Temporal dynamics and functional divergence of the chloroplast division apparatus in Oryza sativa

Chloroplast division is governed by a conserved protein machinery, yet empirical characterization of these regulators remains limited in rice, a primary target for C ₄ engineering. Increased chloroplast occupancy in bundle sheath cells is a hallmark of the C ₄ pathway and so manipulating division is a potential strategy to achieve this goal.

Through developmental transcript profiling and image analysis, we identified a discrete window of active chloroplast proliferation in rice leaves, coinciding with peak expression of conserved plastid division genes. Functional characterization via overexpression revealed regulatory behaviours distinct from those in Arabidopsis thaliana . Overexpression of OsFtsZ1&2 resulted in fewer, enlarged chloroplasts per bundle sheath cell, whereas OsMCD1&OsMinE restricted plastid expansion without altering division rates. Conversely, overexpressing OsPDV1&2 or OsARC6&OsDRP5B increased plastid size without affecting total count. When Os PDV1&2 were co-expressed with transcriptional regulator ZmG2 , we observed modest increases in chloroplast size alongside reduced stomatal aperture, increased stomatal density, and higher intrinsic water-use efficiency.

The results define the temporal landscape of plastid biogenesis in rice and demonstrate divergence across lineages. Our findings suggest that manipulating the division apparatus is insufficient to drive C ₄ -like chloroplast biogenesis in the rice bundle sheath, highlighting the complexity of plastid-host cell coordination in cereals.