Natural variation of DNA methylation and gene expression may determine local adaptations of Scots pine populations.

Long-lived conifers are vulnerable to climate change because classical evolutionary processes are slow in developing adaptive responses. Therefore, the capacity of a genotype to adopt different phenotypes is important. Gene expression is the primary mechanism that converts genome-encoded information into phenotypes, and DNA methylation is employed in the epigenetic regulation of gene expression. We investigated variations in global DNA methylation and gene expression between three Scots pine (Pinus sylvestris L.) populations located in northern and southern Finland using mature seeds. Gene expression levels were studied in six DNA methyltransferase (DNMT) genes, which were characterized in this study, and in 19 circadian clock genes regulating adaptive traits. In embryos, expression diversity was found for three DNMT genes, which maintain DNA methylation. The expression of two DNMT genes was strongly correlated with climate variables, which suggests a role for DNA methylation in local adaptation. For adaptation-related genes, expression levels showed between-population variation in 11 genes in megagametophytes and in eight genes in embryos, and many of these genes were linked to climate factors. Altogether, our results suggest that differential DNA methylation and gene expression contribute to local adaptation in Scots pine populations and may enhance the fitness of trees under rapidly changing climatic conditions.

Scots pine aminopropyltransferases shed new light on evolution of the polyamine biosynthesis pathway in seed plants.

Background and Aims: Polyamines are small metabolites present in all living cells and play fundamental roles in numerous physiological events in plants. The aminopropyltransferases (APTs), spermidine synthase (SPDS), spermine synthase (SPMS) and thermospermine synthase (ACL5), are essential enzymes in the polyamine biosynthesis pathway. In angiosperms, SPMS has evolved from SPDS via gene duplication, whereas in gymnosperms APTs are mostly unexplored and no SPMS gene has been reported. The present study aimed to investigate the functional properties of the SPDS and ACL5 proteins of Scots pine (Pinus sylvestris L.) in order to elucidate the role and evolution of APTs in higher plants. Methods: Germinating Scots pine seeds and seedlings were analysed for polyamines by high-performance liquid chromatography (HPLC) and the expression of PsSPDS and PsACL5 genes by in situ hybridization. Recombinant proteins of PsSPDS and PsACL5 were produced and investigated for functional properties. Also gene structures, promoter regions and phylogenetic relationships of PsSPDS and PsACL5 genes were analysed. Key Results: Scots pine tissues were found to contain spermidine, spermine and thermospermine. PsSPDS enzyme catalysed synthesis of both spermidine and spermine. PsACL5 was found to produce thermospermine, and PsACL5 gene expression was localized in the developing procambium in embryos and tracheary elements in seedlings. Conclusions: 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.

Expression of catalase and retinoblastoma-related protein genes associates with cell death processes in Scots pine zygotic embryogenesis.

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.

Targeted bisulfite sequencing of Scots pine adaptation-related genes.

During environmental changes, epigenetic processes can enable adaptive responses faster than natural selection. In plants, very little is known about the role of DNA methylation during long-term adaptation. Scots pine is a widely distributed coniferous species which must adapt to different environmental conditions throughout its long lifespan. Thus, epigenetic modifications may contribute towards this direction. We provide bisulfite next-generation sequencing data from the putative promoters and exons of eight adaptation-related genes (A3IP2, CCA1, COL1, COL2, FTL2, MFT1, PHYO, and ZTL) in three Scots pine populations located in northern and southern parts of Finland. DNA methylation levels were studied in the two seed tissues: the maternal megagametophyte which contributes to embryo viability, and the biparental embryo which represents the next generation. In most genes, differentially methylated cytosines (DMCs) were in line with our previously demonstrated gene expression differences found in the same Scots pine populations. In addition, we found a strong correlation of total methylation levels between the embryo and megagametophyte tissues of a given individual tree, which indicates that DNA methylation can be inherited from the maternal parent. In conclusion, our results imply that DNA methylation differences may contribute to the adaptation of Scots pine populations in different climatic conditions.

Changing winter climate and snow conditions induce various transcriptional stress responses in Scots pine seedlings.

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.

Atlas of tissue-specific and tissue-preferential gene expression in ecologically and economically significant conifer Pinus sylvestris.

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.

Polyamine Metabolism in Scots Pine Embryogenic Cells under Potassium Deficiency.

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.

Microscopical Detection of Cell Death Processes During Scots Pine Zygotic Embryogenesis.

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.

Genomics of Clinal Local Adaptation in Pinus sylvestris Under Continuous Environmental and Spatial Genetic Setting.

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.

Phenolic compounds in ectomycorrhizal interaction of lignin modified silver birch.

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.

One tissue, two fates: different roles of megagametophyte cells during Scots pine embryogenesis.

In the Scots pine (Pinus sylvestris L.) seed, embryos grow and develop within the corrosion cavity of the megagametophyte, a maternally derived haploid tissue, which houses the majority of the storage reserves of the seed. In the present study, histochemical methods and quantification of the expression levels of the programmed cell death (PCD) and DNA repair processes related genes (MCA, TAT-D, RAD51, KU80, and LIG) were used to investigate the physiological events occurring in the megagametophyte tissue during embryo development. It was found that the megagametophyte was viable from the early phases of embryo development until the early germination of mature seeds. However, the megagametophyte cells in the narrow embryo surrounding region (ESR) were destroyed by cell death with morphologically necrotic features. Their cell wall, plasma membrane, and nuclear envelope broke down with the release of cell debris and nucleic acids into the corrosion cavity. The occurrence of necrotic-like cell death in gymnosperm embryogenesis provides a favourable model for the study of developmental cell death with necrotic-like morphology and suggests that the mechanism underlying necrotic cell death is evolutionary conserved.

Thermospermine Synthase (ACL5) and Diamine Oxidase (DAO) Expression Is Needed for Zygotic Embryogenesis and Vascular Development in Scots Pine.

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.

Recognition of candidate transcription factors related to bilberry fruit ripening by de novo transcriptome and qRT-PCR analyses.

Bilberry (Vaccinium myrtillus L.) fruits are an excellent natural resource for human diet because of their special flavor, taste and nutritional value as well as medical properties. Bilberries are recognized for their high anthocyanin content and many of the genes involved in the anthocyanin biosynthesis have been characterized. So far, neither genomic nor RNA-seq data have been available for the species. In the present study, we de novo sequenced two bilberry fruit developmental stages, unripe green (G) and ripening (R). A total of 57,919 unigenes were assembled of which 80.2% were annotated against six public protein databases. The transcriptome served as exploratory data to identify putative transcription factors related to fruit ripening. Differentially expressed genes (DEGs) between G and R stages were prominently upregulated in R stage with the functional annotation indicating their main roles in active metabolism and catalysis. The unigenes encoding putative ripening-related regulatory genes, including members of NAC, WRKY, LOB, ERF, ARF and ABI families, were analysed by qRT-PCR at five bilberry developmental stages. Our de novo transcriptome database contributes to the understanding of the regulatory network associated with the fruit ripening in bilberry and provides the first dataset for wild Vaccinium species acquired by NGS technology.

Bacterial and fungal communities in boreal forest soil are insensitive to changes in snow cover conditions.

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.

The Snow Must Go On: Ground Ice Encasement, Snow Compaction and Absence of Snow Differently Cause Soil Hypoxia, CO2 Accumulation and Tree Seedling Damage in Boreal Forest.

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.

Moderate stress responses and specific changes in polyamine metabolism characterize Scots pine somatic embryogenesis.

Somatic embryogenesis (SE) is one of the methods with the highest potential for the vegetative propagation of commercially important coniferous species. However, many conifers, including Scots pine (Pinus sylvestris L.), are recalcitrant to SE and a better understanding of the mechanisms behind the SE process is needed. In Scots pine SE cultures, embryo production is commonly induced by the removal of auxin, addition of abscisic acid (ABA) and the desiccation of cell masses by polyethylene glycol (PEG). In the present study, we focus on the possible link between the induction of somatic embryo formation and cellular stress responses such as hydrogen peroxide protection, DNA repair, changes in polyamine (PA) metabolism and autophagy. Cellular PA contents and the expression of the PA metabolism genes arginine decarboxylase (ADC), spermidine synthase (SPDS), thermospermine synthase (ACL5) and diamine oxidase (DAO) were analyzed, as well as the expression of catalase (CAT), DNA repair genes (RAD51, KU80) and autophagy-related genes (ATG5, ATG8) throughout the induction of somatic embryo formation in Scots pine SE cultures. Among the embryo-producing SE lines, the expression of ADC, SPDS, ACL5, DAO, CAT, RAD51, KU80 and ATG8 showed consistent profiles. Furthermore, the overall low expression of the stress-related genes suggests that cells in those SE lines were not stressed but recognized the ABA+PEG treatment as a signal to trigger the embryogenic pathway. In those SE lines that were unable to produce embryos, cells seemed to experience the ABA+PEG treatment mostly as osmotic stress and activated a wide range of stress defense mechanisms. Altogether, our results suggest that the direction to the embryogenic pathway is connected with cellular stress responses in Scots pine SE cultures. Thus, the manipulation of stress response pathways may provide a way to enhance somatic embryo production in recalcitrant Scots pine SE lines.

Water availability influences morphology, mycorrhizal associations, PSII efficiency and polyamine metabolism at early growth phase of Scots pine seedlings.

Scots pine (Pinus sylvestris L.) is adapted to various soil types with diverse water availabilities. However, Scots pine seedlings are vulnerable to abiotic stress during the early growth, when they may be exposed to both dry and wet conditions. Here, we focused on the above and below ground coping strategies of Scots pine seedlings under controlled wet, optimal and dry soil conditions by investigating morphological traits including seedling biomass, number of root tips, proportion of mycorrhizal root tips and brown needles. In addition, we studied metabolic and physiological responses including gene expression involved in biosynthesis and catabolism of polyamines (PA), PSII efficiency and the expression of the catalase (CAT) late-embryogenesis abundant protein (LEA), pyruvate decarboxylase (PDC), glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) genes. We found that seedlings invested in shoots by maintaining stable shoot water content and high PSII efficiency under drought stress. Free and soluble conjugated putrescine (Put) accumulated in needles under drought stress, suggesting the role of Put in protection of photosynthesizing tissues. However, the expression of the PA biosynthesis genes, arginine decarboxylase (ADC), spermidine synthase (SPDS) and thermospermine synthase (ACL5) was not affected under drought stress whereas catabolizing genes diamino oxidase (DAO) and polyamine oxidase (PAO) were down-regulated in shoots. The morphology of the roots was affected by peat water content. Furthermore, both drought stress and water excess restricted the seedling ability to sustain a symbiotic relationship. The consistent pattern of endogenous PAs seems to be advantageous to the Scots pine seedlings also under stress conditions.

Characterization and expression of the human WNT4; lack of associated germline mutations in high--to moderate--risk breast and ovarian cancer.

Wnt4 is needed for correct development of several tissues in the mouse, and WNT4 is found here to be expressed in a temporal manner in human embryonic tissues. In addition, WNT4 mRNA is seen in several adult tissues. The 1.5 kb transcript is dominant in adult tissues, whereas the 2.4 kb transcript is the major one in embryonic tissues. The involvement of WNT4 in normal mammary gland and ovary development suggests that WNT4 germline mutations may be associated with the human cancer predisposition. Their absence in cancer families, however, implies lack of involvement of WNT4 mutations in the etiology of hereditary susceptibility to breast and ovarian cancer. Finally, the chromosomal location of WNT4 is narrowed to 1p36.12.

Does extraction of DNA and RNA by magnetic fishing work for diverse plant species?

An automated nucleic acid extraction procedure with magnetic particles originally designed for isolation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from animal tissues was tested for plant material. We isolated genomic DNA and total RNA from taxonomically diverse plant species representing conifers (Scots pine), broad-leaved trees (silver birch and hybrid aspen), dwarf shrubs (bilberry), and both monocotyledonous (regal lily) and dicotyledonous (Saint John's wort, round-leaved sundew, and tobacco) herbaceous plants. Buffers developed for DNA extraction were successfully used in addition to manufacturer's extraction kits. The quality of RNA was appropriate for many applications, but the quality of DNA was not always sufficient for polymerase chain reaction (PCR) amplification. However, we could strikingly improve the quality by eliminating the adherent compounds during the extraction or later in the PCR phase. Our results show that the use of the procedure could be extended to diverse plant species. This procedure is especially suitable for small sample sizes and for simultaneous processing of many samples enabling large-scale plant applications in population genetics, or in the screening of putative transgenic plants.

Evolution, three-dimensional model and localization of truncated hemoglobin PttTrHb of hybrid aspen.

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.