Transcriptional targets of SPEECHLESS and FAMA control guard cell division and expansion in the late stomatal lineage

A key feature of plant development is the ability to maintain post-embryonic stem cell niches which provide a constant source of new cells for forming organs. In the process, precursor cells can lose division potential in favour of differentiation, acquiring specific identities and functions. A simple example of this trajectory is in the stomatal lineage, where epidermal cells transit different stages as they differentiate into stomatal guard cells. In Arabidopsis, stomatal development is orchestrated by three closely related basic helix-loop-helix (bHLH) transcription factors, SPEECHLESS (SPCH), MUTE and FAMA; which act in sequential steps along the differentiation trajectory. SPCH has a key role at the onset of the lineage, where it promotes asymmetric division and early stomatal identity. At the opposite end of the lineage, FAMA enforces guard cell identity and represses additional divisions of already differentiated cells. Recent data, showing that SPCH is present until late in the lineage where it is necessary for cells to commit to stomatal fate, however, challenges this classic view. How SPCH interacts with FAMA, and what SPCHs targets are at this late stage have remained open questions. Here, we examine the role of SPCH during late stomatal development and show that relative levels and activities of SPCH and FAMA tightly control cell division and expansion. Using a cell-type-specific RNA expression approach, we identify factors downstream of SPCH and FAMA in the late lineage and characterize their temporal and spatial localization. Finally, we demonstrate that timely regulation of late transcriptional targets of SPCH and FAMA is necessary for guard cell morphology and function.