Abstract Flavonoids are a class of secondary metabolites in plants that are involved in many important functions. Various flavonoid compounds have also been reported to be beneficial for human health. Bilberry (Vaccinium myrtillus L.) is the characteristic field layer species in boreal forests and the fruits of bilberry are rich in anthocyanin pigments, a subclass of flavonoids. In the present work, flavonoid biosynthesis was examined in different tissues of bilberry. The focus was on the developing fruits of wild type and natural color mutants of bilberry, and on effect of solar radiation on flavonoid biosynthesis in bilberry leaves. For the isolation of RNA for gene expression analysis, a method was optimized for different tissues of bilberry. The cDNA fragments of five genes from the flavonoid pathway, coding phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavanol 4-reductase and anthocyanidin synthase, were isolated from bilberry using polymerase chain reaction technique, sequenced, and labelled with dioxigenin-dUTP label. These homologous, bilberry-specific probes were used for determining the expression of the flavonoid pathway genes in bilberry fruits, flowers and leaves with a modified non-radioactive method developed in the course of the study. The anthocyanins, catechins, proanthocyanidins, flavonols and hydroxycinnamic acids in fruits, leaves and different fractions of bilberry were identified and quantified with high-performance liquid chromatography combined with a diode array detector and mass spectrometer. The results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. A correlation between flavonol and anthocyanin biosynthesis was detected in bilberry and also in previous literature collected from flavonol and anthocyanin analyses from other fruits. Accordingly, models for the connection between flavonol and anthocyanin synthesis in fruit species were suggested. Activation of the expression of flavonoid pathway genes and accumulation of flavonoids and hydroxycinnamic acids was detected in leaves growing under direct solar radiation, compared to the shadow leaves of the same plants. Based on the results, it is suggested that cyanidin of anthocyanins and flavonol quercetin play a predominant role in the defence against high solar radiation in Vaccinium leaves. The results give new information about the biosynthesis of flavonoids in bilberry at the gene level, in addition to the information of the composition and content of flavonoids during fruit development and in different parts of the bilberry plant. Also, new information was obtained of the roles of flavonoids in protecting plants from excess solar radiation.

Subject of research: different parts – roots, stems, leaves, berries – of bilberries (Vaccinium myrtillus L.), growing near the A1 Vilnius – Kaunas – Klaipėda highway (the 94th kilometre, right side of the road). Objective of research: to determine the amount of lead in different parts of bilberries. Methodology: for research the samples of bilberries were picked near the A1 Vilnius – Kaunas – Klaipėda highway at the 15 – 20 km distance from Kaunas, at 10, 15, 20, 25, 30, 35, 40 and 70 m distance from highway. For experiment – at 100 m distance from highway, bilberries were replanted in laboratory and were grown in the soils of different lead concentration four months. The soil was sprinkling with lead nitrate (Pb(NO3)2) solution of different concentration. The samples of plants were desiccate. The mineralization of different part the plants was carried out, as well as the analysis of soil was made (pH, ratio of hygroscope humidity, concentration of lead in soil). The amounts of lead in plants and soil were determined by the method of atomic absorption spectrometer. Research carried out in accordance with legally approved methodologies. For the statistical evaluation of differences the ANOVA and STATISTICA has been applied. Results of research: by carrying out the research, the amount of lead in different parts of the plants was determined; the dependence of lead amount in different parts of the plant upon the lead amount in solution was analysed; it was evaluated the... [to full text]

<p>I have, in this thesis, studied the interactions between gray-sided voles (Clethrionomys rufocanus) and tundra vegetation, on islands in, and mainland sites close to the lake Iešjávri, in northern Norway. As isolated islands are virtually free of predation, I have been able to compare plant-herbivore interactions in the presence and absence of predators. I transplanted vegetation from an island with predators and voles, to predator-free islands with and with out voles. The results reveal the existence of a terrestrial trophic cascade as voles had a severe impact on the transplanted vegetation on the predator-free islands, but only minor effects on the mainland where predators are present. Moreover, this study shows that plant defence was only a successful strategy when predators were present. Voles reduced the abundance of all available plants during winter on the predator-free islands. The results imply that cascading effects of predation are most important for well-defended plants with grazing-sensitive morphology as these plants escape herbivore impacts in the presence of predators but are vulnerable in their absence</p><p>I studied the recovery of intensively grazed vegetation by building exclosures on islands that have been heavily grazed by voles for almost a decade.This study shows that the collective biomass of vascular plants recovered completely on three years, when voles were excluded. Although most species that are dominating the ungrazed vegetation recovered rapidly in the exclosures, the vegetation did not simply return to its ungrazed state. Herbaceous plants increased and there were pronounced differences in response among evergreen dwarf-shrub species. The semi-prostrate and tannin rich crowberry (Empetrum nigrum), showed the strongest recovery of all species, while the erect lingonberry (Vaccinium vitis-idaea) only showed weak signs of recovery. Thus, growth form determined the potential of plants to recover, whereas the trade-off between defensive investments and capacity to recover was weak or absent.</p><p>I studied the interaction between gray-sided voles and their main winter food plant, bilberry (Vaccinium myrtillus) on islands in and mainland sites close to the lake Iešjávri. I compared the abundance, population structure and palatability of bilberry ramets between vole-free islands, islands with voles but no predators and mainland sites with both voles and predators. Voles decreased the abundance of bilberry on the mainland, but the effect was much stronger on predator-free islands. Bilberry was fairly tolerant to grazing as it partially compensated for the lost tissue by producing more new ramets. Moreover, a cafeteria experiment showed that voles preferred the ramets from predator-free islands, which is inconsistent with conjectures emphasizing inducible plant defenses. The vole-bilberry interaction lacks features of delayed density dependence that could explain the vole cycles. I conducted a clipping and fertilization experiment to further investigate the effects of herbivory on palatability of bilberry shoots. Fertilization decreased the concentration of condensed tannins in shoots of bilberry and voles preferred fertilized and clipped shoots. I found no indication of induced defense that could reduce the palatability of bilberry twigs in response to herbivory.</p><p>The relationships between gray-sided vole densities, levels of invertebrate herbivory and chemical quality of leaves of Northern willow (Salix glauca) were studied on islands and mainland sites with contrasting vole densities. I found a positive correlation between level of invertebrate herbivory and vole density. The number of leaves per shoot, leaf size and leaf nitrogen content were also positively correlated with vole densities, while leaf C/N ratios were negatively correlated with vole densities. The positive correlation between vole densities and level of invertebrate herbivory is probably due to a facilitative effect of voles on invertebrate herbivores, mediated through changes in plant chemistry.</p>

I have, in this thesis, studied the interactions between gray-sided voles (Clethrionomys rufocanus) and tundra vegetation, on islands in, and mainland sites close to the lake Iešjávri, in northern Norway. As isolated islands are virtually free of predation, I have been able to compare plant-herbivore interactions in the presence and absence of predators. I transplanted vegetation from an island with predators and voles, to predator-free islands with and with out voles. The results reveal the existence of a terrestrial trophic cascade as voles had a severe impact on the transplanted vegetation on the predator-free islands, but only minor effects on the mainland where predators are present. Moreover, this study shows that plant defence was only a successful strategy when predators were present. Voles reduced the abundance of all available plants during winter on the predator-free islands. The results imply that cascading effects of predation are most important for well-defended plants with grazing-sensitive morphology as these plants escape herbivore impacts in the presence of predators but are vulnerable in their absence I studied the recovery of intensively grazed vegetation by building exclosures on islands that have been heavily grazed by voles for almost a decade.This study shows that the collective biomass of vascular plants recovered completely on three years, when voles were excluded. Although most species that are dominating the ungrazed vegetation recovered rapidly in the exclosures, the vegetation did not simply return to its ungrazed state. Herbaceous plants increased and there were pronounced differences in response among evergreen dwarf-shrub species. The semi-prostrate and tannin rich crowberry (Empetrum nigrum), showed the strongest recovery of all species, while the erect lingonberry (Vaccinium vitis-idaea) only showed weak signs of recovery. Thus, growth form determined the potential of plants to recover, whereas the trade-off between defensive investments and capacity to recover was weak or absent. I studied the interaction between gray-sided voles and their main winter food plant, bilberry (Vaccinium myrtillus) on islands in and mainland sites close to the lake Iešjávri. I compared the abundance, population structure and palatability of bilberry ramets between vole-free islands, islands with voles but no predators and mainland sites with both voles and predators. Voles decreased the abundance of bilberry on the mainland, but the effect was much stronger on predator-free islands. Bilberry was fairly tolerant to grazing as it partially compensated for the lost tissue by producing more new ramets. Moreover, a cafeteria experiment showed that voles preferred the ramets from predator-free islands, which is inconsistent with conjectures emphasizing inducible plant defenses. The vole-bilberry interaction lacks features of delayed density dependence that could explain the vole cycles. I conducted a clipping and fertilization experiment to further investigate the effects of herbivory on palatability of bilberry shoots. Fertilization decreased the concentration of condensed tannins in shoots of bilberry and voles preferred fertilized and clipped shoots. I found no indication of induced defense that could reduce the palatability of bilberry twigs in response to herbivory. The relationships between gray-sided vole densities, levels of invertebrate herbivory and chemical quality of leaves of Northern willow (Salix glauca) were studied on islands and mainland sites with contrasting vole densities. I found a positive correlation between level of invertebrate herbivory and vole density. The number of leaves per shoot, leaf size and leaf nitrogen content were also positively correlated with vole densities, while leaf C/N ratios were negatively correlated with vole densities. The positive correlation between vole densities and level of invertebrate herbivory is probably due to a facilitative effect of voles on invertebrate herbivores, mediated through changes in plant chemistry.

Abstract Vaccinium berries contain high yields of antioxidant compounds, such as flavonoids and carotenoids, which are recognized to benefit human health. Therefore, commercial interest in cultivated and wild Vaccinium berries is increasing globally. Flavonoids and carotenoids are important secondary metabolites, the biosynthesis of which is regulated by interaction of the genetic background of the plant and the surrounding environment. In fruits the production of secondary metabolites has also a tight linkage with developmental processes. The present thesis is focused on developmental and environmental factors affecting the biosynthesis of carotenoids and flavonoids in berries of the genus Vaccinium. In bilberry altogether eight carotenoids were detected, with lutein and β-carotene being the most abundant compounds, accompanied by minor amounts of xanthophylls, such as antheraxanthin, neoxanthin, violaxanthin and zeaxanthin. During ripening the accumulation of carotenoids decreased. Simultaneously the expression of a carotenoid cleavage dioxygenase (VmCCD1) gene increased, indicating degradation of carotenoids during the fruit development. Simultaneously, flavonols and anthocyanins accumulated during the last stages of berry ripening. Environmental factors had a marked effect on the anthocyanin profile of ripe bilberries. Especially lower temperatures affected more on the accumulation of the delphinidin-based anthocyanidins than on the cyanidin-based ones. However, the spectral light composition also played a role, as an increased proportion in blue wavelengths increased the delphinidin-based anthocyanins at ripe stage. The effect of light quality on other phenolic compounds and carotenoid accumulation was less pronounced. The present results can be applied to cultivation of Vaccinium species in order to produce berries with high nutritional value. / Tiivistelmä Vaccinium-suvun marjat sisältävät runsaasti antioksidatiivisiä yhdisteitä, kuten flavonoideja ja karotenoideja, joilla on viimeaikaisissa tutkimuksissa havaittu lukuisia terveysvaikutuksia. Tämän johdosta kiinnostus sekä viljeltyjä mustikoita että metsämustikoita kohtaan on kasvanut maailmanlaajuisesti. Flavonoidit ja karotenoidit ovat kasvien tuottamia sekundäärimetabolian yhdisteitä, joiden biosynteesin säätelyyn vaikuttavat sekä kasvin geneettinen tausta että ympäristötekijät. Hedelmissä ja marjoissa sekundääriyhdisteiden tuoton säätelyyn vaikuttaa huomattavasti myös kehityksellisten vaiheiden eteneminen. Tässä väitöskirjatyössä selvitettiin marjan kehitykseen liittyvien tekijöiden sekä ympäristötekijöiden vaikutusta flavonoidien ja karotenoidien biosynteesiin sekä viljelyillä mustikoilla että metsämustikoilla. Metsämustikoista tunnistettiin kahdeksan erilaista karotenoidi-yhdistettä, joista luteiini ja β-karoteeni esiintyivät yleisimpinä ksantofyllin, antheraksantiinin, neoksantiinin, violaksantiinin ja zeaksantiinin ohella. Karotenoidien pitoisuudet olivat suurimmillaan marjankehityksen alkuvaiheessa ja laskivat kypsyvissä marjoissa. Vastaavasti karotenoideja pilkkovan VmCCD1 entsyymigeenin ilmeneminen lisääntyi kohosi marjan kypysymisen loppuvaiheessa. Antosyaanien ja myrisetiini-pohjaisten flavonolien pitoisuudet sen sijaan kohosivat kypsymisen aikana. Tässä tutkimuksessa ympäristötekijöillä osoitettiin olevan suuri vaikutus antosyaanien laadulliseen kertymiseen, sillä delfinidiini-tyypin antosyaanien pitoisuudet muuttuivat enemmän ympäristötekijöiden vaikutuksesta verrattuna syanidiini-tyypin antosyaaneihin. Alhaisempi lämpötila yhdessä spesifisten valo-olosuhteiden kanssa lisäsi eniten delfinidiini-tyypin antosyaanien tuottoa. Myös valon laadulla voitiin osoittaa olevan vaikutusta antosyaanien biosynteesiin. Sininen valo lisäsi erityisesti delfinidiini-tyypin antosyaanien tuottoa kypsissä marjoissa. Flavonolien ja karotenoidien tuottoon valon laadun vaikutus oli sen sijaan vähäisempää. Väitöskirjatyön tuloksia voidaan hyödyntää marjojen viljelyssä haluttaessa vaikuttaa marjojen ravitsemukselliseen koostumukseen.

The aim of this work was to compare authenticity analysis in selected food products with fruit component by using, instrumental and molekular methods. Particularly, the presence of blueberries in fruit–based foodstuffs was verified. The theoretical part is focused on the characterization, chemical composition and botanical classification of blueberries (European blueberry and Canadian blueberry). It also contains an overview of instrumental and molecular diagnostic methods that can be used for the analysis of these fruits. The experimental part focuses on the selection of a suitable method of DNA isolation, and primers for the detection of blueberries in commercial products. DNA analysis was performed by qPCR and HRM analysis. In the experimental part, DNA was isolated in sufficient quality for PCR and the presence of blueberries in foodstuffs was verified by qPCR. Using HRM analysis, we were able to differentiate between bilberry (Vaccinium myrtillus) and blueberry (Vaccinium corymbosum) in control samples and in some commercial products. Certain phenolic acids and some flavonoids specific for blueberries were detected by HPLC. The total content of polyphenols and flavonoids was determined by UV / VIS spectrophotometry.