Bringing seed barriers into focus: Optimized clearing and histological staining

Plant reproduction relies on tightly coordinated developmental programs that integrate environmental and endogenous signals. Within seeds, lipid-based extracellular polymers such as cutin and suberin are deposited at defined apoplastic interfaces, where they function as selectively permeable barriers. These structures are actively remodeled by hormonal and metabolic cues to regulate hydration, nutrient exchange, and stress responses. Although the genetic and biochemical pathways underlying these barriers are increasingly well understood, their spatial organization and developmental dynamics remain difficult to investigate because they are embedded within optically dense and multilayered tissues. Existing clearing approaches have substantially improved visualization of internal seed structures, yet systematic whole-mount imaging of multiple lipid-based barriers across development, particularly in combination with fluorescent reporters, remains challenging. To address this, we developed an optimized pipeline that enables rapid and efficient seed tissue clearing and selective visualization of polyaliphatic barriers across four key seed interfaces: the seed surface, seed coat suberin, cuticle at the endosperm/seed coat boundary, and embryo surface cuticle. The method preserves the spatial context of these barriers within intact seeds and enables their co-visualization with fluorescence-based marker lines. Using a common whole-mount imaging framework across successive developmental stages, we follow changes in the spatial organization of distinct barrier domains. The approach is also applicable to more complex seeds, such as those of Camelina sativa, and can be extended to other anatomically complex plant organs by combining the clearing and staining workflow with vibratome sectioning.