Heat Acclimation under Drought Stress Induces Antioxidant Enzyme Activity in the Alpine Plant Primula minima.

Heat and drought stresses are increasingly relevant topics in the context of climate change, particularly in the Alps, which are warming faster than the global average. Previously, we have shown that alpine plants, including Primula minima, can be gradually heat hardened under field conditions in situ to achieve maximum tolerance within a week. Here, we investigated the antioxidant mechanisms of P. minima leaves that had been heat hardened (H) without or with (H+D) additional drought stress. Lower free-radical scavenging and ascorbate concentrations were found in H and H+D leaves, while concentrations of glutathione disulphide (GSSG) were higher under both treatments without any change in glutathione (GSH) and little change in glutathione reductase activity. In contrast, ascorbate peroxidase activity in H leaves was increased, and H+D leaves had >two-fold higher catalase, ascorbate peroxidase and glucose-6-phosphate dehydrogenase activities compared with the control. In addition, the glutathione reductase activity was higher in H+D compared with H leaves. Our results highlight that the stress load from heat acclimation to maximum tolerance is associated with a weakened low-molecular-weight antioxidant defence, which may be compensated for by an increased activity of antioxidant enzymes, particularly under drought conditions.

Frozen mountain pine needles: The endodermis discriminates between the ice-containing central tissue and the ice-free fully functional mesophyll.

Conifer (Pinaceae) needles are the most frost-hardy leaves. During needle freezing, the exceptional leaf anatomy, where an endodermis separates the mesophyll from the vascular tissue, could have consequences for ice management and photosynthesis. The eco-physiological importance of needle freezing behaviour was evaluated based on the measured natural freezing strain at the alpine treeline. Ice localisation and cellular responses to ice were investigated in mountain pine needles by cryo-microscopic techniques. Their consequences for photosynthetic activity were assessed by gas exchange measurements. The freezing response was related to the microchemistry of cell walls investigated by Raman microscopy. In frozen needles, ice was confined to the central vascular cylinder bordered by the endodermis. The endodermal cell walls were lignified. In the ice-free mesophyll, cells showed no freeze-dehydration and were found photosynthetically active. Mesophyll cells had lignified tangential cell walls, which adds rigidity. Ice barriers in mountain pine needles seem to be realised by a specific lignification patterning of cell walls. This, additionally, impedes freeze-dehydration of mesophyll cells and enables gas exchange of frozen needles. At the treeline, where freezing is a dominant environmental factor, the elaborate needle freezing pattern appears of ecological importance.

Winter Frosts Reduce Flower Bud Survival in High-Mountain Plants.

At higher elevations in the European Alps, plants may experience winter temperatures of -30 °C and lower at snow-free sites. Vegetative organs are usually sufficiently frost hardy to survive such low temperatures, but it is largely unknown if this also applies to generative structures. We investigated winter frost effects on flower buds in the cushion plants Saxifraga bryoides L. (subnival-nival) and Saxifraga moschata Wulfen (alpine-nival) growing at differently exposed sites, and the chionophilous cryptophyte Ranunculus glacialis L. (subnival-nival). Potted plants were subjected to short-time (ST) and long-time (LT) freezing between -10 and -30 °C in temperature-controlled freezers. Frost damage, ice nucleation and flowering frequency in summer were determined. Flower bud viability and flowering frequency decreased significantly with decreasing temperature and exposure time in both saxifrages. Already, -10 °C LT-freezing caused the first injuries. Below -20 °C, the mean losses were 47% (ST) and 75% (LT) in S. bryoides, and 19% (ST) and 38% (LT) in S. moschata. Winter buds of both saxifrages did not supercool, suggesting that damages were caused by freeze dehydration. R. glacialis remained largely undamaged down to -30 °C in the ST experiment, but did not survive permanent freezing below -20 °C. Winter snow cover is essential for the survival of flower buds and indirectly for reproductive fitness. This problem gains particular relevance in the context of winter periods with low precipitation and winter warming events leading to the melting of the protective snowpack.

Winter survival of the unicellular green alga Micrasterias denticulata: insights from field monitoring and simulation experiments.

Peat bog pools around Tamsweg (Lungau, Austria) are typical habitats of the unicellular green alga Micrasterias denticulata. By measurement of water temperature and irradiation throughout a 1-year period (2018/2019), it was intended to assess the natural environmental strain in winter. Freezing resistance of Micrasterias cells and their ability to frost harden and become tolerant to ice encasement were determined after natural hardening and exposure to a cold acclimation treatment that simulated the natural temperature decrease in autumn. Transmission electron microscopy (TEM) was performed in laboratory-cultivated cells, after artificial cold acclimation treatment and in cells collected from field. Throughout winter, the peat bog pools inhabited by Micrasterias remained unfrozen. Despite air temperature minima down to -17.3 °C, the water temperature was mostly close to +0.8 °C. The alga was unable to frost harden, and upon ice encasement, the cells showed successive frost damage. Despite an unchanged freezing stress tolerance, significant ultrastructural changes were observed in field-sampled cells and in response to the artificial cold acclimation treatment: organelles such as the endoplasmic reticulum and thylakoids of the chloroplast showed distinct membrane bloating. Still, in the field samples, the Golgi apparatus appeared in an impeccable condition, and multivesicular bodies were less frequently observed suggesting a lower overall stress strain. The observed ultrastructural changes in winter and after cold acclimation are interpreted as cytological adjustments to winter or a resting state but are not related to frost hardening as Micrasterias cells were unable to improve their freezing stress tolerance.

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants.

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam-scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

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.

Cell Wall Reinforcements Accompany Chilling and Freezing Stress in the Streptophyte Green Alga Klebsormidium crenulatum.

Adaptation strategies in freezing resistance were investigated in Klebsormidium crenulatum, an early branching streptophyte green alga related to higher plants. Klebsormidium grows naturally in unfavorable environments like alpine biological soil crusts, exposed to desiccation, high irradiation and cold stress. Here, chilling and freezing induced alterations of the ultrastructure were investigated. Control samples (kept at 20°C) were compared to chilled (4°C) as well as extracellularly frozen algae (-2 and -4°C). A software-controlled laboratory freezer (AFU, automatic freezing unit) was used for algal exposure to various temperatures and freezing was manually induced. Samples were then high pressure frozen and cryo-substituted for electron microscopy. Control cells had a similar appearance in size and ultrastructure as previously reported. While chilling stressed algae only showed minor ultrastructural alterations, such as small inward facing cell wall plugs and minor alterations of organelles, drastic changes of the cell wall and in organelle distribution were found in extracellularly frozen samples (-2°C and -4°C). In frozen samples, the cytoplasm was not retracted from the cell wall, but extensive three-dimensional cell wall layers were formed, most prominently in the corners of the cells, as determined by FIB-SEM and TEM tomography. Similar alterations/adaptations of the cell wall were not reported or visualized in Klebsormidium before, neither in controls, nor during other stress scenarios. This indicates that the cell wall is reinforced by these additional wall layers during freezing stress. Cells allowed to recover from freezing stress (-2°C) for 5 h at 20°C lost these additional cell wall layers, suggesting their dynamic formation. The composition of these cell wall reinforcement areas was investigated by immuno-TEM. In addition, alterations of structure and distribution of mitochondria, dictyosomes and a drastically increased endoplasmic reticulum were observed in frozen cells by TEM and TEM tomography. Measurements of the photosynthetic oxygen production showed an acclimation of Klebsormidium to chilling stress, which correlates with our findings on ultrastructural alterations of morphology and distribution of organelles. The cell wall reinforcement areas, together with the observed changes in organelle structure and distribution, are likely to contribute to maintenance of an undisturbed cell physiology and to adaptation to chilling and freezing stress.

A new technical approach for preparing frozen biological samples for electron microscopy.

BACKGROUND: Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a defined frozen state during the entire fixation procedure is an essential prerequisite. However, current techniques are not able to fix frozen plant tissues for transmission electron microscopy (TEM) without interrupting the cold chain. Chemical fixation by glutaraldehyde and osmium tetroxide is not possible at sub-zero temperatures. Cryo-fixation methods, such as high pressure freeze fixation (HPF) representing the state-of-the-art technique for best structural preservation, are not equipped for freezing frozen samples. In order to overcome this obstacle, a novel technical approach for maintaining the cold chain of already frozen plant samples prior and during HPF is presented. RESULTS: Different algae (Micrasterias denticulata, Klebsormidium crenulatum) and higher plant tissues (Lemna sp., Ranunculus glacialis, Pinus mugo) were successfully frozen and prepared for HPF at freezing temperatures (- 2 °C, - 5 °C, - 6 °C) within a newly developed automatic freezing unit (AFU), that we manufactured from a standard laboratory freezer. Preceding tests on photosynthetic electron transport and ability to plasmolyse show that the temperatures applied did not impair electron transport in PSII nor cell vitality. The transfer of the frozen specimen from the AFU into the HPF-device and subsequently cryo-fixation were performed without intermediate thawing. After cryo-substitution and further processing, the resulting TEM-micrographs showed excellent ultrastructure preservation of the different organisms when compared to specimens fixed at ambient temperature. CONCLUSIONS: The method presented allows preserving the ultrastructure of plant cells in the frozen state during cryo-fixation. The resulting high quality TEM-images represent an important step towards a better understanding of the consequences of extracellular ice formation on cellular ultrastructure. It has the potential to provide new insights into changes of organelle structure, identification of intracellular injuries during ice formation and may help to understand freezing and thawing processes in plant tissues. It may be combined with analytical TEM such as electron energy loss spectroscopy (EELS), X-ray analyses (EDX) and various other electron microscopic techniques.

Non-invasive diagnosis of viability in seeds and lichens by infrared thermography under controlled environmental conditions.

BACKGROUND: Non-invasive procedures for the diagnosis of viability of plant or fungal tissues would be valuable for scientific, industrial and biomonitoring purposes. Previous studies showed that infrared thermography (IRT) enables non-invasive assessment of the viability of individual "orthodox" (i.e. desiccation tolerant) seeds upon water uptake. However, this method was not tested for rehydrating tissues of other desiccation tolerant life forms. Furthermore, evaporative cooling could obscure the effects of metabolic processes that contribute to heating and cooling, but its effects on the shape of the "thermal fingerprints" have not been explored. Here, we further adapted this method using a purpose-built chamber to control relative humidity (RH) and gaseous atmosphere. This enabled us to test (i) the influence of relative humidity on the thermal fingerprints during the imbibition of Pisum sativum (Garden pea) seeds, (ii) whether thermal fingerprints can be correlated with viability in lichens, and (iii) to assess the potential influence of aerobic metabolism on thermal fingerprints by controlling the oxygen concentration in the gaseous atmosphere around the samples. Finally, we developed a method to artificially "age" lichens and validated the IRT-based method to assess lichen viability in three lichen species. RESULTS: Using either 30% or 100% RH during imbibition of pea seeds, we showed that "live" and "dead" seeds produced clearly discernible "thermal fingerprints", which significantly differed by > |0.15| °C in defined time windows, and that RH affected the shape of these thermal fingerprints. We demonstrated that IRT can also be used to assess the viability of the lichens Lobaria pulmonaria, Pseudevernia furfuracea and Peltigera leucophlebia. No clear relationship between aerobic metabolism and the shape of thermal fingerprints was found. CONCLUSIONS: Infrared thermography appears to be a promising method for the diagnosis of viability of desiccation-tolerant tissues at early stages of water uptake. For seeds, it is possible to diagnose viability within the first hours of rehydration, after which time they can still be re-dried and stored until further use. We envisage our work as a baseline study for the use of IR imaging techniques to investigate physiological heterogeneity of desiccation tolerant life forms such as lichens, which can be used for biomonitoring, and for sorting live and dead seeds, which is potentially useful for the seed trade.

Solar irradiation levels during simulated long- and short-term heat waves significantly influence heat survival, pigment and ascorbate composition, and free radical scavenging activity in alpine Vaccinium gaultherioides.

In the 20th century, annual mean temperatures in the European Alps rose by almost 1 K and are predicted to rise further, increasing the impact of temperature on alpine plants. The role of light in the heat hardening of plants is still not fully understood. Here, the alpine dwarf shrub Vaccinium gaultherioides was exposed in situ to controlled short-term heat spells (150 min with leaf temperatures 43-49°C) and long-term heat waves (7 days, 30°C) under different irradiation intensities. Lethal leaf temperatures (LT50 ) were calculated. Low solar irradiation [max. 250 photosynthetic photon flux density (PPFD)] during short-term heat treatments mitigated the heat stress, shown by reduced leaf tissue damage and higher Fv /Fm (potential quantum efficiency of photosystem 2) than in darkness. The increase in xanthophyll cycle activity and ascorbate concentration was more pronounced under low light, and free radical scavenging activity increased independent of light conditions. During long-term heat wave exposure, heat tolerance increased from 3.7 to 6.5°C with decreasing mean solar irradiation intensity (585-115 PPFD). Long-term exposure to heat under low light enhanced heat hardening and increased photosynthetic pigment, dehydroascorbate and violaxanthin concentration. In conclusion, V. gaultherioides is able to withstand temperatures of around 50°C, and its heat hardening can be enhanced by low light during both short- and long-term heat treatment. Data showing the specific role of light during short- and long-term heat exposure and the potential risk of lethal damage in alpine shrubs as a result of rising temperature are discussed.

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.

Chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha.

Under low-light conditions, chloroplasts localize along periclinal cell walls at temperatures near 20 °C, but they localize along anticlinal cell walls near 5 °C. This phenomenon is known as the cold-positioning response. We previously showed that chloroplasts move as aggregates rather than individually during the cold-positioning response in the fern Adiantum capillus-veneris. This observation suggested that chloroplasts physically interact with each other during the cold-positioning response. However, the physiological processes underlying chloroplast aggregation are unclear. In this report, we characterized chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha. Confocal laser microscopy observations of transgenic liverwort plants expressing a fluorescent fusion protein that localizes to the chloroplast outer envelope membrane (OEP7-Citrine) showed that neighboring chloroplast membranes did not fuse during the cold-positioning response. Transmission electron microscopy analysis revealed that a distance of at least 10 nm was maintained between neighboring chloroplasts during aggregation. These results indicate that aggregated chloroplasts do not fuse, but maintain a distance of at least 10 nm from each other during the cold-positioning response.

Formation of chloroplast protrusions and catalase activity in alpine Ranunculus glacialis under elevated temperature and different CO2/O2 ratios.

Chloroplast protrusions (CPs) have frequently been observed in plants, but their significance to plant metabolism remains largely unknown. We investigated in the alpine plant Ranunculus glacialis L. treated under various CO2 concentrations if CP formation is related to photorespiration, specifically focusing on hydrogen peroxide (H2O2) metabolism. Immediately after exposure to different CO2 concentrations, the formation of CPs in leaf mesophyll cells was assessed and correlated to catalase (CAT) and ascorbate peroxidase (APX) activities. Under natural irradiation, the relative proportion of chloroplasts with protrusions (rCP) was highest (58.7 %) after exposure to low CO2 (38 ppm) and was lowest (3.0 %) at high CO2 (10,000 ppm). The same relationship was found for CAT activity, which decreased from 34.7 nkat mg(-1) DW under low CO2 to 18.4 nkat mg(-1) DW under high CO2, while APX activity did not change significantly. When exposed to natural CO2 concentration (380 ppm) in darkness, CP formation was significantly lower (18.2 %) compared to natural solar irradiation (41.3 %). In summary, CP formation and CAT activity are significantly increased under conditions that favour photorespiration, while in darkness or at high CO2 concentration under light, CP formation is significantly lower, providing evidence for an association between CPs and photorespiration.

Application of heat stress in situ demonstrates a protective role of irradiation on photosynthetic performance in alpine plants.

The impact of sublethal heat on photosynthetic performance, photosynthetic pigments and free radical scavenging activity was examined in three high mountain species, Rhododendron ferrugineum, Senecio incanus and Ranunculus glacialis using controlled in situ applications of heat stress, both in darkness and under natural solar irradiation. Heat treatments applied in the dark reversibly reduced photosynthetic performance and the maximum quantum efficiency of photosystem II (Fv /Fm), which remained impeded for several days when plants were exposed to natural light conditions subsequently to the heat treatment. In contrast, plants exposed to heat stress under natural irradiation were able to tolerate and recover from heat stress more readily. The critical temperature threshold for chlorophyll fluorescence was higher under illumination (Tc (')) than in the dark (Tc). Heat stress caused a significant de-epoxidation of the xanthophyll cycle pigments both in the light and in the dark conditions. Total free radical scavenging activity was highest when heat stress was applied in the dark. This study demonstrates that, in the European Alps, heat waves can temporarily have a negative impact on photosynthesis and, importantly, that results obtained from experiments performed in darkness and/or on detached plant material may not reliably predict the impact of heat stress under field conditions.

Chloroplast protrusions in leaves of Ranunculus glacialis L. respond significantly to different ambient conditions, but are not related to temperature stress.

The occurrence of chloroplast protrusions (CPs) in leaves of Ranunculus glacialis L. in response to different environmental conditions was assessed. CPs occur highly dynamically. They do not contain thylakoids and their physiological function is still largely unknown. Controlled in situ sampling showed that CP formation follows a pronounced diurnal rhythm. Between 2 and 27 °C the relative proportion of chloroplasts with CPs (rCP) showed a significant positive correlation to leaf temperature (TL; 0.793, P < 0.01), while irradiation intensity had a minor effect on rCP. In situ shading and controlled laboratory experiments confirmed the significant influence of TL. Under moderate irradiation intensity, an increase of TL up to 25 °C significantly promoted CP formation, while a further increase to 37 °C led to a decrease. Furthermore, rCP values were lower in darkness and under high irradiation intensity. Gas treatment at 2000 ppm CO2/2% O2 led to a significant decrease of rCP, suggesting a possible involvement of photorespiration in CP formation. Our findings demonstrate that in R. glacialis, CPs are neither a rare phenomenon nor a result of heat or light stress; on the contrary, they seem to be most abundant under moderate temperature and non-stress irradiation conditions.

Heat tolerance of early developmental stages of glacier foreland species in the growth chamber and in the field.

In glacier forelands, seeds readily germinate, however, a high proportion of seedlings die shortly after their appearance. We hypothesized that besides drought, frost and missing safe sites, heat on the ground surface could be one of the major threats for seedlings. The heat strain in different ground strata was assessed from 2007 to 2010. The heat tolerance (LT50) of eleven alpine species from different successional stages was tested considering imbibed (G1) and germinated seeds (G2) as well as seedlings (G3). Additionally, the heat hardening capacity of seedlings was determined in the field. Across all species, LT50 decreased significantly by 9 K from G1 (55 °C) to G3 (46 °C), similarly in all species of the successional stages. Field-grown seedlings had mostly an increased LT50 (2K). Intraspecifically, LT50 of seedlings varied between 40.6 and 52.5 °C. Along the chronosequence, LT50 in G1 was similar, but was higher in G2 and G3 of early successional species. The highest temperatures occurred at 0-0.5 cm in air (mean/absolute maximum: 42.6/54.1 °C) posing a significant heat injury risk for seedlings when under water shortage transpirational cooling is prevented. Below small stones (0-0.5 cm), maxima were 4 K lower, indicating heat safer microsites. Maxima >30 °C occurred at 32.3, >40 °C at 6.2 %. Interannually, 2010 was the hottest year with heat exceeding LT50 at all microsites (0-0.5 cm). Temperature maxima on sandy surfaces were lower than on microsites with gravel (diameter <5-10 mm). The hot summer of 2010 may be a small foretaste of in future more severe and frequent heat waves. Ground surface temperature maxima at the pioneer stage are already now critical for heat survival and may partly explain the high seedling mortality recognized on recently deglaciated terrain.

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

BACKGROUND: Heat 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. RESULTS: When 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. CONCLUSIONS: The 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.

Winter frost resistance of Pinus cembra measured in situ at the alpine timberline as affected by temperature conditions.

Winter frost resistance (WFR), midwinter frost hardening and frost dehardening potential of Pinus cembra L. were determined in situ by means of a novel low-temperature freezing system at the alpine timberline ecotone (1950 m a.s.l., Mt Patscherkofel, Innsbruck, Austria). In situ liquid nitrogen (LN2)-quenching experiments should check whether maximum WFR of P. cembra belonging to the frost hardiest conifer group, being classified in US Department of Agriculture climatic zone 1, suffices to survive dipping into LN2 (-196 °C). Viability was assessed in a field re-growth test. Maximum in situ WFR (LT50) of leaves was <- 75 °C and that of buds was less (-70.3 °C), matching the lowest water contents. In midwinter, in situ freezing exotherms of leaves, buds and the xylem were often not detectable. Ice formed in the xylem at a mean of -2.8 °C and in leaves at -3.3 °C. In situ WFR of P. cembra was higher than that obtained on detached twigs, as reported earlier. In situ LN2-quenching experiments were lethal in all cases even when twigs of P. cembra were exposed to an in situ frost hardening treatment (12 days at -20 °C followed by 3 days at -50 °C) to induce maximum WFR. Temperature treatments applied in the field significantly affected the actual WFR. In January a frost hardening treatment (21 days at -20 °C) led to a significant increase of WFR (buds: -62 °C to <- 70 °C; leaves: -59.6 °C to -65.2 °C), showing that P. cembra was not at its specific maximum WFR. In contrast, simulated warm spells in late winter led to premature frost dehardening (buds: -32.6 °C to -10.2 °C; leaves: -32.7 to -16.4 °C) followed by significantly earlier bud swelling and burst in late winter. Strikingly, both temperature treatments, either increased air temperature (+10.1 °C) or increased soil temperature (+6.5 °C), were similarly effective. This high readiness to frost harden and deharden in winter in the field must be considered to be of great significance for future winter survival of P. cembra. Determination of WFR in field re-growth tests appears to be a valuable tool for critically judging estimates of WFR obtained on detached twigs in an ecological context.

Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium.

Leaflets of Sphagnum capillifolium were exposed to temperatures from -5 degrees C to +60 degrees C under controlled conditions while mounted on a microscope stage. The resultant cytological response to these temperature treatments was successfully monitored using a light and fluorescence microscope. In addition to the observable cytological changes during freezing cytorrhysis and heat exposure on the leaflets, the concomitant critical temperature thresholds for inactivation of photosystem II (PS II) were studied using a micro fibre optic and a chlorophyll fluorometer mounted to the microscope stage. Chlorophyllous cells of S. capillifolium showed extended freezing cytorrhysis immediately after ice nucleation at -1.1 degrees C in the water in which the leaflets were submersed during the measurement. The occurrence of freezing cytorrhysis, which was visually manifested by cell shrinkage, was highly dynamic and was completed within 2 s. A total reduction of the mean projected diameter of the chloroplast containing area during freezing cytorrhysis from 8.9 to 3.8 microm indicates a cell volume reduction of approximately -82%. Simultaneous measurement of chlorophyll fluorescence of PS II was possible even through the frozen water in which the leaf samples were submersed. Freezing cytorrhysis was accompanied by a sudden rise of basic chlorophyll fluorescence. The critical freezing temperature threshold of PS II was identical to the ice nucleation temperature (-1.1 degrees C). This is significantly above the temperature threshold at which frost damage to S. capillifolium leaflets occurs (-16.1 degrees C; LT(50)) which is higher than observed in most higher plants from the European Alps during summer. High temperature thresholds of PS II were 44.5 degrees C which is significantly below the heat tolerance of chlorophyllous cells (49.9 degrees C; LT(50)). It is demonstrated that light and fluorescence microscopic techniques combined with simultaneous chlorophyll fluorescence measurements may act as a useful tool to study heat, low temperature, and ice-encasement effects on the cellular structure and primary photosynthetic processes of intact leaf tissues.

A low-temperature freezing system to study the effects of temperatures to -70 {degrees}C on trees in situ.

The ability to determine winter frost resistance of woody plants is limited for two reasons: (1) assessment of frost damage in midwinter is extremely difficult because results obtained by the currently available viability assays deviate greatly and (2) equipment that allows plants to be frozen at controlled freezing and thawing rates to below the midwinter frost resistance of most Northern Hemisphere woody plants is unavailable. To overcome these limitations, we developed a novel low-temperature freezing system (LTFS) that makes it possible to conduct in situ freezing experiments in midwinter with full control of cooling and thawing rates down to -70 degrees C. Frost resistance can be determined unequivocally by the regrowth test. The LTFS was tested on various, mostly subalpine, woody plants. Results obtained demonstrate the importance of conducting frost tests in situ. In needles of Picea abies (L.) Karst., frost injuries were not visible immediately after the frost test but took several weeks to develop fully. The low-freezing temperatures attained and the small control oscillations (typically +/-0.1 K) of the LTFS during cooling permitted in situ detection of low-temperature freezing exotherms in xylem of Quercus robur L. and in buds of P. abies and Rhododendron ferrugineum L., all of which showed supercooling. With the LTFS, the effects of low temperatures on plants can be specified directly by in situ assessment and regrowth tests.