Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing

Abstract

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to −45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to −45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.

Frost Survival Mechanism of Vegetative Buds in Temperate Trees: Deep Supercooling and Extraorgan Freezing vs. Ice Tolerance

“…In 50% of all tested species, translocated ice masses formed exclusively around the premature leaves inside the bud. While in DTA, no freezing exotherms were detectable during formation of translocated ice between the premature leaves, in some species in IDTA occasionally exiguous freezing processes were recorded (e.g., A. alnobetula : Neuner et al, 2019). In temporarily supercooled buds, no preferential location of ice masses could be found (Figure 7).…”

“…The table lists the species examined, the mean temperature (°C) of the high temperature exotherm (HTE) and the low temperature exotherm (LTE) as determined by differential thermal analysis (DTA) and mean midwinter FR (LT 50 ) of winter 2015/16, and the midwinter FR reported by other authors. For supercooling buds, locations of formation of extraorgan ice masses are additionally shown: S, subtending stem; Sc, inside bud scales; L, between premature leaves; nd, not detectable; –, not examined .aBannister and Neuner (2001);bSakai (1982);cSakai and Larcher (1987);dHofmann et al (2015);eCharrier et al (2013);fFilippi (1986),gBenowicz et al (2000);hLenz et al (2016);iNeuner et al (2019);jKuprian et al (2017);kBeuker et al (1998);lBuchner and Neuner (2011);mSchiffmann (2017);nRiikonen et al (2013);oVitra et al (2017);*Dereuddre (1979);** between innermost scale and leaves .…”

“…While reproductive buds of many angiosperms exhibit deep supercooling (Quamme, 1995), their vegetative buds, based upon previous studies, have been suggested to survive freezing temperatures 10 to 15°C lower than reproductive buds, by undergoing extracellular freezing (Sakai and Larcher, 1987). Exceptions, however, have been reported ( Pyrus syriaca : Rajashekar and Burke, 1978) and more recently added ( Acer japonicum : Ishikawa et al, 1997; Malus domestica : Pramsohler and Neuner, 2013; Alnus alnobetula : Neuner et al, 2019).…”

“…While the bud tissues are supercooling, water migrates from the cells to locations that lie outside of the bud, such as the subtending stem or the bud scales. In A. alnobetula , ice masses can even form inside the bud between the premature leaves (Neuner et al, 2019). This process of external formation of ice masses and subsequent freeze dehydration of the bud tissues has been termed extraorgan freezing (Ishikawa and Sakai, 1982; Sakai, 1982).…”

“…While functional frost survival mechanisms of vegetative buds are quite well understood in conifers, they are not in angiosperms. Based on individual findings (Rajashekar and Burke, 1978; Ishikawa et al, 1997; Neuner et al, 2019), we hypothesized that the majority of vegetative angiosperm buds may not be ice tolerant. In case of supercooled buds, we aimed to determine whether, and how lethal, freezing is initiated in the supercooled bud tissue and where translocated ice masses preferentially form.…”