Abstract
In this study, the ice nucleation activity (INA) and ice nucleation temperature (INT) as well as extracellular ice formation within the bark were determined for three woody species with different degrees of frost resistance, Betula nana, Betula albosinensis and Castanea sativa. Current-year stems and at least 2-year old stems of B. nana and C. sativa as well as current-year stems of B. albosinensis were compared, during summer (non-acclimated state) and winter (acclimated state), to evaluate possible ontogenetic and seasonal differences. Acclimated plant parts of the selected species revealed nearly similar results, with an INT from -7.52 to -8.43°C. The current-year stems of B. nana had a somewhat higher INT than the older stems. Microscopic analysis showed that extra-cellular ice formation occurred in the intercellular spaces within the bark of stems of B. nana, B. albosinensis and C. sativa. Size of the intercellular spaces of the bark were species-specific, and B. nana showed the largest intercellular space volume. While freezing behavior and extracellular ice formation thus followed principally the same pattern in all considered species, B. nana is obviously capable of dealing with large masses of extracellular ice which accumulate over extended periods of frost, making B. nana capable of protecting living tissue in colder regions from freezing damage.
INTRODUCTION
Frost resistance plays a crucial role for the survival of plants growing in regions with cold winters and many aspects are not yet completely understood. Full acclimation is necessary for plants to survive freeze-thaw cycles (Améglio et al. 2001), comprising various physiological processes. Acclimation is triggered by environmental factors, particularly decreasing temperature. A common process during frost is extracellular ice formation in plant tissue: extracellular water showing lower solute concentration than the content of living cells freezes first and subsequently draws water from the living tissue, thereby dehydrating it (Mazur 1969; Améglio et al. 2001). This study focuses on the process of freezing initiation in woody stems which is usually triggered by ice nucleating substances which are extrinsic or intrinsic to the plant (Pearce 2001; Kishimoto et al. 2014b).
Different methods for determining the ice nucleation temperatures (INT) exist. Infrared differential thermal analysis (IDTA) is based on image processing and visualizes intra-plant freezing events, as shown by Neuner et al. (2010) for different species such as Castanea sativa. Another method to determine INT is described by Kishimoto et al. (2014a; 2014b). This method also provides the content of ice nucleating particles within a sample, INA (Ice nucleating activity). By calculating the median TINT for different species, a standard value can be determined which allows for interspecific comparison. Additionally, the determination of the cumulative ice nucleation per gram fresh weight or per water volume provides information of ice nucleation activity at defined temperatures. Both values can also show seasonal adaptations. These values are important for analysis of frost hardiness and the comparison of different species, tissues, ontogenetic changes and seasonal differences with respect to frost resistance and susceptibility to freezing.
In this study, INT and INA of three different woody species with different degrees of frost resistance are investigated: the dwarf birch Betula nana, the Chinese red birch Betula albosinensis, and the sweet chestnut Castanea sativa. Betula nana, which can endure the lowest temperatures of these three species, is a small shrub, up to 1 m high, and native especially to northern Europe and the western Asia tundra. Betula nana shows characteristic adaptations to its natural habitat, the cold tundra, such as a reduced height. The stiff and dull dark brown twigs are pubescent and not warty, with resin dots covering the young twigs (de Groot et al. 1997). The older and heavier twigs grow downwards, to the ground. Betula nana does therefore not gain much height during growth but grows mostly in width. This growth habit is interpreted as an adaptation of B. nana to long frost periods and strong winds (de Groot et al. 1997).
Betula albosinensis is a tree with a maximum height of 30 m and is native to China. This spcies is also frost-resistant but does not occur in the extremely cold habitats of B. nana. It has a thin papery bark, ranging from whitish pink to orange red or pink violet. Castanea sativa also reaches a maximal height of 30 m and the bark changes during aging from grey and smooth to grey brown with typical cracks. Castanea sativa is also frost-resistant, but occurs in warm-temperate climates. Its distribution is from southern Europe to northern Africa and the region of the Black Sea (Coombes 2012). All three examined deciduous species need to protect their stems from freezing damage during dormancy in winter, but under different degrees of low temperature and differently long frost periods.
In this study, INA is determined for the stem tissue of the three considered species for the first time, by using the adapted method of Kishimoto et al. (2014a; 2014b). Additionally, also for the first time, stems are inspected with respect to intercellular spaces for extracellular ice formation. Extracellular ice formation in frost hardy plants is usually concentrated at defined intercellular locations (Prillieux 1869; McCully et al. 2004; Roden et al. 2009), and the volume of these spaces comply with the mass of accumulated ice. The results are compared to evaluate possible interspecific differences between INA and capacity for extracellular ice formation.
List of taxa and samples.
Abbreviations: CSOW: Castanea sativa, stems ≥ 2 years “old”, “winter material” = acclimated. – CSYW: Castanea sativa, “young” current-year stems, “winter material” = acclimated. – BAOW: Betula albosinensis, stems ≥ 2 years “old”, “winter material” = acclimated. – BAYW: Betula albosinensis, “young” current-year stems, “winter material” = acclimated. – BNOW: Betula nana, stems ≥ 2 years “old”, “winter material” = acclimated. – BNYW: Betula nana, “young” current-year stems, “winter material” = acclimated. – BNOS: Betula nana, stems ≥ 2 years “old”, “summer material” = not acclimated. – BNYS: Betula nana, “young” current-year stems, “summer material” = not acclimated.
MATERIALS AND METHODS
Plant material, sample collection and acclimation
Samples of Betula nana and Castanea sativa were collected in the Wilhelma (Stuttgart, Germany) and the Hohenheimer Gardens (Stuttgart, Germany). Furthermore, young potted plants, ~5 years old and kept in the inner yard of the State Museum of Natural History Stuttgart (SMNS), were used. The samples of Betula albosinensis were solely provided by the Hohenheimer Gardens. The twigs for the different experiments were collected from the end of summer 2015 till the middle of December 2016, and thus during different stages of natural acclimation. All plant material is listed and described in Tables 1 and 2.
Material from the Wilhelma was used for B. nana in the summer (non-acclimated plants) and in winter for C. sativa (acclimated plants).
All samples were collected fresh at the day of the experiment, and used for imaging or INT as fast as possible. “Summer” twigs which had to be transported over a certain distance (Wilhelma and Hohenheim) were put into water with their cut ends.
In winter, after some nights with frost, material of fully acclimated but yet unfrozen plants was collected. The samples were wetted and transported in closed plastic bags and were used for the experiments as fast as possible.
Potted plants of B. nana were artificially acclimated in November 2016 during one week at 5°C and another week at 0 °C in the custom-built freezer from Fryka, equipped with a triple glazed door and showing a temperature accuracy of ± 1.2 °C at -20 °C (Esslingen, Germany; www.fryka.de). The artificially acclimated plant was used for the INA and the microscopic analysis. The various samples are summarized in Table 1, in which also the acronyms used in the following text are explained.
Determination of ice nucleation temperature (INT)
In this study, the method of Kishimoto et al. (2014a; 2014b) was used, but some alterations were required. The basic procedure is as follows. Glass tubes containing 250 ml ultrapure (Type 1) water were autoclaved at 121°C for 20 minutes. One glass tube each received one of 40 pieces per twig sample. Length and weight of the twig samples for the different species are summarized in Table 2. The limited amount of B. nana material required a reduction of the water volume from originally 500 ml to 250 ml. To get comparable results this adaptation was made for all species. The experiment was repeated 4 times.
Length [mm] and maximum weight [mg] of samples of Betula nana (current-year and 2-years-old stems), Betula albosinensis (current-year stems) and Castanea sativa (current-year and 2-years-old stems).
To be sure that the ultrapure (Type 1) H2O would not influence the results, the ice nucleation temperature of the water control was determined in a pre-experiment: -16.45 °C ± SE 0.05.
For each measurement cycle, eight glass tubes solely filled with autoclaved ultrapure (Type 1) H2O served as control. All glass tubes, with and without tissue samples, were put into the precooled (0 °C) freezer. The samples were cooled down by 1° every 20 minutes while the already frozen tubes were counted during each step. The beginning of the experiment started when the plant parts reached the bottom of the glass tubes.
To support reliable identification of frozen samples, the glass tubes were placed onto a small wooden shelf with a black background. This arrangement provided sufficient contrast between sample and background to observe ice crystal growth. In pre-experiments, the temperature development of the shelf in the freezer was measured to exclude any bias by the equipment.
T1 is the threshold temperature before the content of half of the glass tubes freezes and T2 is the temperature after half of the tubes are frozen. F1 and F2 are the corresponding numbers of frozen tubes at these temperatures, and n is the total number of tubes.
With f = (n– FT) * n-1, M = fresh weight [g], FT is the cumulative number of frozen tubes at temperature T. Further details can be found in Kishimoto et al. (2014b) and Vali (1995).
Image analysis – Light microscopy
Fresh samples collected from the potted Betula nana plants at the SMNS during late summer were analyzed directly with incident light microscopy (Keyence VHX-500F, with VH-Z250R and VH-Z20R). Thicker cross sections were used to obtain suitable pictures for comparison with winter images.
Fresh, acclimated winter samples of all species were collected from an artificially acclimated potted plant of B. nana at the SMNS and later from naturally acclimated potted plants (Castanea sativa) and from plants growing in the Hohenheimer Gardens (C. sativa, B. albosinensis). During transport the stems were handled as explained in the above section “Plant material, sample collection and acclimation”. The cutting ends of the twigs from C. sativa and B. albosinensis were sealed with clay to prevent water loss by evaporation. In this state they were put into the freezer. After this, the stems were cooled down from 0 °C to -10 °C by 2 °/h. After 72 hours images were taken. For this, samples were cut with a precooled razor blade. During the experiments, the frozen samples were stored on five precooled copper plates within a small cooling box.
A: Distribution of ice nucleation activity (INA) shown as cumulative percentage of frozen tubes. – B: Cumulative ice nucleation showing K'(T) (ice nuclei per g fresh weight) for BNYS, BNOS, BNYW, BNOW, BAYW, CSYW and CSOW.
Citation: IAWA Journal 39, 2 (2018) ; 10.1163/22941932-20180201
RESULTS
Betula nana
Ice nucleation activity
Various differences in INT occurred between summer and winter samples as well as between young and older samples (Fig. 1A). Median INT courses against temperature were different for the summer samples, BNYS and BNOS, but were almost identical for both winter samples, BNOW and BNYW (Fig. 1A and Table 3; see Table 1 for description of the acronyms). INA, however, was lower for the winter samples (Fig. 1B). Figure 2 further illustrates the distribution of freezing for the samples of B. nana. Acclimated stems, particularly older material (BNOW), show an early initiation of freezing events, compared to non-acclimated stems. The latter group shows first freezing at -5°C, whereas acclimated samples started to show freezing at -4°C or even -3°C.
Distribution of ice nucleation activity of BNYS, BNOS, BNYW, BNOW, BAYW, CSYW and CSOW.
Citation: IAWA Journal 39, 2 (2018) ; 10.1163/22941932-20180201
Results of ice nucleation temperature and median of ice nuclei active at -8°C for Castanea sativa, Betula albosinensis and Betula nana.
Betula nana INT of current-year stems in October was lower than in November (Table 3).
Intercellular spaces
Since we used younger twig material, the largest part of the bark is represented by cortex in our samples. The following descriptions and discussions refer therefore – for all considered species –to cortex tissues.
During summer, older as well as younger stems of B. nana showed visible intercellular spaces within the bark (Fig 3A, D; 4A, D). During winter, after acclimation, the intercellular spaces within the bark became substantially larger (Fig. 3B, E; 4B, E). After freezing, at -10 °C, ice bodies formed within these intercellular spaces (Fig. 3C, F; 4C, F). For comparison a naturally acclimated potted plant showing the same wide intercellular spaces within the current-year stems was analyzed to exclude freezer depending damage.
Microscopy images of fresh and frozen cross sections of stems. The arrows point at intercellular spaces filled with air (white) or ice (blue). Red arrows indicate sites that are enlarged in Figure 4. – A: fresh BNYS. – B: fresh BNYW. – C: frozen BNYW. – D: fresh BNOS. – E: fresh BNOW. – F: frozen BNOW. – G: fresh BAYW. – H: frozen BAYW. – I: fresh CSYW. – J: frozen CSYW. – K: fresh BAOW. – L: frozen BAOW. – M: fresh CSOW. – N: frozen CSOW. – Scale bars = 250 μm.
Citation: IAWA Journal 39, 2 (2018) ; 10.1163/22941932-20180201
Betula albosinensis
Ice nucleation activity
For acclimated younger stems, freezing of the samples started at -6°C (Fig.1, Fig. 2). Further freezing then proceeded more rapidly, with a peak at -9°C, with decreasing rate of freezing events with further decreasing temperature.
Intercellular spaces
Acclimated young stems of B. albosinensis already showed visible intercellular spaces within the bark (Fig. 3G, K; 4G, K). These became filled with ice during freezing (Fig. 3H, L; 4H, L).
Detailed view of intercellular spaces of the bark (as indicated in Figure 3 by red arrows). Arrows point at intercellular spaces filled with air (white) or ice (blue). – A: fresh BNYS. – B: fresh BNYW. – C: frozen BNYW. – D: fresh BNOS. – E: fresh BNOW. – F: frozen BNOW. – G: fresh BAYW. – H: frozen BAYW. – I: fresh CSYW. – J: frozen CSYW. – K: fresh BAOW. – L: frozen BAOW. – M: fresh CSOW. – N: frozen CSOW. — Scale bars = 100 μm.
Citation: IAWA Journal 39, 2 (2018) ; 10.1163/22941932-20180201
Castanea sativa
Ice nucleation activity
With respect to initiation of freezing, CSYW and CSOW were quite similar (Fig.1). Also, the Median TINT were almost identical for both groups (Table 3). However, there was no pronounced peak for younger stems (Fig. 2). INA results for C. sativa were quite low (≥ 2-years-old stems: 9; current-year stems: 7) (Table 3).
Intercellular spaces
Castanea sativa showed quite small and nearly not detectable intercellular spaces within its bark (Fig. 3I, M; 4I, M). After freezing, ice crystals formed within these spaces (Fig. 3J, N; 4J, N).
Interspecific comparison
INT of Betula albosinensis (current-year stems: -8.06 °C ± 0.20) and C. sativa (≥ 2-years-old stems: -8.14 °C ± 0.14; current-year stems: -8.43 °C ± 0.25) tend to lower values when compared to acclimated material of B. nana (≥ 2-years-old stems: -7.52 °C ± 0.26; current-year stems: -7.56 °C ± 0.19), meaning that freezing was triggered at lower temperatures (Table 3). This was particularly the case for Castanea sativa whose current-year stems showed a very wide peak of INT against temperature. Furthermore, C. sativa (≥ 2-years-old stems: 9; current-year stems: 7) tended to a lower number of INA, compared to all other taxa (B. albosinensis: current-year stems: 12; B. nana: ≥ 2-years-old stems: 21; current-year stems: 12).
All species and sample groups had visible intercellular spaces (Fig. 3 and 4), albeit with different dimensions, within the living tissue which filled with ice during freezing (Fig. 3C, F, H, J, L, N; 4C, F, H, J, L, N). The stems of B. nana (Fig. 3B, E; 4B, E) showed larger intercellular air spaces than the other two species (B. albosinensis, Fig. 3H, L; 4H, L; C. sativa, Fig. 3J, N; 4J, N).
To obtain a quantitative comparison, intercellular air spaces in the cortex were measured in fresh sections, for all species. Non-acclimated plants (“summer state”) were used, to determine pre-existing differences (with respect to acclimation and freezing). Three sections of three individual twigs current-year, and ≥ 2-years-old, for each species were used. Measurements were performed for cortex sectors which appeared to be not deformed or damaged by sectioning. It should be emphasized that this may lead to an underestimation of the total intercellular air spaces, because regions with wider air spaces may be more prone to damage during cutting, and therefore not be included in the measurements. Thus, all values have to be considered to be approximate.
For Castanea sativa, 1.24% ± 0.49 (SD) was obtained, followed by Betula albosinensis with 8.22% ± 3.08. Betula nana showed the largest air space volume, with 16.15% ± 6.24. The area of the intercellular spaces of the current-year stems amounted for C. sativa to 2.04% ± 1.11, while B. albosinensis (9.26% ± 2.43) and B. nana were quite similar (7.19% ± 2.44).
DISCUSSION
In Betula nana, the decreasing number of active ice nuclei from summer to winter as well as the convergence of INT and INA in both BNYW and the BNOW, as a result of acclimation, may represent components of controlled freezing. Some of the INA results reported in Kishimoto et al. (2014a) for blueberry (Vaccinium corymbosum L. cv. Weymouth and Vaccinium ashei Reade cv. Woodard) showed a possible correlation between decrease in INA with freezing damage of the bark in not yet acclimated stems. However, despite the different levels of cold hardiness of the species considered in the present study, ice nucleation activity was in fact very similar for all species in winter. Kishimoto et al. (2014a) published similar results for two blueberry species with different cold hardiness levels, also showing adaptation of their INA during the year with the highest INA in November, albeit with narrower distribution of test tube nucleation. The distribution of test tube nucleation (Fig. 2) for B. nana obtained in the present study slightly shifted to a higher temperature while the temperature span did not change. Compared with Betula albosinensis and Castanea sativa the temperature span was quite similar.
The results for INT and INA of the various considered species are similar to those of Kishimoto et al. (2014a, b). Also, the INT obtained for C. sativa is close to the results obtained by Neuner et al. (2010) based on IDTA. The smaller water volume used in the present study may have influenced the results slightly, because, according to the volume effect, the smaller the amount of water, the higher the probability and level of supercooling. The distribution of freezing temperature may then be shifted towards lower temperature values (Kishimoto et al. 2014a). This volume effect would explain the moderate difference between the results obtained by Neuner et al. (2010) for C. sativa based on IDTA and our results. Neuner et al. (2010) cooled down twigs (about 3 cm long and 1.41 cm wide) in a freezing chamber (-24 °C h-1) which resulted in INTs of -2.8 to -7.4 °C from August to April. The somewhat higher values for INT obtained by Neuner et al. (2010) for C. sativa might therefore be closer to the natural value. Additionally, in the case of B. nana, the potted individual was younger than the plants from Wilhelma and Hohenheim, which could have influenced the results of the INA slightly (Kishimoto et al. 2014a). Despite these various influences, the results found in the present study indicate the magnitude of freezing temperature for the species considered.
Early initiation of ice by INA may avoid excessive supercooling which can lead to damage by internal ice formation (Kishimoto et al. 2014b). In blueberry stems, intrinsic INA is located in bark tissue, and therefore probably promoting the formation of extracellular ice within this tissue (Kishimoto et al. 2014b). In the present study, ice bodies were observed in the intercellular spaces of the bark of the considered species (Pearce 2001; Kishimoto et al. 2014b; Eurich et al. 2016). All acclimated twigs, yet unfrozen, showed larger intercellular spaces than during the summer. This may represent an adaptation to the cold period, caused by a certain degree of dehydration and therefore volume loss of the bark cells. In fact, dehydration is a widely observed component of acclimation (Welling 2003). The Betula species showed larger intercellular spaces in the bark than C. sativa, which may represent an adaptation to longer freezing periods and/or lower temperatures. As described above, freezing will occur firstly in extra-cellular water, due to its low solute content. After its formation, extracellular ice acts as a dehydrating agent, because its water potential decreases about -1.2 MPa for each degree decrease in temperature below zero (Rajashekar & Burke 1982). During an extended period of severe frost, extracellular ice bodies will therefore grow by attracting water from surrounding tissues, making availability of sufficient deposition space mandatory.
In fact, particularly large intercellular spaces could be found in the bark of B. nana which seem to grow during the first winter. Acclimated B. nana plants survived all freeze thaw cycles showing a healthy appearance, indicating that the large intercellular air spaces did not represent freezing damage. Thus, the intercellular space volume in the bark of B. nana may represent an adaptation to the winter conditions of the natural habitat. The results of this study indicate that the observed intercellular spaces for extracellular ice formation are mainly located in the cortex parenchyma. However, the considered material consisted mostly of twigs of about 2 years, or somewhat younger or older. Extracellular ice deposits may possibly also develop at other sites of the bark. Accumulation of ice is therefore another function requiring extended intercellular air spaces, besides aeration paths in species from waterlogged habitats (Angyalossy et al. 2016).
CONCLUSIONS
Despite differences in frost hardiness, INT of Betula nana, Betula albosinensis and Castanea sativa were within the typical range reported for woody plants. However, B. nana tended towards higher values, particularly in the acclimated state which can be interpreted as ensuring controlled freezing during colder temperatures. The two Betula species, especially B. nana, showed larger intercellular spaces than C. sativa, probably to provide sufficient space for external ice buildup within the bark.
ACKNOWLEDGEMENTS
This work has been funded by the German Research Foundation (DFG) as part of the Transregional Collaborative Research Centre (SFB/Transregio) 141 ‘Biological Design and Integrative Structures’/A01. The authors would like to thank Dr. Björn Schäfer (Wilhelma, Stuttgart, Germany), Günter Koch and Dr. Robert Gliniars (Hohenheimer Gardens, Stuttgart, Germany) for providing fresh samples. We thank the editor and two anonomyous reviewers for their constructive comments which improved the manuscript.
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