Hamako Sasamoto

Compatible Solutes and Inorganic Ions in the Mangrove Plant Avicennia marina and Their Effects on the Activities of Enzymes

Naturally grown two-month-old seedlings of Avicennia marina contain high concentrations of Na+ and Cl-.+ Our NMR studies revealed an accumulation of glycinebetaine, asparagine and stachyose in A. marina. The highest concentration of glycinebetaine was observed in young leaves, while the distribution of stachyose was restricted in stems and roots. A sparagine comprised more than 96% of total free amino acids in roots and 84% in leaves from two-year-old plants. Little or no accumulation of proline or polyols, which are proposed as compatible solutes in other plants, could be detected in A. marina. The activities of phosphofructokinase, pyrophosphate:fructose-6-phosphate 1-phosphotransferase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase (decarboxylating), phosphoenolpyruvate carboxylase and NAD:malate dehydrogenase from young leaves of A. marina were inhibited by NaCl, while the activity of fructose-1,6-bisphosphate aldolase was activated by 50-200 m M NaCl. There was little or no effect of high concentrations (up to 500 mᴍ ) of glycinebetaine on the activities of any of these enzymes. No significant protection by glycinebetaine was detected against NaCl inhibition of these enzymatic activities. Based on these results, possible mechanisms for the salt-resistance of A. marina cells are discussed.

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

SummaryEmbryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l−1 glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l−1) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.

Purine metabolism in cells of a mangrove plant, Sonneratia alba, in tissue culture

Summary A tissue culture system was established from pistils of flower buds of a mangrove plant, Sonneratia alba . The optimum growth rate of callus was observed in Murashige and Skoogs medium that contained 50 mM NaCl. In this tissue culture system, we examined the effects of short-term salt stress on the size of the pool of purine nucleotides, as well as the capacity for the salvage and the degradation of purines. At 100 mM NaCl, the size of the total adenylate pool was reduced, but the «adenylate energy charge» was, by contrast, increased. No marked change in the size of guanylate pool occurred in response to NaCl. The rate of conversion of [8- 14 C] adenosine to adenylate was decreased by NaCl, but that to nucleic acids was not significandy affected. In contrast to the results obtained with maize root tips (Peterson et al., 1988), salt stress did not increase the catabolism of [8- 14 C] hypoxanthine. Most of the radioactivity from [8- 14 C] adenosine was accumulated in adenine while that in [8- 14 C] hypoxanthine remained unaffected in Sonneratia alba . The low capacity of the purine-degradation system might ensure a supply of purine bases and nucleosides that can be utilized as substrates for the rapid and efficient biosynthesis of nucleotides by the salvage pathways.

Uptake of inorganic ions and compatible solutes in cultured mangrove cells during salt stress

SummaryCallus of the mangrove plant, Sonneratia alba J. Smith, established from pistils of flower buds were cultured on solid Murashige and Skoog medium supplemented with 0 to 500 mM NaCl. Maximum growth was observed with 50 mM NaCl, and net growth of callus occurred for concentrations up to 200 mM NaCl. At 500 mM NaCl, growth of callus was completely inhibited, although a part of the tissue was still alive after 30 d. Cellular levels of Na+ and Cl− were greatly increased by the treatment with NaCl. Uptake of K+ was also enhanced and was accompanied by increasing levels of Na+ and Cl− so that the Na+/K+ ratio was almost constant (4.1–4.2) in callus grown with 50–200 mM NaCl. Levels of Mg2+ and Ca2+ were not changed significantly with 50–200 mM NaCl, whereas levels of free NH4+, NO3− and SO42− ions, which are convertible to organic compounds, were lowest in callus grown with 50 mM NaCl. The rate of conversion of 15NH4+ into macromolecules during 30 d culture with 0–100 mM NaCl did not vary greatly, but 200 mM NaCl reduced the biosynthesis of macromolecules from this ion. The highest rate of conversion of 15NO3− into macromolecules was observed at 50 mM NaCl. Identification of compatible solutes with NMR-spectroscopy indicated that mannitol is the compatible solute for intact plants of Sonneratia alba, but no accumulation of mannitol was found in calluses, not even in those grown at high concentrations of NaCl.

Somatic embryogenesis from immature and mature zygotic embryos of Cryptomeria japonica I: Embryogenic cell induction and its morphological characteristics

Embryogenic cells (ECs) of sugi (Cryptomeria japonica D. Don) were induced from immature and mature zygotic embryos cultured on different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D)-containing modified Campbell and Durzan medium. The rate of induction of ECs varied depending on the stage of embryos collected. The highest percentage of induction (35%) was obtained with immature zygotic embryos collected July 18 and July 30, 1997, when 1 μM of 2,4-D was added to the induction medium. The ECs easily proliferated when subcultured in a medium of the same composition as the induction medium within 3 weeks. Morphological characteristics of nonembryogenic cells and embryogenic cells of different developmental stages were studied under an inverted fluorescence microscope.

Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1-0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 μg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.

Control of The Development of Somatic Embryo of Japanese Conifers By The Density of Embryogenic Cells in Liquid Culture

The morphogenetic changes of embryogenic tissue at different cell densities were investigated in Japanese conifers, i.e. Larix leptolepis, Picea jezoensis, and Cryptomeria japonica. We found that cell lines generated and maintained in liquid suspensions could take on different morphologies and requirements for somatic embryo development and maturation. Cell density within liquid suspension played an important role in embryogenic tissue proliferation and development. We found that in a high cell density culture of L. leptolepis, the embryogenic tissue continued to proliferate as small spherical cell aggregates; however, no further development occurred. On the other hand, in a low-density cell culture, development of embryogenic tissue into somatic embryos were promoted. In addition, we have established a micro-culture method for culturing single aggregates of embryogenic tissue of all three species. Individual aggregates could be identified and picked up using a micro-manipulator and cultured in 50 μl liquid medium using a 96-well culture plate. In all three species studied, a majority of the cell aggregates actively proliferated within the wells. Maturation of somatic embryos was successful when the newly proliferated cell aggregates were transferred to solid ABA-containing medium.

The Effect of Salt Stress on the Catabolism of Sugars in Leaves and Roots of a Mangrove Plant, Avicennia marina

Abstract Respiration and related aspects of metabolism were investigated in the roots and leaves of 2-year-old trees of the mangrove plant, Avicennia marina in the presence of 100, 250 and 500 mᴍ NaCl. The rate of respiration of leaves increased with increasing concentrations of NaCl in the incubation medium, but respiration of roots was not similarly affected. In order to examine the relative rates of catabolism of glucose by the glycolysis-tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate pathway (PP pathway), we determined the rates of release of 14CO2 from [1-14C]glucose and from [ 6 -14C]glucose in segments of roots and leaves. The ratios of rates (C6/C1) in roots varied from 0.30 to 0.44, while ratios of 0.85 to 0.99 were obtained when leaves were incubated in the presence of various concentrations of NaCl. It appeared that the PP pathway was more involved in sugar catabolism in the roots than in the leaves of A. marina. Uniformaly 14C-labelled sucrose, incubated with segments of roots and leaves for 18 h, was converted to CO2, amino acids (mainly glutamine), organic acids (mainly malic acid), sugars and ethanol-insoluble macromolecules. The incorporation of radioactivity into most of these components was not significantly affected by NaCl. However, in leaves (but not in roots) the release of 14CO2 from [ U -14C]sucrose was en hanced by NaCl at 250 mᴍ and 500 mᴍ, while the rate of incorporation of radioactivity into macromolecules was reduced by high concentrations of NaCl. Incorporation of radioactivity from [ U -14C]sucrose into malic acid was enhanced in both roots and leaves by an increase in the concentration of NaCl from 100 mᴍ to 500 mᴍ (this concentrations is similar to that in sea water). Independent of the concentration of NaCl, more than half of the radioactivity in the neutral fraction from leaves was incorporated into an unidentified sugar, while in the same fraction from roots, the radioactivity was associated with glucose, fructose and sucrose. On the basis of these results, a discussion is presented of the characteristics of catabolism of sugars in A. marina in relation to salt resistance.

Maturation and plant recovery from embryogenic cells of Japanese larch: Effect of abscisic acid in relation to their morphology

Two embryogenic cell lines characterized by different morphology and color, white and red, were obtained from an immature zygotic embryo of Japanese larch (Larix leptolepis Gord.). Mature somatic embryos with cotyledons and regenerated plants were obtained from both cell lines. However optimal concentration of abscisic acid (ABA) for maturation varied depending on morphology of ECs. From the white ECs which intermingled with somatic embryos of very early stage, mature somatic embryos were induced on maturation medium containing 50 μM of ABA. On the other hand, mature somatic embryos with cotyledons were observed from the red ECs which consisted of somatic embryos of more developed stage on hormone-free medium or 0.1 μM ABA containing-medium.

Histological analysis in shoot organogenesis from hypocotyl explants of Kandelia candel (Rhizophoraceae).

In vitro culture of hypocotyl explants from Kandelia candel, a common mangrove species, on hormone-free Murashige and Skoog (MS) medium resulted in shoot formation. Since the hypocotyls showed good potential for in vitro shoot multiplication, the process of bud primordium formation was analyzed from a histological viewpoint. A wound periderm first appeared at the top, exposed cut surface of the explants. The wound-induced meristem continued to divide giving rise to suberized cells oriented towards the cut surface. After formation of the suberized cell layers, the meristem and its inner derivatives differentiated into multilayered, uniformly packed parenchyma cells. Bud primordia differentiated from the dense cytoplasmic cells of the wound-induced meristem just beneath the suberized layer near the severed vascular bundles. Each explant produced several visible shoot buds. Furthermore, histological sections revealed that additional bud primordia were present within the explant just underneath the suberized cells and that these bud primordia appeared to be arrested in their development. The fact that additional bud primordia were present within the explant suggests that further manipulation of the explant is helpful to maximize the potential of this system.