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

C-3-C-4 intermediate photosynthesis has evolved at least five times convergently in the Brassicaceae, despite this family lacking bona fide C-4 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.<br />In this study, we used a pan-genomic association approach to identify genomic features that could confer differential gene expression towards the C-3-C-4 intermediate state by analysing eight C-3 species and seven C-3-C-4 species from five independent origins in the Brassicaceae.<br />We found a strong correlation between transposable element (TE) insertions in cis-regulatory regions and C-3-C-4 intermediacy. Specifically, our study revealed 113 gene models in which the presence of a TE within a gene correlates with C-3-C-4 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 C-3-C-4 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.<br />Our findings hint at a pivotal role of TEs in the evolution of C-3-C-4 intermediacy, especially in mediating differential spatial gene expression.