Tree seedlings at the treeline face various environmental extremes; in particular, their needles can heat up close to thermal limits. Unlike mature-tree needles, treeline conifer seedlings resist heat-induced water loss better, even though their cuticle lacks an outer layer. To determine whether these differences are due to ontogenetic factors or acclimative responses to higher heat loads, conifer seedlings were exposed to a controlled in situ long-term heat treatment. By comparing the rate of water loss through the cuticle (minimum diffusive conductance, gmin), cuticle thickness and chemical micro-composition of control versus heated seedlings of deciduous Larix decidua Mill. and evergreen Picea abies (L.), we intended to assess the acclimative plasticity of these traits. We also aimed to identify specific heat-induced changes in cuticle structure and function. The controlled, in situ, long-term heat treatment lasted for 6 weeks with air temperature set 10-15 K above the ambient air (controls) with a set maximum leaf temperature of 42 °C. This heat treatment significantly reduced gmin in L. decidua but it was not significant in P. abies. In both species, exposure to heat induced an increase in cuticle thickness, as well as provoking species-specific microchemical responses. Lignification of the middle lamella in L. decidua revealed a heat-acclimative potential that likely contributed to reduced cuticular water loss. In P. abies, despite an obvious increase in cuticle thickness, no significant acclimative change was seen in gmin. Flavonoids (kaempferol) were detected throughout the cuticle and outer cell wall of both species, but their contribution to reduced gmin is still unresolved. Our results demonstrate that cuticle and cell wall changes in conifer seedlings are species-specific and trait-dependent, and they highlight the role of epidermal chemistry in shaping resilience to climate warming at the alpine treeline.

The needles of conifer seedlings at the alpine treeline experience hotter near-ground microclimates than adult canopies, prompting the question about how their cuticles maintain water-barrier function under heat. We compared needle cuticle permeability, thickness and microchemistry between seedlings and mature trees of Larix decidua, Picea abies, and Pinus cembra to assess phenotypic plasticity. The minimum leaf diffusive conductance (gmin) was measured two to 24 h after sample detachment during bench drying at temperatures between 25 and 43 degrees C. This procedure should force the stomata to close, providing an indication of the temperature dependent cuticle's water permeability. Raman imaging was used to assess microchemistry and thickness of the cuticles. At the study site, seedling's needles reached temperatures of up to 40 degrees C, which is 14 K higher than that of mature trees. Among species, L. decidua had the highest gmin, followed by P. abies and P. cembra. Seedlings of P. abies and P. cembra exhibited lower gmin at 43 degrees C, indicating greater cuticular resistance to water loss, unlike L. decidua. Raman imaging differentiated species-specific cuticle structures: L. decidua being thinner and aromatic/flavonolrich, P. cembra with a thicker and more lipid/cutin rich cuticle and P. abies was intermediate. Seedlings lacked or had an outer wax layer < 300 nm, though their internal aromatic layers resembled those of mature trees. Despite the rise in cuticular permeability above similar to 35 degrees C, evergreen seedlings maintained lower gmin at 43 degrees C, consistent with acclimative plasticity of cuticle architecture and microchemistry under thermal extremes. These findings link microclimate-driven heat exposure to cuticle traits and highlight that species identity and developmental stage jointly determine water-barrier performance at the treeline.

Climate change is intensifying the frequency and severity of heat waves, posing significant challenges to plant survival, particularly in alpine ecosystems. Elevated leaf temperatures combined with reduced water availability disrupt physiological processes, but the chronology of these events remains unclear. This study investigates the sequence of critical heat thresholds in three alpine plant species from contrasting habitats - Kalmia procumbens, Rhododendron ferrugineum, and Ranunculus glacialis. We assessed stomatal responses (gs), increased cuticular water permeability (gmin, via gas exchange), molecular aggregation and denaturation (via differential scanning calorimetry, DSC), PSII thermotolerance (chlorophyll fluorescence), and tissue heat damage (LT50). Among the investigated species, the earliest detectable physiological response of heat stress was stomatal opening at around 31.9 degrees C, which was followed by functional disturbances of PSII (Tc). With further increase of temperature heat damage progressively developed. Strikingly, an increase of cuticle permeability (gmin) and bulk molecular denaturation via DSC only occurred at significantly higher temperatures than those at which heat damage occurred. Critical heat thresholds were species-specific: R. glacialis showed the lowest heat thresholds, followed by R. ferrugineum and K. procumbens, which corresponds to increasing heat loads in their natural habitat. This study emphasizes the importance of understanding heat stress responses in alpine plants, which face disproportionate impacts from climate change. The findings offer key insights into alpine species' adaptive strategies and suggest that stomatal and molecular heat thresholds may serve as indicators of heat resilience. These insights are critical for improving models of plant responses to global warming, particularly in vulnerable high-altitude ecosystems.

Kalmia procumbens (K. procumbens), a ubiquitous alpine dwarf shrub, thrives at high elevations, particularly on wind-exposed sites. Plants on contrasting north- and southeast-facing slopes at ~2237 m elevation exhibit differences in leaf colour and growth, suggesting acclimative strategies. Leaves from the southeast-facing slope, exposed to higher leaf temperatures (54°C), stronger winds (21 m ∙ s-1), and increased solar irradiation (2319 µMol photons m-2 ∙ s-1), developed thicker cuticles (16 µm) than leaves from north-facing slope (11 µm). Raman imaging revealed that cutin and triterpenoids built the foundation in all cuticles on the adaxial and abaxial leaf sides sampled from the two contrasting slopes, while flavonoids accumulated mostly in the outer adaxial cuticle layer and reached the highest values at the N-site. The thicker cuticle of the S-site was mainly composed of cutin and triterpenoids, while the flavonoids were restricted to a thinner outer layer. Minimum diffusive conductance (gmin) was lower in the S-site leaves, which may be associated to their thicker cuticle. The water permeability (gmin) increased exponentially with temperature in leaves from both slopes. Under heat, above 38°C, north-facing leaves with higher flavonoid content lost increasingly more water. While the flavonoids will defend bacterial and fungal pathogens and have a vital role in enhancing plant resilience, they seem to promote higher water permeability of the cuticle of K. procumbens. By combining physiological, structural and chemical insights, our findings suggest that micro-environmental factors play a significant role in driving acclimative responses in K. procumbens. Cuticle structure, composition and function are finely tuned to alpine microhabitats and illustrate a distinct potential for phenotypic adjustment to environmental and biotic constraints.

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.

Climate change increases the intensity and duration of heatwaves. Heat limits for plants are commonly determined by a 30-minute test. This neglects the effect of heat-dose (intensity x exposure-duration) on heat limits, which has been poorly studied. Heat limits for dysfunction (PSII efficiency) and damage were measured after exposure to various heat-doses (34-64 degrees C x 1-512 min) for five alpine species. The ecological significance of heat-dose was tested based on measured natural heat episodes. With increasing exposure-duration heat limits for 5% damage decreased by 11.2-17.5 K. The same was found for PSII dysfunction, but on average at 7.4 K lower temperatures. Exposure-duration and LT50 followed a slightly species-specific, but logarithmic relationship. This seems very useful for modelling. Natural heat episodes lasted longer than 30 min which questions the 30-minute test. Comparison of natural heat load with the dose-dependent heat limits highlighted that adult trees are safe, but small plants are at high risk for prolonged PSII dysfunction, and even heat damage. Heat responses are not triggered by a single threshold temperature but dose-dependent. The logarithmic relationship found makes it possible to shorten test procedures, extrapolate the response for fully hardened plants and overall more accurately model their heat damage risk in future.

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.

Potato leaves are ice-tolerant but are frost-damaged at −3 °C. Freezing occurs in two steps, a first non-destructive freezing event and a second independent lethal event. Localization of ice, and whether cells freeze-dehydrate after the first freezing event remains unknown. The cause of frost damage during the second freezing event lacks experimental evidence. Cytological responses of mesophyll cells were examined during ice formation using cryo-microscopic techniques after high-pressure freeze-fixation and freeze-substitution. CO2 gas exchange on frozen leaves revealed functional responses, but also frost damage. After the first freezing event, gas exchange was uninterrupted. Consequently, intercellular spaces are free of ice, and ice may be restricted to xylem vessels. The cellular shape of the mesophyll cells was unchanged, cells did not freeze-dehydrate but were supercooled. When thawed after the first freezing event, leaves were initially photoinhibited but regained photosynthesis. During the second freezing event, cells froze intracellularly, and some palisade parenchyma cells remained intact for a prolonged time. Intracellular ice caused complete destruction of cells, and chloroplasts became invisible at the light microscopic level. When thawed after the second freezing, leaves were unable to regain photosynthesis. Consequently, freezing avoidance is the only viable strategy for potatoes to survive frost.

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.

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.

Adaptation strategies in freezing resistance were investigated inKlebsormidium crenulatum, an early branching streptophyte green alga related to higher plants.Klebsormidiumgrows 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 degrees C) were compared to chilled (4 degrees C) as well as extracellularly frozen algae (-2 and -4 degrees 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 degrees C and -4 degrees 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 inKlebsormidiumbefore, 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 degrees C) for 5 h at 20 degrees 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 ofKlebsormidiumto 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.

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.

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.

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.

Abstract 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.

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.

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.

Frost resistance (FR) is a highly adaptive trait and important for plant performance, survival and distribution. While overall seasonal changes in the frost resistance of herbaceous species are well documented, knowledge of the variability during the growth period is scarce. Responses could be expected due to differences in temperature yet investment in frost resistance might be at the expense of plant performance. To analyse temporal and spatial (i.e. same date but differing temperatures) variability, FR of leaves of six herbaceous species on five sampling dates were assessed along an elevational gradient in the northern limestone Alps. We used chlorophyll fluorescence techniques to calculate the lethal temperature of 90% of the population (LT90) thereof. To test the association with plant performance, we measured eco-morphological leaf traits (specific leaf area (SLA), leaf dry matter content, leaf phosphorous and magnesium content as well as stomatal pore area index (SPI)) in parallel. We found that FR as well as leaf traits exhibit a strong temporal variation whereas spatial variability was low. When analysing the relationship of FR to leaf traits we found that SLA as a proxy of growth rate was negatively associated with FR indicating a trade-off between growth and resistance, whereas SPI showed a positive relationship to FR. This finding gives further insight into the variability of traits and will help to improve predictions concerning plant performance and distribution under changing climate regimes.

Alpine environments in Europe are increasingly affected by more erratic precipitation patterns, and more frequent drought and heat waves. Heat-hardening capacity is a key feature for survival of these abiotic stress factors, but it is poorly understood how heat and drought affect plant performance when combined. The main objectives of this study were (1) to determine maximum heat hardening capacity in 14 selected plant species and (2) to study how alpine plants respond to combined heat and drought stress compared to heat alone. (3) For risk assessment maximum leaf temperatures were measured in the field and (4) important methodological aspects of testing heat tolerance were evaluated. Heat hardening capacity was assessed by To the heat threshold of photosystem II (PS II), and by heat tolerance tests based on visual inspection of leaf tissue damage or potential quantum efficiency of PS II (F-v/F-m). A purpose-built Heat Tolerance Testing System (HITS) was used, which allows for controlled heat exposure of whole plants under nearly natural conditions. Additionally, in two species from contrasting habitats, Senecio incanus and Primula minima, the dynamics of heat hardening was studied during and after 8 days exposure to heat (H), or to a combination of heat and severe drought (H+D) within a light-transmissive heat hardening chamber at the alpine field site. In both species, H treatment significantly increased heat tolerance (LT50), determined by the HITS, to 58.0 degrees C and 54.9 degrees C, respectively, and was accompanied by elevated production of abscisic acid (ABA) and salicylic acid (SA), whereas jasmonic acid (JA) levels decreased. Under H+D the LT50 was only 56.5 degrees C and 51.6 degrees C, respectively, and levels of ABA were higher in S. incanus and SA lower in both species in comparison to H. Changes in xanthophyll cycle pigments, alpha-tocopherol and carotenoids:chlorophyll ratio were more pronounced in P. minima than in S. incanus. In P. minima both H and H+D significantly increased singlet oxygen COD scavenging capacity, determined by electron paramagnetic resonance spectroscopy (EPR). In the field, the maximum half-hourly mean (HHM) leaf temperature of P. minima (32.2 degrees C) was significantly lower than of S. incanus (46.5 degrees C, a potentially harmful temperature). We conclude that the investigated species are well adapted to the prevailing temperature conditions in the field. They also possess an outstanding heat hardening capacity, but this can be curtailed when heat is combined with drought. As drought further increases leaf temperatures, the risk of suffering lethal heat damage of some species may increase in the future, particularly at south exposed, ruderal alpine sites with uncertain water supply. (C) 2016 The Author(s). Published by Elsevier B.V.

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.