Compartmentation of photosynthesis gene expression between mesophyll and bundle sheath cells of C ₄ maize is dependent on time of day

Compared with the ancestral C ₃ state, C ₄ photosynthesis enables higher rates of photosynthesis as well as improved water and nitrogen use efficiencies. In both C ₃ and C ₄ plants rates of photosynthesis increase with light intensity and so are maximal around midday. We report that in the absence of light or temperature fluctuations, photosynthesis in maize peaks in the middle of the subjective photoperiod. To investigate molecular processes associated with these changes, we undertook RNA-sequencing of maize mesophyll and bundle sheath strands over a 24-hour time-course. Cell-preferential expression of C ₄ cycle genes was strongest between six and ten hours after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene co-expression analyses suggested members of the DOF and MADS-domain transcription factor families mediate diurnal fluctuations in C ₄ gene expression, and trans -activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators as well as peaks in cell-specific C ₄ gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.