Isolation, Purification, and Subcellular Localization of Isozymes of Superoxide Dismutase from Scots Pine (Pinus sylvestris L.) Needles

Two of four isozymes of superoxide dismutase (SOD) (EC 1.15.1.1) were purified from Scots pine (Pinus sylvestris L.) needles. One form was cytosolic (SOD-1) and the other was associated with chloroplasts (SOD-3). The holoenzyme molecular masses was estimated at approximately 35 kilodaltons by gel filtration. The subunit molecular weight of the dimeric enzymes was estimated to 16.5 kilodaltons (SOD-1) and 20.4 kilodaltons (SOD-3) on sodium dodecyl sulfatepolyacrylamide gels. The NH₂-terminal sequence of the pine enzymes showed similarities to other purified superoxide dismutases located in the corresponding compartment. The cytosolic form revealed two additional amino acids at position 1 and 2 at the NH₂-terminal. Both forms were cyanide- and hydrogenperoxide-sensitive and SOD-3 was found to contain approximately one copper atom per subunit, indicating that they belong to the cupro-zinc SODs. The isoelectric point was 4.9 and 4.5 for SOD-1 and SOD-3, respectively.     

Unique Features of the Loblolly Pine (Pinus taeda L.) Megagenome Revealed Through Sequence Annotation

The largest genus in the conifer family Pinaceae is Pinus, with over 100 species. The size and complexity of their genomes (∼20–40 Gb, 2n = 24) have delayed the arrival of a well-annotated reference sequence. In this study, we present the annotation of the first whole-genome shotgun assembly of loblolly pine (Pinus taeda L.), which comprises 20.1 Gb of sequence. The MAKER-P annotation pipeline combined evidence-based alignments and ab initio predictions to generate 50,172 gene models, of which 15,653 are classified as high confidence. Clustering these gene models with 13 other plant species resulted in 20,646 gene families, of which 1554 are predicted to be unique to conifers. Among the conifer gene families, 159 are composed exclusively of loblolly pine members. The gene models for loblolly pine have the highest median and mean intron lengths of 24 fully sequenced plant genomes. Conifer genomes are full of repetitive DNA, with the most significant contributions from long-terminal-repeat retrotransposons. In depth analysis of the tandem and interspersed repetitive content yielded a combined estimate of 82%.     

The effects of UV-B radiation on epidermal anatomy in loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.)

The effects of enhanced UV‐B radiation on the needle anatomy of loblolly pine (Pinus taeda L.) and Scots pine (Pinus sylvestris L.) were studied in the field under supplemental UV‐B radiation supplied by a modulated irradiation system. The supplemental UV‐B levels were designed to simulate either a 16 or 25% loss of stratospheric ozone over College Park, Maryland. Enhanced UV‐B radiation caused different responses in these two species. The needles of loblolly pine had larger amounts of tannin in the lumen of epidermal cells and more wall‐bound phenolics in the outer epidermal walls of UV‐B‐treated needles, whereas the most pronounced effect on Scots pine needles was increased cutinization. In both species, the outer epidermal cell walls thickened and the needle cross‐sectional and mesophyll areas decreased (statistically significantly only in Scots pine). This suggests that more carbon may have been allocated to the protection mechanisms at the expense of photosynthetic area. The difference in response between these species suggests that the response to UV‐B radiation is not mediated by a single mechanism and that no generalization with regard to the effects of UV‐B on conifers can be made.     

The effect of open-air fumigation with SO2 and O3 on carbohydrate metabolism in Scots pine (Pinus sylvestris) and Norway spruce (Picea abies)

The carbohydrate metabolism of the needles of Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) has been examined in trees that were exposed to SO₂, and O₃, in an open-air fumigation experiment located in the Liphook forest in southern England. Two-year-old seedlings were planted in 1985 in seven experimental plots. Five plots received fumigation treatments of SO₂, O₃ or a combination of these gases to give a 2 × 3 factorial design with one additional ambient plot Fumigation with SO₂, occurred from May 1987 to December 1990 and O₃, fumigation occurred from March to December 1988, May to December 1989 and February to December 1990. Five samples of needles for investigation of carbohydrate metabolism were taken between February and July 1989. The concentrations of soluble carbohydrates (including sucrose and hexoses) were greatly reduced in the needles taken from Scots pine growing in the treated plots, and were also reduced, but to a lesser extent, in the needles taken from Norway spruce. Little variation in the concentration of starch in the needles of either species was detected. The activities of the two final enzymes of sucrose synthesis, sucrose phosphate synthase and sucrose 6-phos-phate phosphatase, were greatly reduced in the needles of Scots pine and were also reduced, but to a lesser extent, in the needles of Norway spruce in the fumigated plots. These reductions could be correlated with decreases in rates of photosynthetic CO₂ assimilation determined by independent groups of researchers working on the Liphook site.     

Purification, Characterization, and Cloning of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine (Pinus taeda L.)

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1. 195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coniferaldehyde (K_m = 1.7 micromolar) compared with sinapaldehyde (K_m in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the λCAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme.     

Photosynthetic capacity of loblolly pine (Pinus taeda L.) trees during the first year of carbon dioxide enrichment in a forest ecosystem

Our objective was to assess the photosynthetic responses of loblolly pine trees (Pinus taeda L.) during the first full growth season (1997) at the Brookhaven National Lab/Duke University Free Air CO₂ Enrichment (FACE) experiment. Gas exchange, fluorescence characteristics, and leaf biochemistry of ambient CO₂ (control) needles and ambient + 20 Pa CO₂ (elevated) needles were examined five times during the year. The enhancement of photosynthesis by elevated CO₂ in mature loblolly pine trees varied across the season and was influenced by abiotic and biotic factors. Photosynthetic enhancement by elevated CO₂ was strongly correlated with leaf temperature. The magnitude of photosynthetic enhancement was zero in March but was as great as 52% later in the season. In March, reduced sink demand and lower temperatures resulted in lower net photosynthesis, lower carboxylation rates and higher excess energy dissipation from the elevated CO₂ needles than from control needles. The greatest photosynthetic enhancement by CO₂ enrichment was observed in July during a period of high temperature and low precipitation, and in September during recovery from this period of low precipitation. In July, loblolly pine trees in the control rings exhibited lower net photosynthetic rates, lower maximum rates of photosynthesis at saturating CO₂ and light, lower values of carboxylation and electron transport rates (modelled from A–C_i curves), lower total Rubisco activity, and lower photochemical quenching of fluorescence in comparison to other measurement periods. During this period of low precipitation trees in the elevated CO₂ rings exhibited reduced net photosynthesis and photochemical quenching of fluorescence, but there was little effect on light- and CO₂-saturated rates of photosynthesis, modelled rates of carboxylation or electron transport, or Rubisco activity. These first-year data will be used to compare with similar measurements from subsequent years of the FACE experiment in order to determine whether photosynthetic acclimation to CO₂ occurs in these canopy loblolly pine trees growing in a forest ecosystem.     

Purification of Two Superoxide Dismutase Isozymes and Their Subcellular Localization in Needles and Roots of Norway Spruce (Picea abies L.) Trees

Two isozymes of superoxide dismutase (SOD; EC 1.15.1.1) were purified from Norway spruce (Picea abies L.) needles to apparent electrophoretic homogeneity. Purification factors were 354 for SOD I and 265 for SOD II. The native molecular mass of both purified enzymes was approximately 33 kD, as determined by gel filtration. The subunit molecular weights, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were 20,000 for SOD I and 16,000 for SOD II in the presence of 2-mercaptoethanol, and 15,800 and 15,000, respectively, in its absence. These results indicate that the native enzymes were homodimers whose subunits contained intrachain disulfide bonds. Isoelectric points determined by nondenaturing isoelectric focusing were 4.5 and 5.5 for SOD I and II, respectively. NH₂-terminal sequence analysis of the first 22 to 23 amino acids revealed 70 to 75% sequence identity with chloroplastic CuZn SODs from other plant species for SOD I, and 75% sequence identity with the cytosolic CuZn SOD from Scots pine for SOD II. SOD I was the major activity in needles and it was associated with chloroplasts. SOD II activity was dominant in roots.     

Variation in fine root biomass of three European tree species: Beech (Fagus sylvatica L.), Norway spruce (Picea abies L. Karst.), and Scots pine (Pinus sylvestris L.)

Abstract

Fine roots (<2 mm) are very dynamic and play a key role in forest ecosystem carbon and nutrient cycling and accumulation. We reviewed root biomass data of three main European tree species European beech, (Fagus sylvatica L.), Norway spruce (Picea abies L. Karst.) and Scots pine (Pinus sylvestris L.), in order to identify the differences between species, and within and between vegetation zones, and to show the relationships between root biomass and the climatic, site and stand factors. The collected literature consisted of data from 36 beech, 71 spruce and 43 pine stands. The mean fine root biomass of beech was 389 g m^?2, and that of spruce and pine 297 g m^?2 and 277 g m^?2, respectively. Data from pine stands supported the hypothesis that root biomass is higher in the temperate than in the boreal zone. The results indicated that the root biomass of deciduous trees is higher than that of conifers. The correlations between root biomass and site fertility characteristics seemed to be species specific. There was no correlation between soil acidity and root biomass. Beech fine root biomass decreased with stand age whereas pine root biomass increased with stand age. Fine root biomass at tree level correlated better than stand level root biomass with stand characteristics. The results showed that there exists a strong relationship between the fine root biomass and the above-ground biomass.     

Effect of open-air fumigation with sulphur dioxide and ozone on phyllosphere and endophytic fungi of conifer needles

The phyllosphere microbial populations inhabiting the needles of three conifer species, Scots pine (Pinus sylvestris L.), Sitka spruce (Picea sitchensis L.) and Norway spruce (Picea abies (L.) Karst.), exposed to SO₂ and O₃, in an open-air fumigation experiment were analysed over a 3 year period using serial dilution after washing, direct plating and a fluorescein diacetate (FDA) enzyme assay. Total fungal populations ranged from 10² to 10⁵ colonyforming units (CPU) g^?1 fresh weight of needles. The dominant fungi isolated from needles varied with tree species and isolation technique; Aureobasidium pullulans (de Bary) Arnaud was most common on Scots pine and Norway spruce and white yeasts on Sitka spruce using the dilution plating method. However, direct plating of needle segments onto culture media indicated that Sclerophoma pythiophila (Corda) Hohnel was dominant on Scots pine and A. pullulans on Sitka and Norway spruce. Green needles of Sitka spruce were found to be endophytically colonized by Rhizosphaera kalkhoffii Bubak, but seldom by Lophodermium piceae (Fuckel) Hohn during extensive sampling in 1990. Statistical analyses revealed significant differences (P<0.05) between plots in the 3 year mean of the total fungal populations or the fungal biomass (FDA assay) on all three tree species. Differences between plots were also observed for a number of dominant component species. Data were also analysed for treatment effects. A significant effect of SO₂ treatment was observed on the total fungal populations on Sitka spruce (P<0.05) which were reduced markedly by the low-SO₂ treatment, while the O₃ treatment caused a significant increase in total fungal numbers on Scots pine (P<0.05). The FDA activity on needles of both Scots pine and Sitka spruce was noticeably higher in the 0₃-only treatment plot, but the overall O₃ effect was not significant. Treatment effects were also detected on the occurrence of component species. The serial dilution method revealed an SO₂ effect (P<0.05) of a reduction in the occurrence of pink yeasts on Sitka spruce and an O₃ effect (P<0.05) of an increase in the occurrence of S. pythiophila on Sitka spruce (P<0.01) but a decrease of Epicoccum nigrum Link and Cladosporium spp. on Scots pine. The direct-plating method revealed an SO₂ effect of an increase in S. pythiophila on Norway spruce (P<0.05). Ozone treatment caused a significant increase in the isolation of a black strain of A. pullulans on Norway spruce (P<0.05). Endophytic colonization of Sitka spruce needles by R. kalkhoffii was found to be increased on two occasions by O₃ exposure.     

Seasonal ionic fluctuations and annual growth rates in stands of Pinus silvestris L. and Picea abies Karst. (L.)

Seasonal variations in potassium, calcium, nitrogen, phosphorus, and magnesium contents of needles from two different stands of Pinus silvestris L. and Picea abies (L.) Karst. were determined monthly during a period of two years, and the growth rates once a year during five years. The stands were growing on sand or on clay. The supposed transport of potassium to the shoot during autumn could not be demonstrated. The nutrient status of pine and spruce growing on sand and clay was very similar, except for calcium and magnesium. There were only small differences in ion contents between pine and spruce, except for calcium. On soils with low water-holding capacity, the available water in dry years is the limiting factor affecting the growth rate of spruce but not of pine. It is concluded that the nutrient status of the leaves, the growth rates and the available water in the soil are factors that should have an important part in any discussion of fertilization of coniferous forests.     

The ion concentration of dew condensed on Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.) needles

Summary Dew droplets collected with pipettes from coniferous needles were analysed for their ionic composition. Almost all samples of dew taken from Scots pine trees (Pinus sylvestris) showed significantly higher ion concentrations than those taken from Norway spruce trees (Picea abies). This can be explained by the micromorphology of the needle surface. The higher microscale roughness of the wax layer of a pine needle causes a more efficient flux of atmospheric aerosol particles compared to the spruce needle surface. Dew on coniferous needles is shown to be capable of maintaining pH values below 3 for several hours.     

Genetic mapping of expressed sequence tag polymorphism (ESTP) markers in loblolly pine (Pinus taeda L.)

The development and mapping of genetic markers based upon expressed sequence tag polymorphisms (ESTPs) in loblolly pine (Pinus taeda L.) are reported. The new markers were generated by PCR-amplification of loblolly pine genomic DNAs with primers designed from sequenced cDNAs. The cDNA libraries were constructed from RNAs expressed in the needles of loblolly pine seedlings or in the xylem from young trees. DNA polymorphisms were identified by analyzing the amplified products for differences in fragment size or restriction sites, or by examining mobility differences using denaturing gradient gel electrophoresis (DGGE). DGGE revealed more DNA polymorphisms than the other two methods. Fifty six ESTPs were mapped using either of two mapping populations and positioned onto a loblolly pine consensus genetic map. Unlike many other markers commonly used in forestry, ESTPs can be used as orthologous markers for comparative mapping, to map genes of known function, or to identify candidate genes affecting important traits in loblolly pine. Received: 10 April 2000 / Accepted: 13 July 2000     

Free Radical Scavengers and Photosynthetic Pigments in Pinus Cembra L. Needles as Affected by Ozone Exposure

The goal of this study was the characterization of the antioxidative protection system of current and 1-year-old needles of a cembran pine (Pinus cembra L.) and its possible responses to elevated concentrations of atmospheric O₃. Twigs of a mature cembran pine at the alpine timberline (1950 m a.s.l.) were exposed in climate-controlled twig chambers for 91 d to charcoal-filtered air (CF), ambient air O₃ concentration (A), and two-fold ambient air O₃ concentration (2A). Additionally, a chamberless control group (AA) was used to examine chamber effects. At the end of the fumigation period the contents of free radical scavengers and photosynthetic pigments were measured in the needles. Independent from O₃ exposure, total ascorbate and -tocopherol contents were higher in 1-year-old needles compared to the current flush while the opposite was found for glutathione. The amounts of pigments and antioxidants in P. cembra needles were comparable to those in other conifers growing at high-elevation sites. The only hint toward O₃ induced changes in the composition of antioxidants was an increase in the glutathione redox state toward more oxidation in 1-year-old needles upon exposure to A or AA conditions, but not upon 2A exposure. Chlorophyll and carotenoid contents were not affected by O₃ neither in current- nor in previous-year needles. The de-epoxidation state of the xanthophyll cycle pigments, however, was significantly increased in 1-year-old needles under A and AA compared to the CF control, but not under 2A. Hence, Pinus cembra, which is well adapted to the extreme environment of the timberline ecotone, exhibited only marginal biochemical changes in response to elevated O₃.     

Genetic Variation in Chinese Pine (Pinus tabulaeformis), a Woody Species Endemic to China

Random amplified polymorphic DNA markers were used to investigate genetic variation of the Chinese pine (Pinus tabulaeformis Carr.), a species endemic to China and the most widely distributed pine species in North China. The results revealed that P. tabulaeformis populations had a relatively high level of genetic diversity (H _t = 0.3268), distributed mainly within (79.2%) rather than among (20.8%) populations. The populations of Lingkong Mountain and Wuling Mountain had a higher level of diversity (0.2687) than the other four populations (0.2537). No statistically significant relationships were found between genetic diversity and climatic factors by correlation analysis and between genetic distance and geographic distance by the Mantel test. These results suggest that the partitioning of genetic diversity in each population might have been influenced not only by water and temperature conditions but also by other factors such as human activities and the Holocene postglacial history of these populations.     

Genetic structure and association mapping of adaptive and selective traits in the east Texas loblolly pine (Pinus taeda L.) breeding populations

First-generation selection (FGS) and second-generation selection (SGS) breeding populations of loblolly pine from east Texas were studied to estimate the genetic diversity, population structure, linkage disequilibrium (LD), signatures of selection and association of breeding traits with a genome-wide panel of 4,264 single nucleotide polymorphisms (SNPs). Relatively high levels of observed (H _o?=?0.178–0.198) and expected (H _e?=?0.180–0.198) heterozygosities were observed in all populations. The amount of inbreeding was very low with many populations exhibiting a slight excess of heterozygotes. The population structure was weak, but F _ST indicated more pronounced differentiation in the SGS populations. As expected for outcrossing natural populations, the genome-wide LD was low, but marker density was insufficient to deduce the decay rate. Numerous associations were found between various phenotypic traits and SNPs, but only a few remained significant after false positive correction. Signatures of diversifying and balancing selection were found in markers representing important biological functions. These results present the first step in the application of marker-assisted selection (MAS) to the Western Gulf Forest Tree Improvement Program (WGFTIP) for loblolly pine and will contribute to the knowledgebase necessary for genomic selection technology.     

Developmental Changes of CuZn- and Mn-Superoxide Dismutase Isozymes in Seedlings and Needles of Norway Spruce (Picea abies L.)

Changes in superoxide dismutase (SOD; EC 1.15.1.1 [EC] ) activityand isozymes were investigated in different developmental stagesof Norway spruce (Picea abies L.). Spruce seeds, seedlings grownin a climate chamber and foliar buds from field-grown treescontained two CuZn-SODs comigrating with SODs I and II previouslyidentified as the chloroplastic and cytosolic SODs in spruceneedles [Krniger et al. (1992) Plant Physiol. 100: 334]. Inaddition one Mn-SOD (SOD III) was identified by insensitivityto cyanide and H₂O₂. Highest total SOD activities were detectedin buds before bud break and in germinating seeds. In seedsand foliar buds SOD II was the major isozyme, whereas SOD Iwas dominant in mature needles. SOD III was present in all developmentalstages of the seedlings, but disappeared in field-grown treesduring bud break and reappeared at the end of summer in matureneedles. These results indicate that the activities of SODsI, II and III in Norway spruce are under independent developmentalcontrol. (Received March 1, 1993; Accepted July 16, 1993)     

Interactive effects of ozone and low UV‐B radiation on antioxidants in spruce (Picea abies) and pine (Pinus sylvestris) needles

To study the role of low UV‐B radiation in modulating the response of antioxidants to ozone, 4‐year‐old pine ( Pinus sylvestris L.) and spruce ( Picea abies L.) seedlings potted in natural soil, were exposed in phytochambers to fluctuating ozone concentrations between 9 and 113 nl 1⁻¹ according to field data recorded at Mt Wank (1175 m above sea level, Bavaria, Germany) and two‐times ambient O₃ levels. UV‐B radiation was either added at a biologically effective level of ca 1.2 kJ m⁻² day⁻¹ , which is close to that found in March at Mt Wank, or was excluded by filters (<0.08 kJ m⁻² day⁻¹). After one growth phase current‐year needles were collected and analysed for antioxidative enzyme activities (superoxide dismutase, SOD, EC 1.15.1.1; catalase, CAT, EC 1.11.1.6; guaiacol peroxidase, POD, EC 1.11.1.7) and soluble antioxidants (ascorbate, glutathione). CAT, POD, ascorbate and glutathione, but not SOD, were increased in needles of both species in response to twice ambient O₃ levels. UV‐B radiation in the presence of ambient O₃ caused an increase in total SOD activity in spruce but had no effects on antioxidants in pine. Twice ambient O₃ levels together with low UV‐B radiation counteracted the O₃‐induced increases in ascorbate and CAT in pine but not in spruce. Under these conditions spruce needles showed the highest antioxidative protection and revealed no indication of lipid peroxidation. Pine needles exposed to UV‐B and elevated O₃ levels showed elevated lipid peroxidation and a 5‐fold increase in dehydroascorbate, suggesting that this species was less protected and suffered higher oxidative stress than spruce.     

Responses in the physiology and biochemistry of Korean pine (Pinus koraiensis) under supplementary UV-B radiation

The effect of supplementary UV-B radiation on Korean pine (Pinus koraiensis Sieb. et Zucc) was investigated. Compared with the control, the T1, T2, and T3 UV-B treatments increased by 1.40, 2.81, and 4.22 kJ m^?2 d^?1, respectively. Gas-exchange parameters, photosynthetic pigment concentrations, contents of secondary metabolites, epicuticular wax, free radical, malondialdehyde (MDA), and the activities of antioxidant enzymes were determined after 40 d of exposure. The concentrations of chlorophyll (Chl) a, Chl b, total Chl, carotenoid (Car), and the ratio Chl a/b in the pine needles were in the following order: T1 > T2 > T3. Compared with the control, the contents of flavonoids and epicuticular wax significantly decreased in all levels of supplementary UV-B radiations (p<0.05). Moreover, the contents of hydrogen peroxide (H₂O₂) and MDA significantly increased with the enhanced UV-B radiations (p<0.05). Korean pine can increase the catalase, ascorbate peroxidase, and superoxide dismutase activities to prevent oxidative stress by supplementary UV-B radiation. However, its defence mechanism is not efficient enough to prevent UV-Binduced damage.     

Influence of elevated ozone and limited nitrogen availability on conifer seedlings in an open-air fumigation system: effects on growth, nutrient content, mycorrhiza, needle ultrastructure, starch and secondary compounds

Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies Karst.) seedlings were exposed to realistically elevated O₃ levels in open‐air experiments over three growing seasons. The total O₃ exposure doses were 1.2 × (1991), 1.5 × (1992) and 1.7 × (1993) ambient levels. During the 1992 and 1993 growing seasons pine and spruce seedlings received two different levels of nitrogen supply. Effects on growth, mycorrhiza formation, needle ultrastructure, primary and secondary compounds were studied. Ozone exposure had only slight effects on biomass production, growth height and nutrient content of studied conifers. Higher nitrogen availability improved growth of the seedlings and resulted in higher concentration of nitrogen in needles. In Scots pine O₃ exposure did not have effects on quantity of total mycorrhizas and short roots, while higher nitrogen availability decreased quantity of mycorrhizas and short roots. In both tree species O₃ exposure induced O₃‐related ultrastructural symptoms, e.g. granulation and dark staining of the chloroplast stroma in the needle mesophyll cells, at both nitrogen availability levels. Ozone exposure and nitrogen availability did not have significant effects on starch concentrations in either tree species. Concentrations of some individual terpenes were higher in O₃‐exposed needles, while concentrations of individual and total resin acids, total phenolics and catechins were not affected by O₃ exposure. Nitrogen availability did not have substantial effects on concentrations of monoterpenes. By contrast, concentrations of some individual and total resin acids were lower in pine needles and higher in spruce needles with higher nitrogen availability, while phenolic concentration in spruce needles decreased at higher nitrogen availability. The results suggest that realistically elevated levels of O₃ in the field can have some negative effects on the mesophyll ultrastructure of conifer needles, but carbon allocation to root and shoot growth and secondary metabolites are not affected substantially.