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Background Global warming is currently occurring at a rapid rate and is having a particularly severe impact on plants, which, as sessile organisms, have a limited ability to escape high temperatures. This requires a better understanding of the thermal limits for different plant species and a better understanding of the processes involved in the development of heat injury in plant leaves. Heat injury results from multiple processes and occurs at the molecular level, involving increased membrane fluidity, lipid peroxidation, and protein aggregation and denaturation. Results We have tested whether the DSC method allows the detection of heat-induced denaturation and aggregation of molecules in intact leaves. During controlled heating a consistent and repeatable pattern was observed in the DSC plot, from which critical heat thresholds could be derived. These critical temperatures were in good agreement with the temperatures determined using classical methods and also clearly mark the thermal limits of molecular structures. The advantage of the DCS method is the precise, rapid and easy detection of heat thresholds. Finally, taken all thresholds together, we can draw a better image of the sequence of events associated with heat injury in plant leaves: heat injury begins with membrane leakage and continues with protein denaturation and aggregation at high (sublethal, lethal) temperatures. Conclusion Since heat injury results from multiple processes, a holistic understanding requires the acquisition of parameters indicative of different processes. The presented DSC method, which allows the detection of denaturation and aggregation of cellular compounds, therefore complements well the classical methods that reflect photosynthetic impairment and whole leaf tissue damage. The new simple and rapid method requires only a minimal amount of leaf material and allows rapid collection of data on damaging temperatures for different plants, which is particularly important in the face of rapidly progressing climatic changes.

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

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.

In temperate-zone mountains, summer frosts usually occur during unpredictable cold spells with snowfalls. Earlier studies have shown that vegetative aboveground organs of most high-mountain plants tolerate extracellular ice in the active state. However, little is known about the impact of frost on reproductive development and reproductive success. In common plant species from the European Alps (Cerastium uniflorum, Loiseleuria procumbens, Ranunculus glacialis, Rhododendron ferrugineum, Saxifraga bryoides, S. moschata, S. caesia), differing in growth form, altitudinal distribution and phenology, frost resistance of reproductive and vegetative shoots was assessed in different reproductive stages. Intact plants were exposed to simulated night frosts between -2 and -14 °C in temperature-controlled freezers. Nucleation temperatures, freezing damage and subsequent reproductive success (fruit and seed set, seed germination) were determined. During all reproductive stages, reproductive shoots were significantly less frost resistant than vegetative shoots (mean difference for LT 50 -4.2 ± 2.7 K). In most species, reproductive shoots were ice tolerant before bolting and during fruiting (mean LT 50 -7 and -5.7 °C), but were ice sensitive during bolting and anthesis (mean LT 50 around -4 °C). Only R. glacialis remained ice tolerant during all reproductive stages. Frost injury in reproductive shoots usually led to full fruit loss. Reproductive success of frost-treated but undamaged shoots did not differ significantly from control values. Assessing the frost damage risk on the basis of summer frost frequency and frost resistance shows that, in the alpine zone, low-statured species are rarely endangered as long as they are protected by snow. The situation is different in the subnival and nival zone, where frost-sensitive reproductive shoots may become frost damaged even when covered by snow. Unprotected individuals are at high risk of suffering from frost damage, particularly at higher elevations. It appears that ice tolerance in reproductive structures is an advantage but not an absolute precondition for colonizing high altitudes with frequent frost events.

Background Freezing resistant plant organs are capable to manage ice formation, ice propagation, and ice accommodation down to variable temperature limits without damage. Insights in ice management strategies are essential for the fundamental understanding of plant freezing and frost survival. However, knowledge about ice management is scarce. Ice crystal localisation inside plant tissues is challenging and is mainly based on optical appearance of ice in terms of colour and shape, investigated by microscopic methods. Notwithstanding, there are major uncertainties regarding the reliability and accuracy of ice identification and localisation. Surface light reflections, which can originate from water or resin, even at non-freezing temperatures, can have a similar appearance as ice. We applied the principle of birefringence, which is a property of ice but not of liquid water, in reflected-light microscopy to localise ice crystals in frozen plant tissues in an unambiguous manner. Results In reflected-light microscopy, water was clearly visible, while ice was more difficult to identify. With the presented polarised cryo-microscopic system, water, including surface light reflections, became invisible, whereas ice crystals showed a bright and shiny appearance. Based on this, we were able to detect loci where ice crystals are accommodated in frozen and viable plant tissues. In Buxus sempervirens leaves, large ice needles occupied and expanded the space between the adaxial and abaxial leaf tissues. In Galanthus nivalis leaves, air-filled cavities became filled up with ice. Buds of Picea abies managed ice in a cavity at the bud basis and between bud scales. By observing the shape and attachment point of the ice crystals, it was possible to identify tissue fractions that segregate intracellular water towards the aggregating ice crystals. Conclusion Cryo-microscopy in reflected-polarised-light allowed a robust identification of ice crystals in frozen plant tissue. It distinguishes itself, compared with other methods, by its ease of ice identification, time and cost efficiency and the possibility for high throughput. Profound knowledge about ice management strategies, within the whole range of freezing resistance capacities in the plant kingdom, might be the link to applied science for creating arrangements to avoid future frost damage to crops.

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.

pH of xylem sap (pHx) was determined in three trees (Malus domestica (apple tree), Picea abies and Pinus cembra) in response to seasonal changes. Conifer trees from lowland (600 m) were compared to trees growing at the alpine timberline (1950 m a.s.l.). Xylem sap was extracted with a Scholander pressure bomb and pHx was measured with a pH microsensor. In all species, pHx changed markedly with season. In spring, pHx was acidic; during winter, the pHx was more alkaline. In apple trees, the pHx did not show a significant correlation with temperature but was rather affected by developmental stage. During flushing in spring, xylem sap acidification took place concomitant to the developmental stage “tight cluster”, when foliar development enables a significant transpiration and a consequent movement of water in the xylem. The xylem sap of the two studied conifers showed a significantly larger seasonal alkalinisation (+2.1) than found in apple trees (+1.2) and was significantly more pronounced at the timberline. Xylem sap acidification took place before bud break. pHx had a significant negative correlation with soil temperatures and corresponded to already reported pHx of angiosperms. Overall, pHx appears to be a sensitive stress marker and indicator of activity status in tree xylem.

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.

In the temperate zone of Europe, plants flowering in early spring or at high elevation risk that their reproductive organs are harmed by episodic frosts. Focusing on flowers of two mountain and three early-flowering colline to montane distributed species, vulnerability to ice formation and ice management strategies using infrared video thermography were investigated. Three species had ice susceptible flowers and structural ice barriers, between the vegetative and reproductive organs, that prevent ice entrance from the frozen stems. Structural ice barriers as found in Anemona nemorosa and Muscari sp. have not yet been described for herbaceous species that of Jasminum nudiflorum corroborates findings for woody species. Flowers of Galanthus nivalis and Scilla forbesii were ice tolerant. For all herbs, it became clear that the soil acts as a thermal insulator for frost susceptible below ground organs and as a thermal barrier against the spread of ice between individual flowers and leaves. Both ice barrier types presumably promote that the reproductive organs can remain supercooled, and can at least for a certain time-period escape from effects of ice formation. Both effects of ice barriers appear significant in the habitat of the tested species, where episodic freezing events potentially curtail the reproductive success.

Infrared thermography has been widely used to study freezing processes in freezing resistant plants but hardly in freezing susceptible species. Solanum tuberosum leaves get frost killed at −3 °C and are unable to frost harden. The basic nature of frost injury to potato leaves is not clear. By employment of infrared differential thermal analysis (IDTA) in combination with viability assessment, we aimed to clarify the mechanistic relationship between ice formation and frost injury. During controlled freezing of potato leaves two distinct freezing events were detected by IDTA. During the first freezing event, the ice wave propagated via the xylem and spread out within 60 s throughout the whole leaf. When leaves were rewarmed after this freezing event, they did not show any frost injury symptoms. We suggest that this non-lethal first ice wave is restricted to the extracellular space. When leaves remained exposed after this exotherm, a second freezing event with a diffuse freezing pattern without a distinct starting point was recorded. When thawed after this second freezing event, leaves always showed frost damage suggesting intracellular freezing. The freezing behavior of potato leaves and its relation to frost damage corroborates that control of ice nucleation is a key for frost protection.