European beech (Fagus sylvatica L.) and Norway spruce (Picea abies (L.) Karst) are the most abundant deciduous and coniferous tree species in the Czech Republic. Both beech and spruce are due to their sensitivity to drought threatened by ongoing climate change that can cause deterioration of their vitality in current habitats. Drought periods are already becoming longer and stronger and previously rare spring droughts become more frequent. The dissertation thesis is based on experiments with Norway spruce and European beech seedling exposed to different levels of drought (well-watered, moderate drought and severe drought). After three to six weeks of drought, the seedlings were well-watered for another two weeks and their response of physiology, anatomy and morphology was evaluated. Experiments were conducted with seedlings at early stage of leaf development and with fully developer leaves to compare response to spring and summer drought. Overall drought response was stronger in spring. Both spring and summer drought reduced gas exchange parameters (in both beech and spruce seedlings up to 100 %), hydraulic conductivity and less strongly also fluorometric parameters. Efficiency of water use in stressed seedlings decreased in spring, while it increased in summer. Shoot and leaves biomass was more reduced by drought than root biomass. Tree-ring width decreased about two times more in beech seedlings than in spruce seedlings. Vessel and tracheids in stem and shoots reduced their number and size, on the contrary, vessel size increased in beech petioles. After rewatering the reduced parameters increased, however, hydraulic conductivity, anatomy and morphology did not reach values of well-watered seedlings. While most of physiological parameters restored in moderately stressed seedlings, in severely stressed seedlings mainly gas exchange parameters remained lower.

Transcriptome reconstruction is an important component in the bioinformatical part of transcriptome studies. It is particulary interesting when a reference genome is missing, highly fragmented or incomplete, since in such situations, a simple alignment (or mapping) would not necessarily tell the full story. One species with such a highly fragmented reference genome is the Norway spruce (Picea abies (L.) H. Karst.) -- a conifer, which is very important for Swedish economy. Given its long juvenile phase and irregular cone setting, the demand of cultivated seeds are larger than the supply. This yields a desire to understand the transcriptomal biology behind the cone setting in P. abies. This thesis presents an introduction to this situation, and the biological and bioinformatical background in general, followed by two papers in which this is applied: Paper I introduces a novel de novo transcriptome assembler, with a focus on recovering isoforms, and paper II makes use of this assembler to be able to detect connections between scaffolds in the P. abies genome. Paper I also studies P. abies var acrocona, a mutant with shorter juvenile phase than the wild type, in order to detect how cone setting is initiated.  From differential expression studies of both mRNA and miRNA, a number of genes potentially involved in cone-setting in P. abies were found, and also a set of miRNAs that could be involved in their regulation. / Transkriptomrekonstruktion är en viktig komponent i den bioinformatiska delen av transkriptomstudier. Särskilt intressant är detta när ett referensgenom saknas, är kraftigt fragmenterat eller ofullständigt, ty i dessa situationer skulle inte en vanlig inpassning (eller mappning) kunna berätta allt. En art med ett kraftigt fragmenterat referensgenom är gran (Picea abies (L.) H. Karst.) -- ett barrträd, som är mycket viktigt för svensk ekonomi. På grund av dess långa uppväxtsfas och oregelbundna kottsättning, så är efterfrågan av förädlade fröer större än utbudet. Detta lämnar en önskan att förstå den transkriptomala biologin bakom granens kottsättning. Denna avhandling presenterar en introduktion till denna situation, den generella biologiska och bioinformatiska bakgrunden, följd av två artiklar i vilket detta är tillämpat: Artikel I introducerar en ny de novo transkriptomassembler med fokus på att återskapa isoformer, och artikel II tillämpar denna assembler för att kunna hitta länkar mellan scaffolder (genom-delar som hittills inte kunnat länkas med varandra) i grangenomet. Artikel II studerar även granmutanten acrocona (kottegran), vilken har kortare uppväxtsfas än vildtypen, för att kunna se vad som initierar kottsättning.  Från differentiella expressionsstudier av såväl mRNA som miRNA, hittades ett antal gener potentiellt involverade i granens kottsättning, samt några miRNA som kan vara involverade i dess reglering. / <p>QC 2021-02-12</p>

The interactions between plants and insects are mediated by volatile molecules. Plants respond to stress by biosynthesis of chemical substances which can deter invading insects or pathogens. Some of these substances are volatile and are emitted to the surroundings and may attract or repel insects. Information about the susceptibility of individual plants to infestation, their volatile emissions and chemical defence is of interest, for example in selecting plants for tree breeding programs. This research was focused on finding volatile chemical markers of resistance in Norway spruce plants that do influence insects associated to conifers. Collection of headspace volatiles by SPME followed by separation and identification with GC-MS is effective in investigating biological systems with a minimum of disturbance. This method has here been used to investigate Norway spruce plants of different ages and stress conditions as well as trapping semiochemicals like nepetalactone emitted by the spruce shoot aphids. It was even possible to analyse the emission of single needles in vivo and obtain a chemical pattern of the site of the stress reaction. Seedlings of different ages showed differences in chemical composition of emitted volatiles, with the pine weevil repellent (S)-(-)-limonene as one of the main compounds. Wounded phloem of conventional plants emitted high amounts of monoterpenes while the phloem of mini plants emitted (3Z)-hexenal and (3Z)-hexen-1-ol. Norway spruce plants did respond to different stress elicitors with similar response, regardless of their genetic origin. The emissions from stressed Norway spruce plants mainly consist of (E)-β-farnesene, (E,E)-α-farnesene, (E)-α-bisabolene, (R)-(-)-linalool and methyl salicylate. Emissions from live spruce shoot aphids were detected during autumn periods, and a method to separate and identify the four diastereomers of nepetalactone by GC-MS and characteristic m/z-fragments was accomplished.

The purpose of this study was to assess the metal content present in pine needles collected from an old shooting range known for elevated lead levels in the soil by different analytical methods. The focus on conifers originated from their evergreen nature year-round, and their ability to absorb potentially harmful elements. Research on needles as biomonitors for environmental contamination, particularly in areas affected by heavy metal pollution, has gained significant global attention. Four trees, two Norway spruces (Picea abies), and two Scots pines (Pinus sylvestris) were identified within the designated area. Samples of conifer needles were collected in plastic bags. The needles underwent washing, drying, and grinding into powder for subsequent digestion method and quantitative determination of metal content using ICP-MS and MPAES. The study focused on elements commonly found in various types of ammunition, specifically manganese, iron, nickel, copper, zinc, molybdenum, antimony, and lead. Results obtained from both analytical methods, which was only manganese, showed statistically significant differences. During the analysis, only manganese could be quantified using MPAES, as the other analytes were below the detection limit. For ICP-MS, the LOD values were much lower, making this method more suitable, as all analytes were above LOD and could be quantified. The recovery values for the CRM were good for all elements when ICP-MS was utilized. However, the calculated recovery value for manganese decreased based on the results obtained from MPAES compared to the calculated recovery value for manganese based on the results obtained from ICP-MS. Interestingly, the analysis of conifer needles revealed no noticeable contamination from the shooting range. Instead, it highlighted variances among different pine needle species. The metal content in the pine needles remained within acceptable limits according to external literature sources.

The composition of lignin in the outer cell-wall layers of spruce and poplar has been studied and the data obtained have been compared with those of the mature reference wood in which the secondary cell wall predominates. Materials with exclusively or predominantly outer cell-wall layers were examined. Accurate data relating to the lignin monomer composition and the number of β-O-4´ bonds were obtained from pure middle lamella/primary cell wall lignin. Firstly, a 10 000 year old white spruce material, with most of the secondary cell wall missing, was studied. The aged lignin was composed of guaiacyl units only, and was slightly more condensed but otherwise similar to the reference lignin. Secondly, the developing xylem of a Norway spruce clone was analyzed during a growth season. In spring and early summer, growth is very rapid and the intention was to sample tissues in which the secondary cell-wall layers had not yet lignified, but where the outer layers at least had started to lignify. Microscopy, Klason lignin and carbohydrate analyses showed that the lignin in the developing xylem of samples from mid-June was located exclusively in the middle lamella. The lignin was more condensed, was composed of guaiacyl units only and contained more end-groups than the reference Norway spruce wood. Thirdly, the cambial tissues of a Balsam poplar clone were surveyed during a growth season. Both the phloem side and the xylem side of the cambial region were examined. The Klason lignin content and carbohydrate monomer distribution showed that in June and August the tissues on the phloem side contained material with mainly middle lamella/primary walls. In June, the xylem side in the cambial region contained mainly middle lamella/primary walls, and in August the secondary cell wall carbohydrates were being deposited. Both tissues contained lignin that was more condensed and had more end-groups than the reference lignin. In mid-June, the developing xylem had a ratio of syringyl to guaiacyl units of 0.6, whereas the ratio for the reference wood was 1.3. In the final study, lignin from the primary cell walls from a hybrid aspen cell suspension culture was investigated. The lignin contained only guaiacyl units which were more condensed than those observed in the reference poplar wood. / <p>QC 20100920</p>

Lignocellulosic plants are the most abundant source of terrestrial biomass and are one of the potential sources of renewable energy that can replace the use of fossil fuels. For a country such as Sweden, where the forest industry accounts for 10% of the total export, there would be large economical benefits associated with increased biomass yield. The availability of research on wood development conducted in conifer tree species, which represent the majority of the forestry in Sweden, is limited and the majority of research has been conducted in model angiosperm species such as Arabidopsis thaliana. However, the large evolutionary distance between angiosperms and gymnosperms limits the possibility to identify orthologous genes and regulatory pathways by comparing sequence similarity alone. At such large evolutionary distances, the identification of gene similarity is, in most cases, not sufficient and additional information is required for functional annotation. In this thesis, two high-spatial resolution datasets profiling wood development were processed; one from the angiosperm tree Populus tremula and the other from the conifer species Picea abies. These datasets were each published together with a web resource including tools for the exploration of gene expression, co-expression and functional enrichment of gene sets. One developed resource allows interactive, comparative co-expression analysis between species to identify conserved and diverged co-expression modules. These tools make it possible to identifying conserved regulatory modules that can focus downstream research and provide biologists with a resource to identify regulatory genes for targeted trait improvement. / Lignocellulosa är den vanligast förekommande källan till markburen biomassa och är en av de förnybara energikällor som potentiellt kan ersätta användningen av fossila bränslen. För ett land som Sverige, där skogsindustrin som står för 10 \% av den totala exporten, skulle därför en ökad produktion av biomassa kunna ge stora ekonomiska fördelar. Forskningen på barrträd, som utgör majoriteten av svensk skog är begränsad och den huvudsakliga forskningen som har bedrivits på växter, har skett i modell organismer tillhörande gruppen gömfröiga växter som till exempel i Arabidopsis thaliana. Det evolutionära avståndet mellan gömfröiga (blommor och träd) och nakenfröiga (gran och tall) begränsar dock möjligheten att identifiera regulatoriska system mellan dessa grupper. Vid sådana stora evolutionära avstånd krävs det mer än att bara identifiera en gen i en modellorganism utan ytterligare information krävs som till exempel genuttrycksdata. I denna avhandling har två högupplösta experiment som profilerar vedens utveckling undersökts; ett från gömfröiga träd Populus tremula och det andra från nakenföriga träd (barrträd) Picea abies. Datat som behandlats har publicerats tillsammans med webbsidor med flera olika verktyg för att bland annat visa genuttryck, se korrelationer av genuttryck och test för anrikning av funktionella gener i en grupp. En resurs som utvecklats tillåter interaktiva jämförelser av korrelationer mellan arter för att kunna identifiera moduler (grupper av gener) som bevaras eller skilts åt mellan arter över tid. Identifieringen av sådana bevarade moduler kan hjälpa att fokusera framtida forskning samt ge biologer en möjlighet att identifiera regulatoriska gener för en riktad förbättring av egenskaper hos träd.

This thesis deals with the influence of microclimatic conditions (air temperature near tree/shrub top, temperature of the stem and root zone) on the wood phenology of trees (Picea abies) and shrubs (Pinus mugo) growing on the same site in the alpine tree line (ATL) ecotone. Phenological and microclimatic research was carried out in the Giant Mountains at 1,370 m a. s. l. in the 2017 vegetation period, with the aim to compare the course of wood formation depending on the microclimate, to determine the temperature thresholds of wood growth and differences in the morphology of trees and shrubs. Based on the microclimate analysis of trees and shrubs, individual variables were determined (average, average minimum, average maximum, daily amplitude of air, stem and root zone temperatures) characterizing the growing season. The collection of micro-drills of trees and shrubs took place from April to October in an interval of 6 - 11 days, which were subsequently processed and examined in the laboratory. The following phenophases of wood development were distinguished: cambial, enlarging, wall-thickening, mature cells and cells of the previous ring. Subsequently, differences in the number of cells of individual phenophases were identified between trees and shrubs. Furthermore, the temperature thresholds for...

This thesis describes aspects of durability of Swedish softwood in above ground applications. The thesis consists of a summary of nine papers, which all aim to explain the existing variation in above ground microbial durability and moisture sensitivity of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). The methods used were two field tests, one accelerated durability test, and three water permeability tests. The main conclusions made in the study were that Norway spruce sapwood is more susceptible to discolouring fungi than heartwood. With respect to Scots pine, the only important factor for its natural durability above ground was whether the samples consisted of heartwood or of sapwood. There was also no systematic variation between pine heartwood from different stand origins in above ground conditions. Origin had no influence on durability in practice for neither spruce nor pine. Furthermore, in contradiction with traditional thinking, annual ring width and density had no influence on durability in any of the tests. The material came from two different samplings. The ‘old’ material was sampled in the beginning of the 1980s and consisted of Scots pine and Norway spruce from three different origins: the north, the central area, and the south of Sweden. The material was subjected to different handling conditions and surface and end-grain treatments. The ‘old’ material was used in a large above ground field test, carried out between 1985 and 1994. Unfortunately, the spruce sapwood and heartwood was not separated in this field test. In the field trial with the ‘old’ material, the most important factor for the durability of Norway spruce was the samples’ surface- and end-grain treatment. It was also observed that untreated spruce showed better durability than samples that were painted but without proper end-grain treatment. Samples originating from one of the stands were more sensitive towards air-drying than samples from the other two, which was reflected in a higher average moisture content and mass loss for the untreated samples. With respect to Scots pine, the most important factor was whether the sample consisted of heartwood or of sapwood. The heartwood samples were durable irrespective of their previous treatment. The Scots pine sapwood samples, on the other hand, had a very fluctuating moisture content if they were not surface- and end-painted. This was also reflected in higher mass losses in these samples. Even when properly surface- and end grain treated, the sapwood samples did not perform as well as the heartwood samples. The ‘new’ material consisted of Scots pine taken from six different stands, and Norway spruce from five different stands, all from areas in southern Sweden. The sampling was performed in order to achieve a large variation in wood properties. Thus, logs from areas with different climate and growth conditions were collected. This material was used for laboratory tests, and also for the second field test, which was evaluated after a test period of two years. In the second field test untreated Scots pine and Norway spruce samples from the ‘new’ material were investigated. Effects of origin and different tree diameters were examined. Furthermore, Scots pine was separated to heartwood and sapwood, and Norway spruce to mature and juvenile wood. The Norway spruce samples were sawn with vertical or horizontal annual rings. For the Norway spruce in the second field test, vertical annual rings were shown to be very beneficial for avoiding crack formation, while samples with horizontal annual rings displayed a large number of cracks. Despite this, the larger number of cracks did not result in a higher moisture content or more fungal discoloration. Juvenile wood had a slightly higher average discolouring fungal growth grading than the other spruce samples. For Scots pine, the only factor of importance was whether the samples consisted of heartwood or of sapwood. Annual ring width, density and origin had no significant effect on either fungal growth or moisture uptake. This observation held for both Norway spruce samples and Scots pine samples. An accelerated test was run in order to investigate Norway spruce sapwood and heartwood with respect to their differences in durability towards discolouring fungi and moisture uptake. The Mycologg method was used to accelerate fungal growth during a number of forced moisture cycles. The results showed that sapwood was much more sensitive to discolouring fungi than heartwood. Sapwood also recorded a higher average moisture content than heartwood. The differences that were observed between the types of samples were not due to annual ring width, density or origin. It was observed that Norway spruce sapwood samples and heartwood samples showed differences in liquid water permeability. This observation was made especially evident in a droplet absorption test. Water droplets were absorbed much faster on the sapwood samples in comparison with the heartwood samples. This higher affinity to water suggested that a sapwood surface would reach a higher moisture content than a heartwood surface. This observation provides a major explanation of why the sapwood samples showed poorer durability towards discolouring fungi in the Mycologg trials.

This thesis describes methods to obtain value–addedcompounds from TMP-turpentine obtained from the spruce, Piceaabies. The methodology focuses on biotransformations using twoapproaches: an oxygenation approach (i.e. oxygenation ofterpene hydrocarbons by cell cultures) and an esterificationapproach (i.e. lipase-catalysed transesterification of vinylacetate with terpene alcohols, and a further fractionation ofthe TMP-turpentine). The main constituents of the turpentine, a-pinene, b-pineneand limonene, were subjected to a P. abies suspension culture.Allylic oxidation formed the major products for α-pineneand β-pinene, which were further oxidised to theirrespective aldehyde or ketone. One of the minor products froma-pinene, cis-verbenol, was not only transformed into verbenonebut also isomerised to trans-verbenol. Limonene gavelimonene-(1,2)-epoxide as the major product. Fractionation of monoterpenes is accomplished throughphysical separation methods, chromatography and distillation,and lipase-catalysed transesterification of vinyl acetate withterpene alcohols. The esters of myrtenol and trans-pinocarveolwere separated from the more slowly reacting alcohols such asborneol and carveol by use of a combination of the Mucor mieheilipase and Candida antarctica lipase A as catalysts.Furthermore, the non-reacting tertiary terpene alcohols wereseparated from the reacting alcohols in a single step byCandida antarctica lipase A. Lipase-catalysed (Candida antarctica lipase B andPseudomonas cepacia lipase) transesterification of vinylacetate with sterically hindered secondary alcoholsunexpectedly yielded hemiacetals or hemiacetal esters. Thereaction conditions required to obtain these side products havebeen studied. <b>Keywords:</b>Picea abies, Pinaceae, Essential oilscomposition; Terpene alcohol; Hemiacetal; Hemiacetal ester,TMP-turpentine; Monoterpene; α-Pinene; β-Pinene;Limonene; Verbenol; Pinocarveol; Borneol; Myrtenol; Suspensioncell culture; Biotransformation; Lipase-catalysed; Oxidation;Allylic oxidation; Transesterification; Autoxidation;Separation.

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