A bipartite transcription factor module controlling expression in the bundle sheath of Arabidopsis thaliana

C₄ photosynthesis evolved repeatedly from the ancestral C₃ state, improving photosynthetic efficiency by ~50%. In most C₄ lineages, photosynthesis is compartmented between mesophyll and bundle sheath cells, but how gene expression is restricted to these cell types is poorly understood. Using the C₃ model Arabidopsis thaliana, we identified cis-elements and transcription factors driving expression in bundle sheath strands. Upstream of the bundle sheath preferentially expressed MYB76 gene, we identified a region necessary and sufficient for expression containing two cis-elements associated with the MYC and MYB families of transcription factors. MYB76 expression is reduced in mutant alleles for these transcription factors. Moreover, downregulated genes shared by both mutants are preferentially expressed in the bundle sheath. Our findings are broadly relevant for understanding the spatial patterning of gene expression, provide specific insights into mechanisms associated with the evolution of C₄ photosynthesis and identify a short tuneable sequence for manipulating gene expression in the bundle sheath.