Single-nuclei sequencing of Moricandia arvensis reveals bundle sheath cell function in the photorespiratory shuttle of C ₃ -C ₄ intermediate Brassicaceae

Spatially confined gene expression determines cell identity and is fundamental to complex plant traits. In the evolutionary transition from C ₃ to the more efficient C ₄ photosynthesis, restricting the glycine decarboxylase reaction to bundle sheath cells initiates a carbon concentrating mechanism via the photorespiratory glycine shuttle. This evolutionary step is generally thought to play an essential role in the progression from ancestral C ₃ to C ₄ photosynthesis. Plants operating this shuttle are often referred to as C ₃ -C ₄ intermediates or C ₂ species. Within the Brassicaceae family, which includes model plants and crops, such species have evolved independently at least five times. However, research on the biochemistry of C ₃ -C ₄ intermediates in the Brassicaceae has been limited to a few case studies of differentially localized proteins between mesophyll and bundle sheath cells. Here, we leveraged recent advances in single-cell transcriptome sequencing to better understand how new cellular specialization affects interconnected pathways. We generated a single-nuclei RNA sequencing dataset for Moricandia arvensis , a Brassicaceae with C ₃ -C ₄ intermediate characteristics, and compared it to a publicly available single-cell transcriptome of leaf tissue of the C ₃ Arabidopsis thaliana . We independently confirmed the localization of selected photorespiratory proteins by electron microscopy of immunogold-labelled leaf sections. Our analysis revealed the shift in expression of genes directly associated with the photorespiratory glycine decarboxylase reaction, but also of related pathways, such as ammonium assimilation, synthesis of specific amino acids, redox regulation, and transport to the M. arvensis bundle sheath. In contrast, the expression of these genes was not restricted to this cell type in the C ₃ plant.