A Three Dimensional View of Damage in Oat Crown Tissue Recovering From Freezing.

The physiology of freezing tolerance in small grains (oats, barley, wheat and rye) is usually studied by analyzing the metabolism and genetics of plants prior to freezing, during cold- and freeze-acclimation. We wanted to study the physiology of plants after freezing, during a period of recovery when the plant either dies or produces new tissue and survives. We froze oat plants at -12C and after thawing, transplanted them under controlled conditions. At 0, 1, 3 7, 14, and 21 days after transplanting we harvested crown tissue and produced over 100 serial sections of the bottom 4mm of each plant. A stain was developed which allowed us to identify freeze-damaged tissue. Photographs of each 0.025mm-thick section was imported into Adobe After Effects and processed to produce a 3D image of the crown.  Using this approach we identified a region of the crown that forms a barrier around dead tissue and apparently prevents the expansion of damage into meristematic regions. A metabolomic analysis to identify the chemical nature of the barrier will be discussed. Color recognition software was used to precisely quantify the volume of the barrier in each crown; this volume increased over time during recovery from freezing. Despite considerable damage to a region of the crown called the crown-core, most tillers of plants frozen at -12°C survived. However, it was discovered that instead of the shoot apex surviving freezing, lateral buds, began to grow during recovery, giving rise to new tillers that allowed the plant to survive the freezing event. This suggests that survival of a crown is dependant on survival of the meristematic tissue called the transition zone where lateral buds survive and begin to grow. Hence, biochemical analysis which attempts to explain survival mechanistically should concentrate on the transition zone instead of the entire crown.