LeafMap · Evolution of leaf shape diversity in Angiosperms using single cell approaches

Evolution of leaf shape diversity in Angiosperms using single cell approaches

The LeafMap project aimed to uncover how genetic networks influence the extraordinary diversity of leaf morphologies in flowering plants. While leaves of different species begin their development as morphologically indistinguishable primordia, they develop into distinct leaf forms through changes in developmental programs. Understanding this process at the resolution of individual cells and across evolutionary lineages represents a significant challenge in plant developmental biology. To address this, the project employed state-of-the-art single-cell and spatial genomics approaches in both Brassicaceae (Arabidopsis thaliana, Cardamine hirsuta) and Solanaceae (Solanum lycopersicum, Capsicum annuum) lineages, which have independently evolved complex leaves. The objectives were threefold: (1) reconstructing the molecular basis of leaf development at cellular resolution in Brassicaceae, (2) identifying spatial transcriptional patterns during leaf morphogenesis, and (3) comparing the evolution of regulatory networks underlying leaf complexity across lineages. Throughout the fellowship, substantial progress was made toward these objectives. High-quality single-cell multiome datasets were generated across four species, providing the first multiome-based comparative atlas of simple versus complex leaf development. These analyses revealed both conserved and lineage-specific cell states and regulators, as well as convergence in the redeployment of meristematic programs to support leaf complexity. In parallel, experimental pipelines for nuclei isolation and whole-mount FISH were established, ensuring robust validation capacity. Progress in spatial transcriptomics was slower than anticipated, but contingency strategies were implemented to address this. Overall, the project has delivered novel insights into the evolution of leaf development, generated valuable datasets and methodological advances for the community, and positioned the fellowship holder at the forefront of plant single-cell biology. 1.1 Objectives Objective 1: Reconstructing the genetic basis of leaf development and complexity at cellular resolution. This objective offers a comprehensive understanding of leaf development from a genome-wide and cellular perspective. Under Objective 1, we successfully established a robust nuclei isolation and single-cell multi-omics pipeline for Arabidopsis thaliana and Cardamine hirsuta, generating high-quality single-cell transcriptome and chromatin accessibility datasets across various developmental stages. The generated data revealed both conserved and species-specific cell populations. Notably, regulators involved in leaf complexity, such as STM and RCO, could be traced within specific cell states, providing mechanistic insights into the developmental divergence between simple and complex leaves. Additionally, the multimodal data allows us to understand not only the gene expression of different cell populations but also their regulatory landscapes, thanks to the paired chromatin accessibility data. However, due to the large volume of data generated, analyzing and interpreting the chromatin accessibility data is still ongoing. Objective 2: Discovery and exploration of GRNs in spatial context during leaf morphogenesis Progress was slower than expected due to technical difficulties, especially the discontinuation of suitable commercial kits for high-resolution spatial transcriptomics in plants. This unexpected problem led us to put contingency plans into action. The host institute invested in new equipment to process frozen sections, including a CryoStar NX70 cryostat from Epredia (approx. 70,000 EUR), which enabled us to explore multiple options to achieve our goal. So far, we have successfully optimized cryosectioning of the target plant materials and performed pilot experiments to support the future success of these methods. Additionally, a multi-color whole-mount RNA in situ hybridization technique was adopted using Cardamine hirsuta shoot and leaf tissues. This pilot study aims to locate cell cycle regulators during leaf development and is currently being used to identify candidate regulators based on the single-cell data generated. This successful optimization highlights our dedication to improving our techniques for the benefit of the project. Objective 3: Understanding leaf complexity from an evolutionary point of view Under Objective 3, we extended the single-cell multiome framework to Solanaceae, generating a cross-lineage dataset of ~180,000 nuclei from four species. Current improvements in machine learning techniques have also enabled us to create a common embedding of the transcriptome space across the four species, which opens up new avenues for comparative single-cell studies. Our findings suggest that the convergent recruitment of meristematic programs in the evolution of complex leaves is a significant step towards understanding leaf complexity from an evolutionary perspective.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз