Frost Hardening Research Articles

Frost hardiness of Norway spruce grown under elevated atmospheric CO 2 and increased nitrogen fertilizing

Summary Frost hardiness was tested on needles from 6-years-old Norway spruces that had been cultivated for 2 years in artificial ecosystems at an atmospheric CO 2 concentration of 280, 420 or 560 ppm and a nitrogen fertilization corresponding to 0, 30, and 90 kg N ha −1 yr −1 (NH 4 NO 3 ) as well as combinations of these treatments. Samples of previous year's needles were taken after completion of frost hardening in autumn, and at three dates during the dehardening process in spring when potentially harmful late frost events naturally occur. The needles were exposed to freezing temperatures down to − 35 °C. Samples were taken at 5 °C intervals and frost injury was estimated by the electrolyte leakage technique. After autumnal hardening, needles were injured only below −25 °C (LT50

Fine Structure of Acid Mist Treated Sitka Spruce Needles: Open-top Chamber and Field Experiments

Sitka spruce grafts (clones DF and 141) grown in open-top chambers (OTC) and ‘mature ’, 6 –8m tall Sitka spruce (clone DF) grown in the field were exposed to acid mist containing an equimolar ion mixture of H 2SO 4and NH 4NO 3at pH2.5. Mist was applied 4 times a week (4 ×1mm) in the OTC experiment and twice a week (2 ×2mm) on average in the field experiment, between May and Oct. 1991. Samples for light and electron microscopy were collected in Nov. and Jan. following acid mist treatment. Acid mist significantly decreased the amount of calcium deposited in the outer epidermal cell walls, the reduction being most pronounced in the OTCs. Ultrastructurally, acid mist caused a significant increase in chloroplast and grana width. Other symptoms associated with acid mist included swelling of chloroplast thylakoids, chloroplast protrusions, cytoplasm vacuolization, increase in large lipid accumulations and sickle-shaped chloroplast thylakoids. In the OTCs, acid mist hastened the acquisition of frost hardening in both clones. In the field, the control trees exhibited more frost injury than the acid mist treated trees suggesting, again, that acid mist had either hastened or enhanced the stage of frost hardiness of treated trees. In general, acid mist induced changes were more pronounced in the OTCs than in the field.

Carbohydrate content of Eucalyptus gunnii leaves along an annual cycle in the field and during induced frost-hardening in controlled conditions

The annual changes in frost hardiness were studied for three Eucalyptus gunnii genotypes. Frost resistance evaluated on leaf discs by the electrolyte leakage method reached a maximum in the coldest period and a minimum in summer demonstrating winter frost hardening. Genotype 634 exhibited a higher intrinsic resistance than the other genotypes both in the hardened and in the non-hardened stages. Plants of this genotype were also frost acclimated in controlled conditions by a progressive decrease of culture temperature (25 to 0 °C) but the degree of hardening appeared to be lower in these conditions. The carbohydrate patterns in leaves varied with acclimation. In controlled conditions the leaves of genotype 634 exhibited a rise in sucrose, fructose and raffinose concentration up to a temperature of 10 to 7 °C which subsequently decreased. In natural conditions a comparison of the three genotypes allowed us to correlate the higher intrinsic resistance of genotype 634 to a higher soluble sugar content. During acclimation fructose and raffinose changes were also correlated to an increase in cold resistance even though the kinetics of these changes differed in controlled and natural conditions. The starch content was very low in the various genotypes in the different conditions but oligosaccharides such as stachyose and possibly verbascose were detected. The results point out the relationships occurring between increased frost resistance and changes in fructose and raffinose concentration in E. gunnii leaves.

The relationship between water content and frost tolerance in shoots of hardwood seedlings

Two experiments were designed to test the relationship between water content and frost tolerance in stems of hardwood seedlings during the frost hardening process. In the first experiment, 3-month-old container-grown yellow birch (Betulaalleghaniensis Britton) seedlings were submitted to a decreasing temperature regime in a 8-h (short day, SD) or 16-h (long day, LD) photoperiod in growth chambers. In the second experiment, 3-month-old container-grown red oak (Quercusrubra L.), bur oak (Quercusmacrocarpa Michx.), and yellow birch seedlings were naturally cold hardened in a production polyhouse. To estimate frost tolerance, freezing tests were performed on whole seedlings (experiment 1) or on the upper 5 cm of the stem apex (experiment 2), and stem damage was visually estimated. Water content of the apex (5 cm) was expressed as the ratio of dry mass over fresh mass (DM/FM) for both experiments. Frost tolerance and DM/FM increased during the course of both experiments. In the growth-chamber experiment, SD seedlings hardened deeper than LD ones (−19.2 °C for SD versus −15.7 °C for LD), and DM/FM was higher for SD after 60 days of photoperiod treatment. In the polyhouse experiment, bur oak, yellow birch, and red oak reached a frost tolerance of −26.7, −25.1, and −20.4 °C, respectively, on November 2. The three species had DM/FM ratios of about 50% on November 2. For both experiments, DM/FM values higher than 45% corresponded to seedlings with a frost tolerance of −10 °C or lower.

Temperature control of the development of frost hardiness in two populations of Leptospermum scoparium.

Seedlings of Leptospermum scoparium J.R. et G. Forst (manuka) originating from seed from a low altitude coastal site (Auckland) and from a high altitude inland site (Desert Road) were grown for 96 days in four controlled environments to compare the relationship between growth temperature and frost hardening. Day/night temperature treatments were 12/6, 12/3, 12/0 and 12/-3 degrees C. Frost hardiness was determined at 14-day intervals by exposing whole seedlings to temperatures ranging from -2 to -8 degrees C. Frost damage differed significantly between the two populations: Desert Road seedlings were less affected than Auckland seedlings. At all growth temperatures, the time courses of frost hardiness of both populations followed curvilinear relationships reaching a maximum hardiness at about Day 50, after which the seedlings spontaneously dehardened. The rate of frost hardening increased linearly with decreasing temperature from 6 to 0 degrees C, but thereafter, no further increase occurred with decreasing temperature to -3 degrees C. The frost hardening process was more sensitive to temperature in the Desert Road seedlings than in the Auckland seedlings, and this difference may account for the intraspecific variation in frost hardening capacity of this species. Comparisons with Pinus radiata D. Don and Lolium perenne L. indicated that interspecific variation in frost hardening capacity can also be accounted for by differences in the sensitivity of the hardening process to temperature.

Photosynthesis in cold acclimated leaves of plants with various degrees of freezing tolerance

The objective of this study was to compare the photosynthetic changes during cold acclimation in various plant types able to acquire different degrees of freezing tolerance. Four herbaceous and six woody plants were hardened under natural or artificial conditions and – after determination of their frost resistance (LT50) – the net photosynthetic rate at an ambient CO2 of 33 Pa (Pn33), the dependencies of Pn to light and to CO2 and the room temperature chlorophyll a fluorescence were recorded under optimal conditions.Herbaceous plants acquired freezing tolerances to temperatures between −10 and −15°C when hardened at temperatures around 0°C. Most leaves fully developed prior to frost hardening exhibited typical symptoms of senescence after frost hardening. In non‐senescing leaves Pn33 was reduced by 15 to 50% mainly due to a reduced stomatal conductance. After hardening at temperatures around −10°C Brassica survived down to −24°C, but Pn33 was almost abolished as a result of disturbances in the chloroplasts. After transferring the plants to 20/15°C Pn33 recovered completely within a few days. Woody plants hardened at temperatures around 0°C tolerated – 15 to −36°C: Pn33 was reduced by 25 to 60% and hardly recovered at 20/15°C. Hardening at −10°C induced a tolerance of −32 to <−80°C. Pn33 was almost totally blocked, but at 20/15°C it returned to the values of the plants hardened at 0°C within a few days. In woody plants disturbances were invariably localized in the chloroplasts.Thus, conifers, and especially Pinus cembra, can survive much lower temperatures than herbaceous plants and, at the same level of freezing tolerance, exhibit appreciably less restriction in relative Pn33.

Seasonal changes in the utilization and turnover of assimilation products in 8-year-old Scots pine (Pinus sylvestris L.) trees

The present study aimed at a physiological understanding of the seasonal changes of the carbohydrate patterns and levels in the various tissues of 8-year-old Scots pine (Pinus sylvestris L.) trees growing under ambient climatic conditions in the botanical garden at Bayreuth. The photosynthates of selected twig sections were labelled by 14CO2 fixation and after chase periods of 1 h up to 8 months, the distribution of radiocarbon in the whole trees was determined and the labelling of identified carbohydrates was compared with the levels of these compounds in the individual tissues. Bud break and sprouting in spring is exclusively supplied by the recent photosynthates of the previous year's needles. During summer assimilates of the old needles were utilized for secondary growth of the axial system while growth of the recent-year's shoots was supported by their own photosynthesis. In autumn, soluble carbohydrates were produced instead of starch, a major part of which in addition to recent photosynthates was utilized for root growth during the cold season. Another part of the autumnal storage material was incorporated into the cell walls of the latest xylem and phloem elements still in winter. A pronounced starch-oligosaccharide interconversion upon frost hardening, and its reversal in spring as has been described for deciduous trees, could not be observed. This was due to maintenance of photosynthetic capability even in the cold season and the replacement of consumed storage material especially in late winter and early spring by new photosynthates.

Effects of freeze-thaw injury on parameters of distributed electrical circuits of stems and needles of Scots pine seedlings at different stages of acclimation

Frost hardening and frost injury of both the stems and needles of Scots pine (Pinus sylvestris L.) seedlings were studied by electrical impedance analysis. This impedance analysis was based on equivalent circuits with distributed circuit elements (DCE). A double-DCE model for stems and two single-DCE models for needles provided excellent fit to the experimental impedance data. However, the single-DCE model for needles which takes into account the asymmetry of the impedance arc proved more appropriate than the symmetric model. Several parameters changed in proportion to frost injury. In stems, extracellular resistance and one relaxation time decreased with increasing damage, whereas intracellular resistance remained relatively unchanged

Autumn frost hardening of one‐year‐old Pinus sylvestris (L.) seedlings: Effect of origin and parent trees

Greenhouse experiments were carried out to study the effects of geographical origin and parent trees on the autumn frost hardening of Pinus sylvestris (L.) seedlings after their first growing seaso...

The Effect of Cold and Sucrose Concentration on its Uptake and on the Respiration Rate of Isolated Embryos of Winter Wheat

AbstractBoth at 5°C and 20°C the sucrose concentrations between 8–14 % appear to be optimal from the point of view of dry mass accumulation by the seedling. The metabolic efficiency was also the highest for these concentrations of sucrose.At both temperatures the hydration of the seedlings was strongly dependent on sucrose concentration in the nutrient, diminishing, respectively, from about 6 to about 2 and from about 10 to about 2 mg H2O × mg DW−1 Such low values of the seedling hydration, however, caused only a 10–20 % decrease of their dry weight. Within the range of the applied sucrose concentration (2–20 %) its beneficial – trophic interaction prevails over the negative – osmotic interaction.At 5°C, as compared with 20°C, a drop in the seedlings hydration was observed, which appears to be the general response of plants to low temperature of growth.An analysis of the 24‐hour respiration rate of the seedlings when grown at 5°C seems to reveal the occurrence of a process requiring additional metabolic energy and, probably, associated with the vernalization process.A method of leveling the temperature dependent hydration of the seedling is proposed to enable the selection of high‐temperature control in the investigations of the biochemical mechanism of frost hardening and vernalization.

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens).

Effects of mineral nitrogen (2, 4, 6 and 8 mM NH4 NO3 ) and nodulation with Rhizobium on frost hardiness in seedlings of white clover (Trifolium repens) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.

Reduced sensitivity to photoinhibition following frost-hardening of winter rye is due to increased phosphate availability

The possibility of a role for phosphate metabolism in the photosynthetic regulation that occurs during frost hardening was investigated in winter rye (Secale cereale L. cv. Musketeer). Leaves of frost-hardened and non-hardened winter rye were studied during photosynthetic induction, and at steady state after being allowed to take up 20 mM orthophosphate through the transpiration stream for 3 h. At the growth irradiance (350 μmol·m-2·s-1) frost-hardening increased the stationary rate of CO2-dependent O2 evolution by 57% and 25% when measured at 5 and 20° C, respectively. Frosthardening also reduced the lag phase to stationary photosynthesis by 40% at 5° C and decreased the susceptibility of leaves to oscillations during induction and after interruption of the actinic beam during steady-state photosynthesis. These responses are all indicative of increased phosphate availability in frost-hardened leaves. As reported previously by Oquist and Huner (1993, Planta 189, 150–156), frost-hardening also decreased the reduction state of QA, the primary, stable quinone acceptor of PSII, and decreased the sensitivity of winter rye to photoinhibition of photosynthesis. Non-hardened rye leaves fed orthophosphate also showed an increased photosynthetic capacity (25% at 20° C and light saturation), lower reduction state of QA, a reduced sensitivity to photoinhibition and lower susceptibility to oscillations resulting from a brief interruption of the actinic light. Thus, the data indicate that phosphate metabolism plays a key role in photosynthetic acclimation of winter rye to low temperatures.

Effects of summer ozone on membrane lipid composition during subsequent frost hardening in Norway spruce [Picea abies (L.) Karst

Norway spruce trees [Picea abies (L.) Karst] were exposed to ozone during the summers of three consecutive years and the fatty acid composition of the chloroplast membrane lipid, monogalactosyl diglyceride (MCDG) was analysed over the last 14 months. Significant variations in the degree of unsaturation and the percentage of linolenic acid (18:3) were not found, either on a seasonal basis or in relation to ozone treatment. The proportion of octadecatetraenoic acid (18:4) and other Δ5 fatty acids increased during autumn in trees grown in filtered air, but this seasonal effect was less pronounced in ozone-treated trees. The ratio of two forms of linoleate, Δ5,9 18:2 and Δ9,12 18:2 also increased significantly during the frost hardening period but not in trees previously exposed to ozone. This implies that two separate biosynthetic pathways exist, one for the Δ12 and one for the Δ12 desaturated series, which operate at different relative rates, depending on environmental conditions. It is suggested that ozone may interfere with the biosynthesis of 18:4 during frost hardening by inhibiting the Δ5 desaturation of oleate.

Sulphate and ammonium in mist impair the frost hardening of red spruce seedlings

summaryTwo‐year‐old seedlings of red spruce [Picea rubens Sarg. syn. P. rubra (Du Roi) Link] were grown in open‐top chambers supplied with charcoal‐filtered air near Edinburgh, Scotland. Between May and November 1988, plants were exposed to mists containing NH4+, SO42− and NO3− ions at concentrations of 1.6 mol m−3 and H+ at 3.2 mol m−3, in pairwise combination and all together. The mists were applied twice weekly at a rate of 2 mm precipitation equivalent per application. Treatment with H2SO4 severely damaged the plants after 3 wk; the concentration for this treatment was therefore reduced from 1.6 mol m−3 to 0.5 mol m3, and new plants were substituted, in June 1988. Frost hardiness was assessed by freeze‐testing detached shoots, then measuring rates of electrolyte leakage. No effects of the mist treatments were detected during the early stages of frost hardening, but on 31 October, shoots that had received sulphate and ammonium ions were less hardy than ‘control’ shoots treated with deionized water. The temperature which killed 20% of shoots was 14 °C higher, and the temperature which killed 56% of shoots was 7 °C higher than for ‘control’ shoots. Acidity per se had no effect on frost hardiness; nitrate ions had no effect and may have mitigated the effects of sulphate when applied together.The results confirm earlier data, and indicate that uptake of NH4+ and SO42− ions can disturb the frost‐hardening process. There are important implications for pollution control strategies in the northeastern United States, if damaging interactions between frost hardiness of red spruce and pollutant deposition are linked to ammonium and sulphate rather than nitrate ions.

Autumn colouration of first year Pinus sylvestris seedlings during frost hardening

Autumn colouration, dry matter content and frost hardiness of Pinus sylvestris seedlings were monitored during their first autumn. Seedlings of southern (62° N, 24° E) and northern (67° N, 26° E) Finland origin were grown under two fertilization regimes in Suonenjoki (63°N, 27°E). Northern seedlings turned purple earlier than southern ones, and low fertilized earlier than normal fertilized. Northern seedlings also hardened earlier than southern ones, while fertilization had no effect on hardening. Neither purple autumn colour nor dry matter content was clearly related to frost hardiness of seedlings when treatment means were compared. Comparison of seedlings within treatments suggested, however, that purple seedlings had higher dry matter content than green seedlings at the beginning of hardening period but the relationship between colouring and frost hardiness was not clear.

Changes in ATP Level in Crowns of Winter Wheat Seedlings during Hardening to Frost

Summary During 8 days of frost hardening of winter wheat seedlings, the ATP level, as well as AEC and phosphorylation potential, increased in the crowns of frost resistant winter wheat cultivars, whereas these values remained unchanged or decreased in the susceptible cultivars.

Prediction of frost hardiness in seedlings of Scots pine (Pinussylvestris) using multivariate analysis of chlorophyll a fluorescence and luminescence kinetics

Seedlings of Scots pine (Pinussylvestris L.) were artificially frost hardened under controlled conditions. To follow the development of hardiness, the frost killing temperature was determined. Four levels of hardiness were defined during the course of frost hardening. After adaptation of the seedlings to dark conditions at room temperature, fluorescence induction and far red induced luminescence decay kinetics were recorded for seedlings of the four defined levels of hardiness. The induction curve of fluorescence and the decay curve of luminescence were digitized for 110 time intervals, and the values were used in a multivariate partial least squares analysis to evaluate the potential of luminescence and fluorescence as predictive probes for cold hardiness. The results showed that both fluorescence induction and luminescence decay kinetics contain information allowing the prediction of frost hardiness of artificially cold-hardened seedlings of Scots pine.

Photoperiod control of the initial phase of frost hardiness development in Pinus radiata

Abstract. The development of frost hardiness in Pinus radiata was investigated to establish whether there is quantitative relationship between photoperiod and the hardening process. Three controlled environment experiments were carried out. In the first, seedlings were exposed to a photoperiod that shortened from 13 h at a rate of 3 min d−1 to 9.5 h. At intervals, the photoperiod was either held constant or lengthened. In the second experiment, seedlings were exposed to one of five constant photoperiods between 9 and 12 h for up to 90 d. In the third, seedlings were exposed to photoperiods shortening at rates of 1 or 5 min d−1. Frost hardiness was also measured during the natural photoperiod‐controlled stage of hardening in outdoor‐grown seedlings. Frost hardiness developed at a constant rate in response to a shortening photoperiod once it had declined to about 12 h. This rate was consistent with the hardening rate that occurred in outdoor‐grown seedlings. Hardening stopped when the photoperiod became constant, indicating a tight coupling between changes in photoperiod and hardiness development. When the photoperiod was held constant, the extent of frost hardiness was directly dependent on the photoperiod but the rate of hardening was apparently independent of the length of photoperiod. However, the rate of hardening was dependent on the rate at which the photoperiod shortened, increasing linearly with increases in the rate of change in photoperiod between 0 and 3 min d−1. These results suggest shortening photoperiods control the first stage of the hardening process by regulating the rate of hardening. Frost hardening was inherently unstable once the maximum hardiness was reached since spontaneous dehardening occurred in spite of the controlled conditions. Dehardening also occurred when the photoperiod was lengthened suggesting that the cue for dehardening to commence was the shift from shortening to lengthening photoperiods.

Light and temperature dependent inhibition of photosynthesis in frost-hardened and un-hardened seedlings of pine

Needles of un-hardened and frost-hardended seedlings of Pinus sylvestris and Pinus contorta were exposed to photoinhibitory photon flux densities at temperatures between 0 and 35°C under laboratory conditions. Photoinhibition of photosynthesis was assayed by measuring oxygen evolution under saturating CO2 in a leaf disc oxygen electrode or by recording of photosystem II fluorescence induction kinetics at 77 K. It was demonstrated that frost hardening of pine did not affect the susceptibility of photosynthesis to short time (2 h) photoinhibition at 15°C. The two pine species irrespective of acclimative state were equally sensitive to photoinhibition as assayed by apparent photon yield analyses of photosynthetic oxygen evolution. Plots of the apparent photon yield of oxygen evolution vs. F v /F m revealed a non-linear relationship.In the temperature range of 15-20°C short term photoinhibition caused a loss of F v without effect on F 0 . However, photoinhibition at temperatures lower or higher caused F 0 to increase and decrease, respectively. In fact the decrease of F v v /F upon lowering the temperature was mainly caused by the temperature effect on F 0 . Besides photoinhibition causing the well established quenching of F v by increased radiationless decay somewhere in the reaction center-antenna complex, it is suggested that F 0 generally increases as a result of loss of functional reaction centers causing decreased trapping of excitation energy. However, the high temperature induced quenching of F 0 suggests that the quenching process (or processes) induced under photoinhibitory conditions is temperature dependent; i.e. it increases with the increase of temperature.In pine the photon yield of photosynthesis was much more sensitive to short term photoinhibition than was the rate of light saturated photosynthesis. This difference is explained by photosystem II and electron transport having surplus capacity relative to that of reductive carbon metabolism.

Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh

Exogenously applied abscisic acid (ABA) induced frost hardening of Arabidopsis thaliana (L.) Heynh. The freezing tolerance of A. thaliana plantlets treated with ABA (15 mg/l) at a non-acclimating temperature (20 °C) appeared to increase even more rapidly than following a low temperature (4 °C) acclimation. Analysis of in vivo-labelled soluble proteins by two-dimensional gel electrophoresis revealed several low temperature - or ABA - induced proteins, which where not produced in non-acclimated plants. A subset of these proteins was induced by both low temperature and ABA treatments, suggesting that they might be directly involved in the frost hardening process in A. thaliana.