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

C3-C4 intermediate photosynthesis has evolved multiple times convergently and independently in the Brassicaceae, although the family lacks bona fide C4 species. Evolution of C3-C4 intermediate photosynthesis requires a reconfiguration of gene-regulatory networks, modified spatial expression patterns of multiple genes and ultrastructural adjustments. Mechanisms underpinning the reconfiguration of these networks are mostly unknown. In this study, we use a pan-genomic association approach to identify genomic features that might confer differential gene expression towards C3-C4 intermediate traits. We found a strong correlation between transposon insertions in cis-regulatory regions and the C3-C4 intermediacy trait. Our study revealed 222 orthogroups where 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. The P-protein of the glycine decarboxylase enzyme complex is known to play a crucial role in the establishment of the photorespiratory glycine shuttle. We found independent transposon insertions in the promoter sequences of the gene encoding the P-protein and hypothesize that these insertions lead to a spatial shift in gene expression. Our findings hint at a pivotal role of TEs in the evolution of C3-C4 intermediacy, especially in mediating differential gene expression.