Transposable elements contribute to the establishment of the glycine shuttle in Brassicaceae species

C₃ -C₄ intermediate photosynthesis has evolved at least five times convergently in the Brassicaceae, despite this family lacking bona fide C₄ species. The establishment of this carbon concentrating mechanism is known to require a complex suite of ultrastructural modifications, as well as changes in spatial expression patterns, which are both thought to be underpinned by a reconfiguration of existing gene-regulatory networks. However, to date, the mechanisms which underpin the reconfiguration of these gene networks are largely unknown. In this study, we used a pan-genomic association approach to identify genomic features that could confer differential gene expression towards the C₃ -C₄ intermediate state by analysing eight C₃ species and seven C₃ -C₄ species from five independent origins in the Brassicaceae. We found a strong correlation between transposable element (TE) insertions in cis-regulatory regions and C₃ -C₄ intermediacy. Specifically, our study revealed 113 gene models in which the presence of a TE within a gene correlates with C₃ -C₄ intermediate photosynthesis. In this set, genes involved in the photorespiratory glycine shuttle are enriched, including the glycine decarboxylase P-protein whose expression domain undergoes a spatial shift during the transition to C₃ -C₄ photosynthesis. When further interrogating this gene, we discovered independent TE insertions in its upstream region which we conclude to be responsible for causing the spatial shift in GLDP1 gene expression. Our findings hint at a pivotal role of TEs in the evolution of C₃ -C₄ intermediacy, especially in mediating differential spatial gene expression.