Carl Gunnar Fossdal

Avdelingsleder

(+47) 481 46 583
carl.gunnar.fossdal@nibio.no

Sted
Ås - Bygg H8

Besøksadresse
Høgskoleveien 8, 1433 Ås

Biografi

Jeg forsker på klimatilpasning hos skogtrær, resistens mot skadesopp, sopp gener/enzym involvert i råte og mikrobiom studier, med et faglig fokus på å forstå de molekylære mekanismene som styrer disse biologiske prosessene. Jeg har jobbet i NIBIO siden 1995. I 1989 ble jeg Cand. Mag med hovedvekt på kjemiske fag ved Universitetet i Oslo. Senere Cand. Scient. i bioteknologi ved Universitetet i Oslo i 1991. I 1999, tok jeg Dr. Scient. graden innen Bioteknologi ved Norges landbrukshøgskole (NLH). Professor kvalifisert innen molekylærbiologi i 2006. Siden 2016 har jeg vært forskningssjef og leder for Avdeling Skoghelse i Divisjon for bioteknologi og plantehelse i NIBIO. Jeg har jobbet som prosjektleder på flere forskningsprosjekter med tema relatert til trærs forsvar mot soppsykdommer, gener som koder for vednedbrytende enzyme og med epigenetiske mekanismer betydning for planters evne til å tilpasse seg endringer i miljøet. Jeg har vært og er involvert i en rekke større forskningsprosjekter finansiert av FRIMEDBIO programmet til Norges forskningsråd, inkludert et pågående TOPPFORSK prosjekt innen fagfeltet epigenetikk.

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Acetylation of wood can provide protection against wood deteriorating fungi, but the exact degradation me- chanism remains unclear. The aim of this study was to determine the effect of acetylation of Pinus radiata wood (weight percent gain 13, 17 and 21%) on the expression of genes involved in decay by brown-rot fungus Rhodonia placenta. Gene expression analysis using qRT-PCR captured incipient to advanced decay stages. As expected the initiation of decay was delayed as a result the degree of acetylation. However, once decay was established, the rate of degradation in acetylated samples was similar to that of unmodi fied wood. This suggests a delay in decay rather than an absolute protection threshold at higher acetylation levels. In accordance with previous studies, the oxidative system of R. placenta was more active in wood with higher degrees of acetylation and expression of cellulose active enzymes was delayed for acetylated samples compared to untreated samples. The reason for the delay in the latter might be because of the slower diffusion rate in acetylated wood or that partially acetylated cellobiose may be less effective in triggering production of saccharification enzymes. Enzymes involved in hemicellulose and pectin degradation have previously not been focused on in studies of degradation of acetylated wood. Surprisingly, CE16 carbohydrate esterase, assumed to be involved in deace- tylation of carbohydrates, was expressed significantly more in untreated samples compared to highly acetylated samples. We hypothesise that this enzyme might be regulated through a negative feedback system, where acetic acid supresses the expression. The up-regulation of two expansin genes in acetylated samples suggests that their function, to loosen the cell wall, is needed more in acetylated wood due the physical bulking of the cell wall. In this study, we demonstrate that acetylation affects the expression of specific target genes not previously re- ported, resulting in delayed initiation of decay. Thus, targeting these degradation mechanisms can contribute to improving wood protection systems.

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Bark beetles and their symbiotic bluestain fungi kill more trees than all other natural factors and cause great economic losses in Norway spruce and other conifers. The tree's natural defenses are the most important factor maintaining bark beetle-fungus complexes at low, endemic levels. Spraying Norway spruce trees with the plant hormone methyl jasmonate (MeJA) primes tree defenses without eliciting notable induced defenses, but enables the trees to respond much more quickly and strongly when challenged by bark beetles or fungi several weeks after treatment. This phenomenon, known as defense priming, is a form of acquired resistance that enables cost-effective and vigorous defense responses. In field experiments with 50-year-old clonal spruce trees terpene concentrations in the bark increased 60-fold within 24 h after mechanical wounding of MeJA primed trees, compared with a 13-fold increase in unprimed control trees. We also observed altered transcriptional patterns in primed trees using Illumina deep transcriptome sequencing. When wounded, primed trees launched vigorous induced defenses with significant differential regulation of gene transcripts, such as those involved in phenylpropanoid synthesis leading to lignification. Resistance-like genes, such as the NB-LRR coding genes, are also more rapidly induced in primed than in unprimed trees. Transcriptome results from primed but unwounded trees indicate an alteration in the state of the chromatin, resembling changes associated with the activity of the epigenetic machinery creating long-lasting epigenetic marks. We do not know yet how long the primed state is activated in Norway spruce, but our data so far indicate that it may last for at least 3 years.

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Plants are exposed to various pathogens in their environment and have developed immune systems with multiple layers of defence to fight-back. However, often pathogens overcome the resistance barriers, infect the plants to cause the disease. Pathogens that cause diseases on economically important crop plants like strawberry incur huge losses to the agriculture industry. For example, The 2016 outbreak of strawberry grey mould (Botrytis cinerea) in Norway caused up to 95% crop losses. Outbreaks like this underline the importance of developing novel and sustainable tools to combat plant diseases, for example by increasing the plants’ natural disease resistance. Priming plant defences using chemical elicitors may be effective in providing the enhanced resistance against multiple pathogens. We have used β-aminobutyric acid (BABA) as a chemical priming agent to induce resistance in Fragaria vesca against Botrytis cinerea. Effects of BABA on disease progression and defence responses of Fragaria are being characterized using molecular tools like RNAseq, RT-PCR and ChIP. As priming chemicals may induce an epigenetic memory in treated plants, we also plan to study the histone methylation patterns in primed plants and the genes that are regulated. Our long-term aim is to understand the duration of the epigenetic memory and its cross-generational transmission to the progeny in Fragaria. Our results will help guide various crop protection strategies in addition to providing new insights to develop novel tools for plant disease management.

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Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28◦C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21–22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21–22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic regulation, which in turn could provide a feedback process leading to the formation of epigenetic marks. We suggest that TIR, NBS and LRR domain containing proteins could fulfill more general functions for signal transduction from external environmental stimuli and conversion them into molecular response. Fine-tuning of the miRNA production likely participates in both developmental regulation and epigenetic memory formation in Norway spruce.

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Forest covers 37% of Norway’s combined area, almost half of which is made up by the tree species called Norway spruce. The rest consists of mostly pine and birch. It is therefore only natural that spruce forests should feature so heavily on black metal album covers and lyrics. The extreme music genre of black metal, as we think of it today, was birthed in Norway. Although it could be said that its place of origin was accidental, the subsequent use and appropriation of that place’s topographic features was not. Since its inception in the early nineties, the genre has spawned countless bands across the globe, many who take on its misanthropic ethos, but also a deep reverence and respect for nature. Within black metal’s aesthetic, photographs of ominous black tree lines and lyrics about disappearing into the depths of the forest abound – it is almost as if the spruce tree has become its own character in the mythology that black metal has become.

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To degrade lignocellulose efficiently, lower termites rely on their digestive tract’s specific features (i.e., hysiological properties and enzymes) and on the network of symbiotic fauna harboured in their hindgut. This complex ecosystem, has different levels of symbiosis, and is a result of diverse co-evolutionary events and the singular social behaviour of termites. The partnership between termites and flagellate protists, together with prokaryotes, has been very successful because of their co-adaptative ability and efficacy in resolving the needs of the involved organisms: this tripartite symbiosis may have reached a physiologically stable, though dynamic, evolutionary equilibrium. The diversity of flagellate protists fauna associated with lower termites could be explained by a division of labour to accomplish the intricate process of lignocellulose digestion, and the ability to disrupt this function has potential use for termite control. Multi-level symbiosis strategy processes, or the cellulolytic capacity of flagellate protists, may lead to innovative pathways for other research areas with potential spin-offs for industrial and commercial use.

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Main conclusion: Epigenetic memory affects the timing of bud burst phenology and the expression of bud burstrelated genes in genetically identical Norway spruce epitypes in a manner usually associated with ecotypes. In Norway spruce, a temperature-dependent epigenetic memory established during embryogenesis affects the timing of bud burst and bud set in a reproducible and predictable manner. We hypothesize that the clinal variation in these phenological traits, which is associated with adaptation to growth under frost-free conditions, has an epigenetic component. In Norway spruce, dehydrins (DHNs) have been associated with extreme frost tolerance. DHN transcript levels decrease gradually prior to flushing, a time when trees are highly sensitive to frost. Furthermore, EARLY BUD BREAK 1 genes (EBB1) and the FT-TFL1- LIKE 2-gene (PaFTL2) were previously suggested to be implied in control of bud phenology. Here we report an analysis of transcript levels of 12 DHNs, 3 EBB1 genes and FTL2 in epitypes of the same genotype generated at different epitype-inducing temperatures, before and during spring bud burst. Earlier flushing of epitypes originating from embryos developed at 18 C as compared to 28 C, was associated with differential expression of these genes between epitypes and between buds and last year’s needles. The majority of these genes showed significantly different expressions between epitypes in at least one time point. The general trend in DHN expression pattern in buds showed the expected reduction in transcript levels when approaching flushing, whereas, surprisingly, transcript levels peaked later in needles, mainly at the moment of bud burst. Collectively, our results demonstrate that the epigenetic memory of temperature during embryogenesis affects bud burst phenology and expression of the bud burst-related DHN, EBB1 and FTL2 genes in genetically identical Norway spruce epitypes.

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One way to protect timber in service against basidiomycete deterioration is by means of acetylation via reaction with acetic anhydride. The reason why acetylated wood (WAc) is resistant against decay fungi is still not exactly understood. The aim of this study was to contribute to this field of science, and Postia placenta colonisation after 4, 12, 20, 28 and 36 weeks was observed at Three acetylation levels of Pinus spp. sapwood. Mass loss (ML) and wood moisture content (MC) data reflected the acetylation levels. The initial equilibrium MC (EMC) proved to be a good indicator of subsequent ML. Genomic DNA quantification showed P. placenta colonisation in all samples, also in samples where no ML were detectable. The number of expressed gene transcripts was limited, but the findings supported the results of previous studies: WAc seems to have some resistance against oxidative mechanisms, which are part of the metabolism of P. placenta. This leads to a delay in decay initiation, a delay in Expression of genes involved in enzymatic depolymerisation, and a slower decay rate. The magnitudes of these effects are presented for each acetylation level. The data also imply that there is no absolute decay threshold at high acetylation levels, but instead a significant delay of decay initiation and a slower decay rate.

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Modified wood can provide protection against a range of wood deteriorating organisms. Several hypotheses have been put forward regarding the protection mechanisms against wood decaying fungi including fungal enzyme inefficiency due to non-recognition, lower micropore size, and insufficient wood moisture content. The aim of this study was to obtain new insight into the protection manner of furfuryl alcohol (FA) modified Scots pine sapwood (WFA), and to examine biochemical mechanisms and adaptive changes in gene expression utilised by Postia placenta during early colonisation of WFA. Samples were harvested after 2, 4, and 8 weeks of incubation. After 8 weeks, the mass loss (0.1%) and wood moisture content (21.0%) was lower inWFA, than in non-modified Scots pine sapwood samples (W), 26.1% and 46.1%, respectively. Microscopy revealed needle-shaped calcium oxalate crystals, at all harvesting points, most prominently present after 4 and 8 weeks, and only in the WFA samples. Among the findings based on gene profiles were indications of a possible shift toward increased expression, or at least no down regulation, of genes related to oxidative metabolism and concomitant reduction of several genes related to the breakdown of polysaccharides in WFA compared to W.

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Epigenetic memory formed in the Norway spruce embryos permanently affect the timing of bud burst and bud set in the progenies, vitally important adaptive traits in this long-lived forest species. Epigenetic memory marks are established in response to the temperature conditions prevailing during embryogenesis; the epitype is fixed by the time the embryo is fully developed and is mitotically propagated throughout the tree’s life span. Somatic embryogenesis closely mimics the natural zygotic embryo formation and results in epigenetically different plants in a predictable temperature-dependent manner with respect to altered phenology. Using RNAseq transcriptome analysis of mRNA and noncodingRNA (ncRNA) changes were monitored in somatic embryos under different temperatures. We found distinct differences in mRNA and ncRNA transcriptomes between the genetically identical embryogenic tissues grown under the epitype-inducing temperatures suggesting temperature-dependent canalizing of gene expression during embryo formation, putatively based on chromatin modifications.

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Embryogenesis is the initial stage of plant life, when the basics of body plan and the post-embryonic development are laid down. Epigenetic memory formed in the Norway spruce embryos permanently affect the timing of bud burst and bud set in progenies, vitally important adaptive traits in this long-lived forest species. The epigenetic memory marks are established in response to the temperature conditions prevailing during zygotic and somatic embryogenesis; the epitype is fixed by the time the embryo is fully developed and is mitotically propagated throughout the tree’s life span. Somatic embryogenesis closely mimics the natural zygotic embryo formation and results in epigenetically different plants in a predictable temperature-dependent manner with respect to altered phenology. Using Illumina-based Massive Analysis of cDNA Ends, the transcriptome changes were monitored in somatic embryos during morphogenesis stage under two different temperatures (18 vs. 30 °C). We found distinct differences in transcriptomes between the genetically identical embryogenic tissues grown under the two epitype-inducing temperatures suggesting temperature-dependent canalizing of gene expression during embryo formation, putatively based on chromatin modifications. From 448 transcripts of genes coding for proteins involved in epigenetic machinery, we found 35 of these to be differentially expressed at high level under the epitype-inducing conditions. Therefore, temperature conditions during embryogenesis significantly alter transcriptional profiles including numerous orthologs of transcriptional regulators, epigenetic-related genes, and large sets of unknown and uncharacterized transcripts.

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To investigate the role of dehydrins (DHNs) in extreme low-temperature (LT) tolerance, we sampled needle tissue of Siberian spruce (Picea obovata Ledeb.) from trees growing in an arboretum in Trondheim, Norway from August 2006 to April 2007 and tracked changes in LT tolerance via relative electrolyte leakage. We used western blotting to estimate relative amounts of proteins binding a DHN K-segment antibody, measured relative amounts of nine transcripts for small (<25 kDa) DHNs by quantitative reverse transcription–polymerase chain reaction (PCR) using primers developed for DHN transcripts in a closely related species, Picea abies (L.) Karsten, and isolated and sequenced PCR products for five P. obovata DHNs. Three protein bands of 53, 35 and 33 kDa were detected on western blots of SDS–PAGE-separated protein extracts. The 53-kDa DHN was already present late in the growing season, but accumulated during acclimation, and levels decreased rapidly during deacclimation. The 33- and 35-kDa proteins, identified as Picg5 class DHNs by mass spectrometry, first appeared in detectable amounts late in the acclimation process and remained at detectable levels throughout the period of maximum LT tolerance. Levels of the 53-kDa DHN correlated with two LT tolerance parameters, while results for the 33- and 35-kDa proteins were equivocal due to limited sample size and variation in LT tolerance during the mid-winter period. Three additional bands of 30, 28 and 26 kDa were detected in extracts from needles collected in November 2010 using an immunity-purified antibody. Immunoblotting of two-dimensional gel electrophoresis gels loaded with proteins extracted from October and November samples corroborated the results obtained by SDS–PAGE western blots. One large spot in the 53 kDa range and two trains of spots in the same size range as the 33 and 35 kDa DHNs were detected using the K-segment antibody. Eight of the nine DHN transcripts closely tracked LT tolerance parameters, whereas the ninth DHN transcripts followed a reverse pattern, decreasing during winter and increasing again during deacclimation. Multiple regression models using principal components of the transcripts to predict two different LT tolerance parameters suggest separate but overlapping functions for different DHNs in establishing and maintaining extreme LT tolerance.

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Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular “memory”. Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate change.

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The pathogenic white-rot basidiomycete Heterobasidion irregulare is able to remove lignin and hemicellulose prior to cellulose during the colonization of root and stem xylem of conifer and broadleaf trees. We identified and followed the regulation of expression of genes belonging to families encoding ligninolytic enzymes. In comparison with typical white-rot fungi, the H. irregulare genome has exclusively the short-manganese peroxidase type encoding genes (6 short-MnPs) and thereby a slight contraction in the pool of class II heme-containing peroxidases, but an expansion of the MCO laccases with 17 gene models. Furthermore, the genome shows a versatile set of other oxidoreductase genes putatively involved in lignin oxidation and conversion, including 5 glyoxal oxidases, 19 quinone-oxidoreductases and 12 aryl-alcohol oxidases. Their genetic multiplicity and gene-specific regulation patterns on cultures based on defined lignin, cellulose or Norway spruce lignocellulose substrates suggest divergent specificities and physiological roles for these enzymes. While the short-MnP encoding genes showed similar transcript levels upon fungal growth on heartwood and reaction zone (RZ), a xylem defense tissue rich in phenolic compounds unique to trees, a subset of laccases showed higher gene expression in the RZ cultures. In contrast, other oxidoreductases depending on initial MnP activity showed generally lower transcript levels on RZ than on heartwood. These data suggest that the rate of fungal oxidative conversion of xylem lignin differs between spruce RZ and heartwood. It is conceivable that in RZ part of the oxidoreductase activities of laccases are related to the detoxification of phenolic compounds involved in host-defense. Expression of the several short-MnP enzymes indicated an important role for these enzymes in effective delignification of wood by H. irregulare.

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Local climate conditions have a major influence on the biological decomposition of wood. To examine the influence of different temperature regimes on wood decay caused by the brown rot fungus Postia placenta in wood with differing natural durability, sapwood (sW) and heartwood (hW) of Scots pine, inoculated mini-blocks were incubated for up to 10 weeks at temperatures conducive or above optimal to wood decay. We profiled mass loss (ML) and wood composition, and accompanying changes in wood colonization and transcript level regulation of fungal candidate genes. The suppressive effect of suboptimal temperature on wood decay caused by P. placenta appeared more pronounced in Scots pine hW with increased durability than in sW with low decay resistance. The differences between sW and hW were particularly pronounced for cultures incubated at 30°C: unlike sW, hW showed no ML, poor substrate colonization and marker gene transcript level profiles indicating a starvation situation. As brown rot fungi show considerable species-specific variation in temperature optima and ability to mineralize components that contribute to wood durability, interactions between these factors will continue to shape the fungal communities associated to wood in service.

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Conifers are evolutionarily more ancient than their angiosperm counterparts, and thus some adaptive mechanisms and features influenced by epigenetic mechanisms appear more highly displayed in these woody gymnosperms. Conifers such as Norway spruce have very long generation times and long life spans, as well as large genome sizes. This seemingly excessive amount of genomic DNA without apparent duplications could be a rich source of sites for epigenetic regulation and modifications. In Norway spruce, an important adaptive mechanism has been identified, called epigenetic memory. This affects the growth cycle of these trees living in environments with mild summers and cold winters, allowing them to adapt rapidly to new and/or changing environments. The temperature during post-meiotic megagametogenesis and seed maturation epigenetically shifts the growth cycle programme of the embryos. This results in significant and long-lasting phenotypic change in the progeny, such as advance or delay of vital phenological processes of high adaptive value, like bud break and bud set. This phenomenon is not only of important evolutionary significance but has clear practical implications for forest seed production and conservation of forest genetic resources. The underlying molecular mechanism that causes the ‘memory’ in long-lived woody species is currently under investigation. Here we summarize the information related to epigenetic memory regulation in gymnosperms, with special emphasis on conifers. The molecular mechanism behind this is still unknown but transcriptional changes are clearly involved. Epigenetic regulation may be realized through several mechanisms, including DNA methylation, histone modification, chromatin remodelling, small non-coding RNAs and transposable element regulation, of which non-coding RNAs might be one of the most important determinants.

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We compared gene expression in Norway spruce secondary phloem (bark) and developing xylem (sapwood) in response to the necrotrophic pathogen Heterobasidion parviporum, wounding and methyl jasmonate (MeJ). The pathogen induced systemic and local up-regulation of PaPX3, PaPX2 and PaChi4 in both bark and sapwood that returned to constitutive levels as the plants recovered from the infection, whereas the local responses to MeJ were similar in both tissues but was longer lasting for PaPX3 and PaChi4. Genes involved in lignin biosynthesis (PaPAL1, PaPAL2, PaC4H3/5 and PaHCT1) were up-regulated locally in the bark in response to pathogen and wounding whereas MeJ induced a similar but stronger local response. The ethylene biosynthesis related transcripts PaACO and PaACS did not increase in response to MeJ treatment or the pathogen, however it increased both locally and systemically as a response to wounding in the sapwood. These results demonstrate that the local and systemic host responses to pathogen infection and wounding largely correspond and reveal striking similarities between the local response to a necrotroph, wounding and MeJ treatment in both bark and living wood.

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Epigenetic memory marks establishment in Norway spruce occur exclusively during embryogenesis in response to environmental impact, and the epitype is fixated by the time the embryo is fully developed without a change in the DNA sequence. We started large scale studies aimed on identifying and characterizing of genes and regulatory elements involved in the initiation, maintenance, and heritability of epigenetic memory using candidate genes and next generation sequencing approaches. Molecular mechanisms of formation of epigenetic memory were studied on the same full-sibs family zygotic embryo in vitro cultures developed in cold (18°C) and warm (30°C) environmental conditions from proliferation till mature embryo stages. Initially we had found large set (64) of Arabidopsis epigenetic regulator gene homologs in spruce. In general, known epigenetic related genes are very well represented among spruce ESTs. Analysis of the transcription patterns of these genes using RT-PCR in epigenetically different embryogenic samples reveal specific transcription patterns on different stages of embryogenic development dependent on epitype. We are expecting to determine certain stages during embryogenesis when epigenetic memory marks are forming. At the same time, nearly no differences in transcription levels of studied genes had been found in seedlings (4 month old), originated from full-sib families clearly differed in epigenetic response. Using MACE (massive cDNA 3-end sequencing) deep mRNA sequencing on the Illumina GSII platform, we analyzed P. abies transcriptomes by comparison warm and cold originated “embryonic epitypes” developed in cold and warm environmental conditions. Significant differences in transcriptomes between epitypes revealed by high-throughput sequencing will be discussed.

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The outcome of a compatible mycorrhizal interaction is different from that in a compatible plant–pathogen interaction; however, it is not clear what mechanisms are used to evade or suppress the host defence. The aim of this work is to reveal differences between the interaction of Norway spruce roots to the pathogen Ceratocystis polonica and the ectomycorrhizal Laccaria bicolor, examine if L. bicolor is able to evade inducing host defence responses typically induced by pathogens, and test if prior inoculation with the ectomycorrhizal fungus affects the outcome of a later challenge with the pathogen. The pathogen was able to invade the roots and caused extensive necrosis, leading to seedling death, with or without prior inoculation with L. bicolor. The ectomycorrhizal L. bicolor colonised primary roots of the Norway spruce seedlings by partly covering, displacing and convoluting the cells of the outer root cortex, leaving the seedlings healthy. We detected increased total peroxidase activity, and staining indicating increased lignification in roots as a response to C. polonica. In L. bicolor inoculated roots there was no increase in total peroxidase activity, but an additional highly acidic peroxidase isoform appeared that was not present in healthy roots, or in roots invaded by the pathogen. Increased protease activity was detected in roots colonised by C. polonica, but little protease activity was detected in L. bicolor inoculated roots. These results suggest that the pathogen efficiently invades the roots despite the induced host defence responses, while L. bicolor suppresses or evades inducing such host responses in this experimental system.

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• Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. • We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. • A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. • Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

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The development of new tools able to select specific plant tissue is crucial for gene expression studies. During the last years, the use of laser microdissection, mainly tested on herbaceous plant tissue, has been found to be a useful technique for these purposes. This method is poorly tested on woody species, and so far no studies of gene expression have been applied on forest trees. For this reason the present work proposes the optimization of a functional protocol using laser microdissection pressure catapulting (LMPC) and real-time reverse transcription–polymerase chain reaction (RT-PCR) in bark stem tissue of Norway spruce (Picea abies). Bark tissue fragments were collected from Norway spruce trees and sliced with a cryostat. RNA was extracted from both whole cross-sections and microdissected bark cells. The feasibility of the method was confirmed by the amplification of the α-tubulin, an endogenous gene of P. abies, with efficiency comparable to that obtained from non-microdissected tissue. The proposed protocol, here adapted for bark tissue of woody species, represents a useful tool in a wide range of hosts that, unlike herbaceous plants, have scarcely been considered up to now.

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Background: NB-LRR resistance proteins are involved in recognizing pathogens and other exogenous stressors in plants. Resistance proteins are the first step in induced defence responses and a better understanding of their regulation is important to understand the mechanisms of plant defence. Much of the post-transcriptional regulation in plants is controlled by microRNAs (miRNA). We examined the expression of five Norway spruce miRNA that may regulate NB-LRR related transcripts in secondary phloem (bark) of resistant Norway spruce after wounding and inoculation with the necrotrophic blue stain fungus Ceratocystis polonica. Results: The plants of this clone recovered from both the pathogen inoculations and wounding alone. We found local and systemic induction of the resistance marker genes PaChi4, PaPAL and PaPX3 indicative of an effective induced host defence response. There were minor local and systemic changes in the expression of five miRNAs and 21 NB-LRRs between healthy and treated plants. Only five putative NB-LRRs (PaLRR1, PaLRR3, PaLRR14, PaLRR15 and PaLRR16) showed significant increases greater than two-fold as a local response to C. polonica. Of all NB-LRRs only PaLRR3, the most highly differentially regulated NB-LRR, showed a significant increase also due to wounding. The five miRNAs showed indications of an initial local and systemic down-regulation at day 1, followed by a later increase up to and beyond the constitutive levels at day 6. However, the initial down-regulation was significant only for miR3693 and miR3705. Conclusions: Overall, local and systemic expression changes were evident only for the established resistance marker genes and PaLRR3. The minor expression changes observed both for the followed miRNAs and their predicted NB-LRR targets suggest that the expression of most NB-LRR genes are maintained close to their constitutive levels in stressed and healthy Norway spruce plants.

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1. Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) is widely used as a viral bio-insecticide against larvae of the European pine sawfly N. sertifer (Geoff.) (Hymenoptera: Diprionidae), which is one of the most harmful defoliators of pines in Northern Europe. A major obstacle to studying this pathogenic virus in nature is the difficulty of confirming and quantifying the presence of NeseNPV. 2. In the present study, we developed real-time polymerase chain reaction (PCR) primers, based on the caspid gene 39 sequence, for the specific and quantitative detection of NeseNPV. The quantitative real-time PCR (qPCR) assay can detect virus from any substrate tested, including different insect life stages (egg, larval, adult), pine foliage, and litter or ground vegetation. The reproducible detection limit for the real-time assay is 0.013 pg of viral DNA (0.013 × 10−12 g), corresponding to 136 viral genomes or approximately one to seven virus occlusion bodies per sample. 3. qPCR is a specific, quantitative, sensitive, reliable and flexible procedure, and is a good supplement to conventional microscopy- or bioassay-based methods for detection of the virus. We have used qPCR to quantify the level of NeseNPV in samples collected in the field after aerial application of the virus, and demonstrated significantly higher virus levels in sawfly larvae from sprayed areas compared with unsprayed control areas 4 weeks after spraying. 4. This qPCR assay can be used to determine important aspects of the biology of NeseNPV (e.g. virus levels in different insect life stages and in their microhabitats on pine foliage and in forest litter).

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A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.

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Pathogen challenge of tree sapwood induces the formation of reaction zones with antimicrobial properties such as elevated pH and cation content. Many fungi lower substrate pH by secreting oxalic acid, its conjugate base oxalate being a reductant as well as a chelating agent for cations. To examine the role of oxalic acid in pathogenicity of white-rot fungi, we conducted spatial quantification of oxalate, transcript levels of related fungal genes, and element concentrations in heartwood of Norway spruce challenged naturally by Heterobasidion parviporum. In the pathogen-compromised reaction zone, upregulation of an oxaloacetase gene generating oxalic acid coincided with oxalate and cation accumulation and presence of calcium oxalate crystals. The colonized inner heartwood showed trace amounts of oxalate. Moreover, fungal exposure to the reaction zone under laboratory conditions induced oxaloacetase and oxalate accumulation, whereas heartwood induced a decarboxylase gene involved in degradation of oxalate. The excess level of cations in defense xylem inactivates pathogen-secreted oxalate through precipitation and, presumably, only after cation neutralization can oxalic acid participate in lignocellulose degradation. This necessitates enhanced production of oxalic acid by H. parviporum. This study is the first to determine the true influence of white-rot fungi on oxalate crystal formation in tree xylem.

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Heterobasidion parviporum, a common pathogenic white-rot fungus in managed Norway spruce forests in northern and central Europe, causes extensive decay columns within stem heartwood of the host tree. Infected trees combat the lateral spread of decay by bordering the heartwood with a fungistatic reaction zone characterized by elevated pH and phenol content. To examine the mode of fungal feeding in the reaction zone of mature Norway spruce trees naturally infected by H. parviporum, we conducted spatial proWling of pectin and hemicellulose composition, and established transcript levels of candidate fungal genes encoding enzymes involved in degradation of the diVerent cell wall components of wood. Colonized inner heartwood showed pectin and hemicellulose concentrations similar to those of healthy heartwood, whereas the carbohydrate proWles of compromised reaction zone, irrespective of the age of fungal activity in the tissue, indicated selective fungal utilization of galacturonic acid, arabinose, xylose and mannose. These data show that the rate of wood decay in the reaction zone is slow. While the up-regulation of genes encoding pectinases and hemicellulases preceded that of the endoglucanase gene during an early phase of fungal interaction with xylem defense, the manganese peroxidase gene showed similar transcript levels during diVerent phases of wood colonization. It seems plausible that the reaction zone components of Norway spruce interfere with both lignin degradation and the associated co-hydrolysis of hemicelluloses and pectin, resulting in a prolonged phase of selective decay.

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In Norway spruce, the temperature during zygotic embryogenesis appears to adjust an adaptive epigenetic memory in the progeny that may regulate bud phenology and cold acclimation. Conditions colder than normal advance the timing whilst temperatures above normal delay the onset of these processes and altered performance is long lasting in progeny with identical genetic background. As a step toward unraveling the molecular mechanism behind an epigenetic memory, transcriptional analysis was performed on seedlings from seeds of six full-sib families produced at different embryogenesis temperature?cold (CE) vs warm (WE) under long and short day conditions. We prepared two suppressive subtractedcDNAlibraries, forward and reverse, representing genes predominantly expressed in plants from seeds obtained after CE and WE embryogenesis following short day treatment (inducing bud set). Sequencing and annotation revealed considerable differences in the transcriptome of WE versus CE originated plants. By using qRT-PCR we studied the expression patterns of 32 selected candidate genes chosen from subtractive cDNA libraries analysis and nine siRNA pathways genes by a direct candidate approach. Eight genes, two transposons related genes, three with no match to Databases sequences and three genes from siRNA pathways (PaDCL1 and 2, PaSGS3) showed differential expression in progeny from CE andWEcorrelated with the family phenotypic differences. These findingsmaycontribute to our understanding of the epigenetic mechanisms underlying adaptive changes acquired during embryogenesis.

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Hvor raskt en organisme kan tilpasse seg forandringer, er avgjørende for hvilket utfall raske endring er i klima vil ha for organismens utbred else og overlevelse. Grana viser en evne til utrolig raskt å tilpasse seg endringer og vår forsk ning knytter denne overlevelsesevnen til det som kalles epigenetikk.

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Gene expression levels (PAL, CCR1, HCT1, and CAD for the phenylpropanoid pathway, PX3 peroxidase, and CHI4 class IV chitinase), lignin, and soluble and cell wall bound phenolic compounds in bark and sapwood of Picea sitchensis clones inoculated with Heterobasidion annosum s.s. were compared before and 3 days after wounding and artificial inoculation, at site of inoculation and 1 cm above the inoculation site. In bark all genes were up-regulated at the site of inoculation but, except for CAD, not in the distal zone. In sapwood all genes were down-regulated, except for PX3 and CHI4; PAL, CCR1, HCT1 and CAD were present at lower levels around the inoculation site than in the distal zone. Compared to wounding only, inoculation with H. annosum triggered different CAD, PX3, and CHI4 levels in bark but not in sapwood. Different concentrations of cell wall bound phenolic compounds (unknown2, unknown3, coniferin, astringin, taxifolin, piceid, and isorhapontin) were found in bark after wounding and inoculation compared to constitutive material (i.e. untreated samples), whereas in sapwood concentrations did not differ following treatment. These results indicate that bark of Sitka spruce has a stronger and earlier response to wounding and pathogen inoculation than sapwood.

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Molecular methods are emerging also as useful tools for wood protection studies. The aim of the present study was to evaluate quantitative real-time polymerase chain reaction (qPCR) as a tool for investigating details of the colonization pattern of basidiomycete decay fungi in wood samples after 6 years of soil exposure. Samples of Pinus sylvestris L. (heartwood without treatment), furfurylated P. sylvestris sapwood and Cu-HDO treated P. sylvestris sapwood was in focus. The qPCR method based on basidiomycete DNA content in the wood had the highest sensitivity, while the ergosterol assay was more sensitive than the chitin assay. Visual rating was compared with laboratory analyses and was found to be correlating well with qPCR. This study demonstrates that qPCR in combination with microscopy provides relevant data about basidiomycete colonization in wooden material.

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Logging residues, branches and treetops after logging, were considered in the past as unsalable portions of the felled trees and remained on the landing. Currently, logging residues are harvested, stored in piles for variable time periods prior to being utilized as a bioenergy source. However, it is still unclear to what extent the colonization by decay fungi during outdoor storage impairs the fuel quality. Our objective was to find out whether the storage method influenced the amount of basidiomycetous fungi, the main wood degraders in logging residues....

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Norway spruce expresses a temperature-dependent epigenetic memory from the time of embryo development, which thereafter influences the timing bud phenology. MicroRNAs (miRNAs)are endogenous small RNAs, exerting epigenetic gene regulatory impacts. We have tested for their presence and differential expression. We prepared concatemerized small RNA libraries from seedlings of two full-sib families, originated from seeds developed in a cold and warm environment. One family expressed distinct epigenetic effects while the other not. We used available plant miRNA query sequences to search for conserved miRNAs and from the sequencing we found novel ones; the miRNAs were monitored using relative real time-PCR. Sequencing identified 24 novel and four conserved miRNAs. Further screening of the conserved miRNAs confirmed the presence of 16 additional miRNAs. Most of the miRNAs were targeted to unknown genes. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in the full-sib family showing distinct epigenetic difference in bud set, but not in the nonresponding full-sib family. Putative miRNA targets were studied. Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel nonconserved miRNAs. The differentially expression of specific miRNAs indicate their putative participation in the epigenetic regulation.

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Rotkjuke, Heterobasidion annosum s.l. er et stort problem i de nordlige barskogsområder. I Norge er hele 20 prosent av grantrærne angrepet av denne skadesoppen. Dette fører ikke bare til at tømmeret blir ubrukelig til de fleste formål, men også til frigjøring av mye CO2 som ellers ville vært bundet i trærne. Rotkjuka angriper via rotkontakter. Dette skjer ofte via stubber av felte nabotrær etter hogst eller tynning. Soppen ødelegger stammen opp til tolv meter fra bakkenivå.

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Hvordan tilpasser trærne seg til klimaendringer? Hva gjør de for å forsvare seg mot sopp- og insektangrep? Hvilken vei vandret grana inn i Norge etter siste istid? Hvordan påviser vi nye sykdommer på trær, eller begynnende råte i trebygninger? Og hvordan kan granas verste fi ende hjelpe oss med å lage framtidens biodrivstoff? Dette er noen av spørsmålene Skog og landskaps forskere stiller seg. I mange tilfeller er moderne bioteknologi en del av løsningen. På Skog og landskap bruker vi ulike bioteknologiske metoder for å løse forskningsoppgaver innen blant annet skoghelse, treteknologi, genetikk og økologi. Denne brosjyren forteller deg mer om våre forskningsaktiviteter knyttet til bioteknologi. Ordet bioteknologi blir ofte brukt synonymt med molekylærbiologi, genteknologi og molekylærgenetikk, og er en integrert del av moderne biologisk forskning. Dyr, planter, sopp eller mikroorganismer som bakterier og virus, eller DNAet (arvestoffet) fra disse, brukes til å produsere nye produkter. Ølbrygging er et tidlig eksempel på bioteknologi, og stiklingsformering av trær og andre nytteplanter er en eldgammel form for kloning.

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Regulation of flowering time in Arabidopsis thaliana is controlled by a network of pathways integrating environmental and internal signals. Two of these pathways, the vernalization and photoperiodic pathways, mediate responses to prolonged cold period and photoperiod, respectively. A number of A. thaliana populations from high-latitude and high-altitude locations in Norway were collected and phenotyped for flowering time in response to 5 photoperiods and 5 vernalization treatments. Vernalization and photoperiodic sensitivity were not correlated with latitude but rather with climatic factors such as winter temperature and precipitation that do not vary with latitude, especially in coastal environments. Coastal populations, both from subarctic and intermediate latitudes, were rather insensitive towards the length of the vernalization treatment but very sensitive towards differences in photoperiods. Stronger photoperiod sensitivity in coastal populations might be a necessary adaptation for sensing the onset of spring in regions with relatively mild and unpredictable winter climates as opposed to continental climates with more stable winters. FLC sequence variation was only partly associated with vernalization response, whereas variation in transcript levels of CRY2, TOC1 and GI was correlated with photoperiodic responses. This suggests that local adaptation of populations may be partly mediated by photoreceptors and circadian clock pathways.

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In woody plants of the temperate zone short photoperiod (SD) leads to growth cessation. In angiosperms CONSTANS (CO) or CO-like genes play an important role in the photoperiodic control of flowering, tuberisation and shoot growth. To investigate the role of CO-like genes in photoperiodic control of shoot elongation in gymnosperms, PaCOL1 and PaCOL2 were isolated from Norway spruce. PaCOL1 encodes a 3.9 kb gene with a predicted protein of 444 amino acids. PaCOL2 encodes a 1.2 kb gene with a predicted protein of 385 amino acids. Both genes consist of two exons and have conserved domains found in other CO-like genes; two zinc finger domains, a CCT and a COOH domain. PaCOL1 and PaCOL2 fall into the group 1c clade of the CO-like genes, and are thus distinct from Arabidopsis CO that belongs to group la. Transcript levels of both PaCOL-genes appear to be light regulated, an increasing trend was observed upon transition from darkness to light, and a decreasing trend during darkness. The increasing trend at dawn was observed both in needles and shoot tips, whereas the decreasing trend in darkness was most prominent in shoot tips, and limited to the late part of the dark period in needles. The transcript levels of both genes decreased significantly in both tissues under SD prior to growth cessation and bud formation. This might suggest an involvement in photoperiodic control of shoot elongation or might be a consequence of regulation by light. (C) 2008 Elsevier Masson SAS. All rights reserved.

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We have made and partially sequenced two subtracted cDNA libraries, one representing genes predominantly expressed in a tree from an early-flushing family of Norway spruce (early-flushing library; EFL) and the second from a late flushing family (late flushing library; LFL), during 4 weeks before bud burst. In the EFL, expressed sequence tags (ESTs) encoding proteins of the photosynthetic apparatus and energy metabolism and proteins related to stress (abiotic and biotic) and senescence were abundant. ESTs encoding metallothionein-like and histone proteins as well as transcription factors were abundant in the LFL. We used quantitative real-time reverse transcription polymerase chain reaction to study the expression patterns of 25 chosen genes and observed that the highest levels of activity for most genes were present when plants were still ecodormant. The results indicate that the late flushing is not a result of a delay in gene activity, but is rather associated with an active transcriptional process. Accordingly, certain metabolic processes may be turned on in order to prevent premature flushing. We discuss the putative role of the studied genes in regulation of bud burst timing. Among the candidate genes found, the most interesting ones were the DNA-binding proteins, water-stress- related genes and metallothioneins. Expression patterns of some genes involved in chemical modification of DNA and histones indicate that epigenetic factors are involved in the timing of bud burst. In the obtained transcriptomes, we could not find genes commonly believed to be involved in dormancy and bud set regulation (PHY, CRY, ABI etc.) in angiosperm plants.

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Polyphenolic parenchyma cells (PP cells) in Norway spruce (Picea abies (L.) Karst.) stem phloem play important roles in constitutive and inducible defenses. To determine whether anatomical and molecular changes in PP cells are correlated with tree resistance, we infected two Norway spruce clones with the pathogenic fungus Ceratocystis polonica (Siem.) C. Moreau. The fungus induced significantly different lesion lengths in the two clones, indicating that one clone was more resistant to the fungus (short lesions) than the other (long lesions). After infection, the cross-sectional area of PP cells and their vacuolar polyphenol bodies increased in the three most recent annual rings of PP cells in both clones. The more resistant clone had larger PP cells with denser polyphenol bodies than the less resistant clone, whereas the less resistant clone accumulated relatively more polyphenols after infection. Compared with the less resistant clone, the more resistant clone contained higher starch concentrations before infection that were reduced more quickly after infection before returning to original values. Low transcript levels of chalcone synthase were detected in uninfected tissues of both clones, but the levels increased dramatically after infection. Transcript levels were higher and peaked 6 days earlier in the more resistant clone than in the less resistant clone. The activity of at least one highly basic peroxidase isoform was greatly enhanced after infection, and this increase occurred earlier in the more resistant clone.

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Introduction: Survival and competitive successes of boreal forest trees depend on a balance between exploiting the full growing season and minimising frost injury through proper timing of hardening in autumn and dehardening in spring. Our research indicates that the female parents of Norway spruce adjust these timing events in their progeny according to the prevailing temperature conditions during sexual reproduction. Reproduction in a cold environment advances bud-set and cold acclimation in the autumn and dehardening and flushing in spring, whereas a warm reproductive environment delays these progeny traits by an unknown non-Mendelian mechanism. We are now looking for molecular mechanisms that can explain this “epigenetic” phenomenon. Material and methods: We have performed identical crosses with the same Norway spruce (Picea abies) parent, as discussed by Skrøppa & Johnsen (1994) and Johnsen et al. (1995), in combination with timed temperature treatments during shorter and longer periods from female meiosis, pollen tube growth, syngamy and embryogenesis and tested the progenies for bud-set and frost hardiness. We have followed the transcription of the spruce phytochromes PHYO, PHYP and PHYN and the class IV chitinase PaChi4 using Quantitative Multiplex Real-Time PCR. Results and conclusions: The effect of temperature on Adaptive properties is most likely a response to accumulated heat during embryogenesis and seed maturation. Our first attempt to look for a molecular mechanism has revealed that transcription of PHYO, PHYP and PHYN and the class IV chitinase PaChi4 (relative to alphaTubulin) all show higher transcription levels in progenies born under cold conditions than their full-sibs born under warmer conditions. This result is consistent with preliminary findings that methylation of cytosine in total DNA is higher in progenies reproduce under warm conditions than their colder full-sib counterparts. If these observations are related to methylation or other epigenetic effects, we may explain why progenies with a memory of a past time cold embryogenesis are more sensitive to short days than their full-sibs with a warmer embryonic history.

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Research indicate that the female parents of Norway spruce adjust these timing events in their progeny according to the prevailing temperature conditions during seed development. Reproduction in a cold environment advances bud-set and cold acclimation in the autumn and dehardening and flushing in spring, whereas a warm reproductive environment delays these progeny traits by an unknown non-Mendelian mechanism. We have performed identical crosses in combination with timed temperature treatments during shorter and longer periods from female meiosis, pollen tube growth, syngamy and embryogenesis, tested the progenies for bud-set and frost hardiness, and concluded that the effect of temperature most likely is a response to accumulated heat during embryogenesis and seed maturation. Our first attempt to look for a molecular mechanism has revealed that transcription of PHYO, PHYP and PHYN and the class IV chitinase PaChi4 (using RealTime PCR) all show higher transcription levels in progenies born under cold conditions than their full-sibs born under warmer conditions. This result is consistent with preliminary findings that methylation of cytosine in total DNA is higher in progenies reproduce under warm conditions than their colder full-sib counterparts. If these observations are related to methylation, we may explain why progenies with a memory of a past time cold embryogenesis are more sensitive to short days than their full-sibs with a warmer embryonic history.

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Utvalgt Forelesning/Selected Talk: Survival and competitive successes of boreal forest trees depend on a balance between exploiting the full growing season and minimising frost injury through proper timing of hardening in autumn and dehardening in spring. Our research has shown that the female parents of Norway spruce adjust these timing events in their progeny according to the prevailing temperature conditions during sexual reproduction. Reproduction in a cold environment advances bud-set and cold acclimation in the autumn and dehardening and flushing in spring, whereas a warm reproductive environment delays these progeny traits by an unknown non-Mendelian mechanism. We have performed identical crosses in combination with timed temperature treatments during shorter and longer periods from female meiosis, pollen tube growth, syngamy and embryogenesis, tested the progenies for bud-set and frost hardiness, and concluded that the effect of temperature most likely is a response to accumulated heat during embryogenesis and seed maturation. Our first attempt to look for a molecular mechanism has revealed that transcription of PHYO, PHYP and PHYN and the class IV chitinase PaChi4 (using RealTime PCR) all show higher transcription levels in progenies born under cold conditions than their full-sibs born under warmer conditions. This result is consistent with preliminary findings that methylation of cytosine in total DNA is higher in progenies reproduce under warm conditions than their colder full-sib counterparts. If these observations are related to methylation, we may explain why progenies with a memory of a past time cold embryogenesis are more sensitive to short days than their full-sibs with a warmer embryonic history.