Epigenetic stress memory in gymnosperms.

Gymnosperms are long-lived, cone-bearing seed plants that include some of the most ancient extant plant species. These relict land plants have evolved to survive in habitats marked by chronic or episodic stress. Their ability to thrive in these environments is partly due to their phenotypic flexibility, and epigenetic regulation likely plays a crucial part in this plasticity. We review the current knowledge on abiotic and biotic stress memory in gymnosperms and the possible epigenetic mechanisms underlying long-term phenotypic adaptations. We also discuss recent technological improvements and new experimental possibilities that likely will advance our understanding of epigenetic regulation in these ancient and hard-to-study plants.

Warmer temperature during asexual reproduction induce methylome, transcriptomic, and lasting phenotypic changes in Fragaria vesca ecotypes.

Plants must adapt with increasing speed to global warming to maintain their fitness. One rapid adaptation mechanism is epigenetic memory, which may provide organisms sufficient time to adapt to climate change. We studied how the perennial Fragaria vesca adapted to warmer temperatures (28°C vs. 18°C) over three asexual generations. Differences in flowering time, stolon number, and petiole length were induced by warmer temperature in one or more ecotypes after three asexual generations and persisted in a common garden environment. Induced methylome changes differed between the four ecotypes from Norway, Iceland, Italy, and Spain, but shared methylome responses were also identified. Most differentially methylated regions (DMRs) occurred in the CHG context, and most CHG and CHH DMRs were hypermethylated at the warmer temperature. In eight CHG DMR peaks, a highly similar methylation pattern could be observed between ecotypes. On average, 13% of the differentially methylated genes between ecotypes also showed a temperature-induced change in gene expression. We observed ecotype-specific methylation and expression patterns for genes related to gibberellin metabolism, flowering time, and epigenetic mechanisms. Furthermore, we observed a negative correlation with gene expression when repetitive elements were found near (±2 kb) or inside genes. In conclusion, lasting phenotypic changes indicative of an epigenetic memory were induced by warmer temperature and were accompanied by changes in DNA methylation patterns. Both shared methylation patterns and transcriptome differences between F. vesca accessions were observed, indicating that DNA methylation may be involved in both general and ecotype-specific phenotypic variation.

Epitype-inducing temperatures drive DNA methylation changes during somatic embryogenesis in the long-lived gymnosperm Norway spruce.

An epigenetic memory of the temperature sum experienced during embryogenesis is part of the climatic adaptation strategy of the long-lived gymnosperm Norway spruce. This memory has a lasting effect on the timing of bud phenology and frost tolerance in the resulting epitype trees. The epigenetic memory is well characterized phenotypically and at the transcriptome level, but to what extent DNA methylation changes are involved have not previously been determined. To address this, we analyzed somatic epitype embryos of Norway spruce clones produced at contrasting epitype-inducing conditions (18 and 28°C). We screened for differential DNA methylation in 2744 genes related mainly to the epigenetic machinery, circadian clock, and phenology. Of these genes, 68% displayed differential DNA methylation patterns between contrasting epitype embryos in at least one methylation context (CpG, CHG, CHH). Several genes related to the epigenetic machinery (e.g., DNA methyltransferases, ARGONAUTE) and the control of bud phenology (FTL genes) were differentially methylated. This indicates that the epitype-inducing temperature conditions induce an epigenetic memory involving specific DNA methylation changes in Norway spruce.

Major transcriptomic differences are induced by warmer temperature conditions experienced during asexual and sexual reproduction in Fragaria vesca ecotypes.

A major challenge for plants in a rapidly changing climate is to adapt to rising temperatures. Some plants adapt to temperature conditions by generating an epigenetic memory that can be transmitted both meiotically and mitotically. Such epigenetic memories may increase phenotypic variation to global warming and provide time for adaptation to occur through classical genetic selection. The goal of this study was to understand how warmer temperature conditions experienced during sexual and asexual reproduction affect the transcriptomes of different strawberry (Fragaria vesca) ecotypes. We let four European F. vesca ecotypes reproduce at two contrasting temperatures (18 and 28°C), either asexually through stolon formation for several generations, or sexually by seeds (achenes). We then analyzed the transcriptome of unfolding leaves, with emphasis on differential expression of genes belonging to the epigenetic machinery. For asexually reproduced plants we found a general transcriptomic response to temperature conditions but for sexually reproduced plants we found less significant responses. We predicted several splicing isoforms for important genes (e.g. a SOC1, LHY, and SVP homolog), and found significantly more differentially presented splicing event variants following asexual vs. sexual reproduction. This difference could be due to the stochastic character of recombination during meiosis or to differential creation or erasure of epigenetic marks during embryogenesis and seed development. Strikingly, very few differentially expressed genes were shared between ecotypes, perhaps because ecotypes differ greatly both genetically and epigenetically. Genes related to the epigenetic machinery were predominantly upregulated at 28°C during asexual reproduction but downregulated after sexual reproduction, indicating that temperature-induced change affects the epigenetic machinery differently during the two types of reproduction.

Methylome, transcriptome, and phenotype changes induced by temperature conditions experienced during sexual reproduction in Fragaria vesca.

Temperature conditions experienced during embryogenesis and seed development may induce epigenetic changes that increase phenotypic variation in plants. Here we investigate if embryogenesis and seed development at two different temperatures (28 vs. 18°C) result in lasting phenotypic effects and DNA methylation changes in woodland strawberry (Fragaria vesca). Using five European ecotypes from Spain (ES12), Iceland (ICE2), Italy (IT4), and Norway (NOR2 and NOR29), we found statistically significant differences between plants from seeds produced at 18 or 28°C in three of four phenotypic features investigated under common garden conditions. This indicates the establishment of a temperature-induced epigenetic memory-like response during embryogenesis and seed development. The memory effect was significant in two ecotypes: in NOR2 flowering time, number of growth points and petiole length were affected, and in ES12 number of growth points was affected. This indicates that genetic differences between ecotypes in their epigenetic machinery, or other allelic differences, impact this type of plasticity. We observed statistically significant differences between ecotypes in DNA methylation marks in repetitive elements, pseudogenes, and genic elements. Leaf transcriptomes were also affected by embryonic temperature in an ecotype-specific manner. Although we observed significant and lasting phenotypic change in at least some ecotypes, there was considerable variation in DNA methylation between individual plants within each temperature treatment. This within-treatment variability in DNA methylation marks in F. vesca progeny may partly be a result of allelic redistribution from recombination during meiosis and subsequent epigenetic reprogramming during embryogenesis.

Transcriptional profiling of defense responses to Botrytis cinerea infection in leaves of Fragaria vesca plants soil-drenched with ß-aminobutyric acid.

Grey mold caused by the necrotrophic fungal pathogen Botrytis cinerea can affect leaves, flowers, and berries of strawberry, causing severe pre- and postharvest damage. The defense elicitor ß-aminobutyric acid (BABA) is reported to induce resistance against B. cinerea and many other pathogens in several crop plants. Surprisingly, BABA soil drench of woodland strawberry (Fragaria vesca) plants two days before B. cinerea inoculation caused increased infection in leaf tissues, suggesting that BABA induce systemic susceptibility in F. vesca. To understand the molecular mechanisms involved in B. cinerea susceptibility in leaves of F. vesca plants soil drenched with BABA, we used RNA sequencing to characterize the transcriptional reprogramming 24 h post-inoculation. The number of differentially expressed genes (DEGs) in infected vs. uninfected leaf tissue in BABA-treated plants was 5205 (2237 upregulated and 2968 downregulated). Upregulated genes were involved in pathogen recognition, defense response signaling, and biosynthesis of secondary metabolites (terpenoid and phenylpropanoid pathways), while downregulated genes were involved in photosynthesis and response to auxin. In control plants not treated with BABA, we found a total of 5300 DEGs (2461 upregulated and 2839 downregulated) after infection. Most of these corresponded to those in infected leaves of BABA-treated plants but a small subset of DEGs, including genes involved in 'response to biologic stimulus', 'photosynthesis' and 'chlorophyll biosynthesis and metabolism', differed significantly between treatments and could play a role in the induced susceptibility of BABA-treated plants.

Immunochemical Detection of Modified Species of Cytosine in Plant Tissues.

Methylated cytosine (5-methylcytosine) is the most studied epigenetic mark involved in the regulation of gene expression. Although it displays highly variable dynamics during plant ontogenesis, it is possible to gain a fine spatial perspective with immunohistochemistry techniques that use specific antibodies and fluorochromes. Besides, there are other cytosine modifications described in plants, although their biological significance is still unknown (i.e., 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine). Here we present a standardized protocol to detect cytosine modifications in plant tissues.

Molecular underpinnings of methyl jasmonate-induced resistance in Norway spruce.

In response to various stimuli, plants acquire resistance against pests and/or pathogens. Such acquired or induced resistance allows plants to rapidly adapt to their environment. Spraying the bark of mature Norway spruce (Picea abies) trees with the phytohormone methyl jasmonate (MeJA) enhances resistance to tree-killing bark beetles and their associated phytopathogenic fungi. Analysis of spruce chemical defenses and beetle colonization success suggests that MeJA treatment both directly induces immune responses and primes inducible defenses for a faster and stronger response to subsequent beetle attack. We used metabolite and transcriptome profiling to explore the mechanisms underlying MeJA-induced resistance in Norway spruce. We demonstrated that MeJA treatment caused substantial changes in the bark transcriptional response to a triggering stress (mechanical wounding). Profiling of mRNA expression showed a suite of spruce inducible defenses are primed following MeJA treatment. Although monoterpenes and diterpene resin acids increased more rapidly after wounding in MeJA-treated than control bark, expression of their biosynthesis genes did not. We suggest that priming of inducible defenses is part of a complex mixture of defense responses that underpins the increased resistance against bark beetle colonization observed in Norway spruce. This study provides the most detailed insights yet into the mechanisms underlying induced resistance in a long-lived gymnosperm.

Priming of inducible defenses protects Norway spruce against tree-killing bark beetles.

Plants can form an immunological memory known as defense priming, whereby exposure to a priming stimulus enables quicker or stronger response to subsequent attack by pests and pathogens. Such priming of inducible defenses provides increased protection and reduces allocation costs of defense. Defense priming has been widely studied for short-lived model plants such as Arabidopsis, but little is known about this phenomenon in long-lived plants like spruce. We compared the effects of pretreatment with sublethal fungal inoculations or application of the phytohormone methyl jasmonate (MeJA) on the resistance of 48-year-old Norway spruce (Picea abies) trees to mass attack by a tree-killing bark beetle beginning 35 days later. Bark beetles heavily infested and killed untreated trees but largely avoided fungus-inoculated trees and MeJA-treated trees. Quantification of defensive terpenes at the time of bark beetle attack showed fungal inoculation induced 91-fold higher terpene concentrations compared with untreated trees, whereas application of MeJA did not significantly increase terpenes. These results indicate that resistance in fungus-inoculated trees is a result of direct induction of defenses, whereas resistance in MeJA-treated trees is due to defense priming. This work extends our knowledge of defense priming from model plants to an ecologically important tree species.

Mass spectrometry reveals the presence of specific set of epigenetic DNA modifications in the Norway spruce genome.

5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene expression in metazoans and plants. Iron-(II)/α-ketoglutarate-dependent dioxygenases can oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although these oxidized forms of 5mC may serve as demethylation intermediates or contribute to transcriptional regulation in animals and fungi, experimental evidence for their presence in plant genomes is ambiguous. Here, employing reversed-phase HPLC coupled with sensitive mass spectrometry, we demonstrated that, unlike 5caC, both 5hmC and 5fC are detectable in non-negligible quantities in the DNA of a conifer, Norway spruce. Remarkably, whereas 5hmC content of spruce DNA is approximately 100-fold lower relative to human colorectal carcinoma cells, the levels of both - 5fC and a thymine base modification, 5-hydroxymethyluracil, are comparable in these systems. We confirmed the presence of modified DNA bases by immunohistochemistry in Norway spruce buds based on peroxidase-conjugated antibodies and tyramide signal amplification. Our results reveal the presence of specific range of noncanonical DNA bases in conifer genomes implying potential roles for these modifications in plant development and homeostasis.

Comparative sensitivity to gamma radiation at the organismal, cell and DNA level in young plants of Norway spruce, Scots pine and Arabidopsis thaliana.

MAIN CONCLUSION: Persistent DNA damage in gamma-exposed Norway spruce, Scots pine and Arabidopsis thaliana, but persistent adverse effects at the organismal and cellular level in the conifers only. Gamma radiation emitted from natural and anthropogenic sources may have strong negative impact on plants, especially at high dose rates. Although previous studies implied different sensitivity among species, information from comparative studies under standardized conditions is scarce. In this study, sensitivity to gamma radiation was compared in young seedlings of the conifers Scots pine and Norway spruce and the herbaceous Arabidopsis thaliana by exposure to 60Co gamma dose rates of 1-540 mGy h-1 for 144 h, as well as 360 h for A. thaliana. Consistent with slightly less prominent shoot apical meristem, in the conifers growth was significantly inhibited with increasing dose rate ≥ 40 mGy h-1. Post-irradiation, the conifers showed dose-rate-dependent inhibition of needle and root development consistent with increasingly disorganized apical meristems with increasing dose rate, visible damage and mortality after exposure to ≥ 40 mGy h-1. Regardless of gamma duration, A. thaliana showed no visible or histological damage or mortality, only delayed lateral root development after ≥ 100 mGy h-1 and slightly, but transiently delayed post-irradiation reproductive development after ≥ 400 mGy h-1. In all species dose-rate-dependent DNA damage occurred following ≥ 1-10 mGy h-1 and was still at a similar level at day 44 post-irradiation. In conclusion, the persistent DNA damage (possible genomic instability) following gamma exposure in all species may suggest that DNA repair is not necessarily mobilized more extensively in A. thaliana than in Norway spruce and Scots pine, and the far higher sensitivity at the organismal and cellular level in the conifers indicates lower tolerance to DNA damage than in A. thaliana.

Wood Modification by Furfuryl Alcohol Resulted in a Delayed Decomposition Response in Rhodonia (Postia) placenta.

The aim of this study was to investigate differential expression profiles of the brown rot fungus Rhodonia placenta (previously Postia placenta) harvested at several time points when grown on radiata pine (Pinus radiata) and radiata pine with three different levels of modification by furfuryl alcohol, an environmentally benign commercial wood protection system. The entire gene expression pattern of a decay fungus was followed in untreated and modified wood from initial to advanced stages of decay. The results support the current model of a two-step decay mechanism, with the expression of genes related to initial oxidative depolymerization, followed by an accumulation of transcripts of genes related to the hydrolysis of cell wall polysaccharides. When the wood decay process is finished, the fungus goes into starvation mode after five weeks when grown on unmodified radiata pine wood. The pattern of repression of oxidative processes and oxalic acid synthesis found in radiata pine at later stages of decay is not mirrored for the high-furfurylation treatment. The high treatment level provided a more unpredictable expression pattern throughout the incubation period. Furfurylation does not seem to directly influence the expression of core plant cell wall-hydrolyzing enzymes, as a delayed and prolonged, but similar, pattern was observed in the radiata pine and the modified experiments. This indicates that the fungus starts a common decay process in the modified wood but proceeds at a slower pace as access to the plant cell wall polysaccharides is restricted. This is further supported by the downregulation of hydrolytic enzymes for the high treatment level at the last harvest point (mass loss, 14%). Moreover, the mass loss does not increase during the last weeks. Collectively, this indicates a potential threshold for lower mass loss for the high-furfurylation treatment.IMPORTANCE Fungi are important decomposers of woody biomass in natural habitats. Investigation of the mechanisms employed by decay fungi in their attempt to degrade wood is important for both the basic scientific understanding of ecology and carbon cycling in nature and for applied uses of woody materials. For wooden building materials, long service life and carbon storage are essential, but decay fungi are responsible for massive losses of wood in service. Thus, the optimization of durable wood products for the future is of major importance. In this study, we have investigated the fungal genetic response to furfurylated wood, a commercial environmentally benign wood modification approach that improves the service life of wood in outdoor applications. Our results show that there is a delayed wood decay by the fungus as a response to furfurylated wood, and new knowledge about the mechanisms behind the delay is provided.

The epigenetic memory of temperature during embryogenesis modifies the expression of bud burst-related genes in Norway spruce epitypes.

MAIN CONCLUSION: Epigenetic memory affects the timing of bud burst phenology and the expression of bud burst-related 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.

Transcriptional profiling of epigenetic regulators in somatic embryos during temperature induced formation of an epigenetic memory in Norway spruce.

MAIN CONCLUSION: A significant number of epigenetic regulators were differentially expressed during embryogenesis at different epitype-inducing conditions. Our results support that methylation of DNA and histones, as well as sRNAs, are pivotal for the establishment of the epigenetic memory. As a forest tree species with long generation times, Norway spruce is remarkably well adapted to local environmental conditions despite having recently, from an evolutionary perspective, recolonized large areas following the last glaciation. In this species, there is an enigmatic epigenetic memory of the temperature conditions during embryogenesis that allows rapid adaptation to changing environment. We used a transcriptomic approach to investigate the molecular mechanisms underlying the formation of the epigenetic memory during somatic embryogenesis in Norway spruce. Nine mRNA libraries were prepared from three epitypes of the same genotype resulting from exposure to epitype-inducing temperatures of 18, 23 and 28 °C. RNA-Seq analysis revealed more than 10,000 differentially expressed genes (DEGs). The epitype-inducing conditions during SE were accompanied by marked transcriptomic changes for multiple gene models related to the epigenetic machinery. Out of 735 putative orthologs of epigenetic regulators, 329 were affected by the epitype-inducing temperatures and differentially expressed. The majority of DEGs among the epigenetic regulators was related to DNA and histone methylation, along with sRNA pathways and a range of putative thermosensing and signaling genes. These genes could be the main epigenetic regulators involved in formation of the epigenetic memory. We suggest considerable expansion of gene families of epigenetic regulators in Norway spruce compared to orthologous gene families in Populus and Arabidopsis. Obtained results provide a solid basis for further genome annotation and studies focusing on the importance of these candidate genes for the epigenetic memory formation.

DNA quantification of basidiomycetous fungi during storage of logging residues.

The demand for bioenergy caused an increased use of logging residues, branches and treetops that were previously left on the ground after harvesting. Residues are stored outdoors in piles and it is unclear to what extent fungi transform this material. Our objective was to quantify the amount of wood degrading fungi during storage using quantitative real-time PCR (qPCR) to detect basidiomycetous DNA in logging residues, a novel approach in this field. We found that the qPCR method was accurate in quantifying the fungal DNA during storage. As the moisture content of the piled logging residues decreased during the storage period, the fungal DNA content also decreased. Scots pine residues contained more fungal DNA than residues from Norway spruce. Loose piles had generally more fungal DNA than bundled ones.

Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem.

The tangentially oriented polyphenolic parenchyma (PP) and radially organized ray parenchyma in the phloem are central in the defense of conifer stems against insects and pathogens. Laser micro-dissection enables examination of cell-specific defense responses. To examine induced defense responses in Norway spruce stems inoculated with the necrotrophic blue-stain fungus Ceratocystis polonica, RNA extracted from laser micro-dissected phloem parenchyma and vascular cambium was analyzed using real-time RT-PCR (qRT-PCR) to profile transcript levels of selected resistance marker genes. The monitored transcripts included three pathogenesis-related proteins (class IV chitinase (CHI4), defensin (SPI1), peroxidase (PX3), two terpene synthesis related proteins (DXPS and LAS), one ethylene biosynthesis related protein (ACS), and a phenylalanine ammonia-lyase (PAL). Three days following inoculation, four genes (CHI4, PAL, PX3, SPI1) were differentially induced in individual cell and tissue types, both close to the inoculation site (5 mm above) and, to a lesser degree, further away (10 mm above). These resistance marker genes were all highly induced in ray parenchyma, supporting the important role of the rays in spruce defense propagation. CHI4 and PAL were also induced in PP cells and in conducting secondary phloem tissues. Our data suggests that different cell types in the secondary phloem of Norway spruce have overlapping but not fully redundant roles in active host defense. Furthermore, the study demonstrates the usefulness of laser micro-dissection coupled with qRT-PCR to characterize gene expression in different cell types of conifer bark.

Dehydrin accumulation and extreme low-temperature tolerance in Siberian spruce (Picea obovata).

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.

Genes associated with lignin degradation in the polyphagous white-rot pathogen Heterobasidion irregulare show substrate-specific regulation.

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.

The pathogenic white-rot fungus Heterobasidion parviporum responds to spruce xylem defense by enhanced production of oxalic acid.

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.

Indications of heightened constitutive or primed host response affecting the lignin pathway transcripts and phenolics in mature Norway spruce clones.

Two mature clones of Norway spruce (Picea abies (L.) Karst.) that have previously been shown to have differential degrees of resistance towards the necrotrophic pathogen Heterobasidion parviporum (Niemelä & Korhonen) were compared with respect to the primed defense expression of transcripts related to biosynthesis of lignin, stilbenes and other phenolic compounds from one year to the next. The host's response to physical wounding and pathogen inoculation was examined in the initial year, whereas indications of heightened basal defense level or primed response, and responses to re-wounding, were examined the following year. The responses of the two clones to wounding and pathogen inoculation, examined in the initial year, differed; the increases in lignin and phenolics were more distinct in response to the pathogen than to wounding alone. The more resistant clone 589 had higher initial lignin concentrations in the cell walls when compared with clone 409, and these remained higher in clone 589 over both years and increased after the treatments. Both clones responded at the transcriptional and chemical levels to wounding; changes were evident both in the initial wounds and when re-wounded the following year. There were distinct differences in the basal transcript levels of the lignin pathway-related genes, phenolics and total lignin levels in healthy tissue from the initial year to the following year indicative of a primed host response or at least altered constitutive level of defense expression.