Advanced hepatic tissue destruction in ablative cryosurgery: potentials of intermittent freezing and selective vascular inflow occlusion

Recent studies indicate that cryosurgery represents a promising approach to treat non-resectable liver tumors. To improve parenchymal tissue destruction, a variety of modifications of the freeze-thaw procedure have been suggested, including repetitive freezing and portal-triad cross-clamping. The aim of the present study was to analyze whether intermittent freezing by application of a double freeze-thaw procedure or selective vascular inflow occlusion are more effective than a single freeze-thaw cycle to achieve complete hepatic tissue destruction. Using a porcine model, intrahepatic cryolesions were induced by freezing the hepatic tissue for a total of 15 min (n=6, SF). Additional animals (n=6) underwent a double freeze-thaw cycle of 7.5 min each (DF). A third group of animals (n=6) was treated by a single 15-min freeze-thaw cycle during selective vascular inflow occlusion (VO-SF). Seven days after freezing, DF did not change the volume of the cryolesion (25.4+/-1.7 cm(3)) compared to SF (29.9+/-3.7 cm(3)), however, resulted in enhanced destruction of hepatocyte nuclear morphology (DF-score: 2.4+/-0.2 versus SF-score: 1.1+/-0.3; p<0.05) and attenuated leukocyte infiltration within the margin of the cryolesion (DF-score: 1.5+/-0.2 versus SF-score: 2.8+/-0.1; p<0.05). VO-SF was also effective to significantly enhance destruction of hepatocyte nuclear morphology (2.8+/-0.1; p<0.05 versus SF), but, additionally, markedly increased the volume of the cryolesions (43.3+/-5.3 cm(3); p<0.05 versus SF and DF). Interestingly, VO-SF further increased the number of apoptotic cells, while leukocyte infiltration (2.3+/-0.3) was not affected compared to that after SF-treatment. Thus, our data indicate that both DF and VO-SF are effective to enhance parenchymal cell destruction within the margin of the cryolesion. VO-SF additionally increases the volume of the lesion and may therefore be most attractive for successful clinical application.     

Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence

Infrared differential thermal analysis (IDTA) and differential imaging chlorophyll fluorescence (DIF) were employed simultaneously to study the two-dimensional pattern of ice propagation in leaves and mesophyll freeze dehydration as detected by a significant increase of basic chlorophyll fluorescence (F(0)). IDTA and DIF technique gave different insights into the freezing process of leaves that was highly species-specific. IDTA clearly visualized the freezing process consisting of an initial fast spread of ice throughout the vascular system followed by mesophyll freezing. While mesophyll freezing was homogeneously in Poa alpina, Rhododendron ferrugineum and Senecio incanus as determined by IDTA, DIF showed a distinct pattern only in S. incanus, with the leaf tips being affected earlier. In Cinnamomum camphora, a mottled freezing pattern of small mesophyll compartments was observed by both methods. In IDTA images, a random pattern predominated, while in DIF images, compartments closer to lower order veins were affected earlier. The increase of F(0) following mesophyll freezing started after a species-specific time lag of up to 26 min. The start of the F(0) increase and its slope were significantly enhanced at lower temperatures, which suggest a higher strain on mesophyll protoplasts when freezing occurs at lower temperatures.     

Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation

Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use‐period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature‐associated change in the pK_as, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well‐studied system, shows the greatest change in pH when going from +25 to ?30°C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to ?30°C, whereas Tris‐hydrochloride had an ～1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0°C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze–thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Farinose fiavonoids are associated with high freezing tolerance in fairy primrose (Primula malacoides) plants

The deposition of surface(farinose)fiavonoids on aerial parts of some Primula species is a well-documented but poorly understood phenomenon.Here,we show thatfiavonoid deposition on the leaves and winter buds may contribute strongly to preventing freezing damage in these plants.The ice nucleation temperature of fairy primrose(Primula malacoides)leaves covered with naturalfiavone was approximately 6°C lower compared to those that had theirfiavone artificially removed.Additionally,farinosefiavonoids on the leaves reduced subsequent electrolyte leakage(EL)from the cells exposed to freezing temperatures.Interestingly,exogenous application offiavone at4 mg/g fresh weight to P.malacoides leaves,which had the originalfiavone mechanically removed,restored freezing tolerance,and diminished EL from the cells to pretreatment values.Our results suggest that farinosefiavonoids may function as mediators of freezing tolerance in P.malacoides,and exogenous application offiavone could be used to reduce freezing damage during sudden but predictable frost events in other plant species.     

Xylem dysfunction caused by water stress and freezing in two species of co-occurring chaparral shrubs

Water transport from the roots to leaves in chaparral shrubs of California occurs through xylem vessels and tracheids. The formation of gas bubbles in xylem can block water transport (gas embolism), leading to shoot dieback. Two environmental factors that cause gas embolism formation in xylem conduits are drought and freezing air temperatures. We compared the differential vulnerabilities of Rhus laurina and Ceanothus megacarpus, co-dominant shrub species in the coastal regions of the Santa Monica Mountains of southern California, to both water stress-induced and freezing-induced embolism of their xylem. Rhus laurina has relatively large xylem vessel diameters, a deep root system, and a large basal burl from which it vigorously resprouts after wildfire or freezing injury. In contrast, Ceanothus megacarpus has small-diameter vessels, a shallow root system, no basal burl and is a non-sprouter after shoot removal by wildfire. We found that R. laurina became 50% embolized at a water stress of –3 MPa and 100% embolized by a freeze–thaw cycle at all hydration levels. In contrast, C. megacarpus became 50% embolized at a water stress of –9 MPa and 100% embolized by freeze–thaw events only at water potentials lower than –3 MPa. Reducing thaw rates from 0·8 °C min^?1 to 0·08 °C min^?1 (the normal thaw rate measured in situ) had no effect on embolism formation in R. laurina but significantly reduced embolism occurrence in well-hydrated C. megacarpus (embolism reduced from 74 to 35%). These results were consistent with the theory of gas bubble formation and dissolution in xylem sap. They also agree with field observations of differential shoot dieback in these two species after a natural freeze–thaw event in the Santa Monica Mountains.     

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.     

Annual ecosystem respiration is resistant to changes in freeze–thaw periods in semi‐arid permafrost

Warming in cold regions alters freezing and thawing (F–T) of soil in winter, exposing soil organic carbon to decomposition. Carbon‐rich permafrost is expected to release more CO₂ to the atmosphere through ecosystem respiration (Re) under future climate scenarios. However, the mechanisms of the responses of freeze – thaw periods to climate change and their coupling with Re in situ are poorly understood. Here, using 2 years of continuous data, we test how changes in F–T events relate to annual Re under four warming levels and precipitation addition in a semi‐arid grassland with discontinuous alpine permafrost. Warming shortened the entire F–T period because the frozen period shortened more than the extended freezing period. It decreased total Re during the F–T period mainly due to decrease in mean Re rate. However, warming did not alter annual Re because of reduced soil water content and the small contribution of total Re during the F–T period to annual Re. Although there were no effects of precipitation addition alone or interactions with warming on F–T events, precipitation addition increased total Re during the F–T period and the whole year. This decoupling between changes in soil freeze – thaw events and annual Re could result from their different driving factors. Our results suggest that annual Re could be mainly determined by soil water content rather than by change in freeze – thaw periods induced by warming in semi‐arid alpine permafrost.     

Freezing avoidance by supercooling in Olea europaea cultivars: the role of apoplastic water,solute content and cell wall rigidity

Plants can avoid freezing damage by preventing extracellular ice formation below the equilibrium freezing temperature (supercooling). We used Olea europaea cultivars to assess which traits contribute to avoid ice nucleation at sub‐zero temperatures. Seasonal leaf water relations, non‐structural carbohydrates, nitrogen and tissue damage and ice nucleation temperatures in different plant parts were determined in five cultivars growing in the Patagonian cold desert. Ice seeding in roots occurred at higher temperatures than in stems and leaves. Leaves of cold acclimated cultivars supercooled down to ?13 °C, substantially lower than the minimum air temperatures observed in the study site. During winter, leaf ice nucleation and leaf freezing damage (LT₅₀) occurred at similar temperatures, typical of plant tissues that supercool. Higher leaf density and cell wall rigidity were observed during winter, consistent with a substantial acclimation to sub‐zero temperatures. Larger supercooling capacity and lower LT₅₀ were observed in cold‐acclimated cultivars with higher osmotically active solute content, higher tissue elastic adjustments and lower apoplastic water. Irreversible leaf damage was only observed in laboratory experiments at very low temperatures, but not in the field. A comparative analysis of closely related plants avoids phylogenetic independence bias in a comparative study of adaptations to survive low temperatures.     

Effect of ice nucleation by droplet of immobilized silver iodide on freezing of rabbit and bovine embryos

Silver iodide was immobilized by applying the insoluble reaction between sodium alginate and calcium chloride. The immobilized silver iodide was immersed into a freezing solution in order to trigger ice nucleation. Temperature change during cooling and postthaw in vitro development of embryos were examined in order to evaluate the effectiveness of the immobilized silver iodide (AgI alginate-gel droplet) on embryo development. Samples containing the AgI alginate-gel droplets released the latent heat of fusion at a higher subzero temperature than samples without the AgI alginate-gel droplets. When the AgI alginate-gel droplet was added to the freezing solution of rabbit and bovine embryos, they were successfully preserved in liquid nitrogen.     

Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae,Eurosta solidaginis (Diptera,Tephritidae)

We examined seasonal changes in freeze tolerance and the susceptibility of larvae of the gall fly, Eurosta solidaginis to inoculative freezing within the goldenrod gall (Solidago sp.). In late September, when the water content of the galls was high (approximately 55%), more than half of the larvae froze within their galls when held at -2.5 degrees C for 24 h, and nearly all larvae froze at -4 or -6 degrees C. At this time, most larvae survived freezing at > or = -4 degrees C. By October plants had senesced, and their water content had decreased to 33%. Correspondingly, the number of larvae that froze by inoculation at -4 and -6 degrees C also decreased, however the proportion of larvae that survived freezing increased markedly. Gall water content reached its lowest value (10%) in November, when few larvae froze during exposure to subzero temperatures > or = -6 degrees C. In winter, rain and melting snow transiently increased gall water content to values as high as 64% causing many larvae to freeze when exposed to temperatures as high as -4 degrees C. However, in the absence of precipitation, gall tissues dried and, as before, larvae were not likely to freeze by inoculation. Consequently, in nature larvae freeze earlier in the autumn and/or at higher temperatures than would be predicted based on the temperature of crystallization (T(c)) of isolated larvae. However, even in early September when environmental temperatures are relatively high, larvae exhibited limited levels of freezing tolerance sufficient to protect them if they did freeze.     

A pig model of hepatic cryotherapy. In vivo temperature distribution during freezing and histopathological changes

We aimed to assess the temperature distribution in the cryolesion during hepatic cryotherapy and the association with postoperative histological changes to optimise the technique and allow better preoperative planning. Hepatic cryolesions were produced in 22 pigs following laparotomy using a CMS-cryosystem and 8mm-AccuProbe-Cryoprobes. The temperature was measured in 1 min intervals at different distances from the probe during freezing. The animals were treated in 5 groups: (i) single freezing of 20 min; (ii) double freezing of 20 min each; (iii) single freezing of 40 min; (iv) single freezing of 20 min (n=4), histology at 1 week p.o., and (v) single freezing of 20 min and Pringle manoeuvre; [(i)-(iii) and (v): histology at 24 h p.o.]. The mean diameter of the -38 degrees C isotherm, i.e., the zone of effective treatment for colorectal metastases was 37 mm for group (i) with a mean iceball diameter of 59 mm and about 46 mm for groups (ii, iii, and v) with mean iceball diameters of 78, 75, and 75 mm, respectively. At 7 days postoperatively secondary necrosis was seen in the largest central part of the lesion, wherever temperatures of -15 degrees C or lower were achieved during cryosurgery. Under the hypothesis that -38 degrees C is the effective temperature for the destruction of colorectal liver metastases, a lesion of 37-mm diameter may be effectively treated with a single 8mm-AccuProbe-Cryoprobe and a 20 min single freeze cycle and a lesion of 46 mm may be effectively treated when a double freeze-thaw cycle of 20 min each, a single freeze cycle of 40 min, or a 20 min single freeze cycle with additional Pringle manoeuvre is used, when it is perfectly placed in the lesion.     

Seasonal changes in the physical state of crown water associated with freezing tolerance in winter wheat

The relationship between freezing tolerance (expressed as LT₅₀, the lethal freezing temperature for 50% of plants) and the amount and physical state (as determined by spin-lattice [T₁] and spin-spin [T₂] relaxation times of protons) of water in crown tissue was examined in contrasting winter wheat (Triticum aestivum L.) varieties grown under field conditions from 1992 to 1994. During acclimation, the LT₅₀ values decreased from around -7 to -17, -20 and -27°C in PI 173438, Chihokukomugi and Valuevskaya, respectively. Tissue water content decreased continuously through autumn to reach a plateau around 3 g H₂O (g dry weight)^-1 in early winter when LT₅₀ was still decteasing, and then gradually increased under snow cover. A significant negative correlation was found between mean minimum air temperatures and freezing tolerance prior to the establishment of continuous snow cover. In contrast, a positive association between mean minimum temperatures and crown tissue water content was significant only when air temperatures were above 0°C, as water content did not decrease further at sub-zero temperatures. Seasonal changes in T₁ were closely related to changes in freezing tolerance. T₁ decreased until January even though water content stopped decreasing. Further tests on 15 field-grown varieties confirmed a strong negative association between freezing tolerance and T₁. The results suggest that cold hardening is comprised of two stages, with the transition occurring at ca 0°C. Development of hardiness was related to (1) a reduction in water content in the first stage (at minimum temperatures > 0°C), and (2) a change in physical state of water without much reduction in water content in the second stage. Varietal differences in hardiness thus arise due to changes in both water content and physical state of water.     

Spring photosynthetic onset and net CO2 uptake in Alaska triggered by landscape thawing

The springtime transition to regional‐scale onset of photosynthesis and net ecosystem carbon uptake in boreal and tundra ecosystems are linked to the soil freeze–thaw state. We present evidence from diagnostic and inversion models constrained by satellite fluorescence and airborne CO₂ from 2012 to 2014 indicating the timing and magnitude of spring carbon uptake in Alaska correlates with landscape thaw and ecoregion. Landscape thaw in boreal forests typically occurs in late April (DOY 111 ± 7) with a 29 ± 6 day lag until photosynthetic onset. North Slope tundra thaws 3 weeks later (DOY 133 ± 5) but experiences only a 20 ± 5 day lag until photosynthetic onset. These time lag differences reflect efficient cold season adaptation in tundra shrub and the longer dehardening period for boreal evergreens. Despite the short transition from thaw to photosynthetic onset in tundra, synchrony of tundra respiration with snow melt and landscape thaw delays the transition from net carbon loss (at photosynthetic onset) to net uptake by 13 ± 7 days, thus reducing the tundra net carbon uptake period. Two global CO₂ inversions using a CASA‐GFED model prior estimate earlier northern high latitude net carbon uptake compared to our regional inversion, which we attribute to (i) early photosynthetic‐onset model prior bias, (ii) inverse method (scaling factor + optimization window), and (iii) sparsity of available Alaskan CO₂ observations. Another global inversion with zero prior estimates the same timing for net carbon uptake as the regional model but smaller seasonal amplitude. The analysis of Alaskan eddy covariance observations confirms regional scale findings for tundra, but indicates that photosynthesis and net carbon uptake occur up to 1 month earlier in evergreens than captured by models or CO₂ inversions, with better correlation to above‐freezing air temperature than date of primary thaw. Further collection and analysis of boreal evergreen species over multiple years and at additional subarctic flux towers are critically needed.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

The relationship between xylem conduit diameter and cavitation caused by freezing

The centrifuge method for measuring the resistance of xylem to cavitation by water stress was modified to also account for any additional cavitation that might occur from a freeze-thaw cycle. A strong correlation was found between cavitation by freezing and mean conduit diameter. On the one extreme, a tracheid-bearing conifer and diffuse-porous angiosperms with small-diameter vessels (mean diameter <30 μm) showed no freezing-induced cavitation under modest water stress (xylem pressure = −0.5 MPa), whereas species with larger diameter vessels (mean >40 μm) were nearly completely cavitated under the same conditions. Species with intermediate mean diameters (30–40 μm) showed partial cavitation by freezing. These results are consistent with a critical diameter of 44 μm at or above which cavitation would occur by a freeze–thaw cycle at −0.5 MPa. As expected, vulnerability to cavitation by freezing was correlated with the hydraulic conductivity per stem transverse area. The results confirm and extend previous reports that small-diameter conduits are relatively resistant to cavitation by freezing. It appears that the centrifuge method, modified to include freeze–thaw cycles, may be useful in separating the interactive effects of xylem pressure and freezing on cavitation.     

Effect of sugars on the thermal and rheological properties of sago starch

Studies have been undertaken to investigate the effect of sugars on the thermal and rheological properties of sago starch. Sugars were found to increase the gelatinization temperature T_gel, and gelatinization enthalpy ΔH. T_gel and ΔH increased in the following order: control (water alone) < ribose < fructose < glucose < maltose < sucrose. The increase in ΔH was greater for 50% starch compared to 10% starch samples. The swelling factors in the presence of sugar were higher compared to the control for sugar concentrations below 25% but were lower at sugar concentration greater than 25%. These effects are discussed in terms of the antiplaticizing effect of the sugars compared to water, the influence of sugar–starch interactions and also the effect of the sugars on water structure. The storage modulus G′, the rate constant of gelation k, and the gel strength were significantly reduced in the presence of sugars. Generally G′ and k decreased in the following order: control (water alone) > hexose > disaccharide > pentoses. This has been attributed to the reduced proportion of amylose leached following gelatinizatison. In the presence of hexoses the freeze–thaw stability of starch gels decreased while in the presence of disaccharides and pentoses the freeze–thaw stability was slightly improved. © 1999 John Wiley & Sons, Inc. Biopoly 50: 401–412, 1999     

Snow melt stimulates ecosystem respiration in Arctic ecosystems

Cold seasons in Arctic ecosystems are increasingly important to the annual carbon balance of these vulnerable ecosystems. Arctic winters are largely harsh and inaccessible leading historic data gaps during that time. Until recently, cold seasons have been assumed to have negligible impacts on the annual carbon balance but as data coverage increases and the Arctic warms, the cold season has been shown to account for over half of annual methane (CH₄) emissions and can offset summer photosynthetic carbon dioxide (CO₂) uptake. Freeze–thaw cycle dynamics play a critical role in controlling cold season CO₂ and CH₄ loss, but the relationship has not been extensively studied. Here, we analyze freeze–thaw processes through in situ CO₂ and CH₄ fluxes in conjunction with soil cores for physical structure and porewater samples for redox biogeochemistry. We find a movement of water toward freezing fronts in soil cores, leaving air spaces in soils, which allows for rapid infiltration of oxygen‐rich snow melt in spring as shown by oxidized iron in porewater. The snow melt period coincides with rising ecosystem respiration and can offset up to 41% of the summer CO₂ uptake. Our study highlights this important seasonal process and shows spring greenhouse gas emissions are largely due to production from respiration instead of only bursts of stored gases. Further warming is projected to result in increases of snowpack and deeper thaws, which could increase this ecosystem respiration dominate snow melt period causing larger greenhouse gas losses during spring.     

Photoperiodic induction of dormancy and freezing tolerance in Betula pubescens. Involvement of ABA and dehydrins

In many woody plants photoperiod signals the initiation of dormancy and cold acclimation. The photoperiod-specific physiological and molecular mechanisms have remained uncharacterised. The role of abscisic acid (ABA) and dehydrins in photope-riod-induced dormancy and freezing tolerance was investigated in birch, Betula pubescens Ehrh. The experiments were designed to investigate if development of dormancy and freezing tolerance under long-day (LD) and short-day (SD) conditions could be affected by manipulation of the endogenous ABA content, and if accumulation of dehydrin-like proteins was correlated with SD and/or the water content of the buds. Experimentally, the internal ABA content was increased by ABA application and by water stress treatment under LD, and decreased by blocking the synthesis of ABA with fluridone under SD. Additionally, high humidity (95% RH) was applied to establish if accidental water stress was involved in SD. ABA content was monitored by gas chromatography-mass spectrometry with selective ion monitoring (SIM). Short days induced a transient increase in ABA content, which was absent in 95% RH, whereas fluridone treatment decreased ABA. Short days induced a typical pattern of bud desiccation and growth cessation regardless of the treatment, and improved freezing tolerance except in the fluridone treatment. ABA content of the buds was significantly increased after spraying ABA on leaves and after water stress, treatments that did not induce cessation of growth and dormancy, but improved freezing tolerance. In addition to several constitutively produced dehydrins, two SD-specific proteins of molecular masses 34 and 36 kDa were found. Photoperiod- and experimentally-induced alterations in ABA contents affected freezing tolerance but not cessation of growth and dormancy. Therefore, involvement of ABA in the photoperiodic control of cold acclimation is more direct than in growth cessation and dormancy. As the typical desiccation pattern of the buds was found in all SD plants, and was not directly related to ABA content or to freezing tolerance, this pattern characterises the onset of photo-period-induced growth cessation and dormancy. The results provide evidence for the existence of various constitutively and two photoperiod-induced dehydrins in buds of birch, and reveal characteristics of dormancy and freezing tolerance that may facilitate further investigations of photoperiodic control of growth in trees.     

Extraorgan freezing in wintering flower buds of Cornus officinalis Sieb. et Zucc.

Abstract. Extraorgan freezing as a mechanism for increasing cold hardiness was shown using flower buds of Cornus officinalis Sieb. et Zucc. Differential thermal analysis (DTA) revealed that florets in flower buds of C. officinalis owed their cold hardiness to deep supercooling and also that slower cooling rates increased the supercooling ability of florets. During slow stepwise cooling (5°C h⁻¹), the water content of florets decreased and that of scales (involucral bracts) increased, which resulted in accumulation of ice within the scales. This was more extensive in early winter and early spring buds than mid-winter ones. Flower buds with silicone oil in the space between florets and scales also showed a similar decrease in water content of florets and an increase in that of scales. This indicated that water migration from the florets to the scales probably took place by way of the peduncles and the receptacle, possibly through their vascular traces, and not directly from the surface of the florets to the ice sink in the form of vapour. Possible mechanisms of extraorgan freezing are postulated along with this finding.