Thermal insulation of the winter cauliflower curd by wrapper-leaves

The role of leaves as insulation for mature curds during exposure to frost has been investigated for winter-harvested cauliflower. The inner, wrapper-leaves and their enclosed air spaces provided significant protection for the curd tissues in mild frost, but under more severe conditions (below – 2.5°C) were ineffective. A relationship between higher wrapper-leaf number and increased insulation was described. The increase in insulation however, was small even with much higher wrapper-leaf numbers. Exposure of the curd surface at maturity was found to prevent the insulating effect of wrapper-leaves, particularly if the diameter of the exposed area was greater than 6 cm. The data also suggested a possible role for the exposed stem of the cauliflower as a site of heat loss that may adversely affect curd temperature. It is postulated that breeding for improved frost resistance by greatly increasing wrapper-leaf number would not be effective, although any reduction in wrapper-leaf number and the presence of tightly wrapped curds should be avoided. Furthermore, exposure of the curd surface at maturity should be kept to a minimum.     

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.     

Supercooling and heterogeneous nucleation of freezing in tissues of tender plants.

The extent to which tissue pieces of several frost tender plant species could be supercooled in the absence of external sources of ice nucleation was determined in a small cold chamber. A considerable range among plant species was revealed in their ability to supercool. This could be expressed as a differential nucleus spectrum that derives the minimum concentration of nucleating sites within the samples. French bean (Phaseolus vulgaris L.) was found to contain nucleators effective in the range ?4 to ?8 °C, whereas spring wheat (Triticum aestivum L.) contained sites active between ?8 and ?16 °C. The data indicate that these plant samples can supercool to temperatures below those normally injurious to them when they are frozen.     

Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to ?45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to ?45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.     

An investigation of the frost hardiness of grapevine (Vitis vinifera) during bud break

The aim of this investigation was to assess ice nucleation and frost resistance of two varieties of grapevine (Siegrebbe and Madeleine Angevine) during bud burst under radiative freezing conditions analogous to those during Spring in the UK. During bud burst, grapevines were almost entirely resistant to freezing during frosts of less than -3°C by virtue of their ability to supercool. The risk of frost damage increased significantly as bud development progressed, and once buds had passed growth stage DS3 they became more sensitive to freezing and freezing damage was more extensive. The two varieties did not differ in frost resistance but, because of its earlier developing habit, variety Siegrebbe could be more prone to frost damage in the field. Buds were more prone to damage after freezing once bud burst had commenced and the damage could not be reversed by acclimating plants for periods of 7 to 21 days at 4°C in an 8 h photoperiod. Such acclimation appeared to predispose frozen buds to more extensive damage.     

Rapid in vitro propagation of cauliflower

A rapid single step method for complete plantlet regeneration in cauliflower is described. Curd explants cultured on Murashige and Skoog (MS) medium supplemented with 1 mg⁻¹ indoleacetic acid (IAA) developed complete plantlets in 25 days. Regeneration potential of curd explants was drastically reduced with increasing storage of cauliflower curds at room temperature beyond 8 days of harvesting. Light intensity of 3000 lux supplied by white fluorescent tubes was optimum to evoke the best response. It was possible to field transfer the hardened plantlets within 35 days of culture initiation.     

The Siberian timberman Acanthocinus aedilis: a freeze-tolerant beetle with low supercooling points

Larvae of the Siberian timberman beetle Acanthocinus aedilis display a number of unique features, which may have important implications for the field of cold hardiness in general. Their supercooling points are scattered over a wide temperature range, and some individuals have supercooling points in the low range of other longhorn beetles. However, they differ from other longhorn beetles in being tolerant to freezing, and in the frozen state they tolerate cooling to below −37°C. In this respect they also differ from the European timberman beetles, which have moderate supercooling capacity and die if they freeze. The combination of freezing tolerance and low supercooling points is unusual and shows that freezing at a high subzero temperature is not an absolute requirement for freezing tolerance. Like other longhorn beetles, but in contrast to other freeze-tolerant insects, the larvae of the Siberian timberman have a low cuticular water permeability and can thus stay supercooled for long periods without a great water loss. This suggests that a major function of the extracellular ice nucleators of some freeze-tolerant insects may be to prevent intolerable water loss in insects with high cuticular water permeability, rather than to create a protective extracellular freezing as has generally been assumed. The freezing tolerance of the Siberian timberman larvae is likely to be an adaptation to the extreme winter cold of Siberia.     

Summer frost resistance and freezing patterns measured in situ in leaves of major alpine plant growth forms in relation to their upper distribution boundary

Summer frost resistance and ice nucleation temperatures for 33 alpine plant species were measured in situ to avoid the shortcomings of laboratory tests. Species were selected to investigate the relationship between plant stature and upper distribution boundary, and frost resistance and freezing patterns. The species tested in situ were on average 1.1 K (± 0.2, SE) frost hardier than in laboratory tests. Frost resistance (LT₅₀) ranged from ?4.5 to ?14.6 °C and appeared insufficient to protect against air temperature minima, corroborating reports of natural frost damage. All species tolerated extracellular ice formation (recorded at ?1.9 ± 0.2 °C; E1). Initial frost damage occurred at average temperatures 4.9 K below E1. In 64% of the species a second exotherm (E2) and frost damage were recorded between ?3.7 and ?9.4 °C. In the highest ranging species E2 was not detectable. Frost resistance increased with increasing upper distribution boundary (0.4 K per 100 m), corresponding well with the altitudinal decrease in air temperature minima. No relationship between plant stature and frost resistance was found. Graminoids were significantly frost hardier than other growth forms. Frost survival at high altitudes will depend not only on altitudinal increase in frost resistance but also on freezing avoidance strategies, snow cover protection and a high recuperation capacity.     

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。     

How endangered is sexual reproduction of high-mountain plants by summer frosts? Frost resistance, frequency of frost events and risk assessment

In temperate-zone mountains, summer frosts usually occur during unpredictable cold spells with snow-falls. 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 LT₅₀ ?4.2 ± 2.7 K). In most species, reproductive shoots were ice tolerant before bolting and during fruiting (mean LT₅₀ ?7 and ?5.7 °C), but were ice sensitive during bolting and anthesis (mean LT₅₀ 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.     

Assessment of the frost sensitivity of Trifolium species by chlorophyll fluorescence analysis

The potential of the chlorophyll fluorescence technique in screening for frost sensitivity in a range of Trifolium species from different geographical origins was assessed by measuring the decrease in variable chlorophyll fluorescence (F_var) of leaves after freezing at - 5°C for 60 min. The method was rapid and the results obtained agreed well with a visual assessment of freezing injury carried out after leaves were returned to optimal growth conditions for 72 h. Trifolium alexandrinum (Berseem clover) cv. Tabor originating from Israel was shown to be the most frost sensitive species studied and Trifolium subterraneum (subterranean clover) cv. Mt. Barker, from temperate regions of Australia, the most frost resistant. On extended periods of freezing, frost damage increased and this was associated with a further reduction in variable chlorophyll fluorescence and in quenching capacity of the thylakoid membranes. These results thus indicate that substantial thylakoid membrane dysfunction is induced at freezing temperatures. Furthermore, it was found that frost hardening of the frost sensitive species T. alexandrinum for 21 days at 5°C reduced the extent of damage sustained by the thylakoid membranes as shown by higher fluorescence quenching capacity, smaller reduction in variable fluorescence (F_var) and higher initial fluorescence (F_o) when leaves of hardened plants were frozen at -5°C and -7°C.     

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 2 μL droplets at 0.2 °C/min by a droplet freezing assay. Freezing temperature of 50% droplets (FT(50)) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT(50) 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 FT(50) 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.     

Ultrastructure before freezing,while frozen,and after thawing in assessing cryoinjury of mouse epididymal spermatozoa

Tails of mouse epididymides were treated as follows: control, unfrozen with and without cryoprotective agents (CPA); frozen (to below ?80 °C), slowly (8 °C/min), and rapidly (18 °C/sec), with and without CPA. Intracellular and/or extracellular location of CPA, at least glycerol, was influenced, respectively, by high (22 °C) or low (0 °C) exposure temperature. Standard procedures in electron microscopy were employed and the frozen state preserved by freeze-substitution. Motility before freezing and after thawing was the criterion of cryosurvival.Results showed no evidence of deleterious ultrastructural effects of freezing at rates compared, or of benefits of CPA, regardless of their cellular location. Differences were noted, however, in the appearance of spermatozoa in the frozen state, as a function of the rate of freezing but not as a function of the presence, absence, or location of either glycerol of DMSO. Rapidly frozen cells showed intracellular ice formation in the acrosome, neck, midpiece, and tail regions; there was no intranuclear ice, and extracellular ice artifacts were small. Slowly frozen cells showed large extracellular ice artifacts with evidence of shrinkage distortion due to the dehydration induced by extracellular ice. No spermatozoa survived any of the freezing treatments, showing the lethal effect of both extracellular ice during slow freezing and of intracellular and/or extracellular ice during rapid freezing.     

Maintenance of the supercooled state in overwintering pyrochroid beetle larvae, Dendroides canadensis : role of hemolymph ice nucleators and antifreeze proteins

Freeze-avoiding fire-colored beetle larvae, Dendroides canadensis, were monitored seasonally to explore the role of endogenous hemolymph ice nucleators and antifreeze proteins on the maintenance of supercooling. In preparation for overwintering, D. canadensis depressed hemolymph ice nucleator activity and increased thermal hysteresis activity [mean value circa 0. 5 °C (summer) versus circa 5 °C (midwinter)] resulting in decreased larval and hemolymph supercooling points [−7 °C (summer) versus −20 °C (midwinter)]. Results of gel filtration chromatography, flotation ultracentifugation and quantitative investigation of ice nucleator activity using hemolymph from summer and winter collected larvae strongly suggest that highly active protein and lipoprotein ice nucleators are removed in preparation for overwintering. Additions of either purified antifreeze proteins or midwinter hemolymph with high antifreeze protein activity to a mixture of protein or lipoprotein ice nucleators isolated from D. canadensis hemolymph inhibited the activity of these nucleators. This suggests that in addition to seasonal removal, inhibition of hemolymph ice nucleators by antifreeze proteins contributes to seasonal increases in hemolymph supercooling capacity. Accepted: 8 August 1996     

Freezing tolerance and avoidance in high-elevation Hawaiian plants

Freezing resistance mechanisms were studied in five endemic Hawaiian species growing at high elevations on Haleakala volcano, Hawaii, where nocturnal subzero (°C) air temperatures frequently occur. Extracellular freezing occurred at around -5°C in leaves of Argyroxiphium sandwicense and Sophora chrysophylla, but these leaves can tolerate extracellular ice accumulation to -15°C and -12°C, respectively. Mucilage, which apparently acted as an ice nucleator, comprised 9 to 11% of the dry weight of leaf tissue in these two species. Leaves of Vaccinium reticulatum and Styphelia tameiameiae were also found to tolerate substantial extracellular freezing. Dubautia menziesii, on the other hand, exhibited the characteristics of permanent supercooling; a very rapid decline in liquid water content associated with simultaneous intracellular and extracellular freezing. However, in those species that tolerate extracellular freezing, the decline in liquid water content during freezing is relatively slow. Osmotic potential was lower at pre-dawn than at midday in four of the species studied. Nocturnal production of osmotically active solutes may have helped to prevent intracellular freeze dehydration as well as to provide non-colligative protection of cell membranes. Styphelia tameiameiae supercooled to -9·3°C and tolerated tissue freezing to below -15°C, a unique combination of physiological characteristics related to freezing. Tolerance of extracellular ice formation after considerable supercooling may have resulted from low tissue water content and a high degree of intracellular water binding in this species, as determined by nuclear magnetic resonance studies. The climate at high elevations in Hawaii is relatively unpredictable in terms of the duration of subzero temperatures and the lowest subzero temperature reached during the night. It appears that plants growing in this tropical alpine habitat have been under selective pressures for the evolution of freezing tolerance mechanisms.     

Increased cold hardiness in eggs of Arcynopteryx compacta (Plecoptera) by dehydration

Eggs of the stonefly, Arcynopteryx compacta, that overwinter in the alpine region of Norwegian mountains, increase their cold-hardiness by dehydration. Eggs enclosed in ice at −22°C survive the loss of about two-thirds of their total water content by shrinkage due to passive diffusion of body water along the concentration gradient. Fully hydrated eggs are killed by freezing at their supercooling point of −26°C, and by direct cooling to −30°C. Dehydrated eggs have a mean supercooling point of −31°C, and survive exposure at −27 and −29°C in ice. Judged from their melting points the eggs do not accumulate low-molecular-weight cryoprotective substances. The difference between freezing and melting points corresponds to a thermal hysteresis of up to 1.8°C. The presence of thermal hysteresis antifreezes may stabilize their supercooled state when enclosed by ice during overwintering. The eggs enter diapause in the autumn, and diapause completion is enhanced both by temperature and time during enclosure in ice.     

Equilibrium freezing of leaf water and extracellular ice formation in Afroalpine ‘giant rosette’ plants

The water potentials of frozen leaves of Afroalpine plants were measured psychrometrically in the field. Comparison of these potentials with the osmotic potentials of an expressed cellular sap and the water potentials of ice indicated almost ideal freezing behaviour and suggested equilibrium freezing. On the basis of the osmotic potentials of expressed cellular sap, the fractions of frozen cellular water which correspond to the measured water potentials of the frozen leaves could be determined (e.g. 74% at -3.0° C). The freezing points of leaves were found to be in the range between 0° C and -0.5° C, rendering evidence for freezing of almost pure water and thus confirming the conclusions drawn from the water-potential measurements. The leaves proved to be frost resistant down to temperatures between -5° C and -15° C, as depending on the species. They tolerated short supercooling periods which were necessary in order to start ice nucleation. Extracellular ice caps and ice crystals in the intercellular space were observed when cross sections of frozen leaves were investigated microscopically at subfreezing temperatures.Symbols T temperature - water potential Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

A chamber for the simulation of radiation freezing of plants

Frost injury to plants can occur following episodic radiation frosts. In the UK this is particularly important to spring sown crops such as potatoes. Most laboratory based frost studies simulate freezing using either conductive or convective freezing chambers. Such frost tests do not simulate overnight freezing events adequately. A freezing chamber based on radiative cooling is described which mimics overnight radiative freezing. The chamber is rectangular in design (1 m × lm × 2 m high) with a radiative cooling plate at the top of the chamber cooled to -40°C to -45°C using HFC coolants, which acts as a cold black body. The sides of the chamber are also cooled to variable temperatures down to -5°C in order to prevent the chamber walls radiating to the plant material during testing. Using thermocouples to measure air temperature and plant temperature the chamber has been characterised to simulate the radiative cooling conditions found in the UK during autumn and spring. Exotherm detection upon plant freezing is simplified by virtue of the reduction in temperature fluctuation normally experienced at the plant surface during natural freezing. Radiation frosts and subsequent frost damage to potatoes have been recorded in the temperature range -4°C to –5°C. The equipment is recommended for studies of frost damage to plants normally caused by episodic radiation frost events.     

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.