Cytological observations of the epithem and the epithem-mesophyll interface in Arabidopsis hydathodes

Hydathodes are specialized leaf tissues that mediate guttation, the release of liquid water droplets from vascular plants. They consist of xylem endings, water pores, and the epithem, a tissue of small cells located between the xylem endings and water pores. Although previous studies have provided anatomical descriptions and emerging molecular insights into Arabidopsis thaliana hydathodes, how epithem cells differ from surrounding cells and how the epithem interfaces with surrounding tissues remain incompletely understood. In this study, we examined the anatomy of A. thaliana hydathodes using complementary imaging approaches. Microfocus-X-ray computed tomography visualized the three-dimensional morphology of hydathodes protruding from the abaxial leaf surface. Light microscopy showed that the epithem consisted of densely packed cells with limited intercellular spaces. Quantitative analyses showed that the cross-sectional area of epithem cells was approximately 25-40% that of mesophyll cells, and that epithem cells exhibited lower plastid occupancy. We further characterized cells located along the epithem-mesophyll boundary, which we provisionally refer to as boundary-like cells based on their position. These cells were closely arranged with limited intercellular spaces, contrasting with the prominent intercellular spaces among surrounding mesophyll cells. Despite this organization, quantitative analyses indicated that boundary-like cells more closely resembled mesophyll cells than epithem cells. To examine whether proteins from different cellular compartments enter guttation fluid, we used transgenic plants expressing GFP targeted to the apoplast, cytosol, or ER lumen. GFP was detected in guttation droplets only from plants expressing apoplastic GFP. Together, these findings refine our understanding of the cellular anatomy of A. thaliana hydathodes and provide a reporter-based approach for examining the movement of apoplastic macromolecules into guttation fluid.