Riitta Tegelberg

Effects of solar UV-A and UV-B radiation on gene expression and phenolic accumulation in Betula pendula leaves

Ultraviolet (UV) radiation is an important environmental factor for plant communities; however, plant responses to solar UV are not fully understood. Here, we report differential effects of solar UV-A and UV-B radiation on the expression of flavonoid pathway genes and phenolic accumulation in leaves of Betula pendula Roth (silver birch) seedlings grown outdoors. Plants were exposed for 30 days to six UV treatments created using three types of plastic film. Epidermal flavonoids measured in vivo decreased when UV-B was excluded. In addition, the concentrations of six flavonoids determined by high-performance liquid chromatography-mass spectrometry declined linearly with UV-B exclusion, and transcripts of PAL and HYH measured by quantitative real-time polymerase chain reaction were expressed at lower levels. UV-A linearly regulated the accumulation of quercetin-3-galactoside and quercetin-3-arabinopyranoside and had a quadratic effect on HYH expression. Furthermore, there were strong positive correlations between PAL expression and accumulation of four flavonols under the UV treatments. Our findings in silver birch contribute to a more detailed understanding of plant responses to solar UV radiation at both molecular and metabolite levels.

Clonal differences in growth and phenolics of willows exposed to elevated ultraviolet-B radiation

Abstract In this study, the effects of elevated ultraviolet-B (UV-B, 280–320 nm) radiation on growth and leaf phenolics were evaluated in clones of dark-leaved willow ( Salix myrsinifolia Salisb.) and tea-leaved willow ( Salix phylicifolia L.). Willows were raised for one growing season in an irradiation field, where they were exposed either to a constant 50% increase in UV-B CIE radiation simulating 20–25% ozone depletion or to a small increase in UV-A radiation (320–400 nm). Control willows were grown in the irradiation field under solar radiation (ambient control). Despite the high constitutive concentrations of a UV-absorbing leaf flavonoid, dihydromyricetin, UV-treatments clearly reduced the biomass and height growth of the shoots of one tea-leaved willow clone. In contrast, the growth of three other tea-leaved willow clones showed no significant sensitivity to UV-radiation. Under elevated UV-B radiation, the leaves of these clones were able to accumulate some of the UV-B-absorbing quercetins, myricetins or luteolins. In dark-leaved willows, biomass production and growth were not decreased by UV-exposures, although the concentrations of leaf flavonoids were clearly lower than those in tea-leaved willows; however, in all the dark-leaved willow clones, elevated UV-radiation increased the concentrations of certain quercetins, dihydromyricetin and phenolic acids. Other willow leaf phenolics, i.e. salicylates, condensed tannins and gallic acid derivatives, were either decreased by the UV-treatments or were unaffected. The results indicate that (1) the constitutive level and quality of secondary chemicals in native willow species or clones does not predict their sensitivity to elevated UV-radiation, (2) secondary chemical responses to UV-radiation in willows are more clone-specific than species-specific and (3) the leaves of field-grown willows treated with UV-B radiation accumulate only those phenolics that screen UV-B efficiently. In dieser Studie wurden die Effekte einer erhohten Ultraviolett-B-Strahlung (UV-B, 280–320 nm) auf das Wachstum und die Blattphenole von Klonen der dunkelblattrigen Weide ( Salix myrsinifolia Salisb.) und der teeblattrigen Weide ( Salix phylicifolia L.) bewertet. Die Weiden wurden fur eine Saison auf Bestrahlungfeldern gezogen, wo sie entweder zur Simulation eines 20–25-prozentigen Ozonverlustes einer konstant um 50% erhohten UV-B CIE -Strahlung oder einer geringfugig erhohten UV-A-Strahlung (320–400 nm) ausgesetzt waren. Die Kontrollweiden wurden in den Bestrahlungsfeldern unter Sonneneinstrahlung gezogen (Ausenkontrolle). Trotz der hohen konstitutiven Konzentrationen des Blattflavonoids Dihydromyricetin verringerten die UV-Behandlungen die Biomasse und das Grosenwachstum der Schosslinge eines teeblattrigen Weidenklons deutlich. Im Kontrast dazu zeigte das Grosenwachstum von drei anderen teeblattrigen Weidenklonen keine signifikante Empfindlichkeit gegenuber UV-Strahlung. Bei erhohter UV-B-Strahlung konnten die Blatter dieser Klone einige der UV-B-absorbierenden Quercetine, Myricetine und Luteoline akkumulieren. Bei den dunkelblattrigen Weiden war die Biomassenproduktion und das Wachstum nicht verringert, obwohl die Konzentrationen der Blattflavonoide deutlich geringer als bei teeblattrigen Weiden waren. Bei allen dunkelblattrigen Weidenklonen steigerte jedoch die erhohte UV-Strahlung die Konzentrationen bestimmter Quercetine, von Dihydromyricetin und phenolischer Sauren. Andere Weidenblattphenole, z. B. Salicylate, kondensierte Tannine und Gallsaurenderivate wurden entweder durch die UV-Behandlung verringert oder nicht beeinflusst. Die Ergebnisse deuten darauf hin, dass (1) der konstitutive Gehalt und die Qualitat der sekundaren Pflanzenstoffe einheimischer Weidenarten oder Klone keine Vorhersage uber die Empfindlichkeit gegenaber erhohter UV-Strahlung erlaubt, (2) die Reaktion sekundarer Pflanzenstoffe auf UV-Strahlung bei Weiden eher klonspezifisch als artspezifisch ist und (3) die Blatter von Weiden, die auf dem Feld unter UV-B-Strahlung gezogen wurden, nur die Phenole akkumulieren, die UV-B effektiv abschirmen.

Assessment of UV Biological Spectral Weighting Functions for Phenolic Metabolites and Growth Responses in Silver Birch Seedlings

In research concerning stratospheric ozone depletion, action spectra are used as biological spectral weighting functions (BSWFs) for describing the effects of UV radiation on plant responses. Our aim was to evaluate the appropriateness of six frequently used BSWFs that differ in effectiveness with increasing wavelength. The evaluation of action spectra was based on calculating the effective UV radiation doses according to 1–2) two formulations of the generalized plant action spectrum, 3) a spectrum for ultraviolet induced erythema in human skin, 4) a spectrum for the accumulation of a flavonol in Mesembryanthemum crystallinum, 5) a spectrum for DNA damage in alfalfa seedlings and 6) the plant growth action spectrum. We monitored effects of UV radiation on the concentration of individual UV absorbing metabolites and chlorophyll concentrations in leaves and growth responses of silver birch (Betula pendula) seedlings. Experiments were conducted outdoors using plastic films attenuating different parts of the UV spectrum. Chlorophyll concentrations and growth were not affected by the UV treatments. The response to UV radiation varied between and within groups of phenolics. In general, the observed responses of phenolic groups and individual flavonoids were best predicted by action spectra extending into the UV‐A region with moderate effectiveness.

Temporal variation in epidermal flavonoids due to altered solar UV radiation is moderated by the leaf position in Betula pendula.

The physiological mechanisms controlling plant responses to dynamic changes in ambient solar ultraviolet (UV) radiation are not fully understood: this information is important to further comprehend plant adaptation to their natural habitats. We used the fluorimeter Dualex to estimate in vivo the epidermal flavonoid contents by measuring epidermal UV absorbance (A(375) ) in Betula pendula Roth (silver birch) leaves of different ages under altered UV. Seedlings were grown in a greenhouse for 15 days without UV and transferred outdoors under three UV treatments (UV-0, UV-A and UV-A+B) created by three types of plastic film. After 7 and 13 days, Dualex measurements were taken at adaxial and abaxial epidermis of the first three leaves (L1, L2 and L3) of the seedlings. After 14 days, some of the seedlings were reciprocally swapped amongst the treatments to study the accumulation of epidermal flavonoids in the youngest unfolded leaves (L3) during leaf expansion under changing solar UV environments. A(375) of the leaves responded differently to the UV treatment depending on their position. UV-B increased the A(375) in the leaves independently of leaf position. L3 quickly adjusted A(375) in their epidermis according to the UV they received and these adjustments were affected by previous UV exposure. The initial absence of UV-A+B or UV-A, followed by exposure to UV-A+B, particularly enhanced leaf A(375) . Silver birch leaves modulate their protective pigments in response to changes in the UV environment during their expansion, and their previous UV exposure history affects the epidermal-absorbance achieved during later UV exposure.

Dark‐leaved willow (Salix myrsinifolia) is resistant to three‐factor (elevated CO2, temperature and UV‐B‐radiation) climate change

Elevated carbon dioxide (CO2 ), temperature (T) and ultraviolet-B (UV-B) radiation may affect plant growth and secondary chemistry in different directions, but the effect of the combination of the three factors has seldom been tested. Here, we grew four dark-leaved willow (Salix myrsinifolia) clones under combinations of ambient or elevated CO2, T and UV-B radiation in top-closed chambers for 7 wk. Elevated UV-B had no effects on growth or phenolic compounds, and there were no significant interactions between UV-B, CO2 and T. CO2 alone increased most growth parameters, but the magnitude of the effect varied among the clones. Total phenolics increased at elevated CO2 , whereas they decreased at elevated T. The responses varied between the clones. The results imply that dark-leaved willow are fairly resistant to the applied three-factor climate change, probably because of high constitutive defense. However, the interactions between clone and climate change factors implies that some clones are more susceptible than the species as a whole.

Does defoliation induce chemical and morphological defenses in the leaves of silver birch seedlings under changing climate

Summary.We examined the effects of defoliation con-currently with elevated temperature and CO2 on some chemical and morphological characteristics in the leaves of silver birch seedlings (Betula pendula). We also analyzed the consequent changes in the palatability of leaves for adult blue alder leaf beetles (Agelastica alni). Under the different climatic treatments, the seedlings were subjected to three fertilizer treatments (0 kg, 130 kg and 270 kg N ha−1) and defoliation treatments (0%, 25% and 50% of the total leaf area). In each climatic treatment, fertilization increased the nitrogen content in the leaves, but decreased total concentrations of soluble phenolics, detected by high-performance liquid chromatography (HPLC), and insoluble condensed tannins. Defoliation, both independently and in combination with elevated temperature and CO2, decreased the concentrations of the phenolics. Compared to the intact controls, the leaves of the defoliated seedlings were smaller and tougher. Under elevated temperature, the beetles consumed a smaller amount of the leaves of plants subjected to the high fertilization, while under ambient climatic conditions, fertilization increased the feeding. The total leaf consumption was higher under the ambient climatic conditions than under elevated temperature, elevated CO2 or the combination of elevated temperature and CO2.

Solar ultraviolet radiation alters alder and birch litter chemistry that in turn affects decomposers and soil respiration

Solar ultraviolet (UV)-A and UV-B radiation were excluded from branches of grey alder (Alnus incana) and white birch (Betula pubescens) trees in a field experiment. Leaf litter collected from these trees was used in microcosm experiments under laboratory conditions. The aim was to evaluate the effects of the different UV treatments on litter chemical quality (phenolic compounds, C, N and lignin) and the subsequent effects of these changes on soil fauna and decomposition processes. We measured the decomposition rate of litter, growth of woodlice (Porcellio scaber), soil microbial respiration and abundance of nematodes and enchytraeid worms. In addition, the chemical quality of woodlice feces was analyzed. The exclusion of both UV-A and UV-B had several effects on litter chemistry. Exclusion of UV-B radiation decreased the C content in litter in both tree species. In alder litter, UV exclusion affected concentration of phenolic groups variably, whereas in birch litter there were no significant differences in phenolic compounds. Moreover, further effects on microbial respiration and chemical quality of woodlice feces were apparent. In both tree species, microbial CO2 evolution was lower in soil with litter produced under exclusion of both UV-A and UV-B radiation when compared to soil with control litter. The N content was higher in the feces of woodlice eating alder litter produced under exclusion of both UV-A and UV-B compared to the control. In addition, there were small changes in the concentration of individual phenolic compounds analyzed from woodlice feces. Our results demonstrate that both UV-A and UV-B alter litter chemistry which in turn affects decomposition processes.

How Realistically Does Outdoor UV-B Supplementation with Lamps Reflect Ozone Depletion: An Assessment of Enhancement Errors

Limitations in the realism of currently available lamps mean that enhancement errors in outdoor experiments simulating UV‐B radiation effects of stratospheric ozone depletion can be large. Here, we assess the magnitude of such errors at two Finnish locations, during May and June, under three cloud conditions. First we simulated solar radiation spectra for normal, compared with 10% and 20% ozone depletion, and convoluted the daily integrated solar spectra with eight biological spectral weighting functions (BSWFs) of relevance to effects of UV on plants. We also convoluted a measured spectrum from cellulose‐acetate filtered UV‐B lamps with the same eight BSWFs. From these intermediate results we calculated the enhancement errors. Differences between locations and months were small, cloudiness had only a minor effect. This assessment was based on the assumption that no extra enhancement compensating for shading of UV radiation by lamp frames is performed. Under this assumption errors between spectra are due to differences in the UV‐B effectiveness rather than differences in the UV‐A effectiveness. Hence, conclusions about plant growth from past UV‐supplementation experiments should be valid. However, interpretation of the response of individual physiological processes is less secure, so results from some field experiments with lamps might need reassessment.

Seasonal fluctuations in leaf phenolic composition under UV manipulations reflect contrasting strategies of alder and birch trees

Seasonal variation in leaf phenolic composition may be important for acclimation of plants to seasonal changes in their biotic and abiotic environment. For a realistic assessment of how plants respond to solar UV-B (280-315 nm) and UV-A (315-400 nm) radiation, seasonal variation in both environment and plant responses needs to be taken into account. This also has implications for studies concerning stratospheric ozone depletion and resulting increased UV-B radiation, as other environmental variables and/or plant phenology could interact with UV radiation. To elucidate this, we established a field experiment using plastic films attenuating different parts of the solar UV spectrum. The concentration of individual phenolic compounds was measured during one growing season in leaves of grey alder (Alnus incana) and white birch (Betula pubescens) trees. Our results showed changes in concentration of, e.g. hydrolyzable tannins in birch that suggest an effect of UV-A alone and e.g. chlorogenic acids in alder indicate a quadratic effect of UV-B irradiance and both linear and quadratic effect for UV-A in second-degree polynomial fits. Further, there was interaction between treatment and sampling time for some individual metabolites; hence, the UV response varied during the season. In addition to the UV effects, three temporal patterns emerged in the concentrations of particular groups of phenolics. Possible implications for both sampling methods and timing are discussed. Moreover, our results highlight differences in responses of the two tree species, which are taken to indicate differences in their ecological niche differentiation.

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.