Ice Nucleation Temperature Research Articles

Analysis of supercooling activity of tannin-related polyphenols

Based on the discovery of novel supercooling-promoting hydrolyzable gallotannins from deep supercooling xylem parenchyma cells (XPCs) in Katsura tree (see Wang et al. (2012) [38]), supercooling capability of a wide variety of tannin-related polyphenols (TRPs) was examined in order to find more effective supercooling-promoting substances for their applications. The TRPs examined were single compounds including six kinds of hydrolyzable tannins, 11 kinds of catechin derivatives, two kinds of structural analogs of catechin and six kinds of phenolcarboxylic acid derivatives, 11 kinds of polyphenol mixtures and five kinds of crude plant tannin extracts. The effects of these TRPs on freezing were examined by droplet freezing assays using various solutions containing different kinds of identified ice nucleators such as the ice nucleation bacterium (INB) Erwinia ananas, the INB Xanthomonas campestris, silver iodide and phloroglucinol as well as a solution containing only unintentionally included unidentified airborne ice nucleators. Among the 41 kinds of TRPs examined, all of the hydrolyzable tannins, catechin derivatives, polyphenol mixtures and crude plant tannin extracts as well as a few structural analogs of catechin and phenolcarboxylic acid derivatives exhibited supercooling-promoting activity (SCA) with significant differences (p>0.05) from at least one of the solutions containing different kinds of ice nucleators. It should be noted that there were no TRPs exhibiting ice nucleation-enhancing activity (INA) in all solutions containing identified ice nucleators, whereas there were many TRPs exhibiting INA with significant differences in solutions containing unidentified ice nucleators alone. An emulsion freezing assay confirmed that these TRPs did not essentially affect homogeneous ice nucleation temperatures. It is thought that not only SCA but also INA in the TRPs are produced by interactions with heterogeneous ice nucleators, not by direct interaction with water molecules. In the present study, several TRPs that might be useful for applications due to their high SCA in many solutions were identified.

Investigations on Polyplex Stability During the Freezing Step of Lyophilization Using Controlled Ice Nucleation—The Importance of Residence Time in the Low-Viscosity Fluid State

The aim of the study was to comprehensively investigate the influence of the freezing step during lyophilization on the stability of gene-delivery particles in order to better understand particle stabilization during freezing. Particle size of plasmid/linear polyethylenimine (LPEI) polyplexes at two DNA concentrations and at increasing sucrose-DNA ratios was investigated separately as a function of freezing procedure, ice-nucleation temperature, residence time of the particles in a partially frozen state, or incomplete freezing. Using a numerical model, the increase in sucrose concentration and system viscosity and corresponding bimolecular reaction rates were theoretically estimated. Freezing with a temperature-hold step after ice nucleation negatively influenced particle stability. Ice-nucleation temperature had an impact only at low DNA concentrations. Particle stability was highly reduced during the early part of freezing (<-3°C), especially at low shelf-ramp rates. In this phase, bimolecular reaction rates increase greatly at still low system viscosity. Below a critical temperature (≤∼-18°C) and at high system viscosity, no further particle aggregation occurred. In conclusion, the initial sample viscosity rather than the unfrozen volume and the residence time of particles in the low-viscosity state are the predominant factors in particle stabilization, which likely apply to aggregation in any system.

Improvement to baking quality of frozen bread dough by novel zein-based ice nucleation films

Freezing deteriorates the baking quality of frozen bread dough. This study revealed the protective effects of zein-based ice nucleation films (INFs) on the baking quality of frozen dough. INFs were prepared by immobilizing biogenic ice nucleators on the surface of zein films, which consequently revealed ice nucleation activity and increased the ice nucleation temperature of water from −15 °C to −6.7 °C. By using these films to wrap frozen dough during five freeze/thaw cycles, the specific volume of bread was increased by up to 25% compared to the bread from control frozen dough. The reason was attributed to 40% more viable yeast cells preserved by INFs. In addition, zein-based INFs also reduced the water loss by frozen dough resulting in higher water content in bread crumb. Combining the protective effects on both specific volume and water content from zein-based INFs, the obtained bread showed 68% lower firmness and fracturability and 2.4 times higher resilience compared to the control. The INFs were also superior in that for zein-based INFs, biogenic ice nucleators showed desirable affinity with the surface to sustain at least fifteen repetitive uses on freezing water.

D214 固体・水界面での氷の核生成に対する接触面積の影響(OS-11:凝固・融解を伴う伝熱現象とその応用(4))

To discuss the uniformity of ice nucleating activity of solid/water interfaces, ice nucleation in water droplets prepared on glass surfaces was investigated for various droplet sizes from micrometer to sub-millimeter. When the interfacial area between water and the glass surface was larger than 1×10^<-8> m^2, the ice nucleation temperature showed large scatter, suggesting that the ice nucleating activity was no longer uniform. When the classical nucleation theory (CNT) is used to analyze surface-induced ice nucleation, the uniformity of interfaces that contribute to ice nucleation must be carefully considered, because CNT cannot be directly applied to non-uniform interfaces.

The frost-resistance mechanism in páramo plants is related to geographic origin

The flora of the South American páramo comprises plants of tropical and temperate origin, which either avoid or tolerate freezing. We tested the hypothesis that the frost-resistance mechanism in páramo plants is determined by their geographic origin, i.e. that plants of tropical origin avoid freezing by supercooling, whereas temperate plants tolerate formation of the extracellular ice. We used original along with published data and analysed a total of 47 species from 14 genera for which the origin in the páramo is inferred from molecular phylogenies. The resistance mechanism was determined by comparing the ice-nucleation temperature in the tissues with the temperature at which 50% injury to the tissue occurred. We found that genera of tropical and south temperate origin mostly avoid freezing, whereas genera of north temperate origin mostly tolerate freezing. Significant correlation between the frost-resistance mechanism and origin of the páramo plants suggests that the geographic origin of these plants determines, to a certain degree, their frost-resistance mechanism. However, in several genera both mechanisms are present which indicates that the frost-resistance mechanism also evolved in response to the selective pressure of the páramo environment.

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

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

Supercooling and vitrification of aqueous glycerol solutions at normal and high pressures

The supercooling and vitrification of aqueous glycerol solutions was studied at high pressures. Homogeneous ice nucleation temperatures (TH) were obtained for aqueous glycerol solutions of R=50, 30, 20, 12, and 10 (R: moles of water/moles of glycerol) up to 300MPa. The R=20 glycerol solution formed a glass above 200MPa at a cooling rate of 200°C/min, indicating that pressure enhances glass-formation of aqueous glycerol solutions. The (dTg/dP) values were obtained for vitrified aqueous glycerol solutions of R=3, 5, 10, and 20. These data can be used for the development of cryo-preservation liquids for living cells at high pressures.

Inactivation of Ice Nucleating Activity of Silver Iodide by Antifreeze Proteins and Synthetic Polymers

Antifreeze proteins (AFPs) and poly(vinyl alcohol) (PVA) are known as anti-ice nucleating agents (anti-INAs), which inhibit ice nucleation initiated by ice nucleating agents (INAs). Although the effectiveness of anti-INAs depends on the type of INA, most previous studies on anti-INAs used only a few types of biological INAs as targets to inactivate. In this study, the effects of fish AFPs (AFP I and AFP III) and PVA on the ice nucleating activity of silver iodide (AgI) were measured by using emulsified solutions. Results showed that AgI was inactivated not only by AFPs and PVA but also by two other polymers previously not considered as anti-INAs, namely, poly(vinylpyrrolidone) and poly(ethylene glycol). Even in the presence of AgI, a non-negligible fraction, typically more than 10%, of emulsified droplets of these anti-INA solutions at 1.0 mg mL(-1) was supercooled to about -37 °C, which corresponds to ice nucleation temperature measured in the absence of AgI.

Frost resistance of reproductive tissues during various stages of development in high mountain plants

Frost resistance of reproductive vs aboveground vegetative structures was determined for six common European high alpine plant species that can be exposed to frosts throughout their whole reproductive cycle. Freezing tests were carried out in the bud, anthesis and fruit stage. Stigma and style, ovary, placenta, ovule, flower stalk/peduncle and, in Ranunculus glacialis, the receptacle were separately investigated. In all species, the vegetative organs tolerated on an average 2-5 K lower freezing temperatures than the most frost-susceptible reproductive structures that differed in their frost resistance. In almost all species, stigma, style and the flower stalk/peduncle were the most frost-susceptible reproductive structures. Initial frost damage (LT₁₀) to the most susceptible reproductive structure usually occurred between -2 and -4°C independent of the reproductive stage. The median LT₅₀ across species for stigma and style ranged between -3.4 and -3.7°C and matched the mean ice nucleation temperature (-3.7 ± 1.4°C). In R. glacialis, the flower stalk was the most frost-susceptible structure (-5.4°C), and was in contrast to the other species ice-tolerant. The ovule and the placenta were usually the most frost-resistant structures. During reproductive development, frost resistance (LT₅₀) of single reproductive structures mostly showed no significant change. However, significant increases or decreases were also observed (2.1 ± 1.2 K). Reproductive tissues of nival species generally tolerated lower temperatures than species occurring in the alpine zone. The low frost resistance of reproductive structures before, during and shortly after anthesis increases the probability of frost damage and thus, may restrict successful sexual plant reproduction with increasing altitude.

Freezing activities of flavonoids in solutions containing different ice nucleators

In this study, we examined the effects on freezing of 26 kinds of flavonoid compounds, which were randomly selected as compounds with structures similar to those of flavonoid compounds existing in deep supercooling xylem parenchyma cells (XPCs) in trees, in solutions containing different kinds of ice nucleators, including the ice nucleation bacterium (INB) Erwinia ananas, INB Xanthomonas campestris, silver iodide, phloroglucinol and unidentified airborne impurities in buffered Milli-Q water (BMQW). Cumulative freezing spectra were obtained in each solution by cooling 2μL droplets at 0.2°C/min by a droplet freezing assay. Freezing temperature of 50% droplets (FT50) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT50 were obtained from more than five separate analyses using more than 100 droplets in total in each flavonoid. Supercooling-promoting activities (SCA) or ice nucleation-enhancing activities (INA) of these flavonoids were determined by the difference in FT50 between control solutions without flavonoids and experimental solutions with flavonoids. In mean values, most of the compounds examined exhibited SCA in solutions containing the INB E. ananas, INB X. campestris, silver iodide, and phloroglucinol although the magnitudes of their activities were different depending on the ice nucleator. In solutions containing the INB E. ananas, 10 compounds exhibited SCAs with significant differences (p<0.05) in the range of 1.4–4.2°C. In solutions containing silver iodide, 23 compounds exhibited SCAs with significant differences in the range of 2.0–7.1°C. In solutions containing phloroglucinol, six compounds exhibited SCAs with significant differences in the range of 2.4–3.5°C. In solutions containing the INB X. campestris, only three compounds exhibited SCAs with significant differences in the range of 0.9–2.3°C. In solutions containing unidentified airborne impurities (BMQW alone), on the other hand, many compounds exhibited INA rather than SCA. In mean values, only four compounds exhibited SCAs in the range of 2.4–3.2°C (no compounds with significant difference at p<0.05), whereas 21 compounds exhibited INAs in the range of 0.1–12.3°C (eight compounds with significant difference). It was also shown by an emulsion freezing assay that most flavonoid glycosides examined did not affect homogeneous ice nucleation temperatures, except for a few compounds that become ice nucleators in BMQW alone. These results suggest that most flavonoid compounds affect freezing temperatures by interaction with unidentified ice nucleators in BMQW as examined by a droplet freezing assay. The results of our previous and present studies indicate that flavonoid compounds have very complex effects to regulate freezing of water.

Video analysis of osmotic cell response during cryopreservation

Cellular response during the freeze–thaw process strongly affects the cryopreservation outcome including cell morphology and cell viability. Cryomicroscopy was used to individually analyze the osmotic response of human pulmonary microvascular endothelial cells (HPMECs) during slow cooling (1°C/min) to −60°C and fast rewarming to 4°C (100°C/min). The ice nucleation temperature was controlled (Tn=−8°C). Different concentrations of different cryoprotectant agents, dimethyl sulfoxide, ethylene glycol, proline, ectoin, and trehalose resulted in various cell volume changes. The described methods for image processing and computer vision allows for a fully automatic and individual analysis of the osmotically driven cell response under a temporal resolution of 2frames per second. As a result, we show that in the presence of dimethyl sulfoxide or ethylene glycol cells shrink during cooling to a high degree, especially at intermediate molar concentrations in the range between 0 and 2M, while during rewarming cells swell to isotonic volumes gradually. Comparative cell vitality tests, membrane integrity, and viability tests after 24h recultivation, under these conditions show a high cell survival. In the absence of cryoprotective agents or with proline, ectoin or trehalose, osmotic shrinkage did not meet our expectations: a freeze-induced swelling was detected during cooling and an extreme swelling was observed after rewarming, which was accompanied by lower comparative cell viability. A linear correlation between the cellular membrane integrity after cryopreservation and the maximal relative cell volume was derived (R2=96). The results clearly show that it is crucial to analyze cells within a sample individually due to their individual different osmotic response.

Summer freezing resistance of high-elevation plant species changes with ontogeny

In high-elevation habitats, an apparent trade-off between high growth-rates and freezing resistance has led some authors to assume that seedlings are less freezing resistant than adults. However, because seedlings are exposed to lower and longer freezing events near the ground, they could be more freezing-resistant than adults. As freezing resistance is related to plant height, ontogenetic changes in the intensity of freezing resistance and the mechanism involved should differ between seedlings and adults of tall vs. short plants, with those changes being greater for tall plants. We evaluated differences between seedlings and adult plants in the ability to resist freezing temperatures in 13 high-Andean species from central Chile. In a field laboratory, we determined the low temperature damage, ice nucleation temperature and freezing resistance mechanisms in their leaves. Seedlings were on average 3.4K less freezing resistant than adults with greater differences found for tall plant species. Six species changed their freezing resistance mechanism with ontogeny, including short and tall plants, although changes were greater for tall plant species. Ontogenetic changes in the freezing resistance of high-Andean species seem to be more related to a trade-off between plant-growth and stress resistance than to differences in thermal microhabitats between seedlings and adults.

J024031 氷核活性を抑制するポリフェノール化合物の効果

Antifreeze proteins (AFPs) and several synthetic polymers are known as anti-ice nuclei (anti-IN), which inactivate ice nuclei (IN) in water and thus inhibit heterogeneous ice nucleation catalyzed by IN. Recently, it has been found that several types of polyphenol compounds also inhibit heterogeneous ice nucleation. In this study, ice nucleation catalyzed by silver iodide (Agl) particles, which are typical inorganic IN, in emulsified solutions of five different polyphenol compounds (a-oligoglucosyl quercetin 3-O-β-D-glucopyranoside (Q3(Glc)n), epigallocatechin gallate (EGCG), tea catechin, tannic acid, and oligonol) was investigated experimentally. Results showed that the ice nucleating activity of Agl was inactivated by all the polyphenols used here. For Q3(Glc)n and EGCG solutions, in the majority of the emulsified droplets, ice nucleation occurred within a narrow temperature range between -17 and -20℃. On the contrary, for tea catechin, tannic acid, and oligonol solutions, ice nucleation occurred within relatively wide temperature range between -17 and -38 ℃. The highest ice nucleation temperatures in the emulsified droplets of the polyphenol solutions were considerably lower than those of AFP and synthetic polymer solutions. Especially, the highest ice nucleation temperature for oligonol solution was -19.4 ℃. Such anti-ice nucleating activities of the polyphenols available at relatively high temperatures would be promising for practical applications in various fields.

Studies of cavitation and ice nucleation in ‘doubly-metastable’ water: time-lapse photography and neutron diffraction

High-speed photographic studies and neutron diffraction measurements have been made of water under tension in a Berthelot tube. Liquid water was cooled below the normal ice-nucleation temperature and was in a doubly-metastable state prior to a collapse of the liquid state. This transition was accompanied by an exothermic heat release corresponding with the rapid production of a solid phase nucleated by cavitation. Photographic techniques have been used to observe the phase transition over short time scales in which a solidification front is observed to propagate through the sample. Significantly, other images at a shorter time interval reveal the prior formation of cavitation bubbles at the beginning of the process. The ice-nucleation process is explained in terms of a mechanism involving hydrodynamically-induced changes in tension in supercooled water in the near vicinity of an expanding cavitation bubble. Previous explanations have attributed the nucleation of the solid phase to the production of high positive pressures. Corresponding results are presented which show the initial neutron diffraction pattern after ice-nucleation. The observed pattern does not exhibit the usual crystalline pattern of hexagonal ice [I(h)] that is formed under ambient conditions, but indicates the presence of other ice forms. The composite features can be attributed to a mixture of amorphous ice, ice-I(h)/I(c) and the high-pressure form, ice-III, and the diffraction pattern continues to evolve over a time period of about an hour.

Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 min, cooled in a Linkam cryostage to −7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to −70 °C. IIF that occurred during the 20 °C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were −34, −40, −35, and −25 °C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10–15 °C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-strandedAqp3RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.

The trade-off between cold resistance and growth determines the Nothofagus pumilio treeline

The upper and poleward limit of tree distribution are usually determined by abiotic factors such as low temperature and strong winds. Thus, cold resistance is a key element for survival in high altitudes and latitudes where conditions can reduce plant growth. A trade-off between resource allocation to cold resistance and growth could emerge in populations frequently exposed to low temperatures like those in the treeline zone. We studied annual height growth and ice nucleation temperature in Nothofagus pumilio (Nothofagaceae) populations growing in its extremes of altitudinal distribution and in 3 sites situated on a latitudinal gradient in the Chilean Andes. Additionally, gas exchange, water and nitrogen use efficiency and total soluble sugar (TSS) were also measured as possible mechanisms for survival in high altitudes. Individuals from the treeline populations showed lower annual height growth and lower ice nucleation temperatures compared with those from lower populations. In the same way, individuals from more poleward populations showed lower annual height growth and lower ice nucleation temperatures. Gas exchange, water and nitrogen use efficiency and TSS were also higher in the high altitude populations. The results obtained support the hypothesis of trade-off, because the upper and poleward populations showed more cold resistance but a lower height growth. Additionally, we show that cold resistance mechanisms do not impact the physiological performance, suggesting possible adaptation of the high altitude populations. Low temperatures may be affecting cellular growth instead of photosynthesis, creating a pool of carbohydrates that could participate in cold tolerance. Other abiotic and biotic factors should be also assessed to fully understand the distributional range of Nothofagus species.

Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees

Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-β-d-glucopyranoside (K3Glc), kaempferol 7-O-β-d-glucopyranoside (K7Glc) and quercetin 3-O-β-d-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist.

Pushed to the limit: consequences of climate change for the Araucariaceae: a relictual rain forest family.

Under predicted climate change scenarios, increased temperatures are likely to predispose trees to leaf and other tissue damage, resulting in plant death and contraction of already narrow distribution ranges in many relictual species. The effects of predicted upward temperatures may be further exacerbated by changes in rainfall patterns and damage caused by frosts on trees that have been insufficiently cold-hardened. The Araucariaceae is a relictual family and the seven species found in Australia have limited natural distributions characterized by low frost intensity and frequency, and warm summer temperatures. The temperature limits for these species were determined in order to help understand how such species will fare in a changing climate. Experiments were conducted using samples from representative trees of the Araucariaceae species occurring in Australia, Agathis (A. atropurpurea, A. microstachya and A. robusta), Arauacaria (A. bidwilli, A. cunninghamii and A. heterophylla) and Wollemia nobilis. Samples were collected from plants grown in a common garden environment. Lower and higher temperature limits were determined by subjecting detached winter-hardened leaves to temperatures from 0 to -17 °C and summer-exposed leaves to 25 to 63 °C, then measuring the efficiency of photosystem II (F(v)/F(m)) and visually rating leaf damage. The exotherm, a sharp rise in temperature indicating the point of ice nucleation within the cells of the leaf, was measured on detached leaves of winter-hardened and summer temperature-exposed leaves. Lower temperature limits (indicated by FT(50), the temperature at which PSII efficiency is 50 %, and LT(50) the temperature at which 50 % visual leaf damage occurred) were approx. -5·5 to -7·5 °C for A. atropurpurea, A. microstachya and A. heterophylla, approx. -7 to -9 °C for A. robusta, A. bidwillii and A. cunninghamii, and -10·5 to -11 °C for W. nobilis. High temperature damage began at 47·5 °C for W. nobilis, and occurred in the range 48·5-52 °C for A. bidwillii and A. cunninghamii, and in the range 50·5-53·5 °C for A. robusta, A. microstachya and A. heterophylla. Winter-hardened leaves had ice nucleation temperatures of -5·5 °C or lower, with W. nobilis the lowest at -6·8 °C. All species had significantly higher ice nucleation temperatures in summer, with A. atropurpurea and A. heterophylla forming ice in the leaf at temperatures >3 °C higher in summer than in winter. Wollemia nobilis had lower FT(50) and LT(50) values than its ice nucleation temperature, indicating that the species has a degree of ice tolerance. While lower temperature limits in the Australian Araucariaceae are generally unlikely to affect their survival in wild populations during normal winters, unseasonal frosts may have devastating effects on tree survival. Extreme high temperatures are not common in the areas of natural occurrence, but upward temperature shifts, in combination with localized radiant heating, may increase the heat experienced within a canopy by at least 10 °C and impact on tree survival, and may contribute to range contraction. Heat stress may explain why many landscape plantings of W. nobilis have failed in hotter areas of Australia.

Ice Nucleation in Emulsified Aqueous Solutions of Antifreeze Protein Type III and Poly(vinyl alcohol)

Antifreeze protein (AFP) III and poly(vinyl alcohol) (PVA) are known as anti-ice nucleating agents (anti-INAs), which inhibit heterogeneous ice nucleation. However, the effectiveness of these anti-INAs in inhibiting ice nucleation in water-in-oil (W/O) emulsions, in which homogeneous ice nucleation can be experimentally simulated, is unclear. In this study, the ice nucleation temperature in emulsified solutions of AFP III, PVA, and other nonanti-INA polymers was measured, and then the nucleation rate was analyzed based on classical nucleation theory. Results showed that ice nucleation was surface-initiated and, except for PVA solutions, probably caused heterogeneously by the emulsifier, SPAN 65, at the droplet surfaces. In this nucleation mode, AFP III had no significant effect on the ice nucleation rate. In contrast, PVA exhibited ice-nucleating activity only at the droplet surfaces, suggesting that the nucleation is due to the interaction between PVA and SPAN 65.

Transcriptional responses of winter barley to cold indicate nucleosome remodelling as a specific feature of crown tissues

We report a series of microarray-based comparisons of gene expression in the leaf and crown of the winter barley cultivar Luxor, following the exposure of young plants to various periods of low (above and below zero) temperatures. A transcriptomic analysis identified genes which were either expressed in both the leaf and crown, or specifically in one or the other. Among the former were genes responsible for calcium and abscisic acid signalling, polyamine synthesis, late embryogenesis abundant proteins and dehydrins. In the crown, the key organ for cereal overwintering, cold treatment induced transient changes in the transcription of nucleosome assembly genes, and especially H2A and HTA11, which have been implicated in cold sensing in Arabidopsis thaliana. In the leaf, various heat-shock proteins were induced. Differences in expression pattern between the crown and leaf were frequent for genes involved in certain pathways responsible for osmolyte production (sucrose and starch, raffinose, γ-aminobutyric acid metabolism), sugar signalling (trehalose metabolism) and secondary metabolism (lignin synthesis). The action of proteins with antifreeze activity, which were markedly induced during hardening, was demonstrated by a depression in the ice nucleation temperature.Electronic supplementary materialThe online version of this article (doi:10.1007/s10142-011-0213-8) contains supplementary material, which is available to authorized users.