Freezing behaviours in wintering Cornus florida flower bud tissues revisited using MRI

How plant tissues control their water behaviours (phase and movement) under subfreezing temperatures through adaptative strategies (freezing behaviours) is important for their survival. However, the fine details of freezing behaviours in complex organs and their regulation mechanisms are poorly understood, and non-invasive visualization/analysis is required. The localization/density of unfrozen water in wintering Cornus florida flower buds at subfreezing temperatures was visualized with high-resolution magnetic resonance imaging (MRI). This allowed tissue-specific freezing behaviours to be determined. MRI images revealed that individual anthers and ovules remained stably supercooled to -14 to -21°C or lower. The signal from other floral tissues decreased during cooling to -7°C, which likely indicates their extracellular freezing. Microscopic observation and differential thermal analyses revealed that the abrupt breakdown of supercooled individual ovules and anthers resulted in their all-or-nothing type of injuries. The distribution of ice nucleation activity in flower buds determined using a test tube-based assay corroborated which tissues primarily froze. MRI is a powerful tool for non-invasively visualizing unfrozen tissues. Freezing events and/or dehydration events can be located by digital comparison of MRI images acquired at different temperatures. Only anthers and ovules preferentially remaining unfrozen are a novel freezing behaviour in flower buds. Physicochemical and biological mechanisms/implications are discussed.