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.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

Effect of polythene covers, a flower inducing treatment, on the content of endogenous gibberellin-like substances in grafts of Norway spruce

In shoots of 12-year-old Norway spruce [Picea abies (L.) Karst.] grafts the content of endogenous gibberellin-like substances was analysed. Covering the crowns with polythene on the turn of June and July stimulated induction of male strobiles and significantly increased the levels of gibberellins. Polar gibberellin (at R_f 0.0–0.2) increased immediately and the less polar ones after one day. After 2 weeks the increased level was maintained only at R _f 0.4–0.6 and 9 days later the differences between covered and not covered grafts disappeared. Removal of the covers did not affect gibberellin levels. It is suggested that covering with polythene stimulates floral induction through an increase in endogenous gibberellin levels.     

Variable growth and reproduction response of the spruce shoot aphid, Cinara pilicornis, to increasing ozone concentrations

Spruce shoot aphid, Cinara pilicornis Hartig (Homoptera: Lachnidae), is an aphid species that has shown enhanced performance on trees exposed to SO₂ or mixtures of air pollutants, whereas results with ozone have been contradictory. Using a 4-week chamber fumigation experiment, we tested how different population dynamic parameters of aphids are affected by ozone (O₃). Mean relative growth rate (MRGR), development time and reproduction rate of C. pilicornis were determined using 7h day-time O₃ concentrations of 0, 40, 80 and 160 ppb. Development period from birth to reproductive stage was fastest at 80 ppb during early shoot elongation, while reproduction and intrinsic rate of population increase was not significantly affected by ozone concentration. There was a significant negative correlation between ozone concentration and the MRGR of first instar nymphs in the third fumigation week. In feeding test performed on seedlings after fumigation, the MRGR was reduced on shoots which had been exposed to 80 ppb O₃. The results suggest that elevated O₃ concentration during early shoot elongation period may stimulate population development of C. pilicornis, but on maturing shoots, high O₃ concentration has a negative effect on aphid performance. This might be due to accelerated ageing of O₃ exposed shoots.     

Extraorgan freezing in wintering flower buds of Cornus officinalis Sieb. et Zucc.

Abstract. Extraorgan freezing as a mechanism for increasing cold hardiness was shown using flower buds of Cornus officinalis Sieb. et Zucc. Differential thermal analysis (DTA) revealed that florets in flower buds of C. officinalis owed their cold hardiness to deep supercooling and also that slower cooling rates increased the supercooling ability of florets. During slow stepwise cooling (5°C h⁻¹), the water content of florets decreased and that of scales (involucral bracts) increased, which resulted in accumulation of ice within the scales. This was more extensive in early winter and early spring buds than mid-winter ones. Flower buds with silicone oil in the space between florets and scales also showed a similar decrease in water content of florets and an increase in that of scales. This indicated that water migration from the florets to the scales probably took place by way of the peduncles and the receptacle, possibly through their vascular traces, and not directly from the surface of the florets to the ice sink in the form of vapour. Possible mechanisms of extraorgan freezing are postulated along with this finding.     

Effect of rapid freezing and thawing on cellular integrity of honey bee sperm

Abstract. Direct observations on the effect of rapid freezing and thawing on honey bee ( Apis mellifera L.) sperm were made by light, scanning and transmission electron microscopy. Rapid freezing of honey bee ejaculated sperm, suspended in freezing diluent, in liquid nitrogen followed by rapid thawing can cause cellular injuries which lead to the death of the sperm. The frozen-thawed sperm, supravitally stained, showed a significant decrease in cell viability compared with that of the control fresh sperm ( P <0.001). Significant uptake of the stain in the dead sperm resulted from damage in the cell membrane. The scanning electron micrographs of frozen-thawed sperm further demonstrated that the injury of cell membrane can lead to the splitting of mitochondrial derivatives from the flagellar axoneme. More cellular injuries including the release of acrosomal content and membrane damage at the acrosome, nucleus and the tail regions were further revealed by transmission electron microscopy. The impact of cellular injuries on the quality of honey bee sperm cryopreserved for artificial insemination of honey bee queens is discussed.     

冻融作用对冻土区微生物生理和生态的影响

不同时空尺度的冻融过程导致冻土温度变化及水的相变和迁移,改变着微生物生境的物理和化学性质。冻融过程可改变细胞内外渗透压平衡,且冰晶生长过程能损伤细胞膜和细胞器。限于营养、氧气等其它极端不利条件,不少细胞逐渐转入休眠状态以度过难关;微生物DNA、蛋白质的合成和能量代谢仅用于维持细胞生存。冻融作用通过改变细胞代谢模式而影响微生物参与的寒区碳氮元素的生物地球化学循环。多年冻土层保存了不同地质时期微生物种群的多样性,作为物理和生物地球化学屏障,可有效削弱地表过程和地壳本底辐射对微生物的影响。在长期的适应过程中,微生物发展了相应的耐受机制,从结构和功能方面,细胞和分子水平上应对冻土环境和冻融过程。这可为寻找地外寒冷星球上可能的生命形式提供一些线索。     

Effects of nutrition and soil warming on stemwood production in a boreal Norway spruce stand

The boreal forest is expected to experience the greatest warming of all forest biomes. The extent of the boreal forest, the large amount of carbon contained in the soil, and the expected climate warming, make the boreal forest a key biome to understand and represent correctly in global carbon models. It has been suggested that an increase in temperature could stimulate the release of CO₂ caused by an increased decomposition rate, more than biomass production, which could convert current carbon sinks into carbon sources. Most boreal forests are currently carbon sinks, but it is unclear for how long in the future the carbon sink capacity of the boreal forest is likely to be maintained. The impact of soil warming on stem volume growth was studied during 6 years, in irrigated (I) and irrigated‐fertilized (IL) stands of 40‐year‐old Norway spruce in Northern Sweden. From May to October heating cables were used to maintain the soil temperature on heated‐irrigated plots (Ih and ILh) 5 °C above that on unheated control plots (Ic and ILc). After six seasons' warming, stem volume production (m³ ha^?1 a^?1) was 115% higher on Ih than on unheated (Ic) plots, and on heated and irrigated‐fertilized plots (ILh) it was 57% higher than on unheated plots (ILc). The results indicate that in a future warmer climate, an increased availability of nitrogen, combined with a longer growing season, may increase biomass production substantially, on both low‐ and high‐fertility sites. It is, however, too early to decide whether the observed responses are transitory or long lasting. It is therefore crucial to gain a better understanding of the responses of boreal forest ecosystems to climate change, and to provide data to test and validate models used in predicting the impact of climate change.     

The role of direct chilling injury and inoculative freezing in cold tolerance of Amblyomma americanum, Dermacentor variabilis and Ixodes scapularis

Abstract. Supercooling points and chill tolerance were compared among nymphs and adults of the ixodid ticks Dermacentor variabilis, Amblyomma americanum and Ixodes scapularis (Acari: Ixodidae).Supercooling points in the range of <-22 to -18°C were observed for nymphs, and -22 to -8°C for adults.The lower lethal temperatures observed under dry conditions, -14 to -10°C, were warmer than the supercooling points, but still much colder than -4.8°C, the lowest temperature recorded from a likely tick habitat in southwestern Ohio.Based on our experiments, spontaneous freezing and direct chilling injury are not significant mortality factors in these species in the field.Mortality was observed between -5 and -3°C for A.americanum and D.variabilis nymphs chilled for 2 h while in direct contact with ice.This mortality is probably due to inoculative freezing.Given the requirement for a rather humid microhabitat for off-host survival, these findings suggest that inoculative freezing is an important cause of overwintering mortality in these medically important species.     

Photoinhibition in the Antarctic moss Grimmia antarctici Card when exposed to cycles of freezing and thawing

Freezing and thawing of the endemic moss species Grimmia antarctici Card, caused photoinhibition. When snow cover was removed from moss in the field, resulting in exposure to fluctuating temperatures and light conditions, photoinhibition, measured as a reduction in the ratio of variable to maximum chlorophyll a fluorescence (F_v/F_m), was observed. The extent of photoinhibition was highly variable and appeared to be reversible during periods of warmer temperatures. A series of controlled laboratory studies found that the light conditions that prevail between freezing and thawing events influenced the recovery from photoinhibition observed during freezing and thawing, with low light conditions facilitating the greatest rates of recovery. After four cycles of freezing and thawing, recovery from photoinhibition in hydrated moss was achieved within 12 h of transfer to 5°C and 15 μmol quanta m^?2 s^?1. These results favour the hypothesis that photoinhibition observed during freezing represents a protective process involving the down-regulation of photo-system II when photosynthetic carbon assimilation is limited by low temperatures.     

Relations between cytokinin level, bud development and apical control in Norway spruce, Picea abies

In conifers such as Norway spruce, the extent of shoot growth is predetermined by the size and number of embryonal organs of the buds laid down the previous year. As it is known that cytokinins have a key role in bud development a possible hypothesis is that the level of cytokinin in the buds during their formation determines their size and complexity. As a first step to test this hypothesis we compared cytokinin levels in buds of different size of annual shoots from 15- to 20-year-old trees of Picea abies (L.) Karst. Apical buds from the leaders, and from branches in lower parts of the trees, were collected in April, July and August. The difference in size of the buds and the shoots growing from them was considerable in these three positions. Extracts were purified by immunoaffinity columns, and the retained compounds were separated by high-performance liquid chromatography (HPLC). Quantification was made by enzyme-linked immunosorbent assay (ELISA), and the accuracy of this method was checked by measurements with liquid chromatography-mass spectrometry (LC-MS) and UV absorption. Zeatin riboside (ZR) was the most abundant cytokinin, but isopentenyladenosine (iPA) was also present in all samples. The large apical bud of the leader contained much higher cytokinin concentrations than the considerably smaller buds from lower positions, and during the period of secondary growth in July, similar relationships were found for annual stem tissue from different positions. The possible role of ZR as a controlling factor in bud development and apical control is discussed. Our conclusion is that the level of zeatin-type cytokinins appears to play an important role in the establishment of differences in bud size and, thereby, the architecture of the tree crown.     

Influence of the wood ant, Formica polyctena, on soil nutrient and the spruce tree growth

The effect of Formica polyctena ant nests on the distribution of soil nutrients, soil pH and the growth of Norway spruce trees was studied in the southern part of the Czech Republic. Soil nutrient content (exchangeable P, N, K and pH) and growth of mature spruce trees were measured at four distances from the nearest ant hill (0–1, 3–5, 10–50 and >200 m). Trees at all distances were visited by ants, except for those >200 m from the nearest nest. Soil pH and of P, K and NO₃ concentrations were higher near ant nests (<1 m), and pH and K at distances of 3–5 m, when compared with distances of 10–50 and >200 m from the nests, where no significant differences in these variables were detected. In contrast, tree ring analyses (1974–2004) showed that trees >200 m from the ant nests grew significantly faster than trees at other distances, followed by trees within 1 m of the nests. No growth differences were found between the growth of trees at 3–5 and 10–50 m from ant nests. We postulate that nutrient and carbohydrate removal of honeydew collected by ant‐tended aphids are slowing growth of tree. However, trees may partly compensate for this depletion by having access to a larger supply of soil nutrients near ant nests.     

Relationship between respiratory depletion of sugars and loss of cold hardiness in coniferous seedlings over-wintering at raised temperatures: indications of different sensitivities of spruce and pine

Long-term effects of elevated winter temperatures on cold hardiness were investigated for Norway spruce (Picea abies L. Karst.), lodgepole pine (Pinus contorta Dougl.) and Scots pine (Pinus sylvestris L.). Two-year-old seedlings with the same pre-history of growth and cold hardening in the field were maintained from early December to late March at two field sites in northern Sweden and in a cold room. The temperatures at these locations averaged –13·5, –8·9 and 5·5°C, respectively. Following treatments, carbohydrate contents and cold tolerances were assessed. Needle respiration was also analysed during the 5·5°C treatment. Cold tolerance of lodgepole pine and Scots pine was much reduced following the 5·5°C treatment. Cold tolerance was somewhat reduced in lodgepole pine following the –8·9 °C treatment, but was essentially maintained in spruce throughout all treatments. The cold tolerance of needles was strongly correlated with their soluble sugar contents. Spruce maintained cold hardiness by having larger reserves of sugars and lower rates of respiration which decreased more rapidly as sugars were depleted. Tolerance of lodgepole pine to frost desiccation was also much reduced following the 5·5°C treatment.     

The effect of nutrition on the seasonal course of needle respiration in Norway spruce stands

 Respiration of 1-year-old needles of 30-year-old Norway spruce trees [Picea abies (L.) Karst.] was studied in a nutrient optimisation experiment in northern Sweden. Respiration rates of detached needles, from ten control (C) and ten irrigated-fertilised (IL) trees, were measured on 16 occasions from June 1992 to June 1993. The aim of the study was to determine the influence of temperature on the seasonal course of needle maintenance respiration, and the effect of nitrogen concentration [N] and carbohydrate content on needle respiration in young Norway spruce trees subjected to long-term fertilisation. The IL treatment significantly affected needle size, in terms of dry mass and length, but not specific needle length (SNL). There was, however, a strong tree-specific effect on SNL (P<10^–9, R ² = 0.75). Needle starch content varied markedly with season (0–25% of total dry mass). This, unless accounted for, would cause erroneous estimates of nutrient concentrations, and of rates of needle respiration, within and between treatments. There was considerable seasonal variation in needle respiration, both in terms of maintenance respiration and temperature dependence (Q₁₀). Q₁₀ had its highest value (2.8) during winter and its lowest (2.0) in the middle of summer. In early autumn (August, September), respiration rate and needle [N] were significantly related (C: P = 0.001, IL: P<0.0005). There was no significant difference in the slope between the two regression lines, but a difference in intercept. At the same needle [N], needles from IL-plots always had a lower respiration rate than needles from control plots. No obvious explanation for the observed difference in intercept was found, but some plausible assumptions are put forward and discussed. Received: 24 January 1997 / Accepted: 1 July 1997     

Effect of local heating and cooling on cambial activity and cell differentiation in the stem of Norway spruce (Picea abies)

BACKGROUND AND AIMS The effect of heating and cooling on cambial activity and cell differentiation in part of the stem of Norway spruce (Picea abies) was investigated. METHODS: A heating experiment (23-25 degrees C) was carried out in spring, before normal reactivation of the cambium, and cooling (9-11 degrees C) at the height of cambial activity in summer. The cambium, xylem and phloem were investigated by means of light- and transmission electron microscopy and UV-microspectrophotometry in tissues sampled from living trees. KEY RESULTS: Localized heating for 10 d initiated cambial divisions on the phloem side and after 20 d also on the xylem side. In a control tree, regular cambial activity started after 30 d. In the heat-treated sample, up to 15 earlywood cells undergoing differentiation were found to be present. The response of the cambium to stem cooling was less pronounced, and no anatomical differences were detected between the control and cool-treated samples after 10 or 20 d. After 30 d, latewood started to form in the sample exposed to cooling. In addition, almost no radially expanding tracheids were observed and the cambium consisted of only five layers of cells. Low temperatures reduced cambial activity, as indicated by the decreased proportion of latewood. On the phloem side, no alterations were observed among cool-treated and non-treated samples. CONCLUSIONS: Heating and cooling can influence cambial activity and cell differentiation in Norway spruce. However, at the ultrastructural and topochemical levels, no changes were observed in the pattern of secondary cell-wall formation and lignification or in lignin structure, respectively.     

The effect of freezing nights on photosynthesis, stomatal conductance, and internal CO2 concentration in seedlings of Engelmann spruce (Picea engelmannii Parry)

Abstract The effect of freezing night temperatures on net photosynthesis, stomatal conductance, and internal CO₂ concentration was investigated in unhardened seedlings of Engelmann spruce. Exposure to – 2.5°C in the dark for 10 h caused a slight and reversible reduction in gas-exchange parameters on the following days. Substantial and irreversible inhibition of photosynthesis occurred after exposure to -4°C or –5°C. Despite a parallel decline in stomatal conductance and net photosynthesis, exposure to a hard freeze caused a decrease in the stomatal limitation to gas exchange. Hard-freeze conditions (less than – 4°C) also caused a decrease in carboxylation efficiency and apparent quantum yield, indicating a freeze-induced failure of the dark reactions and electron transport. There was no significant difference in the photosynthetic response to freezing temperatures in different elevational populations of spruce, although acclimatory adjustments were observed. Gas exchange in seedlings grown under cool conditions (14°C day/9°C night) was less affected and recovered more rapidly after exposure to a hard freeze than in seedlings grown under warm conditions (24°C day/19°C night).     

The effects of SO2 and O3 on the foliar nutrition of Scots pine, Norway spruce and Sitka spruce in the Liphook open-air fumigation experiment

Foliar elements were analysed in Scots pine, Sitka spruce and Norway spruce over a 6 year period before and during continuous exposure to SO₂ and O₃ in an open-air fumigation experiment. Sulphur dioxide treatment elevated foliar sulphur concentration in all species, and there were increases in foliar nitrogen in the two spruce species but not in pine. The concentrations of cations were frequently increased by SO₂ treatment, but there was no correlation between the sulphur concentration of needles and their total cation charge. SO₂-related elevations of foliar magnesium were correlated with the concentration of this element in soil solution, but the mechanism by which other cations were enhanced remains unclear. The only consistent effects on nutrient ratios were for SO₂ treatments to increase sulphur/cation ratios.     

Microbeam analysis of Ca exchange and uptake in the fine roots of spruce: influence of pH and aluminum

Summary A novel stable isotope labelling procedure for microbeam analysis was developed to monitor exchange and uptake of nutrients, primarily Mg, K and Ca, by root tips at the cellular level. Initially root samples were analysed from 2-year-old spruce trees, originating both from a nursery and from a polluted forest site, (1) for the cortex cell wall accessibility and nutrient binding properties, (2) for the influence of low pH and elevated aluminum concentrations on Ca binding to cortex cell walls, and (3) for long-range transport into the secondary xylem, proximal to the labelled root tip. In nursery control plants, Ca is localized mainly in the apoplast of the cortex. Exchange of Mg, K, Ca in the cell wall of the cortex and the primary xylem with label in incubation solutions is almost completed to equilibration within 30 min. In the secondary xylem we could detect Mg, K, and Ca from labelling solutions in minute amounts after 30 min, and as a major fraction after 48 h. This indicates that stable isotope labelling can be used to study both ion-exchange properties of the apoplast and long-range transport. Slight acidification of the labelling incubation media to pH 4.5 reduced Ca binding to the cortex cell walls slightly, but acidification to the extreme value of pH 2.3 reduced binding 41%. A combination of pH 4.5 and increased free aluminum reduced the binding by 83%. In a preliminary attempt to analyse the nutrient binding capability of the root-tip apoplast from pollution affected trees, we exposed fine roots of 2-year-old spruce from an acidified and polluted site showing typical low levels of Ca and Mg in the cortical cell walls to Ca-enriched media. Under these conditions the Ca content of cortex cell walls doubled upon incubation at pH 4.7, reaching 40% of the total binding capacity of our nursey control plants.     

Effect of exposure to fluoride,nitrogen compounds and SO2 on the numbers of spruce shoot aphids on Norway spruce seedlings

Summary Development of spruce shoot aphid (Cinara pilicornis Hartig) populations was monitored in natural and artificial infestations of Norway spruce (Picea abies Karst.) seedlings, exposed to air pollutants in an experimental field. The pollutants, applied both singly and in mixtures, were gaseous sulphur dioxide, NaF (30 mg l^-1 F) and Ca(NO₃)₂ or (NH₄)₂SO₄ in aqueous solutions (200 mg l^-1 N). Aphid numbers on 10 seedlings in each treatment and two control plots were counted at 2-week intervals. At the beginning of the experiment aphid numbers did not differ between treatments. Aphid populations peaked in late June and early July. All the pollutants and their combinations significantly increased the numbers of aphids per seedling. Four apterous females were transferred to spruce seedlings which were growing in containers in the same plots. After 4–5 weeks aphid numbers were significantly higher in the fluoride treatment and in the combined treatment of fluoride, nitrogen and SO₂. The pollution treatments did not have a significant effect on shoot growth. Concentrations of F and S in needles were higher in treatments involving these pollutants. There were no significant differences in the concentrations of free amino acids in shoot stems between control and fluoride treatment. However, the relatively low concentration of arginine in the F treatment at the end of the growing season might indicate disturbances in the nitrogen metabolism of spruce seedlings.