Extracellular ice management in the frost hardy horsetail Equisetum hyemale L.

- Extracellular ice forms in the vallecular canals and the pith cavity.
- For the pith cavity, ice formation depends on position along the shoot.
- Stomatal antechambers are always ice-free due to structured surface.
- Internal canal system provides extensive storage system for extracellular ice.

Formation of extracellular ice at specific positions in the plant interior is a common and probably essential component of plant cold hardiness. Studies on extracellular freezing in spore-bearing plants are, however, scarce. In this study, extracellular ice formation in the cold hardy horsetail Equisetum hyemale L. is analyzed. Horsetails show an extensive system of intercellular air spaces which are probably crucial for internal ice storage during winter. Previous studies emphasized the spacious pith cavity as the main place for ice crystal growth. Shoots were studied during summer and in the frozen state in winter, after natural acclimatization, by using digital (incident light) microscopy, Scanning Electron Microscopy and Cryo Scanning Electron Microscopy. It was shown that the vallecular canals also contain a large share of ice bodies under freezing conditions. The vallecular canals, which are directly seated within the cortex and whose interior is directly connected to the cortex via gaps in the canal wall, were often and rapidly filled with ice. The pith cavity also contained ice, depending on the position along the shoot and the internode. The carinal canals contained almost no ice crystals. Furthermore, some ice crystals were detected in the intercellular spaces of the chlorenchyma and the substomatal chamber. The stomatal antechamber, however, was always ice-free, probably due to the presence of water-repellent wax crystals. The results of this study support available evidence for the crucial role of pre-existing extensive lacunae for extracellular ice formation in E. hyemale. Furthermore, the findings indicate that anatomical details of canal structure and position are important for the pattern of extracellular ice accumulation.