Factors contributing to ice nucleation and sequential freezing of leaves in wheat

Authors: Livingston, David; BERTRAND, ANNICK - Agriculture And Agri-Food Canada; WISNIEWSKI, MICHAEL - Virginia Polytechnic Institution & State University; Tisdale, Ripley; Tuong, Tan; GUSTA, LARRY - University Of Saskatchewan; Artlip, Timothy - Tim

Submitted to: Planta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2021
Publication Date: 5/20/2021
Citation: Livingston, D.P., Bertrand, A., Wisniewski, M., Tisdale, R.H., Tuong, T.D., Gusta, L., Artlip, T.S. 2021. Factors contributing to ice nucleation and sequential freezing of leaves in wheat. Planta. 253:124. https://doi.org//10.1007/s00425-021-03637-w.
DOI: https://doi.org/10.1007/s00425-021-03637-w

Interpretive Summary: It was previously reported that roots and leaves of winter cereal crops such as oats, barley, wheat and rye freeze in a sequential manner with the oldest leaves freezing first and the youngest leaves sometimes never freezing at all. This was a counter-intuitive observation because all parts of the plant were at the same temperature. If the youngest leaves do not freeze then the entire plant will usually recover from freezing. This study was conducted to determine why the leaves of wheat froze in an age-related sequence rather than the entire plant freezing at once. We grew plants in growth chambers until they had 3 tillers and then sampled 6 leaves from the oldest to the youngest for a variety of measurements. 1. It has been shown that plants with larger water conducting vessels freeze first so we measured the diameter of the vessels in wheat and found that the 4 youngest leaves had vessel diameters that were closely related to their freezing order. 2. The vessel sizes in the 2 oldest leaves were not related to freezing order but were related to the amount of water they contained. 3. Sugars that are in solution can make plant tissues freeze at a lower temperature so we measured 15 different sugars in the 6 leaves. Sucrose is known to lower the freezing point of plant tissues and we found that it was related to freezing order in all 6 leaves. Other sugars were related to freezing order in some leaves but not in others 4. The amino acid proline was also directly related to freezing order. Other amino acids were partly related to freezing order. 5. The presence of bacteria and fungi can also affect when a plant freezes and we found numerous species of bacteria and fungi that were directly related to the freezing order of leaves. 6. Genes that code for proteins that initiate freezing were found to explain freezing in some leaves but not in others. This analysis indicates that leaves have different mechanisms that allow them to resist freezing and that they likely use a different combination of effects in different leaves. The results underscore the complexity of freezing tolerance in plants but give breeders several measurements that could be used to select freezing tolerant plants and allow them to generate varieties that are more winter hardy.

Technical Abstract: Previous analyses of freezing in wheat using infrared thermography demonstrated that individual leaves froze independently and in an age-related sequence with older leaves freezing first. This was a counterintuative observation because all parts of the plant were at the same temperature. In some cases the youngest leaves, just above meristematic tissues, supercooled down to -16C. This suggested that the whole plant could survive freezing by supercooling the youngest tissues that would give rise to new, undamaged leaves during recovery from freezing. To determine mechanisms that might explain this sequence of freezing we took several analytical approaches: 1) The size of xylem vessels, where freezing in leaves was initiated, were measured to see if capillary freezing point depression might explain sequential freezing. The four youngest leaves were correlated to the sequence of freezing, with the largest vessels freezing first, but the two oldest leaves were not. 2) Carbohydrate and amino acids were analyzed to determine if solute concentrations as well as interactions with membranes might explain the freezing sequence. Sucrose was highly correlated to the freezing sequence for all leaves but other simple sugars and fructans were not. The amino acid proline, serine and asparagine were correlated to the freezing sequence with older leaves having the lowest concentrations, while others were not. 3) Micro flora within and on leaf surfaces were determined to measure potential freezing initiation. Levels of Bacteria and fungi were correlated to freezing sequence for all leaves with mixed results of individual genera and species. Each of these factors as well as moisture content and transcript expression of ice binding proteins are discussed. As expected, results suggested that no single mechanism explained the freezing sequence observed in infrared analyses. Since each leaf is at a different growth stage, mechanisms that regulate freezing are likely operative in some leaves while not in others. This underscores the complexity of freezing in wheat but provides potential phenotypic characters that could be used by breeders to identify mechanisms that could be combined to develop more winter-hardy genotypes.