Periodic Reporting for period 1 - LAROSSE (Decoding the molecular basis of lateral root stem cell specification in plants: C-fern as a model system.)
Reporting period: 2023-09-01 to 2025-08-31
Plant roots are essential organs that enable efficient uptake of water and nutrients, provide anchorage in the soil, and host mutualistic microbes that enhance mineral acquisition. The rise and diversification of vascular plants—including lycophytes, ferns, and seed plants—across terrestrial ecosystems were greatly facilitated by the evolution of rooting capacity. Fossil evidence indicates that root branching evolved multiple times independently during vascular plant evolution. Ferns occupy a pivotal phylogenetic position as the sister lineage to seed plants and develop roots, including lateral branches, in highly regular patterns driven by ordered divisions of root stem cells. However, the molecular mechanisms governing lateral root stem cell specification in ferns remain largely unknown. The aim of this project was to elucidate the transcriptional changes associated with lateral root development in the fern Ceratopteris at single-cell resolution and to identify key transcription factors involved in lateral root stem cell specification. To achieve this aim, we employed an integrated combination of molecular, cell biological, and genetic approaches.
To address the objectives of LAROSSE , we performed single-cell RNA sequencing in the roots of the fern Ceratopteris. Data analysis including Uniform Manifold Approximation and Projection (UMAP) revealed that fern roots are likely composed of approximately 17 distinct cell clusters. To annotate these clusters, we combined several approaches, including label transfer based on cell type–specific markers from Arabidopsis, the generation of several transcriptional fusion lines, and Hybridization Chain Reaction Fluorescence in situ hybridization (HCR RNA-FISH) techniques. Single-cell transcriptome analysis revealed both divergent and conserved spatial expression patterns of key developmental regulators (objective1). Based on these outcomes, we selected conserved candidate transcription factors to assess their roles in lateral root development. The expression of these candidates has been validated by HCR RNA-FISH (objective 2). Accordingly, we generated negative dominant mutants by fusing the coding sequencing with SRDX domain. The SRDX domain is a short EAR (Ethylene-responsive element binding factor–associated Amphiphilic Repression) motif known to act as a strong transcriptional repressor when fused to transcription factors. This fusion converts normally activating TFs into dominant repressors, thereby facilitating the elucidation of their biological roles. The TF–SRDX fusion constructs were overexpressed under both the CaMV 35S and/or the C-fern EF1A promoters , and T1 progeny were obtained from the transgenic lines. Functional characterization is currently underway to evaluate root developmental defects (objective 3).
The results of this project have been presented at international conferences and meetings. Manuscripts are currently being prepared that will be submitted to open access and high impact journals in the near future. Additionally, the outcomes have been communicated to the general public through outreach activities and further shared via social media.
The results of this project have been presented at international conferences and meetings. Manuscripts are currently being prepared that will be submitted to open access and high impact journals in the near future. Additionally, the outcomes have been communicated to the general public through outreach activities and further shared via social media.
LAROSSE delivered the first single-cell gene expression atlas of fern roots, identifying key gene expression patterns across distinct cell types at single cell resolution. This represents a major advance in understanding root development in a sister lineage of seed plants, ferns. It also provides a valuable resource for future comparative and evolutionary studies. The transcription factor candidates identified in this work will shed light on the molecular regulation of root development and branching strategies in ferns, contributing essential knowledge on stem cell regulation across vascular plants. In the longer term, these insights may inform plant breeding strategies aimed at improving root architecture and enhancing plant resilience to climate change.