Stomatal-based immunity differentiation across vascular plant lineages

Some plants are known to actively close their stomata in the presence of foliar pathogens, inhibiting pathogen entry into leaves, leading to 'stoma-based immunity' as the first line of defense. However, the variation in stoma-based innate immunity across the diversity of vascular plants remains unclear. Here, we investigated the stomatal response and guard cell signaling pathway in various seed plant, fern, and lycophyte species when exposed to the bacterial pathogens or pathogen-associated molecular patterns (PAMPs). We observed active stomatal closure in 10 seed plants when exposed to bacteria or PAMPs, whereas none of the nine fern and one lycophyte species exhibited this response. The PAMP flg22-induced reactive oxygen species burst was observed in all species, but the downstream signaling events, including cytosolic Ca²⁺ accumulation, nitric oxide production, ion fluxes, vacuolar acidification, cytoplasmic pH elevation, vacuolar compartmentation, and disaggregation of the actin cytoskeleton in guard cells, were only observed in seed plants. No such changes were observed in the representatives of ferns and lycophytes. Our findings suggest a major difference in the regulation of stomatal immunity between seed plants and ferns and lycophytes under this study's conditions, unveiling physiological and biophysical mechanisms that may have underpinned the evolutionary adaptation of stomatal responses to pathogen attacks in seed plants.