AOBPreview originally published online on February 7, 2006
Annals of Botany 2006 97(4):593-599; doi:10.1093/aob/mcl008
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Effect of Thawing Time, Cooling Rate and Boron Nutrition on Freezing Point of the Primordial Shoot in Norway Spruce Buds
1 Faculty of Forestry, University of Joensuu, P.O. Box 111, FIN-80101 Joensuu, Finland and 2 Finnish Forest Research Institute, Joensuu Research, P.O. Box 68, FIN-80101 Joensuu, Finland
* For correspondence. E-mail mikko.raisanen{at}joensuu.fi
Received: 11 August 2005 Returned for revision: 5 October 2005 Accepted: 12 December 2005 Published electronically: 7 February 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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• Background Effects of cooling rates on bud frost hardiness have been studied but there is little information on bud responses to thawing. Since the cell wall pore size has been found to increase with boron (B) deficiency, B deficiency may affect the supercooling ability of buds in winter.
• Methods The effects of duration of thawing time and rate of cooling on bud frost hardiness of Norway spruce (Picea abies) were studied in a B fertilization trial in February 2003 and March 2005. Frost hardiness of apical buds was determined by differential thermal analysis (DTA) and visual scoring of damage.
• Key Results In 2003, the freezing point of primordial shoots of buds (Tf), i.e. the low-temperature exotherm (LTE), was, on average, –39 °C when buds were thawed for less than 3 h and the Tf increased to –21 °C after 18 h of thawing. During the first 4 h of thawing, the rate of dehardening was 6 °C h–1. In 2005, buds dehardened linearly from –39 °C to –35 °C at a rate of 0·7 °C h–1. In 2003, different cooling rates of 1–5 °C h–1 had a minor effect on Tf but in 2005 with slow cooling rates Tf decreased. In both samplings, at cooling rates of 2 and 1 °C h–1, Tf was slightly higher in B-fertilized than in non-fertilized trees. By contrast, at very short thawing times in 2003, Tf was somewhat lower in B-fertilized trees.
• Conclusions There was little evidence of reduced frost hardiness in trees with low B status. This study showed that buds deharden rapidly when exposed to above-freezing temperatures in winter, but if cooled again they reharden more slowly. According to this study, rapid dehardening of buds has to be taken into account in assessments of frost hardiness.
Key words: Differential thermal analysis, cold hardiness, Picea abies, apical bud, extra-organ freezing, thawing, winter thaws
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The rate of change in frost hardiness in response to the driving environmental factors is a key factor in climate acclimation of trees. The rate of response depends on the species, organ and physiological stage of the tree. Frost hardiness of wintering buds has been observed to vary according to the ambient air temperature in Norway spruce (Picea abies; Beuker et al., 1997
), peach (Prunus persica ‘Elberta’; Proebsting, 1963) and sweet cherry (Prunus avium; Andrews and Proebsting, 1987). However, the time constant for hardening and dehardening of forest tree species has not been studied. The frost hardiness in buds of Scots pine (Pinus sylvestris) remains rather stable during winter, i.e. the time constant appears to be rather long (Beuker et al., 1997).
In the genus Picea, the frost hardiness of dormant primordial shoots of apical buds is based on avoidance of freezing by deep supercooling and tolerance to freezing by dehydration during extra-organ freezing (Sakai, 1979; Ishikawa et al., 1997; Ide et al., 1998). After initiation of ice crystallization in tissues, ice spreads rapidly via the stem tissues until the collenchymatic plate in the bud axis blocks penetration of ice into the primordial shoot (Quamme, 1995; Jones et al., 2000). Due to the absence of active ice-nucleating agents at the freezing temperature of stem tissues, the primordial shoot in the bud supercools. The developing difference in hydrostatic pressure between the frozen stem and unfrozen bud tissues is the driving force for water efflux from the bud into the bud cavity and bud scales. Consequently, the primordial shoot in the bud dehydrates, and its frost hardiness increases (Sakai, 1979; Sakai and Larcher, 1987). Typically, boreal tree species in the genus Picea have smaller primordial shoots, more bud scales and higher rates of water diffusion to the stem compared to temperate species of the same genus (Sakai, 1983). This implies selective pressure of evolution in Picea species, i.e. dehydration of the shoot primordium during freezing is favoured by ecotypes with minimum winter temperature as an important selection factor.
The divergent pattern of frost hardiness of the buds in spruce and pine in mid-winter is probably due to differences in anatomical structure. As the ice crystallization barrier, spruce has a collenchymatic plate in the bud axis (Bilkova et al., 1999), and consequently, the buds exhibit extra-organ freezing (Sakai, 1979; Pukacki, 1987; Pukacki and Pukacka, 1987). On the other hand, apical buds of the genus Pinus do not have such a barrier, and the primordial shoots freeze extracellularly at about the same temperature as the stem tissues do (Ide et al., 1998). The collenchymatic plate has a fine porous structure, which is permeable to water but less permeable to ice crystals (Sakai and Larcher, 1987; Jones et al., 2000). When the collenchymatic plate of excised buds was degraded using pectinases, the barrier for penetration of ice crystals into the bud disappeared, which facilitated ice nucleation in the primordial shoot (Jones et al., 2000). Similarly, the supercooling ability of xylem parenchymatic cells totally disappeared when pectins in the cell wall were degraded enzymatically, but it was only partially impaired when hemicellulose or cellulose in the outer cell wall was degraded (Wisnievski et al., 1991).
In boron (B)-deficient stands, trees suffer from repeated die-back of leader shoots. One possible reason for this die-back has been suggested to be impaired frost hardiness during dormancy (Aronsson, 1980; Möller, 1983; Pietiläinen, 1984; Jalkanen, 1990). Boron regulates the orientation of pectin chains in cell walls by forming bonds between rhamnogalacturonan-II chains (Ishii and Shimizu, 2001), and thus the availability of B may also affect the rigidity of ice barriers. The rhamnogalacturonan content in cell walls also increases during cold acclimation (Kubacka-Zebalska and Kacperska, 1999). Since in B deficiency the pore sizes of cell walls increase (Fleischer et al., 1999), B may affect penetration of ice crystals through the collenchymatic plate and consequent ice nucleation within the buds (see Jones et al., 2000). We hypothesize that B deficiency changes the fine porous structure of the collenchymatic plate in the buds of Norway spruce, thereby affecting their resistance to freezing stress.
Pre-frozen or slowly cooled stem (Tumanov and Krasavtsev, 1959) and bud samples (Sakai and Larcher, 1987) reach their highest level of frost hardiness in winter. The rate of decline in frost hardiness during thawing has not been studied in great detail for buds of conifers, even though it is known that in fluctuating temperature conditions in mid-winter dormant buds of Norway spruce may be susceptible to frost damage (Beuker et al., 1997). In controlled freezing tests of plants, the rate of decline in frost hardiness also has to be considered because samples are commonly taken first into the laboratory to thaw and to be prepared for testing. If the time constant for dehardening during thawing is short, as compared with the time constant of hardening during consequent cooling, then the duration of thawing may affect the results of frost hardiness tests. Water flow through the collenchymatic plate is the driving force for rapid dehardening during thawing (Andrews and Proebsting, 1987) and rehardening during recooling. Since cell-wall porosity is one of the factors affecting the water resistance of cell walls (Wisnievski, 1995), B deficiency could also have an effect on rates of dehardening and hardening of dormant buds.
The objective of this investigation was to study the effect of thawing on the frost hardiness of apical buds of Norway spruce in a B-fertilization trial. The hypotheses tested were: (1) the highest level of frost hardiness is lost rapidly after thawing and soon regained by cooling; and (2) B deficiency reduces the frost hardiness of dormant buds in terms of maximum hardiness and the rate of dehardening or rehardening.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Study area and sampling
The effects of the duration of thawing time and cooling rate on the frost hardiness of buds were studied in a B-fertilization trial. This trial was established in June 2000 in a 25-year-old Norway spruce (Picea abies L. Karst.) stand in Pyhäselkä, eastern Finland (62°25'N, 29°55'E, 90 m asl.). In 2003, ten of the 148 trees in the experiment were sampled. Five of the sample trees were fertilized with borax (2 kg B ha–1; Borax Decahydrate, Borax Europe Ltd, Brance, France) and five were not fertilized. In trees sampled in 2003, B concentration in the needles of the non-fertilized trees ranged from 1·2–8·0 mg kg–1 dry wt (i.e. from deficient to sub-optimal), while in the B-fertilized trees the B concentration was 14·8–21·9 mg kg–1 dry wt. Because of a slight effect of B on the freezing of buds in sampling 2003 and high variation in the B concentration in the needles of the non-fertilized trees, six different trees from each of the two fertilization treatments were selected for the 2005 sampling on the basis of B concentrations in needles in 2003. The B concentration of the needles of the selected trees ranged from 1·8–2·6 mg kg–1 dry wt in non-fertilized trees and from 17·8–28·5 mg kg–1 dry wt in B-fertilized trees. Twigs on the southern side of the topmost third of the crown were sampled at the end of February 2003 and in March 2005.
After sampling in 2003, the twigs were placed in a refrigerator (5 °C) to thaw overnight. The samples were then cooled to –17 °C at a rate of 5 °C h–1 and stored for 4 weeks. In 2005, samples were not thawed after sampling but were immediately stored at –17 °C for 4 weeks; in 4 weeks the buds were expected to reach their maximum frost hardiness. Frost hardiness of buds was studied by differential thermal analysis (DTA) by means of the low-temperature exotherm (LTE) and visual scoring of the damage in frost-exposed samples (Pukacki and Pukacka, 1987).
DTA measurements
DTA measurements took place in a custom-designed device that consisted of four modules, each having three differentially measuring temperature channels. The samples were set in four aluminium blocks with three differential temperature channels in each block, i.e. a total of 12 samples in one DTA run. The blocks were in a programmable freezing chamber (ARC 300/–55/+20, Arctest, Finland). The temperature difference between the sample and the reference junction was measured by NiCr/Ni thermocouples (diameter 0·25 mm). The temperature of the aluminium block was measured by a Pt-100 thermistor. Before the DTA run, a small hole was punctured longitudinally along the stem xylem for setting of the thermocouple in the stem below the bud. The starting temperature in a DTA run was +5, –5 or –15 °C, depending on the thawing treatment; during the test the temperature was reduced to –49 °C at a rate of 5 °C h–1. Six of the non-thawed samples transferred at –15 °C were cooled to –90 °C at a rate of 5 °C h–1 in a chamber cooled by liquid nitrogen (GCC-30 chamber, Carbolite, UK with XL-180 liquid N2-tank, Taylor-Whatron, UK). The target temperature was maintained for 1 h and then increased to +5 °C at a rate of 10 °C h–1. The freezing point (Tf) of the primordial shoot was calculated according to the ice nucleation temperature (Tn) and the increase in sample temperature (
T) by LTE as Tf = Tn + T.
Effect of thawing time
In 2003, the samples at –17 °C were either exposed to cooling without thawing or were thawed, kept melted for different lengths of times and then gradually cooled again. Sample preparation followed a procedure where duration of thawing was determined as the time between the apoplastic thawing and freezing in the stems below the buds (see Fig. 1). In the pre-treatment procedure at –17 °C, needles and lateral buds were dissected from the twigs, and the remaining apical bud with a 1-cm piece of stem was used for the DTA. Each of the five DTA runs contained six samples for each of the two B treatments. Four of the six samples were randomized to two thawing-time treatments (i.e. two buds to ‘Gradual warming’ and two buds to ‘Stepwise warming’) and two of the six samples were randomized to non-thawing treatments (‘Transfer at –5 °C, and ‘Transfer at –15 °C) from both the fertilized and non-fertilized trees. In the thawing-time treatments, the samples were allowed to thaw for different times, from 1–21 h, before the start of the DTA run. When the thawing time was longer than 7 h, samples were initially warmed to +5 °C at a rate of 5 °C h–1 (‘Gradual warming’). In the case of the shortest thawing times, samples were first kept at –5 °C for at least 2 h and then quickly moved into the DTA device at +5 °C (‘Stepwise warming’). In ‘Stepwise warming’, thawing time was determined as the time between the endotherm and exotherm peaks in the DTA curve (see Fig. 1). The samples in the ‘Gradual warming’ treatment were assumed to thaw when the sample temperature increased above 0 °C. Non-thawed samples were (1) warmed gradually to –5 °C and set in the DTA run at –5 °C (‘Transfer at –5 °C’), or (2) set in the DTA run directly from a freezer at –15 °C (‘Transfer at –15 °C’).
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In 2005, the effect of thawing time on Tf of buds was studied as for the ‘Stepwise warming’ in 2003. Three buds from four trees (two trees from each of two fertilization treatments) were put into one DTA run, and the DTA run was repeated three times with different samples.
In 2003, visual damage in buds was studied by exposing twigs in plastic bags to frost, together with the DTA samples at thawing times. There were unequal numbers of twigs from the different trees. All twigs from different trees were grouped according to fertilization treatments and then randomized into three thawing-time treatments within fertilization treatments. The first set of samples (n = 16) was transferred directly from –17 °C to the freezing test at –15 °C (similar to ‘Transfer at –15 °C’). The second set (n = 15) was taken to the freezing test after gradual warming (5 °C h–1) from –17 °C to 5 °C for 6 h. The third set (n = 13) was gradually warmed (5 °C h–1) from –17 °C to room temperature and used as the reference for scoring the viability of buds stored at –17 °C. After completion of frost exposures and thawing of samples in all treatments, the samples were placed in water in vials and kept on the laboratory table in natural light for one week. Bud injuries were scored on the basis of the colour of the primordial shoots. Brown tissue was scored as dead, pale green tissue as frost-desiccated, and bright green tissue as undamaged.
Effect of cooling rate
In 2003, the effect of cooling rate on Tf of buds in fertilized and non-fertilized trees was tested after 8 weeks of storage at –17 °C. Buds were gradually warmed to 5 °C at a rate of 5 °C h–1 for 24 h and the DTA run was then started with cooling rates of 1, 3 and 5 °C h–1. In 2005, the effect of cooling rate on Tf of buds was measured using one bud from six trees in each of the two fertilization treatments, the cooling rates being 0·5, 1, 2, 3 and 5 °C h–1. In 2005, samples were stored at –17 °C for 5 weeks before measurements were taken.
Statistical analyses
Dependence of the freezing point of the bud primordial shoot (Tf) on thawing time (t in min) was modelled by eqn (1) in 2003 and by linear regression in 2005 (SPSS v.11.5.1 Inc, Chicago, IL, USA):
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The relation between height of the LTE (T) and thawing time (t in min) was modelled with eqn (2) in 2003 and with linear regression in 2005:
 | (2) |
). The difference of the regression coefficients from zero was studied in regression statistics by a t-test. The effect of cooling rate on frost hardiness was analysed by the repeated measures model of ANOVA (SPSS v.11.5.1). Cooling rate of buds was used as the within-subjects factor. Pairwise comparisons between groups in different freezing rates were done by t-tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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After 4 weeks cold-storage at –17 °C, a typical DTA curve for warming and cooling of an apical bud with a piece of stem consisted of an endotherm and a high- and low-temperature exotherm (Fig. 1) in both samplings. In the 2003 sampling, if the samples were thawed for 1–21 h before the start of the DTA run, LTE was observed in 89 % of the samples (n = 36). In 2005, when thawing times were shorter than 4 h, LTE was not observed. In buds that were transferred directly without thawing to the DTA at –5 °C and –15 °C, LTE was observed in 20 % (n = 10) and 0 % (n = 16) of the buds, respectively, even though in the latter case one DTA run continued to –90 °C (n = 6).
In 2003, when thawing time was less than 2 h, the mean freezing point of the primordial shoot was –43 °C, but with prolonged thawing it increased rapidly (Fig. 2). Within the first 4 h of thawing, the mean rate of dehardening was 6 °C h–1, but with prolonged thawing it decreased to 0·36 °C h–1. After 18 h of thawing, the mean Tf of buds was –21 °C. In 2005, after 4 h of thawing, Tf-values of buds were about –40 °C, and after that the buds dehardened linearly at a rate of 0·7 °C h–1 (Fig. 2). In the 2003 sampling, B had a slight effect on dehardening of buds (P = 0·078), as at very short thawing times the Tf of the buds was somewhat lower in B-fertilized than in non-fertilized trees but at long thawing times it was about the same. In 2005, however, the effect of B on the rate of dehardening was not significant (Fig. 2).
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In 2003, the height of the LTE (
T) in buds increased significantly (P < 0·001), from about 0·1 to 0·4 °C between thawing times shorter than 3 h and longer than 20 h (Fig. 3). In 2005, when LTE was observed, T by LTE ranged from 0·03–0·12 °C. In neither 2003 nor 2005 did the height of the LTE differ significantly between fertilized and non-fertilized trees.
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In 2003, when the samples had been incubated at 5 °C for 6 h before the frost exposure, all of buds were visually scored as dead after the freezing test at –49 °C (n = 15). When cooling to –49 °C started at –15 °C, one bud was scored as living, three were frost-desiccated primordial shoots and the rest were dead (n = 16). Most of the primordial shoots (85 %) in the reference samples (freeze-stored at –17 °C but not frozen to –49 °C) were undamaged; two of the primordial shoots from B-fertilized trees (n = 6) were dead while all those from non-fertilized trees were undamaged. After exposure to –49 °C, there were about equal numbers of surviving buds in B-fertilized and non-fertilized trees.
In the 2003 sampling, the cooling rate had a slight effect on the Tf of buds (P = 0·058), but the difference between the means of the extreme groups was only 2·6 °C (Fig. 4). In this comparison, B-fertilization had no significant effect. In non-fertilized trees, the freezing point was somewhat lower at the low cooling rate (1 °C h–1) than it was at the intermediate one (3 °C h–1; P = 0·080, Fig. 4), but at the lowest cooling rate there was no significant difference between fertilized and non-fertilized trees. In 2005, B had no significant effect on the Tf of buds at different cooling rates, as indicated by ANOVA. According to a t-test, however, the Tf of buds in non-fertilized trees was lower when the cooling rate was 2 °C h–1 (P = 0·038). The Tf of buds decreased slightly with slow cooling rates (P = 0·084), being, on average, –31·5 °C when the cooling rate was 5 °C h–1 and –37·6 °C when cooled at 1 °C h–1. When the cooling rate was 1 °C h–1, LTE was detected in only 73 % of the samples, whilst with a cooling rate of 0·5 °C h–1 LTE was not detected in any of the samples. In 2003, mean T was 0·359 °C, while in 2005 it was 0·11 °C.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In the 2003 sampling, frost hardiness of buds decreased rapidly within the first hour of thawing at 5 °C. Thereafter, the dehardening rate decreased markedly after the initial rapid phase, and the frost hardiness stabilized at about –20 °C. In the 2005 sampling, there was no rapid initial phase as the buds dehardened linearly at about the same rate as observed after the rapid initial phase in the 2003 sampling. However, it is possible that in 2005 the same kind of rapid dehardening phase occurred at temperatures lower than the measurable range of the DTA device, since the frost hardiness of buds in 2005 was higher than that in 2003. With short thawing times the LTE of buds may have been beyond the level of detection the DTA device. Different phases in dehardening have been found in the twigs of Norway spruce (Tumanov and Krasavtsev, 1959
) and the flower buds of sweet cherry (Andrews and Proebsting, 1987). In the study of Tumanov and Krasavtsev (1959), artificially hardened twigs first dehardened in 6 h at 2 °C from –100 °C to –45 °C, and with prolonged thawing for 48 h to –30 °C. The short time-constant in the initial phase of dehardening is supported by the observation that in mid-winter the frost hardiness of the apical buds of Norway spruce and the flower buds of some fruit trees fluctuates according to changes in ambient air temperature (Proebsting, 1963; Graham and Mullin, 1976; Andrews and Proebsting, 1987; Beuker et al., 1997).
When the frost hardiness of the buds decreased with prolonged thawing, the height of LTE increased almost linearly during subsequent cooling. This indirectly suggests that more heat was released by ice nucleation in the primordial shoot with prolonged thawing. When the bud is thawed after long-term freezing, water is released from the ice needles, which have previously been found to accumulate during freezing under the collenchymatic plate in the bud cavity (Sakai, 1982; Ishikawa et al., 1997; Ide et al., 1998). This free water rapidly rehydrates the surrounding tissue, including the collenchymatic plate. The primordial shoot, which is dehydrated during freezing (Ide et al., 1998), is, after thawing, rehydrated more slowly than the rate at which the bud dehardens (Andrews and Proebsting, 1987). Similarly, in the present study there was no initial rapid increase in height of LTE after thawing as there was in the freezing point of the primordial shoot. Discontinuity of the liquid fraction (Quamme, 1995; Ishikawa et al., 1997) or the high solute concentration of pores (Ashworth and Abeles, 1984) in the collenchymatic plate protect the primordial shoot against invasion by ice crystallization. Therefore, rapid rehydration of the collenchymatic plate after melting of water facilitated penetration of ice crystals into the primordial shoot. Obviously, the water content of the bud itself is not the main factor in the increase in freezing point, but rather the solutes in the collenchymatic plate region. Although these results corroborate previous hypotheses on ice propagation into buds from stem tissues, they do not disprove the possibility that rehydration of primordial shoot activates some kind of intrinsic ice nucleator in the primordial shoot. Water migration from the stem back to the primordial shoot probably provides the driving force for the initial dehardening of dormant buds during thawing, but is not a consequence of breaking the dormancy of the buds. Consequently, frost hardiness of the buds is decreased only to the level determined in acclimation metabolism. We may expect that the first phase of dehardening is a physical process with rapid water movements, whereas further dehardening would require metabolic changes in cells (Weiser, 1970).
According to visual scoring in 2003, a large proportion (81 %) of unthawed buds were killed by –49 °C, even though no LTE was found. The cause of death of the buds may not have been the intracellular freezing of the primordial shoots but, as suggested previously by Sakai and Larcher (1987), excessive dehydration of the primordial shoot could also be a factor. It is possible that in those samples water was in a bound state without crystallization capability, or that the DTA device was not sensitive enough to detect the small heat pulses of freezing in the strongly dehydrated buds (Sakai and Larcher, 1987). Furthermore, there was no LTE in 11 % of thawed buds in the 2003 sampling. In visual scoring, 15 % of the non-frost-exposed reference buds were scored to be dead. Therefore, apart from damage caused by experimental treatments, buds may have been damaged for other reasons in the field. Damage in buds may affect their supercooling ability.
In the 2003 sampling, no considerable decrease in the freezing point of buds was observed by recooling at different cooling rates. However, the freezing point of buds decreased by 6·1 °C in 2005, when cooling was hastened from 1 °C h–1 to 5 °C h–1. In previous studies, cooling rates that have resulted in a decrease in the freezing points of the buds of conifers have been as slow as 5 °C d–1 (Sakai and Larcher, 1987). The results of those studies probably agree with the results from 2003 obtained in the present study. We may conclude that rehardening is much slower during recooling than is dehardening after thawing, and that when the moisture content of the bud is high the difference may be pronounced. The slower effect of recooling than of thawing on the freezing point of buds is in accordance with the results for sweet cherry flower buds (Andrews and Proebsting, 1987). The results for sweet cherry were explained as being due to the higher osmotic difference between the floret and the surrounding tissue at the beginning of thawing than at the beginning of freezing, and thus more rapid water flux in dehardening compared to the rehardening phase. Presumably, a build-up of either discontinuity in the liquid phase (Quamme, 1995; Ishikawa et al., 1997) or of concentration of solutes in pores during extra-organ freezing does not take place before the flow of liquid water from the primordial shoot to the bud cavity ends and therefore rehardening of buds after thawing requires more time than dehardening. However, the viscosity of water also differs in freezing and thawing temperatures, which may explain part of the difference in rates of hardening and dehardening.
Moisture content of samples could explain differences in frost hardiness between the 2003 and 2005 samplings as well as differences in the dehardening rates of samples. If there is only a little free water after melting of ice crystals in the bud cavity, water flow to the collenchymatic plate is not strong and consequently the ice crystallization temperature in the collenchymatic plate does not increase rapidly. In March 2005, air temperatures in the sampling area were above zero during the daytime but decreased to freezing temperatures at night. Thawing and freezing as well as high daily temperatures can cause dehydration of twigs in late winter (Tranquillini, 1982). Probably due to the lower water content of the bud tissues, the height of LTE was lower in March 2005 than in February 2003. Differences in moisture contents between the years 2003 and 2005 could also explain differences in response to different cooling rates.
The results of this study reveal that, in laboratory assessment of the frost hardiness of supercooling buds, freezing and thawing of buds have to be taken into account. Measurements that are conducted in the laboratory are often begun by thawing the samples before test programs are initiated. Some uncontrolled thawing of samples may also occur during transport and preparation of samples in the laboratory. During thawing, the frost hardiness of buds that have extra-organ freezing ability may change rapidly, and thus the sensitivity of buds to frost damage in nature may be overestimated. Rapid loss of frost hardiness during thawing can increase variance in the frost hardiness data of Norway spruce buds due to even slight differences in the short thawing times.
With B concentrations lower than 4 mg kg–1, the risk of growth disorder induced by deficiency in above-ground part of Norway spruce is distinctly increased (Silfverberg, 1980; Aronsson, 1983). B concentrations in the needles of the non-fertilized trees that were sampled in February 2003 varied from deficient (below 4–5 mg kg–1; Aronsson, 1983) to suboptimal (below 8 mg kg–1; Jukka, 1988). In 2003, B concentration in the needles of non-fertilized trees sampled in 2005 was below 2·6 mg kg–1. In fertilized trees the B concentration in both samplings can be considered optimal.
The freezing point of primordial shoots was slightly higher in non-fertilized trees than in B-fertilized trees only very shortly after thawing in 2003. At slow cooling rates at both sampling times, the freezing point of the buds was also slightly lower in non-fertilized than in B-fertilized trees. This might indicate more rapid water flow between the bud cavity and the primordial shoot through the collenchymatic plate tissues in non-fertilized trees. According to previous studies, if the ice crystallization barrier is seriously damaged, buds have no supercooling ability and exhibit no LTE in the freezing profile, (Jones et al., 2000). In this study, however, there were probably no such serious changes in the porosity of the ice barriers due to B deficiency in non-fertilized trees. In this barrier, pore size is an important factor for regulating the permeability of ice, but anti-freeze agents and dryness of the collenchymatic plate region may also function as protecting factors in ice barriers (Ashworth and Abeles, 1984; Quamme, 1995; Jones et al., 2000). It is also possible that porosity in the collenchymatic plate changes to some extent at low B concentration, but high solute concentration in the pores of the collenchymatic plate can maintain adequate frost hardiness of the buds. B-deficient trees are not necessarily more sensitive to lethal freezing in the primordial shoot, although there were slight differences between non-fertilized and B-fertilized trees in the response of freezing points to different thawing times and cooling rates.
In conclusion, if rapid loss of frost hardiness of buds after thawing occurs in nature at the same rate as in this study, rapid cooling of the air after mid-winter thaws can be harmful to the vegetative buds of Norway spruce, regardless of the B status of the trees. The freezing characteristics of buds in non-fertilized and B-fertilized trees differed, but the importance of this difference for the winter ecology of trees remains to be verified. While the average annual temperature in Fennoscandia has been found to increase over the past century, minimum winter temperatures have become even lower and temperature fluctuation in winter has increased (Tuomenvirta et al., 2000). Warm spells are occasionally followed by cold weather, whereupon the air temperature may fall below –20 °C more rapidly than the lowest rate used in this study (Finnish Meteorological Institute unpubl. data). Thus, buds of Norway spruce may be at risk of damage in mid-winter at the present time, but even more so in a changed future climate. The current study opens up the possibility to model the short-term fluctuations in frost hardiness and to re-evaluate of the frost susceptibility of wintering buds of Norway spruce.
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ACKNOWLEDGEMENTS |
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We thank Ari Venäläinen of the Finnish Meteorological Institute for searching for information from Finnish Meteorological Databases on the natural rates of changes in air temperature, Tommi Kinnunen for assistance in the sampling of trees and Sirkka Sutinen for valuable advice. We also thank Joann von Weissenberg for revising the language of this paper. This research was funded by the Academy of Finland (decision no. 206898) and the Ministry of Agriculture and Forestry of Finland.
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LITERATURE CITED |
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