Stomatal anatomy and gas exchange dynamics in the Brachypodium distachyon complex

Stomata, the epidermal pores enabling gas exchange and transpiration, are crucial for plant fitness and global water and carbon cycling. Grasses possess efficient four-celled, graminoid stomatal complexes, where dumbbell-shaped guard cells (GCs) and lateral subsidiary cells (SCs) enable rapid stomatal movements. Yet, how stomatal anatomical properties, such as size and density, affect stomatal gas exchange capacity and kinetics depends on species and growth conditions. Here, we used the Brachypodium distachyon complex, a trio of wild grass species consisting of diploid B. distachyon (genotype Bd21-3, 2n = 10), diploid B. stacei (genotype ABR114, 2n = 20), and their allotetraploid B. hybridum (genotype ABR113, 2n = 30), which arose from hybridisation between the two diploids, to integrate comparative stomatal anatomy, physiology, and cell biology and dissect the structure–function relationship of Brachypodium stomata. The tetraploid B. hybridum formed larger stomata with reduced density, while diploids both featured similarly small stomata yet higher densities. Steady-state gas exchange measurements reflected anatomical predictions in B. distachyon and B. hybridum. B. stacei, however, severely underperformed and showed significantly lower gas exchange capacity. Ultrastructural and cell wall composition analyses largely revealed conserved GC anatomy and cell wall properties across species. Critically, LM6—targeting pectic (1,5)-α-L-arabinans—showed differential SC wall localisation with signal in B. distachyon and B. hybridum but absence in B. stacei. We propose that altered arabinan distribution could modify SC wall biomechanics, potentially impairing their ability to accommodate GC movement and thus stomatal opening in B. stacei. While arabinans are implicated in stomatal flexibility across species, other factors (e.g., ecological niche, mesophyll photosynthesis, signalling, or unassessed wall components) may also contribute to B. stacei's reduced gas exchange.