-level overview of recent advancements, illustrating how single-cell RNA sequencing (scRNA-seq) has become a pivotal tool in plant biology. scRNA-seq has revolutionized root biology by enabling detailed, cell-specific analysis of gene expression. This has allowed researchers to create comprehensive root atlases, predict cell development, and map gene regulatory networks (GRNs) with unprecedented precision. Complementary technologies, such as multimodal profiling and bioinformatics, further enrich our understanding of cellular dynamics and gene interactions. Innovations in imaging and modeling, combined with genetic tools like CRISPR, continue to deepen our knowledge of root formation and function. Moreover, the integration of these technologies with advanced biosensors and microfluidic devices has advanced our ability to study plant–microbe interactions and phytohormone signaling at high resolution. These tools collectively provide a more comprehensive understanding of root system architecture and its regulation by environmental factors. As these technologies evolve, they promise to drive further breakthroughs in plant science, with substantial implications for agriculture and sustainability.

SUMMARY: Plants grow continuously by developing new organs, a complex process that requires the formation of specific and functional tissue patterns. Tap root systems, as observed in Arabidopsis thaliana , undergo lateral root formation, a developmental mechanism that necessitates the establishment of stem cell lineages. However, the underlying mechanisms remain poorly understood. We have reconstructed a spatiotemporal cell-type-specific transcriptional map of early lateral root organogenesis in Arabidopsis, profiling single and double fluorescent markers across 8 different cell types in the root stem cell lineage. Employing dynamic Bayesian network inference, based on time-course experiments and developmental time, alongside tree-based methods, we investigated lineage developmental progression and precursor stem-cell specification. Our results reveal a morphogenic cascade of hierarchical interdependent transcription factors driving stem cell initiation, and identify the QC/Endodermis transitioning cells as root stem cell progenitors. The associated formative program involves a profound transcriptomic re-arrangement, which, remarkably, precedes the activation of known stem-cell transcriptional signatures. Our data support a model in which root-stem-cell networks do not initiate stem formation, although various stem cell regulators are involved. Collectively, our study identifies core transcriptional signatures associated with stem cell induction and elucidates the dynamic regulatory mechanism driving early stem cell lineage establishment.

Auxin dictates root architecture via the Auxin Response Factor (ARF) family of transcription factors, which control lateral root (LR) formation. In Arabidopsis , ARF7 regulates the specification of prebranch sites (PBS) generating LRs through gene expression oscillations and plays a pivotal role during LR initiation. Despite the importance of ARF7 in this process, there is a surprising lack of knowledge about how ARF7 turnover is regulated and how this impacts root architecture. Here, we show that ARF7 accumulates in autophagy mutants and is degraded through NBR1-dependent selective autophagy. We demonstrate that the previously reported rhythmic changes to ARF7 abundance in roots are modulated via autophagy and might occur in other tissues. In addition, we show that the level of co-localization between ARF7 and autophagy markers oscillates and can be modulated by auxin to trigger ARF7 turnover. Furthermore, we observe that autophagy impairment prevents ARF7 oscillation and reduces both PBS establishment and LR formation. In conclusion, we report a novel role for autophagy during development, namely by enacting auxin-induced selective degradation of ARF7 to optimize periodic root branching.

The root system plays an essential role in plant growth and adaptation to the surrounding environment. The root clock periodically specifies lateral root prebranch sites (PBS), where a group of pericycle founder cells (FC) is primed to become lateral root founder cells and eventually give rise to lateral root primordia or lateral roots (LRs). This clock-driven organ formation process is tightly controlled by modulation of auxin content and signaling. Auxin perception entails the physical interaction of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) or AUXIN SIGNALING F-BOX (AFBs) proteins with AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to form a co-receptor system. Despite the apparent simplicity, the understanding of how specific auxin co-receptors are assembled remains unclear. We identified the compound bis-methyl auxin conjugated with N-glucoside, or BiAux, in Arabidopsis (Arabidopsis thaliana) that specifically induces the formation of PBS and the emergence of LR, with a slight effect on root elongation. Docking analyses indicated that BiAux binds to F-box proteins, and we showed that BiAux function depends on TIR1 and AFB2 F-box proteins and AUXIN RESPONSE FACTOR 7 activity, which is involved in FC specification and LR formation. Finally, using a yeast (Saccharomyces cerevisiae) heterologous expression system, we showed that BiAux favors the assemblage of specific co-receptors subunits involved in LR formation and enhances AUXIN/INDOLE-3-ACETIC ACID 28 protein degradation. These results indicate that BiAux acts as an allosteric modulator of specific auxin co-receptors. Therefore, BiAux exerts a fine-tune regulation of auxin signaling aimed to the specific formation of LR among the many development processes regulated by auxin.