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Morphometric procedures were used to examine peroxisome number and di-stribution in pea (Pisum sativum L.) root nodules under NaCl (50 mM) or HgCl2 (7.3 µM) treatment. Peroxisomes were visualized cytochemically in meristem, invasion zone and prefixing zone of pea root nodules by catalase (EC 1.11.1.6) activity. The observations using light and electron microscopy revealed that the peroxisomes were predominantly spherical in shape and showed catalase activity. In nitrogen fixation zone, catalase active peroxisomes were observed occasionally. Bacteroids of nitrogen fixing zone showed enhanced cata-lase activity probably as a response to higher level of oxidative stress. Fluorescence microscopy investigations revealed enhanced level of (homo)glutathione in prefixing and nitrogen-fixing zone of NaCl- and Hg2+treated nodules, which served as an indicator of antioxidative response. Morphometric measurements revealed that during differentiation of meristematic cells into central tissue (bacteroidal tissue) cells an increase in peroxisome number was observed in unstressed nodules. Peroxisomes located in meristem, invasion zone and prefixing zone of NaCl- and Hg2+-treated nodules outnumbered that in control nodules. A substantial enlargement of peroxisome profiles was detected in NaCl- and Hg2+treated nodules. Peroxisome divisions observed in meristematic and infection thread penetration zone were responsible for an increase in peroxisome number.
Vacuoles play very important physiological roles in plant cells. Pea root nodules, which exhibit distinct zonation (meristematic zone and central tissue zones), may serve as a good experimental model for the investigations of vacuole development and its importance to cell and tissue functioning. Moreover, the nodule central tissue is composed of both infected and uninfected cells which play different physiological roles and differ in the level of vacuolation. Cytological observations revealed that central vacuoles of the infected cells of the effective nodules expand toward cell walls. Thus only thin layers of the cytoplasm separate each central vacuole from plasma membrane and cell wall. This finding is discussed from the viewpoint of improved exchange of solutes and water between the central vacuole and apoplast of the infected cell. Three-dimensional reconstruction of the vacuoles of infected cells within a fragment of effective nodule central tissue, showed their spatial arrangement. Possible advantages coming from the spatial arrangement of vacuoles within the central tissue are discussed. A comparative study of the central tissue (bacteroidal tissue) and meristem vacuolation of the effective and ineffective pea root nodules is also presented. Morphometric measurements revealed that the effective nodule central tissue was more vacuolated than the ineffective one. It was proved that maturation of the infected cells involves dynamic changes in their vacuolation. Having numerous fixing nitrogen bacteroids, the infected cells of effective central tissue were less vacuolated than uninfected cells. On the other hand, both infected and uninfected cells of the effective central tissue showed a much higher level of vacuolation in nitrogen-fixing zone than cells of the same type in ineffective tissue. These results indicate that vacuolation is an important factor in development and functioning of pea root nodule central tissue.
Pea (Pisum sativum L.) plants inoculated with Rhizobium leguminosarum bv. viciae effective strain 248 were irrigated with nitrogen-free medium supplemented with 0, 25, 50 or 75 mM NaCl. The inhibitory effect of salinity on the growth of pea plants treated with 25 mM NaCl occurred 6 weeks post inoculation. In case of 75 mM NaCl treatment, the same effect was observed 2 weeks post inoculation. In contrast to investigations described in the literature our results clearly indicated that 25 mM NaCl stimulated nodule formation, however, in contrast to control nodules (the medium without NaCl), the nodules were considerably smaller. Remaining variants of salt treatment reduced plant growth, nodulation, and total nodule volume calculated per plant. Microscopic observations showed that salinity: (1) caused the loss of turgor of the nodule peripheral cells, (2) changed nodule zonation, (3) stimulated infection thread enlargement and expansion, (4) caused disturbances in bacterial release from the infection threads, and (5) induced synthesis of electron dense material (EDM) and its deposition in vacuoles. It was also found that cisternae of RER were involved in the formation of special cytoplasmic compartments responsible for synthesis of EDM. Autofluorescence study revealed that salinity increased accumulation of phenolics in pea nodules, as well.
Changes in the activity of acid phosphatase (AcPase) in the apoplast of pea root nodule were investigated. The activity was determined using lead and cerium methods. The results indicated a following sequence of AcPase activity appearance during the development of the infection thread: 1) low AcPase activity appears in the outer part of cells of symbiotic bacteria; 2) bacteria show increased AcPase activity, and the enzyme activity appears in the thread walls; 3) activity exhibits also matrix of the infection thread; 4) bacteria just before their release from the infection threads show high AcPase activity; 5) AcPase activity ceases after bacteria transformation into bacteroids. The increase in bacterial AcPase activity may reflect a higher demand for inorganic phosphorus necessary for propagation of the bacteria within the infection threads and/or involved in bacteria release from the infection threads.
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