1,6-Dehydropinidine Is an Abundant Compound in Picea abies (Pinaceae) Sprouts and 1,6-Dehydropinidine Fraction Shows Antibacterial Activity against Streptococcus equi Subsp. equi.

Knowledge about the defensive chemistry of coniferous trees has increased in recent years regarding a number of alkaloid compounds; in addition to phenolics and terpenes. Here, we show that Norway spruce (Picea abies (L.) H. Karst.), an important boreal zone tree species; accumulates 1,6-dehydropinidine (2-methyl-6-(2-propenyl)-1,6-piperideine) in its needles and bark. We reanalyzed previously published GC-MS data to obtain a full picture of 1,6-dehydropinidine in P. abies. 1,6-dehydropinidine appeared to especially accumulate in developing spring shoots. We used solid-phase partitioning to collect the alkaloid fraction of the sprouts and thin-layer chromatography to purify 1,6-dehydropinidine. The antibacterial properties of the 1,6-dehydropinidine fraction were tested using a broth microdilution method; with Streptococcus equi subsp. equi as a model organism. Based on our results 1,6-dehydropinidine is common in alkaloid extractions from P. abies (0.4 ± 0.03 mg g-1 dw in mature needles) and it is especially abundant in young spruce shoots (2.7 ± 0.5 mg g-1 dw). Moreover; 1,6-dehydropinidine extracted from P. abies sprouts showed mild antibacterial potential against Streptococcus equi subsp. equi (MIC 55 µg mL-1). The antibacterial activity of a plant compound thought of as an intermediate rather than an end-product of biosynthesis calls for more detailed studies regarding the biological function of these coniferous alkaloids.

Variability in the composition of phenolic compounds in winter-dormant Salix pyrolifolia in relation to plant part and age.

The phenolic phytochemicals of winter-dormant Salix pyrolifolia were determined from the vegetative buds, and the bark and wood of different-aged twigs by HPLC-DAD and UHPLC-QTOF-MS analyses. All the plant parts were composed of salicylate glucosides and the other Salix-specific, simple phenolic glucosides as well as of phenolic acids, flavonoids and the high molecular-weight condensed tannins. The flavonoid composition was most diverse in buds and they also contained a large amount of chlorogenic acid (5-caffeoylquinic acid IUPAC), while salicylate glucosides and simple phenolic glucosides predominated in bark. The wooden interior part of the twigs contained fewer components and the lowest concentrations of compounds. Salicortin was the main compound in winter-dormant S. pyrolifolia (over 10% of bark biomass), but the concentrations of picein, salireposide, isosalipurposide, catechin and condensed tannins were also high. The flavonoid composition was highly naringenin- and quercetin-biassed. The composition of phytochemicals was organ-specific and remained relatively similar between different-aged trees. However, there were compound-specific fluctuations in the concentrations of phytochemicals with the age of the trees and within plant parts. Generally, the one-year-old plants differed from the older trees in their high concentration of condensed tannins in all the plant parts studied and in the highest concentration of isosalipurposide in bark, while the total amounts of salicylate glucosides in plant parts, and of naringenin glucosides in buds, tended to be highest in 20 year-old-trees.

Effects of long-term UV-exposure and plant sex on the leaf phenoloxidase activities and phenolic concentrations of Salix myrsinifolia (Salisb.).

The accumulation of flavonoids on the leaf surface is a well-characterized protective mechanism against UV-B radiation. Other protective mechanisms, such as the induction of antioxidative enzymes and peroxidase-mediated lignification may also be important. The effects of UV-B radiation have mainly been considered in short-term studies, whereas ecologically more relevant long-term field studies are still rare. Here we examined the effects of long-term exposure to enhanced UV-B radiation on the activities of two antioxidative enzymes, polyphenol oxidase (PPO; EC 1.10.2.2 and EC 1.14.18.1) and guaiacol peroxidase (POD; EC 1.11.1.7), as well as the phenolic concentrations in two sexes of the dioecious species, Salix myrsinifolia. After three consecutive growth seasons with enhanced UV-B radiation, we found that PPO activity was decreased by UV radiation in male plants, which might explain their lower UV-B tolerance when compared to female plants. In addition, male plants had higher specific activity than did female plants under ambient conditions, supporting the idea that males of S. myrsinifolia are generally more growth-oriented than females. By contrast, neither UV treatment nor sex had significant effects on the POD activities of willows. Gender differences in the concentrations of phenolic compounds are in line with the general concept that males are less well defended than females. We suggest that the inability to increase PPO and POD activity, along with lower accumulation of UV-B absorbing compounds under UV-B exposure, might be one of the reasons why males had thinner leaves and were less tolerant of UV-B than were females.

A Seven-Year Study of Phenolic Concentrations of the Dioecious Salix myrsinifolia.

In boreal woody plants, concentrations of defensive phenolic compounds are expected to be at a high level during the juvenile phase and decrease in maturity, although there is variation between plant species. Females of dioecious species, like most of the Salicaceae, are expected to invest their resources in defense and reproduction, while males are expected to be more growth-oriented. We studied age- and sex-dependent changes in leaf and stem phenolics, and in height and diameter growth in a dioecious Salix myrsinifolia plants over a seven-year time period. In addition, we registered flowering as well as rust damage in the leaves. From the first year and throughout ontogenetic development from juvenile to adult phases, there was no significant change in the concentrations of any of the studied compounds in the leaves of S. myrsinifolia. In the stems, the concentrations of six out of 43 identified compounds decreased slightly with age, which may be partly explained by dilution caused by the increment in stem diameter with age. The fairly steady chemistry level over seven years, accompanied by moderate genotypic phenolic variation, indicates important roles of chemical defenses against herbivory for this early-successional species.

Interactive effects of supplemental UV-B and temperature in European aspen seedlings: Implications for growth, leaf traits, phenolic defense and associated organisms.

Past studies reveal opposite effects of elevated UV-B and temperature on plant growth and concentrations of UV-B absorbing compounds, yet few studies have dealt with the combined and interactive effects of these two climate change factors on woody dioecious plants. We investigated the interactive effects of UV-B and temperature treatments on growth, leaf traits and phenolic concentrations in Populus tremula L. (European aspen) seedlings. We also considered the consequences of these effects on their associated organisms: herbivorous insects, rust pathogens, the presence of endophytic fungi and whether or not the responses differ between genders and genotypes. Supplemental temperature and UV-B were modulated to +2 °C and +30.77% above ambient conditions, respectively. Warming increased growth, photosynthesis and foliar nitrogen concentration but reduced leaf thickness and phenolic concentrations. On the other hand, supplemental UV-B increased total phenolic glycosides, mainly flavonols and phenolic acids, and partially counteracted the positive effects of warming on growth. Fast growing genotypes were less susceptible to the growth-reducing effect of combined UVB + T, less infected with rust disease and less prone to insect damage probably due to their higher salicylate and lower nitrogen concentrations. Under ambient temperature, the males of European aspen were taller and had bigger leaves than the females, while under elevated temperature, females grew bigger and, under UV-B, had more tremulacin than males. The multiple interactive effects of UV-B and temperature on growth, leaf traits and phenolic compounds, highlight the importance of multifactor experiments as a realistic predictor of plant responses to climate change.

Sex-related differences in growth and carbon allocation to defence in Populus tremula as explained by current plant defence theories.

Plant defence theories have recently evolved in such a way that not only the quantity but also the quality of mineral nutrients is expected to influence plant constitutive defence. Recently, an extended prediction derived from the protein competition model (PCM) suggested that nitrogen (N) limitation is more important for the production of phenolic compounds than phosphorus (P). We aimed at studying sexual differences in the patterns of carbon allocation to growth and constitutive defence in relation to N and P availability in Populus tremula L. seedlings. We compared the gender responses in photosynthesis, growth and whole-plant allocation to phenolic compounds at different combination levels of N and P, and studied how they are explained by the main plant defence theories. We found no sexual differences in phenolic concentrations, but interestingly, slow-growing females had higher leaf N concentration than did males, and genders differed in their allocation priority. There was a trade-off between growth and the production of flavonoid-derived phenylpropanoids on one hand, and between the production of salicylates and flavonoid-derived phenylpropanoids on the other. Under limited nutrient conditions, females prioritized mineral nutrient acquisition, flavonoid and condensed tannin (CT) production, while males invested more in above-ground biomass. Salicylate accumulation followed the growth differentiation balance hypothesis as low N mainly decreased the production of leaf and stem salicylate content while the combination of both low N and low P increased the amount of flavonoids and CTs allocated to leaves and to a lesser extent stems, which agrees with the PCM. We suggest that such a discrepancy in the responses of salicylates and flavonoid-derived CTs is linked to their clearly distinct biosynthetic origins and/or their metabolic costs.

Combination treatment of elevated UVB radiation, CO2 and temperature has little effect on silver birch (Betula pendula) growth and phytochemistry.

Elevations of carbon dioxide, temperature and ultraviolet-B (UBV) radiation in the growth environment may have a high impact on the accumulation of carbon in plants, and the different factors may work in opposite directions or induce additive effects. To detect the changes in the growth and phytochemistry of silver birch (Betula pendula) seedlings, six genotypes were exposed to combinations of ambient or elevated levels of CO2 , temperature and UVB radiation in top-closed chambers for 7 weeks. The genotypes were relatively similar in their responses, and no significant interactive effects of three-level climate factors on the measured parameters were observed. Elevated UVB had no effect on growth, nor did it alter plant responses to CO2 and/or temperature in combined treatments. Growth in all plant parts increased under elevated CO2 , and height and stem biomass increased under elevated temperature. Increased carbon distribution to biomass did not reduce its allocation to phytochemicals: condensed tannins, most flavonols and phenolic acids accumulated under elevated CO2 and elevated UVB, but this effect disappeared under elevated temperature. Leaf nitrogen content decreased under elevated CO2 . We conclude that, as a result of high genetic variability in phytochemicals, B. pendula seedlings have potential to adapt to the tested environmental changes. The induction in protective flavonoids under UVB radiation together with the positive impact of elevated CO2 and temperature mitigates possible UVB stress effects, and thus atmospheric CO2 concentration and temperature are the climate change factors that will dictate the establishment and success of birch at higher altitudes in the future.

Bioactive polyphenols in leaves, stems, and berries of Saskatoon (Amelanchier alnifolia Nutt.) cultivars.

The Saskatoon berry is currently cultivated in many parts of the world for its suitability for various food products and due to its high content of nutrients and polyphenols. To determine the phytochemical profile of a Saskatoon plant, polyphenols from leaves, stems, and berries were screened from four cultivars grown in Finland using HPLC-DAD and HPLC-ESI/MS. The phenolic composition and concentrations varied among plant parts and cultivars. The main berry components were cyanidin-based anthocyanins (63% of the phenols), quercetin-derived flavonol glycosides, and hydroxycinnamic acids. The total anthocyanin content varied between 258.7 and 517.9 mg/100 fresh weight among cultivars. Protocatechuic acid was found for the first time in Saskatoon berries. The leaves consisted of quercetin- and kaempferol-derived glycosides (41% of the phenols), hydroxycinnamic acids (36%), catechins, and some neolignans. Quercetin 3-galactoside and 3-glucoside, (-)-epicatechin, and chlorogenic acid were the main phenolics in the leaves of all cultivars. The stem components were flavanone and flavonol glycosides (55% of the phenols), catechins (38%), and hydroxybenzoic acids. Concentrations of the main compound, eriodictyol 7-glucoside, varied among cultivars from 3.3 to 6.5 mg/g of stem dry weight. Very high proanthocyanidin contents were found in stems and leaves (10-14% of dry biomass), whereas berries contained a low amount of proanthocyanidins (3% of dry biomass). The findings reveal that leaves and stems of Saskatoon cultivars possess high amounts of various phenolic compounds that may offer new functional raw materials for a wide range of food and health products.

Boron and other elements in sporophores of ectomycorrhizal and saprotrophic fungi.

Fungi are usually thought not to have a boron (B) requirement. It is not known if mycorrhizas take up B from low concentrations that are common in forest soils, as fungi might also immobilise B. Here, we studied the B concentrations in sporophores of 49 ectomycorrhizal and 10 saprotrophic fungi to assess whether B is translocated in mycelium or not. Additionally, P and metal concentrations were measured for comparison. Variability both within species and between species was very large, as the lowest measured B concentration was 0.01 mg kg(-1) in Amanita muscaria, and the highest was 280 mg kg(-1) in Paxillus involutus. There was no clear difference between saprotrophic and mycorrhizal fungi. The majority of species did not accumulate B at more than 0.01-3 mg kg(-1), but there were some species that consistently had median concentration values higher than 5-6 mg kg(-1) and much higher maximum values, particularly Paxillus involutus, Lactarius necator and several Russula species. Most species increased their B concentration in B fertilised plots, but there were exceptions, particularly Rozites caperatus and Lactarius camphoratus. Boron concentrations did not correlate with those of other elements. In conclusion, B is translocated in the mycelia of most of the studied species. The differences between species may be due to differences in their water use, or carbohydrates used in translocation. It remains to be studied, if B concentrations in mycorrhizas or mycelia in soil are in the same order of magnitude as the larger ones found here, and if this has any effects on the host plants.

Growth and defense in deciduous trees and shrubs under UV-B.

Reflection by waxy or resinous surface structures and hairs, repair reactions of biomolecules and induction of different sheltering components provide the means of plant protection from harmful solar UV-B radiation. Secondary products, especially flavonoids and phenolic acids as defense components are also important in plant tolerance to UV-B, fulfilling the dual role as screens that reduce UV-B penetration in plant tissues, and as antioxidants protecting from damage by reactive oxidant species. Plants are sensitive to UV-B radiation, and this sensitivity can be even more clone-specific than species-specific. The results available in the literature for deciduous trees and shrubs indicate that UV-B radiation may affect several directions in the interaction of woody species with biotic (herbivores) and abiotic (CO2 and nutrition) factors depending on the specific interaction in question. These multilevel interactions should have moderate ecological significance via the overall changed performance of woody species and shrubs.

Boron retranslocation in Scots pine and Norway spruce.

We previously traced 10B-enriched boric acid from shoots to roots to demonstrate the translocation of boron (B) in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings. To gain a more detailed understanding of B translocation, we sought: (1) to demonstrate B retranslocation directly, by showing that foliar-applied 10B is located in the new growth after dormancy; and (2) to assess whether shoot-applied B affects growth in the long term. We applied 10B-enriched boric acid to needles of Scots pine and Norway spruce seedlings. After a dormancy period and 9 weeks of growth, small but significant increases in the 10B isotope were found in the new stem and needles of both species. In Scots pine, the total B concentration of the new stem was also increased. Both species contained polyols, particularly pinitol and inositol. Boron-polyol complexes may provide a mechanism for mobilizing B in these species. To determine the long-term effects of applied B, seedlings were grown for two growing seasons after the application of 10B to shoots. In Norway spruce, the proportion of 10B in the root systems and current needles of the harvest year was slightly higher than in the controls, and in Scots pine root systems, marginally so. The B treatment had no effect on growth of Norway spruce seedlings. In Scots pine seedlings, the B treatment caused a 33% increase in total dry mass and significantly increased the number of side branches.

Boron mobility in deciduous forest trees in relation to their polyols.

• Boron (B) has been found to be phloem mobile in species that translocate polyols, whereas it is almost immobile in other species. The objectives of the present study were to survey B mobility in deciduous trees, and to relate it to the presence of polyols. • The stable isotope 10 B was applied as a tracer to mature leaves of seedlings, and growing leaves were subsequently harvested for B isotope analysis. • Extensive B mobility was found in Sorbus aucuparia and Prunus padus, species with high sorbitol content, but also in Ulmus glabra, with only trace amounts of B-complexing polyols. Alnus incana, Fraxinus excelsior, Betula pubescens and Larix sibirica also translocated 10 B into new leaves. Mannitol in Fraxinus and pinitol in Larix probably explain this. A. glutinosa did not remobilize B, although the polyol concentrations were almost identical to A. incana, a closely related species. • B mobility was not as closely related to the presence of polyols as expected, and it appears that to some degree remobilization occurs in many plant species.

Accumulation of flavonoids and related compounds in birch induced by UV-B irradiance.

A growth chamber experiment was conducted to examine the effects of UV-B exposure (4.9 kJ m(-2) day(-1) of biologically effective UV-B, 280-320 nm) on shoot growth and secondary metabolite production in Betula pendula (Roth) and B. resinifera (Britt.) seedlings originating from environments in Finland, Germany and Alaska differing in solar UV-B radiation and climate. Neither shoot growth nor the composition of secondary metabolites was affected by UV-B irradiance, but the treatment induced significant changes in the amounts of individual secondary metabolites in leaves. Leaves of seedlings exposed to UV-B radiation contained higher concentrations of several flavonoids, condensed tannins and some hydroxycinnamic acids than leaves of control seedlings that received no UV-B radiation. At the population level, there was considerable variation in secondary metabolite responses to UV-B radiation: among populations, the induced response was most prominent in Alaskan populations, which were adapted to the lowest ambient UV-B radiation environment. I conclude that solar UV-B radiation plays an important role in the formation of secondary chemical characteristics in birch trees.