Leveraging single-cell transcriptomics to discover new genes in grass stomatal development

Lea S. Berg, Inés Hidalgo Prados, Nathan Lacombe, Paola Ruiz Duarte, Rashmi Tandon, Heike Lindner, Michael T. Raissig
Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern, Switzerland

Graminoid stomata in grasses consist of two dumbbell-shaped guard cells surrounding the central pore and two parallel subsidiary cells (SCs). This unique morphology is linked to faster stomatal movements of grasses contributing to water-use efficiency and fast saturation of assimilation. However, the genetic modules underlying the development of grass stomata remain to be fully understood. Here, we use single-cell RNA sequencing (scRNA-seq) to profile the transcriptome of developmental trajectories in the leaf epidermis of the wild model grass Brachypodium distachyon. Our current scRNA-seq dataset comprises more than 28’000 cells from the vegetative meristem and leaf primordia to differentiating leaves. Analyzing the epidermal subclusters readily identified the stomatal lineages and inferred pseudo-temporal trajectory of its developmental stages. Importantly, scRNA-seq of wild-type and bdmute mutants lacking SCs facilitated the bona fide identification of SC clusters. Many epidermal clusters and developmental stages were successfully confirmed in planta using reporter lines and whole-mount RNA in-situ hybridization. Finally, leveraging a resequenced mutant population identified several novel players in grass stomatal development. Together, our single-cell leaf atlas of B. distachyon can facilitate the discovery of novel genetic modules required for the formation and function of the morphologically innovative and functionally improved stomatal complexes of grasses.