Spatial and Temporal Freezing Dynamics of Leaves Revealed by Time-Lapse Imaging.

Freezing air temperatures kill most leaves, yet the leaves of some species can survive these events. Tracking the temporal and spatial dynamics of freezing remains an impediment to characterizing frost tolerance. Here we deploye time-lapse imaging and image subtraction analysis, coupled with fine wire thermocouples, to discern the in situ spatial dynamics of freezing and thawing. Our method of analysis of pixel brightness reveals that ice formation in leaves exposed to natural frosts initiates in mesophyll before spreading to veins, and that while ex situ xylem sap freezes near 0°C, in situ xylem sap has a freezing point of -2°C in our model freezing-resistant species of Lonicera. Photosynthetic rates in leaves that have been exposed to a rapid freeze or thaw do not recover, but leaves exposed to a slow, natural freezing and thawing to -10°C do recover. Using this method, we are able to quantify the spatial formation and timing of freezing events in leaves, and suggest that in situ and ex situ freezing points for xylem sap can differ by more than 4°C depending on the rate of temperature decline.