FtsZ3 governs chloroplast division by regulating assembly and constriction of the chloroplast division machinery.

Chloroplast division, crucial for maintaining organelle homeostasis, relies on the chloroplast division machinery (CDM). However, the mechanisms underlying CDM assembly and constriction in non-vascular plants remain elusive. FtsZ3, a unique homolog of the bacterial cell division protein Filamentous temperature-sensitive Z (FtsZ), is specific to streptophyte algae, bryophytes, and lycophytes. Here, our data showed that FtsZ3 proteins from hornwort, moss, and lycophyte localize to chloroplasts and function as stromal components of the CDM. Using CRISPR/Cas9 gene editing in the moss Physcomitrium patens and conducting genetic and cytological analyses of targeted mutants, we identified a mechanism governing constriction of the chloroplast envelope membrane. Central to this mechanism is the temporospatial regulation of CDM assembly and constriction by FtsZ3. Notably, FtsZ3 exhibits functional divergence from FtsZ2, which serves as the structural backbone for FtsZ-ring formation. FtsZ3 has distinct domain-specific functions: its GTPase domain is crucial for self-assembly and restraining FtsZ2 assembly beneath the inner envelope membrane (IEM), while the conserved C-terminal motif mediates its interaction with the IEM protein Accumulation and Replication of Chloroplasts 6 (ARC6), which is essential for CDM constriction. This study reveals a mechanism of chloroplast division and offers valuable insights into the evolutionary adaptation of organelle division machinery across plant lineages.