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Title: Evidence of cyclical light/dark-regulated expression of freezing tolerance in young winter wheat plants

item Skinner, Daniel
item Bellinger, Brian
item HISCOX, WILLIAM - Washington State University
item HELMS, GREGORY - Washington State University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/2018
Publication Date: 6/18/2018
Citation: Skinner, D.Z., Bellinger, B.S., Hiscox, W., Helms, G. 2018. Evidence of cyclical light/dark-regulated expression of freezing tolerance in young winter wheat plants. PLoS ONE. 13(6).

Interpretive Summary: Tolerance of subfreezing temperatures is essential for winter wheat plants, seeded in the autumn and harvested the following summer. The environmental influence on the expression of freezing tolerance is poorly understood. This study demonstrated that freezing tolerance of the plants varies dramatically throughout the 24-hour day when the plants were grown at constant 4 degrees Celcius and 12 hours light/12 hours dark. Two cycles of high-to-low tolerance were found, one in the light and one in the dark. Specific genes known to regulate a major part of tolerance also showed two cycles of high, then low expression in the 24-hour period, as was glutamine, a cellular metabolite. These results provide a new level of understanding of the expression and control of freezing tolerance and the influence of environmental conditions. From a practical standpoint, these results provide guidance on the standardization of freezing tolerance testing conditions in order to facilitate meaningful comparisons of results.

Technical Abstract: The ability of winter wheat (Triticum aestivum L.) plants to develop freezing tolerance through cold acclimation is a complex trait that responds to many environmental cues including day length and temperature. A large part of the freezing tolerance is conditioned by the C-repeat binding factor (CBF) gene regulon. We investigated whether the level of freezing tolerance of 12 winter wheat lines varied throughout the day and night in plants grown under a constant low temperature and a 12-hour photoperiod. Freezing tolerance was significantly greater (P<0.0001) when exposure to subfreezing temperatures began at the midpoint of the light period, or the midpoint of the dark period, compared to the end of either period, with an average of 21.3% improvement in survival. Thus, freezing survival was related to the photoperiod, but cycled from low, to high, to low within each 12-hour light period and within each 12-hour dark period, indicating ultradian cyclic variation of freezing tolerance. Quantitative real-time PCR analysis of expression levels of CBF genes 14 and 15 indicated that these two genes also were regulated in ultradian fashion, but essentially 180° out of phase with each other. Proton nuclear magnetic resonance analysis (1H-NMR) showed that the chemical composition of the wheat plants' cellular fluid varied diurnally, with consistent separation of the light and dark phases of growth. A compound identified as glutamine was consistently found in greater concentration in a strongly freezing-tolerant wheat line, compared to moderately and poorly freezing-tolerant lines. The glutamine also varied in ultradian fashion in the freezing-tolerant wheat line, consistent with the ultradian variation in freezing tolerance, but did not vary in the less-tolerant lines. These results suggest at least two distinct signaling pathways, one conditioning freezing tolerance in the light, and one conditioning freezing tolerance in the dark, both are at least partially under the control of the CBF regulon, and a response pathway involving glutamine appears to be active in strongly freezing-tolerant lines, but not less-tolerant lines.