UV-B induction of flavonoid biosynthesis in Scots pine (Pinus sylvestris L . ) seedlings

 Cultivation of Scots pine (Pinus sylvestris L.) seedlings under simulated global radiation including the UV-B band (280 – 320 nm; 220 mW m^–2 UV-B_BE) led to increased formation of the diacylated flavonol glucosides 3″,6″-di-p-coumaroyl-astragalin and 3″,6″-di-p-coumaroyl-isoquercitrin in primary and cotyledonary needles, respectively. 3″,6″-Di-p-coumaroyl-astragalin was also the main constitutive diacylated flavonol glucoside in both needle types. This compound predominantly accumulated in primary needles upon UV-B irradiation, and reached concentrations of 2.4 μmol g^–1 fresh weight (fw). Its concentration was only weakly affected in cotyledonary needles. 3″,6″-Di-p-coumaroyl-isoquercitrin was mainly induced in cotyledonary needles with maximum concentrations of 0.8 to 0.9 μmol g^–1 fw, but was virtually unaffected in primary needles under the same irradiation conditions. Pulse labelling with L-(U-¹⁴C)phenylalanine revealed that these metabolites were formed de novo. Phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone synthase (EC 2.3.1.74) were only slightly induced by the UV-B treatment. The results described here represent the first report on UV-B-induced flavonoid biosynthesis in a conifer species. Received: 5 December 1995 / Accepted: 20 March 1996     

Flavonol 3-O-glycoside hydroxycinnamoyltransferases from Scots pine (Pinus sylvestris L.)

Flavonol 3-O-glucosides esterified with ferulic or p-coumaric acid at positions 3' and 6' are the major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles. The last steps in the biosynthesis of these compounds are catalyzed by enzymes that transfer the acyl part of hydroxycinnamic acid CoA esters to flavonol 3-O-glucosides. A newly developed enzyme assay revealed three flavonol 3-O-glucoside hydroxycinnamoyltransferases (HCTs) in Scots pine needles with specificities for positions 3', 4' or 6'. The positions of the acyl groups were identified by cochromatography with reference compounds and by NMR spectroscopy. The enzymes were characterized by molecular mass, isoelectric point, and also pH and temperature optima. Substrate specificities for flavonol glycosides and hydroxycinnamic acid CoA esters as well as kinetic properties of 3'- and 6'HCT suggested that acylation preferably occurs with glucosides and p-coumaroyl-CoA. In addition, acylation takes place in a well-defined order, beginning at position 6' followed by acylation at position 3'. These results give the first detailed characterization of flavonol 3-O-glycoside HCTs involved in the protection of plant tissues against UV-B (280-315 nm) radiation.     

Seasonal accumulation of ultraviolet-B screening pigments in needles of Norway spruce (Picea abies (L.) Karst.)

Conifer needles are highly effective in screening ultraviolet-B radiation (280–320 nm). This ability is mainly attributed to the presence of flavonoids and hydroxycinnamic acids in the epidermal tissue. In two field cabinet experiments with two different clones of Norway spruce we assessed the seasonal accumulation of UV-B screening pigments under near-ambient, and close-to-zero UV-B irradiation. At the beginning of needle development, i.e. in June, kaempferol 3- O -glucoside was the dominant UV-B screening pigment. It was replaced during needle differentiation by the more effective diacylated flavonol glucosides, particulary kaempferol 3- O -(3",6"- O -di- p -coumaroyl)-glucoside, which reached highest concentrations in July. In addition to the soluble pool of diacylated flavonol glucoside derivatives, a cell wall-bound UV-B screen in the epidermal cell walls was formed during needle differentiation, consisting mainly of p -coumaric acid and kaempferol 3- O -glucoside. An effect of UV-B radiation on the accumulation of diacylated flavonol glucosides was only observed in 1996 with clone 2, when the concentrations of kaempferol 3- O -(3",6"- O -di- p -coumaroyl)-glucoside were significantly higher in July and August under field, and near-ambient than under close-to-zero UV-B irradiance. For wall-bound p -coumaric acid and kaempferol 3- O -glucoside UV-B radiation enhanced the concentrations of these compounds by approximately 20% in relation to the concentrations in close-to-zero UV-B-treated plants in both field cabinet experiments.     

Effects of ozone on organic acids in needles of Norway spruce and Scots pine

Summary The effect of ozone, needle age, and season on the pH of homogenate and acid contents of Scots pine and Norway spruce needles is presented. In addition enzyme activities of cytochrome C-oxidase (cyt. C-ox), phosphoenolpyruvate-carboxylase (PEPC), shikimic acid-dehydrogenase (SHDH) and malate-dehydrogenase (MDH) were measured in Scots pine needles. In freshly sprouted spruce needles the level of quinic acid is high and the pH of the needle homogenate is low. Shikimic acid starts at low levels, increases with increasing needle age and becomes dominant, whereas the quinic acid content decreases. Malic acid has a marked seasonal trend; no trend was found in citric acid. Ozone (200 g/m³) decreased shikimic acid and quinic acid, whereas pH, malic acid and citric acid increased. Ozone (100 g/m³) had a similar effect, except in the current-year spruce needles. In Scots pine needles ozone led to increased enzymatic activities of cyt. C-ox, PEPC and SHDH, and a decrease in the activity of MDH. This effect was more pronounced in summer than in autumn, but the visible damage was greater in autumn. These effects can be found with other stresses and are not specific for ozone.     

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.     

Neodiprion sertifer defoliation causes long-term systemic changes of oxidative enzyme activities in Scots pine needles

The aim of the experiments reported here was to study possible long-term effects of Neodiprion sertifer Geoff. (Hymenoptera: Diprionidae) herbivory, or artificial defoliation, on oxidative enzyme activities in Scots pine (Pinus sylvestris L.) needles as a consequence of induced defense responses. During year 1 (the first season), defoliation by N. sertifer, which feeds on previous season’s needles, did not result in statistically significant changes in polyphenol oxidase activity in the current year’s needles. In contrast, defoliation did lead to increased peroxidase activity in those needles. In the second season (year 2) N. sertifer defoliation of pine seedlings, also defoliated in the previous season either by larvae or artificially, resulted in a decrease of peroxidase activity in the current year’s needles. No significant differences between treatments carried out in year 1 were found in year 2 for peroxidase activity in the previous year’s needles. However, defoliation in year 1 by N. sertifer resulted in decreased needle consumption, and higher mortality of larvae, in year two. These results indicate the existence of long-term changes in needle oxidative enzyme activities as a consequence of N. sertifer feeding.     

Nitrogen metabolism of Douglas fir and Scots pine as affected by optimal nutrition and water supply under conditions of relatively high atmospheric nitrogen deposition

Nitrogen metabolism of the needles of 40-year-old Douglas fir and Scots pine trees, growing in two forest stands on cation-poor and acidic sandy soil with a relatively high atmospheric nitrogen deposition was studied. The composition of the free amino acid (FAA) pool, the concentrations of total nitrogen and soluble protein and the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were determined in the needles. An excessive nitrogen supply by a high atmospheric nitrogen deposition in both forest stands was indicated by the high concentrations of total nitrogen and the amino acids arginine, glutamic acid, glutamine and aspartic acid in control trees. In addition the effect of optimal nutrition and water supply (fertigation) on the needle nitrogen metabolism was evaluated. The total concentration of the FAA pool in needles of both tree species was lower in the fertigated than in the non-fertigated (control) trees, except for 1-year-old needles of Scots pine, in which the concentration after fertigation did not differ from the control. The lower total FAA concentration in the fertigated trees could be attributed to arginine, the concentration of which was on average 60% lower than in the control. Neither the concentration of soluble protein nor the activity of GS were influenced by fertigation. The activity of GDH in fertigated trees only differed significantly from the control in October. Scots pine needles had higher concentrations of protein (50%) and higher activities of GS (44%) and GDH (25%) than Douglas fir needles. Possible explanations for the lower vitality of Douglas fir compared to Scots pine are given.     

Needle ontogeny of mature Scots pines under enhanced UV-B radiation

The aim of this work was to test our hypothesis that pine needles protect themselves against UV-B radiation via anatomical changes in the epidermal layer. This could lead to needle growth reductions if large quantities of assimilates are allocated for the epidermal protective mechanisms at the expense of photosynthetic area. Effects of enhanced UV-B radiation on the needle ontogeny of mature Scots pines (Pinus sylvestris L.) were studied during the second season of a field experiment. Depending on the season and the time of the year (1996-1997), the enhanced UV-B irradiance varied from 0.92 to 5.09 kJ m^-2 day^-1 UV-B_BE compared to 0.54-2.44 kJ m^-2 day^-1 UV-B_BE of ambient radiation. It was found that UV-B treatment accelerated the early development of needles. In 6-day-old enhanced UV-B-treated needles, mesophyll and hypodermic cells were fully differentiated, whereas in ambient-treated needles, no lobate mesophyll cells were seen and hypodermic cells had not yet developed. In fully grown needles, no accelerated differentiation was seen, except that the epidermal cross-sectional area was smaller. The continuation of the experiment will show if such a significant difference only occurs irregularly and incidentally or if it is of consistent significance for needles.     

Effects of long-term open-air exposure to fluoride,nitrogen compounds and SO2 on visible symptoms,pollutant accumulation and ultrastructure of Scots pine and Norway spruce seedlings

 Effects of SO₂, aqueous fluoride (NaF) and a solution of nitrogen compounds (NH₄NO₃) on the visible symptoms, pollutant accumulation and ultrastructure of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.] seedlings were studied in an open-air experiment lasting for 3 consecutive years. Visible injury symptoms were most pronounced in combination exposures and whenever F was applied. Visible symptoms correlated well with needle pollutant concentrations. Exposure to NaF increased needle F contents particularly when F was applied with SO₂ or NH₄NO₃. This suggests that a reduction in N or SO₂ emissions, in F polluted areas, could improve the condition of conifers via decreased accumulation of phytotoxic F in the needles. Norway spruce needles accumulated 2 – 10 times as much S and F as those of Scots pine. Microscopic observations showed various changes in the needle mesophyll cell ultrastructure. In both species, exposure to SO₂ increased significantly the amount of cytoplasmic vacuoles, suggesting detoxification of excess sulphate or low pH. F treatments resulted in a significant enlargement of plastoglobuli in Scots pine and a darkening of plastoglobuli in Norway spruce. All exposures enhanced the accumulation of lipid bodies. An increased portion of translucent plastoglobuli was most pronounced in N treatments. Many of the ultrastructural changes and visible symptoms appeared only as number of years exposed increased, indicating that long-term experiments are needed. Both visible symptoms and ultrastructural changes pointed to the more pronounced sensitivity of Norway spruce compared to Scots pine. Ultrastructural results mostly supported earlier qualitative observations of F, N and SO₂ effects on needle mesophyll cell ultrastructure. However, no reduction of thylakoids in SO₂ containing exposure or curling of thylakoids in F exposure could be detected in the present study. Received: 5 December 1994 / Accepted: 28 April 1995     

Changes in leaf expansion and epidermal screening effectiveness in Liquidambar styraciflua and Pinus taeda in response to UV-B radiation

Absorption or screening of ultraviolet-B (UV-B) radiation by the epidermis may be an important protective method by which plants avoid damage upon exposure to potentially harmful UV-B radiation. In the present study we examined the relationships among epidermal screening effectiveness, concentration of UV-absorbing compounds, epidermal anatomy and growth responses in seedlings of loblolly pine (Pinus taeda L.) and sweetgum (Liquidambar styraciflua L.). Seedlings of each species were grown in a greenhouse at the University of Maryland under either no UV-B radiation or daily supplemental UV-B radiation levels of 4, 8 or 11 kJ m^?2 of biologically effective UV-B (UV-B_BE) radiation. Loblolly pine seedlings were subsequently grown in the field under either ambient or supplemental levels of UV-B radiation. At the conclusion of the growing season, measurements of epidermal UV-B screening effectiveness were made with a fiber-optic microprobe. In loblolly pine, less than 0.5% of incident UV-B radiation was transmitted through the epidermis of fascicle needles and about 1% was transmitted in primary needles. In contrast, epidermal transmittance in sweetgum ranged from about 20% in leaves not preconditioned to UV-B exposure, to about 10% in leaves grown under UV-B radiation. The concentration of UV-absorbing compounds was unaffected by UV-B exposure, but generally increased with leaf age. Increases in epidermal thickness were observed in response to UV-B treatment in loblolly pine, and this accounted for over half of the variability in UV-B screening effectiveness. In spite of the low levels of UV-B penetration into the mesophyll, delays in leaf development (both species) and final needle size (loblolly pine) were observed. Seedling biomass was reduced by supplemental UV-B radiation in loblolly pine. We hypothesize that the UV-induced growth reductions were manifested by changes in either epidermal anatomy or epidermal secondary chemistry that might negatively impact cell elongation.     

Variation in needle terpenoids among Pinus sylvestris L. (Pinaceae) provenances from Turkey

Variation of terpenes and resin acids in needles of young Scots pine (Pinus sylvestris L.) seedlings from nine different provenances in Turkey was investigated. The provenances represent 1200-km West to East and 400-km South to North transects. Seven monoterpenes and two sesquiterpenes were reported in the needles of pines studied. Generally, the kinds of terpenes were similar but the relative amount of some compounds differed among the origins. The major components of the monoterpene fraction in Turkish sources were α-pinene (84.8%), β-pinene (4.1%) and limonene (3.0%), corresponding to 91.9% of the crude needle extract. In a PCA-analysis, 3-carene, myrcene and terpinolene in seedlings from Turkish provenances were quite low and thus, they were clearly different from a Northern European Scots pine provenance from Finland. In the resin acid fraction, abietic acid (62.4%) and dehydroabietic acid (16.1%) were the most abundant constituents in the needles of the Scots pine from Turkish provenances.     

The influence of elevated ultraviolet-B radiation (UV-B) on tissue quality and decomposition of loblolly pine (Pinus taeda L.) needles

Stratospheric ozone depletion is expected to elevate the influx of ultraviolet-B radiation (UV-B) to the biosphere. Increased levels of UV-B may, in turn, alter important ecosystem processes such as decomposition. Previous studies have shown that growth under elevated UV-B can alter leaf quality in angiosperm species and thereby indirectly change subsequent rates of leaf decay. In this experiment, we determined if elevated UV-B would alter the chemical composition and decay of needle tissue from two seed sources of the gymnosperm Pinus taeda L. Maryland and Virginia seed sources of P. taeda were grown in the field for 3 years beneath lampbanks supplying either ambient, low elevated or high elevated UV-B. These levels of UV-B corresponded to 0, 16 and 25% stratospheric ozone depletion at the experimental site in Beltsville, MD (39 degrees N). Needles were collected from six randomly chosen plants for each combination of seed source and UV-B level. The needle samples were analyzed for total C and N, UV-B absorbing compounds, and carbon fractions. Decay rates were also determined by measuring rates of CO(2) evolution from needle material decomposed under laboratory conditions. UV-B did not significantly alter the chemical composition of needles from the Virginia seed source. In contrast, needles from the Maryland seed source tended to have elevated lignin/N ratios and a lower holocellulose content when grown under the highest level of UV-B. Furthermore, while needles from the Virginia pines did not have UV-B altered decay rates, Maryland needles grown under low elevated UV-B conditions decomposed 36% more rapidly than needles from other treatments. Results from this experiment illustrate at least three characteristics about the indirect effect of UV-B on decomposition, (1) UV-B can modify decomposition of tissue from gymnosperms as well as angiosperms; (2) UV-B effects on tissue chemistry and decay may not only be species-specific but also seed-source specific; and (3) UV-B effects on decomposition may not increase with increasing UV-B dose.     

The effects of enhanced UV-B radiation on physiological activity and growth of Norway spruce planted outdoors over 5 years

The responses of Norway spruce [Picea abies (L.) Karst.] to enhanced UV-B radiation during the 5-year treatment performed outdoors have been subjected to ecophysiological and growth analysis. The plants were exposed to UV-B radiation, simulating 17% ozone depletion. Ecophysiological parameters were monitored three times a year on three needle age classes, while growth was analysed at the end of each growth season. Spruce exhibited great variability in the amounts of photosynthetic pigments and methanol-soluble UV-B absorbing compounds, light use efficiency, photosynthesis and respiratory potential. The needle, branch and plant biomass production was not significantly affected during the 5-year treatment. The repeated-measures procedure comparing growth parameters through subsequent seasons, revealed a decrease of branch diameter under enhanced UV-B, which could be interpreted as a cumulative UV-B effect. The effects of UV-B radiation depended on needle development stage, interaction with environmental conditions and stresses. A reduced negative effect of UV-B radiation was observed during the prolonged drought in 2003, which was hypothesised as an alleviating effect. The tolerance of Norway spruce to elevated UV-B was to a large extent due to the high content of methanol-soluble UV-B absorbing compounds that was related neither to environmental conditions, including UV-B dose, nor to the developmental stage of the needles. The current year needles exhibited a tendency to increased production of UV-B absorbing compounds under elevated UV-B radiation. The outdoor study performed under variable environmental conditions showed great complexity of spruce response to enhanced UV-B.     

Flavonoids from Pinus sylvestris needles and their variation in trees of different origin grown for nearly a century at the same area

Flavonoids in needles of Scots pine planted in 1912–1914 in Poland from seeds originating from different parts of Europe, were isolated, chemically characterised and analysed by HPLC. It was shown that flavonoid profiles were similar in all tested populations and were different from those previously reported for Scots pine seedlings. They included taxifolin, taxifolin 3′-O-glucoside, quercetin as well as quercetin 3-O-glucoside and 3′-O-glucoside. The quercetin 3-O-glucoside could be found only in a trace amount in all samples and quercetin 3′-O-glucoside appeared in all samples regardless their origin. The relative concentration of taxifolin 3′-O-glucoside, quercetin, taxifolin and total flavonoids showed dependence on the origin of seeds; needles from high latitude populations contained smaller amounts of these compounds. Presented data clearly indicate that Scots pine contain glycosidases specific for glycosylation at C-3′ rather than at C-3. Besides, they indicate that long lasting influence of similar environmental factors is not able to change genetic regulatory systems responsible for flavonoid biosynthesis.     

Long-term effects of exogenous methyl jasmonate application on Scots pine (Pinus sylvestris) needle chemical defence and diprionid sawfly performance

Scots pine [ Pinus sylvestris L. (Pinaceae)] trees with four different seed origins were exposed to exogenous applications of the elicitor, methyl jasmonate (MeJA), for three consecutive years. We studied the effects of MeJA on needle chemistry (including monoterpenes, sesquiterpenes, and tricyclic resin acids), plant growth, and the performance of two diprionid sawflies, the European pine sawfly ( Neodiprion sertifer Geoffr.) and the common pine sawfly ( Diprion pini L.) (both Hymenoptera: Diprionidae). In general, foliar MeJA application affected the whole range of needle secondary chemistry with significantly higher concentrations of two monoterpenes, β-pinene and limonene, in particular. Furthermore, for some seed origins the growth rates of N. sertifer and D. pini larvae were lower on needles of MeJA-treated plants with either high total terpene or high resin acid concentrations. However, inconsistencies in diprionid sawfly performance within each studied Scots pine origin suggest genetic variance in needle secondary chemistry. The differences between selected seed origins and notably variable responses to MeJA application imply that adaptation of the seed to new conditions may have had an impact on secondary chemistry and, thus, on insect performance. Finally, our results suggest that modification of Scots pine defence by a low-concentration exogenous elicitor affects the production of terpenoids in the newly growing needles, leading to poorer pine sawfly performance in origins with high terpenoid content, while not harming the growth of Scots pine trees.     

A review of ozone responses in Scots pine (Pinus sylvestris)

The data on Scots pine responses to elevated ozone (O₃) mainly come from experimental studies with young seedlings and trees. Based on the 38 experiments reviewed here, Scots pine may be considered as an O₃-sensitive conifer species, with mature pines more sensitive than younger trees. This is due to their relatively small proportion of current (c) year needles with the highest photosynthetic capacity. Moreover, young seedlings and trees seem to acclimate to slightly elevated realistic O₃ exposures, and hence do not often exhibit growth and biomass reductions in spite of the visible and microscopic needle injuries and changes in needle chemistry. The O₃ sensitivity in Scots pine is thought to relate to impaired water status due to the malfunction of stomata and subsequent increase in transpiration. This may lead to reduced wood biomass in the long term, if Scots pines try to maximise the biomass of c needles and root biomass to maintain efficient water and nitrogen (N) supply to support the photosynthesis of c needles. Tree water status also contributes to the spring-time recovery of photosynthesis. We call especially for studies on atmosphere–needle surface interaction that would yield novel information on the impact of O₃ on epicuticular waxes and stomatal functioning, which both regulate O₃ flux and tree water status and hence also modify photosynthesis. The need for flux-based field studies is especially important in the light of future climatic change, since the risk presented by O₃ to Scots pine forests in Northern and Central Europe seems to be equal.     

The effects of ultraviolet-B radiation on loblolly pine. I. Growth, photosynthesis and pigment production in greenhouse-grown seedlings

One-year old loblolly pine ( Pinus taeda L.) seedlings were grown in an unshaded greenhouse for 7 months under 4 levels of ultraviolet-B (UV-B) radiation simulating stratospheric ozone reductions of 16, 25 and 40% and included a control with no UV-B radiation. Periodic measurements were made of growth and gas exchange characteristics and needle chlorophyll and UV-B-absorbing-compound concentrations. The effectiveness of UV-B radiation on seedling growth and physiology varied with the UV-B irradiance level. Seedlings receiving the lowest supplemental UV-B irradiance showed reductions in growth and photosynthetic capacity after only 1 month of irradiation. These reductions persisted and resulted in lower biomass production, while no increases in UV-B-absorbing compounds in needles were observed. Seedlings receiving UV-B radiation which simulated a 25% stratospheric ozone reduction showed an increase in UV-B-absorbing-compound concentrations after 6 months, which paralleled a recovery in photosynthesis and growth after an initial decrease in these characteristics. The seedlings grown at the highest UV-B irradiance (40% stratospheric ozone reduction) showed a more rapid increase in the concentration of UV-B-absorbing compounds and no effects of UV-B radiation on growth or photosynthetic capacity until after 4 months at this irradiance. Changes in photosynthetic capacity were probably the result of direct effects on light-dependent processes, since no effects were observed on either needle chlorophyll concentrations or stomatal conductance. Further studies are necessary to determine whether these responses persist and accumulate over subsequent years.     

Seasonality of Glutathione Dynamics in Scots Pine and Bilberry

Abstract: Seasonal changes in the glutathione status of Scots pine ( Pinus sylvestris L .) needles and bilberry ( Vaccinium myrtillus L.) stems and leaves were investigated during 1995 and 1997–1998, respectively. The glutathione concentration, the proportion of reduced glutathione (GSH%) and glutathione reductase (GR) activity were measured at monthly intervals throughout one year. The glutathione concentration and GSH% in current year's Scots pine needles were highest during late summer, but decreased during autumn. It is suggested that exchange reactions between free and protein-bound glutathione may be involved in this reduction. Both metabolites decreased towards summer in the previous year's needles. GR activity exhibited the lowest values in summer and the highest values in winter in both needle generations. In bilberry stems, glutathione peaked during midwinter, while GSH% remained high and relatively constant throughout the year. GR activity was highest in August, after which it gradually declined. In bilberry leaves, the total glutathione concentration was high in early summer. This peak was followed by a sharp decrease during midsummer. GR activity in the leaves was very low, but showed a clear increase towards autumn. Obviously, glutathione metabolism showed considerable seasonal variation in Scots pine needles and in bilberry leaves and stems, but the pattern of variation differed between the studied species and organs.