Winter Nights during Summer Time: Stress Physiological Response to Ice and the Facilitation of Freezing Cytorrhysis by Elastic Cell Wall Components in the Leaves of a Nival Species.

Ranunculus glacialis grows and reproduces successfully, although the snow-free time period is short (2-3 months) and night frosts are frequent. At a nival site (3185 m a.s.l.), we disentangled the interplay between the atmospheric temperature, leaf temperatures, and leaf freezing frequency to assess the actual strain. For a comprehensive understanding, the freezing behavior from the whole plant to the leaf and cellular level and its physiological after-effects as well as cell wall chemistry were studied. The atmospheric temperatures did not mirror the leaf temperatures, which could be 9.3 °C lower. Leaf freezing occurred even when the air temperature was above 0 °C. Ice nucleation at on average -2.6 °C started usually independently in each leaf, as the shoot is deep-seated in unfrozen soil. All the mesophyll cells were subjected to freezing cytorrhysis. Huge ice masses formed in the intercellular spaces of the spongy parenchyma. After thawing, photosynthesis was unaffected regardless of whether ice had formed. The cell walls were pectin-rich and triglycerides occurred, particularly in the spongy parenchyma. At high elevations, atmospheric temperatures fail to predict plant freezing. Shoot burial prevents ice spreading, specific tissue architecture enables ice management, and the flexibility of cell walls allows recurrent freezing cytorrhysis. The peculiar patterning of triglycerides close to ice rewards further investigation.

Complex bud architecture and cell-specific chemical patterns enable supercooling of Picea abies bud primordia.

Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to -50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood. Bud architecture was assessed by 3D-reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell-specific chemical patterns by Raman microscopy and mass spectrometry imaging. A bowl-like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At -18 °C, break-down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature-dependent freeze dehydration (-0.1 MPa K-1 ) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin-rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides. Bud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell-specific biochemical analysis.

Acquisition of Freezing Tolerance in Vaccinium macrocarpon Ait. Is a Multi-Factor Process Involving the Presence of an Ice Barrier at the Bud Base.

Bud freezing survival strategies have in common the presence of an ice barrier that impedes the propagation of lethally damaging ice from the stem into the internal structures of buds. Despite ice barriers' essential role in buds freezing stress survival, the nature of ice barriers in woody plants is not well understood. High-definition thermal recordings of Vaccinium macrocarpon Ait. buds explored the presence of an ice barrier at the bud base in September, January, and May. Light and confocal microscopy were used to evaluate the ice barrier region anatomy and cell wall composition related to their freezing tolerance. Buds had a temporal ice barrier at the bud base in September and January, although buds were only freezing tolerant in January. Lack of functionality of vascular tissues may contribute to the impedance of ice propagation. Pith tissue at the bud base had comparatively high levels of de-methyl-esterified homogalacturonan (HG), which may also block ice propagation. By May, the ice barrier was absent, xylogenesis had resumed, and de-methyl-esterified HG reached its lowest levels, translating into a loss of freezing tolerance. The structural components of the barrier had a constitutive nature, resulting in an asynchronous development of freezing tolerance between anatomical and metabolic adaptations.

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

In temperate climates, overwintering buds of trees are often less cold hardy than adjoining stem tissues or evergreen leaves. However, data are scarce regarding the freezing resistance (FR) of buds and the underlying functional frost survival mechanism that in case of supercooling can restrict the geographic distribution. Twigs of 37 temperate woody species were sampled in midwinter 2016 in the Austrian Inn valley. After assessment of FR, infrared-video-thermography and cryo-microscopy were used to study the freezing pattern in and around overwintering vegetative buds. Only in four species, after controlled ice nucleation in the stem (-1.6 ± 0.9°C) ice was observed to immediately invade the bud. These buds tolerated extracellular ice and were the most freezing resistant (-61.8°C mean LT50). In all other species (33), the buds remained supercooled and free of ice, despite a frozen stem. A structural ice barrier prevents ice penetration. Extraorgan ice masses grew in the stem and scales but in 50% of the species between premature supercooled leaves. Two types of supercooled buds were observed: in temporary supercooling buds (14 species) ice spontaneously nucleated at -20.5 ± 4,6°C. This freezing process appeared to be intracellular as it matched the bud killing temperature (-22.8°C mean LT50). This response rendered temporarily supercooled buds as least cold hardy. In 19 species, the buds remained persistently supercooled down to below the killing temperature without indication for the cause of damage. Although having a moderate midwinter FR of -31.6°C (LT50), some species within this group attained a FR similar to ice tolerant buds. The present study represents the first comprehensive overview of frost survival mechanisms of vegetative buds of temperate trees. Except for four species that were ice tolerant, the majority of buds survive in a supercooled state, remaining free of ice. In 50% of species, extraorgan ice masses harmlessly grew between premature supercooled leaves. Despite exposure to the same environmental demand, midwinter FR of buds varied intra-specifically between -17.0 and -90.0°C. Particularly, species, whose buds are killed after temporary supercooling, have a lower maximum FR, which limits their geographic distribution.