Extracellular ice formation in special intercellular air spaces in Stachys byzantina C. Koch

Abstract

In the present study, the sites of extracellular ice formation within leaves and petioles of Stachys byzantina C. Koch were identified and anatomically studied by different imaging techniques. Naturally acclimated plant parts were analyzed during the winter season in various states (fixed, native, before freezing, frozen and after thawing) by using digital microscopy, cryo scanning electron microscopy or via µ‐CT. Besides ice bodies occurring in normal intercellular spaces within the mesophyll, larger ice accumulations developed at special sites close to the vascular bundle of the petiole and the adaxial leaf veins. At those sites, ice accumulated in mainly four continuous intercellular spaces, which are reversibly expandable. In those spaces, various “connective zones” allowed the cells to remain connected during the freezing process. Two of these large intercellular spaces are located on the abaxial side, the other two on the adaxial side. This symmetrical arrangement continues into the petiole. Due to the dehydrating effect of extracellular ice formation, the living tissue showed considerable shrinkage during freezing. However, almost no freezing induced wilting could be detected, probably due to the stabilizing effect of collenchyma and lignified vascular tissue.