Higher Freezing Tolerance Research Articles

Potassium-induced freezing tolerance is associated with endogenous abscisic acid, polyamines and soluble sugars changes in grapevine

Crop nutrition is a practical method against freezing injury which may improves grapevine freezing tolerance (FT) potential by increasing bud nitrogenous and carbohydrates storages. In this study the effect of foliar application of potassium sulfate (PS; K2SO4; 0, 1, 2 and 3%) on some physico-chemical properties of fruit and leaf mineral contents of ‘Sultana’ grapevine (Vitis vinifera L.) were studied. Moreover, to further elucidate the efficiency of foliar PS on FT and some relevant morpho-physiological changes were evaluated at four sampling dates: Nov., Jan., Mar., and Apr. following exposure to artificial freezing using electrolyte leakage and bud browning bioassay. In summer 2013 and 2014, PS was sprayed at fruit set, pea-sized berry, verison and maturity on 16 years old grapevines located in Malayer Grape Research Station (Iran) under a randomized complete block design. Based on results, foliar PS significantly affected yield, cluster weights, and berry weight and some fruit quality indices including soluble solids, titratable acidity, pH and phenolic compounds. Foliar PS significantly changed the leaf N, K, P, Mg, Zn and Fe concentrations. The effect of nutrition treatments was also significant on FT of grapevines at four sampling stages. In Jan., the highest FT (LT50=−25.97°C) and the lowest FT (LT50=−20.16°C) was found in 3% PS- treated and control untreated vines, in respectively. Moreover, PS especially at 3% resulted in higher increments in abscisic acid, total phenol, soluble sugars, and polyamines concentrations. Potassium spray increased membrane stability and decreased electrolyte leakage. The ability of Potassium in FT improvement (lower electrolyte leakage) was found to be related to change in endogenous abscisic acid concentration as a stress hormone in preliminary and subsequent changes in other metabolites such as accumulation of soluble sugars, polyamines, and phenolic acids.

Arginine and proline applied as food additives stimulate high freeze tolerance in larvae of Drosophila melanogaster.

The fruit fly Drosophila melanogaster is an insect of tropical origin. Its larval stage is evolutionarily adapted for rapid growth and development under warm conditions and shows high sensitivity to cold. In this study, we further developed an optimal acclimation and freezing protocol that significantly improves larval freeze tolerance (an ability to survive at -5°C when most of the freezable fraction of water is converted to ice). Using the optimal protocol, freeze survival to adult stage increased from 0.7% to 12.6% in the larvae fed standard diet (agar, sugar, yeast, cornmeal). Next, we fed the larvae diets augmented with 31 different amino compounds, administered in different concentrations, and observed their effects on larval metabolomic composition, viability, rate of development and freeze tolerance. While some diet additives were toxic, others showed positive effects on freeze tolerance. Statistical correlation revealed tight association between high freeze tolerance and high levels of amino compounds involved in arginine and proline metabolism. Proline- and arginine-augmented diets showed the highest potential, improving freeze survival to 42.1% and 50.6%, respectively. Two plausible mechanisms by which high concentrations of proline and arginine might stimulate high freeze tolerance are discussed: (i) proline, probably in combination with trehalose, could reduce partial unfolding of proteins and prevent membrane fusions in the larvae exposed to thermal stress (prior to freezing) or during freeze dehydration; (ii) both arginine and proline are exceptional among amino compounds in their ability to form supramolecular aggregates which probably bind partially unfolded proteins and inhibit their aggregation under increasing freeze dehydration.

Comparison of cold hardiness in the leaves of various grape cultivars based on photochemical reflectance index

We compared cold hardiness in the leaves of 12 grape cultivars, including two wild species, two Vitis vinifera × V. labrusca , three HPDs (interspecific crosses) and five V. vinifera L. cultivars using PRI -T curves and analysis of variance (ANOVA). The photochemical reflectance index ( PRI ) of most grape leaves decreased linearly when frozen progressively in darkness, but these patterns varied. The PRI of diploid cultivars and blanc cultivars at the standard temperature ( PRI 4°C) remained relatively steady during exposure to successively lower temperatures (0, -2, -4 and -6°C) compared with polyploid and noir cultivars, representing a boundary dividing grape cultivars into frost tolerant and vulnerable. According to this principle, which was tested by ANOVA, the cold hardiness of the four species was ranked (from high to low) as V. vinifera L. > HPD = wild species > V. vinifera × V. labrusca . The cold hardiness of the 12 cultivars was divided into three classes: Resistant: PRI increased markedly compared with the control, R PRI (( PRI - PRI control )/ PRI control ) > 0 (p < 0.05), indicating high freezing tolerance and wide temperature adaption; Tolerant: PRI changed little compared with the control (p < 0.05), R PRI trended to 0, indicating relative stability when exposed to a short period of freezing temperatures; Vulnerable: PRI decreased dramatically compared with the control, R PRI < 0 (p < 0.05), indicating that photosynthesis was inhibited or damaged due to freezing. We also observed seasonal differences in the cold hardiness of the cultivars; grape leaves were more vulnerable to cold in fall than in spring. This study provides a practical method for estimating cold hardiness in grape.

Freezing tolerance and its relationship with soluble carbohydrates, proline and water content in 12 grapevine cultivars

The objectives of this study were to compare freezing tolerance (FT) of 12 field-grown grapevine cultivars at four stages: Nov., Jan., Mar., and Apr., and to investigate the relationship between FT and soluble carbohydrates, proline and water content changes of buds during acclimation and deacclimation stages. After exposure to various freeze test temperatures bud LT50 values were estimated using electrolyte leakage and bud browning measurement. Bud FT of all cultivars studied increased with the overall trend of declining temperatures from Nov. through Jan., after which the buds began to deacclimate from Mar. and gradually lost FT in Apr.; overall LT50 means were −13.79, −21.74, −20.38, and −10.06, respectively. However, cultivars did not show alike acclimation and deacclimation rates and, for example, ‘Yaquti’, an early ripening cultivar, showed relatively higher FT in early autumn, but was one of the least hardy cultivars in later stages. In Jan. (full hardening), cultivars were classified as hardy (‘Red Sultana’, ‘Fakhri’, ‘Shahani’ and White Sultana), moderately hardy (‘Tabarzeh’, ‘Gaznei’, ‘Thompson Seedless’, Laal, ‘Sahebi’ and ‘Rishbaba’) and least hardy (‘Yaquti’ and ‘Ruby Seedless’). The pattern of soluble carbohydrates and proline changes were highly consistent with the LT50 profile. Bud water content, especially in early autumn, had a stronger correlation with FT, compared to the concentration of cytoplasmic osmoregulants. Likewise, soluble carbohydrates proved to be a superior indicator of FT, as compared to proline. Our results showed osmoregulants as well as bud water content are useful indices to screen FT in a range of grapevine germplasm resources.

JcCBF2 gene from Jatropha curcas improves freezing tolerance of Arabidopsis thaliana during the early stage of stress.

High chilling-susceptibility is becoming the bottleneck for cultivation and commercialization of Jatropha curcas L. For insights to chilling resistance ability of this plant species, a cold response transcription factor, JcCBF2, was cloned and studied. It codes a 26kDa protein, which contains all conserved motifs unique to the C-repeat binding factor (CBF) family and has high similarity to CBFs of Ricinus communis and Populus. Its transcripts express specifically in leaves of Jatropha at cold temperature. After transmitting the report vector, 35S::JcCBF2-GFP, into Arabidopsis thaliana, JcCBF2 protein is main detected in cell nucleus, being consistent to the nuclear orientation signal in its N-terminal. Compared to the control Arabidopsis, the frozen leaves of JcCBF2-overexpressed seedlings grow stronger with less malondialdehyde, smaller leaf conductivity and activer superoxide dismutase, showing their higher freezing tolerance. RT-PCR tests revealed that JcCBF2 functioned mainly at the early stage (0-6h) of resistance events in Arabidopsis, and its transcripts reduced after 6h. In addition, JcCBF2 could quickly regulate transcripts of some cold-responsive (COR) genes such as RD29A, COR105A and COR6.6, also during the early stage of frozen treatment. This study not only proves the chilling resistance roles of JcCBF2, but also presents a candidate gene engineering for improvement of chilling tolerance in J. curcas.

THE DEVELOPMENT OF TECHNIQUES AND MOLECULAR MARKERS FOR INTERSPECIFIC BREEDING OF GRAPE FOR FREEZING TOLERANCE

The availability of winegrapes with a higher freezing tolerance than currently used V. vinifera cultivars is desirable for the Wine Industry in Canada. One way to accomplish this is by crossing V. riparia with V. vinifera. This study reports on the analysis of techniques that might be used in such a program. Electrolyte leakage values obtained from grapes either before or after a low temperature treatment did not show a significant difference between the two species. Arabidopsis DNA extracted by a quick ethanol-based method could be amplified but this was not the case for grape DNA. PCR primers were developed to identify different alleles of Vitis CBF1 and Vitis CBF4. Analyses of 2 V. vinifera cultivars, 17 V. riparia accessions and 6 winegrapes from Italy and Germany showed that these primers could be useful for marker assisted selection.

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

The deposition of surface (farinose) flavonoids on aerial parts of some Primula species is a well-documented but poorly understood phenomenon. Here, we show that flavonoid 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 natural flavone was approximately 6 °C lower compared to those that had their flavone artificially removed. Additionally, farinose flavonoids on the leaves reduced subsequent electrolyte leakage (EL) from the cells exposed to freezing temperatures. Interestingly, exogenous application of flavone at 4 mg/g fresh weight to P. malacoides leaves, which had the original flavone mechanically removed, restored freezing tolerance, and diminished EL from the cells to pretreatment values. Our results suggest that farinose flavonoids may function as mediators of freezing tolerance in P. malacoides, and exogenous application of flavone could be used to reduce freezing damage during sudden but predictable frost events in other plant species.

Evaluation of freezing tolerance of winter wheat (Triticum aestivum L.) under controlled conditions and in the field

Freezing tolerance of winter wheat (Triticum aestivum L.) is one of the main factors governing winter survival. To avoid winterkill it is very important to choose freezing tolerant wheat genotypes. Experiments were carried out to evaluate freezing tolerance of winter wheat varieties and breeding lines under field and controlled conditions with cold acclimation and with no cold acclimation stage. Field experiments were done for two years during the winters of 2010–2011 and 2011–2012. The evaluation of winter hardiness was conducted in April of 2011 and 2012 by visual scoring on a 1–9 score basis (score 1 – all plants were killed, score 9 – all plants survived). Wheat genotypes under investigation experienced very different climatic conditions in the field during the two winters. Consequently, no correlation between winter hardiness scores of 2010–2011 and 2011–2012 winters was observed. Winter hardiness scores ranged from 1.0 (WW-8) to 7.6 (‘Zentos’) after 2010–2011 winter, whereas winter hardiness scores varied from 5.8 (WW-31) to 7.8 (‘Kovas DS’) after 2011–2012 winter. Freezing tolerance of winter wheat genotypes was also assessed in the controlled conditions by measuring chlorophyll fluorescence parameter Fv/Fm in leaves and percentage survival of plants after the freezing test. High correlation (r = 0.88, p < 0.01) between freezing test survival of non-acclimated wheat and Fv/Fm ratio showed that this method can speed up the selection for freezing tolerance by evaluating wheat genotypes immediately after the test. Results of our study showed low negative, but significant relationship (r = −0.41, p < 0.05) between 2010–2011 winter hardiness scores in the field and freezing test survival of non-acclimated plants in the laboratory experiment. Wheat genotypes that quickly lose winter hardiness have higher freezing tolerance without hardening stage. Our results support the hypothesis of two separate genetic systems of freezing tolerance in winter wheat which are determined by duration and rate of gene expression during cold acclimation.

Development of a Model System to Identify Differences in Spring and Winter Oat

Our long-term goal is to develop a Swedish winter oat (Avena sativa). To identify molecular differences that correlate with winter hardiness, a winter oat model comprising of both non-hardy spring lines and winter hardy lines is needed. To achieve this, we selected 294 oat breeding lines, originating from various Russian, German, and American winter oat breeding programs and tested them in the field in south- and western Sweden. By assaying for winter survival and agricultural properties during four consecutive seasons, we identified 14 breeding lines of different origins that not only survived the winter but also were agronomically better than the rest. Laboratory tests including electrolytic leakage, controlled crown freezing assay, expression analysis of the AsVrn1 gene and monitoring of flowering time suggested that the American lines had the highest freezing tolerance, although the German lines performed better in the field. Finally, six lines constituting the two most freezing tolerant lines, two intermediate lines and two spring cultivars were chosen to build a winter oat model system. Metabolic profiling of non-acclimated and cold acclimated leaf tissue samples isolated from the six selected lines revealed differential expression patterns of 245 metabolites including several sugars, amino acids, organic acids and 181 hitherto unknown metabolites. The expression patterns of 107 metabolites showed significant interactions with either a cultivar or a time-point. Further identification, characterisation and validation of these metabolites will lead to an increased understanding of the cold acclimation process in oats. Furthermore, by using the winter oat model system, differential sequencing of crown mRNA populations would lead to identification of various biomarkers to facilitate winter oat breeding.

Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata , survive cryopreservation in liquid nitrogen

The larva of the drosophilid fly, Chymomyza costata, is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (-196 °C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury.

Freezing tolerance of Porphyra yezoensis (Bangiales, Rhodophyta) gametophyte assessed by chlorophyll fluorescence

In January and February 2010, heavy sea ice formed along the coast of the Bohai Sea and the northern Yellow Sea, China. Intertidal organisms were subjected to serious freezing stress. In this study, we investigated the freezing tolerance of the upper intertidal economic seaweed Porphyra yezoensis. The maximum photochemical efficiency of PS II (F v/F m) in undehydrated thalli remained high after 24 h at −2°C and that in dehydrated thalli decreased in a proportion to thallial water loss. F v/F m dropped sharply after 24 h at −20°C, regardless of absolute cellular water content (AWC). The F v/F m in frozen thalli recovered rapidly at 0–20°C. A wide range of water loss in the thalli enhanced their tolerance to freezing. F v/F m values in undehydrated thalli dropped sharply after 3 d at −2°C or 10 d at −20°C while those in dehydrated thalli (20–53% AWCs) remained at high levels after 9 d at −2°C or 30 d at −20°C. These results indicate that P. yezoensis has high freezing tolerance by means of dehydration during the ebb tide and rapid recovery of F v/F m from freezing. A strategy of P. yezoensis industry to avoid heavy loss during freezing season is discussed based on these findings.

Generation of an evolved Saccharomyces cerevisiae strain with a high freeze tolerance and an improved ability to grow on glycerol

Glycerol is a residue generated during biodiesel production and represents around 10% of the total product output. Biodiesel production is currently having a significant impact on glycerol price, leading to an increased interest in the use of glycerol as a cheap substrate for fermentation processes. We have analysed the growth kinetics of two wild-type strains of Saccharomyces cerevisiae grown on synthetic media containing glycerol as the sole carbon and energy source. Both strains were initially unable to grow when cultivated under these conditions, and an unusually long lag phase was necessary prior to the appearance of slow-growing cells. Following the application of an "evolutionary engineering" approach, we obtained S. cerevisiae strains with an improved ability to grow on glycerol. We report here the isolation of an evolved strain that exhibits a reduction of the lag phase, a threefold increase of the specific growth rate and a higher glycerol consumption rate compared to wild-type strains. The evolved strain has retained its fermentative activity, producing ethanol at the same rate and yield as the wild type. Interestingly, the yeast biomass obtained by cultivating the evolved strain on synthetic glycerol-based media also showed a high viability after prolonged storage at -20°C. The strategy adopted in our study could be easily applied to obtain S. cerevisiae strains with new industrially relevant traits, such as an improved ability to use cheap substrates and high resistance to freeze and thaw procedures.

Accumulation of theanderose in association with development of freezing tolerance in the moss Physcomitrella patens

Mosses are known to have the ability to develop high degrees of resistance to desiccation and freezing stress at cellular levels. However, underlying cellular mechanisms leading to the development of stress resistance in mosses are not understood. We previously showed that freezing tolerance in protonema cells of the moss Physcomitrella patens was rapidly increased by exogenous application of the stress hormone abscisic acid (ABA) [Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., Takezawa, D., 2003a. Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475–483]. Herein it is shown that protonema cells with acquired freezing tolerance specifically accumulate low-molecular-weight soluble sugars. Analysis of the most abundant trisaccharide revealed that the cells accumulated theanderose (G 6-α-glucosyl sucrose) in close association with enhancement of freezing tolerance by ABA treatment. The accumulation of theanderose was inhibited by cycloheximide, an inhibitor of nuclear-encoded protein synthesis, coinciding with a remarkable decrease in freezing tolerance. Furthermore, theanderose accumulation was promoted by cold acclimation and treatment with hyperosmotic solutes, both of which had been shown to enhance cellular freezing tolerance. These results reveal a novel role for theanderose, whose biological function has been obscure, in high freezing tolerance in moss cells.

Differential responses of silver birch (Betula pendula) ecotypes to short-day photoperiod and low temperature

We investigated interrelations of dormancy and freezing tolerance and the role of endogenous abscisic acid (ABA) in the development of silver birch (Betula pendula Roth) ecotypes in controlled environments. Short-day treatment induced growth cessation, bud set and dormancy development, as well as initiation of cold acclimation and an increase in freezing tolerance. Subsequent low temperature and short days (12-h photoperiod) resulted in a significant increase in freezing tolerance, whereas bud dormancy was gradually released. The concentration of ABA increased in response to short days and then remained high, but ABA concentrations fluctuated irregularly when the dormant plants were subsequently exposed to low temperature during short days. Although there was a parallel development of freezing tolerance and bud dormancy in response to short days, subsequent exposure to low temperature had opposite effects on these processes, enhancing freezing tolerance and releasing dormancy. Compared with the southern ecotype, the northern ecotype was more responsive to short days and low temperature, exhibiting earlier initiation of cold acclimation, growth cessation and an increase in ABA concentrations in short days, and higher freezing tolerance, faster dormancy release and greater alteration in ABA concentrations when subsequently exposed to low temperature during short days. The rates and extent of the increases in ABA concentration may be related to increases in freezing tolerance and dormancy development during short days, whereas the extent of the fluctuations in ABA concentration may play an important role in enhancing freezing tolerance and releasing dormancy during a subsequent exposure to low temperature during short days.

Sexual differences in cold acclimation and freezing tolerance development in sea buckthorn ( Hippophae rhamnoides L.) ecotypes

Sexual differences in cold acclimation and freezing tolerance development of two contrasting sea buckthorn ( Hippophae rhamnoides L.) ecotypes from northern and southern regions in China were recorded after exposure to short day photoperiod (SD) and low temperature (LT). The results demonstrated that cold acclimation could be triggered by exposing the plants to SD or LT alone, and that a combination of both treatments had an additive effect on freezing tolerance in all plants tested. However, development of freezing tolerance was dependent on the sex of plants under SD and LT, the males were clearly more responsive to SD and LT than the females in both ecotypes studied. On the other hand, development of freezing tolerance was also ecotype-dependent, the northern ecotype was more responsive to SD and LT than the southern ecotype, resulting in earlier cold acclimation under SD and higher freezing tolerance under LT. Moreover, development of freezing tolerance induced by SD and LT was accompanied by changes in ABA levels. These alterations in ABA levels were different in different treatments, ecotypes and sexes. Therefore, the differences in SD and LT-induced physiological responses showed that the different ecotypes and the different sexes might employ different survival strategies under environmental stress.

Development of a Novel Microbial Sensor with Baker's Yeast Cells for Monitoring Temperature Control during Cold Food Chain

A novel microbial sensor containing a commercial baker's yeast with a high freeze tolerance was developed for visibly detecting inappropriate temperature control of food. When the yeast cells fermented glucose, the resulting gas production triggered the microbial sensor. The biosensor was a simple, small bag containing a solution of yeast cells, yeast extract, glucose, and glycerol sealed up with multilayer transparent film with barriers against oxygen and humidity. Fine adjustment of gas productivity in the biosensor at low temperatures was achieved by changing either or both concentrations of glucose and yeast cells. Moreover, the amount of time that food was exposed to inappropriate temperatures could be deduced by the amount of gas produced in the biosensor. The biosensor was stable without any functional loss for up to 1 week in frozen storage. The biosensor could offer a useful tool for securing food safety by maintaining low-temperature control in every stage from farm to fork, including during transportation, in the store, and at home.

A lipid transfer protein gene BG-14 is differentially regulated by abiotic stress, ABA, anisomycin, and sphingosine in bromegrass ( Bromus inermis)

The objective was to investigate the expression of a lipid transfer protein gene (LTP) both in bromegrass (Bromus inermis) cells and seedlings after exposure to abiotic stresses, abscisic acid (ABA), anisomycin, and sphingosine. A full-length cDNA clone BG-14 isolated from bromegrass suspension cell culture encodes a polypeptide of 124 amino acids with typical LTP characteristics, such as a conserved arrangement of cysteine residues. During active stages of cold acclimation LTP expression was up-regulated, whereas at the final stage of cold acclimation LTP transcript level declined to pre-acclimation level. A severe drought stress induced the LTP gene; yet, LTP expression doubled 3 d after re-hydration. Both temperature and heat shock duration influence LTP induction; however temperature is the primary factor. Treatment with NaCl stimulated accumulation of LTP mRNA within 15 min and the transcripts remained at elevated levels for the duration of the salinity stress. Most interestingly, Northern blots showed LTP was rapidly induced not only by ABA, but also by anisomycin and sphingosine in suspension cell cultures. Of the three chemicals, ABA induced the most rapid and highest response in LTP expression as well as highest freezing tolerance, whereas sphingosine was the least active for both LTP expression and freezing tolerance.

CHANGES IN FREEZING TOLERANCE AND PROTEIN CONSTITUENTS OF LONICERA CAERULEA DURING COLD ACCLIMATION

Lonicera caerulea var. emphyllocalyx is an edible honeysuckle and a deciduous shrub native to the northern areas of Japan. In this study, we evaluated the freezing tolerance of twigs, based on the bud sproutings, collected from October to March from five different habitats in Hokkaido, Japan, and of calli. Differences in the freezing tolerance of twigs were recorded in February, and may possibly be ascribed to differences in air temperature and the depth of snow in the study areas. As evaluated by the 2,3,5-triphenyltetrazolium chloride (TTC) reduction test, the time needed to develop freezing tolerance began earlier and lasted longer in buds than in stems. Remarkably, there were no differences in freezing tolerance between buds and stems from December to January, when both tissue types showed a high freezing tolerance. During the period of decreasing freezing tolerance from winter to spring, buds showed greater tolerance than stems. Calli exposed to the low temperature of 5 °C increased their cold tolerance after 8 days of treatment, reaching a plateau on day 12; however, cold-treated calli lost their freezing tolerance within only 2 days of incubation at 25 °C. The proteins extracted from these calli were analyzed by two-dimensional polyacrylamide gel electrophoresis. Our results showed abundant accumulations of a 42 kDa (pI 5.4) protein during cold acclimation. This protein decreased during incubation at 25 °C. Manipulating the level of the 42 kDa (pI 5.4) protein through breeding may help to develop higher freezing tolerance in Lonicera caerulea var. emphyllocalyx.

Rearrangement of the Cytoskeleton in Triticum aestivum Cells during Cold Hardening and after Treatment with Abscisic Acid

Immunocytochemical study of the basic characteristics of the tubulin and actin cytoskeleton (total content, orientation, structure, and stability) was performed for various root zones of the seedlings of winter wheat cultivars contrasting in their freezing tolerance. Plant cold hardening (3°C, 7 days) and ABA treatment (30 μM, 3 days) increased the stability of tubulin microtubules (MT), that is, reduced the depolymerizing action of oryzalin in vivo. However, the mechanisms of hardening and ABA stabilizing action on the cytoskeleton were different: low temperature enhanced spatial MT aggregation and resulted in the formation of a dense network of thick MT bundles, whereas ABA reduced the content of tubulin components and induced microfilament (MF) depolymerization. Most pronounced temperature- and ABA-induced cytoskeleton changes were observed in the differentiation zone, which indicates an important role of this root zone in plant adaptation and development of root freezing tolerance. Low temperatures reduced the hormonal effect on the structural arrangement and stability of MT and MF in wheat cultivars of high and moderate freezing tolerance but increased hormonal effects in the slightly tolerant cultivar. MF depolymerization and an increase in the proportion of stable MT are supposed to be a necessary condition for seedling growth retardation after their treatment with ABA and for seedlings at the initial phase of their adaptation to low temperature. At the final phase of cold hardening, some growth acceleration is evidently determined by the accumulation of highly labile MT and greater actin polymerization.

Cold acclimation in silver birch (Betula pendula). Development of freezing tolerance in different tissues and climatic ecotypes

A number of environmental cues including short day photoperiod (SD) and low temperature (LT) are known to interact in triggering growth cessation, cold acclimation and other adaptive responses in temperate‐zone tree species. Proper timing of these responses is particularly important for survival of trees in the boreal and subarctic regions. Therefore, we used a northern tree species, silver birch (Betula pendula Roth) as an experimental model to investigate the effect of SD and LT on development of freezing tolerance and on levels of endogenous abscisic acid (ABA) in short‐term experiments under controlled conditions. We characterized differences in SD and LT‐induced cold acclimation between three different climatic ecotypes from southern, central and northern habitats. The results demonstrated that cold acclimation was rapidly triggered by exposing the plants to SD or LT, and that a combination of the different treatments had an additive effect on freezing tolerance. Freezing tolerance induction was not uniform in the different tissues, the buds and leaves developed freezing tolerance more rapidly than the stem, and the young leaves had a higher freezing tolerance than the old leaves. The ability of the leaves to respond to SD and LT and similarity of the bud and leaf responses indicate that birch leaves provide a rapid and convenient system for studies on molecular mechanisms of cold acclimation. Development of freezing tolerance was dependent on the climatic ecotype, the northern ecotype was clearly more responsive to both SD and LT than the two more southern ecotypes. Development of freezing tolerance induced by SD and LT was accompanied by transient changes in ABA levels. These alterations in ABA levels were ecotype‐dependent, the northern ecotype reacting more strongly to the environmental cues.