Conserved spatial patterning of gene expression in independent lineages of C₄ plants

C₄ photosynthesis enhances carbon fixation efficiency by reducing photorespiration through the use of an oxygen-insensitive carboxylase and spatial separation of photosynthesis between mesophyll and bundle sheath cells. The C₄ pathway has evolved independently in > 60 plant lineages, but molecular mechanisms underpinning this convergence remain unclear. To explore this, we generated high-resolution transcriptome atlases for two independently evolved C₄ dicotyledonous species - Gynandropsis gynandra (NAD-malic enzyme subtype) and Flaveria bidentis (NADP-malic enzyme subtype). We used both single-cell and single-nucleus RNA sequencing to capture gene expression profiles from individual leaf cells, enabling a detailed comparison between cell types and transcriptional signatures. While both approaches produced biologically comparable data for major leaf cell types, transcriptomes from single-nucleus sequencing showed lower stress signatures and were more representative of tissue proportions in the leaf. The single-nucleus data revealed that bundle sheath cells from both C₄ species share a gene expression pattern associated with mesophyll cells of C₃ plants. A conserved set of transcription factors, including members of the C₂H₂ and DOF families, was identified in the bundle sheath cells of both species. This study presents the first single-cell-resolution transcriptomes for two independent C₄ dicot lineages and provides a valuable resource, including a web-based portal for data visualisation.