Seasonal variations in the properties of ribosome assemblies from vegetative buds of Scots pine

The amount of total ribosome assemblies extractable from the vegetative buds of 2 m high Scots pine ( Pinus sylvestrís L.) plants remained more or less constant throughout the sampling period from September to May. The stability of the ribosomes, the shape of the polysome profiles obtained after sucrose density gradient centrifugation and the clustering of material as seen in the scanning electron micrographs suggested the presence of storage formations during the winter.
All samples of isolated ribosomes were able to synthesize proteins in vitro. During midwinter the translation capacity, when calculated on a ribosome unit basis, was about one third of that found in September and May. This reflects not only the occurrence of storage formation during the winter, but also the amount of initiated translation processes at any given time. The decrease in the in vitro translation capacity in the autumn ceases around the end of November. Ribosome activity starts to increase as early as the end of January or beginning of February. It seems that the reactions are triggered either by an endogenous clock or by the change in the daylength.     

Variation in the polysome assembly and incorporation of [3H]-uridine in the cells of pine buds during the cold season

The ribosome assemblies isolated from buds of Scots pine ( Pinus sylvestris L.) containing microsporangiate strobili varied both quantitatively and qualitatively in samples collected from October to April. The seasonal fluctuation in the amount of ribosonnes was more evident in the cytosolic fraction than in the smaller membrane-bound fraction. The profiles obtained after sucrose density gradient centrifugation were of two types. One type was commonly obtained from samples collected late in the autumn and early in the spring, and this type was characterized by a relatively high peak for the large subunits, a low or negligible peak for the dimers, and an even or ascending series of peaks for the polymers. The other type was obtained from samples collected during the winter, and was characterized by small peaks for both subunits, a moderate to large peak for the dimers and a descending series of peaks for the polymers. However, the scanning electron microscope investigations indicated that the winter-time samples did not lack polysomes and clusters of ribosomes. They did not become visible in the polysome profiles because they pelleted too tightly at the bottom of the centrifuge tubes to be removed with gradient fractionation. The au-toradiographic analyses suggested that the cells were capable of synthesizing mRNA throughout the winter, whereas rRNA synthesis was arrested. On the basis of the above results, we postulate that the synthesis of the enzyme proteins needed for the maintenance of winter-time metabolism takes place in the cytosolic ribosome fraction. The possible existence of winter-time polysome stores is also pointed out.     

Use of the scanning electron microscope in the study of isssolated ribosomes and polysomes

A procedure is described by which the ribosome assemblies isolated from the buds of Scots pine ( Pinus sylvestris L.) as well as the ribosome and polysome fractions purified by sucrose density gradient centrifagation can be prepared for study by scanning electron microscopy. The technique has also been used to illustrate ribosomes extracted froai leaves and roots of Nicotiana tabacum L., from roots of Tuiipa gesneriana hybr. and from commercial wheat germ. The sample under study is spread on a piece of coverslip', frozen and dried at room temperature. The coverslip, with attached material, is taken through the critical point drying procedure, glued on a specimen retainer aod coated with gold-palladium. The coverslip serves as a sufficiently stable specimen support to allow the study of ribosomes at magnifications of several hundred thousand. Use of the method makes it possible to study the structure of polysomes, to check the purity of fractioned samples and to investigate differences in the ribosome assembly of the developing plant tissues.     

In vitro translation capacity of ribosomes isolated from the cells of pine buds during the cold season

The cytosolic and membrane-bound ribosome assemblies were isolated from the microsporangiate strobili of Scots pine ( Pinus sylvestris L.). Both assemblies studied were able to incorporate [³H]-leucine into trichloroacetic acid-insoluble material throughout the sampling period from October to April, indicating that the isolation procedure employed preserves the ability of the ribosomes to synthesize protein. Samples of identical size, taken from cytosolic as well as from membrane-bound ribosomes isolated from the same source, behaved similarly in vitro: they were both active, their activity was arrested by ribonnuclease and, as shown by aurintricarboxylic acid treatment, most of their mRNA molecules were complexed with the ribosomes. Variation in translation capacities seemed to depend on the season and physiological stage of the bud tissue.     

Metabolic and structural responses of pine buds under climate chamber conditions

Scots pine ( Pinus sylvetris L.) plants, about 2 m high, were placed in controlled conditions for 2 weeks in January, April and November. During the experiments made in January, the conditions in the climate chambers simulated either a gradual or abrupt advancement of spring. In April they simulated either the advancement of the season or its reversal back to January. In November the plants were transferred to conditions that resembled spring. In January, pieces of buds collected at the end of the experiment were also fixed for electron microscope studies.
Isolation of the ribosomes and the determination of their in vitro translation capacity revealed that in January the response to environmental changes was evident. An increase in synthesized proteins was caused by a rise in the translation capacity of ribosome assemblies rather than by an increase in their quantity. The cellular ultras-structure changed in conformity with the changes characteristic of the spring. In April, the plants transferred to the climate chambers maintained their ability to synthesize proteins, but the buds were judged to be under stress. In November die ability of the buds to respond to environmental changes was retarded or inhibited.     

Morphology and ultrastructure of isolated gemmae of Drosera pygmaea and their in vitro germination

Gemma morphology, histology and ultrastructure before and after germination in vitro were studied in Drosera pygmaea. The histology of the gemma is similar to that of a seed, being characterized by an embryo-like structure and storage tissue, although no seed coat is formed. One embryo-like structure within the gemma, which gives origin to a new plant, expresses polar organisation with distinct meristematic regions. Storage tissue surrounding the embryo-like structure resembles endosperm and it is built of parenchyma cells possessing plastids with starch grains and dense material within vacuoles. The regeneration from the gemma may provide useful system to study plant morphogenesis under stress conditions including in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dependence of alpha-synuclein aggregate morphology on solution conditions

Alpha-synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from alpha-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type alpha-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl(2) were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalenesulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating alpha-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions approximately 100 microm) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2M NaCl or 10mM MgCl(2) to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of alpha-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions.     

Inhibitors of endogenous proteinases in the seeds of Scots pine, Pinus sylvestris

Extracts of resting pine seeds inhibited the proteinase activities present in extracts of endosperms of germinating seeds (hydrolysis of haemoglobin at pH 3.7 and hydrolysis of casein at pH 5.4 and 7.0). Heating the extracts of resting seeds at 60°C destroyed their own proteinase activity but their proteinase inhibitor activity decreased by only 25 to 30%. Some properties of the inhibitor(s) were studied using extracts treated at 60°C. The inhibitor activities were non-dialysable. the inhibition increased linearly with increasing inhibitor concentration up to 80% of total proteinase activity, and the maximal inhibition was 80% at pH 3.7. 90% at pH 5.4. and 97% at pH 7.0. The extracts of resting seeds did not inhibit the pepsin-like acid pine proteinase that accounts for a minor part of the proteolytic activity of endosperm extracts at pH 3.7. Neither did they have any effect on the acid pine carboxypeptidase or trypsin and chymotrypsin. Fresh extracts of endosperms of germinating seeds contained relatively high proteinase activity (assayed directly) and moderate inhibitor activity (assayed after treatment at 60°C). When fresh extracts were dialysed at 50°C for 48 h their proteinase activities increased considerably while the corresponding inhibitor activities disappeared. It is concluded that the decrease of inhibitors during dialysis is due to enzymatic inactivation and that the corresponding increase of proteinase activities is at least partly due to the destruction of the inhibitors.     

Identification of the photosynthetic CO2 fixation inhibitors in isolated pine chloroplasts as resin acids

Thus far all attempts to isolate CO, fixing chloroplasts from pine have failed. In this paper it is proposed that resin acids present in pine needles partition into membranes during chloroplast isolation and interfere with specific reactions of the Calvin cycle. CO, fixation by isolated spinach chloroplasts was strongly inhibited by the introduction of a suspension of chloroplasts isolated from Pinus sylvestris L. A partially purified organic extract obtained from chloroplasts of this pine species also strongly inhibited CO, fixation by the spinach chloroplasts. The major inhibitory compounds from the organic extract were identified as a mixture of resin acids by gas-liquid chromatography and mass spectrometry. Two resin acids, abietic acid and dehydroabietic acid, were tested for inhibitory activity. Both resin acids were potent inhibitors of photosynthetic CO₂fixation, with dehydroabietic acid being about three times more potent than abietic acid.     

Ribulose bisphosphate carboxylase, protein and nitrogen in Scots pine seedlings cultivated at different nutrient levels

Seedlings of Scots pine (Pinus sylvestris L.) of a northern provenance were cultivated in nutrient solution for 10 weeks in a climate chamber. The nutrient solution (renewed by solution exchange) contained 2.5, 10 or 50 mg N I^?1. All other essential elements were added in optimal proportion to the nitrogen. Seedlings cultivated at 10 and 50 mg N I^?1 were similar with respect to all characteristics studied. Seedlings cultivated at 2.5 mg N I^?1 showed a lower growth rate, especially for the shoot, and an altered morphology, with high root:shoot ratios and long, slender roots. The nitrogen concentrations in shoot and needles as well as in whole seedlings were not significantly affected by the nitrogen supply, while the nitrogen concentrations in the roots were somewhat lower at 2.5 mg N I^?1. Ribulose bisphosphate carboxylase (EC 4.1.1.39) activity and the concentrations of carboxylase, total and soluble protein and of chlorophyll in the needles were consistently much lower for seedlings cultivated at 2.5 mg N I^?1, than for seedlings grown at higher nutrient levels. A close correlation was observed between activity and concentration of the carboxylase (r=0.95). Carboxylase activity and protein were more sensitive to a low nutrient supply than was chlorophyll. The data show how activity and concentration of ribulose bisphosphate carboxylase and the concentrations of soluble and total protein and of chlorophyll in needles of pine seedlings can be negatively affected by the nutrient supply, also when the nitrogen concentrations in the needles are close to those observed at optimal nutrient supply. It is suggested that pine seedlings store assimilated non-protein nitrogen in the needles when protein synthesis is under restraint. The nitrogen concentration in needles and seedlings could not be used as a measure of the physiological state of the seedlings.     

Primers designed to amplify a mitochondrial nad1 intron in ponderosa pine, Pinus ponderosa, limber pine, P. flexilis, and Scots pine, P. sylvestris

The b/c intron of the mitochondrial nad1 gene, was sequenced to characterize the indel region of ponderosa pine, Pinus ponderosa. The sequence in ponderosa pine was aligned with the sequence in Scots pine, Pinus sylvestris, to design seven primers that are useful for sequencing and for revealing size variation in amplified fragments in ponderosa pine, Scots pine, and limber pine, Pinus flexilis. These primers reveal variability in all three species, and the pattern of variability within ponderosa pine is described by a preliminary survey. The indel region of ponderosa pine contains three distinct elements with lengths of 31, 32, and 34 bp. Received: 1 March 2000 / Accepted: 14 April 2000<@head-com-p1a.lf>Communicated by P.M.A. Tigerstedt     

Direct plant regeneration from leaf explants of Drosera rotundifolia cultured in vitro

Shoot regeneration was obtained from isolated leaves of Drosera rotundifolia L. cultured on MS media with various concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). The best direct shoot organogenesis was obtained on growth regulator-free medium or medium supplemented with 10^-8 M NAA. Liquid culture medium significantly increased regeneration capacity of leaf tissue. Histological and scanning electron microscopy investigations verify direct plant regeneration without intermediate callus formation. Leaf epidermal cells showed the highest regeneration potential leading to the regeneration of buds. Young shoots with three to seven leaflets rooted spontaneously on the growth regulator-free medium within 38 days of culture and isolated mature plants produced fertile seeds.Abbreviations BA 6-benzyladenine - FAA 40% formalin (5%) +90% acetic acid (5%) +70% ethanol (90%) - ME Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid - plumbagin 5-hydroxy-2-methyl-1,4-naphthoquinone - 7-methyljuglone 7-methyl-5-hydroxy-1,4-naphthoquinone - SEM scanning electron microscopy - TEM transmission electron microscopy - PPF photosynthetic photon flux     

Interaction between flight,reproductive development and oviposition in the pine weevil Hylobius abietis

• 1 The development of reproductive and flight capacity of pine weevils Hylobius abietis during the spring and their dispersal to, and subsequent development at, new clearfell oviposition sites comprise key phases in their life cycle in managed forests. At an old clearfell site where autumn‐emerging weevils had overwintered, weevils were trapped as they re‐emerged in the spring and tested for their ability to fly and then dissected to determine the degree of wing muscle and egg development.
• 2 Re‐emerging weevils were most abundant in pine growing at the edge of the clearfell and, over most of the trapping period (April to June), their capacity for flight (proportion flying and wing muscle width) was more advanced than in weevils from the clearfell itself, with a similar trend in the degree of reproductive development (proportion with mature eggs and egg volume).
• 3 In weevils from the clearfell, flight capacity and reproductive development increased concurrently to a peak around mid‐May. In weevils from pine, wing muscles were already well developed at the start of trapping, although few of them flew. Their more advanced development was attributed to the increased opportunities for maturation feeding after emergence in the previous autumn.
• 4 In the spring, weevils reached the canopy of trees for maturation feeding by walking and, to a lesser extent, by flight. Weevils dispersed by flight to oviposition sites in mid‐May when most of them were reproductively mature. After arrival, flight ability and wing muscle size declined rapidly but egg production was maintained until most weevils had stopped flying. When wing muscles reached their minimum size, there was a marked decline in egg size, suggesting that wing muscle breakdown is important in maintaining egg production at oviposition sites. Prospects for further wing muscle and reproductive development are discussed.

     

Light microscopic structure of needles of Scots pine (Pinus sylvestris L.) in relation to air pollution and needle element concentrations in S.E. Finland and the Karelian Isthmus, N.W. Russia

 The mesophyll structure of current and second year needles of mature Scots pines (Pinus sylvestris L.), growing on a survey line from the Karelian Isthmus (N.W. Leningrad region, Russia) to S.E. Finland, was studied at the light microscopic level in 1992 and 1994. The area is affected by sulphur dioxide and nitrogen oxide emissions from Russia (200  000–285 000 ton/year SO₂ and about 100  000 ton/year NO_X), Estonia (140  000–320  000 ton/year SO₂ and 30  000–50  000 ton/year NO_X) and Finland (6000–9000 ton/year SO₂ and 23 000–25 000 ton/year NO_X) and by unknown amounts of neutralizing compounds from Estonia. Mean concentrations of SO₂ were between 1.0 and 1.5 μg m^–3 and those of ozone between 52 and 61 μg m^–3 during growing seasons of 1991–1994. The concentrations tended to be lower in 1993/94 than in 1991/92. The results showed cellular changes related to exposure to sulphur dioxide, ozone and acid wet deposition. The percentage of cells with injuries related to acid deposition was higher in 1994 than in 1992, possibly due to higher reduction of neutralizing than of acidifying compounds in the main emission areas, while the percentage of cells with injuries related to sulphur dioxide correlated positively with needle sulphur concentrations and showed a declining trend between Karelian Isthmus and S.E. Finland in the needles sampled in 1992. These changes and those related to ozone were considerably less numerous in 1994 than in 1992, possibly on account of the lower concentrations of SO₂ and ozone in 1993/94 than in 1991/92, especially in early summer, the time of the most sensitive phase of needle growth. In addition, periods of lower wet deposition in summer 1992, and considerably lower temperatures in autumn/winter 1992 than in 1994 may have enhanced the injuries in the needles collected in October/December 1992, relative to those collected in 1994. The results of the microscopic work agreed well with the simultaneous chemical measurements, pointing to the usefulness of cellular analysis for diagnostic work in the field. Received: 26 September 1997 / Accepted: 24 November 1997     

樟子松人工固沙林稳定性的研究

提出了人工林稳定性的内涵及其评价标准．分析了影响沙地樟子松人工林稳定性的各种干扰因子,通过对沙地樟子松人工林造林成活、生长情况、对不良外界环境的抗御能力、林分寿命、林分结构和生产力的综合评价分析,认为沙地樟子松引种人工林基本上是稳定的;而生长在立地较好的丘间低地的林分,其稳定性大于沙丘顶部的林分;从抗枯梢病角度看,幼林的抗性大于中林;从林分密度和生产力角度来看．红花尔基沙地樟子松林稳定性高于章古台．     

Monitoring the European pine sawfly with pheromone traps in maturing Scots pine stands

Abstract

• 1 During 1989–93, field studies were conducted in Finland to develop a method based on pheromone traps to monitor and forecast population levels of the European pine sawfly (Neodiprion sertifer Geoffr.) and tree defoliation.
• 2 Three traps per site were baited with 100 µg of the N. sertifer sex pheromone, the acetate ester of (2S,3S,7S)‐3,7‐dimethyl‐2‐pentadecanol (diprionol), in maturing pine stands in southern and central Finland. In addition, three different dosages (1, 10 and 100 µg) of the pheromone were tested in 1991–92.
• 3 The highest number of males was observed in traps baited with the highest dose. On average, there was a 10‐fold increase in trap catch between lure doses.
• 4 Density of overwintering eggs was used to evaluate the effectiveness of pheromone traps in predicting sawfly populations. The proportion of healthy overwintering eggs was determined each year. A model based on the number of current shoots on sample trees, diameter at breast height and tree height was formulated to estimate eggs per hectare.
• 5 Linear regression analysis produced high coefficients of determination between number of males in traps and density of total eggs in the subsequent generation, when populations were at peak densities. The relationships were not significant for low population densities. The results indicate a risk of moderate defoliation when the seasonal trap catch is 800–1000 males per trap or higher.

     

Radial growth of Scots pine in urban and rural populations of Ekaterinburg megalopolis

Eleven stands of Scots pine (Pinus sylvestris L.) from the city of Ekaterinburg and its surroundings were sampled and analyzed using dendrochronological methods to detect the effects of climate, biotic and anthropogenic factors on the annual growth of trees. Tree-ring chronologies were developed for six sites within the city and for five control sites. All chronologies were highly and positively correlated before the 1940s. However, after this period, there was a significant decrease in the correlation among chronologies from urban and rural sites. Divergence lasted about 20 years. This firstly has an anthropogenic cause, mainly due to the evacuation in 1941 of more than 60 industrial factories to Sverdlovsk (now Ekaterinburg), which generated a significant increase in air pollution. Environmental pollution seems to negatively affect tree growth. In the early 1950s, trees in the region also suffered from severe droughts. The results of climate and historical data analysis suggest that the trees on urban sites were weakened by both climate and air pollution factors, which led to a massive nun moth (Lymantria monacha L.) infestation of trees. Defoliation led to a drastic reduction in tree-ring width and, in some cases, to the complete loss of annual rings. The recovery period lasted 10–15 years on average. Rural populations were much less affected by the insect outbreak. After urban populations of pine recovered in the 1960s, radial growth of urban and rural populations became synchronized again.     

Flight ability and reproductive development in newly‐emerged pine weevil Hylobius abietis and the potential effects of climate change

• 1 Adult pine weevils Hylobius abietis emerge from conifer root‐stumps, on which larvae develop, over an extended period during summer and autumn. Newly‐emerged weevils were tested for their ability to fly and assessed for wing muscle and reproductive development. In addition, the effect of summer–autumn maturation feeding on reproductive development was assessed in field bioassays.
• 2 There was considerable variation in development between newly‐emerged weevils that was related to the timing of emergence. The first weevils, emerging in early July, weighed less than later‐emerging ones, had undeveloped flight muscles and did not fly. Over the emergence period, wing muscle size and flight ability increased markedly, with 50–60% flying by mid‐September. Differences between emerging adults are likely to have been affected by temporal changes in the quality of the bark on which the larvae feed.
• 3 Reproductive development lagged behind that of wing muscles but, in early August, there was a rapid increase in the proportion of weevils with immature eggs and a corresponding increase in oocyte size. However, although wing muscles were fully formed in later‐emerging weevils, immature eggs were only approximately 10% of the volume of mature eggs.
• 4 In field bioassays of summer–autumn maturation feeding, eggs continued to develop and some weevils laid mature eggs. Feeding and development during the pre‐overwinter period is likely to influence winter survival and also dispersal and reproduction in the following spring.
• 5 The potential effects of climate change on the weevil life cycle are briefly discussed. Weevils are likely to benefit from the higher temperatures and later autumns predicted under climate change, resulting in an increase in damage to transplants.

     

Gas exchange in a 20-year-old stand of Scots pine

The rates of net photosynthesis and transpiration of one-year-old shoots were measured in situ in five different positions within the crown of a young Scots pine ( Pinus sylvestris L.). Measurements were carried out on south- and north-facing shoots on the third and sixth whorls, respectively, and on an east-facing shoot on the ninth whorl. In another investigation the rates of gas exchange of one-year-old shoots on the third whorl of eight different trees were studied. The measurements were made during June and July, 1977, under non-limiting conditions of soil water. The daily rates of net photosynthesis in whorls three and six followed the light conditions closely, with higher rates for the south side of each whorl and higher for whorl three than six. On whorl nine the shoot had a higher light compensation point and a low rate of photosynthesis at light saturation compared to the other shoot positions. The quantum yield for the shoot on the lowest whorl, as estimated from the linear part of the light response curve, was 50% lower than for shoots on whorl three and six.
The variation in transpiration rates was pronounced within the crown as an effect of differences in the absolute value and diurnal course of stomatal conductance. The variation in net photosynthesis was small between different trees while the variation in transpiration was much higher. Thus the variation in water use efficiency was great. It is concluded that it is possible to extrapolate measurements of net photosynthesis from individual trees up to a stand level without introducing large errors in the estimate. More caution must be paid before extrapolating tree transpiration up to stand transpiration. However, before an extrapolation of gas exchange is made from tree to stand level the variation in net photosynthesis and transpiration rate within the crown must be known.