Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.Abbreviation CF1 peripheral part of chloroplast coupling factor ATPase     

Phytochrome action and frost hardening in black spruce seedlings

Black spruce [ Picea mariana (Mill.) B.S.P.] development is sensitive to photoperiod. To date the implication of photoperiod, and especially phytochrome, in the frost hardening process of black spruce has not been fully tested. Two light fluence rates, night interruption of darkness, and red vs far-red radiation treatments were applied to black spruce seedlings, followed by freezing at –6°C. Parallel to the freezing test, growth measurements, bud formation and the xylem water potential estimates of the seedlings were done. While dry weight accumulation depends on the irradiation energy level, bud formation and freezing tolerance are photoperiodically sensitive. Furthermore, bud formation and frost hardening are dependent upon whether phytochrome is in the active form or inactive form, as demonstrated by the positive effect of short days, far-red radiation and the reversal of the red effect by far-red radiation. Also, xylem water potential appears to be influenced by short day and far-red conditioning.     

Assessment of the frost sensitivity of Trifolium species by chlorophyll fluorescence analysis

The potential of the chlorophyll fluorescence technique in screening for frost sensitivity in a range of Trifolium species from different geographical origins was assessed by measuring the decrease in variable chlorophyll fluorescence (F_var) of leaves after freezing at - 5°C for 60 min. The method was rapid and the results obtained agreed well with a visual assessment of freezing injury carried out after leaves were returned to optimal growth conditions for 72 h. Trifolium alexandrinum (Berseem clover) cv. Tabor originating from Israel was shown to be the most frost sensitive species studied and Trifolium subterraneum (subterranean clover) cv. Mt. Barker, from temperate regions of Australia, the most frost resistant. On extended periods of freezing, frost damage increased and this was associated with a further reduction in variable chlorophyll fluorescence and in quenching capacity of the thylakoid membranes. These results thus indicate that substantial thylakoid membrane dysfunction is induced at freezing temperatures. Furthermore, it was found that frost hardening of the frost sensitive species T. alexandrinum for 21 days at 5°C reduced the extent of damage sustained by the thylakoid membranes as shown by higher fluorescence quenching capacity, smaller reduction in variable fluorescence (F_var) and higher initial fluorescence (F_o) when leaves of hardened plants were frozen at -5°C and -7°C.     

Simulation of in situ freezing damage of the photosynthetic apparatus by freezing in vitro of thylakoids suspended in complex media

Chloroplast thylakoid membranes were isolated from leaves of unhardened and cold-acclimated spinach (Spinacia oleracea L.). For freezethaw treatment, the membranes were suspended in complex media composed to simulate the solute concentrations in the chloroplast stroma in the unhardened and hardened states of the leaves. In particular, high concentrations of amino acids were applied for simulating the hardened state. After frost treatment, photosynthetic activities and chlorophyll fluorescence parameters of the thylakoids were tested to determine the degree of freezing damage. The results revealed a pattern of freezing injury similar to that observed upon frost treatment of thylakoids in situ. A major manifestation of damage was the inhibition of photosynthetic electron transport. Uncoupling of photophosphorylation, which is the dominating effect of freezing of thylakoids suspended in binary solutions (e.g., containing one sugar and one inorganic salt), was also visible but less pronounced in the complex media. Thylakoids obtained from cold-acclimated leaves did not exhibit an increased frost tolerance in vitro, as compared with thylakoids from unhardened plants. The results, furthermore, indicated a strong protective effect of free amino acids at the concentrations and composition found in chloroplasts of hardened leaves. The presence of inorganic salts in the complex media slightly stabilized rather than damaged the membranes during freezing. It is concluded that inactivation of thylakoids in situ may be understood as the destabilizing action of the combined solutes surrounding the thylakoids, occurring when solute concentration is raised due to freezing of water.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Hepes 4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid - PSI photosystem I - PSII photosystem II     

Effects of frost hardening, dehardening and freezing stress on in vivo chlorophyll fluorescence of seedlings of Scots pine (Pinus sylvestris L.)

Abstract. The kinetics of in vivo chlorophyll fluorescence of photosystem II (PS II) was measured at room temperature and 77 K during frost hardening of seedlings of Scots pine (Pinus sylvestris L.), and after exposure of frost-hardened shoots to sub-freezing temperatures. A more pronounced decrease in variable fluorescence yield for the upper exposed than for the lower shaded surface of the needles suggested that some photoinhibition occurred during prolonged frost hardening at 50 μmol photons m^?2 s^?1 and 4°C. Reversible inhibition of photosynthesis after exposure to sub-freezing temperatures was initially manifested as an increase of steady-state energy-dependent fluorescence quenching (q_E) and a reduction in the rate of O₂ evolution. Further inhibition after treatment at still lower temperatures caused a progressive decline of steady-state photochemical quenching (q_Q) and the rate of O₂ evolution, whereas q_E remained high. This implies an inactivation of enzymes in the photosynthetic carbon reduction cycle decreasing the consumption of ATP and NADPH, which is likely to cause an increase of membrane energization and a reduction of the primary electron acceptor (Q_A) of PS II. Alternatively, the changes in q_Q and q_E might be attributed to an inhibition of photophosphorylation. Severe, irreversible damage to photosynthesis resulted in a suppression of q_E and of variable fluorescence yield, probably because the photochemical efficiency of PS II was impaired. Changes in the fast fluorescence kinetics at room temperature after severe freezing damage were interpreted as an inhibition of the electron flow from Q_A to the plastoquinone pool. It is suggested that irreversible freezing injury to needles of frost-hardened P. sylvestris causes damage to the Q_B,-protein.     

The relation between growth cessation and frost hardening in Scots pines of different origins

The cessation of shoot elongation, diameter growth and needle elongation were compared with the initiation of frost hardening of the stems and needles in an 8-year-old provenance trial of Scots pine (Pinus sylvestris L.) established in central Finland. The saplings were of six different origins ranging from Estonia to northern Finland, forming a latitudinal gradient of ca. 10°N. The frost hardiness of the stems of current-year shoots was assessed by electrical impedance analysis and that of current-year needles by electrolyte leakage and visual scoring of damage. Artificial freezing tests were used in the assessments. The pattern of growth cessation (shoot and needle elongation, diameter growth) tended to follow the latitude of origin, i.e. growth ceased in the northernmost provenance first and in the southernmost one last. Both stems and needles of the northern provenances hardened earlier than the southern ones, but the differences in hardiness disappeared as hardening progressed. Growth cessation and initial hardening to -15°C were clearly correlated at the provenance level, indicating that growth must cease prior to hardening, and that earlier cessation of growth predicts earlier frost hardening of stems and needles. No differences in frost hardiness of stems were found at the provenance level at the end of the growing period in August. At that time, the frost hardiness of needles of the northernmost provenance was higher than that of other origins. Within the provenance, the stems were less hardy than the needles.     

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 (P_n33), the dependencies of P_n 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 P_n33 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 P_n33 was almost abolished as a result of disturbances in the chloroplasts. After transferring the plants to 20/15°C P_n33 recovered completely within a few days. Woody plants hardened at temperatures around 0°C tolerated – 15 to ?36°C: P_n33 was reduced by 25 to 60% and hardly recovered at 20/15°C. Hardening at ?10°C induced a tolerance of ?32 to n33 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 P_n33.     

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO₂-dependent O₂ evolution (representing net photosynthetic CO₂ fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.Abbreviations Chl chlorphyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Hepes 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PS I photosystem I - PS II photosystem II     

Differences in frost hardiness of two Norway spruce morphotypes growing at Mt. Brocken, Germany

Norway spruce (Picea abies (L.) Karst.) exhibits strong ecotypic variation along altitudinal gradients in morphological traits, e.g. slenderness of crowns or arrangement of second-order branches. We were interested whether montane and lowland morphotypes differ in a key trait for the survival in cold environments, i.e. frost hardiness, and asked: (i) are montane morphotypes more resistant to frost damage and (ii) do they have a lower risk of frost damage by late frosts in spring than lowland morphotypes?We used the electrolyte leakage-method to measure frost hardiness on a monthly basis from October 2006 to May 2007 in stands of the montane and lowland morphotypes at Mt. Brocken in the Harz Mountains, Germany.LT₅₀ (i.e. the temperature that results in 50% of maximum electrolyte leakage) was assessed by freezing treatments in a frost chamber and was significantly influenced by morphotype, month and minimum ambient temperatures. LT₅₀ was significantly lower in the montane than in the lowland morphotype, with −107 °C and −49 °C, respectively. However, the interactions between morphotype with minimum ambient temperature or month were not significant. Thus, as frost hardiness of the two morphotypes responded to temperature in the same way, both morphotypes can be supposed to be exposed to the same risk of frost damage during hardening in autumn and dehardening in spring.     

Cold acclimation was partially impaired in boron deficient Norway spruce seedlings

Susceptibility of trees to freezing injury has been suggested to increase in boron (B) deficiency but there is no experimental evidence to support this proposition. In this study, Norway spruce (Picea abies L. Karst.) seedlings were cultivated for two growing seasons in deficient, intermediate and ‘optimal’ B levels. Cold hardening of the seedlings was measured after the second growing season. Freezing tolerance in tips of shoots, needles, stems and roots was determined by controlled freezing tests and electrolyte leakage method, and that of buds, in addition, by differential thermal analysis (DTA). Electrical impedance was used to monitor changes in the apoplastic space during cold acclimation. Root dry weight and shoot height growth were lower in B deficiency. Cold acclimation of buds and stems was reduced by B deficiency. When hardened seedlings were subjected to subzero temperatures for 3 weeks, extracellular electrical resistance of stems became the highest at the lowest B supply which was probably due to decreased desiccation tolerance. As a conclusion, susceptibility to freezing damage may be increased by B deficiency in Norway spruce trees.     

Seasonal and Experimentally Induced Changes in the Ultrastructure of Chloroplasts of Pinus silvestris

The ultrastructure of chloroplasts in mesophyll cells of Pinus silvesris was examined under the electron microscope. Secondary needles were regularly sampled from a tree in a natural stand for one year. Primary needles from one-year-old seedlings exposed to frost hardening and dehardening conditions in a controlled environment chamber were also studied. These seedlings were exposed to 8 or 55 W m^-2. All needles were put in fixative at the different sampling dates and stored in a refrigerator until they were prepared for electron microscopy at the end of the experimental period. During the summer the choroplasts were symmetrically shaped and heavily loaded with starch. The membrane systems were well developed and consisted of both grana and stroma thylakoids. In autumn and during early artificial frost hardening the starch content was reduced, the chloroplasts appeared amoeboid and membrane-free stroma regions were seen. Later the chloroplasts became swollen and aggregated in one part of the cell. Starch was lost and the chloroplasts aggregated earlier at 8 W m^-2 than at 55 W m^-2. During winter the stroma thylakoids were first reduced in number and later even the grana thylakoids were damaged, resulting in mostly disorganized single membranes. Also the chloroplast envelope disappeared. In spring and early summer the chloroplasts migrated to the proximity of the cell walls. The membrane systems were reorganized and starch accumulated. During the first days of artificial dehardening the photosynthetic membranes were severely damaged, especially at 55 W m^-2, but soon new membranes were formed. Starch accumulated earlier at 55 than at 8 W m^-2. The reported ultrastructural variations are discussed in relation to functional and biochemical fluctuations caused by the season or by artificial variations in the climate as demonstrated earlier.     

Anthocyanins and glutathione S-transferase activities in response to low temperature and frost hardening in Vaccinium myrtillus (L.)

Anthocyanin (Acy) contents and GST activities of bilberry (Vaccinium myrtillus L.) were investigated in two experiments conducted in June (Exp. I: active growth) and in August September (Exp. II: beginning of frost hardening) in Northern Finland (65 degrees N). Bilberry plants were subjected to +2 degrees C and +18 degrees C in Exp. I or +5/0 degrees C (day/night) and +18/+13 degrees C (day/night) in Exp. II. GST activities were assessed using either 1-chloro-2,4-dinitrobenzene (CDNB) or trans-cinnamic acid (tCA) as substrates. We found temperature to have no effect on Acy during either active growth or frost hardening. Acy increased several-fold from active growth to the beginning of frost hardening, but no increment was observed during the development of frost hardening. This suggests a role of Acy in photoprotection at low temperatures rather than their direct involvement in the development of freezing tolerance. The lack of response of GST activity to frost hardening and to temperature in autumn may indicate an indirect role of GSTs in frost hardening as protective enzymes. GST activity was the same with the two substrates studied (CDNB, tCA), supporting the assumption that GSTs could catalyze reactions with endogenous phenylpropanoids.     

Intracellular resistance correlates with initial stage of frost hardening in willow (Salix viminalis)

Distributed model parameters of shoots of five clones of willow ( Salix viminalis ) were examined with electrical impedance analysis at the end of the growing season and with cold acclimation. The parameters were compared with regard to frost hardiness, linoleic (18:2) and linolenic (18:3) fatty acids, unsaturation/saturation ratio of fatty acids and dry weight content. The intracellular resistance (r_i) correlated best with changes in frost hardiness. The value of r_i rose from 1–2 Ω m in non-hardened to about 12 Ω m in hardened samples. In the initial stages of frost hardening, a linear relationship was found between r_i and frost hardiness and levels of 18:2 fatty acid, and an inverse relationship between r_i and levels of 18:3 fatty acid. The unsaturation/saturation ratio of fatty acids rose fairly rapidly in the initial stage of hardening. The dry weight content increased stepwise during the experimental period, and less steadily than r_i. In addition, equivalent circuit parameters changed in the prehardening phase, and were probably connected with cell differentiation and lignification. Frost hardiness by the visual method and by extracellular resistance, determined after controlled freezing tests, correlated well in the initial stages of hardening until about − 10°C but deviated upon further hardening.     

The relationship between cell injury and osmotic volume reduction: III. Freezing injury and frost resistance in winter wheat

In order to distinguish between several possible mechanisms of frost hardening in winter wheat (Triticum aestivum L.) cells from two hardy and two tender cultivars were plasmolyzed in CaCl₂ solution at room temperature and cell volumes estimated by microscopic examination. Analyses of Boyle-van't Hoff plots of these data reveal that all cells from cultivars progressively increase their intracellular solute concentration up to 20 days hardening. This increase, which we had predicted from published calorimetric data to be the sole mechanism of hardening explained less than half of the increase in hardening seen in the most hardy cultivar, Kharkov. Hardening also increased the osmotically inactive volume.At CaCl₂ concentrations greater than 5%, plasmolyzed protoplasts departed further from the Boyle-van't Hoff prediction, remaining larger than expected until some higher concentration of CaCl₂, where protoplast volume again sharply decreased. In all cultivars except hardened Kharkov, the concentration of CaCl₂ producing this abrupt volume decrease had a freezing point corresponding to the killing temperature. If this concentration was exceeded during plasmolysis, then the protoplasts burst during deplasmolysis at some volume less than their original volume.We interpret these data to mean that, in addition to the often described hardening mechanism of increased cell solute and water binding, winter wheat shows a third mechanism, a mechanical resistance to protoplast shrinkage which produces volumes larger than those predicted during osmotic stress. The resisting element appears to be the plasma membrane itself. Shrinkage brings the membrane under compressive stress, developing tangential pressure within it. Cell injury occurs when the cell membrane area has been reduced to the point at which irreversible loss of membrane material is inevitable. Cell death occurs during deplasmolysis when the protoplast bursts because its membrane contains insufficient material to subtend the area of the cell wall.Of the cultivars tested, hardened Kharkov was unique in avoiding injury. Hardened Kharkov was injured only after the volume inflection had been greatly exceeded. Refractile droplets of lipid appeared in the cytoplasm of hardened Kharkov protoplasts during plasmolysis but disappeared during deplasmolysis suggesting that hardy Kharkov was able reversibly to store membrane lipids in cytoplasmic vesicles and return them to the membrane on deplasmolysis.     

Effects of frost hardening on the composition of galactolipids and phospholipids occurring during isolation of chloroplast thylakoids from needles of scots pine

Frost hardening of seedlings of Scots pine (Pinus sylvestris) at a non-freezing temperature of 4°C resulted in a 2-fold increase of the acyl lipids of the needles. This was because of increases in phospholipids and triglycerides. The galactolipid content of the needles was almost the same in unhardened and frost-hardened seedlings. In unhardened seedlings the mol ratio of monogalactosyl diacylglycerol (MGDG) to digalactosyl diacylglycerol (DGDG) was 1.7 ± 0.3 and 0.9 ± 0.2 in needles and isolated thylakoids, respectively. Corresponding ratios for frost-hardened seedlings were 1.5 ± 0.2 and 0.3 ± 0.03. The lower ratios found in isolated thylakoids, particularly in thylakoids from frost-hardened seedlings, are suggested to depend on the enzyme galactolipid: galactolipid galactosyltransferase being active during the isolation procedure. This is deduced from the result that the content of MGDG decreased and that of DGDG and 1.2 diglycerides increased. Needles of Scots pine also contain phospholipidase D. This enzyme was active during thylakoid preparation, particularly after frost hardening, as judged from the large amount of phosphatidic acid found the in thylakoid fraction isolated from frost-hardening needles. The fatty acid composition of the acyl lipids showed no major changes due to hardening at non-freezing temperature.     

Changes in free amino acid content and frost resistance in Nothofagus dombeyi leaves

The annual course of frost resistance and free proline content was studied in leaves at different stages of development of a woody species (Nothofagus dombeyi) from Southern Chile. The freezing resistance reached a minimum in late spring or summer and a maximum in the autumn-winter period. Adult and juvenile trees showed a similar degree of resistance; meanwhile, cold resistance was maximum at the seedling stage. Free proline levels and frost resistance in leaves changed throughout the seasonal cycle, increasing in winter and decreasing in summer. Artificial hardening caused changes in amino acid content of leaves; while valine, proline, lysine, histidine, serine and alanine increased upon hardening, aspartic acid, glutamic acid and arginine decreased. The nature of cold-induced metabolic adjustments is discussed as well as its ecological significance.     

Freezing injury in cold-acclimated and unhardened spinach leaves

Spinach plants (Spinacia oleracea L.) were frost-hardened by cold-acclimation to 1° C or kept in an unhardy state at 20°/14° C in phytotrons. Detached leaves were exposed to temperatures below 0°C. Rates of photosynthetic CO₂ uptake by the leaves, recorded after frost treatment, served as a measure of freezing injury. Thylakoid membranes were isolated from frost-injured leaves and their photosynthetic activities tested. Ice formation occurred at about-4° to-5° C, both in unhardened and cold-acclimated leaves. After thawing, unhardened leaves appeared severely damaged when they had been exposed to-5° to-8° C. Acclimated leaves were damaged by freezing at temperatures between-10° to-14° C. The pattern of freezing damage was complex and appeared to be identical in hardened and unhardened leaves: 1. Inactivation of photosynthesis and respiration of the leaves occurred almost simultaneously. 2. When the leaves were partly damaged, the rates of photosynthetic electron transport and noncyclic photophosphorylation and the extent of light-induced H⁺ uptake by the isolated thylakoids were lowered at about the same degree. The dark decay of the proton gradient was, however, not stimulated, indicating that the permeability of the membrane to-ward protons and metal cations had not increased. 3. As shown by partial reactions of the electron transport system, freezing of leaves predominantly inhibited the oxygen evolution, but photosystem II and photosystem I-dependent electron transport were also impaired. 4. Damage of the chloroplast envelope was indicated by a decline in the percentage of intact chloroplasts found in preparations from injured leaves. The results are discussed in relation to earlier studies on freezing damage of thylakoid membranes occurring in vitro.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenol indophenol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES 2(N-morpholino) ethane sulfonic acid     

Carbohydrate distribution and cellular injuries in acid rain and cold-treated spruce needles

Summary The cellular structures of acid rain-irrigated needles of several provenances of Norway spruce (Picea abies L. Karst) seedlings were studied after winter experimental freezing. Frost injuries and recovery were characterized by visual damage scoring and classification of mesophyll cell alterations, also using histochemical methods for carbohydrate fluorescent staining. The treatment with-30° C during the late dormancy period was sufficient to cause significant injuries and intracellular degradation in the tissues of the green needles. The most affected seedlings in terms of visual injury scoring were found among those treated with clean water or at pH 3, while freezing injury, defined as an occlusion of phenolic substances in the central vacuole of the mesophyll cells, was most abundant in the needles from spruces irrigated either with clean water or at pH 4 or pH 3. Electron microscopy revealed the details of the injury, e. g. thinning out of the cytoplasm and chloroplast stroma, darkening of the chloroplasts and eventually swelling of the chloroplasts and protoplast. PAS and ConA reactions in the needle tissue revealed intense starch accumulation in the mesophyll and transfusion tissues as early as in March, with a tendency to increase, especially in the untreated needles during the recovery period. Plasma membrane disturbances were indicated by histochemical identification of callose deposits in the mesophyll cell walls, these being most abundant in the acid rain-treated needles. All these findings suggest that freezing at –30° C was more deleterious to the seedlings pretreated with acid or clean water than to those not given additional irrigation.     

Assessing frost hardiness of Pinus bungeana shoots and needles by electrical impedance spectroscopy with and without freezing tests

Aims Nursery and forest operations require that frost hardiness results be produced faster than can be provided by controlled freezing tests. There is a great challenge to develop a rapid method for predicting frost hardiness that might not necessitate controlled freezing tests. The aim of this study was to examine the assessment of the frost hardiness of shoots and needles of Pinus bungeana by electrical impedance spectroscopy (EIS) with and without controlled exposure to freezing.Methods The frost hardiness of current-year shoots and needles of P. bungeana in an 8-year-old provenance field trial was measured at Shisanlin Nursery in Beijing, China, from September 2006 to January 2007 by means of EIS and conventional electrolyte leakage (EL). In the same plants, but without controlled freezing test, were monitored the EIS parameters in current-year shoots and needles.Important findings The results showed that (i) after controlled freezing tests, the frost hardiness estimated by EIS parameters (extracellular resistance, r e, and membrane time constant, τ m) was significantly correlated with the frost hardiness assessed by EL method (r = 0.95) and (ii) for the samples not exposed to controlled freezing treatment, the relaxation time τ 1 for shoots and β for needles had greater correlations with the frost hardiness estimated by EL after controlled freezing tests relative to the other parameters (r = ?0.90 for shoots and r = 0.84 for needles, respectively). The parameters r e of shoots and needles and τ m of needles might be applied for measuring frost hardiness of samples after exposed to controlled freezing tests. The frost hardiness results can be obtained within 48 h. The parameters τ 1 of shoots and β of needles could be used for estimating the frost hardiness of samples without using a controlled freezing test. The frost hardiness results can be obtained within 24 h.     

Does availability of potassium affect cold hardening of Scots pine through polyamine metabolism?

The effect of different potassium availability on the polyamines and frost resistance of Scots pine seedlings ( Pinus sylvestris L.) during cold hardening was studied. Scots pine seedlings were grown applying different rates of potassium by using the relative addition rate technique followed by a 2- or 9-week hardening period with decreased light intensity, day length and temperature. After 2 weeks of treatment the seedlings were not hardened (LT₅₀, =−11°C) and showed no differences in frost resistance, although differences in the polyamine levels between the K levels were observed. After 9 weeks of hardening the seedlings at the low, medium and high K levels showed a mean frost resistance (LTs.₅₀) of −81, −63, and −47°C, respectively. A negative effect of K on the frost resistance of the needles was also found in adult trees in September. The results indicate that at the early stage of cold hardening, potassium or free polyamine levels do not affect the frost resistance of Scots pine needles. However, in hardened seedlings and adult trees potassium displays a negative and putrescine a positive correlation with frost resistance, whereas spermidine and spermine show no correlation.