ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

Summary We investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. NRCC no. 37345.     

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

T-DNA presence and opine production in tumors of Picea abies (L.) Karst induced by Agrobacterium tumefaciens A281

The hypervirulent Agrobacterium tumefaciens strain A281 formed frequent tumors (31%) on Picea abies (Norway spruce), an economically important tree species in Swedish forests. Three-month-old seedlings were inoculated and tumors were established that grew hormone-independently in culture. Tumors contained agropine and mannopine/mannopinic acid as determined by acid pH paper electrophoresis. In addition, DNA hybridization studies showed that the DNA from these tumor lines contained sequences homologous to Ti plasmid T-DNA, whereas wild-type spruce seedling DNA did not. These results suggest that Agrobacterium vectors can be used for gene transfer into this important forest species.     

Genetic control of root exudation

The literature on genetics of root exudation and on genotypic differences in qualitative and quantitative composition of root exudates in crop and native plant species was critically assessed. Differences in exudation have been reported for genotypes that differ in tolerance to nutrient deficiencies, ion toxicities, and pathogen attack. The exudation profile of a limited number of genotypes (frequently only two genotypes with the contrasting response to the environmental stress) have been reported to date. Little is known about the variability in larger samples of the germplasm or about actual genetics behind differential qualitative and quantitative composition of root exudates. Changing the exudation profile of a given genotype may be achieved by manipulating the biosynthetic capacity and by increasing the capacity of the plasma membrane to transport the specific compound out into the rhizosphere. Overexpression of the bacterial citrate synthase gene in the cytoplasm of tobacco plants resulted in exudation of large quantities of citrate into the rhizosphere and partial alleviation of the aluminium (Al) toxicity stress. A similar strategy of transforming plants with citrate synthase gene is being tried as a way of improving plant capacity to extract phosphorus (P) from soils with notoriously low P availability.More research into the genetic basis of qualitative and quantitative differences in root exudation is warranted. Understanding the genetic control of root exudation, followed by manipulation of qualitative and quantitative composition of root exudates, will result in better adaptation of plants to environmental conditions and a greater yield of crops.     

Negative effects on survival and performance of Norway spruce seedlings colonized by dark septate root endophytes are primarily isolate-dependent

Root endophytes are common and genetically highly diverse suggesting important ecological roles. Yet, relative to above-ground endophytes, little is known about them. Dark septate endophytic fungi of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) are ubiquitous root colonizers of conifers and Ericaceae, but their ecological function is largely unknown. Responses of Norway spruce seedlings of two seed provenances to inoculations with isolates of four PAC species were studied in vitro. In addition, isolates of Phialocephala subalpina from two populations within and one outside the natural range of Norway spruce were also included to study the effect of the geographic origin of P. subalpina on host response. The interaction of PAC with Norway spruce ranged from neutral to highly virulent and was primarily isolate-dependent. Variation in virulence was much higher within than among species, nonetheless only isolates of P. subalpina were highly virulent. Disease caused by P. subalpina genotypes from the native range of Norway spruce was more severe than that induced by genotypes from outside the natural distribution of Norway spruce. Virulence was not correlated with the phylogenetic relatedness of the isolates but was positively correlated with the extent of fungal colonization as measured by quantitative real-time PCR.     

Karyotype of Norway spruce by multicolor FISH

The chromosomes (2n = 2x = 24) of Norway spruce are very large since their size reflects the huge amount of genomic DNA (2C = 30 × 10⁹ bp). However, the identification of homologous pairs is hampered by their high degree of similarity at the morphological level. Data so far presented in the literature were not sufficient to solve all the ambiguities in chromosome identification. Several genomic Norway spruce DNA clones containing highly repetitive sequences have been identified and characterised in our laboratory. Three of them were selected for fluorescent in situ hybridization (FISH) experiments because of their strong signals and suitability for chromosome identification: PATR140 hybridized at the centromeric site of three chromosome pairs; PAF1 hybridized in six subtelomeric and two centromeric sites; 1PABCD6 co-localized with the subtelomeric sites identified by PAF1. The statistical analysis of microscopic measurements of chromosomes in combination with the FISH signals of these probes allowed the unambigous construction of Norway spruce karyotype. We also compared the karyotype of Norway spruce with that of other spruce species to infer the number and kind of rearrangements that have occurred during the evolution of these species.Communicated by D.B. Neale     

Phenolic compounds in Norway spruce as affected by boron nutrition at the end of the growing season

The increase in concentrations of phenolic compounds in boron (B) deficiency has been demonstrated in many herbaceous plant species, but information on woody plants is scarce. It has been suggested that accumulation of phenolic compounds plays a role in the development of cold hardiness in herbaceous plants but also that B deficiency decreases winter hardiness. Here we study the effects of B nutrition on phenolic compounds in Norway spruce (Picea abies L.) in the course of cold acclimation. Phenolic compounds were analysed in Norway spruce seedlings from three different B-fertilisation treatments in two harvests: non-acclimated and cold-acclimated seedlings. Norway spruce phenolic compounds consisted mainly of condensed tannins. During B deficiency, condensed tannins and monocoumaroyl–astragalin der. 1 increased in non-acclimated seedlings. The increase in tannins was 21%, which was nearly significant. However, the effect of B on phenolic compounds was almost absent in cold-acclimated seedlings. The condensed tannin concentration increased much more with time in the simulated autumn than due to B deficiency, and we conclude that the B effect was probably not large enough to be important for the hardening of the seedlings. The total phenolic concentrations more than doubled during the course of cold hardening suggesting that phenolics have a role in the winter hardiness in Norway spruce.     

Interactions between green spruce aphid (Elatobium abietinum (Walker)) and Norway and Sitka spruce under high and low nutrient conditions

1 Green spruce aphid (Elatobium abietinum) is a serious pest of spruce (Picea spp.) in north‐west Europe, causing defoliation of one‐year‐old and older needles. 2 Relationships between population development of E. abietinum, needle loss and tree growth were compared for five pure genotypes of Sitka spruce and mixed‐genotype material of Sitka and Norway spruce, grown under high and low nutrient conditions. 3 Despite wide differences in flushing date between spruce genotypes, E. abietinum populations peaked on the same date on each genotype and on the mixed‐genotype material, irrespective of nutrient supply. 4 Larger aphid populations developed on trees grown under high nutrient conditions than under low nutrients. However, more needles were lost per aphid in the low nutrient treatment and overall defoliation rates in the two nutrient treatments were similar. 5 Total aphid numbers differed significantly between Sitka spruce genotypes within nutrient treatments, but not in relation to bud‐burst or needle terpene content. Reductions in height growth caused by infestation were greater (15–44%), and related to mean aphid densities and defoliation, in the low nutrient treatment, but were smaller (11–27%) and not related to aphid density and defoliation in the high nutrient treatment. 6 Development of E. abietinum populations was similar on Norway and Sitka spruce, but Norway spruce lost fewer needles. However, the effects of infestation on tree growth were more closely related to aphid density and were similar for Norway and Sitka spruce. 7 Infestation caused a decrease in total root dry weight of Norway and Sitka spruce in proportion to the reductions observed in above‐ground growth.     

Induced responses in stilbenes and terpenes in fertilized Norway spruce after inoculation with blue-stain fungus, Ceratocystis polonica

In conifers, attacks by bark beetles and associated pathogenic fungi cause an induced wound response, which is characterized by accumulation of antifungal compounds and morphological changes that aid wound healing. In this article the stilbene and terpene concentrations of Norway spruce phloem were monitored as symptoms of induced wound responses in relation to changed nutrient conditions caused by fertilization. Plots of mature Norway spruce were fertilized with N, P or NPK. One year after fertilization the trees were artificially infected with Ceratocystis polonica, a pathogenic fungus associated with the bark beetle Ips typographus. The response of stilbenes to fungal inoculation was mainly qualitative. The concentration of stilbene glycosides in the phloem decreased, and in the immediate vicinity of the site of fungal inoculation, stilbene glycosides were less frequent than in mechanically wounded or unwounded phloem. Corresponding stilbene aglycones were most frequent inside the reaction lesion. The concentration of total stilbene aglycones near the inoculation site was significantly lower in N-fertilized trees than in unfertilized trees. Fungal inoculation caused a strong quantitative response in terpenes. The total terpene concentration of the phloem increased significantly, to almost 100 times greater near the inoculation site compared to the constitutive values. N fertilization significantly reduced the total terpene and total stilbene aglycone concentrations near the inoculation sites. Thus, N fertilization may reduce the ability of Norway spruce to defend itself against fungal pathogens.     

Cytomixis in shoot apex of Norway spruce [<Emphasis Type="Italic">Picea abies</Emphasis> (L.) Karst.]

Atypical cell walls and nuclei were observed in the apex of Norway spruce shoots from late April to early May on the material collected from a few grafts of a clone of Norway spruce growing on an experimental area. Images of ultrastructure attest to cytomixis. The phenomenon of cytomixis has previously been described in various plant material, both in the meiotic and mitotic cells, but this is the first report of cytomixis in gymnosperms.     

Host resistance elicited by methyl jasmonate reduces emission of aggregation pheromones by the spruce bark beetle, Ips typographus

We treated Norway spruce (Picea abies) stems with methyl jasmonate (MeJA) to determine possible quantitative and qualitative effects of induced tree defenses on pheromone emission by the spruce bark beetle Ips typographus. We measured the amounts of 2-methyl-3-buten-2-ol and (S)-cis-verbenol, the two main components of the beetle's aggregation pheromone, released from beetle entrance holes, along with phloem terpene content and beetle performance in MeJA-treated and untreated Norway spruce logs. As expected, phloem terpene levels were higher and beetle tunnel length was shorter (an indication of poor performance) in MeJA-treated logs relative to untreated logs. Parallel to the higher phloem terpene content and poorer beetle performance, beetles in MeJA-treated logs released significantly less 2-methyl-3-buten-2-ol and (S)-cis-verbenol, and the ratio between the two pheromone components was significantly altered. These results suggest that host resistance elicited by MeJA application reduces pheromone emission by I. typographus and alters the critical ratio between the two main pheromone components needed to elicit aggregation. The results also provide a mechanistic explanation for the reduced performance and attractivity observed in earlier studies when bark beetles colonize trees with elicited host defenses, and extend our understanding of the ecological functions of conifer resistance against bark beetles.     

A Norway spruce FLOWERING LOCUS T homolog is implicated in control of growth rhythm in conifers

Growth in perennial plants possesses an annual cycle of active growth and dormancy that is controlled by environmental factors, mainly photoperiod and temperature. In conifers and other nonangiosperm species, the molecular mechanisms behind these responses are currently unknown. In Norway spruce (Picea abies L. Karst.) seedlings, growth cessation and bud set are induced by short days and plants from southern latitudes require at least 7 to 10 h of darkness, whereas plants from northern latitudes need only 2 to 3 h of darkness. Bud burst, on the other hand, is almost exclusively controlled by temperature. To test the possible role of Norway spruce FLOWERING LOCUS T (FT)-like genes in growth rhythm, we have studied expression patterns of four Norway spruce FT family genes in two populations with a divergent bud set response under various photoperiodic conditions. Our data show a significant and tight correlation between growth rhythm (both bud set and bud burst), and expression pattern of one of the four Norway spruce phosphatidylethanolamine-binding protein gene family members (PaFT4) over a variety of experimental conditions. This study strongly suggests that one Norway spruce homolog to the FT gene, which controls flowering in angiosperms, is also a key integrator of photoperiodic and thermal signals in the control of growth rhythms in gymnosperms. The data also indicate that the divergent adaptive bud set responses of northern and southern Norway spruce populations, both to photoperiod and light quality, are mediated through PaFT4. These results provide a major advance in our understanding of the molecular control of a major adaptive trait in conifers and a tool for further molecular studies of adaptive variation in plants.     

Dendrochronology of Norway spruce (Picea abies (L.) Karst.) from two range centres in lowland Poland

In Europe, spruce grows in two main regions which meet in Poland, one to the north and east, the other to the south and west. The northeastern area ranges from the northern treeline extending from Norway to Siberia, to southern Sweden, north-eastern Poland and the southern Ural mountains. The southwestern spruce region reaches the mountainous areas of the Alps and the Balkans, the mountains and uplands of the Sudety Mountains, the Carpathians and neighbouring lowlands. Opinions about the distribution of Norway spruce have changed over the years, and its scarcity in the centre of Poland has been strongly debated. The favoured current theory is that Norway spruce once had a continuous distribution in Poland. It is assumed that the rare occurrence in the central Polish lowland is due to a combination of unfavourable soil conditions and previous management activity. The main aim of this work was to analyse climate–growth relationships of Norway spruce in eastern Poland and distinguish regions with similar increment patterns with regards to spruce range. Spruce growth in northern Polish sites is positively correlated with rainfall from May to July. Tree-ring widths in southern sites are more correlated with March temperature. Selected homogenous regions are the same as range types. Trees from the so-called “spruceless area” seem to have similar climate–growth relationships to trees from the southern region. This finding does not settle the question of the origin of the trees, but it does indicate that similar environmental conditions exist in these two areas and proves that the climate was not a limiting factor there.

Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies)

Plant class III peroxidases (POXs) take part in the formation of lignin and maturation of plant cell walls. However, only a few examples of such peroxidases from gymnosperm tree species with highly lignified xylem tracheids have been implicated so far. We report here cDNA cloning of three xylem-expressed class III peroxidase encoding genes from Norway spruce (Picea abies). The translated proteins, PX1, PX2 and PX3, contain the conserved amino acids required for heme-binding and peroxidase catalysis. They all begin with putative secretion signal propeptide sequences but diverge substantially at phylogenetic level, grouping to two subclusters when aligned with other class III plant peroxidases. In situ hybridization analysis on expression of the three POXs in Norway spruce seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm of young, developing tracheids within the current growth ring where lignification is occurring. Function of the putative N-terminal secretion signal peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions with EGFP (enhanced green fluorescent protein) and expressing them in tobacco protoplasts. Full-length coding region of px1 was also heterologously expressed in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1 peroxidase is processed via the endoplasmic reticulum (ER) most likely for secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal localization for participation in the maturation of the spruce tracheid secondary cell wall.     

Reliable and practical methods for cryopreservation of embryogenic cultures and cold storage of somatic embryos of Norway spruce

Somatic embryogenesis (SE) is considered as the most-effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). For mass propagation, a cryopreservation method able to handle large numbers of embryogenic tissues (ETs) reliably and at low costs is needed. The aim of the present study was to compare pretreatments, cryoprotectants and slow-cooling devices for cryopreservation of Norway spruce ETs, with 12 variations of methods and a total of 136 spruce genotypes. Secondly, possible applications for cold storage of mature somatic embryos were studied with the aim of developing a flexible time window for embling production. At best, 100% of the embryogenic lines were recovered following cryopreservation, but the results varied among the sets of lines. Also physiological condition of the tissues, pre-treatment and cryoprotectant applied, as well as the slow-cooling device used were found to affect the recovery. The best option for cryopreservation of Norway spruce is to select fresh growth from young ETs as samples, pretreat them on semi-solid medium with increasing sucrose concentration (0.1 M for 24 h; 0.2 M for another 24 h), apply a mixture of polyethylene glycol 6000, glucose, and dimethylsulfoxide, 10% w/v each, as cryoprotectant and use a programmable freezer with a slow cooling rate (0.17 °C/min). On average, 87% of the genotypes can be recovered, without any effect on their genetic fidelity, as shown by microsatellite markers and embryo production capacity. Mature somatic embryos of Norway spruce can also be safely cold-stored at +4 °C, without adverse effects on their germination ability.     

Circumnutation oscillations of the hypocotyl and hypocotyl hook formation: A comparison of Norway spruce (Picea abies (L.)Karst.) and Scots pine (Pinus silvestris L.) seedlings

Circumnutation oscillations of hypocotyls were studied in Norway spruce and Scots pine using time-lapse cinematography. It was found that the circumnutations were specific in species. The analysis of hypocotyl ontogeny (100 h) revealed a different duration of the phases I, II and III of nutation oscillations in the first and second taxon. From the quantitative point of view it can be stated that during the phase I of nutation oscillations the Norway spruce hypocotyls exhibited higher frequency activity (corrections in direction of the hypocotyl by an elongation-controlling system corrected by a feedback mechanism), while in the phase III of nutation oscillations higher growth rate and larger amplitudes were observed in the Scots pine hypocotyl when compared with those of Norway spruce. The hypocotyl hook appeared only in Norway spruce.