Targeting editing of tomato SPEECHLESS cis-regulatory regions generates plants with altered stomatal density in response to changing climate conditions

Flexible developmental programs enable plants to customize their organ size and cellular composition. In leaves of eudicots, the stomatal lineage produces two essential cell types, stomata and pavement cells, but the total numbers and ratio of these cell types can vary. Central to this flexibility is the stomatal lineage initiating transcription factor, SPEECHLESS (SPCH). Here we show, by multiplex CRISPR/Cas9 editing of SlSPCH cis-regulatory sequences in tomato, that we can identify variants with altered stomatal development responses to light and temperature cues. Analysis of tomato leaf development across different conditions, aided by newly-created tools for live-cell imaging and translational reporters of SlSPCH and its paralogues SlMUTE and SlFAMA, revealed the series of cellular events that lead to the environmental change-driven responses in leaf form. Plants bearing the novel SlSPCH variants generated in this study are powerful resources for fundamental and applied studies of tomato resilience in response to climate change.