Analysis of supercooling activities of surfactants

Supercooling-promoting activities (SCAs) of 25 kinds of surfactants including non-ionic, anionic, cationic and amphoteric types were examined in solutions (buffered Milli-Q water, BMQW) containing the ice nucleation bacterium (INB) Erwinia ananas, silver iodide (AgI) or BMQW alone, which unintentionally contained unidentified ice nucleators, by a droplet freezing assay. Most of the surfactants exhibited SCA in solutions containing AgI but not in solutions containing the INB E. ananas or BMQW alone. SCAs of many surfactants in solutions containing AgI were very high compared with those of previously reported supercooling-promoting substances. Cationic surfactants, hexadecyltrimethylammonium bromide (C16TAB) and hexadecyltrimethylammonium chloride (C16TAC), at concentrations of 0.01% (w/v) exhibited SCA of 11.8 °C, which is the highest SCA so far reported. These surfactants also showed high SCAs at very low concentrations in solutions containing AgI. C16TAB exhibited SCA of 5.7 °C at a concentration of 0.0005% (w/v).     

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.     

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.     

Zero-sized effect of nano-particles and inverse homogeneous nucleation. Principles of freezing and antifreeze

It was found that freezing of water in terms of homogeneous nucleation of ice never occurs even in ultra-clean micro-sized water droplets under normal conditions. More surprisingly, at sufficiently low supercoolings, foreign nano-particles exert no effect on the nucleation barrier of ice; it is as if they physically "vanished." This effect, called hereafter the "zero-sized" effect of foreign particles (or nucleators), leads to the entry of a so-called inverse homogeneous-like nucleation domain, in which nucleation is effectively suppressed. The freezing temperature of water corresponds to the transition temperature from the inverse homogeneous-like nucleation regime to foreign particle-mediated heterogeneous nucleation. The freezing temperature of water is mainly determined by (i) the surface roughness of nucleators at large supercoolings, (ii) the interaction and structural match between nucleating ice and the substrate, and (iii) the size of the effective surface of nucleators at low supercoolings. Our experiments showed that the temperature of -40 degrees C, commonly regarded as the temperature of homogeneous nucleation-mediated freezing, is actually the transition temperature from the inverse homogeneous-like nucleation regime to foreign particle-mediated heterogeneous nucleation in ultra-clean water. Taking advantage of inverse homogeneous-like nucleation, the interfacial tensions between water and ice in very pure water and antifreeze aqueous solutions were measured at a very high precision for the first time. The principles of freezing promotion and antifreeze and the selection for the biological ice nucleation and antifreeze proteins are obtained. The results provide completely new insights into freezing and antifreeze phenomena and bear generic implications for all crystallization systems.     

The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate

Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.     

Characterization and Quantification of Intrinsic Ice Nucleators in Winter Rye (Secale cereale) Leaves

Extracellular ice formation in frost-tolerant organisms is often initiated at specific sites by ice nucleators. In this study, we examined ice nucleation activity (INA) in the frost-tolerant plant winter rye (Secale cereale). Plants were grown at 20[deg]C, at 5[deg]C with a long day, and at 5[deg]C with a short day (5[deg]C-SD). The threshold temperature for INA was -5 to -12[deg]C in winter rye leaves from all three growth treatments. Epiphytic ice nucleation-active bacteria could not account for INA observed in the leaves. Therefore, the INA must have been produced endogenously. Intrinsic rye ice nucleators were quantified and characterized using single mesophyll cell suspensions obtained by pectolytic degradation of the leaves. The most active ice nucleators in mesophyll cell suspensions exhibited a threshold ice nucleation temperature of -7[deg]C and occurred infrequently at the rate of one nucleator per 105 cells. Rye cells were treated with chemicals and enzymes to characterize the ice nucleators, which proved to be complexes of proteins, carbohydrates, and phospholipids, in which both disulfide bonds and free sulfhydryl groups were important for activity. Carbohydrates and phospholipids were important components of ice nucleators derived from 20[deg]C leaves, whereas the protein component was more important in 5[deg]C-SD leaves. This difference in composition or structure of the ice nucleators, combined with a tendency for more frequent INA, suggests that more ice nucleators are produced in 5[deg]C-SD leaves. These additional ice nucleators may be a component of the mechanism for freezing tolerance observed in winter rye.     

The freezing characteristics of cauliflower curd

Cultivar differences in frost resistance and the heritable nature of resistance were demonstrated using seedling cauliflower plants. Such cultivar differences were not however expressed in the curd. Selection for frost resistance in cauliflower should therefore use whole plant screening techniques. Curd material when frozen as isolated florets, supercooled over the range – 1°C to – 12°C and the mean freezing point of all curds tested was -6°C to -7.25°C (overall mean -6.44°C). Curd florets which supercooled but did not freeze were completely undamaged, whereas freezing always led to cell damage and death observed as water-soaking of the floret surface and measured using an electrical conductivity method. The large range of freezing points measured suggests a range of active ice nucleators either on or within the florets. When curds were frozen intact the ability of florets to supercool was severely restricted which was attributed to the seeding of freezing by the internal growth of ice crystals. A crop protection strategy needs to identify and control or modify warm temperature nucleators in cauliflower curd.     

Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells

Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold’s beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 °C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C. japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E. ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed.     

Identification of a novel ice-nucleating bacterium of Antarctic origin and its ice nucleation properties

A novel ice-nucleating bacterium (INB) was isolated from Ross Island, Antarctica. INBs could be isolated more frequently than was generally thought. INB strain IN-74 was found in the white colony group. Strain IN-74 was identified from its taxonomic characteristics as a novel INB, Pseudomonas antarctica IN-74. When strain IN-74 was cultured aerobically in a medium consisting of the ice-nucleating broth (pH 7.0) for 6 days at 4 degrees C, the ice-nucleating activity of strain IN-74 cells was obtained. Strain IN-74 cells produced ice nuclei only at extremely low growth temperatures. The nuclei appeared to be less thermolabile than those of INB Pseudomonas fluorescens KUIN-1. The freezing difference spectra in D2O and H2O at ice-nucleating temperature for strain IN-74 cells and conventional INBs (Pseudomonas fluorescens KUIN-1, Pseudomonas viridiflava KUIN-2, and Pseudomonas syringae C-9) exhibited different curves.     

Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells

Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold’s beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 °C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C. japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E. ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed.     

Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

Ice nucleation and freezing tolerance in New Zealand alpine and lowland weta, Hemideina spp. (Orthoptera; Stenopelmatidae)

Abstract.The alpine tree weta Hemidiena maori Pictet et Saussure (Orthoptera: Stenopelmatidae) is a large, flightless insect found above the treeline on many of the mountain ranges of the South Island of New Zealand. The population found on the Rock and Pillar Range, Central Otago has been identified as freezing tolerant with a haemolymph ice nucleating agent. The ability of H. maori to survive freezing is compared to the lowland weta Hemideina thoracica Walker and H. crassidens Blanchard, both of which are able to survive the formation of some ice in their bodies. Mortality is associated with time spent frozen in H. thoracica , and it is hypothesized that this species is killed when a critical proportion of its body water is frozen. All five subalpine and alpine populations of H. maori surveyed were found to be freezing tolerant.
Comparison of temperatures of first nucleation and mean supercooling point of haemolymph droplets suggest that haemolymph ice nucleating activity varies between populations of H. maori. Hemideina maori collected from the Mt Cook region appear to lack a haemolymph ice nucleator. This population is nevertheless freezing tolerant, suggesting that the haemolymph ice nucleating agent described in H. maori is not essential for freezing tolerance. Hemideina crassidens and H. ricta Hutton, both of which are found in lowland habitats, also had high mean supercooling point and temperatures of first nucleation of haemolymph droplets, suggesting that these species also have a haemolymph ice nucleator.
Comparison of ice nucleation characteristics of haemolymph and faecal material (representing gut contents) suggests that gut nucleators in H. maori may be at least as efficient as the haemolymph nucleator. It is concluded that freezing tolerance is probably not an adaptation to the alpine environment. This highlights the need for inter- and intraspecific comparative studies if physiological data are to be used to draw evolutionary conclusions.     

Biological control of an insect pest by gut-colonizing Enterobacter cloacae transformed with ice nucleation gene

The ice nucleation (IN) gene inaA of epiphytic Erwinia (Pantoea) ananas IN10 was transformed into Enterobacter cloacae WBMH-3-CMr originated from the faeces of silkworms. The transformant designated as Ent. cloacae WBMH-3-CMr(pICE6S13) exhibited IN activity, unlike the parent strain. The transgenic strain was ingested by mulberry pyralid larvae, fed on detached mulberry leaves, and the supercooling capacity and cold hardiness of these larvae were examined. The mean supercooling point (SCP) of the larvae ingesting the transgenic strain was - 3.3 degrees C, 8 degrees C higher than that of larvae treated with distilled water (control) and 1.5 C higher than an ice nucleation active (INA) strain of Erw. ananas. The SCPs of the larvae were stably maintained over the 9 d after ingestion. The maintenance of these high SCPs was due to transgenic Ent. cloacae having a more stable and efficient gut colonization than Erw. ananas, which is identified by the distribution of a narrower range of SCPs (-2 to -5 degrees C) in larvae treated with the transgenic stain. Furthermore, most of the larvae ingesting the transgenic strain froze and died when they were exposed to cold conditions of -5 degrees C for 18 h, 3 or 7 d after ingestion. In contrast, most of the larvae ingesting no bacterium did not die under similar conditions. On the other hand, the growth ability of Ent. cloacae WBMH-3-CMr on mulberry leaves tended to be lower than that of epiphytic Erw. ananas, as assayed by pot tests. These findings would expand the possibility of biological control using INA bacteria since Ent. cloacae would harbour a broader host (insect) range for gut colonization and a smaller affinity to plants to benefit from prevention of plant frost injury.     

Gene Expression and Signal Transduction in Water-Stress Response

We evaluated the use of infrared (IR) video thermography to observe directly ice nucleation and propagation in plants. An imaging radiometer with an HgCdTe long-wave (8-12 [mu]m) detector was utilized to image the thermal response of plants during freezing. IR images were analyzed in real time and recorded on videotape. Information on the videotape was subsequently accessed and analyzed utilizing IR image analysis software. Freezing of water droplets as small as 0.5 [mu]L was clearly detectable with the radiometer. Additionally, a comparison of temperature tracking data collected by the radiometer with data collected with thermocouples showed close correspondence. Monitoring of an array of plant species under different freezing conditions revealed that ice nucleation and propagation are readily observable by thermal imaging. In many instances, the ice nucleation-active bacterium Pseudomonas syringae placed on test plants could be seen to initiate freezing of the whole plant. Apparent ice nucleation by intrinsic nucleators, despite the presence of ice nucleation-active bacteria, was also evident in some species. Floral bud tissues of peach (Prunus persica) could be seen to supercool below the temperature of stem tissues, and ice nucleation at the site of insertion of the thermocouple was frequently observed. Rates of propagation of ice in different tissues were also easily measured by thermal imaging. This study demonstrates that IR thermography is an excellent method for studying ice nucleation and propagation in plants.     

Ice nucleation and antinucleation in nature

Plants and ectothermic animals use a variety of substances and mechanisms to survive exposure to subfreezing temperatures. Proteinaceous ice nucleators trigger freezing at high subzero temperatures, either to provide cold protection from released heat of fusion or to establish a protective extracellular freezing in freeze-tolerant species. Freeze-avoiding species increase their supercooling potential by removing ice nucleators and accumulating polyols. Terrestrial invertebrates and polar marine fish stabilize their supercooled state by means of noncolligatively acting antifreeze proteins. Some organisms also depress their body fluid melting point to ambient temperature by evaporation and/or solute accumulation.     

A method for quantitative determination of ice nucleating agents in insect hemolymph

The relationship between the concentration of insect hemolymph ice nucleators in samples of 0.9% NaCl solution and the supercooling points of the samples was determined by using a dilution technique. The supercooling points were only moderately reduced following dilution by a factor of up to 10³, whereas dilution beyond this point caused a marked drop in the supercooling points. The dilution factor corresponding to a 50% reduction in the nucleating activity of native hemolymph is taken as a measure of the concentration of ice nucleators in native hemolymph.This method was used to determine the concentration of ice nucleators in the hemolymph of Eurosta solidaginis larvae from Minnesota and Texas, acclimated to different temperatures. Significant levels of nucleators were found only in larvae from Minnesota, and +5 °C was found to be the optimal temperature for nucleator formation. This comparatively high temperature optimum is interpreted as a physiological adaptation, ensuring sufficient nucleator levels in the hemolymph by the time of the first exposure to freezing temperatures in the winter.     

Low-temperature conditioning of the ice nucleation active bacterium,Erwinia herbicola

Rapid “low-temperature conditioning” and “solute conditioning” of the ice nucleation active bacterium Erwinia herbicola No. 26 are described. Conditioning is the process by which the ability to initiate ice at high temperatures is gained in these bacteria. The cumulative ice nucleator concentration, N[T], was used to measure the number of ice nucleators present in the bacterial systems. N[T] was determined at temperatures from −2 ° to −10 °C and was measured under varying conditioning temperature, time, and solute regimes. Values of N[T] increased rapidly on cooling samples from 30 to 5 °C. The optimum low temperature for conditioning was 5 °C. The conditioning process followed first-order reaction kinetics and time constants (1/rate constant) were between 43 and 62 min at 5 °C. Individual ice nucleators were isolated in droplets and were stable for at least 2 hr. Low-temperature conditioning did not occur when protein synthesis was inhibited by eliminating amino acids in the low-temperature conditioning media or by using the protein synthesis inhibitors chloramphenicol and streptomycin. Analysis of low-temperature conditioning, using heterogeneous ice nucleation theory predicted that ice nucleators are large and have diameters ranging from 80 Å (active at −8 °C) to 300 Å (active at −3 °C). In conclusion, it was predicted that conditioning resulted from growth of the nucleator from about 80 to 300 Å, from a change in the surface properties of 300 Å nucleator making it more similar to ice, or from a combination of these.     

Physiological and ecological significance of biological ice nucleators

When a pure water sample is cooled it can remain in the liquid state at temperatures well below its melting point (0 degrees C). The initiation of the transition from the liquid state to ice is called nucleation. Substances that facilitate this transition so that it takes place at a relatively high sub-zero temperature are called ice nucleators. Many living organisms produce ice nucleators. In some cases, plausible reasons for their production have been suggested. In bacteria, they could induce frost damage to their hosts, giving the bacteria access to nutrients. In freeze-tolerant animals, it has been suggested that ice nucleators help to control the ice formation so that it is tolerable to the animal. Such ice nucleators can be called adaptive ice nucleators. There are, however, also examples of ice nucleators in living organisms where the adaptive value is difficult to understand. These ice nucleators might be structures with functions other than facilitating ice formation. These structures might be called incidental ice nucleators.     

Complex bud architecture and cell‐specific chemical patterns enable supercooling of Picea abies bud primordia

Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to ?50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood. Bud architecture was assessed by 3D—reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell‐specific chemical patterns by Raman microscopy and mass spectrometry imaging. A bowl‐like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At ?18 °C, break‐down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature‐dependent freeze dehydration (?0.1 MPa K^?1) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin‐rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides. Bud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell‐specific biochemical analysis.     

细菌冰核提高印度谷螟过冷却点的研究

印度谷螟(Plodia interpunctella)是一种不耐结冰的昆虫,在冬季它通过降低过冷却 点以避免结冰。现已查明,冰核活性细菌能显著提高植物的过冷却点,导致许多作物在较高 的温度下发生霜冻害。本文也证明细菌冰核能显著提高印度谷螟虫的过冷却点。对照的平均过冷却点是-17.6℃;分别用0.1g和1g细菌冰核与1kg面粉混合后进行处理,平均过冷却点分别比对照提高了12.8℃和13.6℃。研究结果支持这样的观点：细菌冰核有可能成为一种在冬季使用的、杀灭不耐结冰害虫的生物制剂。