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Byline: Yoshitaka Arai (2,4), Masayoshi Kawaguchi (1), Kunihiko Syono (3), Akira Ikuta (2) Keywords: Amidohydrolase; Enzyme purification; Indole-3-acetamide; Rice callus Abstract: The activity of indole-3-acetamide (IAM) hydrolase from rice cells was enriched ca. 628-fold by gel filtration and anion exchange column chromatography. The molecular masses of the IAM hydrolase estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis were approximately 50.5 kD and 50.0 kD, respectively. The enzyme exhibited maximum activity at pH 6.0--6.5. The enzyme was stable against heat treatments between 4 and 50degC and works optimally at 52degC. The activity remained constant at 4degC for at least 143 days. The purified enzyme fraction hydrolyzed indoleacetic acid ethyl ester (Et-IAA) in addition to IAM and its homologue, 1-naphthalene-acetamide, but not indole-3-acetonitrile. Km values of the enzyme were 0.96 mM and 0.55 mM for IAM and Et-IAA, respectively. Although the molecular mass of the enzyme was very similar to that of IAM hydrolase of Agrobacterium tumefaciens involved in tumor formation, the biochemical properties of the enzyme including its high Km value were considerably different from those of the A. tumefaciens enzyme. Based on these enzyme properties, we will discuss whether the amidohydrolase is involved in auxin biosynthesis in rice cells. Author Affiliation: (1) Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (2) Department of Biological Science and Technology, Science University of Tokyo, Noda, Chiba, Japan (3) Graduate School of Science, Japan Women's University, Tokyo, Japan (4) Torii Pharmaceutical Institute, Sakura, Chiba, Japan Article History: Registration Date: 17/02/2004 Received Date: 17/04/2003 Accepted Date: 30/01/2004 Online Date: 23/03/2004

Byline: Tomomasa Takase (2), Yuki Yanagawa (3), Setsuko Komatsu (1), Hiroki Nakagawa (2), Junji Hashimoto (1) Keywords: Cell cycle; MG132; Rice; (Oryza sativa L.); Proteome; [beta]-Tubulin Abstract: To understand the cell cycle process in plants, we searched for proteins that quantitatively change during the cell cycle in suspension-cultured rice (Oryza sativa L.) cells. The proteins were analyzed by a two-dimensional polyacrylamide gel electrophoresis image-analysis system. We detected 11 proteins that quantitatively changed during the cell cycle, among which [beta]-tubulins and a calreticulin-like protein were identified. The amounts of [beta]-tubulin proteins were low in the M phase and high in the G1 phase. In contrast, mRNAs for two of the three types of [beta]-tubulin were high in the M phase of the cell cycle. The addition of protease inhibitors MG132 or E64d to the cells decreased the [beta]-tubulin proteins during 24 h, suggesting that [beta]-tubulin proteins are degraded in vivo by proteases other than those whose activities are inhibited by MG132 or E64d. Author Affiliation: (1) National Institute of Agrobiological Science, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan (2) Department of Bioproduction Science, Faculty of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan (3) Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520--8104, USA Article History: Received Date: 14/05/2003 Accepted Date: 19/08/2003 Online Date: 01/10/2003

Byline: Takashi Aoyama (1), Atsuhiro Oka (1) Keywords: His-Asp phosphorelay; Intracellular signal transduction; Phytohormone; Signal perception; Transcriptional regulation; Two-component regulatory system Abstract: Cytokinins regulate various events in plant development according to the intrinsic developmental program and in response to environmental stimuli. Recent genetic and molecular biological studies have revealed the framework of the intracellular signal transduction pathway from cytokinin perception to transcriptional regulation of primary cytokinin-responsive genes in Arabidopsis thaliana. Membrane-bound histidine kinases, including CRE1/AHK4, AHK2, and AHK3, perceive cytokinins. The signal is then transferred via histidine-containing phosphotransfer factors, AHPs, to transcription-factor-type response regulators, such as ARR1, which execute the signal-dependent transactivation of primary cytokinin-responsive genes, including those for other types of response regulator. Simply stated, the cytokinin signal is mediated by the His-Asp phosphorelay, which was originally found in bacterial two-component regulatory systems. However, many details, especially those that are essential for elucidating the regulatory mechanisms underlying complicated cytokinin responses, remain unknown. Author Affiliation: (1) Laboratory of Molecular Biology, Institute for Chemical Research, Kyoto University, Gokasho, Uji , Kyoto 611-0011, Japan Article History: Received Date: 04/02/2003 Accepted Date: 08/03/2003 Online Date: 17/04/2003

Byline: Osami Misumi (1), Yoshiki Nishimura (1), Tuneyoshi Kuroiwa (1) Keywords: Keywords: Aging, Cell proliferation, Chlamydomonas reinhardtii, Chloroplast DNA, Chloroplast DNA content, Novobiocin Abstract: is an unicellular green alga that contains one cup-shaped chloroplast with about 60 copies of cpDNA. Chloroplasts (cp) multiply in the cytoplasm of the plant cell by binary division, with multiple copies of cpDNA transmitted and maintained in successive generations. The effect of cpDNA copy number on cell proliferation and aging was investigated using a C. reinhardtii moc mutant, which has an undispersed cp-nucleoid and unequal segregation of cpDNA during cell division. When the mother cell divided into four daughters, one moc daughter cell chloroplast contained about 60 copies of cpDNA, and the chloroplasts in the three other daughter cells contained the 4--7 copies of cpDNA. In liquid medium, the number of moc cells at the period of stationary phase was about one-third that of the wild type. To observe the process of proliferation and aging in the mother cell, we used solid medium. Three out of four moc cell spores were preferentially degenerated 60 days after cell transfer. To confirm this, wild-type and moc mother cells containing four daughter cells were treated with novobiocin to inhibit cpDNA replication. Cell degeneration increased only in the moc strain following novobiocin introduction. In total, our results suggest that cells possessing smaller amounts of cpDNA degenerate and age more rapidly. Author Affiliation: (1) Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo, 113--0033 Japan, JP Article note: Received 7 September 2000/ Accepted in revised form 14 February 2001

Byline: Ichiro Terashima (1), Shin-Ichi Miyazawa (1), Yuko T Hanba (2) Keywords: Keywords: Cell size, CO2 diffusion, Cost/benefit analysis, Leaf photosynthesis, Mesophyll Abstract: ) depend not only on photosynthetic biochemistry but also on mesophyll structure. Because resistance to CO.sub.2 diffusion from the substomatal cavity to the stroma is substantial, it is likely that mesophyll structure affects A through affecting diffusion of CO.sub.2 in the leaf. To evaluate effects of various aspects of mesophyll structure on photosynthesis, we constructed a one-dimensional model of CO.sub.2 diffusion in the leaf. When mesophyll thickness of the leaf is changed with the Rubisco content per unit leaf area kept constant, the maximum A occurs at an almost identical mesophyll thickness irrespective of the Rubisco contents per leaf area. On the other hand, with an increase in Rubisco content per leaf area, the mesophyll thickness that realizes a given photosynthetic gain per mesophyll thickness (or per leaf cost) increases. This probably explains the strong relationship between A and mesephyll thickness. In these simulations, an increase in mesophyll thickness simultaneously means an increase in the diffusional resistance in the intercellular spaces (R .sub.ias), an increase in the total surface area of chloroplasts facing the intercellular spaces per unit leaf area (S .sub.c), and an increase in construction and maintenance cost of the leaf. Leaves can increase S .sub.c and decrease R .sub.ias also by decreasing cell size. Leaves with smaller cells are mechanically stronger. However, actual leaves do not have very small cells. This could be because actual leaves exhibiting considerable rates of leaf area expansion, adequate heat capacitance, high efficiency of N and/or P use, etc, are favoured. Relationships between leaf longevity and mesophyll structure are also discussed. Author Affiliation: (1) Department of Biology, Graduate School of Science, Osaka University, Machikaneyama-cho, Toyonaka, Osaka, 560--0043 Japan, JP (2) Research Institute for Bioresources, Okayama University, Chuo, Kurashiki, 710--0046 Japan, JP Article note: Received 20 September 2000/ Accepted in revised form 4 January 2001

Byline: Toru Tokuoka (1), Hiroshi Tobe (2) Keywords: Keywords: Embryology, Euphorbiaceae, Ovule, Phyllanthoideae, Seed Abstract: ) are distinct from the rest of the subfamily in having a thick inner integument (over six cells thick), an exotegmen composed of cuboidal cells (type II), and vascular bundles in the outer integument and, as molecular evidence also suggests, should be transferred to a separate family Putranjivaceae. Hymenocardieae (Didymocistus and Hymenocardia), whose positions have been controversial, are monophyletic in sharing endotestal seeds with a collapsed exotegmen which is unknown elsewhere in Euphorbiaceae. The genera seem to require separation from the Euphorbiaceae. In addition, a morphological heterogeneity of the two large genera Cleistanthus and Phyllanthus, as well as of tribe Antidesmeae subtribe Scepinae were also discussed. Author Affiliation: (1) Department of Natural Environmental Sciences, Faculty of Integrated Human Studies, Kyoto University, Kyoto, 606--8501 Japan, JP (2) Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606--8502 Japan, JP Article note: Received 20 October 2000/ Accepted in revised form 14 January 2001

Byline: Yoshihiro Ozeki (1), Yoshio Ito (1), Nobuhiro Sasaki (1), Mikiko Oyanagi (1), Hirofumi Akimoto (1), Yukie Chikagawa (1), Junko Takeda (2) Keywords: Keywords: Anthocyanin, Carrot, Differentiation, myb, Phenylalanine ammonia-lyase (PAL) Abstract: and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Author Affiliation: (1) Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184--8588 Japan, JP (2) Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606--8502 Japan, JP Article note: Received 10 June 2000/ Accepted in revised form 1 July 2000

Byline: Sodmergen (1), Jian-Gong Niu (1), Lin-Jiang Li (2), Jiang-Xun He (3), Feng-Li Guo (4) Keywords: Keywords: Epifluorescence microscopy, Laser confocal microscopy, Organelle DNA, Plant cells, YO-PRO-1 Abstract: plastid and mitochondrial DNA in the plant cells such as the sperm cell of Jasminum nudiflorum, the generative cell of Pharbitis lim-bata, the cultured cell of Nicotiana tabacum and the root cell of Vicia faba with epifluorescence microscopy and laser confocal microscopy using YO-PRO-1 as a fluorescent dye. The excitation for YO-PRO-1 was blue light in epifluorescence microscopy and 488 nm Kr/Ar ion laser in confocal microscopy. Dimorphic epifluorescent spots that corresponded plastid DNA and mitochondrial DNA were distinctly detected in the cells of each species examined. In this report, we introduce YO-PRO-1 as a new epifluorescent dye for successful in situ detection of small amount DNA in plant live cells and cell sections with perticular emphasis on the importance of sample preparation. Author Affiliation: (1) College of Life Sciences, Peking University, Beijing 100871, China, CN (2) Institute of Zoology, the Chinese Academy of Sciences, Beijing 100080, China, CN (3) Department of Biology, Jiujiang Teachers College, Jiujiang 332000, China, CN (4) College of Biological Sciences, Beijing Agricultural University, Beijing 100094, China, CN Article note: Received 10 November 1998/ Accepted in revised form 13 January 1999