Anne Jokela

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.

The structure and hardening status of Scots pine needles at different potassium availability levels

Abstract Scots pine (Pinus sylvestris L.) seedlings were exposed to three levels of potassium (low, medium and high) and their needle morphology, the cellular structure of the mesophyll and transfusion parenchyma, and the hardening status of the mesophyll cells were examined by light and transmission electron microscopy. The higher the potassium level the greater was the growth of the needles. The area of the mesophyll tissue increased slightly and those of the phloem, xylem and resin ducts decreased in the needles of the seedlings grown at the high K level. Cellular studies revealed that swelling of the chloroplast thylakoids, accumulation of starch in the chloroplasts, translucency of the cytoplasm and plasmolysis in the mesophyll cells were related to a low K level. The hardening status of the mesophyll cells was enhanced after 5 weeks of hardening treatment at high K as seen in changes in chloroplast shape and position and the structure of the endoplasmic reticulum, but the pines showed no major differences in the hardening status of their mesophyll cells between K levels at the end of the experiment, after 9 weeks of hardening. Frost resistance, as shown by the electrolyte leakage test, was nevertheless highest at low K, being related to the increase in the concentration of polyamine putrescine at this potassium level.

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.

Pine embryogenesis: many licences to kill for a new life.

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.

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.

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.

Effects of root damage on the nutritional status and structure of Scots pine needles

Summary In this experiment root damage was induced artificially in 35-year-old Scots pine trees growing on a nutrient-poor pine heath in northern Finland by cutting their surface roots at the beginning of the growing season. Nutrient concentrations in the needles were measured during the next growing season and the development of changes in the inner structure of the needles was observed. The needles were examined by light microscopy and at the ultrastructural level to reveal early symptoms of possible nutrient imbalances. Needle nutrient concentrations were lowered after root cutting, and changes in needle microscopic structure caused by nutrient imbalance were detected. Root reduction induced shorter needle length growth, and morphometric measurements with an image analyzer showed that the needle cross-sectional area, areas of various tissues and the sizes of certain cell types were reduced. Injury to the phloem tissue in the vascular bundle was indicative of a nutritional imbalance in the needles. The injuries to the mitochondrial structure and abnormally arranged chloroplast thylakoids at ultrastructural level pointed to P deficiency and the translucency of the chloroplast stroma to N deficiency. Vacuolization of the cytoplasm was induced, possibly indicating cold damage.

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

BackgroundThe 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.ResultsWe 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.ConclusionsOur 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.

Dealing with the problem of non-specific in situ mRNA hybridization signals associated with plant tissues undergoing programmed cell death

BackgroundIn 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.ResultsIn 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.ConclusionsIn 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.

Ozone-induced free polyamine response in Scots pine in northern Finland

The study aimed to assess the ozone-induced response in Scots pine (Pinus sylvestris L.) needles by measuring free polyamine concentrations. An open-top chamber experiment with realistically elevated ozone concentrations was carried out in northern Finland. A carry-over effect was detected: the concentrations of free polyamines, especially putrescine, were decreased at the beginning of the next growing season in the ozone-exposed trees. This indicates that the free polyamine pathway was not activated by ozone stress in Scots pines in northern conditions.