Polyamine (PA) metabolism was studied in liquid cultures of Scots pine (Pinus sylvestris L.) embryogenic cells. The focus of the study was on the metabolic changes at the interphase between the initial lag phase and the exponential growth phase. PA concentrations fluctuated in the liquid cultures as follows. Putrescine (Put) concentrations increased, whereas spermidine (Spd) concentrations decreased in both free and soluble conjugated PA fractions. The concentrations of free and soluble conjugated spermine (Spm) remained low, and small amounts of excreted PAs were also found in the culture medium. The minor production of secondary metabolites reflected the undifferentiated stage of the embryogenic cell culture. Put was produced via the arginine decarboxylase (ADC) pathway. Futhermore, the gene expression data suggested that the accumulation of Put was caused neither by an increase in Put biosynthesis nor by a decrease in Put catabolism, but resulted mainly from the decrease in the biosynthesis of Spd and Spm. Put seemed to play an important role in cell proliferation in Scots pine embryogenic cells, but the low pH of the culture medium could also, at least partially, be the reason for the accumulation of endogenous Put. High Spd concentrations at the initiation of the culture, when cells were exposed to stress and cell death, suggested that Spd may act not only as a protector against stress but also as a growth suppressor, when proliferative growth is not promoted. All in all, Scots pine embryogenic cell culture was proved to be a favourable experimental platform to study PA metabolism and, furthermore, the developed system may also be beneficial in experiments where, e.g., the effect of specific stressors on PA metabolism is addressed.
Contrary to previous views, our results demonstrate that SPMS activity is not a novel feature developed solely in the angiosperm lineage of seed plants but also exists as a secondary property in the Scots pine SPDS enzyme. The discovery of bifunctional SPDS from an evolutionarily old conifer reveals the missing link in the evolution of the polyamine biosynthesis pathway. The finding emphasizes the importance of pre-existing secondary functions in the evolution of new enzyme activities via gene duplication. Our results also associate PsACL5 with the development of vascular structures in Scots pine.
Polyamines (PAs) are ubiquitous polycations involved in many physiological processes in plants, including somatic embryogenesis, cellular growth and stress reactions. In the present study, we focus on the consequences in PA metabolism caused by polyethylene glycol (PEG) in proembryogenic Scots pine (Pinus sylvestris L.) liquid cultures. The growth and viability of the cell masses and changes in PA concentrations and phenolic secondary metabolites were investigated under control, 5 and 10 % PEG treatments. The effect of osmotic stress responses was investigated at the gene expression level including stress, cell division, programmed cell death and PA-related genes and PA metabolites. Moreover, the expression of ethylene and proline biosynthesis genes and phenylalanine ammonia lyase and stilbene synthase (psSTS) was analyzed. Under osmotic stress conditions, we found a consistent pattern of endogenous PAs in Scots pine proembryogenic cells. However, accumulation of free spermine (Spm) and methyl putrescine under osmotic stress might indicate their specific role in stress protection. Expression of polyamine oxidase was down-regulated under osmotic stress, suggesting the role of PA catabolism in regulation of Spm levels. Scots pine proliferating proembryogenic cells are in a developmentally undifferentiated stage where the content of secondary metabolites is generally low. However, in the present study the total content of phenolic compounds increased but the biosynthesis of phenylpropanoids and stilbenes, generally considered as stress-protective molecules, was not affected by osmotic stress.
Unlike in flowering plants, the detailed roles of the enzymes in the polyamine (PA) pathway in conifers are poorly known. We explored the sequence conservation of the PA biosynthetic genes and diamine oxidase (DAO) in conifers and flowering plants to reveal the potential functional diversification of the enzymes between the plant lineages. The expression of the genes showing different selective constraints was studied in Scots pine zygotic embryogenesis and early seedling development. We found that the arginine decarboxylase pathway is strongly preferred in putrescine production in the Scots pine as well as generally in conifers and that the reduced use of ornithine decarboxylase (ODC) has led to relaxed purifying selection in ODC genes. Thermospermine synthase (ACL5) genes evolve under strong purifying selection in conifers and the DAO gene is also highly conserved in pines. In developing Scots pine seeds, the expression of both ACL5 and DAO increased as embryogenesis proceeded. Strong ACL5 expression was present in the procambial cells of the embryo and in the megagametophyte cells destined to die via morphologically necrotic cell death. Thus, the high sequence conservation of ACL5 genes in conifers may indicate the necessity of ACL5 for both embryogenesis and vascular development. Moreover, the result suggests the involvement of ACL5 in morphologically necrotic cell death and supports the view of the genetic regulation of necrosis in Scots pine embryogenesis and in plant development. DAO transcripts were located close to the cell walls and between the walls of adjacent cells in Scots pine zygotic embryos and in the roots of young seedlings. We propose that DAO, in addition to the role in Put oxidation for providing H 2 O 2 during the cell-wall structural processes, may also participate in cell-to-cell communication at the mRNA level. To conclude, our findings indicate that the PA pathway of Scots pines possesses several special functional characteristics which differ from those of flowering plants.
Endophytes are microorganisms living inside plant hosts and are known to be beneficial for the host plant vitality. In this study, we isolated three endophytic fungus species from the roots of Scots pine seedlings growing on Finnish drained peatland setting. The isolated fungi belonged to dark septate endophytes (DSE). The metabolic profiles of the hot water extracts of the fungi were investigated using Ultrahigh Performance Liquid Chromatography with Diode Array Detection and Electron Spray Ionization source Mass Spectrometry with Orbitrap analyzer (UPLC–DAD–ESI–MS–Orbitrap). Out of 318 metabolites, we were able to identify 220, of which a majority was amino acids and peptides. Additionally, opine amino acids, amino acid quinones, Amadori compounds, cholines, nucleobases, nucleosides, nucleotides, siderophores, sugars, sugar alcohols and disaccharides were found, as well as other previously reported metabolites from plants or endophytes. Some differences of the metabolic profiles, regarding the amount and identity of the found metabolites, were observed even though the fungi were isolated from the same host. Many of the discovered metabolites have been described possessing biological activities and properties, which may make a favorable contribution to the host plant nutrient availability or abiotic and biotic stress tolerance.
Bryophytes, including Sphagnum, are common species in alpine and boreal regions especially on mires, where full sunlight exposes the plants to the damaging effects of UV radiation. Sphagnum species containing UV-protecting compounds might offer a biomass source for nature-based sunscreens to replace the synthetic ones. In this study, potential compounds and those linked in cell wall structures were obtained by using methanol and alkali extractions and the UV absorption of these extracts from three common Sphagnum moss species Sphagnum magellanicum, Sphagnum fuscum and Sphagnum fallax collected in spring and autumn from western Finland are described. Absorption spectrum screening (200–900 nm) and luminescent biosensor (Escherichia coli DPD2794) methodology were used to examine and compare the protection against UV radiation. Additionally, the antioxidant potential was evaluated using hydrogen peroxide scavenging (SCAV), oxygen radical absorbance capacity (ORAC) and ferric reducing absorbance capacity (FRAP). Total phenolic content was also determined using the Folin-Ciocalteu method. The results showed that methanol extractable compounds gave higher UV absorption with the used methods. Sphagnum fallax appeared to give the highest absorption in UV-B and UV-A wavelengths. In all assays except the SCAV test, the methanol extracts of Sphagnum samples collected in autumn indicated the highest antioxidant capacity and polyphenol content. Sphagnum fuscum implied the highest antioxidant capacity and phenolic content. There was low antioxidant and UV absorption provided by the alkali extracts of these three species.
Wood as a construction material affects indoor environmental quality by moisture buffering, good acoustic properties and by the wood specific volatile organic compounds (VOC). The most abundant VOCs of soft wood are volatile monoterpenes (VM) giving the typical odor of wood. In the present study, long-term VM emissions of wood of two northern coniferous tree species, Scots pine and Norway spruce were observed regularly during one year in storage. Fresh and dried 20 cm long wood blocks were placed in a test chamber; VMs were collected with the solid phase micro extraction fiber and analyzed by gas chromatography-mass spectrometer. Average emission of nine different VMs, α- and β-pinene, 3-carene, limonene, terpinolene, myrcene, camphene, ortho-cymene and ƴ-terpinene, decreased 68–87% during one-year long storage. Moisture content (MC), knots, tree species and tree individual affected the VM emissions from wood. When a certain level of MC (12%) was reached, the VM levels decreased, but the method of drying (industrial or dried in storage) did not affect the amount of released VMs. Rehydration and dehydration increased and decreased VM emissions, respectively. Moreover, two of the eight Scots pine heartwood planks contained almost no 3-carene while being the most abundant monoterpene in the others.
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