Periodic Reporting for period 1 - PLANTID (Conserved develoPmentaL trAjectories chaNnelling laTeral root prImorDium morphogenesis)
Reporting period: 2023-09-01 to 2025-08-31
One of the most pressing challenges of the 21st century is sustaining agricultural productivity in the face of mounting environmental stress. Root systems are central to this challenge, as they anchor plants in the soil and control the uptake of water and nutrients. Their spatial configuration, referred to as root system architecture, is a key determinant of a plant’s capacity to adapt to drought, nutrient limitation, and other stressors. A crucial driver of root system architecture plasticity is the formation of lateral roots. By branching from the main root axis, lateral roots dramatically expand the soil volume that plants can explore. While this process has been extensively studied at the cell and molecular levels in the model dicot plant Arabidopsis thaliana, little is known about its operation in cereals and legumes, despite their significant agricultural relevance. These groups of plants have distinct anatomies and cell wall compositions, which likely require different developmental strategies. A critical aspect of LR formation is spatial accommodation, the process by which a newly initiated lateral root primordium breaches several pre-existing cell layers to emerge. This process requires a tightly coordinated program of cell wall remodelling, balancing the softening of overlying tissues with the reinforcement of the emerging organ. In monocots, this process is further complicated by the presence of the exodermis, a reinforced outer cell layer whose role during organ formation is not yet clear. The overall objective of PLANTID is to identify conserved genetic programs and key cell wall regulators that control root branching in two evolutionarily and agriculturally important models: the wild grass Brachypodium distachyon and the cultivated legume white lupin (Lupinus albus). By linking single-cell transcriptomic data and spatial transcriptomic data with detailed maps of cell wall composition in both species, the project aims to uncover how these plants coordinate the balance between growth and resistance during organogenesis. The long-term pathway to impact lies in translating this knowledge into breeding strategies for crops with more efficient, resilient root systems. Such crops could maintain growth under drought or poor soils, directly contributing to EU goals for sustainable agriculture, food security, and climate resilience.
The project is structured into two complementary work packages (WPs) mainly focused on elucidating the cellular and molecular mechanisms of lateral root (LR) development in Brachypodium distachyon. We combined high-resolution transcriptomics with immunolocalisation and biomechanical analyses to create an integrated model of LR emergence.
*WP1 aimed to map the developmental trajectories of cells during LR formation. A high-resolution transcriptomic atlas was generated using single-cell RNA sequencing (scRNA-seq) on root tips at various time points after LR synchronisation. Main Achievements so far are:
- Generated a comprehensive scRNA-seq atlas of Brachypodium roots during lateral root development.
- Identified major tissue-specific clusters, including pericycle, endodermis, cortex, epidermis, and the lateral root cap.
- Reconstructed cellular developmental pathways, suggesting that endodermal cells can dedifferentiate and contribute to lateral root cap formation.
- Discovered a strong enrichment of cell wall remodelling gene families (XTHs, PMEs, expansins, polygalacturonases) in lateral root-associated cell clusters.
- An ongoing spatial transcriptomic analysis is being performed to validate candidate regulators at key time points, in collaboration with IJPB in Paris
- A collaboration is applying kinetic single-nucleus data to model the gene regulatory network and predict upstream master regulators of cell wall remodelling.
*WP2 focused on creating a detailed "cell wall atlas" to link the genetic programs identified in WP1 to the physical changes that occur during lateral root emergence. High-resolution immunolocalisation was performed using a comprehensive panel of glycan-directed antibodies. Main Achievements so far are:
- We built a detailed “cell wall atlas” revealing the spatiotemporal dynamics of hemicellulose and pectin epitopes during lateral root formation.
- We demonstrated the sequential deposition and removal of specific cell wall polysaccharide epitopes in both the lateral root primordia and the overlying cortical cells, consistent with a role in softening the emergence path, while also confirming the coordinated reinforcement of the outer cell layers of the primordia.
*Additional Achievements and Resources:
- Efficient Brachypodium CRISPR/Cas and full regeneration protocols have been established in the lab.
- A method article detailing the toolkit developed for studying Brachypodium LR development has been submitted to Methods in Molecular Biology and will soon be accessible to the plant biologist community
- Mutants affecting cell wall remodeling (NaN3 mutants collections from the JGI center in the US and INRAE Nantes) are currently under investigation.
*Future Work and New Directions
- Functional characterizations of the candidate genes involved in cell wall remodelling during lateral root development are ongoing.
- Building on these foundational studies, the project will now functionally connect gene expression programs with the biomechanical properties of the root tissue. We plan on establishing a direct link between genetic regulation and tissue mechanics. The project will employ techniques like extensometers (in collaboration with the Majada Lab, University of Lausanne, CH). The goal is to create an integrated model showing how cell-type-specific transcriptional activities lead to cell wall modifications that determine the overall mechanical properties of the root during lateral root emergence.
- The project is expanding to include the white lupin (Lupinus albus). A new “cell wall atlas” for this species is in progress. This will enable a comparative analysis to identify orthologous cell wall genes from Brachypodium and determine if they have a conserved function in the organogenesis of both lateral roots and the specialised cluster roots unique to lupin.
- The recent study Exodermis lignification impacts lateral root emergence in Brachypodium distachyon (Bellande et al., 2025, bioRxiv preprint under review at New Phytologist) shows how stress-induced lignification acts as a mechanical brake on LR emergence. This reveals a mechanistic link between stress adaptation and root system architecture plasticity in grasses. The pathway to impact builds on translating this knowledge into breeding strategies for crops with more efficient, resilient root systems. Already, a collection of 332 wild Brachypodium accessions from the Mediterranean basin has been gathered and is now available in the Vermeer lab for genome-wide association studies (GWAS) focusing on this specific response to root tip excision and exodermis lignification. Partnerships with the University of Utrecht and Forschungszentrum Jülich have been initiated to phenotype this diversity at scale. Furthermore, the project contributes to an emerging international consortium that shares single-cell and single-nucleus datasets of Brachypodium roots, ensuring maximum integration of discoveries. Together, these efforts directly support the EU Green Deal and Horizon Europe Cluster 6 objectives for sustainable agriculture, food security, and climate resilience.
*WP1 aimed to map the developmental trajectories of cells during LR formation. A high-resolution transcriptomic atlas was generated using single-cell RNA sequencing (scRNA-seq) on root tips at various time points after LR synchronisation. Main Achievements so far are:
- Generated a comprehensive scRNA-seq atlas of Brachypodium roots during lateral root development.
- Identified major tissue-specific clusters, including pericycle, endodermis, cortex, epidermis, and the lateral root cap.
- Reconstructed cellular developmental pathways, suggesting that endodermal cells can dedifferentiate and contribute to lateral root cap formation.
- Discovered a strong enrichment of cell wall remodelling gene families (XTHs, PMEs, expansins, polygalacturonases) in lateral root-associated cell clusters.
- An ongoing spatial transcriptomic analysis is being performed to validate candidate regulators at key time points, in collaboration with IJPB in Paris
- A collaboration is applying kinetic single-nucleus data to model the gene regulatory network and predict upstream master regulators of cell wall remodelling.
*WP2 focused on creating a detailed "cell wall atlas" to link the genetic programs identified in WP1 to the physical changes that occur during lateral root emergence. High-resolution immunolocalisation was performed using a comprehensive panel of glycan-directed antibodies. Main Achievements so far are:
- We built a detailed “cell wall atlas” revealing the spatiotemporal dynamics of hemicellulose and pectin epitopes during lateral root formation.
- We demonstrated the sequential deposition and removal of specific cell wall polysaccharide epitopes in both the lateral root primordia and the overlying cortical cells, consistent with a role in softening the emergence path, while also confirming the coordinated reinforcement of the outer cell layers of the primordia.
*Additional Achievements and Resources:
- Efficient Brachypodium CRISPR/Cas and full regeneration protocols have been established in the lab.
- A method article detailing the toolkit developed for studying Brachypodium LR development has been submitted to Methods in Molecular Biology and will soon be accessible to the plant biologist community
- Mutants affecting cell wall remodeling (NaN3 mutants collections from the JGI center in the US and INRAE Nantes) are currently under investigation.
*Future Work and New Directions
- Functional characterizations of the candidate genes involved in cell wall remodelling during lateral root development are ongoing.
- Building on these foundational studies, the project will now functionally connect gene expression programs with the biomechanical properties of the root tissue. We plan on establishing a direct link between genetic regulation and tissue mechanics. The project will employ techniques like extensometers (in collaboration with the Majada Lab, University of Lausanne, CH). The goal is to create an integrated model showing how cell-type-specific transcriptional activities lead to cell wall modifications that determine the overall mechanical properties of the root during lateral root emergence.
- The project is expanding to include the white lupin (Lupinus albus). A new “cell wall atlas” for this species is in progress. This will enable a comparative analysis to identify orthologous cell wall genes from Brachypodium and determine if they have a conserved function in the organogenesis of both lateral roots and the specialised cluster roots unique to lupin.
- The recent study Exodermis lignification impacts lateral root emergence in Brachypodium distachyon (Bellande et al., 2025, bioRxiv preprint under review at New Phytologist) shows how stress-induced lignification acts as a mechanical brake on LR emergence. This reveals a mechanistic link between stress adaptation and root system architecture plasticity in grasses. The pathway to impact builds on translating this knowledge into breeding strategies for crops with more efficient, resilient root systems. Already, a collection of 332 wild Brachypodium accessions from the Mediterranean basin has been gathered and is now available in the Vermeer lab for genome-wide association studies (GWAS) focusing on this specific response to root tip excision and exodermis lignification. Partnerships with the University of Utrecht and Forschungszentrum Jülich have been initiated to phenotype this diversity at scale. Furthermore, the project contributes to an emerging international consortium that shares single-cell and single-nucleus datasets of Brachypodium roots, ensuring maximum integration of discoveries. Together, these efforts directly support the EU Green Deal and Horizon Europe Cluster 6 objectives for sustainable agriculture, food security, and climate resilience.
PLANTID advances the state of the art in multiple dimensions:
- We show how stress-induced lignification acts as a mechanical brake on lateral root emergence. This reveals a mechanistic link between stress adaptation and root system architecture plasticity in grasses.
– We produced the first single-cell atlas of lateral root development in Brachypodium, identifying cell-type state transitions and cell-type-specific wall remodeling programs. The generation of the first spatial transcriptomic covering lateral root development in Brachypodium distachyon is ongoing.
- We delivered the most comprehensive immunohistochemical atlas of lateral root emergence in a monocot.
- We are helping to establish a global consortium for sharing single-cell and single-nucleus Brachypodium datasets, ensuring reproducibility and accelerating cross-team discoveries.
- We formulated a kinetic model in which the reinforced lateral root primordia (“push”) emerge through cortical layers softened by targeted wall degradation (“yield”).
- A DPPN access grant has been submitted with the Brachypodium Initiative Consortium to further explore the natural diversity of the root system architecture of 175 wild Mediterranean accessions and how the variability in root system architecture influences the plant's responses to a changing environment.
- We show how stress-induced lignification acts as a mechanical brake on lateral root emergence. This reveals a mechanistic link between stress adaptation and root system architecture plasticity in grasses.
– We produced the first single-cell atlas of lateral root development in Brachypodium, identifying cell-type state transitions and cell-type-specific wall remodeling programs. The generation of the first spatial transcriptomic covering lateral root development in Brachypodium distachyon is ongoing.
- We delivered the most comprehensive immunohistochemical atlas of lateral root emergence in a monocot.
- We are helping to establish a global consortium for sharing single-cell and single-nucleus Brachypodium datasets, ensuring reproducibility and accelerating cross-team discoveries.
- We formulated a kinetic model in which the reinforced lateral root primordia (“push”) emerge through cortical layers softened by targeted wall degradation (“yield”).
- A DPPN access grant has been submitted with the Brachypodium Initiative Consortium to further explore the natural diversity of the root system architecture of 175 wild Mediterranean accessions and how the variability in root system architecture influences the plant's responses to a changing environment.