Free amino acid and protein levels,and γ‐glutamyltransferase activity in Pinus sylvestris apical buds and shoots during the growing season

Free amino acid and protein levels, and γ‐glutamyltransferase activity in apical buds and shoots of Scots pine during the growing season. The aim of the study was to obtain the basic information about nitrogen mobilization needed in carrying out studies on the optimal nitrate and ammonium ratios in the metabolism of Scots pine (Pinus sylvestris L.). Considerable seasonal changes in the concentrations of free amino acids and other ninhydrin‐positive low molecular‐weight compounds were observed in the buds and shoots of Scots pine. 43 different amino compounds were identified, the concentrations of arginine, glutamine, glutamic acid, γ‐aminobutyric acid, alanine and aspartic acid being highest at the break of dormancy. The amounts of certain amino compounds decreased during the growing season, those of arginine, ethanolamine and various ammonium compounds in particular. The amount of glutamic and aspartic acids, glycine, alanine and γ‐aminobutyric acid, however, remained relatively constant. The protein concentration and the specific activity of γ‐glutamyltransferase increased towards the end of the growing period. Slight differences were found between the fertilized trees and the control trees.     

Seasonal variations in location and population structure of endophytes in buds of Scots pine

We studied the location and distribution of a bacterial isolate, a Mycobacterium sp., in buds of Scots pine (Pinus sylvestris L.). Using a probe specific for the 16S rRNA of the Mycobacterium sp., the bacterium was found by in situ hybridization in the meristematic tissues of 40% of all bud samples examined. Because we had previously found other bacterial and fungal endophytes in the meristematic tissues of Scots pine buds, we studied their occurrence in buds during shoot development and dormancy. Using probes targeted to the 16S or 18S rRNA of the endophytes Mycobacterium sp., Methylobacterium spp., Pseudomonas spp. and Rhodotorula minuta, endophytes were found in association with growing tissues, with Methylobacterium spp. being the dominant species. Endophytes were detected in abundance before elongation or differentiation of a bud, but once a tissue was fully developed, endophytes were no longer detected. Metabolic activity of the endophytes was suppressed at the onset of, and during, dormancy of Scots pine, but recovered before the following growing season.     

Preliminary Studies on Establishment of Genetic Transformation System in Loblolly Pine

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Responses of Pinus sylvestris and Picea abies Seedlings to Limited Phosphorus Fertilization and Treatment with Elevated Ozone Concentrations

Scots pine ( Pinus sylvestris L.) and Norway spruce [ Picea abies (L.) Karst.] seedlings were exposed to high phosphorus (HP) or low phosphorus (LP) availability for one growing season in the open field, and to combined P availability and elevated ozone (O 3 ) concentrations (0, 55, 110 and 210 ppb for Scots pine and 0, 40, 75 and 150 ppb for Norway spruce, respectively) for 28 days in controlled laboratory chambers. Compared with HP, the LP treatment reduced Scots pine current-year (C) shoot and root dry masses and Norway spruce total dry mass, whereas the highest O 3 concentrations increased the magnesium concentration of Scots pine C needles and P concentrations of the C needles of both tree species. Chlorophyll a, a+b and carotenoid concentrations of Scots pine C needles were significantly higher in the LP treatment compared with HP under the highest O 3 concentration (210 ppb). In the mesophyll tissue of C needles of both tree species, LP treatment increased the size of mitochondria and elevated O 3 -induced granulation of chloroplast stroma and disintegration of cytoplasm. Exposure to elevated O 3 concentrations increased swelling of chloroplast thylakoids and reduced the amount of vacuolar tannin in the LP Scots pine C needles. The results suggest disturbances in needle photosynthetic machinery due to acute exposure to the combination of elevated O 3 and low P availability. However, clear additive effects were found only in needle P concentrations < 1 mg g -1 in short-term O 3 exposure.     

Photosynthetic utilization efficiency of absorbed photosynthetically active radiation by Scots pine and birch forest stands in the southern Taiga

Absorption and utilization of photosynthetically active radiation (PAR) were investigated in Scots pine (Pinus sylvestris L.) and birch (Betula pendula Roth.) stands that were 41 years old at the end of the experimental period. Canopy depth of the Scots pine stand was about half that of the birch stand (6.5 versus 11.0 m), but absorption of PAR was similar in the two stands. The Scots pine forest canopy, with a leaf area index of 8.9, absorbed 90% of the incoming PAR (APAR), whereas the birch forest canopy, with a leaf area index of 5.9, absorbed 92% of APAR. During maximum foliage development, the upper Scots pine canopy absorbed more PAR than the upper birch canopy (75 versus 66%). The upper, middle and lower layers of the Scots pine canopy contained 37, 48 and 15% of the total needle surface area, respectively. The corresponding distribution of foliage surface area in the three layers of the birch canopy was 50, 30 and 20%, respectively. Measurements of photosynthetic rate were combined with estimates of leaf area index and stand phytomass to determine rates of primary production on a sunny day, a cloudy day, and on an annual basis. The energy equivalents of short- and long-term carbon gain were used with determinations of APAR to calculate photosynthetic utilization efficiency. Throughout the growing season, photosynthetic utilization efficiency of APAR in the upper canopy layer of the Scots pine forest was almost twice that in the lower canopy layer. In the birch forest, photosynthetic utilization efficiency was greater in the lower canopy layer than in the upper canopy layer. In all cases, utilization efficiency was higher in the birch stand than in the Scots pine stand (52 versus 29 J kJ(-1)). Taking account of respiration of the non-photosynthetic parts of each stand (night respiration of needles or leaves; respiration of branches, trunk and roots), estimated utilization efficiency of APAR for net primary production was 11 J kJ(-1) for Scots pine and 19 J kJ(-1) for birch. Solar conversion ratios, expressed as whole-plant net primary productivity per unit of APAR for the growing season, were 0.81 g MJ(-1) for Scots pine and 0.93 g MJ(-1) for birch.     

Seedling shoot,needle and bud development in three provenances of Pinus sylvestris and Pinus contorta cultivated in northern Sweden

The patterns of current‐year shoot, needle and terminal bud elongation in seedlings of three Scots pine (Pinus sylvestris L.) and three lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) provenances were compared during the third and fourth growing seasons after planting. Lodgepole pine produced longer shoots and buds than did Scots pine, mainly because lodgepole pine formed more stem units and elongated at a faster rate. Stem unit length and the duration of shoot and bud elongation differed relatively little between species and provenances. Lammas or polycyclic growth occurred in some lodgepole pine provenances, but not in any Scots pine provenance, and was associated with enhanced shoot elongation. Needle elongation commenced earlier, proceeded at a faster rate, and was greater in lodgepole pine than in Scots pine, but ceased about the same time in all species and provenances. The heat sum required to attain 50% of final length was lower for shoots and needles in lodgepole pine than in Scots pine, and for shoots in northern provenances than in southern ones. Mitotic activity in the apical meristem of the terminal bud, which occurred less than one week after the seedlings were free from snow, started and ceased about the same time in each species, but was higher in lodgepole pine than in Scots pine early in the shoot elongation period.     

Susceptibility of four conifer species to Gremmeniella abietina

G. abietina causes severe dieback in pole-stage stands of Scots pine in Britain. The susceptibility of ll provenances of Scots pine and of three other conifer species was tested by inoculations. Significant differences in susceptibility were found amongst Scots pine provenances, with those originating from a native Scottish pinewood at Loch Maree showing the highest levels of disease. Corsican pine was shown to be the most susceptible species tested although sporulation was greatest on Scots pine. Norway spruce was also shown to be susceptible when inoculated late in the growing season. Infection in lodgepole pine was negligible. High levels of β-phellandrene were tentatively linked with resistance among trees in a single stand of Scots pine.     

Preliminary studies on establishment of genetic transformation system in loblolly pine

A transformation system was established for loblolly pine (Pinus taeda L.) mature zygotic embryos usingAgrobacterium tumefaciens. The gene coding for the β-glucuronidase (GUS) gene was introduced into loblolly pine tissues and its transient expression was detected with histochemical staining. The influences of different genotypes.Agrobacterum concentrations. and cocultivation time on GUS expression and kanamycin resistant callus and shoot regeneration were investigated. The results showed that the highest GUS expression frequency (16.3%) and shoot regeneration frequency (78%) were obtained from genotype 9–1003 withAgrobacterium concentration decreased 9 times and cocultivation time of 56 hours respectively. GUS expression was obtained in all genotypes tested. The successful expression of the GUS gene in different genotypes suggested that it will be a useful transformation system for loblolly pine. (Responsible Editor: Chai Ruihai)     

Foliar application of GA3 during terminal long-shoot bud development stimulates shoot apical meristem activity in Pinus sylvestris seedlings

The effect of exogenous gibberellin (GA3) on shoot apical meristem activity in conifer vegetative buds was investigated by spraying 0 or 0.1% GA3 on the foliage of first-year Scots pine (Pinus sylvestris L.) seedlings twice weekly for 9 weeks during development of the terminal long-shoot bud. Exogenous GA3 promoted mitotic activity in the apical zone, thereby increasing both the rate and duration of cataphyll formation and giving rise to a higher and wider apical meristem. The increase in number of cataphylls increased the number of axillary meristems, which developed as short-shoot buds.     

Seasonal dynamics of wood formation: a comparison between pinning,microcoring and dendrometer measurements

Three different methods were evaluated for analysing wood formation of Norway spruce [Picea abies (L.) Karst.] and Scots pine (Pinus sylvestris L.) in Finland. During two growing seasons, wood formation dynamics were determined both by wounding the cambium with a needle followed by localisation of the wound-associated tissue modification after the growing season (pinning), and by extracting small increment cores during the growing season (microcoring). Stem radius was additionally monitored with band dendrometers. For Norway spruce, pinning and microcoring yielded similar dates for the onset of wood formation. The timing of wood production during the growing season was also similar for pinning and microcoring. For Scots pine, the onset of wood formation was recorded from microcores almost 2 weeks later than from pinning samples. In Scots pine, microcore measurements also produced somewhat later cessation dates for tracheid formation than the pinning samples. For both tree species, the total number of tracheids formed during the growing season was, however, about the same for pinning and microcoring. Dendrometer results clearly differed from those of pinning and microcoring. In particular, the dendrometers showed an increase of stem radius considerably earlier in spring, when the other methods did not detect wood formation. Thus, pinning and microcoring currently represent the most reliable techniques for detailed monitoring of wood formation.     

A mycobacterium isolated from tissue cultures of mature Pinus sylvestris interferes with growth of Scots pine seedlings

We isolated a rapidly growing, pigment-producing mycobacterium from senescent tissue cultures derived from mature Scots pine (Pinus sylvestris L.). The bacterium was found in three senescent suspension cultures and in a senescent protoplast culture. Growth of Scots pine cells had ceased in all of these cultures. Exogenous contamination was eliminated by rigorous surface sterilization of the buds with hypochlorite before aseptic removal of the bud scales. Based on biochemical and physiological properties and DNA sequence comparisons, the isolated mycobacterium did not belong to any known species. Its sequence most closely resembled those of Mycobacterium obuense (97%) and M. aichiense (96%). Tissue browning was frequently observed in callus or suspension culture of Scots pine. Because the effect of the mycobacterium on growth of undifferentiated tissues that were browning was difficult to evaluate, we applied the bacterium to Scots pine seeds in aseptic conditions. Seedlings grown in the presence of the mycobacterium had shorter hypocotyls than control seedlings and seedlings cocultivated with a Pseudomonas strain known to be harmless to plants. However, hypocotyl growth of seedlings cocultivated with another mycobacterium, M. chlorophenolicum, was similar to that observed in the presence of the isolated mycobacterium. Phenylalanine ammonia-lyase activity of seedlings cocultivated with the mycobacterium isolate was significantly higher than that of control seedlings or seedlings cocultivated with M. chlorophenolicum or Pseudomotnas fluorescens. We believe that this is the first report of the isolation of mycobacteria from tissue cultures of a tree. Our finding that the mycobacterium may interfere with the growth of Scots pine in vitro warrants further study.     

Nitrate metabolism in Scots pine seedlings during their first growing season

Fluctuations in nitrate reductase activity (NRA), nitrate, nitrite, protein and total nitrogen content of bare-root Scots pine seedlings (Pinus sylvestris L.) raised outdoors were investigated during the first growing season. Nitrate reductase activity was higher in roots than in needles, whereas NO(3) (-) content was higher in needles than in roots and increased in both from June to October. Nitrate reductase activity in roots correlated more closely with NO(3) (-) N in the soil than did NO(3) (-) in the roots. In autumn, there was a closer correlation between foliar NRA and NO(3) (-) in the needles than with NO(3) (-)-N in the soil. Nitrite was not detected in the seedlings during the growing season. Total nitrogen content decreased toward the autumn, whereas protein content initially decreased but increased again in autumn. Acrylic netting placed above the seedlings increased both air and soil temperatures and apparently accelerated the use of nitrate.     

Transient gene expression in western white pine using agroinfiltration

A method for transient gene expression was developed for western white pine(WWP, Pinus monticola Dougl. ex D.Don) using reporter gene uid A encoding bglucuronidase(GUS). GUS was transiently expressed in cross sections of primary and secondary needles, cotyledons, and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens. Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy. In fluorometric assays, GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles, while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly. Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons, all tested WWP tissues using the protocol had high levels of transient GUS expression. Thus, heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach. The current protocol for efficient transient gene expression willaid functional genomics study of WWP and its pathogens and related conifer species.     

Differences in Agrobacterium infections in silver birch and Scots pine

The aim of this study was to investigate the differences in infections caused by Agrobacterium tumefaciens in a conifer, Scots pine (Pinus sylvestris), and in a non-host deciduous species, silver birch (Betula pendula). All the Agrobacterium tumefaciens strains tested caused crown-gall formation in both tree species, but the infection rates varied remarkably. In Scots pine, the development of galls was rare, and slower than in silver birch. Inoculation into the base of the stem were the most successful in gall induction. Silver-birch galls were large, often surrounding the whole stem, in contrast to Scots pine galls, which were characterized by their small size and neck-like connection with the host plant. In silver birch, no other morphological changes could be seen. In Scots pine, abnormal phenotypes with proliferating short shoots above the galls were observed during the second and third growing season. The results indicate that, of the two non-host tree species, the deciduous one, silver birch, is more susceptible to an A. tumefaciens infection than the conifer, Scots pine. The matrix for A. tumefaciens infection in silver birch differs from that in Scots pine, since the terpene compounds of Scots pine seem either to kill the agrobacteria or to suppress their growth. The differences between the species could be partly caused by their difference in sensitivity to phytohormones. These features reflect evolutionary incompatibility between A. tumefaciens and a gymnosperm.     

Structure and abundance of arachnid communities in Scots and lodgepole pine plantations

Between April and September 1994, six plots within Aberfoyle Forest in Scotland, three of Scots (Pinus sylvestris) and three of lodgepole pine (Pinus contorta), were surveyed for spiders and harvestmen, as these are predators of several forest invertebrate pests.A total of 64 species of spider were trapped in Scots and lodgepole pine; 56 in Scots pine and 32 in Lodgepole pine. The most abundant spiders, both in Scots and Lodgepole pine, were Linyphiidae.Scots pine plots were floristically more diverse and structurally more complex than lodgepole pine plots. More species of spiders and harvestmen (midsummer only) were found at ground level in Scots pine than lodgepole pine plots. More species and a greater numbers of spiders were found in the canopy of Scots pine than in that of lodgepole pine. However, no differences in the abundance of harvestmen were found between the canopies of Scots and lodgepole pine. Spider species diversity was greatest in Scots pine.     

Canopy reduction as a possible measure for adaptation of young Scots pine stand to insufficient precipitation in Central Europe

Climate change towards a warmer and dryer vegetation period may negatively impact growing conditions for Scots pine monocultures situated on dry, sandy soils in Central Europe. The purpose of the study was to evaluate the effect of thinning on precipitation throughfall in young Scots pine stands on typical pine sites. In 1992, observation of precipitation and throughfall started at the Tyniště research site (lowland of Eastern Bohemia) in a 7-year-old pine stand planted in rows at a stocking of ca 10,000 trees ha⁻¹. Throughfall was measured at weekly intervals during the growing season (April-September) by gauges randomly located in two treatments - variant 1C - Control without thinning and variant 2T - Thinned - and compared to precipitation at an open space outside of the canopy. The results demonstrated the positive effects of heavy low thinning (removal of 47% of the total number of trees and 31% of total pre-treatment basal area) on the water supply of young Scots pine stands. On the Thinned treatment, throughfall increased by 2-8% compared to Control plot. This positive effect persisted for six years after the first canopy reduction and the differences were significant for the first four years after thinning. After the second treatment (high thinning), throughfall on the Thinned treatment showed a nominal, but statistically insignificant increase. The likely reason for this result is that the application of a different type of thinning increased the variability of the canopy and, consequently, the effect of released crowns could not be detected.     

Allometry and evaluation of in situ optical LAI determination in Scots pine: a case study in Belgium

We evaluated several optical methods for in situ estimation of leaf area index (LAI) in a Belgian Scots pine (Pinus sylvestris L.) stand. The results obtained were compared with LAI determined from allometric relationships established in the same stand. We found high correlations between branch cross-sectional area, diameter at breast height (DBH) and basal area as dependent variables, and leaf mass, needle area and crown projection as independent variables. We then compared LAI estimated by allometry with LAI determined by three optical methods (LAI-2000, TRAC and digital hemispherical photography) both before and after corrections for blue light scattering, clumping and non-leafy material. Estimates of stand LAI of Scots pine ranged from 1.52 for hemispherical photography to 3.57 for the allometric estimate based on DBH. There was no significant difference (alpha = 0.01) between the allometric LAI estimates and the optical LAI values corrected for blue light scattering, clumping and interception by non-leafy material. However, we observed high sensitivity of the optical LAI estimates to the various conversion factors, particularly to the clumping factor, indicating the need for caution when correcting LAI measured by optical methods.     

Evidence that longer needle retention of spruce and pine populations at high elevations and high latitudes is largely a phenotypic response

There is abundant evidence that evergreen conifers living at high elevations or at high latitudes have longer-lived needles than trees of the same species living elsewhere. This pattern is likely caused by the influence of low temperature in combination with related factors such as a short growing season and low nutrient availability. Because it is not known to what degree such patterns result from phenotypic versus genotypic variation, we evaluated needle longevity for common-garden-grown lowland populations of European Scots pine (Pinus sylvestris L.) of wide latitudinal origin and Norway spruce (Picea abies L.) of wide elevational origin. Nine-year-old trees of 16 Scots pine populations ranging in origin from 47 degrees to 60 degrees N were studied in Kórnik, Poland (52 degrees N) and 18-year-old trees of 18 Norway spruce populations ranging in origin from 670 to 1235 m elevation in southwestern Poland were studied near Morawina, Poland (51 degrees N, 180 m elevation). There was no tendency in either species for populations from northern or high elevation origins to retain needles longer than other populations. All of the Scots pine populations had between 2.5 to 3.0 needle age cohorts and all of the Norway spruce populations had between 6.4 and 7.2 needle age cohorts. Thus, extended needle retention in Scots pine and Norway spruce populations in low-temperature habitats at high elevations and high latitudes appears to be largely an environmentally regulated phenotypic acclimation.     

The effect of girdling on the moisture content of small-sized trees

In this study, the effect of girdling on the moisture content of small-sized trees for heat energy production was clarified. The moisture content was measured for Scots pine (Pinus sylvestris), Norway spruce (Picea abies), and Downy birch (Betula pubescens) during two growing seasons after girdling. The trees were girdled at breast height for around 30 cm by removing the bark, phloem, and cambium from around the stem. At the beginning of the growing season the mean moisture content of the living Scots pine (P. sylvestris) and Norway spruce (P. abies) was 60%, and for Downy birch (B. pubescens) it was 50%. During the first growing season the effect of girdling on the moisture content was low, but during the second growing season the moisture content decreased significantly. The moisture content of the Norway spruce (P. abies) (23%) and Downy birch (B. pubescens) (33%) was at its lowest point at 14 months after girdling. There were no significant changes in the moisture content of the Scots pine (P. sylvestris) in this study. The results of this study can be used in basic research and in the development of energy wood production.