Repeated freeze–thaw cycles induce embolism in drought stressed conifers (Norway spruce,stone pine)

Freezing and thawing lead to xylem embolism when gas bubbles caused by ice formation expand during the thaw process. However, previous experimental studies indicated that conifers are resistant to freezing-induced embolism, unless xylem pressure becomes very negative during the freezing. In this study, we show that conifers experienced freezing-induced embolism when exposed to repeated freeze-thaw cycles and simultaneously to drought. Simulating conditions at the alpine timberline (128 days with freeze-thaw events and thawing rates of up to 9.5 K h(-1) in the xylem of exposed twigs during winter), young trees of Norway spruce [Picea abies (L.) Karst.] and stone pine (Pinus cembra L.) were exposed to 50 and 100 freeze-thaw cycles. This treatment caused a significant increase in embolism rates in drought-stressed samples. Upon 100 freeze-thaw cycles, vulnerability thresholds (50% loss of conductivity) were shifted 1.8 MPa (Norway spruce) and 0.8 MPa (stone pine) towards less negative water potentials. The results demonstrate that freeze-thaw cycles are a possible reason for winter-embolism in conifers observed in several field studies. Freezing-induced embolism may contribute to the altitudinal limits of conifers.     

Analysis of freeze-thaw embolism in conifers. The interaction between cavitation pressure and tracheid size

Ice formation in the xylem sap produces air bubbles that under negative xylem pressures may expand and cause embolism in the xylem conduits. We used the centrifuge method to evaluate the relationship between freeze-thaw embolism and conduit diameter across a range of xylem pressures (Px) in the conifers Pinus contorta and Juniperus scopulorum. Vulnerability curves showing loss of conductivity (embolism) with Px down to -8 MPa were generated with versus without superimposing a freeze-thaw treatment. In both species, the freeze-thaw plus water-stress treatment caused more embolism than water stress alone. We estimated the critical conduit diameter (Df) above which a tracheid will embolize due to freezing and thawing and found that it decreased from 35 microm at a Px of -0.5 MPa to 6 microm at -8 MPa. Further analysis showed that the proportionality between diameter of the air bubble nucleating the cavitation and the diameter of the conduit (kL) declined with increasingly negative Px. This suggests that the bubbles causing cavitation are smaller in proportion to tracheid diameter in narrow tracheids than in wider ones. A possible reason for this is that the rate of dissolving increases with bubble pressure, which is inversely proportional to bubble diameter (La Place's law). Hence, smaller bubbles shrink faster than bigger ones. Last, we used the empirical relationship between Px and Df to model the freeze-thaw response in conifer species.     

Water stress induced cavitation and embolism in some woody plants

A comparison was made of the relative vulnerability of xylem conduits to cavitation and embolism in three species [ Thuja occidentalis L., Tsuga canadensis (L.) Carr. and Acer saccharum Marsh.]. Waterlogged samples of wood were air dehydrated while measuring relative water loss, loss of hydraulic conductance, cumulative acoustic emissions (= cavitations) and xylem water potential. Most cavitation events and loss of hydraulic conductance occurred while water potential declined from – 1 to –6 MPa. There were differences in vulnerability between species. Other people have hypothesized that large xylem conduits (e.g. vessels) should be more vulnerable to cavitations than small conduits (e.g. tracheids). Our findings are contrary to this hypothesis. Under water stress, the vessel bearing wood retained water better than tracheid bearing wood. However, within a species large conduits were more prone to cavitation than small conduits.     

木本植物木质部的冻融栓塞应对研究进展

冻融栓塞在中高纬度地区木本植物中普遍存在。抗冻融栓塞能力对在寒冷环境中木本植物的生长和安全越冬十分关键, 这直接决定植物分布范围。冻融栓塞是由于冰中气体溶解度低, 木质部水分在低温下冷冻, 使之前水中溶解的气体逸出到导管中, 随后木质部中的冰融化又使气泡扩张而引发的栓塞现象。木质部解剖结构的差异会影响植物的抗冻融栓塞能力, 植物还可以通过调节木质部正压、代谢耗能等方式主动修复冻融栓塞, 也可通过增加树液溶质含量等逃避冷冻, 以减少低温损伤。然而, 与干旱栓塞相比, 目前对木质部冻融栓塞的形成以及植物响应和调节机制的理解不足。为此, 该文首先综述了木质部冻融栓塞的形成机制和植物的逃避、忍耐、修复等3种冻融栓塞的应对策略, 然后总结了木质部抗低温胁迫能力的生理表现、影响因子和评价指标, 并在此基础上讨论了低温抗性、干旱抗性和水力效率之间的多元权衡关系, 最后提出今后该领域中的5个优先研究问题: (1)不同植物冰冻的最低温度阈值; (2)是否存在应对低温胁迫的水力脆弱性分割机制; (3)冻融栓塞修复与代谢消耗的关系; (4)低温抗性、干旱抗性和水力效率之间的权衡关系; (5)抗冻融栓塞性状是否能够纳入经济性状谱系。     

Durstige Pflanzen senden Signale. Trockenstress produziert Ultraschall

Drought‐stressed plants produce ultrasonic signals Ultrasonic emission analysis, a method for non‐destructive material testing, can also be used for acoustic measurements on plants. Audible or ultrasound acoustic signals are produced in the water transport system of plants when high tensions cause a sudden breakage of water columns. These cavitations are induced by entry of air from adjacent conduits. Ultrasonic emissions are also observed in freezing samples of dehydrated wood. Ultrasonic emission analysis enables a non‐destructive monitoring of cavitation induced by tension, although interpretation of registered events is difficult. Future technological developments and research activities will improve our understanding of plant acoustic emissions.     

Interpretation of seasonal changes of xylem embolism and plant hydraulic resistance in Fagus sylvatica

The annual course of xylem embolism in twigs of adult beech trees was monitored, and compared to concurrent changes of tree water status and hydraulic resistances. Xylem embolism was quantified in 1-year-old apical twigs by the hydraulic conductivity as a percentage of the maximum measured after removal of air emboli. Tree and root hydraulic resistances were estimated from water potential differences and sap flux measurements. The considerable degree of twig embolism found in winter (up to 90% loss of hydraulic conductivity) may be attributed to the effect of freeze-thaw cycles in the xylem. A partial recovery from winter embolism occurred in spring, probably because of the production of new functional xylem. Xylem embolism fluctuated around 50% throughout the summer, without significant changes. Almost complete refilling of apical twigs was observed early in autumn. A significant negative correlation was found between xylem embolism and precipitation; thus, an active role of rainfall in embolism reversion is hypothesized. Tree and root hydraulic resistances were found to change throughout the growing period. A marked decrease of hydraulic resistance preceded the refilling of apical twigs in the autumn. Most of the decrease in total tree resistance was estimated to be located in the root compartment.     

Freeze-Thaw Stress: Effects of Temperature on Hydraulic Conductivity and Ultrasonic Activity in Ten Woody Angiosperms

Freeze-thaw events can affect plant hydraulics by inducing embolism. This study analyzed the effect of temperature during the freezing process on hydraulic conductivity and ultrasonic emissions (UE). Stems of 10 angiosperms were dehydrated to a water potential at 12% percentage loss of hydraulic conductivity (PLC) and exposed to freeze-thaw cycles. The minimal temperature of the frost cycle correlated positively with induced PLC, whereby species with wider conduits (hydraulic diameter) showed higher freeze-thaw-induced PLC. Ultrasonic activity started with the onset of freezing and increased with decreasing subzero temperatures, whereas no UE were recorded during thawing. The temperature at which 50% of UE were reached varied between −9.1°C and −31.0°C across species. These findings indicate that temperatures during freezing are of relevance for bubble formation and air seeding. We suggest that species-specific cavitation thresholds are reached during freezing due to the temperature-dependent decrease of water potential in the ice, while bubble expansion and the resulting PLC occur during thawing. UE analysis can be used to monitor the cavitation process and estimate freeze-thaw-induced PLC.Xylem embolism is a limiting factor for plant survival and distribution (Choat et al., 2012; Charrier et al., 2013). Two major factors can induce embolism in the xylem of plants: drought and freeze stress. Freeze-thaw-induced embolism is caused by bubbles formed during freezing that then expand on thawing (Lemoine et al., 1999; Hacke and Sperry, 2001; Cruiziat et al., 2002; Tyree and Zimmermann, 2002). As wider conduits contain more gas and form larger bubbles, which expand at less negative tension, conduit diameter and xylem sap tension are critical for the formation of freeze-thaw-induced embolism (Davis et al., 1999; Pittermann and Sperry, 2003). Accordingly, Mayr and Sperry (2010) observed a loss of conductivity only when samples were under critical tension during thawing. Under drought stress, tension in the xylem sap increases the sensitivity to embolism generated by successive freeze-thaw cycles (Mayr et al., 2003, 2007).Ultrasonic emissions (UE) analysis can be used to detect cavitation events in wood. It is unclear how well related UE are to cavitation events, as they are extracted from continuous acoustic emissions and depend on set definitions. However, UE analysis has been proven effective for monitoring drought-induced embolism in the laboratory (Pena and Grace, 1986; Salleo and Lo Gullo, 1986; Borghetti et al., 1993; Salleo et al., 2000) as well as in field experiments (Ikeda and Ohtsu, 1992; Jackson et al., 1995; Jackson and Grace, 1996; Hölttä et al., 2005; Ogaya and Penuelas, 2007). In a cavitating conduit, signals are probably produced by the disruption of the water column and subsequent tension relaxation of cell walls.UE have also been detected during freezing events, but the origin of these signals was less clear. In some cases, UE were observed during thawing, which are thus probably related to embolism formation according to the classic thaw-expansion hypothesis (Mayr and Sperry, 2010); however, all species studied have produced UE on freezing, which cannot yet be explained (Raschi et al., 1989; Kikuta and Richter, 2003; Mayr et al., 2007; Mayr and Sperry, 2010; Mayr and Zublasing, 2010). The low solubility of gases in ice prompted the idea that air bubbles expulsed from the ice structure produce UE near the ice-liquid interface (Sevanto et al., 2012). As the water potential of ice is strongly temperature dependent, the minimum temperature during freezing might be a relevant factor. Numerous studies have analyzed UE patterns during freeze-thaw cycles in conifers (Mayr et al., 2007; Mayr and Sperry, 2010; Mayr and Zublasing, 2010) or angiosperms (Weiser and Wallner, 1988; Kikuta and Richter, 2003), but few of them measured embolism concomitantly. Percentage loss of hydraulic conductivity (PLC) was only measured in a few studies and only in conifers (Mayr et al., 2007; Mayr and Sperry, 2010).In this study, we analyzed the effect of freeze-thaw cycles on the hydraulic conductivity and ultrasonic activity in 10 angiosperm species. We hypothesized that (1) the extent of freeze-thaw-induced embolism depends on xylem anatomy (related to conduit diameter) and minimal temperature (related to the water potential of ice); (2) ultrasonic activity is also influenced by anatomy and temperature; and (3) PLC and UE are positively correlated. PLC was measured in 10 angiosperm species after freeze-thaw cycles at different minimal temperatures (−10 to −40°C). Furthermore, UE were recorded during a freeze-thaw cycle down to −40°C.     

Ultrasound acoustic emissions from freezing xylem*

Rachides of Juglans regia L. (Juglandaceae) and one‐year‐old twigs of Evonymus latifolia (L.) Mill. (Celastraceae) were cooled in air to ?25 °C, with an ultrasound detector attached to the xylem where peripheral tissues had been peeled off. Ultrasound acoustic emissions started between ?4·5 and ?14·3 °C, as measured with a thermocouple inserted into the xylem. The number of emissions was significantly lower from saturated plant parts than from those frozen at field water potentials. Bench‐drying of saturated samples produced significantly less signals than the freezing protocols. These findings are in accordance with the hypothesis that freezing of xylem under tension induces cavitation events. They corroborate earlier work which tried to provide a logical explanation for the seemingly paradoxical cryo‐scanning electron microscope observations of changing vessel contents during a daycourse in the field.     

Freezing of xylem sap without cavitation

Freezing of stem sections and entire twigs of hemlock (Tsuga canadensis) has been demonstrated to occur without increasing the resistance to the movement of water through the frozen part after rewarming. This was interpreted to mean that freezing did not produce cavitation in the xylem sap even though A) the sap was unquestionably frozen; B) it contained dissolved gases; and C) it was under tension before freezing and after. Freezing stem sections of some other evergreen gymnosperms during the summer again produced no evidence for cavitation of the xylem sap. On the other hand, freezing stem sections of some angiosperms invariably increased the resistance to sap flow leading to wilting and death in a few hours when the sap tension was at normal daytime values at the time of freezing. These results were interpreted to mean that the bordered pits on the tracheids of gymnosperms function to isolate the freezing sap in each tracheid so that the expansion of water upon freezing not only eliminates any existing tension but also develops positive pressure in the sap. Dissolved gases frozen out of solution may then be redissolved under this positive pressure as melting occurs. As the bubbles are reduced in size by this ice pressure developed in an isolated tracheid, further pressure is applied by the surface tension of the water against air. If the bubbles are redissolved or are reduced to sufficient small size by the time the tension returns to the sap as the last ice crystals melt, then the internal pressure from surface tension in any existing small bubbles may exceed the hydrostatic tension of the melted sap and the bubbles cannot expand and will continue to dissolve.     

Water storage in the wood and xylem cavitation in 1-year-old twigs of Populus deltoides Bartr.

The possible role of water expelled from cavitated xylem conduits in the rehydration of water-stressed leaves has been studied in one-year-old twigs of populus deltoides Bartr. Twigs were dehydrated in air. At desired values of leaf water potential (Ψ_l) (between near full turgor and -1.62 MPa), twigs were placed in black plastic bags for 1–2h. Leaf water content was measured every 3–5 min before bagging and every 10 min in the dark. Hydraulic conductivity and xylem cavitation were measured both in the open and in the dark. Cavitation was monitored as ultrasound acoustic emissions (AE). A critical Ψ_l value of -0.96 MPa was found, at which AE increased significantly while the leaf water deficit decreased by gain of water. Since the twigs were no longer attached to roots, it was concluded that water expelled from cavitated xylem conduits was transported to the leaves, thus contributing to their rehydration. Xylem cavitation is discussed in terms of a ‘leaf water deficit buffer mechanism’, under not very severe water stress conditions.     

Three Different Methods for Measuring Xylem Cavitation and Embolism: A Comparison

Three different methods for measuring xylem embolism due towater cavitation were compared—the acoustic method, thehydraulic method and the anatomical method. Young plants ofCeratonia siliqua L. were water stressed for 9, 16 and 23 d. Xylem cavitation was detected by counting ultrasound (100–300kHz) acoustic emissions (AE) from 1-year-old twigs (acousticmethod). Xylem embolism was detected by measuring the loss ofhydraulic conductivity of twigs of the same age (hydraulic method).The blockage of single xylem conduits was detected by perfusingSafranin into the xylem of 1-year-old twigs of stressed plantsand measuring the number and the diameters of non-conductingxylem conduits, under the microscope (anatomical method). It was noted that: (a) the number of AE and the loss of conductivityincreased with the water stress applied; (b) a linear relationseemed to exist between the number of AE and the loss of conductivity,suggesting that the AE counted could be only (or mainly) producedin the xylem conduits; (c) the vulnerability of the xylem conduitsto embolism was a direct function of their diameter; and (d)the measured loss of conductivity was of the same order of magnitudeas the theoretical one. The three methods gave fairly similar results. Nonetheless,they are not alternative to one another in that: (a) the acousticmethod allows continuous recordings to be made but does notprovide information about the actual damage suffered by plants;(b) the hydraulic method is very informative but destructive;and (c) the anatomical method is very useful both in phytogeographicaland in genetic improvement studies. Ceratonia siliqua L, Carob tree, water stress, xylem embolism, acoustic method, hydraulic method, anatomical method     

Three Different Methods for Measuring Xylem Cavitation and Embolism: A Comparison

Three different methods for measuring xylem embolism due towater cavitation were compared—the acoustic method, thehydraulic method and the anatomical method. Young plants ofCeratonia siliqua L. were water stressed for 9, 16 and 23 d. Xylem cavitation was detected by counting ultrasound (100–300kHz) acoustic emissions (AE) from 1-year-old twigs (acousticmethod). Xylem embolism was detected by measuring the loss ofhydraulic conductivity of twigs of the same age (hydraulic method).The blockage of single xylem conduits was detected by perfusingSafranin into the xylem of 1-year-old twigs of stressed plantsand measuring the number and the diameters of non-conductingxylem conduits, under the microscope (anatomical method). It was noted that: (a) the number of AE and the loss of conductivityincreased with the water stress applied; (b) a linear relationseemed to exist between the number of AE and the loss of conductivity,suggesting that the AE counted could be only (or mainly) producedin the xylem conduits; (c) the vulnerability of the xylem conduitsto embolism was a direct function of their diameter; and (d)the measured loss of conductivity was of the same order of magnitudeas the theoretical one. The three methods gave fairly similar results. Nonetheless,they are not alternative to one another in that: (a) the acousticmethod allows continuous recordings to be made but does notprovide information about the actual damage suffered by plants;(b) the hydraulic method is very informative but destructive;and (c) the anatomical method is very useful both in phytogcographicaland in genetic improvement studies. Ceratonia siliqua L., Carob tree, water stress, xylem embolism, acoustic method, hydraulic method, anatomical method     

Detection of xylem cavitation in field-grown pine trees using the acoustic emission technique

The relationship between the ultrasonic acoustic emission (AE) and the needle water potential in field-grown Japanese black pine,Pinus thunbergii, and excised-pine shoots was investigated in summer. The AE technique was employed as a reliable indicator of cavitation events in the water columns of xylem tracheids even under field conditions. In excised shoots. AE events detected when the needle water potential fell below about-0.9 MPa. In the case of field-grown pine trees, however, relatively few AE event were detected when needle water potential was lower than-0.9 MPa. This suggests that the occurrence of cavitation events in Japanese black pine growing under field conditions is comparatively rare, even in summer.     

Mechanism of freeze-induced embolism in Fagus sylvatica L.

 The mechanism of freeze stress-induced embolism in Fagus sylvatica L. branches was analyzed under controlled conditions. Excised branches were exposed to successive freeze-thaw cycles in temperature controlled chambers. Thermocouples were placed on the bark to detect sap freezing exotherms. The degree of xylem embolism was estimated after each cycle by the loss of hydraulic conductivity. After one freeze-thaw cycle the degree of embolism was found to decrease with xylem specific hydraulic conductivity, small apical shoots being more susceptible to embolism. Exotherms revealed that distal shoots were freezing first and exuded sap as a result of water expansion. The lower water content in apical shoots upon freezing probably induced higher sap tensions which promoted air bubble expansion and vessel cavitation preferentially near the apices. When the decrease in water content was experimentally prevented, embolism developed to a lesser extent. The higher vulnerability of shoot apices may protect the rest of the branch from winter damage. Received: 29 May 1998 / Accepted: 15 August 1998     

Xylem embolism in response to freeze-thaw cycles and water stress in ring-porous, diffuse-porous, and conifer species

Vulnerability to xylem embolism by freeze-thaw cycles and water stress was quantified in ring-porous (Quercus gambelii Nutt.), diffuse-porous (Populus tremuloides Michx., Betula occidentalis Hook.), and conifer species (Abies lasiocarpa Nutt., Juniperus scopulorum Sarg.). Embolism was measured by its reduction of xylem hydraulic conductivity; it was induced by xylem tension (water-stress response) and by a tension plus a freeze-thaw cycle (freeze response). Conifers showed little (Juniperus) or no (Abies) freeze response even to repeated cycles. In contrast, Quercus embolized more than 90% by freezing at tensions below 0.2 MPa, whereas similar embolism without freezing required tensions above 4.5 MPa. Diffuse-porous trees (Betula, Populus) showed an intermediate freeze response. The magnitude of the freeze response was correlated with conduit volume but occurred at higher tensions than predicted from theory. Large early-wood vessels (2.8 × 10⁻⁹ m³) in oak were most vulnerable to embolism by freezing, small vessels in Populus and Betula were intermediate (approximately 7 × 10⁻¹¹ m³), and tracheids in conifers (about 3 × 10⁻¹³ m³) were most resistant. The same trend was found within a stem: embolism by freeze-thawing occurred preferentially in wider conduits. The water-stress response was not correlated with conduit volume; previous work indicates it is a function of interconduit pit membrane structure. Native embolism levels during winter corroborated laboratory results on freezing: Quercus embolized 95% with the first fall freeze, Populus and Betula showed gradual increases to more than 90% embolism by winter's end, and Abies remained below 30%.     

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.     

Ultrasonic acoustic emissions from the sapwood of cedar and hemlock : an examination of three hypotheses regarding cavitations

Measurements are reported of ultrasonic acoustic emissions (AEs) measured from sapwood samples of Thuja occidentalis L. and Tsuga canadensis (L.) Carr. during air dehydration. The measurements were undertaken to test the following three hypotheses: (a) Each cavitation event produces one ultrasonic AE. (b) Large tracheids are more likely to cavitate than small tracheids. (c) When stem water potentials are >−0.4 MPa, a significant fraction of the water content of sapwood is held by `capillary forces.' The last two hypotheses were recently discussed at length by M. H. Zimmermann. Experimental evidence consistent with all three hypotheses was obtained. The evidence for each hypothesis respectively is: (a) the cumulative number of AEs nearly equals the number of tracheids in small samples; (b) more water is lost per AE event at the beginning of the dehydration process than at the end, and (c) sapwood samples dehydrated from an initial water potential of 0 MPa lost significantly more water before AEs started than lost by samples dehydrated from an initial water potential of about −0.4 MPa. The extra water held by fully hydrated sapwood samples may have been capillary water as defined by Zimmerman.

We also report an improved method for the measurement of the `intensity' of ultrasonic AEs. Intensity is defined here as the area under the positive spikes of the AE signal (plotted as voltage versus time). This method was applied to produce a frequency histogram of the number of AEs versus intensity. A large fraction of the total number of AEs were of low intensity even in small samples (4 mm diameter by 10 mm length). This suggests that the effective `listening distance' for most AEs was less than 5 to 10 mm.

     

Freezing of Conifer Xylem and the Cohesion-Tension Theory

The research explored the apparent contradiction between the cohesion-tension theory and the lack of blockage following freezing in conifers. Five to ten cm lengths of the main stem of actively transpiring Thuja occidentalis, Picea glauca and Pinus sylvestris were frozen. On thaw, there was no evidence of blockage in the formerly frozen xylem. Microscopic examination of frozen wood and hydrodynamic considerations showed that air was present in the xylem water and that bubbles were probably present upon freezing. On freezing about 9 per cent of the water irreversibly migrated into the unfrozen plant parts. Lack of blockage in the presence of air bubbles was reconciled to the cohesion-tension theory by a model in which only the largest bubble in a number of interconnected cells expands. As this bubble expands tension is released allowing all bubbles in adjoining tracheids to redissolve. Potential rate of resolution of unexpanded bubbles following release of tension was calculated to be less than 0. 1 second.     

The mechanism of freezing injury in xylem of winter apple twigs

In acclimated winter twigs of Haralson apple (Pyrus Malus L.), a lag in temperature during cooling at a constant rate was observed at about −41 C by differential thermal analysis. The temperature at which this low temperature exotherm occurred was essentially unaffected by the cooling rate. During thawing there was no lag in temperature (endotherm) near the temperature at which the low temperature exotherm occurred, but upon subsequent refreezing the exotherm reappeared at a somewhat higher temperature when twigs were rewarmed to at least −5 C before refreezing. These observations indicate that a small fraction of water may remain unfrozen to as low as −42 C after freezing of the bulk water in stems. The low temperature exotherm was not present in twigs freeze-dried to a water content below 8.5% (per unit fresh weight), but it reappeared when twigs were rehydrated to 20% water. When freeze-dried twigs were ground to a fine powder prior to rehydration, no exotherm was observed. Previous work has shown that the low temperature exotherm arises from xylem and pith tissues, and that injury to living cells in these tissues invariably occurs only when twigs are cooled below, but not above the temperature of the low temperature exotherm. This study revealed that the low temperature exotherm resulted from the freezing of a water fraction, that the freezing of this water was independent of the freezing of the bulk water, that the exotherm was associated with some gross structural feature but not the viability of the tissue, and that injury to living cells in the xylem and pith was closely and perhaps causally related to the initial freezing of this water.     

Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing

Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.