Understanding the response of winter cereals to freezing stress through freeze-fixation and 3D reconstruction of ice formation in crowns

One of the more difficult aspects of discovering mechanisms involved in winterhardiness is detecting where ice is formed and how it interacts with tissues in the frozen state. Many tissues recover and change shape during thawing which prevents a clear picture of ice formation and how individual cells might have responded to this form of stress. Cryo-sectioning and related techniques, while providing valuable information, only allow a two-dimensional view of what is in fact, a three-dimensional phenomenon. In this study, an established freeze-fixation protocol was used in conjunction with histology to visualize empty spaces or voids created by ice within crowns of oat. Images of sections were aligned and background color was cleared to provide 3D visualization of voids that had formed within tissues as a result of freezing. Reconstruction in 3 dimensions revealed that ice had formed continuously in the roots but terminated at the root-shoot junction. This supports previous research that a barrier exists at the base of the crown in freezing tolerant cultivars of winter cereals. In addition, ice-induced voids within the crown were narrow and vertically inclined; they did not form large spherical shapes as had previously been suggested from two-dimensional analysis. Within apical regions of the crown, voids always formed just below the epidermis on what would eventually become the lower surface of the leaf. The 3D structure of these formations resembled a curtain with a termination point at the top of the transition zone and which extended continuously into the leaves. These results suggest that multiple mechanisms must be operative concurrently for the crown to survive. This underscores the need for a variety of approaches that includes clear and detailed observational data to fully comprehend winter survival of cereal crops.