Gilbert Neuner

Frost Resistance and the Distribution of Conifers

The distribution of plants is well correlated with climate. For example, Koppen’s (1936) climate classification is based on major biomes which are subdivided with respect to various relationships between temperature and precipitation, whilst Thornthwaite’s (1948) classification is based on potential evapo-transpiration. The distribution of major vegetation types is explained and even predicted by such classifications. Resistance to low temperatures, however, operates at the species level, and the distribution of species is strongly related to their resistance to winter frosts (Sakai and Larcher 1987; Larcher 1995). Species from cold climates are likely to have high resistance to frost while those from warmer climates are less frost hardy. Consequently, a classification of climate that takes minimum temperatures into account is most likely to be correlated with frost resistance of species. In horticulture, the concept of plant hardiness zones is well established. These zones are based on the lowest mean air temperatures of the coldest month and, as their first usage was in the USA (Rehder 1940), are based on the Fahrenheit scale of temperature. The U.S. Department of Agriculture (USDA) refined the method and adopted 11 hardiness zones — from Zone 1 with mean minimum air temperatures of less than −50°F with incremental bands of 10°F until Zone 10 (30–40°F) and finally Zone 11 (>40°F) (see Appendix; Rehder 1940; Huxley et al. 1992).

Ice Propagation in Dehardened Alpine Plant Species Studied by Infrared Differential Thermal Analysis (IDTA)

Infrared differential thermal analysis (IDTA) was used to study ice propagation and whole plant freezing patterns in dehardened intact individuals of various alpine plant species, including a shrub (Rhododendron ferrugineum), a herbaceous plant (Senecio incanus), a cushion plant (Silene acaulis), and two graminoids (Poa alpina and Juncus trifidus). Freezing patterns differed markedly among species and reflected peculiarities of the shoot structure and the vascular system. In graminoids, each single leaf required a separate ice nucleation event, as the polystele prevents ice propagation between leaves via the stem. Additionally, enhanced supercooling resulted in a temperature range of whole plant freezing of up to 10°C, which corroborates the high summer frost resistance of graminoids. This could have ecological significance for frost survival. In contrast, in dicotyledonous species one nucleation event was usually sufficient for whole plant freezing. Controlled ice-seeding experiments on leaves with droplets of water and bacterial water suspension (Pseudomonas syringae) showed that ice propagation into the leaf tissue from the surface was inhibited as long as the leaves were undamaged. The rate of ice propagation in veins was significantly higher at lower temperatures and reached up to 24 cm s−1 in J. trifidus, which is much higher than reported in earlier findings. Ice propagation in graminoids was much faster, which may indicate that ice propagates within the protoxylem lacunae of large vessels.

Inflorescences of alpine cushion plants freeze autonomously and may survive subzero temperatures by supercooling

Freezing patterns in the high alpine cushion plants Saxifraga bryoides, Saxifraga caesia, Saxifraga moschata and Silene acaulis were studied by infrared thermography at three reproductive stages (bud, anthesis, fruit development). The single reproductive shoots of a cushion froze independently in all four species at every reproductive stage. Ice formation caused lethal damage to the respective inflorescence. After ice nucleation, which occurred mainly in the stalk or the base of the reproductive shoot, ice propagated throughout that entire shoot, but not into neighboring shoots. However, anatomical ice barriers within cushions were not detected. The naturally occurring temperature gradient within the cushion appeared to interrupt ice propagation thermally. Consequently, every reproductive shoot needed an autonomous ice nucleation event to initiate freezing. Ice nucleation was not only influenced by minimum temperatures but also by the duration of exposure. At moderate subzero exposure temperatures (−4.3 to −7.7 °C) the number of frozen inflorescences increased exponentially. Due to efficient supercooling, single reproductive shoots remained unfrozen down to −17.4 °C (cooling rate 6 K h−1). Hence, the observed freezing pattern may be advantageous for frost survival of individual inflorescences and reproductive success of high alpine cushion plants, when during episodic summer frosts damage can be avoided by supercooling.

Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence

Infrared differential thermal analysis (IDTA) and differential imaging chlorophyll fluorescence (DIF) were employed simultaneously to study the two-dimensional pattern of ice propagation in leaves and mesophyll freeze dehydration as detected by a significant increase of basic chlorophyll fluorescence (F(0)). IDTA and DIF technique gave different insights into the freezing process of leaves that was highly species-specific. IDTA clearly visualized the freezing process consisting of an initial fast spread of ice throughout the vascular system followed by mesophyll freezing. While mesophyll freezing was homogeneously in Poa alpina, Rhododendron ferrugineum and Senecio incanus as determined by IDTA, DIF showed a distinct pattern only in S. incanus, with the leaf tips being affected earlier. In Cinnamomum camphora, a mottled freezing pattern of small mesophyll compartments was observed by both methods. In IDTA images, a random pattern predominated, while in DIF images, compartments closer to lower order veins were affected earlier. The increase of F(0) following mesophyll freezing started after a species-specific time lag of up to 26 min. The start of the F(0) increase and its slope were significantly enhanced at lower temperatures, which suggest a higher strain on mesophyll protoplasts when freezing occurs at lower temperatures.

Frost resistance in alpine woody plants

This report provides a brief review of key findings related to frost resistance in alpine woody plant species, summarizes data on their frost resistance, highlights the importance of freeze avoidance mechanisms, and indicates areas of future research. Freezing temperatures are possible throughout the whole growing period in the alpine life zone. Frost severity, comprised of both intensity and duration, becomes greater with increasing elevation and, there is also a greater probability, that small statured woody plants, may be insulated by snow cover. Several frost survival mechanisms have evolved in woody alpine plants in response to these environmental conditions. Examples of tolerance to extracellular freezing and freeze dehydration, life cycles that allow species to escape frost, and freeze avoidance mechanisms can all be found. Despite their specific adaption to the alpine environment, frost damage can occur in spring, while all alpine woody plants have a low risk of frost damage in winter. Experimental evidence indicates that premature deacclimation in Pinus cembra in the spring, and a limited ability of many species of alpine woody shrubs to rapidly reacclimate when they lose snow cover, resulting in reduced levels of frost resistance in the spring, may be particularly critical under the projected changes in climate. In this review, frost resistance and specific frost survival mechanisms of different organs (leaves, stems, vegetative and reproductive over-wintering buds, flowers, and fruits) and tissues are compared. The seasonal dynamics of frost resistance of leaves of trees, as opposed to woody shrubs, is also discussed. The ability of some tissues and organs to avoid freezing by supercooling, as visualized by high resolution infrared thermography, are also provided. Collectively, the report provides a review of the complex and diverse ways that woody plants survive in the frost dominated environment of the alpine life zone.

Freezing pattern and frost killing temperature of apple (Malus domestica) wood under controlled conditions and in nature

The freezing pattern and frost killing temperatures of apple (Malus domestica Borkh.) xylem were determined by differential thermal analysis and infrared differential thermal analysis (IDTA). Results from detached or attached twigs in controlled freezing experiments and during natural field freezing of trees were compared. Non-lethal freezing of apoplastic water in apple xylem as monitored during natural winter frosts in the field occurred at -1.9 ± 0.4 °C and did not change seasonally. The pattern of whole tree freezing was variable and specific to the environmental conditions. On detached twigs high-temperature freezing exotherms (HTEs) occurred 2.8 K below the temperature observed under natural frosts in the field with a seasonal mean of -4.7 ± 0.5 °C. Microporous apple xylem showed freezing without a specific pattern within a few seconds in IDTA images during HTEs, which is in contrast to macroporous xylem where a 2D freezing pattern mirrors anatomical structures. The pith tissue always remained unfrozen. Increasing twig length increased ice nucleation temperature; for increased twig diameter the effect was not significant. In attached twigs frozen in field portable freezing chambers, HTEs were recorded at a similar mean temperature (-4.6 ± 1.0 °C) to those for detached twigs. Upon lethal intracellular freezing of apple xylem parenchyma cells (XPCs) low-temperature freezing exotherms (LTEs) can be recorded. Low-temperature freezing exotherms determined on detached twigs varied significantly between a winter minimum of -36.9 °C and a summer maximum -12.7 °C. Within the temperature range wherein LTEs were recorded by IDTA in summer (-12.7 ± 0.5 to -20.3 ± 1.1 °C) various tiny clearly separated discontinuous freezing events could be detected similar to that in other species with contrasting XPC anatomy. These freezing events appeared to be initially located in the primary and only later in the secondary xylem. During the LTE no freezing events in the bark and central pith tissue were recorded. Attached twigs were exposed to various freezing temperatures at which LTEs occur. Even if 60% of XPCs were frost-damaged twigs were able to recuperate and showed full re-growth indicating a high regeneration capacity even after severe frost damage to XPCs.

A novel system for in situ determination of heat tolerance of plants: first results on alpine dwarf shrubs

BackgroundHeat stress and heat damage to plants gain globally increasing importance for crop production and plant survival in endangered habitats. Therefore the knowledge of heat tolerance of plants is of great interest. As many heat tolerance measurement procedures require detachment of plants and protocols expose samples to various heat temperatures in darkness, the ecological relevance of such results may be doubted. To overcome these constraints we designed a novel field compatible Heat Tolerance Testing System (HTTS) that opens the opportunity to induce controlled heat stress on plants in situ under full natural solar irradiation. Subsequently, heat tolerance can be evaluated by a variety of standard viability assays like the electrolyte leakage test, chlorophyll fluorescence measurements and visual assessment methods. Furthermore, recuperation can be studied under natural environmental conditions which is impossible when detached plant material is used. First results obtained on three alpine dwarf - shrubs are presented.ResultsWhen heat tolerance of Vaccinium gaultherioides Bigelow was tested with the HTTS in situ, the visual assessment of leaves showed 50% heat injury (LT50) at 48.3°C, while on detached leaves where heat exposure took place in small heat chambers this already happened at 45.8°C. Natural solar irradiation being applied during heat exposure in the HTTS had significantly protective effects: In Loiseleuria procumbens L. (Desv.), if heat exposure (in situ) took place in darkness, leaf heat tolerance was 50.6°C. In contrast, when heat exposure was conducted under full natural solar irradiation heat tolerance was increased to 53.1°C. In Rhododendron ferrugineum L. heat tolerance of leaves was 42.5°C if the exposure took place ex situ and in darkness, while it was significantly increased to 45.8°C when this happened in situ under natural solar irradiation.ConclusionsThe results obtained with the HTTS tested in the field indicate a mitigating effect of natural solar irradiation during heat exposure. Commonly used laboratory based measurement procedures expose samples in darkness and seem to underestimate leaf heat tolerance. Avoidance of detachment by the use of the HTTS allows studying heat tolerance and recuperation processes in the presence of interacting external abiotic, biotic and genetic factors under field conditions. The investigation of combined effects of heat exposure under full solar irradiation, of recuperation and repair processes but also of possible damage amplification into the results with the HTTS appears to be particularly useful as it allows determining heat tolerance of plants with a considerably high ecological significance.

Ice Formation and Propagation in Alpine Plants

Low atmospheric temperatures are among the well-known common features of the alpine macroclimate (see Korner 2003). Absolute low temperature extremes at high altitude sites are no greater than at low altitude sites. The lowest absolute air temperature minimum at high altitude in the Austrian Alps was −37.4°C (Mt. Sonnblick, 3,105 m, 1 Jan. 1905). This temperature minimum is only marginally lower than the absolute air temperature minimum recorded at low altitude sites in Austria, which was −36.6°C (Zwettl, 520 m, 11 Feb. 1929). Strikingly, the lowest air temperature record in Austria originates from the bottom of a doline at an altitude of 1,270 m (Grunloch, Lunz am See, Lower Austria, 19 Feb. 1932), where due to temperature inversion an absolute minimum of −52.6°C was recorded (Aigner 1952).

Readiness to frost harden during the dehardening period measured in situ in leaves of Rhododendron ferrugineum L. at the alpine timberline

Summary The readiness to frost harden during the dehardening period can be crucial to the frost survival of Rhododendron ferrugineum leaves. When already dehardened plants occasionally fall snow free in late winter or spring still heavy night frosts can occur in the subalpine environment. R. ferrugineum shrubs were treated in situ using a newly developed field portable freezing chamber. They were exposed to controlled night frosts of close to the lowest temperature sustained without frost damage (LT 0 ) to determine the potential frost hardening response under otherwise completely natural conditions. There was a lag period of 3 days during which no significant increase of frost resistance was observed. After three days frost resistance increased suddenly by 5.9°C within 24 hours. High daytime leaf temperatures (+19°C) combined with night frosts further retarded the rate of frost hardening. The in situ frost treatment generally yielded frost resistances (LT 10 ) approaching the highest ever measured in that season. In early spring the in situ frost hardening response was three times greater than early reports for detached twigs with a total hardiness gain between 7.8°C and 9.2°C. It is suggested that the limiting step in frost hardening in leaves of Rhododendron ferrugineum in spring is not the extent of frost resistance achieveable but rather the slow rate of frost hardening.

Velocity and pattern of ice propagation and deep supercooling in woody stems of Castanea sativa, Morus nigra and Quercus robur measured by IDTA

Infrared differential thermal analysis (IDTA) was used to monitor the velocity and pattern of ice propagation and deep supercooling of xylem parenchyma cells (XPCs) during freezing of stems of Castanea sativa L., Morus nigra L. and Quercus robur L. that exhibit a macro- and ring-porous xylem. Measurements were conducted on the surface of cross- and longitudinal stem sections. During high-temperature freezing exotherms (HTEs; -2.8 to -9.4°C), initial freezing was mainly observed in the youngest year ring of the sapwood (94%), but occasionally elsewhere (older year rings: 4%; bark: 2%). Initially, ice propagated rapidly in the largest xylem conduits. This resulted in a distinct freezing pattern of concentric circles in C. sativa and M. nigra. During HTEs, supercooling of XPCs became visible in Q. robur stems, but not in the other species that have narrower pith rays. Intracellular freezing of supercooled XPCs of Q. robur became visible by IDTA during low-temperature freezing exotherms (<-17.4 °C). Infrared differential thermal analysis revealed the progress and the two-dimensional pattern of XPC freezing. XPCs did not freeze at once, but rather small cell groups appeared to freeze at random anywhere in the xylem. By IDTA, ice propagation and deep supercooling in stems can be monitored at meaningful spatial and temporal resolutions.