Molecular mechanisms of plant low temperature stress tolerance

生物体在冷冻条件下的存活取决于其耐受或减轻冷冻/解冻过程产生的应力的能力。获得抗冻性的机制确保在细胞水平上将损伤降至最低。在耐寒植物如小麦中，低温暴露导致数百种蛋白质的积累，因此其中许多蛋白质可能与抗冻性有关。目前的挑战是确定哪些蛋白质在耐受性中具有特定的作用，以及它们如何在细胞保护中发挥作用以免受冷冻损伤。这些蛋白质可能需要在冷驯化过程中（在秋季冷冻前）和/或在去驯化和恢复（在春季解冻后）。在这两个阶段都需要有效的机制来优化植物的越冬能力。对于储存在超低温下的动物细胞，也需要防止冷冻引起的损伤。细胞和组织的长期储存是生物技术应用如药物毒性测试和移植所需要的。这种方法还需要使用冷冻保护物质，以保护细胞和组织免受脱水，渗透压变化和冷冻期间冰形成引起的损伤。我们已经鉴定了四种小麦蛋白（WCS 120，TaIRI-2，TaENO和TaBAS 1），它们可以在冷冻过程中有效地保护肝脏和胰腺来源的细胞，另一种蛋白（TaCPN 60）仍有待鉴定。这些蛋白质显示出不同的结构特征，并可能通过不同的作用机制作为冷冻保护剂。TaENO、TaBAS 1和TaCPN 60蛋白尚未在小麦中表征。我的研究计划的长期目标是了解调节植物耐冻性的细胞和分子机制。我未来五年的短期目标是：1-确定TaENO，TaBAS 1和TaCPN 60是否与小麦中的FT相关; 2-确定TaENO和TaBAS 1在小麦中的功能; 3-通过蛋白质设计鉴定新的冰结合结构域并设计冷冻活性蛋白。交付成果是基础性的，并在范围内得到应用：鉴定与植物抗冻性相关的蛋白质将为标记辅助育种提供工具，以促进新遗传系的开发;鉴定蛋白质/结构域并产生具有哺乳动物细胞抗冻活性的嵌合蛋白质。我的多学科研究计划将在未来5年内为2名博士，2名硕士和5-10名本科生提供培训，在符合公平，多样性和包容性原则的环境中。从我提出的研究计划中获得的知识将有助于保持加拿大在植物研究和细胞冷冻保存方面的竞争优势，并将使加拿大植物农业和生物技术部门受益。

The survival of organisms subjected to freezing conditions depends on their ability to tolerate or alleviate stresses resulting from the freeze/thaw processes. The mechanisms involved in the acquisition of freezing tolerance ensure that damage is minimized at the cellular level. In cold-hardy plants such as wheat, low temperature exposure leads to the accumulation of hundreds of proteins, and many of them are therefore potentially associated with freezing tolerance. The current challenge is to identify which proteins have a specific role in tolerance, and how they function in cellular protection against freezing damage. These proteins are likely required in the cold acclimation process (pre-freezing in the fall) and/or in deacclimation and recovery (post-thaw in the spring). Efficient mechanisms are needed at both stages for optimal overwintering capacity of the plants. Protection against freezing-induced injuries is also needed for animal cells that are stored at ultra-low temperatures. Long-term storage of cells and tissues is needed for biotechnological applications such as drug toxicity testing and transplantation. This approach also requires the use of cryoprotective substances that protect cells and tissues from dehydration, changes in osmolarity and damage caused by ice formation during freezing. We have identified four wheat proteins (WCS120, TaIRI-2, TaENO and TaBAS1) that can efficiently protect liver and pancreas-derived cells during freezing, and another that remains to be characterized (TaCPN60). These proteins show different structural features and likely act as cryoprotectants via distinct mechanisms of action. The TaENO, TaBAS1 and TaCPN60 proteins have not been characterized in wheat. The long-term objective of my research program is to understand the cellular and molecular mechanisms that regulate freezing tolerance in plants. My short-term objectives for the next five years are to: 1- Determine if TaENO, TaBAS1 and TaCPN60 are associated with FT in wheat; 2- Determine the functions of TaENO and TaBAS1 in wheat; and 3- Identify novel ice-binding domains and engineer cryoactive proteins via protein design. The deliverables are fundamental and applied in scope: the identification of proteins associated with freezing tolerance in plants will provide tools for marker-assisted breeding, to facilitate the development of new genetic lines; and the identification of proteins/domains and generation of chimeric proteins with cryoprotective activity for mammalian cells. My multidisciplinary research program will provide training for 2 PhD, 2 MSc and 5-10 undergraduate students during the coming 5-year period, in an environment that complies with the equity, diversity and inclusion principles. The knowledge gained from my proposed research program will contribute to maintaining Canada's competitive edge in plant research and cell cryopreservation, and will benefit the Canadian plant agriculture and biotechnology sectors.