Know Your Neighbours: Drought Response of Norway Spruce,Silver Fir and Douglas Fir in Mixed Forests Depends on Species Identity and Diversity of Tree Neighbourhoods

Norway spruce is a widely cultivated species in Central Europe; however, it is highly susceptible to droughts, which are predicted to become more frequent in the future. A solution to adapt spruce forests to droughts could be the conversion to mixed-species stands containing species which are less sensitive to drought and do not increase the drought stress in spruce. Here we assessed the drought response of spruce and the presumably more drought-tolerant silver fir and Douglas fir in mixed-conifer stands. We measured tree ring widths of 270 target trees, which grew in mixed and mono-specific neighbourhoods in 18 managed stands in the Black Forest, to quantify the complementarity effects caused by species interactions on growth during the extreme drought event of 2003 and for a number of years with “normal” growth and climatic conditions. Mixed-species neighbourhoods did not significantly affect tree ring growth in normal years. However, during the drought, silver fir benefitted from mixing, while Douglas fir was more drought-stressed in the mixture. The drought response of spruce was dependent on the density and species composition of the neighbourhood, showing both positive and negative mixing effects. Mixed stands containing these tree species could improve adaptation to drought because the risks of extreme events are spread across species, and the performance of individual species is improved. Our knowledge about specific species interactions needs to be improved to manage tree mixtures more effectively with regard to the participating species and stand density.     

Root strength and density decay after felling in a Silver Fir-Norway Spruce stand in the Italian Alps

Aims

Forests induce a mechanical reinforcement of soil, generally quantified in terms of additional root cohesion (c _r ), which decreases due to root decay after felling. The aim of this work is providing new field data on soil reinforcement by roots after trees cutting.

Methods

The present work investigated c _r decay in a mixed Silver Fir-Norway Spruce (Abies alba Mill. Picea abies (L.) Karst.) stand in the Italian Alps over a period of 3 years after felling by monitoring the two c _r driving variables: root tensile resistance and root density.

Results

Results showed that a significant difference in root resistance occurred only 3 years after felling, whereas the decrease in the number of roots was significant in the second year. The degradation process was more rapid in shallower layers and for thinner roots, as a consequence of the pattern of biological activity rate. The reduction of c _r after felling was, for a reference profile depth of 70 cm, 55 % in the first 2 years and another 16 % in the third year.

Conclusions

The findings of this study, providing new data on the decrease of c _r after felling, can be introduced into geotechnical models allowing a better estimation of the stability of forest hillslopes.

     

Oxygen and Hydrogen Stable Isotope Ratios of Bulk Needles Reveal the Geographic Origin of Norway Spruce in the European Alps

Background

Tracking timber is necessary in order to prevent illegal logging and protect local timber production, but there is as yet no suitable analytical traceability method. Stable isotope ratios in plants are known to reflect geographical variations. In this study we analysed four stable isotope ratios in order to develop a model able to identify the geographic origin of Norway spruce in the European Alps.

Methodology and Principal Findings

δ¹⁸O, δ²H, δ¹³C and δ¹⁵N were measured in bulk needles of Picea abies sampled in 20 sites in and around the European Alps. Environmental and spatial variables were found to be related to the measured isotope ratios. An ordinary least squares regression was used to identify the most important factor in stable isotope variability in bulk needles. Spatial autocorrelation was tested for all isotope ratios by means of Moran’s I. δ¹⁸O, δ²H and δ¹⁵N values differed significantly between sites. Distance from the coast had the greatest influence on δ²H, while latitude and longitude were strongly related to δ¹⁸O. δ¹³C values did not appear to have any relationship with geographical position, while δ¹⁵N values were influenced by distance from the motorway. The regression model improved the explanatory power of the spatial and environmental variables. Positive spatial autocorrelations were found for δ¹⁸O and δ²H values.

Conclusions

The δ ¹⁸O, δ²H and δ¹⁵N values in P. abies bulk needles are a suitable proxy to identify geographic origin as they vary according to geographical position. Although the regression model showed the explanatory variables to have significant power and stability, we conclude that our model might be improved by multivariate spatial interpolation of the δ ¹⁸O and δ²H values.     

Climate continentality and treeline species distribution in the Alps

Abstract

The distribution of tree species and the elevation of the alpine treeline are strongly affected by climate continentality. In the present work we performed a detailed survey of the upper limits of tree vegetation in two areas with contrasting climate located in the central Italian Alps, in order to evaluate the structure of the treeline under different degrees of continentality. Tree and krummholz (stunted) individual position, their dimension and life form were recorded from the upper limit of the closed forest to the species limit. The results were compared with an estimation of tree species distribution at the treeline in the whole Lombardy Alps, performed by a survey of tree species occurrence in areas of known climatic traits. The structure of the treeline (upper limits, life form altitudinal arrangement) and its ongoing dynamics were different in the two areas: climate continentality assessed by hygric and thermal continentality indices influenced the distribution of some treeline species. Although the influence of human and geomorphologic disturbance could not be excluded, the importance of the degree of continentality must be stressed when evaluating the response of the treeline to past and present climatic change.     

Recovery of Aboveground Plant Biomass and Productivity After Fire in Mesic and Dry Black Spruce Forests of Interior Alaska

Plant biomass accumulation and productivity are important determinants of ecosystem carbon (C) balance during post-fire succession. In boreal black spruce (Picea mariana) forests near Delta Junction, Alaska, we quantified aboveground plant biomass and net primary productivity (ANPP) for 4 years after a 1999 wildfire in a well-drained (dry) site, and also across a dry and a moderately well-drained (mesic) chronosequence of sites that varied in time since fire (2 to ∼116 years). Four years after fire, total biomass at the 1999 burn site had increased exponentially to 160 ± 21 g m⁻² (mean ± 1SE) and vascular ANPP had recovered to 138 ± 32 g m⁻² y⁻¹, which was not different than that of a nearby unburned stand (160 ± 48 g m⁻² y⁻¹) that had similar pre-fire stand structure and understory composition. Production in the young site was dominated by re-sprouting graminoids, whereas production in the unburned site was dominated by black spruce. On the dry and mesic chronosequences, total biomass pools, including overstory and understory vascular and non-vascular plants, and lichens, increased logarithmically (dry) or linearly (mesic) with increasing site age, reaching a maximum of 2469 ± 180 (dry) and 4008 ± 233 g m⁻² (mesic) in mature stands. Biomass differences were primarily due to higher tree density in the mesic sites because mass per tree was similar between sites. ANPP of vascular and non-vascular plants increased linearly over time in the mesic chronosequence to 335 ± 68 g m⁻² y⁻¹ in the mature site, but in the dry chronosequence it peaked at 410 ± 43 g m⁻² y⁻¹ in a 15-year-old stand dominated by deciduous trees and shrubs. Key factors regulating biomass accumulation and production in these ecosystems appear to be the abundance and composition of re-sprouting species early in succession, the abundance of deciduous trees and shrubs in intermediate aged stands, and the density of black spruce across all stand ages. A better understanding of the controls over these factors will help predict how changes in climate and fire regime will affect the carbon balance of Interior Alaska. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of Management on Economic Profitability of Forest Biomass Production and Carbon Neutrality of Bioenergy Use in Norway Spruce Stands Under the Changing Climate

We analyzed the effects of management on the economic profitability of forest biomass production and carbon neutrality of bioenergy use in Norway spruce (Picea abies L. Karst) stands under the changing climate. We employed a forest ecosystem model and life cycle assessment tool. In particular, we studied the effects of thinning, nitrogen fertilization, and rotation length on: (1) the production of timber and energy biomass, and its economic profitability (net present value), (2) carbon stock in the forest ecosystem and carbon balance in forestry, and (3) carbon dioxide (CO₂) emissions from the use of biomass in energy production. Results showed that the current Finnish baseline management with and without nitrogen fertilization resulted in the highest mean annual timber production and net present value (NPV) for long rotations (60 to 80 years), regardless of climate scenario. Mean annual production of energy biomass was enhanced by increasing stocking by 20–30 % compared to the baseline management, and/or use of nitrogen fertilization. Such management gave lower CO₂ emissions per unit of energy compared to the baseline management, as the carbon stock in the forest ecosystem and the carbon balance in forestry increased. Overall, the carbon neutrality and net present value were, on average, the highest in the baseline management or with a 20 % increase in stocking, with nitrogen fertilization and 60- to 80-year rotation lengths, regardless of the climate applied. However, it was not possible to simultaneously maximize the NPV of forest biomass production and the carbon neutrality of bioenergy use.     

Indirect Measurement of Pore Size and Permeability in Scots Pine and Norway Spruce

Five experiments to investigate the water relationships of Scotspine and Norway spruce sapwood are described. Field exposureof stakes, and their moisture characteristic relating wood moisturecontent to the water potential of the surroundings showed thatspruce had a lower moisture content than pine under the sameconditions. A pore size distribution derived from the characteristicsshowed more large pores in spruce than in pine, but in sprucethere were also more small pores. The inference was that thelarge pores of spruce were readily accessible to water, allowinga fast rate of water uptake, as found in the wick action andswelling investigations, while the small pores were not accessible,resulting in a low rate of water uptake and a low rate of swelling.The possibility is briefly discussed that it is the pore sizedistribution of the two woods which determines their water relationships,and the difference in pore size which determines their treatability. Key words: Pinus sylvestris L, Picea abies (L) Karst, Pore size, Water uptake     

Coarse and Fine Control and Annual Changes of Sucrose-Phosphate Synthase in Norway Spruce Needles

Annual changes of activity of sucrose-phosphate synthase (SPS) from spruce (Picea abies [L.] Karst.) needles were studied with respect to three regulatory levels: metabolic fine control, covalent modification (phosphorylation), and protein amount. Glucose-6-phosphate served as an allosteric activator of spruce SPS by shifting the Michaelis constant for the substrate fructose-6-phosphate from 4.2 to 0.59 mM, whereas inorganic phosphate competitively inhibited this activation. The affinity for the other substrate, UDP-glucose, was unaffected. Incubation of the crude extract with ATP resulted in a time- and concentration-dependent decrease of the maximal velocity of SPS. This inactivation was sensitive to staurosporine, a potent protein kinase inhibitor, indicating the participation of a protein kinase. Probing SPS protein with heterologous antibodies showed that the subunit of spruce SPS is an approximately 139-kD protein and that changes in the extractable activity during the course of a year were correlated with the amount of SPS protein. High SPS activities in winter were paralleled by increased levels of the activator glucose-6-phosphate and the substrate fructose-6-phosphate, indicating a high capacity for sucrose synthesis that may be necessary to maintain photosynthetic CO2 fixation in cold-hardened spruce needles.     

Micro- and Macro-Geographic Scale Effect on the Molecular Imprint of Selection and Adaptation in Norway Spruce

Forest tree species of temperate and boreal regions have undergone a long history of demographic changes and evolutionary adaptations. The main objective of this study was to detect signals of selection in Norway spruce (Picea abies [L.] Karst), at different sampling-scales and to investigate, accounting for population structure, the effect of environment on species genetic diversity. A total of 384 single nucleotide polymorphisms (SNPs) representing 290 genes were genotyped at two geographic scales: across 12 populations distributed along two altitudinal-transects in the Alps (micro-geographic scale), and across 27 populations belonging to the range of Norway spruce in central and south-east Europe (macro-geographic scale). At the macrogeographic scale, principal component analysis combined with Bayesian clustering revealed three major clusters, corresponding to the main areas of southern spruce occurrence, i.e. the Alps, Carpathians, and Hercynia. The populations along the altitudinal transects were not differentiated. To assess the role of selection in structuring genetic variation, we applied a Bayesian and coalescent-based F _ST-outlier method and tested for correlations between allele frequencies and climatic variables using regression analyses. At the macro-geographic scale, the F _ST-outlier methods detected together 11 F _ST-outliers. Six outliers were detected when the same analyses were carried out taking into account the genetic structure. Regression analyses with population structure correction resulted in the identification of two (micro-geographic scale) and 38 SNPs (macro-geographic scale) significantly correlated with temperature and/or precipitation. Six of these loci overlapped with F _ST-outliers, among them two loci encoding an enzyme involved in riboflavin biosynthesis and a sucrose synthase. The results of this study indicate a strong relationship between genetic and environmental variation at both geographic scales. It also suggests that an integrative approach combining different outlier detection methods and population sampling at different geographic scales is useful to identify loci potentially involved in adaptation.     

Apoplastic Peroxidases and Lignification in Needles of Norway Spruce (Picea abies L.)

The objective of the present study was to investigate the correlation of soluble apoplastic peroxidase activity with lignification in needles of field-grown Norway spruce (Picea abies L.) trees. Apoplastic peroxidases (EC 1.11.1.7) were obtained by vacuum infiltration of needles. The lignin content of isolated cell walls was determined by the acetyl bromide method. Accumulation of lignin and seasonal variations of apoplastic peroxidase activities were studied in the first year of needle development. The major phase of lignification started after bud break and was terminated about 4 weeks later. This phase correlated with a transient increase in apoplastic guaiacol and coniferyl alcohol peroxidase activity. NADH oxidase activity, which is thought to sustain peroxidase activity by production of H2O2, peaked sharply after bud break and decreased during the lignification period. Histochemical localization of peroxidase with guaiacol indicated that high activities were present in lignifying cell walls. In mature needles, lignin was localized in walls of most needle tissues including mesophyll cells, and corresponded to 80 to 130 [mu]mol lignin monomers/g needle dry weight. Isoelectric focusing of apoplastic washing fluids and activity staining with guaiacol showed the presence of strongly alkaline peroxidases (isoelectric point [greater than or equal to] 9) in all developmental stages investigated. New isozymes with isoelectric points of 7.1 and 8.1 appeared during the major phase of lignification. These isozymes disappeared after lignification was terminated. A strong increase in peroxidase activity in autumn was associated with the appearance of acidic peroxidases (isoelectric point [less than or equal to] 3). These results suggest that soluble alkaline apoplastic peroxidases participate in lignin formation. Soluble acidic apoplastic peroxidases were apparently unrelated to developmentally regulated lignification in spruce needles.     

Patterns of Nucleotide Diversity at Photoperiod Related Genes in Norway Spruce [Picea abies (L.) Karst.]

The ability of plants to track seasonal changes is largely dependent on genes assigned to the photoperiod pathway, and variation in those genes is thereby important for adaptation to local day length conditions. Extensive physiological data in several temperate conifer species suggest that populations are adapted to local light conditions, but data on the genes underlying this adaptation are more limited. Here we present nucleotide diversity data from 19 genes putatively involved in photoperiodic response in Norway spruce (Picea abies). Based on similarity to model plants the genes were grouped into three categories according to their presumed position in the photoperiod pathway: photoreceptors, circadian clock genes, and downstream targets. An HKA (Hudson, Kreitman and Aquade) test showed a significant excess of diversity at photoreceptor genes, but no departure from neutrality at circadian genes and downstream targets. Departures from neutrality were also tested with Tajima''s D and Fay and Wu''s H statistics under three demographic scenarios: the standard neutral model, a population expansion model, and a more complex population split model. Only one gene, the circadian clock gene PaPRR3 with a highly positive Tajima''s D value, deviates significantly from all tested demographic scenarios. As the PaPRR3 gene harbours multiple non-synonymous variants it appears as an excellent candidate gene for control of photoperiod response in Norway spruce.     

The Origin and Dynamics of Subalpine White Spruce and Balsam Fir Stands in Boreal Eastern North America

Associations among the few tree species in the North American boreal landscape are the result of complex interactions between climate, biota, and historical disturbances during the Holocene. The closed-crown boreal forest of eastern North America is subdivided into two ecological regions having distinct tree species associations; the balsam fir zone and the black spruce zone, south and north of 49°N, respectively. Subalpine old-growth stands dominated by trees species typical of the balsam fir forest flora (either balsam fir or white spruce) are found on high plateaus, some of which are isolated within the black spruce zone. Here we identified the ecological processes responsible for the distinct forest associations in the subalpine belt across the eastern boreal landscape. Extensive radiocarbon dating, species composition, and size structure analyses indicated contrasted origin and dynamics of the subalpine forests between the two ecological regions. In the black spruce zone, the subalpine belt is a mosaic of post-fire white spruce or balsam fir stands coexisting at similar elevation on the high plateaus. With increasing time without wildfire, the subalpine forests become structurally similar to the balsam fir forest of the fir zone. These results concur with the hypothesis that the subalpine forests of this area are protected remnants of an historical northern expansion of the fir zone. Its replacement by the fire-prone black spruce forest flora was caused by recurrent fires. In the subalpine belt of the fir zone, no fire was recorded for several millennia. Harsh climate at high altitude is the primary factor explaining white spruce dominance over balsam fir forming a distinct subalpine white spruce belt above the balsam fir dominated forest.     

Movement of Cell Organelles and the Geotropic Curvature in Roots of Norway Spruce (Picea abies)

The geotropic development in roots of Norway spruce [(Picea abies (L.)] H. Karst, has been followed by light and electron microscopy and compared with the movement of cell organelles (statoliths) in the root cap cells. The geotropic curvature develops in two phases: (a) an initial curvature in the root cap region, which results in an asymmetry in the extreme root tip and which appears after about 3 h stimulation in the horizontal position; and (b) the geotropic curvature in the basal parts of the root tip, which after 8 h is distributed over the entire elongation zone. A graphic extrapolation, based on measurements of the root curvatures after various stimulation periods, indicates a presentation time in the range of 8 to 10 min. The root anatomy and ultrastructure have been examined in detail in order to obtain information as to which organelles may act as gravity receptors. The root cap consists of a central core (columella) distinct from the peripheral part. The core contains three to four rows of parenchymatic cells each consisting of 15 to 18 storeys of statocyte cells with possibly mobile cell organelles. Amyloplasts and nuclei have been found to be mobile in the root cap cells, and the movement of both types of organelles has been followed after inversion of the seedlings and stimulation in the horizontal position for various periods of time at 4°C and 21°C. Three-dimensional reconstructions of spruce root cap cells based on serial sectioning and electron microscopy have been performed. These demonstrate that the endoplasmic reticulum (ER)-system and the vacuoles occupy a considerable part of the statocyte cell. For this reason the space available for free movement of single statolith particles is highly restricted.     

Inhibition of Apoplastic and Symplastic Peroxidase Activity from Norway Spruce by the Photooxidant Hydroxymethyl Hydroperoxide

Young, clonal Norway spruce trees (Picea abies L.) were exposed for 2 years at high altitudes to ambient atmospheric concentrations of photooxidants containing hydroxymethyl hydroperoxide (HMHP) as an important constituent. In spruce needles from a site with higher concentrations of organic peroxides in air, the apoplastic peroxidase activities were significantly lower than in needles exposed to lower organic peroxide concentrations. Guaiacol peroxidase activities in total needle extracts were not affected. In vitro HMHP at a concentration of 35 [mu]M inhibited apoplastic and total needle guaiacol peroxidase activities by 50% at pH 5.25. At the same pH, ascorbate-specific peroxidase activity required about 100 [mu]M HMHP for 50% inhibition. At pH 7, 1.46 mM HMHP caused a 50% reduction in guaiacol peroxidase and a 13% reduction in ascorbate peroxidase activity. The present results suggest that HMHP in ambient air may affect peroxidase activity in spruce needles. Peroxidases located in the relatively acidic aqueous phase of the cell walls appear to be more susceptible to HMHP inhibition than those present in neutral or slightly alkaline symplastic compartments of cells such as the cytosol or chloroplasts.     

Changes in the Content of Indole-3-Acetic Acid and Cytokinins in Spruce, Fir and Oak Trees after Herbicide Treatment

Treatment of spruce, fir and oak trees with herbicides, which may be one of the forest damage inducing agents, caused pronounced changes in the contents and distribution of indole-3-acetic acid (IAA) and cytokinins (CKs) one year after treatment, i.e. at the time of the first microscopically visible damage in treated trees. In Picea pungens IAA content increased in the terminal buds by about 105 % and in the apical buds of the first order branches by 220 %. The same was true for young sprouts of Abies nordmanniana, while in leaves of oak trees IAA content was decreased by 15 % after glyphosate treatment and by 30 % after 2,4-dichlorophenoxyacetic acid (2,4-D) treatment. Another striking feature was a significantly decreased content of IAA in the lower parts of roots in Picea pungens (50 % of the control), which is accompanied by an increase in IAA content in the middle part of the roots (130 %). On the other hand, the IAA content of both sprouts and roots of A. nordmanniana was significantly increased after herbicide treatment.In P. pungens, the content of free cytokinins (sum of zeatin, zeatin riboside, isopentenyladenine and isopentenyladenosine) decreased due to herbicide treatment. The strongest decrease was seen in roots, especially in their upper and middle parts (the average reduction of cytokinin content in roots was 63 %). In the above-ground organs the reduction was seen namely for isopentenyladenine and isopentenyladenosine, while the abundance of zeatin riboside was, on the other hand, higher in treated plants. In Quercus robur leaves, the total content of cytokinins also decreased, namely after glyphosate treatment. In consequence of these changes, CK/IAA ratio decreased pronouncedly in all organs of herbicide-treated trees, with the exception of oak leaves treated by 2,4-D.     

Control of Nitrous Oxide Emissions in European Beech,Norway Spruce and Scots Pine Forests

Elevated nitrogen deposition has increased tree growth, the storage of soil organic matter, and nitrate leaching in many European forests, but little is known about the effect of tree species and nitrogen deposition on nitrous oxide emission. Here we report soil N₂O emission from European beech, Scots pine and Norway spruce forests in two study areas of Germany with distinct climate, N deposition and soils. N₂O emissions and throughfall input of nitrate and ammonium were measured biweekly during growing season and monthly during dormant season over a 28 months period. Annual N₂O emission rates ranged between 0.4 and 1.3 kg N ha^?1 year^?1 among the stands and were higher in 1998 than in 1999 due to higher precipitation during the growing season of 1998. A 2-way-ANOVA revealed that N₂O fluxes were significantly higher (p<0.001) at Solling than at Unterlüß while tree species had no effect on N₂O emissions. Soil texture and the amount of throughfall explained together 94% of the variance among the stands, indicating that increasing portions of silt and clay may promote the formation of N₂O in wet forest soils. Moreover, cumulative N₂O fluxes were significantly correlated (r² = 0.60, p<0.001) with cumulative NO ₃ ^? fluxes at 10 cm depth as an indicator of N saturation, however, the slope of the regression curve indicates a rather weak effect of NO ₃ ^? fluxes on N₂O emissions. N input by throughfall was not correlated with N₂O emissions and only 1.6–3.2% of N input was released as N₂O to the atmosphere. Our results suggest that elevated N inputs have little effect on N₂O emissions in beech, spruce and pine forests.     

Modeling Response of N Addition on C and N Allocation in Scandinavian Norway Spruce Stands

Because nitrogen is considered to be the major growth-limiting element in boreal forests, the increasing nitrogen availability from deposition should lead to increasing growth. We have tested this assumption by simulating, with a simple model, carbon and nitrogen development in seven long-term fertilization experiments in three Nordic countries. The only differences between sites in the model are climate, the ambient nitrogen deposition, nitrogen fertilization regimes, and the initial conditions at the start of the experiment. The model simulates the observed stand development well as long as nitrogen remains the limiting factor. The simulated retention of deposited nitrogen is in general low (less than 50%), whereas retention of fertilizer nitrogen is higher. This seems to imply that the higher production in the fertilized stands will not be maintained once the fertilization is stopped. The model results also indicate that the major effect of climate on site productivity is through soil processes, not tree physiology.     

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.Anna Karlgren and Jenny Carlsson contributed equally to this study.Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se     

Antioxidants and Manganese Deficiency in Needles of Norway Spruce (Picea abies L.) Trees

Chlorotic and green needles from Norway spruce (Picea abies L.) trees were sampled in the Calcareous Bavarian Alps in winter. The needles were used for analysis of the mineral and pigment contents, the levels of antioxidants (ascorbate, glutathione), and the activities of protective enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate radical reductase, dehydroascorbate reductase, glutathione reductase). In addition, the activities of two respiratory enzymes (glucose-6-phosphate dehydrogenase, NAD-malate dehydrogenase), which might provide the NADPH necessary for functioning of the antioxidative system, were determined. We found that chlorotic needles were severely manganese deficient (3 to 6 micrograms Mn per gram dry weight as compared with up to 190 micrograms Mn per gram dry weight in green needles) but had a similar dry weight to fresh weight ratio, had a similar protein content, and showed no evidence for enhanced lipid peroxidation as compared with green needles. In chlorotic needles, the level of total ascorbate and the activities of superoxide dismutase, monodehydroascorbate radical reductase, NAD-malate dehydrogenase, and glucose-6-phosphate dehydrogenase were significantly increased, whereas the levels of ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, and glutathione were not affected. The ratio of ascorbate to dehydroascorbate was similar in both green and chlorotic needles. These results suggest that in spruce needles monodehydroascorbate radical reductase is the key enzyme involved in maintaining ascorbate in its reduced state. The reductant necessary for this process may have been supplied at the expense of photosynthate.