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In northern boreal forests the warming winter climate leads to more frequent snowmelt, rain-on-snow events and freeze-thaw cycles. This may be harmful or even lethal for tree seedlings that spend even a half of the year under snow. We conducted a snow cover manipulation experiment in a natural forest to find out how changing snow conditions affect young Scots pine (Pinus sylvestris L.) seedlings. The ice encasement (IE), absence of snow (NoSNOW) and snow compaction (COMP) treatments affected ground level temperature, ground frost and subnivean gas concentrations compared to the ambient snow cover (AMB) and led to the increased physical damage and mortality of seedlings. The expression responses of 28 genes related to circadian clock, aerobic and anaerobic energy metabolism, carbohydrate metabolism and stress protection revealed that seedlings were exposed to different stresses in a complex way depending on the thickness and quality of the snow cover. The IE treatment caused hypoxic stress and probably affected roots which resulted in reduced water uptake in the beginning of the growing season. Without protective snowpack in NoSNOW seedlings suffered from cold and drought stresses. The combination of hypoxic and cold stresses in COMP evoked unique transcriptional responses including oxidative stress. Snow cover manipulation induced changes in the expression of several circadian clock related genes suggested that photoreceptors and the circadian clock system play an essential role in the adaptation of Scots pine seedlings to stresses under different snow conditions. Our findings show that warming winter climate alters snow conditions and consequently causes Scots pine seedlings various abiotic stresses, whose effects extend from overwintering to the following growing season. [ABSTRACT FROM AUTHOR]

Despite their ecological and economical importance, conifers genomic resources are limited, mainly due to the large size and complexity of their genomes. Additionally, the available genomic resources lack complete structural and functional annotation. Transcriptomic resources have been commonly used to compensate for these deficiencies, though for most conifer species they are limited to a small number of tissues, or capture only a fraction of the genes present in the genome. Here we provide an atlas of gene expression patterns for conifer Pinus sylvestris across five tissues: embryo, megagametophyte, needle, phloem and vegetative bud. We used a wide range of tissues and focused our analyses on the expression profiles of genes at tissue level. We provide comprehensive information of the per-tissue normalized expression level, indication of tissue preferential upregulation and tissue-specificity of expression. We identified a total of 48,001 tissue preferentially upregulated and tissue specifically expressed genes, of which 28% have annotation in the Swiss-Prot database. Even though most of the putative genes identified do not have functional information in current biological databases, the tissue-specific patterns discovered provide valuable information about their potential functions for further studies, as for example in the areas of plant physiology, population genetics and genomics in general. As we provide information on tissue specificity at both diploid and haploid life stages, our data will also contribute to the understanding of evolutionary rates of different tissue types and ploidy levels. [ABSTRACT FROM AUTHOR]

Abstract Background In situ hybridization is a general molecular method typically used for the localization of mRNA transcripts in plants. The method provides a valuable tool to unravel the connection between gene expression and anatomy, especially in species such as pines which show large genome size and shortage of sequence information. Results In the present study, expression of the catalase gene (CAT) related to the scavenging of reactive oxygen species (ROS) and the polyamine metabolism related genes, diamine oxidase (DAO) and arginine decarboxylase (ADC), were localized in developing Scots pine (Pinus sylvestris L.) seeds. In addition to specific signals from target mRNAs, the probes continually hybridized non-specifically in the embryo surrounding region (ESR) of the megagametophyte tissue, in the remnants of the degenerated suspensors as well as in the cells of the nucellar layers, i.e. tissues exposed to cell death processes and extensive nucleic acid fragmentation during Scots pine seed development. Conclusions In plants, cell death is an integral part of both development and defence, and hence it is a common phenomenon in all stages of the life cycle. Our results suggest that extensive nucleic acid fragmentation during cell death processes can be a considerable source of non-specific signals in traditional in situ mRNA hybridization. Thus, the visualization of potential nucleic acid fragmentation simultaneously with the in situ mRNA hybridization assay may be necessary to ensure the correct interpretation of the signals in the case of non-specific hybridization of probes in plant tissues.

Abstract Background The 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. Results Silver 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. Conclusion The 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.

Understanding the consequences of local adaptation at the genomic diversity is a central goal in evolutionary genetics of natural populations. In species with large continuous geographical distributions the phenotypic signal of local adaptation is frequently clear, but the genetic basis often remains elusive. We examined the patterns of genetic diversity in Pinus sylvestris, a keystone species in many Eurasian ecosystems with a huge distribution range and decades of forestry research showing that it is locally adapted to the vast range of environmental conditions. Making P. sylvestris an even more attractive subject of local adaptation study, population structure has been shown to be weak previously and in this study. However, little is known about the molecular genetic basis of adaptation, as the massive size of gymnosperm genomes has prevented large scale genomic surveys. We generated a both geographically and genomically extensive dataset using a targeted sequencing approach. By applying divergence-based and landscape genomics methods we identified several loci contributing to local adaptation, but only few with large allele frequency changes across latitude. We also discovered a very large (ca. 300 Mbp) putative inversion potentially under selection, which to our knowledge is the first such discovery in conifers. Our results call for more detailed analysis of structural variation in relation to genomic basis of local adaptation, emphasize the lack of large effect loci contributing to local adaptation in the coding regions and thus point out the need for more attention toward multi-locus analysis of polygenic adaptation. [ABSTRACT FROM AUTHOR]

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 H2O2 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. [ABSTRACT FROM AUTHOR]

The northern regions are experiencing considerable changes in winter climate leading to more frequent warm periods, rain-on-snow events and reduced snow pack diminishing the insulation properties of snow cover and increasing soil frost and freeze-thaw cycles. In this study, we investigated how the lack of snow cover, formation of ice encasement and snow compaction affect the size, structure and activities of soil bacterial and fungal communities. Contrary to our hypotheses, snow manipulation treatments over one winter had limited influence on microbial community structure, bacterial or fungal copy numbers or enzyme activities. However, microbial community structure and activities shifted seasonally among soils sampled before snow melt, in early and late growing season and seemed driven by substrate availability. Bacterial and fungal communities were dominated by stress-resistant taxa such as the orders Acidobacteriales, Chaetothyriales and Helotiales that are likely adapted to adverse winter conditions. This study indicated that microbial communities in acidic northern boreal forest soil may be insensitive to direct effects of changing snow cover. However, in long term, the detrimental effects of increased ice and frost to plant roots may alter plant derived carbon and nutrient pools to the soil likely leading to stronger microbial responses. Snow-manipulation experiment indicated that bacterial and fungal communities in northern boreal forest soil are dominated by stress-resistant taxa that are insensitive to changes in snow cover conditions. [ABSTRACT FROM AUTHOR]

At high latitudes, the climate has warmed at twice the rate of the global average with most changes observed in autumn, winter and spring. Increasing winter temperatures and wide temperature fluctuations are leading to more frequent rain-on-snow events and freeze-thaw cycles causing snow compaction and formation of ice layers in the snowpack, thus creating ice encasement (IE). By decreasing the snowpack insulation capacity and restricting soil-atmosphere gas exchange, modification of the snow properties may lead to colder soil but also to hypoxia and accumulation of trace gases in the subnivean environment. To test the effects of these overwintering conditions changes on plant winter survival and growth, we established a snow manipulation experiment in a coniferous forest in Northern Finland with Norway spruce and Scots pine seedlings. In addition to ambient conditions and prevention of IE, we applied three snow manipulation levels: IE created by artificial rain-on-snow events, snow compaction and complete snow removal. Snow removal led to deeper soil frost during winter, but no clear effect of IE or snow compaction done in early winter was observed on soil temperature. Hypoxia and accumulation of CO2 were highest in the IE plots but, more importantly, the duration of CO2 concentration above 5% was 17 days in IE plots compared to 0 days in ambient plots. IE was the most damaging winter condition for both species, decreasing the proportion of healthy seedlings by 47% for spruce and 76% for pine compared to ambient conditions. Seedlings in all three treatments tended to grow less than seedlings in ambient conditions but only IE had a significant effect on spruce growth. Our results demonstrate a negative impact of winter climate change on boreal forest regeneration and productivity. Changing snow conditions may thus partially mitigate the positive effect of increasing growing season temperatures on boreal forest productivity. [ABSTRACT FROM AUTHOR]

Background: The cell cycle and cellular oxidative stress responses are tightly controlled for proper growth and development of Scots pine (Pinus sylvestris L.) seed. Programmed cell death (PCD) is an integral part of the embryogenesis during which megagametophyte cells in the embryo surrounding region (ESR) and cells in the nucellar layers face death. In the present study, we show both the tissue and developmental stage specific expression of the genes encoding the autophagy related ATG5, catalase (CAT), and retinoblastoma related protein (RBR) as well as the connection between the gene expressions and cell death programs. Results: We found strong CAT expression in the cells of the developing embryo throughout the embryogenesis as well as in the cells of the megagametophyte and the nucellar layers at the early embryogeny. The CAT expression was found to overlap with both the ATG5 expression and hydrogen peroxide localization. At the late embryogeny, CAT expression diminished in the dying cells of the nucellar layers as well as in megagametophyte cells, showing the first signs of incipient cell death. Accumulation of starch and minor RBR expression were characteristic of megagametophyte cells in the ESR, whereas strong RBR expression was found in the cells of the nucellar layers at the late embryogeny. Conclusions: Our results suggest that ATG5, CAT, and RBR are involved in the Scots pine embryogenesis and cell death processes. CAT seems to protect cells against hydrogen peroxide accumulation and oxidative stress related cell death especially during active metabolism. The opposite expression of RBR in the ESR and nucellar layers alongside morphological characteristics emphasizes the different type of the cell death processes in these tissues. Furthermore, the changes in ATG5 and RBR expressions specifically in the megagametophyte cells dying by necrotic cell death suggest the genetic regulation of developmental necrosis in Scots pine embryogenesis. [ABSTRACT FROM AUTHOR]

Thus far, research on plant hemoglobins (Hbs) has mainly concentrated on symbiotic and non-symbiotic Hbs, and information on truncated Hbs (TrHbs) is scarce. The aim of this study was to examine the origin, structure and localization of the truncated Hb (PttTrHb) of hybrid aspen (Populus tremula L. × tremuloides Michx.), the model system of tree biology. Additionally, we studied the PttTrHb expression in relation to non-symbiotic class1 Hb gene (PttHb1) using RNAi-silenced hybrid aspen lines. Both the phylogenetic analysis and the three-dimensional (3D) model of PttTrHb supported the view that plant TrHbs evolved vertically from a bacterial TrHb. The 3D model suggested that PttTrHb adopts a 2-on-2 sandwich of α-helices and has a Bacillus subtilis -like ligand-binding pocket in which E11Gln and B10Tyr form hydrogen bonds to a ligand. However, due to differences in tunnel cavity and gate residue (E7Ala), it might not show similar ligand-binding kinetics as in Bs-HbO (E7Thr). The immunolocalization showed that PttTrHb protein was present in roots, stems as well as leaves of in vitro -grown hybrid aspens. In mature organs, PttTrHb was predominantly found in the vascular bundles and specifically at the site of lateral root formation, overlapping consistently with areas of nitric oxide (NO) production in plants. Furthermore, the NO donor sodium nitroprusside treatment increased the amount of PttTrHb in stems. The observed PttTrHb localization suggests that PttTrHb plays a role in the NO metabolism. [ABSTRACT FROM AUTHOR]

Background: In situ hybridization is a general molecular method typically used for the localization of mRNA transcripts in plants. The method provides a valuable tool to unravel the connection between gene expression and anatomy, especially in species such as pines which show large genome size and shortage of sequence information. Results: In the present study, expression of the catalase gene (CAT) related to the scavenging of reactive oxygen species (ROS) and the polyamine metabolism related genes, diamine oxidase (DAO) and arginine decarboxylase (ADC), were localized in developing Scots pine (Pinus sylvestris L.) seeds. In addition to specific signals from target mRNAs, the probes continually hybridized non-specifically in the embryo surrounding region (ESR) of the megagametophyte tissue, in the remnants of the degenerated suspensors as well as in the cells of the nucellar layers, i.e. tissues exposed to cell death processes and extensive nucleic acid fragmentation during Scots pine seed development. Conclusions: In plants, cell death is an integral part of both development and defence, and hence it is a common phenomenon in all stages of the life cycle. Our results suggest that extensive nucleic acid fragmentation during cell death processes can be a considerable source of non-specific signals in traditional in situ mRNA hybridization. Thus, the visualization of potential nucleic acid fragmentation simultaneously with the in situ mRNA hybridization assay may be necessary to ensure the correct interpretation of the signals in the case of non-specific hybridization of probes in plant tissues. [ABSTRACT FROM AUTHOR]

Polyamines (PA) have a protective role in maintaining growth and development in Scots pine during abiotic stresses. In the present study, a controlled liquid Scots pine embryogenic cell culture was used for studying the responses of PA metabolism related to potassium deficiency. The transcription level regulation of PA metabolism led to the accumulation of putrescine (Put). Arginine decarboxylase (ADC) had an increased expression trend under potassium deficiency, whereas spermidine synthase (SPDS) expression decreased. Generally, free spermidine (Spd) and spermine (Spm)/ thermospermine (t-Spm) contents were kept relatively stable, mostly by the downregulation of polyamine oxidase (PAO) expression. The low potassium contents in the culture medium decreased the potassium content of the cells, which inhibited cell mass growth, but did not affect cell viability. The reduced growth was probably caused by repressed metabolic activity and cell division, whereas there were no signs of H2O2-induced oxidative stress or increased cell death. The low intracellular content of K+ decreased the content of Na+. The decrease in the pH of the culture medium indicated that H+ ions were pumped out of the cells. Altogether, our findings emphasize the specific role(s) of Put under potassium deficiency and strict developmental regulation of PA metabolism in Scots pine. [ABSTRACT FROM AUTHOR]

Programmed cell death (PCD) processes are essential in the plant embryogenesis. To understand how PCD operates in a developing seed, the dying cells need to be identified in relation to their surviving neighbors. This can be accomplished by the means of in situ visualization of fragmented DNA-a well-known hallmark of PCD. In the developing Scots pine (Pinus sylvestris L.) seed, several tissues die via morphologically different PCD processes during the embryogenesis. Here, we describe the protocols for the characterization of Scots pine seeds at the early and late developmental stages and, further, the localization of nucleic acids and DNA fragmentation by the acridine orange staining and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling) assay in the dying seed tissues.

In plants, programmed cell death (PCD) is an important mechanism that controls normal growth and development as well as many defence responses. At present, research on PCD in different plant species is actively carried out due to the possibilities offered by modern methods in molecular biology and the increasing amount of genome data. The pine seed provides a favourable model for PCD because it represents an interesting inheritance of seed tissues as well as an anatomically well-described embryogenesis during which several tissues die via morphologically different PCD processes.