Molecular mechanisms of plant low temperature stress tolerance

生物体在冰冻条件下的生存取决于它们耐受或缓解冻结/解冻过程产生的压力的能力。获得冰冻耐受性所涉及的机制确保在细胞水平上将损害降至最低。在小麦等耐寒植物中，低温暴露会导致数百种蛋白质的积累，因此其中许多蛋白质可能与耐寒性有关。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免受冰冻损害方面发挥作用。这些蛋白质可能在冷驯化过程中(秋季冷冻前)和/或脱驯化和恢复过程中(春季解冻后)需要。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，也需要防止冰冻造成的伤害。细胞和组织的长期储存是生物技术应用所必需的，例如药物毒性测试和移植。这种方法还需要使用低温保护物质，以保护细胞和组织免受脱水、渗透压变化和冷冻过程中结冰造成的损害。我们已经鉴定了四种小麦蛋白(WCS120、Tairi-2、TaENO和TaBAS1)，它们可以在冷冻过程中有效地保护肝和胰腺来源的细胞，另一种蛋白(TaCPN60)还有待鉴定。这些蛋白质表现出不同的结构特征，可能通过不同的作用机制起到低温保护剂的作用。TaENO、TaBAS1和TaCPN60蛋白在小麦中尚未被鉴定。 我的研究计划的长期目标是了解调节植物耐寒性的细胞和分子机制。我未来五年的短期目标是：1-确定TaENO、TaBAS1和TaCPN60是否与小麦中的FT有关；2-确定TaENO和TaBAS1在小麦中的功能；以及3-识别新的冰结合结构域，并通过蛋白质设计设计低温活性蛋白。交付成果具有基础性和广泛的应用范围：鉴定与植物耐冻性有关的蛋白质将为分子标记辅助育种提供工具，以促进新遗传系的发展；鉴定蛋白质/结构域和产生对哺乳动物细胞具有低温保护活性的嵌合蛋白质。我的多学科研究项目将在未来5年内为2名博士、2名硕士和5-10名本科生提供培训，培训环境符合公平、多样性和包容性原则。从我提议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，并将使加拿大的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.