Ice and air: visualisation of freezing spread and freeze–thaw embolism in young <i>Liriodendron tulipifera</i> leaves

Spring freezing is an unforgiving stress for young leaves, often leading to death and with consequences for tree productivity and survival. While both the water-transport system and living tissues are vulnerable to freezing, we do not currently know whether damage to one or both of these systems causes death in leaves exposed to freezing. In this study, whole saplings of Liriodendron tulipifera were exposed to freezing and thawing trajectories designed to mimic natural spring freezes. We monitored the formation of freeze-thaw xylem embolism and damage to photosynthetic tissues and found a predictable progression of ice formation across the leaf surface that was strongly influenced by leaf- vein architecture, notably the presence or absence of bundle-sheath extensions. Our results also showed that freeze-thaw embolism occurred only in the lowest vein orders where mean vessel diameter exceeded 30 µm. With evidence of both freeze-thaw embolism and damage to photosynthetic tissue, we conclude that this dual-mode of lethality in leaves might be common among other wide-vesseled angiosperm leaves, potentially playing a role in limiting geographic distributions, and demonstrate that bundle sheath extensions might stall or even prevent freezing spread.