Oral Presentation International Plant Molecular Biology Conference 2024

Getting into shape - a grass-specific factor guides morphogenesis and pore formation in the grass stomatal complex (#548)

Roxane Patricia Spiegelhalder 1 , Anakine Prizins 2 , Dan Zhang 2 , Paola Ruiz-Duarte 1 , Heike Lindner 1 , Michael T. Raissig 1
  1. Institute for plant science (IPS), University of Bern, Bern, Switzerland
  2. Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany

Stomata are epidermal valves at the leaf surface that open and close to absorb photosynthetic carbon dioxide and restrict water loss through transpiration. Grasses form morphologically innovative stomata, consisting of two dumbbell-shaped guard cells (GCs) flanked by two lateral subsidiary cells (SCs). This “graminoid” morphology is associated with faster stomatal movements and more water use-efficient gas exchange. Yet, the developmental programs that shape GC morphogenesis and pore formation are unknown. A forward genetic screen in the model grass Brachypodium distachyon identified a mutant phenotype that showed stocky and undifferentiated (“pouty”) or collapsed GCs (“thin-lipped”). We mapped the mutation to a grass-specific gene of unknown function and were able to reproduce the “thin-lipped and pouty” (tlp) phenotype using CRISPR/Cas9-based gene editing. In developing GCs, fluorescently tagged TLP reporter proteins accumulate at the plasma membrane at apical and basal sides of the GMC and, following the symmetric division, in a highly polarized manner on apical and basal ends of the newly formed central GC wall. Overexpression of the TLP reporter was able to rescue the tlp mutant phenotype. Careful 3D microscopy of tlp stomata revealed impaired pore formation and aberrant morphogenesis of the dumbbell-shaped grass GCs. Finally, disrupting polarized localization of TLP in the developing GCs and forcing TLP equally to the whole plasma membrane showed strong dominant negative effects with misformed pores and missing or misshapen central cell walls. In conclusion, a grass-specific, polarized factor shapes the unique morphogenesis of the dumbbell-shaped GCs and guides pore formation in grasses.