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.

Multiple roles for UV RESISTANCE LOCUS8 in regulating gene expression and metabolite accumulation in Arabidopsis under solar ultraviolet radiation.

Photomorphogenic responses triggered by low fluence rates of ultraviolet B radiation (UV-B; 280-315 nm) are mediated by the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8). Beyond our understanding of the molecular mechanisms of UV-B perception by UVR8, there is still limited information on how the UVR8 pathway functions under natural sunlight. Here, wild-type Arabidopsis (Arabidopsis thaliana) and the uvr8-2 mutant were used in an experiment outdoors where UV-A (315-400 nm) and UV-B irradiances were attenuated using plastic films. Gene expression, PYRIDOXINE BIOSYNTHESIS1 (PDX1) accumulation, and leaf metabolite signatures were analyzed. The results show that UVR8 is required for transcript accumulation of genes involved in UV protection, oxidative stress, hormone signal transduction, and defense against herbivores under solar UV. Under natural UV-A irradiance, UVR8 is likely to interact with UV-A/blue light signaling pathways to moderate UV-B-driven transcript and PDX1 accumulation. UVR8 both positively and negatively affects UV-A-regulated gene expression and metabolite accumulation but is required for the UV-B induction of phenolics. Moreover, UVR8-dependent UV-B acclimation during the early stages of plant development may enhance normal growth under long-term exposure to solar UV.

The development of a digitising service centre for natural history collections.

Digitarium is a joint initiative of the Finnish Museum of Natural History and the University of Eastern Finland. It was established in 2010 as a dedicated shop for the large-scale digitisation of natural history collections. Digitarium offers service packages based on the digitisation process, including tagging, imaging, data entry, georeferencing, filtering, and validation. During the process, all specimens are imaged, and distance workers take care of the data entry from the images. The customer receives the data in Darwin Core Archive format, as well as images of the specimens and their labels. Digitarium also offers the option of publishing images through Morphbank, sharing data through GBIF, and archiving data for long-term storage. Service packages can also be designed on demand to respond to the specific needs of the customer. The paper also discusses logistics, costs, and intellectual property rights (IPR) issues related to the work that Digitarium undertakes.

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.

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.

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.

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.

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 CO(2) 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.

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.

Light sources with different spectra affect root and mycorrhiza formation in Scots pine in vitro.

We studied the effects of broad-spectrum light quality on the interaction between the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch and Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings cultured in vitro. The light sources were cool white (CW), warm white (WW) and red-rich daylight (RD) fluorescent lamps. Inoculation with P. tinctorius enhanced adventitious root formation of the cuttings in all light treatments. Rooting of the inoculated cuttings was highest in WW light (89%), followed by CW (73%) and RD light (66%). During 6 weeks of in vitro culture, rooted cuttings formed only a few lateral roots. The fungus grew over lateral roots, but the Hartig net was absent in all light treatments. In non-inoculated cuttings, neither root formation nor subsequent root growth was affected by light quality. In the seedling experiment, inoculation in the WW treatment resulted in a significantly (P < 0.05) greater number of lateral roots than inoculation in the RD treatment. The percentage of lateral roots covered with fungal hyphae was also highest in WW light (62%), followed by CW (50%) and RD (27%) light. A similar pattern was observed in the intensity of Hartig net formation. We conclude that effects of broad-spectrum light quality on the ectomycorrhizal fungus-root interaction are dependent on the developmental stage of the root.

Effects of long-term, elevated ultraviolet-B radiation on phytochemicals in the bark of silver birch (Betula pendula).

Long-term outdoor experiments were conducted to investigate the effects of elevated ultraviolet-B (UV-B, 280-320 nm) radiation on secondary metabolites (phenolics and terpenoids) and the main soluble sugars (sucrose, raffinose and glucose) in the bark of silver birch (Betula pendula Roth) saplings. Saplings were exposed to a constant 50% increase in erythemal UV irradiance (UV-B(CIE); based on the CIE (International Commission on Illumination) erythemal action spectrum) and a small increase in UV-A radiation (320-400 nm) for three growing seasons in an irradiation field in central Finland. Two control groups were used: saplings exposed to ambient radiation and saplings exposed to slightly increased UV-A radiation. Concentrations of sucrose, raffinose and glucose in bark were higher in UV-treated saplings than in saplings grown in ambient radiation, indicating that stem carbohydrate metabolism was changed by long-term elevated UV radiation. Saplings in the elevated UV-A + UV-B radiation treatment and the UV-A radiation control treatment had significantly increased concentrations of certain UV-absorbing phenolics, such as salidroside, 3,4'-dihydroxypropiophenone-3-glucoside, (+)-catechin and (-)-epicatechin compared with saplings in ambient radiation. In contrast, the radiation treatments had no effect on the non-UV-B-absorbing terpenoids, papyriferic acid and deacetylpapyriferic acid. We conclude that plant parts, in addition to leaves, accumulate specific phenolic UV-filters in response to UV radiation exposure.