Single-cell multi-omics reveals distinct regulatory architectures coordinating carbon assimilation and cyclic...

C₄ photosynthesis arose repeatedly through redeployment of pre-existing regulatory circuitry, yet how a shared regulatory system coordinates its interdependent photosynthetic functions remains unresolved. Here we integrate single-nucleus RNA-seq and sci-ATAC-seq with bulk histone profiles from mesophyll- and bundle sheath-enriched fractions and transcription factor binding profiles obtained in protoplasts. We compare the C₄ shuttle and Calvin-Benson-Bassham cycle with NDH-mediated cyclic electron flow in bundle sheath cells. Selected carbon assimilation loci were associated with differential H3K27me3, whereas the NDH-associated branch showed regulatory inputs linked to DNA binding with one finger (DOF) transcription factors. Dissection of the ZmNdhU promoter identifies a 49 bp DOF-responsive cis element whose activity is sensitive to local sequence context and insertion position in the promoter constructs tested and that supports vascular bundle-associated reporter expression in a heterologous promoter context. Bundle sheath-enriched expression of ZmDOF30 increases NDH-associated expression, cyclic electron flow, leaf ATP content and photosynthetic performance. In a field trial, the transgenic lines showed increased growth and biomass. Comparative analyses revealed lineage-associated variation in predicted promoter-proximal DOF-binding potential within a broader H3K27me3-associated transcription factor background. These findings distinguish regulatory configurations among maize photosynthetic modules and identify DOF-NDH regulation as a candidate route for improving photosynthetic performance in maize, with broader relevance to NADP-ME grasses.