Tapio Laakso

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.

Does lignin modification affect feeding preference or growth performance of insect herbivores in transgenic silver birch (Betula pendula Roth)

Transgenic silver birch (Betula pendula Roth) lines were produced in order to modify lignin biosynthesis. These lines carry COMT (caffeate/5-hydroxyferulate O-methyltransferase) gene from Populus tremuloides driven by constitutive promoter 35S CaMV (cauliflower mosaic virus) or UbB1 (ubiquitin promoter from sunflower). The decreased syringyl/guaiacyl (S/G) ratio was found in stem and leaf lignin of 35S CaMV-PtCOMT transgenic silver birch lines when compared to non-transformed control or UbB1–PtCOMT lines. In controlled feeding experiments the leaves of transgenic birch lines as well as controls were fed to insect herbivores common in boreal environment, i.e., larvae of Aethalura punctulata, Cleora cinctaria and Trichopteryx carpinata (Lepidoptera: Geometridae) as well as the adults of birch leaf-feeding beetles Agelastica alni (Coleoptera: Chrysomelidae) and Phyllobius spp. (Coleoptera: Curculionidae). The feeding preferences of these herbivores differed in some cases among the tested birch lines, but these differences could not be directly associated to lignin modification. They could as well be explained by other characteristics of leaves, either natural or caused by transgene site effects. Growth performance of lepidopteran larvae fed on transgenic or control leaves did not differ significantly.

FT-NIR spectroscopy in predicting the decay resistance related characteristics of solid Scots pine (Pinus sylvestris L.) heartwood

Abstract FT-NIR spectroscopy equipped with a fiber optic probe was used to predict the mass loss caused by a brown-rot fungus (Coniophora puteana) in Scots pine heartwood. Because decay tests are impractical for generating reference data for the calibration of prediction models, the possibilities of using the concentration of heartwood extractives as a reference variable instead of mass loss was studied. The material investigated covered a wide range of natural variation in durability and diffuse reflectance infrared Fourier transform spectra were recorded from the cross section of 41 pines. The partial least square (PLS) regression models were found to be satisfactory for prediction of the mass loss and the concentration of extractives (total phenolics, resin acids, pinosylvin and pinosylvin monomethyl ether). It was concluded that FT-NIR spectroscopy has the potential to become a tool for the decay resistance grading of Scots pine heartwood timber, especially if the prediction models will be based on heartwood extractives.

Seasonal variation in formation, structure, and chemical properties of phloem in Picea abies as studied by novel microtechniques

AbstractMain conclusionPhloem production and structural development were interlinked with seasonal variation in the primary and secondary metabolites of phloem. Novel microtechniques provided new perspectives on understanding phloem structure and chemistry. To gain new insights into phloem formation in Norway spruce (Picea abies), we monitored phloem cell production and seasonal variation in the primary and secondary metabolites of inner bark (non-structural carbohydrates and phenolic stilbene glucosides) during the 2012 growing season in southern and northern Finland. The structure of developing phloem was visualised in 3D by synchrotron X-ray microtomography. The chemical features of developing phloem tissues isolated by laser microdissection were analysed by chemical microanalysis. Within-year phloem formation was associated with seasonal changes in non-structural carbohydrates and phenolic extractive contents of inner bark. The onset of phloem cell production occurred in early and mid-May in southern and northern Finland, respectively. The maximal rate of phloem production and formation of a tangential band of axial phloem parenchyma occurred in mid-June, when total non-structural carbohydrates peaked (due to the high amount of starch). In contrast, soluble sugar content dropped during the most active growth period and increased in late summer and winter. The 3D visualisation showed that the new axial parenchyma clearly enlarged from June to August. Sub-cellular changes appeared to be associated with accumulation of stilbene glucosides and soluble sugars in the newest phloem. Stilbene glucosides also increased in inner bark during late summer and winter. Our findings may indicate that stilbene biosynthesis in older phloem predominantly occurs after the formation of the new band(s) of axial parenchyma. The complementary use of novel microtechniques provides new perspectives on the formation, structure, and chemistry of phloem.

Resin-Salve from Norway Spruce - A Potential Method to Treat Infected Chronic Skin Ulcers?

The home-made resin salve from Norway spruce is traditionally and widely used in folk medicine to heal various skin infections and wounds in Northern Finland. We have performed laboratory studies to solve the mechanism of resin salve. The resin salve exhibited a bacteriostatic effect against all tested Gram-positive bacteria important in human medicine including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE), but was not effective against Gram-negative bacteria. An exception among the Gram-negative bacteria was Proteus vulgaris against which resin salve was effective. High amounts of lipophilic extractives, like resin acids were dissolved into water from the resin salve. Also, a large proportion of lignans and cinnamic acid were found in the water extract.

Knotwood as a window to the indirect measurement of the decay resistance of Scots pine heartwood

Abstract There is wide variation in the extractive content and decay resistance of Scots pine (Pinus sylvestris L.) heartwood. The heartwood is not visible in standing trees and only poorly visible in timber. Therefore, it is difficult to identify extractive-rich trees, and consequently the most decay-resistant heartwood. On the other hand, knots are clearly visible in standing trees and timber. In the present paper we studied the possibility of measuring the decay resistance of Scots pine heartwood indirectly on the basis of the extractive concentration of knotwood. The material investigated consisted of 40 felled trees with a wide between-tree variation for extractive content and decay resistance of their heartwood. The extractive content of knotwood was found to be four- to five-fold higher than that of heartwood. Statistically significant correlations were found between the mass loss of heartwood and the concentrations of total phenolics and stilbenes in knotwood (r=-0.54, P<0.001 and r=-0.40, P=0.011, respectively), and for the concentration of total phenolics (r=0.42, P=0.008) and stilbenes (r=0.39, P=0.012) between heartwood and knotwood. We suggest further development of this technique in the context of rapid industrial screening of durable pine heartwood.

Wounding response in xylem of Scots pine seedlings shows wide genetic variation and connection with the constitutive defence of heartwood

In this greenhouse experiment, 3-year-old Scots pine (Pinus sylvestris L.) seedlings were wounded by drilling holes through the stem. In the xylem next to the wound, the concentration of resin acids (RAC) increased, and the production of extractives typical for heartwood (stilbenes) and knotwood (stilbenes and lignans) of mature trees was induced. The induced stilbenes were pinosylvin (PS) and pinosylvin monomethyl ether (PSM), and the lignans nortrachelogenin (NTG) and matairesinol (MR). There was positive phenotypic correlation between concentrations of the different extractives. Except for the RAC, the extractive concentrations showed no correlation with the size of the seedlings. The treated seedlings belonged to half-sib families, which enabled the estimation of the genetic parameters for the response variables. The proportion of heritable variation (heritability, h(2)) in the concentration of PS, NTG and MR varied between 0.71 and 1.03, whereas for PSM and RAC the heritability was lower (0.35 and 0.31). Genetic correlation was significant between PS and PSM (r = 0.55, P = 0.018), and between NTG and MR (r = 0.50, P = 0.033). Heritabilities were also estimated on the basis of the regression of the offspring on their mothers h(2)(0P). These estimates were assessed for the concentration of PS, PSM and RAC in the wound response area of the seedlings and correspondingly in the heartwood of their mothers. The heritability was highest for the concentration of PS h(2)(0P). The findings of this study support the suggestion that the wounding of Scots pine seedlings may facilitate the development of an early testing method for breeding heartwood durability.

Decay resistance, extractive content, and water sorption capacity of Siberian larch (Larix sibirica Lebed.) heartwood timber

Abstract The aim of this study was to find chemical or physical properties of Siberian larch heartwood timber that correlate with the variation in decay resistance. Juvenile heartwood from 24-year-old grafts of 15 clones was exposed to three brown-rot fungi according to the standard in vitro decay test (European standard EN 113). The mass losses caused by the brown rot fungi Coniophora puteana, Poria placenta, and Gloeophyllum trabeum were 20%, 28% and 17% of the dry mass, respectively. The average mass loss over the three fungi had a strong negative correlation with the concentration of taxifolin (r=–0.673, P=0.006), as well as with the concentration of total phenolics determined by the Folin-Ciocalteu assay (r=–0.677, P=0.006). Thus, the concentration of flavonoids is a promising property for indirect measurement of the decay resistance of Siberian larch timber. The most abundant heartwood extractives, arabinogalactans, had a non-significant relationship with the decay resistance, but their concentration correlated positively with the capacity of the wood to adsorb water (r=0.736, P=0.002). The hygroscopic properties of the wood or the wood density were not associated with the decay resistance.

Phenolic compounds in ectomycorrhizal interaction of lignin modified silver birch.

BackgroundThe monolignol biosynthetic pathway interconnects with the biosynthesis of other secondary phenolic metabolites, such as cinnamic acid derivatives, flavonoids and condensed tannins. The objective of this study is to evaluate whether genetic modification of the monolignol pathway in silver birch (Betula pendula Roth.) would alter the metabolism of these phenolic compounds and how such alterations, if exist, would affect the ectomycorrhizal symbiosis.ResultsSilver birch lines expressing quaking aspen (Populus tremuloides L.) caffeate/5-hydroxyferulate O-methyltransferase (PtCOMT) under the 35S cauliflower mosaic virus (CaMV) promoter showed a reduction in the relative expression of a putative silver birch COMT (BpCOMT) gene and, consequently, a decrease in the lignin syringyl/guaiacyl composition ratio. Alterations were also detected in concentrations of certain phenolic compounds. All PtCOMT silver birch lines produced normal ectomycorrhizas with the ectomycorrhizal fungus Paxillus involutus (Batsch: Fr.), and the formation of symbiosis enhanced the growth of the transgenic plants.ConclusionThe down-regulation of BpCOMT in the 35S-PtCOMT lines caused a reduction in the syringyl/guaiacyl ratio of lignin, but no significant effect was seen in the composition or quantity of phenolic compounds that would have been caused by the expression of PtCOMT under the 35S or UbB1 promoter. Moreover, the detected alterations in the composition of lignin and secondary phenolic compounds had no effect on the interaction between silver birch and P. involutus.

Root neck of Norway spruce as a source of bioactive lignans and stilbenes.

Abstract Bioactive compounds of acetone-water extracts obtained from the root neck (RN) of Norway spruce (Picea abies [L.] Karst.) have been studied for the first time. RN samples were divided into different zones and cut, and wood and bark were extracted separately with acetone-water. The extracts were analyzed by gas chromatography-mass spectrometry. The extractives have a tissue-specific distribution. Several lignans were identified in the heartwood. High concentrations (10%) of hydroxymatairesinol were found from the heartwood at the lowermost part of the RN. The inner part of the bark contained the highest concentrations of stilbene glucosides (20%), which were present in the outer bark only to a minor extent (0.2%). Pressurized hot water extraction was also applied and was found to be effective in separating lignans from biomass. Norway spruce roots and stumps are usually utilized for gaining bioenergy. The results presented here show that these abundant residual materials of forest economy may be a valuable source of highly bioactive compounds such as the lignan hydroxymatairesinol and stilbenes.