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.

Transcript profiling for early stages during embryo development in Scots pine.

BACKGROUND: Characterization of the expression and function of genes regulating embryo development in conifers is interesting from an evolutionary point of view. However, our knowledge about the regulation of embryo development in conifers is limited. During early embryo development in Pinus species the proembyo goes through a cleavage process, named cleavage polyembryony, giving rise to four embryos. One of these embryos develops to a dominant embryo, which will develop further into a mature, cotyledonary embryo, while the other embryos, the subordinate embryos, are degraded. The main goal of this study has been to identify processes that might be important for regulating the cleavage process and for the development of a dominant embryo. RESULTS: RNA samples from embryos and megagametophytes at four early developmental stages during seed development in Pinus sylvestris were subjected to high-throughput sequencing. A total of 6.6 million raw reads was generated, resulting in 121,938 transcripts, out of which 36.106 contained ORFs. 18,638 transcripts were differentially expressed (DETs) in embryos and megagametophytes. GO enrichment analysis of transcripts up-regulated in embryos showed enrichment for different cellular processes, while those up-regulated in megagametophytes were enriched for accumulation of storage material and responses to stress. The highest number of DETs was detected during the initiation of the cleavage process. Transcripts related to embryogenic competence, cell wall modifications, cell division pattern, axis specification and response to hormones and stress were highly abundant and differentially expressed during early embryo development. The abundance of representative DETs was confirmed by qRT-PCR analyses. CONCLUSION: Based on the processes identified in the GO enrichment analyses and the expression of the selected transcripts we suggest that (i) processes related to embryogenic competence and cell wall loosening are involved in activating the cleavage process; (ii) apical-basal polarization is strictly regulated in dominant embryos but not in the subordinate embryos; (iii) the transition from the morphogenic phase to the maturation phase is not completed in subordinate embryos. This is the first genome-wide transcript expression profiling of the earliest stages during embryo development in a Pinus species. Our results can serve as a framework for future studies to reveal the functions of identified genes.

Developmental Changes in Scots Pine Transcriptome during Heartwood Formation.

Scots pine (Pinus sylvestris L.) wood is desired in woodworking industries due to its favorable timber characteristics and natural durability that is contributed by heartwood extractives. It has been discussed whether the Scots pine heartwood extractives (mainly stilbenes and resin acids) are synthesized in the cells of the transition zone between sapwood and heartwood, or if they are transported from the sapwood. Timing of heartwood formation during the yearly cycle has also not been unambiguously defined. We measured steady-state mRNA levels in Scots pine transition zone and sapwood using RNA sequencing. Year-round expression profiles of selected transcripts were further investigated by quantitative RT-PCR. Differentially accumulating transcripts suggest that, of the Scots pine heartwood extractives, stilbenes are synthesized in situ in the transition zone and gain their carbon-skeletons from Suc and triglycerides. Resin acids, on the other hand, are synthesized early in the spring mainly in the sapwood, meaning that they must be transported to the heartwood transition zone. Heartwood formation is marked by programmed cell death that occurs during the summer months in the transition zone.

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.

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.

Variation among matsutake ectomycorrhizae in four clones of Pinus sylvestris.

Tricholoma matsutake is an ectomycorrhizal fungus that forms commercially important mushrooms in coniferous forests. In this study, we explored the ability of T. matsutake to form mycorrhizae with Pinus sylvestris by inoculating emblings produced through somatic embryogenesis (SE) in an aseptic culture system. Two months after inoculation, clones with less phenolic compounds in the tissue culture phase formed mycorrhizae with T. matsutake, while clones containing more phenols did not. Effects of inoculation on embling growth varied among clones; two of the four tested showed a significant increase in biomass and two had a significant increase in root density. In addition, results suggest that clones forming well-developed mycorrhizae absorbed more Al, Fe, Na, P, and Zn after 8 weeks of inoculation. This study illustrates the value of SE materials in experimental work concerning T. matsutake as well as the role played by phenolic compounds in host plant response to infection by mycorrhizal fungi.

Fertility variation and status number in clonal seed orchards of Pinus sylvestris.

The present study was carried out to evaluate fertility variation, status number and gene diversity based on strobili productions in two clonal seed orchards of Scots pine (Pinus sylvestris L.). There were large differences among clones for the female and male strobili productions in the orchards. Positive and significant (p< or =0.05) correlations were found between female and male strobili production (r = 0.76, 0.55). Female fertility variation (1.03, 1.07) was larger than male fertility variation (1.02, 1.03) in the orchards. The status numbers estimated based on the total fertility were very high (97 and 98% of census numbers). The large fertility variation could be balanced by different treatments such as mixing seed equally from clones or genetinc thinning.

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.

Developmental and genetic variation in nuclear microsatellite stability during somatic embryogenesis in pine.

Genotypic instability is commonly observed in plants derived from tissue culture and is at least partly due to in vitro-induced stress. In this work, the issues of whether genetic instability induced by in vitro stress varies among families and if genetic instability influences the adaptation to in vitro conditions and embryo development have been addressed. By comparing the stability of four variable nuclear microsatellite loci in embryogenic cultures and zygotic embryos of Pinus sylvestris, a significant difference in genetic stability among families was found. In six out of 10 families analysed, the level of genetic stability was similar between somatic and zygotic embryos. However, for the rest of the families, the mutation rate was significantly higher during somatic embryogenesis. Families showing a low genetic stability during establishment of embryogenic cultures had a higher embryogenic potential than those which were genetically more stable. In contrast, embryo development was suppressed in genetically unstable families. The relatively high mutation rates found for some families might reflect the plasticity of the families to adapt to stress, which is important for widely distributed species such as Pinus sylvestris.

Spermidine and the ectomycorrhizal fungus Pisolithus tinctorius synergistically induce maturation of Scots pine embryogenic cultures.

Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi.

Consistency of polyamine profiles and expression of arginine decarboxylase in mitosis during zygotic embryogenesis of Scots pine.

In this study, we show that both arginine decarboxylase (ADC) protein and mRNA transcript are present at different phases of mitosis in Scots pine (Pinus sylvestris) zygotic embryogenesis. We also examined the consistency of polyamine (PA) profiles with the effective temperature sum, the latter indicating the developmental stage of the embryos. PA metabolism was analyzed by fitting statistical regression models to the data of free and soluble conjugated PAs, to the enzyme activities of ADC and ornithine decarboxylase (ODC), as well as to the gene expression of ADC. According to the fitted models, PAs typically had the tendency to increase at the early stages but decrease at the late stages of embryogenesis. Only the free putrescine fraction remained stable during embryo development. The PA biosynthesis strongly preferred the ADC pathway. Both ADC gene expression and ADC enzyme activity were substantially higher than putative ODC gene expression or ODC enzyme activity, respectively. ADC gene expression and enzyme activity increased during embryogenesis, which suggests the involvement of transcriptional regulation in the expression of ADC. Both ADC mRNA and ADC protein localized in dividing cells of embryo meristems and more specifically within the mitotic spindle apparatus and close to the chromosomes, respectively. The results suggest the essential role of ADC in the mitosis of plant cells.

[Patterns of protein biosynthesis in the embryo and endosperm during embryogenesis in Pinus sylvestris L]. / Zakonomernosti biosinteza belkov zarodysha i éndosperma v protsesse émbriogeneza u Pinus sylvestris L.

The patterns of protein biosynthesis in the embryo and endosperm during embryogenesis in the Scots pine were studied using electrophoresis and biochemistry methods. Proteins of the albumin-globulin fraction were visualized already at the early embryonic stages. The main polypeptide components (48-60, 37-39, and 20-22 kDa) were gradually accumulated in the course of maturation. A high synchrony was noted between the stages of embryogenesis and molecular events related to protein biosynthesis and accumulation in the developing seed.