The Transpiration Rate of Unhardened, Hardened, and Dehardened Seedlings of Spruce and Pine

Tissue injuries in winter are sometimes interpreted as caused by drought damages. The possibility that the tolerance of conifers of winter and spring conditions is increased through decrease in transpiration rate has been little investigated. The transpiration rate of 3 or 6 months old unhardened, hardened, and dehardened seedlings of Pinns silvestris L. and Picea abies (L.) Karst. was assessed gravimetrically, at 20°C. The transpiration rates of seedlings of spruce and pine which had been hardened for 3 months were about ¹/₂ the rates of dehardened seedlings. Transpiration during a dehardening period increased at different rates in pine and spruce. After a dehardening period of 3–5 days the transpiration rate of spruce reached a maximum, whereas pine reached the maximum transpiration rate after a dehardening period of 10–14 days. Transpiration of seedlings of spruce and pine hardened for 3 months showed only a very slight reaction to light and darkness. This indicated that stomata of hardened seedlings remained closed. The results are discussed in connection with frost and drought injuries in pine and spruce during early spring.     

Frost tolerance and biochemical changes during hardening and dehardening in contrasting white clover populations

Successful winter survival of perennial plants, like white clover, is dependent on proper timing of both hardening and dehardening. The purpose of this study was to investigate the regulation of these processes in two cultivars (AberCrest and AberHerald) and two Norwegian ecotypes (Særheim collected at 58°46′N lat. and Bodø at 67°20′N lat.) of white clover (Trifolium repens L.). For hardening and dehardening, plants were exposed to controlled temperature conditions and frost hardiness of stolons was tested by programmed freezing at the rate of 3°C per hour. In addition, stolons were analysed for starch, soluble sugars and soluble amino acids. Cultivars AberCrest and AberHerald, selected for growth at low temperature and winter hardiness in the United Kingdom, were significantly less hardy than the Norwegian populations. After six weeks of hardening (2 weeks at 6°C and 4 weeks at 0.5°C), estimated LT₅₀ values were ?13.8, ?13.0, ?17.8 and ?20.3°C for AberCrest, AberHerald, Saerheim and Bodø, respectively. The rate of dehardening increased with increasing temperature. At low temperature (6°C), the northern ecotype from Bodø was more resistant to dehardening than AberHerald. However, at 18°C the absolute rate of dehardening (°C day^?1) was twice as high in Bodø as in AberHerald plants. Stolon elongation during dehardening was initiated at lower temperatures in AberHerald than in plants of the Bodø ecotype. The content of total soluble sugars, sucrose and the amino acids proline and arginine were significantly higher in hardy plants of Bodø than in those of AberHerald. Sucrose levels decreased during dehardening and correlations between sucrose content and LT₅₀ during this process were statistically highly significant for both Bodø and AberHerald. The least hardy populations of white clover were characterized by thick stolons, long internodes and large leaves.     

The importance of hardening and winter temperature for growth in mountain birch populations

Seedlings of five mountain birch populations (Betula pubescens Ehrh. ssp. czerepanovii) from Fennoscandia and Iceland were raised and grown at natural daylengths at Tromsø, Norway (69°N) and different temperatures during late summer and fall season, followed by winter temperature treatment at ambient and +4 °C above ambient temperatures at Bergen, Norway (60°N). The experiment took place during two seasons (2000/01 and 2001/02). The following summer shoot and biomass growth were reduced as a result of winter warming and subsequent premature dehardening in early flushing provenances and treatments. Biomass increased in plants grown at low hardening temperature when compared with high temperature treatment. As a conclusion, increased winter temperatures would tend to increase the risk of spring frost damage and reduce growth in birch seedlings, because the differences between the frost hardening and ambient temperatures are decreasing, and because the time from budbreak to dehardening is shortened. The results are discussed in relation to simultaneous experiments with frost hardiness in the same populations and treatments.     

Regulation of the loss of frost hardiness in Pinus radiata by photoperiod and temperature

Abstract. In controlled environments, the interactive effects of warm (16: 8°C, day: night) and cool (12: 4°C, day: night) temperatures and long (13.5 h) and short (10 h) photoperiods on the dehardening of seedlings of Pinus radiata D. Don were investigated. In another experiment, the effect of four photoperiods from 9 to 14 h was examined. In a third, dehardening at constant temperatures from 5 to 17°C was followed. There was no evidence for an interaction between photoperiod and temperature. Dehardening was temporarily delayed by photoperiods below about 10 h, but there was no other quantitative effect of photoperiod. At constant temperatures, the rate of dehardening was initially constant but declined as the minimum summer frost hardiness was reached. In the initial phase the rate of dehardening was a linear function of temperature, increasing from 0.05°C day⁻¹ at 8°C to 0.30 °C day⁻¹ at 17°C. Temperature controlled the loss of frost hardiness by regulating the rate of dehardening.     

Hardening, abscisic acid, proline and freezing resistance in two winter wheat varieties

Two varieties of winter wheat ( Triticum aestivum L.) differing in freezing resistance ("Holme" from Sweden, freezing resistant, and "Amandus" from Germany, less freezing resistant) were hardened for five weeks by gradually reducing the day/night temperature from 20°C/15°C during the first week to 2° C/0° C during the fifth week and the photoperiod from 15 to 9 h. This treatment increased the freezing resistance of both varieties in comparison to unhardened control plants. Hardening caused an increase in osmolarity of cell sap and in the levels of proline and abscisic acid (ABA). Increase in osmolarity preceded the increase in ABA level, and proline levels increased later than ABA levels. Holme had higher values of osmolarity as well as higher levels of ABA and proline. but the differences between the two varieties were significant only for proline. Since the pressure potential remained constant or increased slightly during the hardening period, it is suggested that the accumulation of ABA is due to the hardening process and not to simple water stress caused by cold-induced inhibition of water uptake by the root.
Spraying hardened plants with 10⁻⁴ M ABA 24 h before a freezing test increased freezing resistance in both varieties, but did not obliterate the differences in freezing resistance between the two varieties. Spraying hardened plants with an aqueous proline solution (10%, w/v) was without effect on freezing resistance. It is concluded that the hardening procedure causes an accumulation of ABA in winter wheat leaves and that ABA is involved in the chain of events leading to freezing resistance.     

The relationship between the differences in frost resistance of Arabidopsis and Thellungiella and heat shock proteins and dehydrins

Plants of extremophile Thellungiella (Thellungiella salsuginea (Pall.) OE Schulz) withstood freezing at ?15°C for 2 h without hardening, whereas plants of Arabidopsis (Arabidopsis thaliana (L.) Heinh.) were damaged at ?10°C and died at ?15°C under these conditions. The content of heat shock proteins (HSPs) HSP101, HSP60 and constitutive HSC70 was significantly higher in unhardened Thellungiella plants than in unhardened Arabidopsis plants. The spectrum of dehydrins (DHNs) in unhardened Thellungiella plants was more diverse and their total content was higher than in unhardened Arabidopsis plants. Frost resistance of Arabidopsis increased after hardening (4°C, 7 days), and there was an increase in the content of HSP101 and HSP60, as well as in the content of the DHN with a mol wt of 70 kD. Thellungiella plants survived after hardening at ?18°C, and the increase in the content of HSP101, HSP70, and HSP60 was significantly less pronounced than in Arabidopsis. At the same time, the content of DHNs in Thellungiella increased significantly during the hardening primarily because of the appearance of two DHNs (mol wts of 42 and 45 kD). It is assumed that an increased content of HSPs and DHNs and their greater diversity can be one of the factors of Thellungiella resistance to low temperatures as compared to Arabidopsis.     

Fungicidal and Cytotoxic Activity of a Capsicum chinense Defensin Expressed by Endothelial Cells

Plant defensins are antimicrobial peptides that exhibit mainly antifungal activity against a broad range of plant fungal pathogens. However, their actions against Candida albicans have not been extensively studied. The mRNA for γ-thionin, a defensin from Capsicum chinense, has been expressed in bovine endothelial cells. The conditioned medium of these cells showed antifungal activity on germ tube formation (60–70% of inhibition) and on the viability of C. albicans (70–80% of inhibition). Additionally, C. albicans was not able to penetrate transfected cells. Conditioned medium from these cells also inhibited the viability (80%) of the human tumor cell line, HeLa.     

Uptake and Growth-promoting Activity of Indoleacetic Acid in Segments of Cold-hardened Wheat Coleoptiles

Seedings of winter wheat (Triticum aestivum L. cv. Kharkov MC 22) were grown at 24 C (unhardened) and 4 C (hardened). Indoleacetic acid (IAA) was added to excised coleoptile segments after lengthy incubation and their responses were determined by photometric auxanometry at both 25 C and 5 C. The segments' rates of uptake of ¹⁴CIAA were also compared at both temperatures. Cold hardening had no significant effect on the rates of elongation and uptake in a saturating concentration of IAA (2 to 10 μM) at either temperature. Elongation was more sensitive to temperature of measurement than was uptake. At suboptimal concentrations of IAA and 25 C, hardened coleoptiles took up [2-¹⁴C]-IAA twice as fast but elongated half as fast as unhardened coleoptiles. This and the lack of effect of cold hardening on apparent uptake of [1-¹⁴C]-IAA raised the possibility that a higher rate of IAA-decarboxylation was coupled with the higher rate of uptake of IAA by hardened coleoptiles. Homeostatic hormonal regulation was also evident in the same endogenous rates of elongation of segments of cold-hardened and unhardened coleoptiles.     

Frost hardiness development in Pinus silvestris seedlings in response to fertilization

The effects of different levels of nitrogen on freezing survival, the amounts of chlorophylls and β-carotene, the ratios Chl a/Chl b, and (Chl a + Chl b)/β-carotene in hardened and unhardened seedlings of Scots Pine (Pinus silvestris L.) have been investigated. There was. no correlation between frost hardiness and nitrogen content in the unhardened seedlings. However, the hardy seedlings with the highest nitrogen content, showed a decrease in frost hardiness of 3°C. The amounts of chlorophylls and β-carotene increased for all the treatments during hardening, except for the seedlings that had the highest nitrogen content; so that the effect of fertilizer was more evident in non-hardened than in hardened plants. The ratios Chl a/Chl b decreased during hardening in all the treatments. This was an effect of a larger increase of Chl b than Chl a. The ratios of Chl a + Chl b/β-carotene also decreased during hardening except for the plants that were fertilized with the second lowest amount of nitrogen. Since the amounts of Chl a and Chl b increase during hardening the amount of β-carotene increases even more.
The growth rates of the seedlings were closely related to the nitrogen content for the different treatments. All plants were infected with ectotrophic mycorrhizae, which resulted in an enhancement of ion transportation from the soil to the plants. This is important when the supply of ions is low.     

Metabolic changes associated with cold-acclimation in contrasting cultivars of barley

Cereal plants become more resistant to freezing when first exposed to a period of cold-acclimation. Many physiological and molecular changes have been shown to occur at low temperatures, but the role and the contribution of each to frost resistance is still poorly understood. Two cultivars of barley ( Hordeum vulgare L.), the winter barley Onice and the spring barley Gitane, were acclimated under controlled conditions under an 8-h photoperiod at 4°C (light) and 2°C (dark) for 21 days. Changes in free proline, ABA, water-soluble carbohydrates and free fatty acids were measured to assess their involvement in cold-acclimation and to explain the different frost-resistant capacities of the two cultivars. Exposure of barley plants to low temperature resulted in an equal increase in proline in both cultivars. During the first days of cold acclimation, ABA levels showed a peak in the frost-resistant cultivar, lasting about 24 h, followed by a decrease. The water soluble carbohydrates reached their highest content after 3 days of hardening, although after 14 to 21 days of acclimation the carbohydrate content was similar to that of unhardened plants. The frost-resistant Onice had a much higher free fatty acid content than the frost-sensitive Gitane. Furthermore in Onice 86% of free farty acids was represented by unsaturated molecular species. Inolenic acid alone being 71%. In contrast, in the frost-sensitive cultivar only 31% of free fatty acids was unsaturated and a large amount of 9-oxo-nonanoic acid, a product present in the linolenic acid cascade, was also detected.
The ABA content after 2 days of hardening and the free fatty acid composition were clearly different between the two cultivars and may explain, at least in part, the different frost-resistant capacities of Onice and Gitane.     

Changes in the level of MACC during cold hardening and dehardening in winter wheat

Changes in the level of 1-(malonylamino)-cyclopropane-1-carboxylic acid (MACC) were determined in 6 winter wheat cultivars during cold hardening at 4°C. The cultivars differed by one degree of frost resistance within the range of degree II to VII of the COMECON scale. The time-course of changes in MACC level showed a similar pattern in all 6 cultivars; i.e. increase till day 6, no changes for the next 10 days, and then a steady decrease till the end of the hardening period. There was little difference between the final and the initial levels. The increase of MACC level, expressed as per cent of the original level, was not directly correlated with either the degree of frost resistance of the actual percentage of survival. In some cultivars. mean errors exceeded the difference in MACC accumulation between cultivars closest on the resistance scale.
The fate of MACC during the second half of hardening and after transfer of plants to 25°C was studied in cultivars Bezostaya and San Pastore. During the second half of the hardening period the level of MACC decreased in the leaves of both cultivars, but increased significantly in the roots. Within two days of transfer of the hardened plants to 25°C, the MACC level in leaves increased again, while that in the roots decreased. This finding, together with the preliminary evidence of very low MACC metabolism, strongly suggest that MACC accumulates in roots during the hardening period and when transferred to 25°C, it moves from roots to leaves.     

Plant resistance to cold stress: Mechanisms and environmental signals triggering frost hardening and dehardening

This introductory overview shows that cold, in particular frost, stresses a plant in manifold ways and that the plant’s response, being injurious or adaptive, must be considered a syndrome rather than a single reaction. In the course of the year perennial plants of the temperate climate zones undergo frost hardening in autumn and dehardening in spring. Using Scots pine (Pinus sylvestris L.) as a model plant the environmental signals inducing frost hardening and dehardening, respectively, were investigated. Over 2 years the changes in frost resistance of Scots pine needles were recorded together with the annual courses of day-length and ambient temperature. Both act as environmental signals for frost hardening and dehardening. Climate chamber experiments showed that short day-length as a signal triggering frost hardening could be replaced by irradiation with far red light, while red light inhibited hardening. The involvement of phytochrome as a signal receptor could be corroborated by respective night-break experiments. More rapid frost hardening than by short day or far red treatment was achieved by applying a short period (6 h) of mild frost which did not exceed the plant’s cold resistance. Both types of signals were independently effective but the rates of frost hardening were not additive. The maximal rate of hardening was − 0.93°C per day and frost tolerance of < − 72°C was achieved. For dehardening, temperature was an even more effective signal than day-length.     

Hardening and Dehardening of Lolium perenne in Response to Fluctuating Temperatures

Hardening and dehardening responses of two contrasting varietiesof Lolium perenne, measured as LT50 estimates, were followedin fluctuating temperature environments. Unhardened seedlingswere exposed to hardening environments for 7, 14, and 21 d inall combinations of 2, 4, 6, 8 and 10 C with either high dayand low night temperatures or low day and high night temperatures.Seedlings hardened for 28 d at 2 C were exposed to dehardeningenvironments in all combinations of 4, 6, 8, 10, and 12 C withhigh day and low night temperatures. A low day, or night, temperature of 2 C in combination withany other temperature increased hardening compared with theconstant higher temperature. For Premo, a hardy variety, thisincrease was 3 C when night temperature was reduced from 10to 2 C in combination with a day temperature of 10 C. Similarly,a low night temperature reduced the dehardening response ofPremo to higher day temperatures. At 12 C this effect on LT60was greater than 2 C. Much smaller responses to daily periodsof low temperature were found for the less hardy variety, GrasslandsRuanui. During each 24-h period, exposures to 2 C of longer than 4h were required to achieve greater hardening than that achievedin continuous 10 C treatments. Hardiness was not improved furtherby exposures longer than 8 h. Responses to diurnal temperature fluctuations were discussedin relation to possible mechanisms and to changes in hardinessduring the winter under different weather systems. Lolium perenne, cold hardening, cold dehardening, diurnal temperature fluctuations, varieties     

Effects of climatic warming on cold hardiness of some northern woody plants assessed from simulation experiments

Effects of climatic warming on cold hardiness were investigated for some northern woody plants. In the first experiment, seedlings of Norway spruce ( Picea abies [L.] Karst.), Scots pine ( Pinus sylvestris L.) and lodgepole pine ( Pinus contorta Dougl. var. latifolia Engelm.) were exposed to naturally fluctuating temperatures averaging −6°C (ambient) and 0°C (elevated) for 16 weeks in midwinter before they were thawed and re-saturated with water. In lodgepole pine, needle sugar concentrations had decreased by 15%, and the temperature needed to induce 10% injury to needles in terms of electrolyte leakage had increased by 6°C following treatment to elevated as compared with control temperatures. In contrast, Norway spruce and Scots pine showed no effects. The lack of an effect for Scots pine was ascribed to seedlings containing unusually large energy reserves that buffered respiratory expenditure of sugars. A strong, linear relationship between levels of cold hardiness, assessed by the electrolyte leakage method, and sugars was found when combining data from this and previous, similar experiments. In the second experiment, the evergreen dwarf shrub Empetrum hermaphroditum Hagerup was analysed for leaf cold hardiness, using the electrolyte leakage method, and sugar concentrations in late spring and late autumn during the third year of a warming experiment in a subarctic dwarf shrub community. The objective was to test the hypothesis that warming in the growing season alters hardening/dehardening cycles by increasing soil nitrogen mineralization and plant growth. Data found, however, suggested that cold hardening/dehardening cycles were unaffected by warming.     

Carbohydrate content and glycosidase activities following cold hardening in two grass species

The freezing resistance of the grass species Phleum pratense L. (timothy) and Phalaris arundinaces L. increases significantly after cold hardening. The content and composition of soluble carbohydrates were determined in the plants after short day treatment, cold hardening and dehardening. The amounts of mono-, di- and trisaccharides were reduced during the short day treatment, increased during cold hardening and decreased again during dehardening. The total amounts of soluble carbohydrates (mono-, di-, tri- and polysaccharides) were the same in hardened and dehardened plants, indicating that during hardening soluble polysaccharides (fructose polymers, fructans) were converted to mono- and oligosaccharides. Sucrose increased most after hardening conditions and, in P. arundinacea , a significant increase in 1-F-fructosylsucrose (isokestose) was also observed.
Invertase (β-fructofuranosidase. EC 3.2.1.26) activity increased following cold hardening and decreased following dehardening, while the α-galactosidase (EC 3.2.1.22) activity seemed to increase after dehardening. The glycosidases are probably involved in the mobilisation of polysaccharides during cold hardening.     

AN INCREASE OF ORNITHINE δ-AMINOTRANSFERASE-MEDIATED PROLINE SYNTHESIS IN RELATION TO HIGH-TEMPERATURE INJURY IN GRACILARIA TENUISTIPITATA (GIGARTINALES, RHODOPHYTA)

Changes of proline biosynthesis in relation to high-temperature (35° C) injury were investigated in Gracilaria tenuistipitata var. liui Zhang et Xia. On exposure to 35° C, the specific growth rate decreased after 5 days while free proline levels increased gradually after 2 days and reached the maximal level on days 4–6 but decreased at day 7. The repair ability of thalli treated at 35° C by measuring the growth rate after transfer to 25° C for another 5 days decreased in thalli that had been grown at 35° C for more than 2 days, and the extent increased as treatment at 35° C was prolonged. After 4 days of treatment at 35° C, the activities of both ornithine δ-aminotransferase (δ-OAT; EC 2.6.11.3) and Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) increased, but that of γ-glutamyl kinase (γ-GK; EC 2.7.2.11) remained unchanged, and that of glutamate-5-semialdehyde dehydrogenase (GSAd; EC 1.4.1.3) decreased. The application of 10 μM gabaculine, an irreversible inhibitor of δ-OAT, at 35° C recovered the growth ability but inhibited the increase of both δ-OAT activity and free proline level; its effects were reversed by 1 mM proline. G. saliconia, which is relatively tolerant to high temperature, showed a decrease of both δ-OAT activity and free proline level at 35° C. It seems that a stimulation of proline synthesis from the ornithine pathway via an increase in both δ-OAT and P5CR activities might be associated with high-temperature injury in G. tenuistipitata.     

Genetic and environmental contributions to the winter hardiness of conifers

Summary

Both genetic and environmental components are involved in the processes of winter hardening and dehardening which permit needles of conifers like Norway spruce to survive very low temperatures (< - 30°C) over winter and to recover fully in time for the following and subsequent seasons. One of the major environmental effects of increasing concentrations of atmospheric ozone (O₃) in recent summers has been to affect detrimentally the ability of conifer needles to harden properly and at the correct rate the following autumn. Part of the mechanism by which this occurs in the cytoplasm of needle cells has been traced to detrimental effects on both the Δ¹² fatty acid desaturase and the unusual Δ⁵ desaturase which appears to be part of the low temperature survival mechanism of conifers. The genetic component of winter hardening also involves needle lipids. Studies of lipids in the needles of Norway spruce trees of different provenances growing for many years in the UK have shown that they change during winter hardening as though they were still on trees in the original sites throughout Europe from which the seeds of these trees were initially collected.