Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants

Species specific LTR retrotransposons were first cloned in five rare relic species of drug plants located in the Perm’ region. Sequences of LTR retrotransposons were used for PCR analysis based on amplification of repeated sequences from LTR or other sites of retrotransposons (IRAP). Genetic diversity was studied in six populations of rare relic species of plants Adonis vernalis L. by means of the IRAP method; 125 polymorphic IRAP markers were analyzed. Parameters for DNA polymorphism and genetic diversity of A. vernalis populations were determined.

Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants

Species specific LTR retrotransposons were first cloned in five rare relic species of drug plants located in the Perm’ region. Sequences of LTR retrotransposons were used for PCR analysis based on amplification of repeated sequences from LTR or other sites of retrotransposons (IRAP). Genetic diversity was studied in six populations of rare relic species of plants Adonis vernalis L. by means of the IRAP method; 125 polymorphic IRAP markers were analyzed. Parameters for DNA polymorphism and genetic diversity of A. vernalis populations were determined.

Monitoring of Fungal Growth and Degradation of Wood

Valko- ja ruskolahosienet tunnetaan luonnossa tehokkaimpina puun ja karikkeen lignoselluloosan lahottajina. Valkolahosienet pystyvät hajottamaan kaikkia puun osia: ligniiniä, selluloosaa ja hemiselluloosaa. Selektiivisesti ligniiniä hajottavat sienet lahottavat puusta suhteessa enemmän vaikeasti hajoavaa ligniiniä kuin selluloosaa tai hemiselluloosaa, jolloin jäljelle jää valkoista ja miltei puhdasta selluloosaa. Bioteknisissä sovelluksissa juuri selektiviiviset valkolahottajat ovat kiinnostavia. Niiden avulla voidaan puuhaketta esikäsitellä esimerkiksi paperinvalmistuksessa haitallisen ligniinin poistamiseksi. Ruskolahosienet ovat huomattavia puun, puutavaran ja puisten rakenteiden lahottajia, kuten tässä työssä käytetty Gloeophyllum trabeum (saunasieni ) ja Poria (Postia) placenta (istukkakääpä). Ruskolahosienet hajottavat puusta hemiselluloosan lisäksi selluloosaa, jolloin jää jäljelle ruskea ja jauhomaiseksi mureneva ligniini. Ruskolahosienet muovaavat ligniiniä jonkin verran. Kahden ruskolahosienen G. trabeumin ja P. placentan lisäksi tutkittiin valkolahosieniä, joista Ceriporiopsis subvermispora (karstakääpä) ja harvinainen Physisporinus rivulosus -sieni (talikääpä) hajottavat ligniiniä erittäin selektiivisesti. Phanerochaete chrysosporium on kaikkialla paljon tutkittu sieni, ja Phlebia radiata valkolahosientä (rusorypykkä) on tutkittu paljon mikrobiologian osastolla. Lisäksi tutkittiin Phlebia tremellosa -sienten (hytyrypykkä) ligninolyyttisten entsyymien tuottoa ja 14C-leimatun synteettisen ligniinin (DHP) hajotusta. P. radiata ja P. tremellosa -sienten on todettu aiemmin hajottavan ligniiniä selektiivisesti. Työssä selvitettiin miten sienten kasvua voi mitata, miten vertailukelpoisia eri mittaamismenetelmillä saadut tulokset ovat ja ilmenevätkö sienten aktiivisimmat kasvuvaiheet samaan aikaan eri menetelmillä mitattuna. Tärkeimmät tulokset olivat seuraavat havainnot: (i) P. radiata ja P. tremellosa -sienikannat tuottivat ligniini- ja mangaaniperoksidaasientsyymejä (LiP ja MnP) sekä lakkaasia, ja sienistä puhdistettiin 2-3 LiP- ja P. radiatasta yksi MnP-entsyymi; (ii) P. tremellosa -sienet hajottivat leimattua synteettistä ligniiniä (DHP) yhtä hyvin kuin paljon tutkitut P. chrysosporium ja P. radiata -sienet; (iii) puu, sienen luonnollinen kasvualusta, lisäsi valkolaho- ja ruskolahosienten demetoksylaatiota [O14CH3]-leimatusta ligniinin malliyhdisteestä 14CO2:ksi ilman puuta olleeseen alustaan verrattuna; (iv) demetoksylaatio (14CO2:n tuotto) oli normaalissa ilma-atmosfäärissä useimmiten parempi happeen verrattuna; (v) hapessa paras 14CO2:n tuotto saatiin puupalakasvatuksissa, joihin oli lisätty ravinnetyppeä tai typen lisäksi glukoosia sekä valkolaho- että ruskolahosienillä; (vi) ilmassa 14CO2:n tuotto oli puulla voimakkainta valkolahosienillä ilman lisäravinteita, kun taas G. trabeum -sienellä se oli yhtä hyvä eri alustoissa; (vii) biomassan muodostuminen rihmastojen ergosterolipitoisuuksista mitattuna oli ruskolahosienillä parempi kuin valkolahosienillä; (viii) ja biomassojen huippupitoisuudet olivat 6:lla sienellä eri suuruisia ja niiden maksimimäärien ajankohdat vaihtelivat viiden viikon kasvatusten kuluessa. Mikrobiologian osastolla Viikissä eristetty ja paljon tutkittu P. radiata -valkolahosieni oli mukana kaikissa tehdyissä kokeissa. Sienen LiP-aktiivisuus ja 14CO2:n tuotto 14C-rengas-leimatusta synteettisestä ligniinistä (DHP) korreloivat erittäin hyvin. Biomassan muodostuminen ergosterolilla määritettynä tuki hyvin entsyymiaktiivisuusmittauksilla ja isotooppikasvatuksilla saatuja tuloksia.

Archaea in the Mycorrhizosphere of Boreal Forest Trees

Archaea were long thought to be a group of ancient bacteria, which mainly lived in extreme environments. Due to the development of DNA sequencing methods and molecular phylogenetic analyses, it was shown that the living organisms are in fact divided into three domains; the Archaea, Bacteria and the Eucarya. Since the beginning of the previous decade, it was shown that archaea generally inhabit moderate environments and that these non-extremophilic archaea are more ubiquitous than the extremophiles. Group 1 of non-extreme archaea affiliate with the phylum Crenarchaeota. The most commonly found soil archaea belong to the subgroup 1.1b. However, the Crenarchaeota found in the Fennoscandian boreal forest soil belong to the subgroup 1.1c. The organic top layer of the boreal forest soil, the humus, is dominated by ectomycorrhizal fungal hyphae. These colonise virtually all tree fine root tips in the humus layer and have been shown to harbour distinct bacterial populations different from those in the humus. The archaea have also been shown to colonise both boreal forest humus and the rhizospheres of plants. In this work, studies on the archaeal communities in the ectomycorrhizospheres of boreal forest trees were conducted in microcosms. Archaea belonging to the group 1.1c Crenarchaeota and Euryarchaeota of the genera Halobacterium and Methanolobus were detected. The archaea generally colonised fungal habitats, such as ectomycorrhizas and external mycelia, rather than the non-mycorrhizal fine roots of trees. The species of ectomycorrhizal fungus had a great impact on the archaeal community composition. A stable euryarchaeotal community was detected especially in the mycorrhizas, of most of the tested Scots pine colonising ectomycorrhizal fungi. The Crenarchaeota appeared more sporadically in these habitats, but had a greater diversity than the Euryarchaeota. P. involutus mycorrhizas had a higher diversity of 1.1c Crenarchaeota than the other ectomycorrhizal fungi. The detection level of archaea in the roots of boreal trees was generally low although archaea have been shown to associate with roots of different plants. However, alder showed a high diversity of 1.1c Crenarchaeota, exceeding that of any of the tested mycorrhizas. The archaeal 16S rRNA genes detected from the non-mycorrhizal roots were different from those of the P. involutus mycorrhizas. In the phylogenetic analyses, the archaeal 16S rRNA gene sequences obtained from non-mycorrhizal fine roots fell in a separate cluster within the group 1.1c Crenarchaeota than those from the mycorrhizas. When the roots of the differrent tree species were colonised by P. involutus, the diversity and frequency of the archaeal populations of the different tree species were more similar to each other. Both Cren- and Euryarchaeota were enriched in cultures to which C-1 substrates were added. The 1.1c Crenarchaeota grew anaerobically in mineral medium with CH4 and CO2 as the only available C sources, and in yeast extract media with CO2 and CH4 or H2. The crenarchaeotal diversity was higher in aerobic cultures on mineral medium with CH4 or CH3OH than in the anaerobic cultures. Ecological functions of the mycorrhizal 1.1c Crenarchaeota in both anaerobic and aerobic cycling of C-1 compounds were indicated. The phylogenetic analyses did not divide the detected Crenarchaeota into anaerobic and aerobic groups. This may suggest that the mycorrhizospheric crenarchaeotal communities consist of closely related groups of anaerobic and aerobic 1.1c Crenarchaeota, or the 1.1c Crenarchaeota may be facultatively anaerobic. Halobacteria were enriched in non-saline anaerobic yeast extract medium cultures in which CH4 was either added or produced, but were not detected in the aerobic cultures. They may potentially be involved in anaerobic CH4 cycling in ectomycorrhizas. The CH4 production of the mycorrhizal samples was over 10 times higher than for humus devoid of mycorrhizal hyphae, indicating a high CH4 production potential of the mycorrhizal metanogenic community. Autofluorescent methanogenic archaea were detected by microscopy and 16S rRNA gene sequences of the genus Methanolobus were obtained. The archaeal community depended on both tree species and the type of ectomycorrhizal fungus colonising the roots and the Cren- and Euryarchaeota may have different ecological functions in the different parts of the boreal forest tree rhizosphere and mycorrhizosphere. By employing the results of this study, it may be possible to isolate both 1.1c Crenarchaeota as well as non-halophilic halobacteria and aerotolerant methanogens from mycorrhizospheres. These archaea may be used as indicators for change in the boreal forest soil ecosystem due to different factors, such as exploitations of forests and the rise in global temperature. More information about the microbial populations with apparently low cell numbers but significant ecological impacts, such as the boreal forest soil methanogens, may be of crucial importance to counteract human impacts on such globally important ecosystems as the boreal forests.

Digital Photography in the Diagnosis and Follow-up of Ocular Diseases

Purpose: The aim of the present study was to develop and test new digital imaging equipment and methods for diagnosis and follow-up of ocular diseases. Methods: The whole material comprised 398 subjects (469 examined eyes), including 241 patients with melanocytic choroidal tumours, 56 patients with melanocytic iris tumours, 42 patients with diabetes, a 52-year old patient with chronic phase of VKH disease, a 30-year old patient with an old blunt eye injury, and 57 normal healthy subjects. Digital 50° (Topcon TRC 50 IA) and 45° (Canon CR6-45NM) fundus cameras, a new handheld digital colour videocamera for eye examinations (MediTell), a new subtraction method using the Topcon Image Net Program (Topcon corporation, Tokyo, Japan), a new method for digital IRT imaging of the iris we developed, and Zeiss photoslitlamp with a digital camera body were used for digital imaging. Results: Digital 50° red-free imaging had a sensitivity of 97.7% and two-field 45° and 50° colour imaging a sensitivity of 88.9-94%. The specificity of the digital 45°-50° imaging modalities was 98.9-100% versus the reference standard and ungradeable images that were 1.2-1.6%. By using the handheld digital colour video camera only, the optic disc and central fundus located inside 20° from the fovea could be recorded with a sensitivity of 6.9% for detection of at least mild NPDR when compared with the reference standard. Comparative use of digital colour, red-free, and red light imaging showed 85.7% sensitivity, 99% specificity, and 98.2 % exact agreement versus the reference standard in differentiation of small choroidal melanoma from pseudomelanoma. The new subtraction method showed growth in four of 94 melanocytic tumours (4.3%) during a mean ±SD follow-up of 23 ± 11 months. The new digital IRT imaging of the iris showed the sphincter muscle and radial contraction folds of Schwalbe in the pupillary zone and radial structural folds of Schwalbe and circular contraction furrows in the ciliary zone of the iris. The 52-year-old patient with a chronic phase of VKH disease showed extensive atrophy and occasional pigment clumps in the iris stroma, detachment of the ciliary body with severe ocular hypotony, and shallow retinal detachment of the posterior pole in both eyes. Infrared transillumination imaging and fluorescein angiographic findings of the iris showed that IR translucence (p=0.53), complete masking of fluorescence (p=0.69), presence of disorganized vessels (p=0.32), and fluorescein leakage (p=1.0) at the site of the lesion did not differentiate an iris nevus from a melanoma. Conclusions: Digital 50° red-free and two-field 50° or 45° colour imaging were suitable for DR screening, whereas the handheld digital video camera did not fulfill the needs of DR screening. Comparative use of digital colour, red-free and red light imaging was a suitable method in the differentiation of small choroidal melanoma from different pseudomelanomas. The subtraction method may reveal early growth of the melanocytic choroidal tumours. Digital IRT imaging may be used to study changes of the stroma and posterior surface of the iris in various diseases of the uvea. It contributed to the revealment of iris atrophy and serous detachment of the ciliary body with ocular hypotony together with the shallow retinal detachment of the posterior pole as new findings of the chronic phase of VKH disease. Infrared translucence and angiographic findings are useful in differential diagnosis of melanocytic iris tumours, but they cannot be used to determine if the lesion is benign or malignant.

Overwintering ecology of northern field layer plants : snow and photosynthesis in Vaccinium vitis-idaea L.

Winter is a significant period for the seasonality of northern plants, but is often overlooked when studying the interactions of plants and their environment. This study focuses on the effects of overwintering conditions, including warm winter periods, snow, and snowmelt on boreal and sub-Arctic field layer plants. Wintertime photosynthesis and related physiological factors of evergreen dwarf shrubs, particularly of Vaccinium vitis-idaea, are emphasised. The work combines experiments both in the field and in growth chambers with measurements in natural field conditions. Evergreen dwarf shrubs are predominantly covered by snow in the winter. The protective snow cover provides favourable conditions for photosynthesis, especially during the spring before snowmelt. The results of this study indicate that photosynthesis occurs under the snow in V. vitis-idaea. The light response of photosynthesis determined in field conditions during the period of snow cover shows that positive net CO2 exchange is possible under the snow in the prevailing light and temperature. Photosynthetic capacity increases readily during warm periods in winter and the plants are thus able to replenish carbohydrate reserves lost through respiration. Exposure to low temperatures in combination with high light following early snowmelt can set back photosynthesis as sustained photoprotective measures are activated and photodamage begins to build up. Freezing may further decrease the photosynthetic capacity. The small-scale distribution of many field layer plants, including V. vitis-idaea and other dwarf shrubs, correlates with the snow distribution in a forest. The results of this study indicate that there are species-specific differences in the snow depth affinity of the field and ground layer species. Events and processes taking place in winter can have a profound effect on the overall performance of plants and on the interactions between plants and their environment. Understanding the processes involved in the overwintering of plants is increasingly important as the wintertime climate in the north is predicted to change in the future.

A matter of taste : genetic and environmental influences on responses to sweetness

Diet is a major player in the maintenance of health and onset of many diseases of public health importance. The food choice is known to be largely influenced by sensory preferences. However, in many cases it is unclear whether these preferences and dietary behaviors are innate or acquired. The aim of this thesis work was to study the extent to which the individual differences in dietary responses, especially in liking for sweet taste, are influenced by genetic factors. Several traits measuring the responses to sweetness and other dietary variables were applied in four studies: in British (TwinsUK) and Finnish (FinnTwin12 and FinnTwin16) twin studies and in a Finnish migraine family study. All the subjects were adults and they participated in chemosensory measurements (taste and smell tests) and filled in food behavior questionnaires. Further, it was studied, whether the correlations among the variables are mediated by genetic or environmental factors and where in the genome the genes influencing the heritable traits are located. A study of young adult Finnish twins (FinnTwin16, n=4388) revealed that around 40% of the food use is attributable to genetic factors and that the common, childhood environment does not affect the food use even shortly after moving from the parents home. Both the family study (n=146) and the twin studies (British twins, n=663) showed that around half of the variation in the liking for sweetness is inherited. The same result was obtained both by the chemosensory measurements (heritability 41-49%) and the questionnaire variables (heritability 31-54%). By contrast, the intensity perception of sweetness or the responses to saltiness were not influenced by genetic factors. Further, a locus influencing the use-frequency of sweet foods was identified on chromosome 16p. A closer examination of the relationships among the variables based on 663 British twins revealed that several genetic and environmental correlations exist among the different measures of liking for sweetness. However, these correlations were not very strong (range 0.06-0.55) implying that the instruments used measure slightly different aspects of the phenomenon. In addition, the assessment of the associations among responses to fatty foods, dieting behaviors, and body mass index in twin populations (TwinsUK n=1027 and FinnTwin12 n=299) showed that the dieting behaviors (cognitive restraint, uncontrolled eating, and emotional eating) mediate the relationship between obesity and diet. In conclusion, the work increased the understanding of the background variables of human eating behavior. Genetic effects were shown to underlie the variation of many dietary traits, such as liking for sweet taste, use of sweet foods, and dieting behaviors. However, the responses to salty taste were shown to be mainly determined by environmental factors and thus should more easily be modifiable by dietary education, exposure, and learning than sweet taste preferences. Although additional studies are needed to characterize the genetic element located on chromosome 16 that influences the use-frequency of sweet foods, the results underline the importance of inherited factors on human eating behavior.

Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

Pathogen-Induced Defense Signaling and Signal Crosstalk in Arabidopsis

Erwinia carotovora subsp. carotovora is a bacterial phytopathogen that causes soft rot in various agronomically important crop plants. A genetically specified resistance to E. carotovora has not been defined, and plant resistance to this pathogen is established through nonspecific activation of basal defense responses. This, together with the broad host range, makes this pathogen a good model for studying the activation of plant defenses. Production and secretion of plant cell wall-degrading enzymes (PCWDE) are central to the virulence of E. carotovora. It also possesses the type III secretion system (TTSS) utilized by many Gram-negative bacteria to secrete virulence- promoting effector proteins to plant cells. This study elucidated the role of E. carotovora HrpN (HrpNEcc), an effector protein secreted through TTSS, and the contribution of this protein in the virulence of E. carotovora. Treatment of plants with HrpNEcc was demonstrated to induce a hypersensitive response (HR) as well as resistance to E. carotovora. Resistance induced by HrpNEcc required both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense signaling in Arabidopsis. Simultaneous treatment of Arabidopsis with HrpNEcc and PCWDE polygalacturonase PehA elicited accelerated and enhanced induction of defense genes but also increased production of superoxide and lesion formation. This demonstrates mutual amplification of defense signaling by these two virulence factors of E. carotovora. Identification of genes that are rapidly induced in response to a pathogen can provide novel information about the early events occurring in the plant defense response. CHLOROPHYLLASE 1 (AtCLH1) and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) are both rapidly triggered by E. carotovora in Arabidopsis. Characterization of AtCLH1 encoding chlorophyll-degrading enzyme chlorophyllase indicated that it might have a role in chlorophyll degradation during plant tissue damage. Silencing of this gene resulted in increased accumulation of reactive oxygen species (ROS) in response to pathogen infection in a light-dependent manner. This led to enhanced SA-dependent defenses and resistance to E. carotovora. Moreover, crosstalk between different defense signaling pathways was observed; JA-dependent defenses and resistance to fungal pathogen Alternaria brassicicola were impaired, indicating antagonism between SA- and JA-dependent signaling. Characterization of ERD15 suggested that it is a novel, negative regulator of abscisic acid (ABA) signaling in Arabidopsis. Overexpression of ERD15 resulted in insensitivity to ABA and reduced tolerance of the plants to dehydration stress. However, simultaneously, the resistance of the plants to E. carotovora was enhanced. Silencing of ERD15 improved freezing and drought tolerance of transgenic plants. This, together with the reducing effect of ABA on seed germination, indicated hypersensitivity to this phytohormone. ERD15 was hypothesized to act as a capacitor that controls the appropriate activation of ABA responses in Arabidopsis.

Cytokinin signalling in the regulation of cambial development

Secondary growth of plants is of pivotal importance in terrestrial ecosystems, providing a significant carbon sink in the form of wood. As plant biomass accumulation results largely from the cambial growth, it is surprising that quite little is known about the hormonal or genetic control of this important process in any plant species. The central aim of my thesis studies was to explore the function of cytokinin in the regulation of cambial development. Since their discovery as regulators of plant cell divisions, cytokinins have been assumed to participate in the control of cambial development. Evidence for this action was deduced from hormone treatment experiments, where exogenously applied cytokinin was shown to enhance cambial cell divisions in diverse plant organs and species. In my thesis work, the conservation of cytokinin signalling and homeostasis genes between a herbaceous plant, Arabidopsis, and a hardwood tree species, Populus trichocarpa. Presumably reflecting the ancient origin of cytokinin signalling system, the Populus genome contains orthologs for all Arabidopsis cytokinin signalling and homeostasis genes. Thus, genes belonging to five main families of isopentenyl transferases (IPTs), cytokinin oxidases (CKXs), two-component receptors, histidine containing phosphotransmitters (HPts) and response regulators (RRs) were identified from the Populus genome. Three subfamilies associated with cytokinin signal transduction, the CKI1-like family of two-component receptors, the AHP4-like HPts, and the ARR22-like atypical RRs, were significantly larger in Populus genome than in Arabidopsis. Potential contribution to the extensive secondary development of Populus by the members of these considerably expanded gene families will be discussed. Representatives of all cytokinin signal transduction elements were expressed in the Populus cambial zone, and most of the expressed genes appeared to be slightly more abundant on the phloem side of the meristem. The abundance of cytokinin related genes in the cambium emphasizes the important role of this hormone in the regulation of the extensive secondary growth characteristic of tree species. The function of the pseudo HPts in primary vascular development was studied in Arabidopsis root vasculature. It was demonstrated that the pseudo HPt AHP6 has a role in locally inhibiting cytokinin signalling in the protoxylem position in the Arabidopsis root, thus enabling differentiation of the protoxylem cell file. The possible role of pseudo HPts in cambial development will be discussed. The expression peak of cytokinin signalling genes in the tree cambial zone strongly indicates that cytokinin has a role in the regulation of this meristem function. To address whether cytokinin signalling is required for cambial activity, transgenic Populus trees with modified cytokinin signalling were produced. These trees were expressing a cytokinin catabolic gene from Arabidopsis, CYTOKININ OXIDASE 2, (AtCKX2) under the promoter of a Betula CYTOKININ RECEPTOR 1 (BpCRE1). The pBpCRE1::CKX2 transgenic Populus trees showed a reduced concentration of a biologically active cytokinin, correlating with their impaired cytokinin response. Furthermore, the radial growth of these trees was compromised, as illustrated by a smaller stem diameter than in wild-type trees of the same height. Moreover, the level of cambial cytokinin signalling was down-regulated in these thin-stemmed trees. The reduced signalling correlated with a decreased number of meristematic cambial cells, implicating cytokinin activity as a direct regulator of cambial cell division activity. Together, the results of my study indicate that cytokinins are major hormonal regulators required for cambial development.

Mitochondrial helicase Twinkle in mtDNA copy number regulation and a late-onset disease model

Mitochondrial diseases are caused by disturbances of the energy metabolism. The disorders range from severe childhood neurological diseases to muscle diseases of adults. Recently, mitochondrial dysfunction has also been found in Parkinson s disease, diabetes, certain types of cancer and premature aging. Mitochondria are the power plants of the cell but they also participate in the regulation of cell growth, signaling and cell death. Mitochondria have their own genetic material, mtDNA, which contains the genetic instructions for cellular respiration. Single cell may host thousands of mitochondria and several mtDNA molecules may reside inside single mitochondrion. All proteins needed for mtDNA maintenance are, however, encoded by the nuclear genome, and therefore, mutations of the corresponding genes can also cause mitochondrial disease. We have here studied the function of mitochondrial helicase Twinkle. Our research group has previously identified nuclear Twinkle gene mutations underlying an inherited adult-onset disorder, progressive external ophthalmoplegia (PEO). Characteristic for the PEO disease is the accumulation of multiple mtDNA deletions in tissues such as the muscle and brain. In this study, we have shown that Twinkle helicase is essential for mtDNA maintenance and that it is capable of regulating mtDNA copy number. Our results support the role of Twinkle as the mtDNA replication helicase. No cure is available for mitochondrial disease. Good disease models are needed for studies of the cause of disease and its progression and for treatment trials. Such disease model, which replicates the key features of the PEO disease, has been generated in this study. The model allows for careful inspection of how Twinkle mutations lead to mtDNA deletions and further causes the PEO disease. This model will be utilized in a range of studies addressing the delay of the disease onset and progression and in subsequent treatment trials. In conclusion, in this thesis fundamental knowledge of the function of the mitochondrial helicase Twinkle was gained. In addition, the first model for adult-onset mitochondrial disease was generated.

Children’s Relationship to Plants among Primary School Children in Finland

There is considerable evidence that children in modern society are losing
their contact with nature and, more precisely, with green plants. Is this also the case in Finland, a northern country famous for its forests and wild nature? This study examines the relationship of 9- to 10-year-old Finnish schoolchildren with the green environment and plants. The data were gathered by a questionnaire
comprising structured and open-ended questions. The focus of the research was on two comparisons: first, on the nature and child relationship in rural and urban neighborhoods and, second, among boys and girls. Participants in the study amounted to a total of 76 children, 42 in the Helsinki suburb area and 34 in Paltamo. The results suggested that the children in rural surroundings had closer contact with nature than their urban associates. For example, the children of Paltamo reported to know the trees better, and considered human beings to be part of nature more often (100% vs. 76% of the pupils in Helsinki, P = 0,003). Similarly, the results showed that girls in the study (N = 48) were more interested in plants than boys (N = 28). For the girls, the beauty and joy of plants was important, whereas the boys appreciated plants as the source of life. After the pre-questioning, the children of Helsinki participated in an in-class horticultural intervention and 10 days later, answered a similar questionnaire again. The results of the open-ended questions revealed that equally to children in other Western countries, Finnish children may also be in danger of losing their direct contact with the natural environment. It was common to pass free time in organized sports such as hockey or football (boys), or by just walking and talking with friends (girls). Rural children told that they still built huts, pick berries, and climb trees, whereas urban children played in parks and city groves. The results suggest that it is essential to research
further the children’s own experiences if we are to understand, and subsequently, to enhance, the crucial role of the green environment in their lives. Horticultural interventions can be effective starting points to add to children’s knowledge, affection, and interest toward greenery, but it is highly recommended that they take place outdoors rather than indoors.

Children’s Relationship to Plants among Primary School Children in Finland

There is considerable evidence that children in modern society are losing
their contact with nature and, more precisely, with green plants. Is this also the case in Finland, a northern country famous for its forests and wild nature? This study examines the relationship of 9- to 10-year-old Finnish schoolchildren with the green environment and plants. The data were gathered by a questionnaire
comprising structured and open-ended questions. The focus of the research was on two comparisons: first, on the nature and child relationship in rural and urban neighborhoods and, second, among boys and girls. Participants in the study amounted to a total of 76 children, 42 in the Helsinki suburb area and 34 in Paltamo. The results suggested that the children in rural surroundings had closer contact with nature than their urban associates. For example, the children of Paltamo reported to know the trees better, and considered human beings to be part of nature more often (100% vs. 76% of the pupils in Helsinki, P = 0,003). Similarly, the results showed that girls in the study (N = 48) were more interested in plants than boys (N = 28). For the girls, the beauty and joy of plants was important, whereas the boys appreciated plants as the source of life. After the pre-questioning, the children of Helsinki participated in an in-class horticultural intervention and 10 days later, answered a similar questionnaire again. The results of the open-ended questions revealed that equally to children in other Western countries, Finnish children may also be in danger of losing their direct contact with the natural environment. It was common to pass free time in organized sports such as hockey or football (boys), or by just walking and talking with friends (girls). Rural children told that they still built huts, pick berries, and climb trees, whereas urban children played in parks and city groves. The results suggest that it is essential to research
further the children’s own experiences if we are to understand, and subsequently, to enhance, the crucial role of the green environment in their lives. Horticultural interventions can be effective starting points to add to children’s knowledge, affection, and interest toward greenery, but it is highly recommended that they take place outdoors rather than indoors.

Children’s Relationship to Plants among Primary School Children in Finland

There is considerable evidence that children in modern society are losing
their contact with nature and, more precisely, with green plants. Is this also the case in Finland, a northern country famous for its forests and wild nature? This study examines the relationship of 9- to 10-year-old Finnish schoolchildren with the green environment and plants. The data were gathered by a questionnaire
comprising structured and open-ended questions. The focus of the research was on two comparisons: first, on the nature and child relationship in rural and urban neighborhoods and, second, among boys and girls. Participants in the study amounted to a total of 76 children, 42 in the Helsinki suburb area and 34 in Paltamo. The results suggested that the children in rural surroundings had closer contact with nature than their urban associates. For example, the children of Paltamo reported to know the trees better, and considered human beings to be part of nature more often (100% vs. 76% of the pupils in Helsinki, P = 0,003). Similarly, the results showed that girls in the study (N = 48) were more interested in plants than boys (N = 28). For the girls, the beauty and joy of plants was important, whereas the boys appreciated plants as the source of life. After the pre-questioning, the children of Helsinki participated in an in-class horticultural intervention and 10 days later, answered a similar questionnaire again. The results of the open-ended questions revealed that equally to children in other Western countries, Finnish children may also be in danger of losing their direct contact with the natural environment. It was common to pass free time in organized sports such as hockey or football (boys), or by just walking and talking with friends (girls). Rural children told that they still built huts, pick berries, and climb trees, whereas urban children played in parks and city groves. The results suggest that it is essential to research
further the children’s own experiences if we are to understand, and subsequently, to enhance, the crucial role of the green environment in their lives. Horticultural interventions can be effective starting points to add to children’s knowledge, affection, and interest toward greenery, but it is highly recommended that they take place outdoors rather than indoors.