Freezing dynamics and injury in overwintering buds

Theoretical framework: Survival of overwintering buds is critical for reproduction, growth, species distribution, and crop productivity. Buds have diverse architectures, but their role in cold hardiness remains poorly understood. Cold hardiness is a dynamic trait. Climate warming increases frost risk as buds fail to develop sufficient cold hardiness without cold cues. Molecular and phenological studies exist, but mechanistic understanding of freezing at the bud level is limited. Understanding how architecture governs ice formation, accommodation, and injury is essential to predict frost risk. Hypotheses: We hypothesize that bud survival depends on bud architecture, freeze-induced dehydration, and biochemical traits. We will reassess freezing survival typologies, identify structural and chemical features enabling supercooling or ice segregation, and determine whether injury arises from intracellular ice or dehydration. This will improve understanding of cold hardiness and frost vulnerability under climate change. Approach: Woody species will be grown at three locations with distinct temperature regimes under the same photoperiod. Cold hardiness will be linked to structural and functional traits. Freezing dynamics (ice formation, supercooling vs. freeze dehydration, water frozen per temperature) will be studied with differential scanning calorimetry and psychrometry. Sublethal and lethal ice masses will be visualized with thermography and cryomicroscopy. Regions where ice forms or is inhibited will be analyzed with quantitative microscopy, Raman spectroscopy, and atomic force microscopy. Seasonal carbohydrate deposition will be mapped to assess its role in supercooling. Differential scanning calorimetry and cold hardiness tests will determine whether intracellular freezing or critical dehydration causes injury. Transmission electron microscopy of frozen samples will reveal membrane or organelle dysfunction. Innovation: This project is innovative because it addresses how buds remain ice-free despite ice in surrounding tissues. Integrating thermal, structural, and biochemical analyses will clarify whether injury is driven by intracellular ice or dehydration. Outcomes will identify protective metabolites and structural traits for breeding resilient crops and trees, and improve models predicting frost damage, biodiversity shifts, and forest stability under climate change.