Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.)

Authors: Livingston, David; Henson, Cynthia; Wise, Mitchell; Tuong, Tan; TALLURY, SHYAM - North Carolina State University; DUKE, STAN - University Of Wisconsin

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/15/2012
Publication Date: 12/2/2012
Citation: Livingston, D.P., Henson, C.A., Wise, M.L., Tuong, T.D., Tallury, S., Duke, S. 2012. Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.). PLoS One. 8(1): e53468.

Interpretive Summary: Rye, wheat, barley and oats are crops which can be planted in the fall and harvested before summer the following year. Growing the fall-planted form of these crops always results in a significant increase in yield over their spring-planted counterparts. Winter survival of the below-ground portion of the stem, called the crown, is the only restriction to growing a fall planted crop. Numerous studies have been conducted on the acclimation of winter crops during fall that allow them to withstand winter conditions. Very little research has been done on mechanisms which plants use to regrow after winter. In this study the 4 winter cereals listed above were frozen and histologically studied during a period of recovery from freezing. Oat had a unique response to freezing that none of the other crops had. It was a barrier that stained deep red and separated live from dead tissue within the crown. We used color recognition, fluorescence microscopy and 3D reconstruction to characterize this barrier in preparation for a study of the change in metabolism of the crown during freezing. This analysis will help scientists better understand the ability of crops to adapt to climate change and enable farmers to have more choices as they manage the numerous difficulties associated with global warming.

Technical Abstract: The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown with relatively normal cells on the outside edge of the crown from 1 to 14 days after freezing. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissue immediately inside this ring of cells were dead and those outside were alive. This boundary layer or barrier between living and dead cells became visible at day 3 and by day 7 formed a continuous Safranin staining structure surrounding the core of the crown. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. HPLC analysis of crown extracts indicated significant differences in fluorescing compounds between unfrozen samples and those that resumed growth after freezing. The percentage of Safranin staining tissue in frozen plants was significantly higher than in unfrozen plants at 3, 7 and 14 days after freezing. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown.