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

Authors: Livingston, David; Tuong, Tan

Submitted to: Environmental and Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/16/2013
Publication Date: 12/25/2014
Citation: Livingston, D.P., Tuong, T.D. 2014. Understanding the response of winter cereals to freezing stress through freeze-fixation and 3d reconstruction of ice formation in crowns. Environmental and Experimental Botany. 106:24-33.

Interpretive Summary: Fixing plants that are in the frozen state allows researchers to process the plants using histology techniques and observe the effects of ice inside tissues. We froze a winter hardy oat cultivar and fixed the below ground portion of the stem called the crown while it was frozen. After processing the plant histologically we performed a 3 dimensional reconstruction of the images. We found that ice grew into the roots but ice did not migrate into the lower part of the crown. This supports a previously reported freezing tolerance mechanism of a root-shoot junction that keep ice from growing into the crown. Very little ice was found inside the center of the crown despite this being the most freezing sensitive part of the plant in the fall. The upper part of the crown had ice formations that resembled curtains rather than large spherical crystals as has previously been proposed using 2D visualization of ice formations. These findings demonstrate the need for visual data to understand complex interactions of plant with their environment such as in winter hardiness.

Technical Abstract: One of the most difficult aspects of understanding mechanisms involved in winterhardiness is knowing 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 respond to this form of stress. Cryo-sectioning and related techniques, while providing valuable information, only allow a two dimensional view of 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. Sections were aligned and background cleared to provide 3D visualization of voids that had formed within tissues as a result of freezing. The reconstruction in 3 dimensions revealed that ice had formed continuously in the roots but terminated at the root-shoot junction supporting previous research that a barrier exists at the base of the crown in freezing tolerant cultivars of winter cereals. In addition, where present within the crown, ice-induced voids were narrow and vertically inclined; they did not form large spherical shapes as has 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 up 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.