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Yasuda, H., Kanda, K., Koiwa, H., Suenaga, K., Kidou, S. and Ejiri, S. (2005)
Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells.
Planta, 222: 118-129

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 α-type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phallodin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.

Onda, Y. and Ito, K. (2005)
Changes in the composition in xylem sap during development of the spadix of skunk cabbage (Symplocarpus foetidus).
Bioscience, Biotechnology, and Biochemistry, 69:1156-1161.

The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20℃ even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of post-thermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31.2±24.7 μl h-1g-1) and post-thermogenic (50.5±30.4 μl h-1g-1) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus.

Ito, T. and Ito, K.(2005)
Nonlinear dynamics of homeothermic temperature control in skunk cabbage, Symplocarpus foetidus.
Physical Review E, 72, 051909.

Certain primitive plants undergo orchestrated temperature control during flowering. Skunk cabbage, Symplocarpus foetidus, has been demonstrated to maintain an internal temperature of around 20 ℃ even when the ambient temperature drops below freezing. However, it is not clear whether a unique algorithm controls the homeothermic behavior of S. foetidus, or whether such an algorithm might exhibit linear or nonlinear thermoregulatory dynamics. Here we report the underlying dynamics of temperature control in S. foetidus using nonlinear forecasting, attractor and correlation dimension analyses. It was shown that thermoregulation in S. foetidus was governed by low-dimensional chaotic dynamics, the geometry of which showed a strange attractor named the "Zazen attractor." Our data suggest that the chaotic thermoregulation in S. foetidus is inherent and that it is an adaptive response to the natural environment.

Sarker, B. C., Hara, M. and Uemura, M. (2005)
Proline synthesis, physiological responses and biomass yield of eggplants during and after repetitive soil moisture stress.
Scientia Horticulturae 103: 387-402.

Eggplants (Solanum melongena L. cv. Senryo No. 2) were grown at different levels of soil moisture stress in pots under glasshouse conditions in two separate years. Stress was applied at short-term repetitive (T1), long-term repetitive (T2), and prolonged severe stress (T3) during different growing periods compared with a control (T0). The volumetric water content (VWC) of soil, leaf water potential (Cp), proline content, transpiration rate (Tr), stomatal conductance (GS) and photosynthesis rate (Pn) of leaves and biomass yield were investigated to verify the extent of injury caused during and after moisture stress. The Cp decreased in response to stress increase, and it increased to the initial level after stress recovery. Leaf proline synthesis as a compatible solute greatly increased by intensified stress either in the short-term or long-term; the highest value was 51.6 mg g-1 fresh weight (FW) recorded for T3 plants that showed irreversible wilting at 115 times greater than the initial value (0.45 mg g-1 FW). T1 and T2 plants showed a reduced pattern of proline synthesis as they produced 18.8 and 39.9 mg g-1 FW, respectively, which were 42 and 89 times greater than the initial level; but the proline synthesis in these plants was markedly reduced to 1.1 and 5.6 mg g-1 FW, respectively, within a day of stress recovery by rewatering. The Tr, GS and Pn were significantly reduced and varied among the eggplants with stress severity and duration. GS and Pn decreased because of stress and increased again after stress, but not necessarily fully, and did not return to previous levels within a day due to stress injury. Biomass yield was also significantly decreased as the moisture stress retarded the physiological functions. Under short-term and long-term stress conditions, eggplants synthesized proline significantly, but in contrast the net photosynthesis remained less affected and maintained its activity. The results of this study suggest that proline synthesis increases during stress increase and returns to the initial level after stress recovery, which seems to act as part of a survival mechanism. Therefore, eggplants (Solanum melongena L. cv. Senryo No. 2) have an adaptive potential to acclimate under stress conditions.

Wagatsuma, T., Uemura, M., Mitsuhashi, W., Maeshima, M., Ishikawa, S., Kawamura, T., Maruyama, T., Shiono, Y., Khan, Md. S. H. and Tawaraya, K. (2005)
A new and simple technique for the isolation of plasma membrane lipids from root-tips.
Soil Science and Plant Nutrition, 51: 135-139.

It has been suggestedthat plasma membrane (PM) 1ipids play a major role in aluminum (Al) tolerance;however, no direct investigations have been carried out using PM lipids from root-tips. Here we report a new technique for PM isolation as an alternative to the laborious two-polymer phase partitioning method that is commonly applied, as follows:1) separation of protoplasts from l-cm root-tips portions by enzymatic digestion, 2) attachment of the purified protoplasts to glass plates coated with polylysine, 3) preparation of PM ghosts by successive burst of the attached protoplasts using separate buffer solutions (25 mM PIPES, 5 mM EDTA, and 2 mM MgCl2, at pH 7.0) with slow stirring for 60 s. The PMs were confirmed to be devoid of organelle membranes by fluorescence microscopy, thin layer chromatography (TLC) and western blot analysis. The PM lipids obtained were found to be useful for studies on their differential permeability and lipid composition between lines of triticale or cultivars of maize under Al stress.

Ito, K. and Seymour, R. S.(2005)
Expression of uncoupling protein and alternative oxidase depends on lipid or carbohydrate substrates in thermogenic plants.
Biology Letters, 1: 427-430.

Thermogenesis, in which cellular respiratory activity is considerably stimulated, requires mitochondrial uncoupling protein (UCP) in mammals and an alternative oxidase (AOX) in plants. Here, we show that the genes for both proteins are expressed in thermogenic plants, but the type correlates with the respiratory substrate. A novel gene termed PsUCPa encoding a variant of UCP was specifically expressed in thermogenic flowers of Philodendron selloum, which uses lipids as substrates. However, a gene termed DvAOX encoding for AOX protein was expressed in thermogenic flowers of Dracunculus vulgaris, which presumably uses carbohydrates as substrates. These findings suggest that cellular metabolism is a major determinant in selective expression of appropriate thermogenic genes in plants.

Sakurai, J., Ishikawa, F., Yamaguchi, T., Uemura, M. and Maeshima, M. (2005)
Identification of 33 rice aquaporin genes and analysis of their expression and function.
Plant & Cell Physiology, 46:1568-1577.

Plant aquaporins form a large protein family including plasma membrane-type (PIPs) and tonoplast-type aquaporins (TIPs), and facilitate osmotic water transport across membranes as a key physiological function. We identified 33 genes for aquaporins in the genome sequence of rice (Oryza sativa L. cv. Nipponbare). We investigated their expression levels in leaf blades, roots and anthers of rice (cv. Akitakomachi) using semi-quantitative reverse transcription-PCR (RT-PCR). At both early tillering (21 d after germination) and panicle formation (56 d) stages, six genes, including OsPIP2;4 and OsPIP2;5, were expressed predominantly in roots, while 14 genes, including OsPIP2;7 and OsTIP1;2, were found in leaf blades. Eight genes, such as OsPIP1;1 and OsTIP4;1, were evenly expressed in leaf blades, roots and anthers. Analysis by stopped-flow spectrophotometry revealed high water channel activity when OsPIP2;4 or OsPIP2;5 were expressed in yeast but not when OsPIP1;1 or OsPIP1;2 were expressed. Furthermore, the mRNA levels of OsPIP2;4 and OsPIP2;5 showed a clear diurnal fluctuation in roots; they showed a peak 3 h after the onset of light and dropped to a minimum 3 h after the onset of darkness. The mRNA levels of 10 genes including OsPIP2;4 and OsPIP2;5 markedly decreased in roots during chilling treatment and recovered after warming. The changes in mRNA levels during and after the chilling treatment were comparable with that of the bleeding sap volume. These results suggested the relationship between the root water uptake and mRNA levels of several aquaporins with high water channel activity, such as OsPIP2;4 and OsPIP2;5.

Wagatsuma, T., Ishikawa, S., Uemura, M., Mitsuhashi, W., Kawamura, T., Khan, Md. S. H. and Tawaraya, K. (2005)
Plasma membrane lipids are the powerful components for early stage aluminum tolerance in triticale.
Soil Science and Plant Nutrition, 51: 701-704.

The order of aluminum (Al) tolerance in triticale lines (ST2>ST22) after re-elongation in an Al-free 0.2 mM calcium (Ca) solution for 9 h (Ca period) following 1 h pretreatment with 20 μM Al (Al period) agreed with that after 24 h of Al treatment. Permeability of the plasma membrane (PM) of root-tip cells after the Ca period was significantly increased in Al-sensitive ST22. Al was acculated more heavily in the root-tip portion of ST22 than in that of ST2m although similar amounts of malic and citric acid anions were released from both triticale lines. We established a new system examining lipid permeability using synthesized nylon-2,8 ultrathin and porous capsules trapped previously with 0.1% (w/v) methylene blue solution and coated thereafter with PM lipid isolated from root tips by a newly developed technique. Permeability of the PM lipid measured with time in 0.2 mM Ca or without 50 μM Al photometrically (A680) was significantly greater in Al-sensitive ST22 after 5 min of Al treatment. This is the first report to directly show the primary and early role of PM lipid in AL tolerance in triticale.

Koide, S., Atungulu, G., Uemura, M. and Nishiyama, M.(2005)
Mechanical properties and viability of Japanese radish cylinders immersed in sodium chloride solutions.
Biosystems Engineering 92: 335-340.

Mechanical properties (rupture properties and Young's modulus) of Japanese radish samples (Raphanus sativus L.) were determined following immersion in 2.5, 5.0 and 10.0% w/w NaCl solutions. Upon immersion in NaCl solutions, rupture energy increased while Young's modulus decreased. Influences of NaCl concentration on rupture energy and Young's modulus of Japanese radish cylinders were not apparent in this study. These results were expressed with empirical equations. The calculated values from the empirical equation agreed well with the experimental values. In this article, triphenyl tetrazolium chloride (TTC) reduction and moisture content changes when Japanese radish samples are immersed under various NaCl solutions were also investigated. A decrease in TTC reduction corresponded to an increase of the rupture energy. A decrease in Young's modulus corresponded to increased rupture energy and moisture reduction.

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