In Synechocystis sp. PCC 6803, the pmgA-disrupted mutant has defect in decrease of photosystem I (PSI) content under high-light conditions. This mutant also showed higher chlorophyll a content compared to wild-type cells under high-light conditions. When the chlorophyll a content in the mutant was repressed to the extent of that in wild-type cells by the addition of levlinic acid, the inhibitor of the chlorophyll biosynthesis, PSI content in the mutant became as low as that in wild-type cells. This suggests that the high levels of chlorophyll a content lead to the excess production of PSI complex under high-light conditions. The activity of the biosynthesis of 5-amino levulinic acid in the mutant under high-light conditions was higher than that in wild-type cells, indicating that the early step of the chlorophyll a biosynthesis might play an important role in the regulation of PSI content under high-light conditions.

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In cyanobacteria, small LuxR-type transcriptional regulators having three cysteines in their C-termini are highly conserved. We found that a small LuxR-type regulator, Ssl0564, in Synechocystis sp. PCC 6803 regulates the expression of a set of genes such as ndhD2 encoding a subunit of NAD(P)H dehydrogenase, rpe encoding pentose phosphate epimerase, and chlL, chlN, and chlB encoding subunit of light-independent protochlorophyllide reductase. Northern blot analysis showed that the expression of these target genes is regulated in response to changes of light intensity and this regulation is dependent on the redox state of acceptor side of photosystem I. When photosynthetic electron transport activity is lowered, cysteine residues in Ssl0564 are oxidized, leading to the up- or down-regulation of the target genes. We are going to identify cysteine residue(s) involved in redox sensing by the characterization of cysteine variants.

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Many cyanobacterial species possess two copies of genes encoding putative transcriptional regulators having an AbrB-type DNA binding domain at their C-termini. In this study, we made the disrupted mutants of sll0359 and sll0822 encoding AbrB-type regulators in Synechocystis sp. PCC 6803. Under low-light conditions, the content of chlorophyll and phycocyanin in sll0359 disruptant was about 70% of that in the wild-type cells. The doubling time of the mutant was 1.75 times longer than that of the wild type. Northern blot analysis revealed that the expression levels of genes encoding subunits of photosystem I and enzymes for pigment biosynthesis were lower in the mutant. Under high-light conditions, growth rate and pigment content were similar between the wild type and the mutant, indicating that the ability of high-light acclimation was not affected by the disruption of sll0359. Characterization of sll0822 disruptant will be also reported.

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Secretory Carrier Membrane Proteins (SCAMPs) are ubiquitous components of recycling vesicles that shuttle between the plasma membrane, endosomes, and trans-Golgi complex in mammalian cells. SCAMPs are consisted with a cytoplasmic N-terminal domain with NPF repeats, four central transmembrane regions, and a cytoplasmic tail. We obtained tobacco homolog cDNA for SCAMP (NtSCAMP) from EST collection of tobacco culture BY-2 cells. Using this clone and antibodies against encoding protein, we have been analyzing about localization and the function of SCAMP in plant cells. We investigated the localization of NtSCANP with YFP fusion (NtSCAMP-YFP) in BY-2 cells by video and laser scanning fluorescent microscopy. SCAMP-YFP was localized in plasma membrane, and endosome, which is a FM4-64 positive compartment. In dividing cells, it was detected predominantly on phragmoplast. By immunocytochemical analyses using anti-NtSCAMP antibody, NtSCAMP were localized along divided plate.

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Seed storage proteins synthesized on rough ER are transported to protein storage vacuoles (PSVs) via vesicle-mediated pathway. AtVSR1, one of the vacuolar sorting receptors in Arabidopsis, plays an important role in the recognition of the vacuolar-targeting signal of the storage proteins.¹⁾ In atvsr1 seeds, the storage proteins are abnormally secreted to accumulate in the extracellular space. When a GFP-fusion protein with vacuolar-targeting signal was expressed in atvsr1 seeds, GFP fluorescence was observed in the extracellular space. By monitoring the GFP fluorescence, we screened for Arabidopsis mutants that had defects in the sorting machinery of storage proteins from EMS-mutagenized M2 seed population. These mutant seeds abnormally accumulated the precursors of storage proteins, pro12S globulins and pro2S albumins.
¹⁾Shimada, T. Fuji, K. et al. (2003) Proc. Natl. Acad. Sci. USA. 100(26): 16095-100.

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In maturing seeds, storage protein precursors are synthesized on rER and are sorted to PSVs, where they are processed into mature forms and accumulated. Arabidopsis mag2 mutants abnormally accumulate the precursors of 12S globulins and 2S albumins. Novel structures with high electron-dense core were found in mag2 seed cells. Some of them are surrounded with ribosomes. Immunogold analysis suggests that the high electron-dense core is an aggregate of pro2S albumins and that pro12S globulins are localized around the core. ER chaperons, BiP and PDI, are abnormally accumulated and colocalized with precursors of 12S globulins around the pro2S core. These results suggest that mag2 mutants have defect(s) in the exit step from ER, resulting in ER stress. MAG2 gene encodes a novel protein exhibiting homology to mammalian RINT-1 and yeast TIP20. MAG2 might function in membrane traffic between Golgi and ER for seed storage proteins.

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Approximately 10% of genes coded on the plant genome are estimated as potentially chloroplast transport proteins. However all proteins transported into the chloroplast have not been known yet. We developed cell-free translational system that worked more than 14 days. Translation is one of the most popular events in cytoplasm, and then we have an idea as development of genome-wide approach for the protein analysis based on the cell-free system. Ninety-six genes in RAFL clones with highly ChloroP score were selected and generated DNA templates by the Split-primer PCR. After transcription, these proteins were synthesized in the cell-free system and used to transport assay with isolated pea chloroplasts. Twenty-two of 42 synthesized proteins were confirmed transport into chloroplast. This indicates that this approach is suitable for genome-wide analysis of chloroplast transport proteins. As next step, phosphorylation on transit peptide (TP) of the transported proteins is investigated.

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Rice α-amylaseI-1 (AmyI-1) is a glycoprotein bearing N-linked carbohydrate chain, that is secreted from the secretory tissues. We have recently demonstrated thatα-amylaseI-1 was localized inside of plastids in rice leaf cells. To clarify targeting mechanism of α-amylaseI-1 to plastids, transient expression analyses of α-amylaseI-1 fused with GFP were performed in onion epidermal cells. Simultaneous expression of AmyI-1-GFP and plastid marker (WxTP-DsRed) indicated that AmyI-1-GFP co-localized with the plastid marker. Furthermore, the targeting of AmyI-1-GFP into plastids was arrested by the dominant negative mutants of AtArf1 and AtSar1 those are involved in the traffic of ER-to-Golgi. In addition, when expression of Golgi marker (ST-mRFP) and WxTP-GFP with or without overexpression of AmyI-1 was carried out, incorporation of ST-mRFP to WxTP-GFP frequently occurred under overexpression AmyI-1. Based on the overall experimental results, we concluded that α-amylaseI-1 targets into the plastids through the ER-Golgi system.

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We investigate ADPglucose hydrolyzing nucleotide pyrophosphatase/phosphodiesterase (NPP) in rice (Oryza sativa L.). An NPP enzyme was purified and characterized from the shoot tissues of rice germinating seeds. It was shown to be glycosylated, since the enzyme protein bound to Con A, stained with periodic acid-Schiff reagent and could be digested by Endo-H. The complete NPP encoding cDNA, designated as OsNPP1, was isolated and characterized. OsNPP1 contains numerous N-glycosylation sites and a cleavable hydrophobic signal sequence that does not match with the N-terminal part of the mature protein. A confocal-fluorescence microscopy of rice cells expressing OsNPP1-GFP revealed that it occurs in the plastidial compartment, which was confirmed by immunocytochemical studies. Endo-H digestibility studies revealed that both OsNPP1 and OsNPP1-GFP occurring in the chloroplast are glycosylated. These data show the occurrence of a traffic route of glycosylated proteins from the endoplasmic reticulum-Golgi system to the chloroplast in higher plants.

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We previously reported that deletion of an Arabidopsis ATG6, a component of phosphatidylinositol 3-kinase (PI3K) complex, causes male gametophytic phenotypes. We have recently found that each component of PI3K complex, such as VPS34 and VPS15, is also essential for pollen germination, which supported our previous findings. To reveal a role of ATG6-PI3K complex in pollen germination, we examined function and localization of ATG6. Expression of Arabidopsis ATG6 allowed yeast atg6/vps30 mutant cells to accumulate autophagic bodies and to transport CPY protein into vacuoles. This suggested that Arabidopsis ATG6 has dual functions in autophagy and in vacuolar protein sorting. In Arabidopsis suspension cells, GFP-ATG6 and GFP-VPS34 resides on the punctate structure, which involves ATG8, a marker protein of pre-autophagosomal structure. PI3K-complex is likely to play a role in autophagosome formation in plant cells.

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Most peroxisomal matrix proteins are synthesized in the cytosol with targeting signal at C (PTS1) or N terminus (PTS2). PTS1 and PTS2 proteins are recognized by recepters, Pex5p and Pex7p, respectively, and carried to peroxisomes, and then imported by the import machinery (Pex10p, Pex12p, etc.) on the peroxisomal membrane. Catalase, an enzyme degrading hydrogen peroxide, is highly accumulated in peroxisomes. Perhaps, there is the specific pathway for catalase because catalase has neither typical PTS1 nor PTS2.
In this study, we analyzed focusing Pex5p, Pex7p, Pex10p and Pex12p. We generated transgenic tobacco BY-2 cells in which they were suppressed by RNAi method. When the expression of Pex5p or Pex7p was suppressed, however, catalase could be imported into peroxisomes. There is a possibility that other proteins besides Pex5p are involved in catalase import. We isolated the proteins binding to tobacco catalase by yeast two-hybrid system, and analyzed the binding proteins.

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We have analyzed ozone (O₃) tolerance in two Arabidopsis ecotypes: Col (O₃-tolerance) and Ws (O₃-sensitive). An approach of QTL mapping in RI lines had performed to detect O₃-sensitive loci. Two main QTLs were identified, one is located chr.4 and the other is chr.5. The former locus was located near (AtACS6) gene that involve in ethylene biosynthesis. The (AtACS6) expression and ethylene evolution in Ws was higher than in Col. Comparison of entire sequence of (AtACS6) genome showed that Ws has 13bp extra sequence in its promoter region. To know function of the extra sequence for (AtACS6) expression, we made two transgenic plants; one was excluded the extra sequence from Ws promoter and the other was inserted the extra sequence into Col promoter. Expression levels of (AtACS6) depend on presence of the extra sequence, suggesting that this sequence involve in O₃-induced ethylene production level and determines part of O₃-sensitivity in Ws.

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Recent studies showing that overexpression of aldehyde-detoxifying enzymes improved stress tolerance of the transgenic plants demonstrate the cytotoxicity of the lipid peroxide-derived aldehydes in plants. In order to identify the chemical species of the aldehydes responsible for cellular injury in plants under environmental stresses, we analyzed aldehyde profiles in oxidatively stressed leaves, using HPLC after derivatizing the acetonitrile extracts of leaves with 2,4-dinitrophenylhydrazine. Non-stressed tobacco leaves contained at least 12 aldehyde species, ca. 20 nmol aldehydes/g fresh weight in total. n-Hexanal and 3Z-hexenal were the major components. Very short-chain aldehydes like propionaldehyde and butyraldehyde were also detected. In the ozone-treated leaves, the contents of these aldehydes were 10-fold higher, and additional aldehydes such as acetaldehyde and 2E-hexenal, which is a potent cytotoxin, were observed. The content of malondialdehyde was less than 1% of the total aldehydes in both stressed and non-stressed leaves.

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Lipid peroxides in cells are degraded to form cytotoxic α,β-unsaturated aldehydes (reactive aldehydes). We examined the effects of reactive aldehydes on photosynthesis. When spinach chloroplasts were incubated in either acrolein, 4-hydroxynonenal or crotonaldehyde at 2 mM for 8 min at 25 degree C in darkness, the CO₂ photoreduction was inhibited by 100%, 75% or 50%, respectively. The electron transport activity (H₂O -> NADP⁺) was inhibited by only 5%. Compared with the reactive aldehydes of the same carbon chain length, aldehydes without the α,β-unsaturated bond showed less than half extents of inhibition of the CO₂ photoreduction. When the CO₂ photoreduction was totally lost by acrolein, the Calvin cycle enzymes were inhibited to the following extents: ribulose-5-phosphate kinase, 90%; glyceraldehydes-3-phosphate dehydrogenase, 73%; aldolase, 47.3%; sedoheptulose-1,7-bisphosphatase, 32%; Rubisco, 23%; fructose-1,6-bisphosphatase, 22%. The total inhibition of the CO₂ photoreduction can be explained by the accumulation of the partial inactivation of these enzymes.

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We isolated high-light (HL) stress-inducible genes, including a heat shock transcription factor (HsfA2) from Arabidopsis. The transcript level of HsfA2 was significantly increased under the several types of stress condition. In the HsfA2-overexpressing Arabidopsis (35S:HsfA2) plants, forty-six genes, including the stress-related genes, were highly expressed compared with those in the wild-type plants. Here we studied the gene regulation system via HsfA2 that might be associated with the response to various types of environmental stress. The transcript levels of HsfA2 target genes as well as that of HsfA2 were induced under the HL stress or the H₂O₂ treatment. In knockout-HsfA2 plants, the expression of HsfA2 target genes was strongly reduced under HL conditions. Furthermore, the 35S:HsfA2 plants showed increased tolerance to severe oxidative stress. Our findings indicate that HsfA2 acts as a key regulator in the induction of the defense system to several stresses.

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Nudix hydrolases are a family of proteins that hydrolyze a variety of nucleoside diphosphate derivatives. E.coli MutT hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the oxidized form of free guanine nucleotide, to prevent the mutation induced by such nucleotides. We found that Arabidopsis thaliana contains a number of cytosolic MutT/Nudix hydrolases (AtNUDX1-11). In this study, we characterized AtNUDX1, a MutT homolog, in Arabidopsis thaliana. The recombinant AtNUDX1 protein had the ability to hydrolyze not only 8-oxo-dGTP but also 8-oxo-GTP. In addition, AtNUDX1 completely suppressed increased frequency of spontaneous mutations in the E.coli mutT strain. These results indicate that AtNUDX1 functions to prevent the mutation of both DNA and RNA in A. thaliana. No phenotypic difference was observed between the wild-type plants and AtNUDX1-KO mutant under the oxidative stress caused by the treatment with 2μM paraquat. We are progressing toward to examine the 8-oxo-dG content in their plants.

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Erythroascorbic acid (eAsA) seen in budding yeast has been supposed to be synthesized by the successive reactions of NADP-dependent arabinose dehydrogenase (ARA) and arabinonolactone oxidase (ALO1). Here, we found NAD-dependent ARA and identified its corresponding gene.
First, we showed the presence of NAD-dependent ARA activity with a higher affinity against arabinose than NADP-dependent ARA in protein extracts of yeasts. Next, we found that the uncharacterized YMR041c gene is the ortholog of plant galactose dehydrogenase, and that its recombinant protein possesses NAD-dependent ARA activity. YMR041c coupled with ALO1 produced eAsA in vitro. Moreover, the gene disruptant of YMR041c abolished the increase in intracellular eAsA by arabinose feeding, while wild-type and NADP-dependent ARA disruptant showed more than 10-fold. These results clearly shows the similarity between the biosynthetic pathway of eAsA in yeasts and that of ascorbic acid in higher plants also in terms of enzyme structures.

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Our previous research has shown that Mn²⁺ and Zn²⁺ inhibit the La³⁺-induced superoxide generation in tobacco (Nicotiana tabacum L., cell-line BY-2) cells. In this study, we observed the Al³⁺-induced generation of superoxide in tobacco BY-2 cell suspension culture, using a superoxide-specific Cypridina luciferin analog (CLA). We examined the effect of culture age on the Al³⁺-induced response and found that higher level of superoxide generation was induced in the young cell cultures (2 - 4 day-old) when the range of culture age between 2 and 12 days after subculturing was examined. We also examined the effects of ZnSO₄ and MnSO₄ on Al³⁺-induced superoxide generation. Results indicated that both Zn²⁺ and Mn²⁺ inhibit the superoxide generation. Zn²⁺ at low dose (ca. 0.1 mM) and Mn²⁺ at high dose (10 - 30 mM) inhibited the Al-induced response by 50%.

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Superoxide radicals are toxic by-products of oxidative metabolism. Superoxide dismutase (SOD) catalyzes the disproportionation of superoxide radicals to give dioxygen and hydrogen peroxide. Bryophytes are considered as the first land plants and adaptive responses to oxidative stress are crucial in their evolution. We found that copper repressed FeSOD activity in chloroplasts of a moss, Barbula unguiculata. This copper mediated repression was achieved at mRNA. We showed that the 5' flanking region of FeSOD gene from -318 to -199 was involved in the copper-mediated transcriptional repression. To examine the function of the 120-bp sequence in copper responsiveness, we performed the gain-of-function experiment. The 120-bp DNA sequence was cloned upstream of the constitutive actin promoter and GUS reporter gene. The DNA fragment endowed the reporter with a remarkable property of copper-dependent repression, suggesting that the sequence is sufficient for copper-mediated transcriptional repression.

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One of our isolates MBIC11204 is green coccoid micoroalgae, and shares the morphological features with the genus Choricystis. But 18S rDNA and rbcL sequences clearly discriminate with MBIC11204 and Choricystis spp. so far reported. The alga possesses the vesicles stainable by Nile Red (NR), and hydrocarbons (C17-20) are also detected in n-hexane-extract. Therefore, we have reported that this alga is a light-oil-producing new algal species of the new genus as Pseudochoricystis ellipsoidea (tentative name) (Japan Society for Culture Collections 2005, Annual Meeting 2005 The Society for Biotechnology). We investigated in this study growth and hydrocarbon producing characteristics of P. ellipsoidea. Growth rate and maximal cell density of P. ellipsoidea are as high as those of Chlorococcum littorale, a strain suitable for high-density culture. Optimum pH and temperature for growth is 3-4.5 and 25^oC, respectively. Oil accumulation was greatly stimulated by nitrogen starvation, and required light energy and carbon dioxide.

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Glutamate decarboxylase (GAD) catalyzes the conversion of L-glutamate to γ-aminobutyric acid (GABA). Interestingly, the GAD in angiosperm has a calmodulin-binding domain (CaMBD) at the C-terminus, whose enzymatic activity is regulated by a Ca²⁺/CaM. We have reported that rice has a novel GAD (OsGAD2) that is unable to bind to a CaM (Akama et al, 2001) and OsGAD2ΔC lacking a C-terminal region, over-expressed in E. coli, was Ca²⁺/CaM independent, surprisingly its enzymatic activity increased five times higher than that of wild-type OsGAD2. We report here production of rice plants where OsGAD2ΔC is over-expressed under the control of the promoter of a rice glutelin gene (GluB-1) to analyze ten transgenic rice lines. Amino acid analysis extracted from these seeds indicated more than ten-fold GABA compared with those of wild-type plants. Western blot analysis with an anti-GAD2 antibody showed high level of the enzyme expression in GABA-accumulated seeds.

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Introduction of the Dof1 gene was shown to yield augmentation of nitrogen assimilation in Arabidopsis (1). The Dof1-introduced Arabidopsis plants showed the improved growth in the nitrogen-deficiency environment. To evaluate effects of Dof1 in another plant species, we generated Dof1-introduced potato plants and established eight independent transgenic lines. In leaves of these transgenic plants, increased amounts of free amino acids, especially asparagine and glutamine, were detected. In addition, transgenic potato plants also showed the increased number and weight of tubers and the elevated starch content in tubers. Approximately a double amount of starch appeared to be synthesized in each potato plant. These results implicated that Dof1 could function and enhance nitrogen assimilation in potato as well as in Arabidopsis. Enhanced biosynthesis in source organs might stimulate material deposition in sink organs of transgenic potato plants. (1) Yanagisawa, Akiyama, Kisaka, Uchimiya, Miwa (2004) PNAS, 101, 7833-7838.

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Japanese cedar (Cryptomeria japonica D. Don) is one of the major forest tree species in Japan, but recently more than 10 % of the population are suffered from its pollinosis. Genetic modification might be a powerful tool to minimize breeding period of time and can attach only our favorable traits. We have succeeded in regenerating Japanese cedar plantlets by somatic embryogenesis via embryogenic tissues derived from immature embryo collected in June to July, and in producing transgenic Japanese cedar by infection of the embryogenic tissues with Agrobacterium which harbouring a binary vector that contain Maize Ubiquitin Promoter -sGFP(S65T) construct.
Eleven weeks after the infection three transgenic lines those which GFP fluorescence can observed were isolated and propagated tissues were transferred to embryo induction media. Regenerated plantlets were obtained finally 7 months after the infection and they were supplied for further analysis to confirm integration of the genes to the genome.

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To obtain evolutionary insights into gene regulation in dicots, we transferred Arabidopsis genomic DNA fragments to tomato and Lotus japonicus using TAC (Transformation competent artificial chromosome) vector. Our previous results showed that, using DNA array, the expression patterns of the transferred genes in tomato were similar to those in Arabidopsis. In this study, we analyzed the correlation of the normalized expression levels of the transferred genes in tomato and those of the endogenous expression of Arabidopsis genes. The result showed that there was a moderate positive correlation in each organs analyzed. Additionally, quantification of transcripts using real-time RT-PCR demonstrated that some of the transferred genes in tomato showed similar transcript levels with the endogenous Arabidopsis genes. Based on the observations and the results of expression analysis of Lotus japonicus, we discuss the conservation of quantitative gene regulation between dicot plants.

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It is difficult to extend the phenotyping of mutants to genome-wide scale because mutants require the suitable condition to exhibit their phenotype. To find as much information as possible, we focused on chlorophyll fluorescence emitted from cyanobacteria. Cyanobacterial photosynthetic system is not separated from other metabolic systems. Therefore chlorophyll fluorescence that is indicator for photosynthesis could reflect the state of other metabolic systems. The kinetics of 303 mutants grown under high and low light conditions was observed. Among them, 203 mutants exhibited distinctly different phenotypes from the wild type. The percentage of unknown gene disruptants that showed phenotype only when grown under high light condition was higher than that of annotated gene disruptants. The result might imply that unknown gene disruptant group contains a high percentage of the mutant whose symptoms of gene disruption might be observed only under specific condition.

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By using a variety of research resources, it is now able to investigate any knockout mutants for almost every gene in Arabidopsis. Arabidopsis is, then, not only a model plant for plant research, but also a model organism in which it is possible to carry out saturation mutagenesis in an organism. We constructed thousands of Ds transposon-tagged lines, which have a single insertion because of an advantage of Ac/Ds system. In this resource, we selected 4000 transposon-tagged lines with insertions in their gene-coding regions and systematically observed the visible phenotype of each line. Totally about 200 clear visible phenotypes were classified into 43 categories of morphological phenotypes. Phenotypic images have been entered into a searchable database (http://rarge.gsc.riken.jp/phenome/). Even if we could not detect visible phenotypes, we have selected homozygous transposon-tagged lines systematically. We think that this would be another useful resource for a collection of gene-knockout lines.

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Endocytosis plays important roles in various functions of plants, such as an establishment of cell polarity and a polar transport of auxin in many tissues. To understand molecular mechanisms of endocytosis in plants, we have been focusing our attention on Rab5 GTPases. Arabidopsis thaliana contains three Rab5-related GTPases (Ara7, Rha1, and Ara6) in the genome. These three Rab5 GTPases are localized on endosomes and regulate endosomal membrane fusion. We have found that all three Rab5 members are activated by only one guanine nucleotide exchange factor (GEF), AtVps9. Arabidopsis genome appears to have two genes encoding Rab5 GEF marked by the Vps9 domain, but one of them seems to be non-functional. Results of biochemical characterization and functional analysis of AtVps9 will be presented.

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Ara6, Ara7 and Rha1 are Rab5 GTPase members of Arabidopsis thaliana, which are involved in the vesicular transport through endosomes. We identified AtVps9a as a guanine nucleotide exchange factor (GEF) which activates these three GTPases. AtVps9a should be an essential protein in Arabidopsis because we could not establish a plant homozygous for a T-DNA insertion in the AtVPS9a gene. Immature siliques from heterozygous plants contained approximately 3:1 green/brown seeds. A genome fragment containing AtVPS9a complemented this phenotype. Light microscopy revealed that most of homozygous embryos were arrested at torpedo stage, and that approximately 10% of the homozygous embryos were aborted before 8-cell stage. We observed arrested embryos by SEM and TEM. On the basis of our ultrastructural analyses, the relationship between vesicular transport through endosomes and the activation of Rab5 in plant early embryogenesis will be discussed.

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ER body, which is derived from endoplasmic reticulum, is a spindle-shaped structure (~10 μm long and ~1 μm wide). ER bodies are easily detected in the transgenic Arabidopsis expressing the ER-localized green fluorescent protein (GFP). A major component of ER bodies is β-glucosidase (PYK10). To clarify the molecular mechanism underlying formation of the unique shaped ER bodies, we applied forward genetic approach. We isolated an Arabidopsis mutant that had abnormally elongated ER bodies. The mutation was found in the PYK10 molecule, in which Cys-5 was replaced with Tyr at the N-terminal region. This suggests that a major component of ER bodies is responsible for determination or modulation of ER body shape. We succeeded in induction of ER bodies in Arabidopsis suspension cultured cells. This experimental system will make it possible to investigate the formation process of ER bodies.

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ER body is an ER derived organelle, which might be responsible for defense systems, because they are induced by wounding. A proteomic analysis showed that both PYK10 and PBP1 were decreased in their levels in Arabidopsis nai1 mutant, which has no ER bodies. PYK10, which is localized in ER bodies, is a β-glucosidase. On the other hand, PBP1 is a cytosolic jacalin like lectin (JAL). PBP1 interacts with and activate PYK10 in damaged tissue (1). We could identify candidates of other activation factors of PYK10 from JALs by analysis of public DNA microarray dataset. Analysis of T-DNA insertion mutants of these candidate genes showed that some of them were actually involved in the efficient activation PYK10.
(1) Nagano et al. (2005) Plant & Cell Physiology, 46, 1140-1148.

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Oil seeds store a large amount of lipid, which is used during seed germination and seedling growth. Oil body, which accumulates mainly triacylglycerol, is an organelle that is surrounded by a half membrane structure. It has been shown that oil bodies are formed from endoplasmic reticulum during seed maturation and the content lipids are degraded by glyoxysomes after seed germination. To investigate biogenensis of oil body in maturing seeds, we isolated Arabidopsis mutants that had different densities of the seeds from wild-type seeds. Electron micrographic analysis revealed that oil bodies in the mutant seeds exhibited abnormal size and shape. Detailed analysis with the mutant seeds will provide valuable information of the molecular mechanism for biogenensis of oil bodies in maturing seeds.

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In higher plants, peroxisomes are subdivided functionally into glyoxysomes, leaf peroxisomes and unspecialized peroxisomes. We extensively surveyed gene expression profile of peroxisomal proteins in various organs of Arabidopsis thaliana. In seedlings, genes of glyoxysomal proteins were expressed. In green cotyledons and rosette leaves, genes of leaf peroxisomal proteins were expressed. These results agree with the previous reports and indicate that our data of gene expression profile was reliable in categorizing functional profiles of peroxisomal genes. In our results, a group of genes that encoded peroxisomal proteins with unknown function were expressed specifically in roots. It suggests that peroxisomes in roots are functionally differentiated from those in other organs. One of the genes expressed in roots encoded putative polyamine oxidase. GFP fusion protein of the gene product was localized in peroxisomes. We discuss function of the root peroxisomes in Arabidopsis thaliana based on the analyses of peroxisomal polyamine oxidase.

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More than 30 peroxisome biogenesis factors (peroxin) have been identified in several organisms. It has been suggested that peroxin function for various phases of peroxisome biogenesis including peroxisome assembly, matrix protein import, and peroxisome proliferation. The Arabidopsis genome is predicted to encode about 20 proteins homologous to the yeast or mammal peroxins, but their functions remain unclear. In this study, we generated transgenic plants which were suppressed each peroxin gene expression using RNAi method and analyzed whether these homologues actually function for peroxisome biogenesis. We used the plant that stably expressed GFP-PTS1 (peroxisome targeting signal 1) as a parent plant for RNAi plant, and therefore could observe peroxisomes by GFP fluorescent. In the results, we could classified two groups of Arabidopsis peroxin, one group is that GFP was localized to cytosol and the other is that size of peroxisome was large. We discuss function of peroxin homologues in peroxisome biogenesis.

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RelA/SpoT has been known to work as a stringent factor synthesizing and/or hydrolyzing unusual nucleotide ppGpp in response to changes in nutrient conditions in bacteria. ppGpp is bound to RNA polymerase, which result in activation or repression of many kind of genes. Recently, several RelA/SpoT homologs (RSH) have been identified in alga and higher plants. To understand the role of the stringent factor in higher organisms, we performed genetic and biochemical analyses of RSHs from Arabidopsis. Complementation analysis by use of Escherichia coli and Rhodobacter capsulatus RelA and/or SpoT mutant strains indicated that two of RSHs could complement the loss of bacterial RelA, but not SpoT. This indicates that each RSH is specialized to synthesize and hydrolyze ppGpp in Arabidopsis.

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Trichomes (epidermal hairs) of plants have diverse functions, one of which is a structural barrier against herbivorous insects. Interestingly, several plant species including Arabidopsis thaliana are known to develop new leaves with increased trichome density in response to the damage to older leaves. This would be an induced defensive response of plants against herbivores. To reveal the mechanisms involved in the regulation of epidermal patterning of the new leaves by previous wounding, which in turn determine the trichome density, we have been taking a molecular genetic approach using Arabidopsis. We found that both wound-induced biosynthesis, and SCF^COI1 complex-mediated signaling of the wound hormone, jasmonic acid, are essential for trichome density upregulation. Next, we established an assay system to easily detect changes in trichome density. Using this system, we examined the role of known trichome-related transcription factors during trichome density shift. We also identified several mutants defective in trichome density upregulation.

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In wild-type Arabidopsis roots, epidermal cells which located in a cleft between two underlying cortical cells (designated H cells) differentiate into root hair cells and the other cells (designated N cells) into non-hair cells. CAPRICE (CPC), a positive regulator of differentiation into root hair cell, is transcribed and translated in N cells and then CPC protein moves to H cells. To elucidate the molecular mechanism that regulates intercellular movement of CPC protein, we isolated a mutant (FN20-4) in which intercellular movement of CPC:GFP fusion protein was suppressed. In FN20-4, root hairs were still formed from H cells, suggesting that the native CPC protein moves from N cells to H cells in this mutant. In FN20-4, genomic rearrangement has occurred and at least 11 genes are disrupted. Identification of gene that regulates intercellular movement of CPC:GFP fusion protein is in progress.

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The physical properties of xylem cell wall provide mechanical strength to the stems and allow the efficient transport of water and solutes. We focused on Glycine Rich Protein (GRP), a kind of cell wall structural proteins, which is expressed mainly in xylem of the inflorescence stem of Arabidopsis. The loss-of-function mutation of the GRP gene displayed several morphological phenotypes in the upper portion of the inflorescence stem. The GRP could not be detected in the upper portion of the inflorescence stem, and was restricted to the wall of tracheary element of protoxylem in basal part of the inflorescence stem. The morphological phenotypes in the grp mutant were rescued by applying increased amount of fertilizer. The results suggest that GRP modifies mechanical strength of the cell wall in xylem in basal part of the stem, thereby providing a conduit for the translocation of nutrients and water.

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During xylem vessel formation it is known that various events, such as secondary wall thickenings and programmed cell death, occur synchronously. We have identified many genes involved in these events by using microarray analyses during in vitro vessel element formation. However, little is known about the regulatory mechanisms underlying the synchronous expression of these genes. In this study, the FOX-hunting system was used to screen for regulatory factors of xylem vessel formation, which may regulate the expression of xylem vessel marker genes, PaspA3 and Atxyn3 encoding an aspartic proteinase and a xylanase, respectively, based on these promoter activity. As a result, we identified a mutant showing the ectopic activity of PaspA3 promoter but not showing that of Atxyn3 promoter. We also found that the overexpression of a bHLH gene caused the mutant phenotype. Now we are analyzing intrinsic functions of the bHLH and other bHLH included in the same subfamily.

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In higher plants, stem cells are maintained in the shoot apical meristem (SAM) and the root meristem (RM), thus allowing post-embryonic organogenesis. In SAM,WUSCHEL is shown to contribute to stem cell maintenance non-cell-autonomously. In RM, the quiescent center (QC), which is a small group of mitotically less active cells, has been postulated to prevent differentiation of surrounding stem cells, but little is known about the molecular mechanisms underlying this non-cell-autonomous cellular signaling.
Recently, WUSCHEL-related Homeobox 5 (WOX5) was reported to be expressed specifically in the QC. In this study, we investigated the role of WOX5 in stem cell maintenance. Analyses of loss-of-function wox5 mutants and the transgenic plants that ectopically express WOX5 suggested that WOX5 maintains undifferentiated status of the stem cells located distal to QC, but that those located basal to QC are maintained by pathways that act either separately from or redundantly with WOX5.

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In order to identify genes involved in early stage of procambium development, we screened 51,000 Arabidopsis gene trap lines. We isolated a line that exhibited GUS staining pattern preferentially along procambial cells and meristems. We identified Gamma-Glutamyl Hydorolase (GGH) gene, which is known to cleave the polyglutamate chain of folates, as the causal gene. In Arabidopsis genome, three GGH genes are tandemly placed. Overexpression of any of three GGH genes caused similar developmental abnormalities indicating reduction of meristematic activity. Conversely, suppression of all GGH genes caused developmental abnormalities indicating enhancement of meristematic activity. To know the roles of folates in tissue development, we examined the effect of a folate biosynthesis inhibitor and glutamyl conjugated folates on the development of Arabidopsis plant and Zinnia culture cells. Based on the data obtained, we suggest that the GGH gene may regulate the developmental phase transition by cleaving poly-glutamate residue of folates.

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We have developed a new activation tagging system in which Arabidopsis plants expressing a GAL4:VP16 transcription activator in a tissue-specific manner were transformed with a T-DNA that harbors GAL4-binding sequences (UAS). By using this method we isolated eight UAS-tagged root patterning (urp) mutants, urp1D through urp8D. Tagged genes were identified by determining T-DNA insertion sites, followed by co-segregation and recapitulation experiments. Seven of the eight genes were found to encode putative transcription factors belonging to AP2, NAC, C2H2 Zinc finger, R2R3 MYB, DOF and RWP-RK families. Expression studies indicated that at least some of these genes are expressed specifically in certain cell types in the root meristem region. Two of these genes have been implicated in root patterning by other groups through an elaborated marker-based screening or homology-based functional studies, indicating that our method is effective in identifying novel genes that have escaped from conventional mutant screenings.

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Cell type-specific expression of microRNAs (miRNAs) plays important roles in development of multicellular organisms. It is reported that the spatial restriction of miRNA, miR165 and/or miR166 (miR165/166) of Arabidopsis thaliana, plays a critical role in the polarity-dependent leaf development along the adaxial (central)-abaxial (peripheral) axis. miRNAs are transcribed as long precursors and progressively processed by the factors including Dicer-family protein. However, the cell type-specific regulatory mechanism of miRNA is poorly understood. Here, we show several lines of evidence that ASYMMETRIC LEAVES2 (AS2), ASYMMETRIC LEAVES1 HDT1/HD2A and HDT2/HD2B control the distribution of miR165/166 in Arabidopsis. The combination of the as2 mutation and the inhibition of histone deacetylase(s) led plants to form abaxialized leaves through the altered distribution pattern of miR165/166. Ectopic and/or over-production of AS2 reduced the accumulation level of miR165/166. This negative regulation of the accumulation of miR165/166 by AS2 required endogenous AS1.

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Petals are formed between sepals and stamens. Processes of petal maturation were described precisely (Smyth et al., 1990), which contain three phases. 1) Petal primordia appear between sepal primordia and bases of stamen primordia. 2) Petals elongate along the sepals through narrow space between sepals and the anthers. 3) Petals elongate rapidly to mature when flower opens.
To investigate the mechanism underlying the processes, we screened mutants with defects in petal morphology. We isolated a mutant, folded petals (fop). Histological analysis showed that folding of the petals was observed at the second phase shown above. The petals in fop showed no difference in size and shape from those in wild type. FOP encodes a putative transmembrane protein with unknown function. Analysis of pFOP:GUS transgenic plants revealed that FOP was expressed in petals. We will discuss about the function of FOP in the context of the petal maturation.

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The Arabidopsis SHEPHERD (SHD) gene encodes an Hsp90-like molecular chaperone resident in the ER. The shd-1 mutant from Ws shows clavata (clv)-like phenotype, whereas the shd-2 from Columbia (Col) does not. When we crossed shd-1 with Col, only less than a half of shd homozygotes in F2 population showed the clv-like phenotype. We speculated an enhancer is present in the Ws genome that makes shd-1 be such phenotype. A linkage analysis revealed the enhancer tightly linked to the CLV2 gene. Furthermore, the Col-type CLV2 introduced into shd-1 suppressed the clv-like phenotype. Therefore, we conclude that only Ws-type CLV2 requires the SHD for its function. The CLV2 has been reported to harbor many polymorphisms accompanying amino acid substitutions. Our results suggest the chaperone function of SHD allows these polymorphisms. We think CLV2 seems to be an example of the theory, in which Hsp90 allows the storage and release of genetic variation.

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CRES-T is an efficient and useful gene silencing system in which a chimeric repressor of specific transcription factor suppresses the expression of its target gene dominantly. A transgenic Torenia expressing the chimeric AGAMOUS repressor (1069-51) showed serrated petal margin comparable to that treated with cytokinin (Nishijima, 2004). 1069-51 also showed the irregular development of vascular bundle in petals as well as ectopic and intensive vascular tissue formation in pistils, although these phenotypes were not observed in cytokinin-treated wild-type (wt) plants. Cytokinin treatment on 1069-51 caused marginal white variegation, deeper serration on petal edges, and multiplication of vascular bundle in pistils. The backcross population (wt x 1069-51) only showed the multiplication of vascular bundle in pistils. These results suggest that the signaling pathway involved in the cytokinin-regulated petal development in 1069-51 is somewhat affected by the chimeric AGAMOUS repressor. We will discuss the relationship between the 1069-51 phenotype and AGAMOUS functions.

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Determination of floral organs in rice is controlled by mechanisms similar to the ABC model of Arabidopsis, but the detail is still unknown. OsMADS6, a member of rice MADS-box gene family, is classified in class G genes, and is expressed in lodicules, stamens, carpels, and so on. Also OsMADS6 is reported to interact with class A, B, E, G MADS-box proteins. In this study, we confirmed the expression profile of OsMADS6 using GUS reporter gene. OsMADS6 RNAi lines showed many defects in floral organs such like claw-shaped lemma/palea, palea-like lodicules, increase of lodicules and stigmas, decrease of stamens, and reduced fertility. In addition, the knocked-down mutants with the Tos17 insertion in OsMADS6 showed the phenotypes similar to those of the OsMADS6 RNAi lines. These results suggest that OsMADS6 participates in higher order MADS complexes with other MADS-box proteins to determine the identity and numbers of various floral organs.

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The Arabidopsis ERECTA (ER) gene plays an important role in regulating the shape of organs derived from the shoot apical meristem. The ER gene encodes a putative receptor protein kinase and has been implicated to mediate a signal transduction, which is required for proper development.
Recent researches have revealed some aspects of downstream events of the ER pathway. These results predicted the role of ER in auxin biosynthesis or epidermis differentiation. However, we have comparatively little information about the upstream factors controlling the ER pathway. Toward understanding the molecular nature of the ER signaling pathway, we have focused on identifying components of the regulatory network of the transcription of ER. To this end, we have performed an EMS mutagenesis screen using a line expressing GUS reporter under the control of the endogenous ER promoter. We will report recent findings and future perspectives obtained from the analysis of these mutants.

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The Arabidopsis ACL5 gene, whose inactivation causes a severe defect in the stem elongation, encodes a polyamine biosynthetic enzyme, spermine synthase. Although polyamines are recognized as being necessary for cell proliferation, how they control the plant development remains to be understood. To elucidate the mechanism, we have focused on suppressor mutants of acl5-1 (sac51-d-54-d). In this study, we performed cDNA microarray analysis with RNAs extracted from acl5-1, sac51-d acl5-1, and wild-type seedlings. To better view the expression profiles, genes were grouped based on their expression pattern. We identified a large number of genes whose transcript levels were increased in acl5-1, but not in sac51-d acl5-1. They include genes involved in vascular differentiation. In addition, we found that a hAT-like transposase gene was expressed at lower levels in both acl5-1 and sac51-d acl5-1 than in the wild type, and that AtMRU1 and At5g35480 transcripts were significantly increased by the sac51-d mutation.

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We developed 2-dimensional real-time imaging system for beta-ray emitters within a plant to analyze chemical translocation or signal transmission. When a radioactive tracer was applied to a plant, a beta-ray emitted from the plant was converted to light through a scintillator and then, the weak light was detected by a photon-counting camera. The image obtained by this system had a very high sensitivity for ³²P, about ten times higher than that of an imaging plate (IP) with similar resolution. It was noted that even ⁴⁵Ca and ¹⁴C, which beta-ray energy was very low was also able to apply for this system. It is also important for precise imaging analysis that the image is quantitative or not. Now we are trying to make this system allows to take quantitative images with ³²P and young soybean plants.

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Phosphate is one of the most important essential nutrients for plants. In rhizosphere, plants have many adaptations for the uptake of limited phosphate.
The response for the phosphate deficiency was not clear how the plants could detect the low phosphate concentration and the way of signal transmission in phosphate deficiency. To analyze the quick response for phosphate deficiency and successive signaling reaction, we tried real-time imaging of phosphate translocation within a plant.
Using the imaging system we developed, real-time ³²P translocation image of Arabidopsis thaliana, Lotus japonicus and Glycine max was obtained in different concentration of phosphate in culture solution. In addition of the ³²P image analysis, now we are trying to analyze the expressions of high-affinity phosphate transporters for provide more knowledge of the response for phosphate deficiency.

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