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.

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.

Dry-air drying at room temperature - a practical pre-treatment method of tree leaves for quantitative analyses of phenolics?

INTRODUCTION: In ecological experiments, storage of plant material is often needed between harvesting and laboratory analyses when the number of samples is too large for immediate, fresh analyses. Thus, accuracy and comparability of the results call for pre-treatment methods where the chemical composition remains unaltered and large number of samples can be treated efficiently. OBJECTIVE: To study if a fast dry-air drying provides an efficient pre-treatment method for quantitative analyses of phenolics. METHODOLOGY: Dry-air drying of mature leaves was done in a drying room equipped with dehumifier (10% relative humidity, room temperature) and results were compared to freeze-drying or freeze-drying after pre-freezing in liquid nitrogen. The quantities of methanol-soluble phenolics of Betula pendula Roth, Betula pubescens Ehrh., Salix myrsinifolia Salisb., Picea abies L. Karsten and Pinus sylvestris L. were analysed with HPLC and condensed tannins were analysed using the acid-butanol test. RESULTS: In deciduous tree leaves (Betula, Salix), the yield of most of the phenolic compounds was equal or higher in samples dried in dry-air room than the yield from freeze-dried samples. In Picea abies needles, however, dry-air drying caused severe reductions in picein, stilbenes, condensed tannin and (+)-catechin concentrations compared to freeze-drying. In Pinus sylvestris highest yields of neolignans but lowest yields of acetylated flavonoids were obtained from samples freeze-dried after pre-freezing. CONCLUSION: Results show that dry-air drying provides effective pre-treatment method for quantifying the soluble phenolics for deciduous tree leaves, but when analysing coniferous species, the different responses between structural classes of phenolics should be taken into account.

Combined effect of elevated UVB, elevated temperature and fertilization on growth, needle structure and phytochemistry of young Norway spruce (Picea abies) seedlings.

Simultaneously with warming climate, other climatic and environmental factors are also changing. Here, we investigated for the first time the effects of elevated temperature, increased ultraviolet-B (UVB) radiation, fertilization and all combinations of these on the growth, secondary chemistry and needle structure of 1-year-old Norway spruce (Picea abies (L.) Karst.) seedlings in an outdoor experiment. After one growing season, elevated temperature increased root : shoot ratio and concentrations of needle piperidine alkaloids, while concentrations of needle catechins and acetophenones and bark flavonoids decreased compared with ambient temperature seedlings. UVB-radiation increased concentrations of bark condensed tannins, while fertilization increased total biomass and concentrations of needle catechins. In addition to the main effects, concentrations of some individual phenolic compounds showed UV × temperature or UV × temperature × fertilization interactions, and fertilization modified temperature response on root : shoot ratio. All the treatments described here affected the defence chemistry profiles of the seedlings, which may imply some changes in plant-herbivore interactions in connection with changing climate. The interactions between treatments indicate a need for further experiments involving several simultaneously affecting environmental changes.

Differences in vole preference, secondary chemistry and nutrient levels between naturally regenerated and planted Norway spruce seedlings.

Field voles (Microtus agrestis) cause severe damage to young Norway spruce (Picea abies) plantations during wintertime in Fennoscandia. We experimentally investigated vole preference for winter-dormant, naturally regenerated seedlings; spring-planted seedlings; or autumn-planted seedlings; and how preference corresponds with seedling chemistry. Voles showed the highest preference for autumn-planted seedlings and the second highest for spring-planted seedlings, while naturally regenerated seedlings were avoided. The stems of the autumn-planted seedlings contained higher concentrations of nitrogen and piperidine alkaloids and lower concentrations of stilbenes than did the other groups. In addition to differences between naturally regenerated and planted seedlings, we investigated seasonal differences in naturally regenerated P. abies needle and bark secondary chemistry. While piperidine alkaloid concentrations did not vary with season, the soluble non-tannin phenolics of needles and the condensed tannins of bark were lower in May than in November or January. At the time of planting, the concentration of bark piperidine alkaloids was higher in autumn-planted than in spring-planted seedlings. We detected two alkaloids not previously found in P. abies, 2-methyl-6-propyl-1,6-piperideine and a tentatively identified pinidine-isomer. Our results demonstrate that vole choice of spruce seedlings is promoted by high nitrogen and low stilbene content, both associated with seedlings planted late in the season. As vole damage is linked to seedling chemistry, damage potentially could be mitigated by advancing planting or by manipulating plant chemistry in nurseries.

Responses in growth and emissions of biogenic volatile organic compounds in Scots pine, Norway spruce and silver birch seedlings to different warming treatments in a controlled field experiment.

We investigated the responses in growth and emissions of biogenic volatile organic compounds (BVOCs) in Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies [L.] Karst.) and silver birch (Betula pendula Roth) seedlings to a simulated climate warming of +2 °C (T2) and +4 °C (T4), compared to the ambient conditions, during two growing seasons (2019-2020) in a controlled field experiment in central Finland. In all seedlings, height was measured weekly. Diameter was measured continuously for one seedling from each tree species per plot. For shoot and root biomass measurements, half of the seedlings were harvested at end of the first growing season and the rest at the end of the second growing season. Foliage BVOC emission rates were measured at the end of the second growing season. Biomass, height, and diameter growth of silver birch did benefit the most from warming in both growing seasons. In the Scots pine and Norway spruce seedlings, height and diameter growth increased with increasing temperature in the second growing season, more so in Scots pine. Overall, the shoot and root biomass of conifer seedlings increased with increasing temperature. In the conifer seedlings, warming increased biomass and diameter growth more than height growth, due to their predetermined height growth pattern. The warming increased BVOC emissions more clearly in silver birch, whilst the BVOC emissions were in conifers less sensitive to temperature variation. Based on our findings, silver birch seedlings could be expected to benefit the most from warmer growing conditions and Norway spruce the least.

Effects of deficiency and overdose of group 2 sigma factors in triple inactivation strains of Synechocystis sp. strain PCC 6803.

Acclimation of cyanobacteria to environmental changes includes major changes in the gene expression patterns partly orchestrated by the replacement of a particular σ subunit with another in the RNA polymerase holoenzyme. The cyanobacterium Synechocystis sp. strain PCC 6803 encodes nine σ factors, all belonging to the σ(70) family. Cyanobacteria typically encode many group 2 σ factors that closely resemble the principal σ factor. We inactivated three out of the four group 2 σ factors of Synechocystis simultaneously in all possible combinations and found that all triple inactivation strains grow well under standard conditions. Unlike the other strains, the ΔsigBCD strain, which contains SigE as the only functional group 2 σ factor, did not grow faster under mixotrophic than under autotrophic conditions. The SigB and SigD factors were important in low-temperature acclimation, especially under diurnal light rhythm. The ΔsigBCD, ΔsigBCE, and ΔsigBDE strains were sensitive to high-light-induced photoinhibition, indicating a central role of the SigB factor in high-light tolerance. Furthermore, the ΔsigBCE strain (SigD is the only functional group 2 σ factor) appeared to be locked in the high-fluorescence state (state 1) and grew slowly in blue but not in orange or white light. Our results suggest that features of the triple inactivation strains can be categorized as (i) direct consequences of the inactivation of a particular σ factor(s) and (ii) effects resulting from the higher probability that the remaining group 2 σ factors associate with the RNA polymerase core.

Genetic modification of the flavonoid pathway alters growth and reveals flexible responses to enhanced UVB - Role of foliar condensed tannins.

Accumulation of certain phenolics is a well-known response of plants to enhanced UVB radiation (280-315 nm), but few experiments have compared the relative importance of different phenolic groups for UVB resilience. To study how an altered phenolic profile affects the responses and resilience of silver birch (Betula pendula) to enhanced UVB, we used RNA interference (RNAi) targeting dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), or anthocyanidin reductase (ANR) to change the accumulation of phenolics. The unmodified control line and RNAi-modified plants were grown for 51 days under ambient or +32% enhanced UVB dose in a greenhouse. RNAi greatly affected phenolic profile and plant growth. There were no interactive effects of RNAi and UVB on growth or photosynthesis, which indicates that the RNAi and unmodified control plants were equally resilient. UVB enhancement led to an accumulation of foliar flavonoids and condensed tannins, and an increase in the density of stem glands and glandular trichomes on upper leaf surfaces in both the control and RNAi-modified plants. Our results do not indicate a photoprotective role for condensed tannins. However, decreased growth of high-flavonoid low-tannin DFRi and ANRi plants implies that the balance of flavonoids and condensed tannins might be important for normal plant growth.

Accumulation of phenolics and growth of dioecious Populus tremula (L.) seedlings over three growing seasons under elevated temperature and UVB radiation.

Accumulation of secondary metabolites may exhibit developmentally regulated variation in different plant organs. Moreover, prevailing environmental conditions may interact with development-related variations in plant traits. In this study, we examined developmentally regulated variation in phenolic accumulation in the twigs of dioecious Populus tremula (L.) and how the effects of elevated temperature and ultraviolet B (UVB) radiation on growth and phenolics accumulation varied as the plants get older. In an open-field experiment, six female and six male genotypes were exposed to single and combined elevated temperature and UVB radiation treatments for three consecutive growing seasons. The concentrations of low molecular weight phenolics and condensed tannins did not show age-dependent variation in the twigs. In temperature-treated plants, diameter growth rate decreased, and concentration of condensed tannins increased as plants aged; there were no cumulative effects of elevated UVB radiation on growth and phenolic accumulation. Females maintained a higher concentration of low molecular weight phenolics throughout the experimental period; however, growth and phenolic concentration did not vary over time in females and males. Our results suggest that phenolic accumulation in perennial plants may not necessarily always exhibit age-dependent variation and the effects of elevated temperature on growth and phenolic may diminish as plants get older.

Responses in growth and phenolics accumulation to lateral bud removal in male and female saplings of Populus tremula (L.) under simulated climate change.

The responses in growth and defense after tissue damage are highly variable in plants depending on species, damaged-tissue type and the intensity of damage. The prevailing abiotic conditions can also influence these responses. In this study, our aim was to examine how the removal of lateral vegetative buds affects the growth and accumulation of phenolics in saplings of the dioecious Populus tremula grown under simulated climate change. For three growing seasons, the saplings were grown under ambient conditions (control), elevated temperature (+2°C) and elevated UV radiation (30%) (UVB and UVA as its control), or a combination of these. In the fourth growing season, all saplings were grown under ambient conditions. The bud removal was performed twice - in summer and autumn - in the third year. The responses of growth and the accumulation of phenolics to the bud removal were measured at the end of the fourth growing season. Removal of 5% of the lateral buds resulted in higher leaf, stem and total plant biomass in both sexes of P. tremula saplings, compared to intact plants. The effects were greater in the temperature-treated plants, especially in the temperature-treated females. The concentrations of flavonoids and condensed tannins were higher in the bud-removed individuals. The concentration of condensed tannins was also higher in the males than in the females, opposite to the concentration of phenolic acids. There was no significant interaction between bud removal and UVB treatment on either growth or phenolics. Our results suggest that plants can allocate resources to both growth and defense simultaneously in response to tissue loss, and that global warming can modify the responses to some extent.

Effects of soil pyrene contamination on growth and phenolics in Norway spruce (Picea abies) are modified by elevated temperature and CO2.

With the constant accumulation of polycyclic aromatic hydrocarbons (PAHs) in soil and increasing temperature and CO2 levels, plants will inevitably be exposed to combined stress. Studies on the effects of such combined stresses are needed to develop mitigation and adaptation measures. Here, we investigated the effects of soil pyrene contamination (50 mg kg-1) on growth and phenolics of 1-year-old Norway spruce seedlings from five different origins in Finland at elevated temperature (+ 2 °C) and CO2 (+ 360 ppm). Pyrene significantly decreased spruce height growth (0-48%), needle biomass (0-44%), stem biomass (0-43%), and total phenolic concentrations in needles (2-13%) and stems (1-19%) compared to control plants. Elevated temperature alone did not affect growth but led to lower concentrations of total phenolics in needles (5-29%) and stems (5-18%) in both soil treatments. By contrast, elevated CO2 led to higher needle biomass (0-39%) in pyrene-spiked soils and higher concentrations of stem phenolics (0-18%) in pyrene-spiked and control soils compared to ambient treatments. The decrease in height growth and phenolic concentrations caused by pyrene was greater at elevated temperature, while elevated CO2 only marginally modified the response. Seedlings from different origins showed different responses to the combined environmental stressors. The changes in growth and in the quantity and quality of phenolics in this study suggest that future climate changes will aggravate the negative influence of soil pyrene pollution on northern conifer forest ecosystems.

Sex-related responses of European aspen (Populus tremula L.) to combined stress: TiO2 nanoparticles, elevated temperature and CO2 concentration.

The combined effects of climate change and chemical contaminants on plant performance are still not well understood. Especially, whether different sexes of dioecious plants respond differently to combined stresses is unknown. In order to study the sex-related responses of European aspen to soil nTiO2 contamination (0, 50, 300 mg kg-1) under elevated temperature (+1.6 °C) and CO2 (730 ppm), we conducted a study in greenhouses. Ti accumulated in roots exposed to nTiO2 (1.1-3.3 and 2.7-21.1 mg kg-1 in 50 and 300 mg kg-1 treatments, respectively). Elevated CO2 had no effects on Ti uptake, while elevated temperature increased it in the 300 mg kg-1 treatment. Males grew taller than females under ambient conditions, but females had greater height and biomass increment under elevated temperature. In all climate treatments, nTiO2 increased leaf phenolics in females by 12-19% and 15-26% at 50 and 300 mg kg-1, respectively. Leaf phenolics decreased under elevated temperature, but increased under elevated CO2 in both sexes. Results suggest that females have better chemical defense against nTiO2 than males under future climate conditions. In the longer run, this may cause changes in the competitive abilities of both sexes, which again may affect sex ratios and genetic variation in nature.

Elevated temperature and CO2 affect responses of European aspen (Populus tremula) to soil pyrene contamination.

High northern latitudes are climatic sensitive areas, and are also regions to which polycyclic aromatic hydrocarbons (PAHs) easily transport and accumulate with potential risk to natural ecosystems. However, the effect of PAHs on northern woody plant growth and defense under climate change is very little studied. Here, we conducted a unique experiment in greenhouses to investigate sex-related responses of the dioecious Populus tremula to pyrene (50mgkg-1) and residue of pyrene in soils under ambient and elevated temperature (+1.8°C on average) and CO2 (740ppm). Pyrene decreased stem biomass and leaf area by 9% and 6%, respectively under ambient conditions, and the reduction of leaf area was more severe under elevated temperature (38%), elevated CO2 (37%), and combined T+CO2 (42%). Other growth parameters were unchanged by pyrene. Pyrene did not affect the concentration of leaf total phenolics under ambient conditions, but increased it by 16%, 1%, and 20% compared to controls under elevated temperature, elevated CO2, and T+CO2, respectively. Pyrene had only minor sex-specific effects on plant growth and phenolics. The concentration of residual pyrene in pyrene-spiked soils was higher under elevated CO2 than under ambient, elevated temperature, and combined T+CO2. The results suggest that both sexes of P. tremula have the capacity to regulate growth and metabolism to adjust to the stress of the tested pyrene contamination under elevated temperature and CO2, but potential risk of pyrene to plants still exists in the future changing climate.

Climate Change Effects on Secondary Compounds of Forest Trees in the Northern Hemisphere.

Plant secondary compounds (PSCs), also called secondary metabolites, have high chemical and structural diversity and appear as non-volatile or volatile compounds. These compounds may have evolved to have specific physiological and ecological functions in the adaptation of plants to their growth environment. PSCs are produced by several metabolic pathways and many PSCs are specific for a few plant genera or families. In forest ecosystems, full-grown trees constitute the majority of plant biomass and are thus capable of producing significant amounts of PSCs. We summarize older literature and review recent progress in understanding the effects of abiotic and biotic factors on PSC production of forest trees and PSC behavior in forest ecosystems. The roles of different PSCs under stress and their important role in protecting plants against abiotic and biotic factors are also discussed. There was strong evidence that major climate change factors, CO2 and warming, have contradictory effects on the main PSC groups. CO2 increases phenolic compounds in foliage, but limits terpenoids in foliage and emissions. Warming decreases phenolic compounds in foliage but increases terpenoids in foliage and emissions. Other abiotic stresses have more variable effects. PSCs may help trees to adapt to a changing climate and to pressure from current and invasive pests and pathogens. Indirect adaptation comes via the effects of PSCs on soil chemistry and nutrient cycling, the formation of cloud condensation nuclei from tree volatiles and by CO2 sequestration into PSCs in the wood of living and dead forest trees.

Epidihydropinidine, the main piperidine alkaloid compound of Norway spruce (Picea abies) shows promising antibacterial and anti-Candida activity.

This study reports for the first time promising antibacterial and antifungal effects of epidihydropinidine, the major piperidine alkaloid in the needles and bark of Norway spruce, Picea abies (L.) Karsten. Epidihydropinidine was growth inhibitory against all bacterial and fungal strains used in our investigation, showing the lowest MIC value of 5.37µg/mL against Pseudomonas aeruginosa, Enterococcus faecalis, Candida glabrata and C. albicans. Epidihydropinidine was nearly three times more active than tetracycline against P. aeruginosa and E. faecalis. Promising antibacterial effects were also recorded against Staphylococcus aureus and Bacillus cereus (MIC 10.75µg/mL) as well as against Salmonella enterica (MIC and MBC 43µg/mL). Our preliminary results suggest that epidihydropinidine as well related alkaloids of Norway spruce could be powerful candidates for new antibiotics and for preventing food spoilage.

Screening bioactivity and bioactive constituents of Nordic unifloral honeys.

The objective of this study was to screen the antibacterial and antioxidant activity of thirty nine honey samples from Finland, Sweden, Norway and Denmark. Their physicochemical properties were analysed, antioxidant activity was evaluated by DPPH assay and antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by microdilution assay. The honey samples obtained were buckwheat, caraway, clover, dandelion, fireweed, heather, lime tree, lingonberry, rape, raspberry, sweet clover, willow, mire, honeydew and polyfloral. Eleven honey samples showed high antioxidant activity. With 15% honey dilution, three unifloral honeys had over 85% inhibition against growth of P. aeruginosa and ten honey samples against S. aureus. The buckwheat, raspberry and honeydew honeys showed the highest antibacterial and antioxidant activity. An unexpectedly high amount of methylglyoxal was found in mire and forest honeys. Some phenolic compounds are shown to be plant species-specific floral markers due to their appearance in specific unifloral honey samples.

Effect of climate change on bud phenology of young aspen plants (Populus tremula. L).

Boreal tree species are excellent tools for studying tolerance to climate change. Bud phenology is a trait, which is highly sensitive to environmental fluctuations and thus useful for climate change investigations. However, experimental studies of bud phenology under simulated climate change outdoors are deficient. We conducted a multifactorial field experiment with single (T, UVA, UVB) and combined treatments (UVA+T, UVB+T) of elevated temperature (T, +2°C) and ultraviolet-B radiation (+30% UVB) in order to examine their impact on both male and female genotypes of aspen (Populus tremula L.). This study focuses on the effect of the treatments in years 2 and 3 after planting (2013, 2014) and follows how bud phenology is adapting in year 4 (2015), when the treatments were discontinued. Moreover, the effect of bud removal was recorded. We found that elevated temperature played a key role in delaying bud set and forcing bud break in intact individuals, as well as slightly delaying bud break in bud-removed individuals. UVB delayed the bud break in bud-removed males. In addition, both UVA and UVB interacted with temperature in year 3 and even in year 4, when the treatments were off, but only in male individuals. Axillary bud removal forced both bud break and bud set under combined treatments (UVA+T, UVB+T) and delayed both under individual treatments (T, UVB). In conclusion, male aspens were more responsive to the treatments than females and that effect of elevated temperature and UV radiation on bud set and bud break of aspen is not disappearing over 4-year study period.