UV-B and temperature enhancement affect spring and autumn phenology in Populus tremula.

Perennial plants growing at high latitudes synchronize growth and dormancy to appropriate seasons by sensing environmental cues. Autumnal growth cessation, bud set and dormancy induction are commonly driven by the length of photoperiod and light quality, and the responses are modified by temperature. However, although ultraviolet (UV)-B radiation is well known to affect plant growth and development, information on the effects on bud phenology is scarce. We examined the separate and combined effects of enhanced temperature and UV-B on autumnal bud set and spring bud break in female and male clones of Populus tremula in an outdoor experiment in Joensuu, Eastern Finland. Enhancements of UV-B and temperature were modulated to +30% and +2 °C, respectively, from June to October 2012. Enhanced UV-B accelerated bud set, while increased temperature delayed it. For both UV-B and temperature, we found sex-related differences in responsiveness. Temperature increase had a stronger delaying effect on bud maturation in male compared with female clones. Also, male clones were more responsive to UV-B increase than female clones. Increasing autumnal temperature enhanced bud break in spring for both sexes, while UV-B enhanced bud break in male clones. In conclusion, we found that UV-B affected phenological shifts in P. tremula, and that temperature and UV-B affected genders differently.

Responses of soil-grown Scots pine seedlings to experimental warming, moderate nitrogen addition and bark herbivory in a three-year field experiment.

Increased soil nitrogen (N), warming and bark herbivory all are expected to affect boreal forests in the future. We studied the effects of warming (0.5 °C and 4.0 °C above ambient air and soil temperature, respectively), moderate N addition (30 kg N ha-1 y-1) and bark herbivory by large pine weevil (Hylobius abietis L.) on soil-grown Scots pine (Pinus sylvestris L.) seedlings in a three-year (2014-2016) open-air field experiment. Seedling dry mass, root mass fraction (RMF), root morphology, mycorrhizal colonization, mycorrhizal morphotypes, root phenolics and microbial abundance in the rhizosphere area were studied. We observed that both moderate N addition and warming showed interactive effects, and generally improved seedling growth after the three consecutive growing seasons. However, soil dryness was increased due to combined warming and N addition treatment in 2016, and it seemed to limit the shoot growth stimulation as well as increase the dependence of the non-herbivory seedlings on the mycorrhizas. Moderate N addition generally reduced herbivory damage intensity and increased RMF. It also decreased total mycorrhizal colonization rate and increased SRL of the seedlings in 2016, but only in the absence of other factors. In 2016, herbivory affected soil exploration efficiency and mycorrhizal colonization without other factors, and had a tendency to increase root phenolics. There were only minor effects of N addition and herbivory on soil microbial abundances. We conclude that warming and N addition to soil may increase growth in young Scots pine if soil drought or herbivory do not start to limit it; and that in young Scots pine stands moderate bark herbivory are likely to affect roots more than shoots.

Performance and secondary chemistry of two hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) clones in long-term elevated ozone exposure.

The effects of moderately elevated ozone (ca. 35 ppb) on the growth and secondary chemistry of the leaves of two soil-grown Finnish hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) clones with different ozone sensitivities were studied at an open-air exposure field in Kuopio, Finland. Stomatal conductance, photosynthetic rate, and chlorophyll fluorescence were measured during the third growing season. Foliar phenolic concentrations, ergosterol concentration of fine roots, and final dry mass of the trees were determined at the end of the third growing season. Elevated ozone increased the ectomycorrhizal status of the fine roots but had no effect on gas exchange or on the final biomass of either of the clones, indicating equal sensitivity to ozone and no effect of elevated ozone on the intraspecific competitive ability of the clones after three growing seasons. However, in agreement with the data from potted plants of the same clones after two growing seasons, significant differences between the clones were found in all parameters measured. A negative correlation between growth and high concentrations of foliar phenolics indicated that allocation to secondary chemistry also was costly in terms of growth under high resource availability.

Leaf biochemical adjustments in two Mediterranean resprouter species facing enhanced UV levels and reduced water availability before and after aerial biomass removal.

Effects of supplemented UV radiation and diminished water supply on the leaf concentrations of phenols and antioxidants of two Mediterranean resprouter species, Arbutus unedo and Quercus suber, were assessed before and after entire aerial biomass removal. Potted seedlings of both species were grown outdoors for 8 months with enhanced UV-A + UV-B, enhanced UV-A or ambient UV, in combination with two watering conditions (field capacity or watering reduction). After this period, all aerial biomass was removed and new shoots (resprouts) developed for a further 8 months under the two treatments. In general, the investment in leaf phenols was substantially greater in A. unedo than in Q. suber, while Q. suber allocated more resources to non-phenolic antioxidants (ascorbate and glutathione). In response to enhanced UV-B radiation, Q. suber leaves rose their UV-screening capacity mainly via accumulation of kaempferols, accompanied by an increased concentration of rutins, being these effects exacerbated under low-watering conditions. Conversely, A. unedo leaves responded to UV-B radiation reinforcing the antioxidant machinery by increasing the overall amount of flavonols (especially quercetins) in seedlings, and of ascorbate and glutathione, along with catalase activity, in resprouts. Nevertheless, UV effects on the amount/activity of non-phenolic antioxidants of A. unedo resprouts were modulated by water supply. Indeed, the highest concentration of glutathione was found under the combination of enhanced UV-B radiation and reduced watering, suggesting an enlargement of the antioxidant response in A. unedo resprouts. Different biochemical responses to enhanced UV and drier conditions in seedlings and resprouts of these two species might modulate their competitive interactions in the near future.

Interannual variation in UV-B and temperature effects on bud phenology and growth in Populus tremula.

Warming affects phenological processes such as spring bud break and autumnal bud set, and also growth rates of trees. Recently, it has been shown that these physiological processes also may be influenced by the ultraviolet-B (UV-B) part of the solar spectrum, and there are reasons to expect that the two environmental factors induce interactive effects when acting in concert. In this study, our aim was to elucidate how experimental enhancements in temperature and UV-B, alone and in combination, affect growth and seasonal phenology of Eurasian aspen (Populus tremula) over several growing seasons (three years). Moreover, we tested how environmentally induced changes in phenology affect the growth achieved over each season, that is, the importance of a prolonged growing season for growth yield. The plants grew in an outdoor experiment with modulated enhancements of temperature and UV-B during the growing season. Both UV-B and temperature enhancement affected bud set dates, while bud break dates were only affected by temperature enhancement. Temperature delayed bud set in all years, but gradually less over years, while UV-B yielded earlier bud set the first year but showed a delayed response the following years. Bud break was always earlier under temperature enhancement. The experimentally induced extension of the growing season in both ends had a positive effect of growth throughout the three-year period. However, the reduced responsiveness of bud set to both enhancement treatments suggest that the plants gradually acclimated to the modified climate, a finding that should also be investigated for other tree species.

Changes in the concentrations of phenolics and photosynthates in Scots pine (Pinus sylvestris L.) seedlings exposed to nickel and copper.

Studies were done on the effects of elevated soil concentrations of copper (Cu) and (Ni) on foliar carbohydrates and phenolics in Scots pine (Pinus sylvestris L.). Four year-old seedlings were planted in pots filled with metal-treated mineral forest soil in early June. The experimental design included all combinations of four levels of Cu (0, 25, 40 and 50 mg kg(-1) soil dw) and Ni (0, 5, 15 and 25 mg kg(-1) soil dw). Current year needles were sampled for soluble sugar, starch and phenolics at the end of September. Ni increased sucrose concentration in the needles, indicating disturbances in carbohydrate metabolism. Trees exposed to Ni had higher concentrations of condensed tannins compared with controls. In contrast, concentrations of several other phenolic compounds decreased when seedlings were exposed to high levels of Cu or to a combination of Ni and Cu. The results suggest that concentrations of phenolics in Scots pine needles vary in their responses to Ni and Cu in the forest soil.

The effect of elevated carbon dioxide and fertilization on primary and secondary metabolites in birch,Betula pendula (Roth).

Seedlings of European white birch (Betula pendula Roth) were grown in growth chambers for one growth season under four carbon dioxide regimes (350, 700, 1050 and 1400 ppm) and at three fertilization levels (0, 100 and 500 kg ha-1 monthly). The soluble carbohydrates and secondary phenolics in the leaves and stems were analysed. It was found that fertilizer addition reduced the amounts of glucose and fructose while sucrose remained almost unaffected. The sugar content of leaves increased at 700 ppm and 1050 ppm of CO2 and decreased at the highest CO2 concentration (1400 ppm). The amounts of proanthocyanidins and flavonoids in leaves decreased with fertilization addition and increased with CO2 enrichment. The production of simple phenolic glucosides varied according to the fertilization and CO2 treatments. The triterpenoid content of stems seemed to increase with fertilization and CO2-addition. Our results indicate that the production of phytochemicals in the birch seedlings is very sensitive to both fertilization and CO2 addition, which is in agreement with earlier studies, and thus provide some support for the hypothesis of carbon allocation to plant defence when there is an excess of carbon and nutrient. The considerable variation in the production of secondary components may indicate that the synthesis of these defensive metabolites can be regulated by a plant to certain extent, depending on the ability of the plant to acclimate to changes in the physical environment.

Phenolic glycosides govern the food selection pattern of willow feeding leaf beetles.

The relationship between the food selection of four leaf beetle species (Phratora vitellinae, Plagiodera versicolora, Lochmaea capreae, Galerucella lineola) and the phenolic glycosides of willow (Salix spp.) leaves was tested in laboratory food choice experiments. Four willow species native to the study area (Eastern Finland) and four introduced, cultivated willows were tested.The willow species exhibited profound differences in their phenolic glycoside composition and total concentration. The food selection patterns of the leaf beetles followed closely the phenolic glycoside spectra of the willow species. Both the total amount and the composition of phenolic glycosides affected the feeding by the beetles. Phenolic glycosides apparently have both stimulatory and inhibitory influences on leaf beetle feeding depending on the degree of adaptation of a particular insect. Very rare glycosides or exceptional combination of several glycoside types seem to provide certain willow species with high level of resistance against most herbivorous insects. Analogously the average absolute amount of leaf beetle feeding was lower on the introduced willows than on the native species to which the local herbivores have a good opportunity to become adapted.

Increased CO2 and nutrient status changes affect phytomass and the production of plant defensive secondary chemicals in Salix myrsinifolia (Salisb.).

The effect of CO2 enrichment (700 and 1050 ppm) on phytomass, soluble sugars, leaf nitrogen and secondary chemicals of three Salix myrsinifolia clones was studied in plants cultivated at very poor (sand seedlings) and moderate (peat seedlings) nutrient availability and under low illumination. The total shoot phytomass production of sand scedlings was less than 10% of that of the peat seedlings. Carbon dioxide increased the total shoot phytomass of peat seedlings. When the ambient carbon supply was doubled (to 700 ppm) the growth of sand seedlings was slightly enhanced but 1050 ppm CO2 gave growth figures similar to those at the control CO2 level. Leaf nitrogen content and total soluble sugar contents were significantly higher in peat seedlings than in sand seedlings. Leaf nitrogen showed a decreasing trend in relation to CO2 increase. On the other hand, CO2 did not have any clear-cut effect on total sugars. At the control CO2 level the content of salicortin, which is a dynamic phenolic, was higher in the peat seedlings than in the sand seedlings, but salicin showed the opposite trend. CO2 enrichment considerably decreased these phenolics in the peat seedlings. At the control CO2 level, the content of more static phenolics, such as proanthocyanidins, was higher in sand seedlings. An increased carbon supply considerably increased static phenolics in the peat seedlings. Willow defence against generalist herbivores is moderately decreased by enhancement of atmospheric carbon dioxide.

Phenolic compounds of willow bark as deterrents against feeding by mountain hare.

Overwintering mountain hares (Lepus timidus) fed selectively on the shoots of a number of northern willow (Salix spp.) species. The hares preferred certain species over others and mature shoots over juvenile ones. There was a negative correlation between the phenolic glycoside concentration in the twigs and the hares' feeding. This correlation was substantiated by feeding experiments in which oat grains treated with purified phenoglycoside and catechin fractions of willow bark were offered along with untreated control oat grains to free-ranging mountain hares. Both fractions in concentrations normally found in willow twigs inhibited hare feeding. The results suggest that these phenolic compounds play a crucial role in the building up of resistance patterns among the willows. The decline in resistance in mature shoots of tall willows indicates that the juvenile resistance can be, perhaps secondarily, an adaptation against mammals browsing from ground level. Accordingly, low willow species retain a high level of resistance also in maturity.

Slight tissue wounding fails to induce consistent chemical defense in three willow (Salix spp.) clones.

We studied the effects of natural wounding by insects and artificial wounding by clipping with scissors on the phenolic chemistry of two willows, Salix myrsinifolia and Salix pentandra. Half of the blade of a mature leaf was removed from each experimental plant either by allowing insects (chrysomellid beetles) to feed on the leaf or by clipping off half the blade of a leaf with scissors. We also examined the ability of wounded plants to warn neighboring plants of imminent wounding by an airborne signal by maintainign one set of control plants in the room containing the wounded plants and another set of control plants in a room hermetically sealed from the room containing the wounded plants. After 48 h, the experimental leaf and the fourth leaf and eighth leaf upwards in the leaf sequence from the experimental leaf were analyzed for phenols by high-pressure liquid chromatography. The same leaves in the leaf sequence from each control plant were similarly analyzed for phenols. Only one phenol, salicortin in leaves of S. myrsinifolia, increased in concentration in response to defoliation, and the observed response was small. The type of wounding affected this increase in salicortin, with natural wounding by insects causing a greater response than artificial wounding in one S. myrsinifolia clone, and artificial wounding causing a greater response than insect wounding in the other clone. This result indicates that S. myrsinifolia cannot control the effects of diffeeent types of wounding on its leaf secondary chemistry. We also found no indication of airborne warning signals between wounded and unwounded plants that trigger an elevation of leaf defenses in unwounded plants in anticipation of herbivore attack.

Altitudinal and seasonal changes of phenolic compounds in Buxus sempervirens leaves and cuticles.

The variation in the leaf content of phenolic compounds has been related to the UV-B changes of the environment in which plants grow. In this context, we aimed to investigate: a) whether the seasonal and altitudinal changes in the content of phenolic compounds of Buxus sempervirens L. leaves and cuticles could be related to the natural fluctuations in UV-B levels and b) the possible use of specific phenolic compounds as biomarkers of ambient UV-B levels. To achieve these goals we sampled, every three months during one year, leaves of B. sempervirens along an altitudinal gradient. At the lowest and the highest altitudes, we also conducted a UV-exclusion experiment to discern whether the observed changes could be attributed to the natural variation in UV-B. Results show that total phenolic content of leaves was lower in June than in the other sampling dates, which suggests a leaf ontogenic rather than a UV-B effect on the leaf content of these compounds. Regarding the elevational gradient, the overall amount of phenolic acids and neolignan of entire leaves increased with altitude while the total amount of flavonoids in leaf cuticles decreased. However, the lack of a significant effect of our UV-exclusion treatment on the content of these compounds suggests that the observed variations along the altitudinal gradient would respond to other factors rather than to UV-B. Concomitantly, we did not find any phenolic compound in leaves or cuticles of B. sempervirens that could be considered as a biomarker of ambient UV-B levels.

Do elevated temperature and CO2 generally have counteracting effects on phenolic phytochemistry of boreal trees?

Global climate change includes concomitant changes in many components of the abiotic flux necessary for plant life. In this paper, we investigate the combined effects of elevated CO2 (720 ppm) and temperature (+2 K) on the phytochemistry of three deciduous tree species. The analysis revealed that elevated CO(2) generally stimulated increased carbon partitioning to various classes of phenolic compounds, whereas an increase in temperature had the opposite effect. The combined effects of both elevated CO2 and temperature were additive, i.e., canceling one another's individual effects. Obviously, the effects of global climate change on leaf chemistry must simultaneously consider both temperature and CO2. If these results are generally applicable, then the counteracting effect of the temperature is likely to play a major role in alpine, boreal, and arctic zones in determining the balance between populations of plants and herbivores.

The effect of genotype and cultivation method on the total salicylate yield of dark-leaved willows (Salix myrsinifolia).

Ten clones of dark-leaved willows ( Salix myrsinifolia Salisb.) were grown for two years on two different sites, Luikonlahti and Punkaharju, using various combinations of soil cultivation, fertilization and mulch treatments, with the objective of comparing the effect of different cultivation methods on the growth and total salicylate yield. In cultivated soil, the total salicylate yield ranged from 20 to 220 kg/ha, depending on the clone and cultivation method. The use of black polythene mulch and the addition of mineral fertilizer reduced the total salicylate concentrations of several clones. On the other hand, polythene mulch noticeably increased the hectare yield of total salicylates by promoting biomass accumulation.

The effect of the sample preparation method of extractable phenolics of Salicaceae species.

The effect of different tissue prehandling methods on the phenolic content of willow bark, leaves, and twigs was studied. The phenolics were extracted at room temperature, purified, and analyzed by high resolution capillary gas chromatography. Neither oven-drying at a low temperature nor room-drying of fresh leaves and oven-drying of bark produced any qualitative changes in the glucoside composition and only a minor binding effect was seen on the amounts of each glucoside. On the other hand, oven-drying of the intact long twigs and room-drying of the bark are prehandling methods to avoid. Freeze-drying or immediate analysis of frozen leaves lowered the total amount of glucosides and caused considerable qualitative changes to the glucoside composition. 80% acetone was found to be slightly more effective and gentle extraction solvent for all glucosides compared with methanol.

The enzymatic decomposition of salicin and its derivatives obtained from Salicaceae species.

The enzymatic catalysis of the decomposition of Salicaceae phenolic glucosides was tested using almond beta-glucosidase and rabbit and porcine liver esterases. The beta-glucosidase catalyzed the complete hydrolysis of salicin and salicortin, yielding saligenin and glucose. Salicortin also produced (+)-6-hydroxycyclohexen-2-one (6-HCH). The acylglucosides were not decomposed by the beta-glucosidase. Both esterases catalyzed the decomposition of tremulacin, salicortin, and 2'-O-acetylsalicortin, releasing tremuloidin, salicin, and 2'-O-acetylsalicin as the main products, accompanied by 6-HCH and catechol. Tremuloidin and 2'-O-acetylsalicin were quite stable under the esterase hydrolysis, and salicin was not decomposed at all.

Variation in Growth and Secondary Phenolics Among Field-Cultivated Clones of Salix myrsinifolia.

Nine S. MYRSINIFOLIA clones were studied for secondary phenolics and phytomass production in an old, unfertilized hayfield after the second growing season. S. MYRSINIFOLIA is one of the most promising willow species for the production of herbal drug material. Its salicylate content is moderately high in all parts of the shoot. The leaf and stem phytomass varied significantly among clones, but more than 70% of the total variation could be explained by within-clonal phytomass variation. Similarly, the accumulation of salicin, salicortin, chlorogenic acid (+)-catechin, proanthocyanidins, and unknowns varied significantly according to source (tissue, individual, or clone), and in most cases the environmental (within-clonal) variation was nearly as high as the genetic variation (between-clones). In the selection of the best clones for the production of herbal drug material three main factors should be taken account: costs of cultivation, costs for transportation, and costs for production of extracts. A high phytomass clone may be a low-quality clone as far as salicylates are concerned.

Manipulation of the phenolic chemistry of willows by gall-inducing sawflies.

The ability to induce galls on plants has evolved independently in many insect orders, but the adaptive significance and evolutionary consequences of gall induction are still largely unknown. We studied these questions by analyzing the concentrations of various plant defense compounds in willow leaves and sawfly galls. We found that the galls are probably nutritionally beneficial for the sawfly larvae, because the concentrations of most defensive phenolics are substantially lower in gall interiors than in leaves. More importantly, changes in chemistry occur in a similar coordinated pattern in all studied willow species, which suggests that the insects control the phenolic biosynthesis in their hosts. The resulting convergence of the chemical properties of the galls both within and between host species indicates that the role of plant chemistry in the evolution of host shifts may be fundamentally less significant in gallers than in other phytophagous insects.

Testing the effects of drying methods on willow flavonoids, tannins, and salicylates.

In this study, we compared the effects of several preservation methods on the secondary phenolics of the mature leaves of purple willow (Salix purpurea L., salicaceae) with results obtained with fresh leaf analyses. Conventional freeze-drying, in which the leaves were first frozen with liquid nitrogen and then placed in a freeze-dryer, produced substantial qualitative and quantitative changes in purple willow flavonoids and salicylates. Modified freeze-drying, in which leaves were put into a freeze-dryer without being prefrozen, gave concentrations that, for most secondary components, were comparable with those found in fresh leaves. Reducing the freeze-dryer chamber temperature hindered the decomposition of phenolics in prefrozen leaves and in leaves dried without prefreezing. Heat drying induced substantial changes in the composition of all phenolics, except for apigenin-7-glucoside. Vacuum drying at room temperature gave the highest concentrations for nearly all phenolics, while room-drying with desiccation gave results that were comparable with those obtained by fresh leaf analyses.

Ontogenic development of chemical defense by seedling resin birch: Energy cost of defense production.

Whether production of chemical defenses by plants is or is not an energetically costly process is an important, but unresolved, question in chemical ecology. We suggest studies of the ontogenetic development of plant defense systems can help resolve the question. As an example of this approach to the cost question, we explore the problems associated with production of immobile chemical defenses that defend juvenile resin birches against browsing by mammals. From this exploration we draw two conclusions: (1) Shortly after germination, production of chemical defenses by small-seeded species, such as birch, is energetically costly. (2) Opposing selection for defense versus competitive ability in the seedling stage of birch has resulted in a trade-off in allocation of carbon to production of immobile chemical defense versus allocation of carbon to production of storage reserves. We suggest this trade-off results in a large indirect cost of defense because carbon used for production of immobile chemical defenses is unavailable for support of growth in the future, but stored carbon can be used to support future growth.