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.

Ice barriers promote supercooling and prevent frost injury in reproductive buds, flowers and fruits of alpine dwarf shrubs throughout the summer.

Over-wintering reproductive buds of many woody plants survive frost by supercooling. The bud tissues are isolated from acropetally advancing ice by the presence of ice barriers that restrict ice growth. Plants living in alpine environments also face the risk of ice formation in summer months. Little knowledge exists, how reproductive structures of woody alpine plants are protected from frost injury during episodic summer frosts. In order to address this question, frost resistance of three common dwarf shrubs, Calluna vulgaris, Empetrum hermaphroditum and Loiseleuria procumbens was measured and ice formation and propagation were monitored in twigs bearing reproductive shoots during various stages of reproductive development (bud, anthesis, and fruit) throughout the alpine summer. Results indicated that, in the investigated species, ice barriers were present at all reproductive stages, isolating the reproductive shoots from ice advancing from the subtending vegetative shoot. Additionally, in the reproductive stems ice nucleating agents that are active at warm, sub-zero temperatures, were absent. The ice barriers were 100% effective, with the exception of L. procumbens, where in 13% of the total observations, the ice barrier failed. The ice barriers were localized at the base of the pedicel, at the anatomical junction of the vegetative and reproductive shoot. There, structural aspects of the tissue impede or prevent ice from advancing from the frozen stem into the pedicel of the reproductive shoot. Under the experimental conditions used in this study, ice nucleation initially occurred in the stem of the vegetative shoot at species-specific mean temperatures in the range of -4.7 to -5.8 °C. Reproductive shoots, however, remained supercooled and ice free down to a range of -7.2 to -18.2 °C or even below -22 °C, the lowest temperature applied in the study. This level of supercooling is sufficient to prevent freezing of reproductive structures at the lowest air temperature occurring at the altitude of the upper distribution boundary of the natural habitat of the investigated species which is between -8 and -10 °C in summer. Frost resistance assays indicated that reproductive shoots are much less frost resistant than vegetative stems, and in contrast to vegetative shoots, are not ice tolerant. Supercooling of reproductive shoots in alpine, woody plant species is an effective mechanism that protects developing offspring from potential frost damage resulting from episodic summer freezing events.

Does winter desiccation account for seasonal increases in supercooling capacity of Norway spruce bud primordia?

Bud primordia of Picea abies (L.) H. Karst. remain ice free at subzero temperatures by supercooling. Once ice forms inside the primordium, it is immediately injured. Supercooling capacity increases seasonally from ~-5 °C to as much as -50 °C by currently unknown mechanisms. Among other prerequisites, dehydration of tissues over the winter months has been considered to play a key role in freezing tolerance. In this regard, the water content of bud primordia may be crucial, especially in reference to supercooling. In order to assess the role of dehydration in supercooling capacity, seasonal changes in supercooling capacity and the water potential of bud primordia of Picea abies (L.) H. Karst were measured at two sites that differed by 1298 m in elevation, after artificial frost hardening and dehardening treatments and after controlled bench drying. The extent of supercooling of bud primordia varied from -7 °C in summer to -24.6 °C in winter, a difference of 17.6 -19.3 K. Total actual water potential (Ψtact) of bud primordia was -2 MPa in summer and decreased to a mean of -3.8 MPa in midwinter. The decline involved dehydration, and to a lesser extent, osmoregulation. At decreased Ψtact values (<3.0 MPa), supercooling capacity significantly increased <-19.5 °C, however, the correlation between actual water potential and supercooling capacity was poor. Frost-hardening treatments increased the supercooling capacity of bud primordia (-0.6 K day-1) and lowered Ψtact (-0.2 MPa day-1). Frost-dehardening treatments reduced supercooling capacity (+1.1 K day-1), and at the same time, increased Ψtact (+0.3 MPa day-1). In contrast, artificial drying of bud primordia in the range observed seasonally (-2.0 MPa) had no effect on supercooling capacity. These results suggest that there is no causal relationship between desiccation and the supercooling capacity of bud primordia in P. abies, but rather it involves other compounds within the cells of the bud primordium that reduce the water potential.

Persistent Supercooling of Reproductive Shoots Is Enabled by Structural Ice Barriers Being Active Despite an Intact Xylem Connection.

Extracellular ice nucleation usually occurs at mild subzero temperatures in most plants. For persistent supercooling of certain plant parts ice barriers are necessary to prevent the entry of ice from already frozen tissues. The reproductive shoot of Calluna vulgaris is able to supercool down to below -22°C throughout all developmental stages (shoot elongation, flowering, fruiting) despite an established xylem conductivity. After localization of the persistent ice barrier between the reproductive and vegetative shoot at the base of the pedicel by infrared differential thermal analysis, the currently unknown structural features of the ice barrier tissue were anatomically analyzed on cross and longitudinal sections. The ice barrier tissue was recognized as a 250 µm long constriction zone at the base of the pedicel that lacked pith tissue and intercellular spaces. Most cell walls in this region were thickened and contained hydrophobic substances (lignin, suberin, and cutin). A few cell walls had what appeared to be thicker cellulose inclusions. In the ice barrier tissue, the area of the xylem was as much as 5.7 times smaller than in vegetative shoots and consisted of tracheids only. The mean number of conducting units in the xylem per cross section was reduced to 3.5% of that in vegetative shoots. Diameter of conducting units and tracheid length were 70% and 60% (respectively) of that in vegetative shoots. From vegetative shoots water transport into the ice barrier must pass pit membranes that are likely impermeable to ice. Pit apertures were about 1.9 µm x 0.7 µm, which was significantly smaller than in the vegetative shoot. The peculiar anatomical features of the xylem at the base of the pedicel suggest that the diameter of pores in pit membranes could be the critical constriction for ice propagation into the persistently supercooled reproductive shoots of C. vulgaris.

Infrared thermal analysis of plant freezing processes.

Infrared thermal analysis is an invaluable technique to study the plant freezing process. In the differential mode infrared thermal analysis allows to localize ice nucleation and ice propagation in whole plants or plant samples at the tissue level. Ice barriers can be visualized, and supercooling of cells, tissues, and organs can be monitored. Places where ice masses are accommodated in the apoplast can be identified. Here, we describe an experimental setting developed in the laboratory in Innsbruck, give detailed information on the practical procedure and preconditions, and give additionally an idea of the problems that can be encountered and how they by special precautions may be overcome.