Mapping of 583,053 EST originated from 382,787 full-length cDNA clones identified 29,642 transcription units (TU) on rice genome. Comparison of 57,535 TU originated from 59,712 predicted CDS revealed 25,249 of Annotated Expressed genes (AE), 32,286 of Annotated Non-expressed genes and 5132 of Non-annotated Expressed genes. InterPro analysis of the amino acid sequences of corresponding CDS showed that AE and NAE genes have the similar profiles to the profile of the completely sequenced 32,127 clones, while ANE genes have rare hit of InterPro domains and occupied by the domains related to the transposable elements.

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We are in the process of several large-scale sequence analyses aimed
at elucidating the whole genetic system involved in nitrogen fixation
and photosynthesis. One of such projects is the genome analysis of
the model leguminous plant, Lotus japonicus. Using the
information on ESTs and cDNA markers of legume plants, genomic clones
corresponding to the multiple seed points of the genome have been
chosen as initial targets. As the accumulation of the seed sequences
has progressed, clone selection by walking has also initiated. To
analyze long-range genomic sequence rapidly, we developed an
annotation pipeline to automate gene-finding. The sequence data,
annotation and mapping information are available through the WWW at
http://www.kazusa.or.jp/lotus/. In this presentation, we will review
on the current status of our intensive genomic studies including
generation of DNA markers and a genetic linkage map, construction of
genome and cDNA libraries and their sequencing of Lotus
japonicus genome.

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With the accumulating genome sequences and their positional information, comparative genomics has become feasible in Lotus japonicus. As a first trial, synteny analysis of L. japonicus was carried out against Arabidopsis thaliana and Medicago truncatula.
Synteny analysis between L. japonicus and A. thaliana was performed on the basis of gene-by-gene comparison. Currently, syntenic relations consisting of 3 or more genes were found in 65% of annotated L. japonicus clones. On the other hand, when the assigned syntenic regions were plotted on each chromosomal position, no clear macrosyntenic relationship was observed.
Regarding to the synteny between L. japonicus and M. truncatula, syntenic relations was found in 45% of annotated L. japonicus clones. Significant level of macrosyntenic relation was found when these syntenic relations were plotted on the linkage map.
The latest status of the synteny analyses of L. japonicus will be presented.

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Cyanidioschyzon merolae is a unicellular red alga that lives in sulphate-rich hot springs. It has 20 chromosomes which vary from 422 kbp to 1.6 Mbp in size. The entire C. merolae genome of about 16.5 Mbp was determined (Matsuzaki et al. 2004). The architecture of telomeres is related to the integrity of eukaryotic genome. To determine telomeres in C. merolae, two PCR methods were used. One is a PCR amplification using polyC-tailing by terminal transferase and anchor primers. Another one is a inverse PCR method. These results suggested that sequence of telomere repeat is 5'-AATGGGGGG-3'. Using primer including this telomere sequence and primer near the end of each chromosome, end regions of other chromosomes were amplified by PCR and cloned. Their sequences suggested that C. merolae has a few complete telomere repeats at all chromosome ends.

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The chloroplast genome of some green plants, green algae, bryophytes and the plastid genome of some red algae and the apicoplast of some apicomplexan parasites have been determined. They are circular double-stranded DNAs, which share a quadripartite structure and characterized by an inverted repeat separated by large and small single-copy regions. Some legume, conifer and red algae are exceptions. We try to determine the chloroplast genome of the cotton, which is the most important natural fiber-producing plant. The Tobacco chloroplast DNA was used as a reference for the design of primers. Primer-walking strategy was adopted. Now almost around 140 kilo-base pairs of the cotton chloroplast DNA sequence were determined, depending on that, it is possible to say the cotton chloroplast DNA follow the rule of the quadripartite chloroplast structure, in addition to the well conserved sequence of the putative genes and genes arrangement.

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A chloroplast is the most important organella in plant involved in photosynthesis and production of various metabolites, but the most proteins are the nuclear-encoded. In Arabidopsis, a large-scale analysis of mutants is possible because many tag lines exist. For the functional analysis of the nuclear-encoded chloroplast proteins, we systematically collected these tag lines. 2,090 chloroplast proteins are identified by at least 3 of the 4 predictors (iPSORT, PCLR, Predotar, and TargetP), and 1,277 genes were disrupted by insertion of Ds or T-DNA, which is about 61 % of 2,090 chloroplast proteins. This collection of tag lines become larger as numbers of tag lines increase. We have not only isolated albino or pale-green mutants (apg), but also collected homo lines without clear phenotypes when plated on agar medium. Using these homo lines, various screening will be performed to identify novel functions of chloroplast protein.

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The primitive unicellular red alga Cyanidium caldarium, as well as Cyanidioschyzon merolae, grows in acidic and high temperature conditions around hot springs. Japanese Cyanidium caldarium RK-1 is found and well studied by Japanese researchers, and is known to be different from strain RK-1 used by the group of Germany on plastid genome sequence (Ohta et al. 1997). The phylogenetic analyses of some plastid genes data indicate that Japanese C. caldarium RK-1 is more closely related to C. merolae rather than German C. caldarium RK-1. We aim at complete analysis of the plastid genome sequence of Japanese C. caldarium RK-1.
The plastid genome of C. caldarium RK-1 is known to be a circular DNA composed of about 150kbp on previous study and we have analyzed ~60% of it. Although many of genes are very high similarity to C. merolae, we found a little differences of gene order.

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Now, many researchers that study a higher plant are using Arabidopsis thariana as the subjects of an experiment. All the genome information on a col ecotype was determined by the genome project in 2000. Although many ecotype and close species exist in Arabidopsis, so far, research has been advanced only within some kinds of ecotypes such as col, ler and etc. Although there is a merit that can share the experiment result by this studying a standard ecotype intensively, it is thought that many non-identified factors also exist now. We think that can use for other ecotypes and close species analyzing these new factors. It decided that genotyping of ecotypes is tried. In the beginning of study, we are genotyping for about 350 ecotypes using 16 SSLP (Simple Sequence Length Polymorphism) markers. We want to increase the number of SSLP markers as much as possible from now.

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Although cis element prediction from gene expression data is generally used, there are few reports in plant science for a comprehensive analysis of regulatory mechanisms of gene expression using bioinformatics approach. We comprehensively predicted cis elements from gene expression data obtained by microarray. Based on the results of cis element prediction, we estimated regulatory mechanisms of gene expression by computational analysis. As an example of estimation of regulatory mechanisms, genes for ribosome proteins will be presented.
<Flow of analysis>
1. classify genes using gene expression data
2. extract conserved sequences from promoter regions of the gene groups
3. evaluate effects of the conserved sequences on gene expression
4. extract non-redundant cis elements
5. assess the cis element predictions
6. estimate regulatory mechanisms of gene expression

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The Fox Hunting System (Full-length cDNA over-expresser gene-hunting system), a system for random over-expression of a normalized Arabidopsis full-length cDNA library, was applied for the functional analysis of stress-inducible transcription factors. An agro-library containing forty three stress-inducible transcription factors was constructed and used for transformation of Arabidopsis plants. Among 224 T1 transgenic plants, eight transformants expressed DREB1A gene as an internal control, with typical DREB1A-overexpressing phenotypes. Five plants expressing a zinc-finger protein gene showed hyponastic leaves and downward-pointing flowers. In these transgenic plants, expression of several genes involved in SAM development were altered suggesting that the zinc-finger protein has a important role in morphological development as well as in stress responses. In this presentation, we will demonstrate that the Fox Hunting System is useful for the screening of valuable functions from a small pool of genes such as transcription factors showing similar expression pattern.

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Arabidopsis is one of the most important model plants at post-genome era. Several public databases, which rapidly grow every day, are available to researchers to explore Arabidopsis genetic information (e.g., sequence, annotation, GO, metabolic pathway and gene expression). However, it is time-consuming to frequently traverse every distinct database sites. As these interfaces and database schemata are not uniform, researchers have to learn how to use each database. Moreover, GO analysis is also troublesome to find the ancestor nodes defined by the brief GO_terms, since the majority of GO_terms appears at more than one node in Dag (directed acyclic graphs). To avoid these difficulties, we have developed a database KATANA (Kazusa Arabidopsis thaliana Annotation Abstract. http://www.kazusa.or.jp/katana/). Our database can simultaneously and quickly give the answer of the distinct databases to researchers' queries. KATANA accelerates the data-mining in plant functional genomics.

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Enormous numbers of tomato EST sequences have been deposited in the public databases (e.g., 153,820 in dbEST, Nov., 2004). We have been working on EST sequencing of the miniature tomato cultivar Micro-Tom, which is easy to grow well, even on a laboratory shelf with artificial light, similar to Arabidopsis. Our laboratory had sequenced >40,000 cDNA clones from leaf and fruit cDNA libraries and obtained 35,824 ESTs. In this study, we combined the ESTs with the publicly available tomato ESTs (186,405 ESTs in total) and then assembled them into 26,363 unigenes with a computer program, Phrap. Out of the unigenes we identified 660 and 137 unigenes that have single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs), respectively. The unigenes that shared homology with Arabiopdis genes were further assigned with GO terms via the Arabidopsis GO-terms. We deposited these results in our Micro-Tom database, which will facilitate functional genomics in tomato.

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The sequences around translation initiation codons (from -20 to +13) were compared among 271 cDNA clones obtained from peach fruit. Following features were found: 1) three or more consecutive bases containing single kind or repetitive sequences which is made up of six or more bases are frequently existed in 5' untranslated regions. 2) from -4 to -1, "A" appears frequently than others, namely 43% at -1 and "AA" at -2 to -1 is 25%. 3) "G" appears 72% frequency at +4, "C" at +5 is 55%. Accordingly, most frequently found amino acid residue at second is Alanine with a 40% frequency. 4) in frame stop codon is found in 49 clones (18%) and out of frame in 109 clones (40%). Collectively, many different features are found in the sequences before and after the initiation codons, which may be correlated with establishment of translation initiation point or with efficiency of translation.

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The NAC domain transcription factor is plant specific and forms a large family in Arabidopsis. To analyze the function of this family, we applied the chimeric repressor gene silencing technology (CRES-T system) and suppressed the expression of the NAC target genes. We show here that the two novel NAC genes, NAS1 and NAS2, are the regulator for anther dehiscence. Expression of the chimeric repressor of the NAC transcription factor driven by its own promoter resulted in undehiscent anther and male sterile. This defect was due to loss of secondary wall thickenings in anther endothecium. Analysis using the promoter-reporter genes showed that promoter activities of NAS1 and NAS2 were detected in anther wall. Ectopic expression of NAS1 and NAS2 induced ectopic secondary wall thickenings in various aboveground tissues. These suggest that NAS1 and NAS2 are involved to anther dehiscence by promoting secondary wall thickenings in anther wall.

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An active transposon in intact plants has been identified in rice. The 607-bp transposon, termed nonautonomous DNA-based active rice transposon (nDart), has no coding capacity and was found in the gene encoding Mg-protoporphyrin IX methyltransferase in a chlorophyll-deficient albino mutant isolated from the backcrossed progeny of the cross between japonica varieties. nDart has 19-bp terminal inverted repeats (TIRs) and generates an 8-bp target-site duplication (TSD). At least, 13 nDart elements were found in the rice genome of Nipponbare. We also found larger elements, termed DNA-based active rice transposon (Dart) , that contained one ORF. Dart shares several features with nDart, including identical TIRs, similar subterminal sequences and the generation of an 8-bp TSD. We conclude that these active transposons in intact rice plant, nDart and Dart, belong to the hAT superfamily of class 2 transposons.

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Rice full length cDNA sequence data were used to design probes of 22K rice oligoarray (Agilent Technologies), and the reproducibility and reliability of the oligoarray has been confirmed, and reported by many researchers.
Advantage of microarray experiment is that many gene expression profiles can be obtained by only a few experiments, and researchers can select many candidate genes that are important to some phenotypes. However, for the further analysis of gene function, "many" genes are problem. If number of candidate genes were more than a few hundreds, almost researchers could not extract important genes from candidate genes. Therefore, tools of finding important genes are needed. In this talk, we will present about data sharing and utilization of QTL information for microarray data analysis.

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Bacterial wilt is caused by a soil borne pathogen, Ralsotonia solanacearum, and damaging the production of major crops such as potato, tomato, and eggplant. The pathogen infection occurs mainly at roots via wounds or secondary roots. In early stages of infection, the pathogen multiply in the vascular element and produce extracellular polysaccharides clogging their vascular system. The pathogenicity and molecular mechanism of the infection of R. solanacearum have been well studied through molecular genetic approaches and the genome sequencing project. On the other hand, only a limited amount of information is reported for plant responses during the infection or development of disease resistance. Here, we report gene expression profiles of Tobacco plants during the infection of either an incompatible- or a compatible-strain of R. solanacearum, using a DNA array filter set with about 11,000 non-redundant tomato EST sequences.

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Regulatory genes, such as transcription factors, protein kinases and F-box proteins play critical roles in life cycle of higher plants. Although extensive studies have been carried out for functional analysis of individual regulatory genes, the function of only a small fraction of these regulatory genes has been revealed so far.
It is valuable to learn how the regulatory genes are expressed and regulated at the whole-genome scale. Therefore, we prepared Arabidopsis whole-genome regulatory gene oligo DNA microarray containing all transcription factors, protein kinase and F-box protein genes in Arabidopsis genome recently. The 60-mer oligo DNA corresponding to 1979 transcription factors, 1060 protein kinase and 551 F-box protein genes are spotted on slide glass as a custom array of Agilent Co. In this meeting, we present expression profiles of regulatory genes in various treatments and plant tissues using the Arabidopsis regulatory gene oligo DNA microarray.

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AtGenExpress is a multinational project to collect and distribute transcriptome-data sets of Arabidopsis thalinana. using a highly standardized platform Affymetrix GeneChip, ATH1, which covers most Arabidopsis genes in the genome (about 23,000 probe sets corresponding to about 25,000 genes). The consortium collected data of developmental series, organ specificity series, light response series, stress response series, pathogen response series, hormone response series, nutrition response series, and etc. The data consists of >1,000 GeneChips (hybridizations). More than 20,000 genes were detected to be expressed significantly in any of the experiments. This is one of the largest numbers of gene expression detected in an organism. All collected data are integrated, displayed, distributed for public access at TAIR, http://www.arabidopsis.org. RIKEN-data set is also available at http://pfg.psc.riken.jp/AtGenExpress/index.html. In this presentation, we will mainly focus on how to use the data and results from large-scale clustering analyses.

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We are running various experiments using the rice 22K oligoarray based on the sequences of full-length cDNA clones. In this presentation, we will discuss about a preliminary cluster analysis performed to understand the relationship between gene expression data in callus formation or regeneration process.
Thirty-four data set of microarray experiments were combined, and K-means clustering was performed based on similarity of the expression pattern obtained from these experiments. Clones were classified into 100 clusters by this clustering. The smallest cluster contained 5 clones, while the largest cluster contained 384 clusters.
The result was stored in a newly constructed relational database, which also includes various annotation information from rice full-length cDNA database (KOME), and Gene Ontology datasets, which is an standard reference in integration of gene annotation data. Based on this database, we surveyed the possibility of the genes as contributors of callus formation or regeneration process.

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Gene expression in Nipponbare and two lines (HW1 and HW5) overexpressing a mutant gene for anthranilate synthase alpha subunit (OASA1D) were evaluated in relation to methyl jasmonate treatment.
Two- factorial ANOVA of microarray data revealed that some (1.7% = 371/21495) genes other than OASA1D were differentially expressed in these three genotypes. Genes involved in the biosynthesis of tryptophan from D-arabino-heptulosonate 7-phosphate were upgregulated by exposure to methyl jasmonate but downstream genes that process tryptophan into other metabolites did not show a similarly coordinated response. This may be a reason for the accumulation of tryptophan in OASA1D lines.
Genes showing significant differences among genotypes were observed in stress signal transduction pathways and in the biosynthesis of other amino acids, hormones, indoles and flavonoids. Pathway analysis of gene expression in transgenic rice has revealed important insights into the effects of genetic transformation and exposure to methyl jasmonate on the rice transcriptome.

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Plastids are semiautonomous higher plant organelles possessing own genome and exhibiting their own gene expression system. Genes encoded in plastid genome are transcribed by PEP and/or NEP. PEP (plastid-encoded plastid RNA polymerase) is composed of the core enzyme including catalytic subunits and σ factors which regulate the transcriptional initiation. Furthermore, it is considered that transcriptional regulator proteins and additional PEP subunits which modulate enzymatic activity also exist in plastids.
We have been already prepared the PEP fraction, which excludes σ factors-related activities, from wheat plastids. Proteins contained within this fraction were analyzed by mass spectrometry (LC-MS/MS). As expected, all PEP core subunits (α, β, β', β") were included in this fraction, however any σ factor proteins were not detected. This fraction also contains several proteins related to the plastid gene expression and protein maturation, e.g., transcription-related proteins, ribosomal proteins and molecular chaperones. This study was partly supported by METI/NEDO.

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We performed the proteomic analysis of the Golgi membranes in rice cell to clarify the structure and function of rice Golgi complex. We isolated the IDPase-associated Golgi membranes by a discontinuous sucrose density gradient centrifugation. The proteins existed in this fraction were analyzed by 2-D electrophoresis-MS and shot gun analyses. We wre able to detect seventy well-known Golgi-localized proteins such as N-acetylglucosaminyltransferase Ι-like protein in the fraction. But many ptoteins other than Golgi-localized were also identified. In order to obtain the further purified Golgi membranes,rice cells were transformed with 35S::AtSYP-31(cis-Golgi marker)/GFP,and then the AtSYP-31/GFP-labeled Golgi membranes were separated by floating in a sucrose density gradient. The ratio of GFP/total proteins of the Golgi membranes was found to increase 20-fold compared with that of the original micorosomal membranes. The identification of proteins associated with the AtSYP-31/GFP-labeled Golgi membranes will be reported.

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Phytopathogenic fungi such as Fusarium species synthesize trichothecene family phytotoxins. We are investigating the mode of action of trichothecene in Fusarium-susceptible Arabidopsis. The necrotic lesion formations were observed in these trichothecene-infiltrated leaves. Those lesions exhibited induction of cell deaths, generation of hydrogen peroxides, activation of two MAPKs and induction of defense genes in Arabidopsis leaves. In this study, we perfomed a 2D-LC method for differential display of protein expression during trichothecene-induced cell death in Arabidopsis. The method involves fractionation according to pI using chromatofocusing in the first dimension followed by separation of proteins in each pI fraction using nonporous reversed phase HPLC. A 2D map of protein content of each sample based on pI versus hydrophobicity as detected by UV absorption was generated. A differential display 2D map indicated the presence of up- or down-regulated proteins during these cell deaths. Furthermore, we tried to identify these proteins by MALDI-TOF/MS.

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Cystathionine γ-synthase (CGS) of Arabidopsis catalyzes the key step of methionine biosynthesis. Expression of the CGS gene is negatively feedback-regulated at the step of mRNA stability in response to S-adenosylmethionine (SAM). The regulation was reproduced in an in vitro translation system of wheat germ extract. Studies using the wheat germ extract revealed that, prior to the CGS mRNA degradation, translational pausing was induced in response to SAM application. In order to further analyze the regulation mechanism, an in vitro system derived from Arabidopsis will make an ideal system. In this study, we developed an in vitro translation system from Arabidopsis callus cultures. As analyzed by reporter assay, the Arabidopsis cell extract system shows translation activity comparable to that of wheat germ extract, and the level of downregulation by SAM was also at the same level as in wheat germ extract.

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Metabolic profiling (metabolomics) has emerged as a new research area in functional genomics and is expected to provide biochemical information for identifying physiological roles of novel genes of unknown functions. In this report, we treated Arabidopsis seedlings with known enzyme inhibitors (active ingredients of commercial herbicides) and analyzed the changes in metabolic profiles in such herbicide-treated Arabidopsis tissues using our FT-ICR MS metabolomics platform. After mass correction by internal standards, acquired data was subjected to PCA, and the Arabidopsis metabolites were classified into several herbicide-treated metabolome clusters. Analyses of T-DNA insertion mutants for the herbicide target enzymes are also underway. We discuss potential to use the FT-ICR MS metabolomics platform as a new tool for finding novel enzyme inhibitors or characterization of genes of unknown functions.

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A metabolomics platform on a basis of Fourier Transform-Ion Cyclotron Mass Spectrometry (FT-ICR MS) was implemented for simultaneous analysis of a wide array of metabolites present in crude plant tissue extract. A big advantage to use FT-ICR MS is the applicability for the analysis of crude tissue extracts without any chromatographic separation processes. Another important benefit is the very high resolution, which could lead to the prediction of molecular identity of each metabolite present in such crude extracts. Tools for MS data handling and statistical analysis were also developed to obtain metabolic fingerprinting patterns from FT-ICR MS analyses. In this study, we extracted Arabidopsis cultured cell with 100% methanol, and the crude extracts were subjected to MS analysis using electrospray ionization method. Metabolic fingerprinting patterns were obtained from T87 cells of different culture conditions. Further analysis is underway to identify specific compounds found in cultured cells of P450 gene overexpression.

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We produced F₁ hybrid tomato lines by crossing the miniature tomato cultivar Micro-Tom and the wild tomato Lycopersicon pimpinellifolium and analyzed the metabolites using HPLC and GC-TOF-MS (gas chromatography time-of-flight mass spectrometry). We showed that lycopene content of L. pimpinellifolium in fruit was significantly higher than in Micro-Tom. Lycopene contents of the F₁ hybrid fruit were between those of L. pimpinellifolium and Micro-Tom. Moreover, we performed GC-TOF-MS analyses of the metabolites of these tomato lines in biological triplicates. Principal component analysis (PCA) showed that the tomatoes with the same genetic backgrounds were distinguished in their metabolite profiles, suggesting that metabolome profiling might be useful for evaluating genetic traits in breeding.

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Flavonoids derived from organs of Lotus japonicus accessions Gifu and Miyakojima were identified using reversed-phase HPLC with on-line photodiode array detection and electrospray ionization mass spectrometry method (HPLC/PDA/ESI/MS). Several flavonoid glycosides including four types of flavonol and two types of anthocyanin were detected in leaves, stems and flowers, while isoflavonoid derivatives from daidzein, formononetin and coumestrol were detected exclusively in roots. The two accessions exhibited different accumulation pattern and the contents of flavonoids in organs. We will also present the metabolite data obtained by GC/TOF/MS (gas chromatography coupled to time-of-flight mass spectrometry) and CE/MS (capillary electrophoresis coupled to mass spectrometry).

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Cryopreservation in liquid nitrogen is the only method available to ensure safe and cost-effective preservation of various bioresources, including plant cultured cells. The cryopreservation protocol for cultured cells requires high cell viability after storage in liquid nitrogen to ensure the regeneration of suspension cultures and to avoid the selection of particular types of cell. In this study, we tried to cryopreserve tobacco BY-2 by encapsulation simple prefreezing method and encapsulation vitrification method.
BY-2 cells were successfully cryopreserved by both encapsulation simple prefreezing method and encapsulation vitrification method. Many of cells immobilized in alginate gel beads survived the storage in liquid nitrogen and recovered their growth ability after rewarming. The encapsulation simple prefreezing method achieved higher cell viability and more rapid regeneration of suspension cell cultures than the encapsulation vitrification method. These results suggest that the encapsulation simple prefreezing method is better for cryopreservation of BY-2 cells.

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We are currently working on producing transgenic suspension-cultured cell lines that over-express or suppress Arabidopsis metabolism-related genes under strong promoters. We chose more than one thousand of metabolism-related genes, especially whose cDNA clones were available as full-length ones from the RIKEN Gene Bank. The full-length cDNA fragments were transferred into binary vectors at the downstream of the CaMV 35S promoter with a high-troughput protocol we developed. The gene constructs were introduced into the Arabidopsis suspension-cultured T87 cells with the Agrobacterium-mediated transformation protocol we developed (see Ogawa et al. in this annual meeting). In most cases, more than 70% of hygromycin-resistant calli exhibited high levels of transcripts derived from the transgenes. Unless developmental phenomena are concerned, the suspension-cultured T87 cells would be ideal for functional genomics, especially of the metabolisms observed in the cells.

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An efficient method to characterize functions of many novel genes, which are discovered by genome analysis, is to overexpress the genes in cells and to analyze the phenotype of transformed cells using suspension culture system. However, because the growth of large numbers of independent plant cells in suspension usually require large facilities, a novel method to culture many independent cell lines with small quantity at the same time should be developed. Therefore, we developed a 96-well culture system using a 96-well deep plate, a gas permeable filter and a newly developed rotator for the 96-well deep plates. By using this system, we could cultivate tobacco BY-2 cells over six weeks without any growth defect under a condition to subculture once a week into flesh medium using a multi-channel pipette. Currently we are testing if this method can also be used to grow rice Oc cells in suspension.

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PAP1 encodes the MYB transcription factor that enhances expression of a set of anthocyanin biosynthesis genes in Arabidopsis thaliana. To elucidate the biosynthesis mechanism, we overexpressed the gene under the strong promoter in the Arabidopsis suspension-cultured T87 cells. Metabolome analyses using high-performance liquid chromatography-mass spectrometry (MS) and gas chromatography time-of-flight MS revealed accumulation of anthocyanins, cyanidin derivatives in the transgenic cells. The transcriptome analysis of the cells using the Agilent 21,500 DNA array chips indicated induction of some anthocyanin biosynthesis genes. In addition, putatively assigned genes for glycosyltransferase and acyltranserase were also induced in the cells, suggesting the involvement of these genes in the anthocyanin biosynthesis. Induction of some genes encoding glutathione S-transferase and transcription factors, which was not shown in transgenic plants, suggests a distinct control mechanism of PAP1 in suspension-cultured cells.

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Despite of considerable contributions of the model plant Arabidopsis thaliana in plant molecular biology, it has a limit in the secondary metabolism research, as the number of metabolites found in Arabidopsis is lower than those of other plants. Therefore, other model plants could be useful. The legume model Lotus japonicus could be one of the candidates as about 30% of the whole genes show no significant homology with the Arabidopsis genes. We have used suspension-cultured cells of L. japonicus for functional analyses of metabolism-related genes. Our previous study showed that yeast extracts induced expression of several genes. Here we analyzed metabolites of the elicitor-treated cultured cells using LC-MS, GC-TOF-MS and CE-MS, and found that isoflabonoid-like metabolites increased in the cells, but most of other metabolites remained unchanged. DNA array analysis of the cells and subsequent analysis using the Kazusa Plant Pathway Viewer program showed specific activation of isoflavonoid biosynthesis.

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Comprehensive analyses of plant transcripts and metabolites with DNA array technology and chromatographic separation techniques coupled to mass spectrometry such as gas chromatography-mass spectrometer (GC-TOF-MS), respectively, produce a vast amount of quantitative data. It is crucial to have computational tools to mine meaningful data from huge databases of transcriptome and metabolome. Thus, we have been developing the web-based plant metabolic pathway viewer, Kazusa Plant Pathway Viewer (Kappa Viewer) for displaying transcript and/or metabolite data on plant metabolic pathway maps. We prepared 140 metabolic pathway maps for covering major metabolism of Arabidopsis thaliana. We will introduce the latest version of Kappa Viewer that has summarized indicator map for over-viewing the whole changes of transcripts and metabolites as a new function.

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RIKEN BioResource Center (RIKEN BRC) has joined with National Bioresource Project conducted by Ministry of Education, culture, sports, Science and Technology. Through the project, RIKEN BRC collects, examines, preserves and distributes Arabidopsis seeds, plant DNA materials and plant cultured cell lines. Until now, 880 laboratories from 39 countries have registered as our user. By the end of this September, we have collected approx. 250,000 and distributed approx. 50,000 materials.
RIKEN BRC also promotes projects on the development of novel resource and technology. With this project, we are going to establish the techniques required for genotyping of natural accessions of Arabidopsis as well as cryopreservation of plant cultured cells. These techniques will be provided to research community through the training course that has started from this year.
In near future, we will develop and collect more plant DNA materials and cultured cell lines to contribute plant science.

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RIKEN Arabidopsis Genome Encyclopedia (RARGE) database provides information of basically Arabidopsis resources which includes full-length cDNAs, their promoter regions, Ds transposon-tagged lines and microarray expression profiles, and implements searching tools using resource code and words as a query. We have isolated 245 946 of cDNAs and collected 11 933 of transposon-tagged lines. Recently, we added a novel data on alternative splicing to the database. Data on 1,794 TU of alternative splicing variants are available in RARGE. Users can browse these data categorized by splicing type with gene alignment and genome mapping view. In this meeting, we describe about function enhancement of searching service for integrated analysis supporting biology researchers
RARGE (http://rarge.gsc.riken.jp/)

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RNA isolation from siliques and dry seeds of Arabidopsis thaliana requires complex methods due to the high contents of polysaccharides and secondary metabolites. We herein report an RNA isolation method that basically consists of use of high-sodium extraction buffer, two-step extraction with organic solvents, isopropanol precipitation alone. LiCl precipitation was additionally conducted if necessary. The yields of total RNA from siliques, dry seeds, flower buds, leaves, roots and stems were 286 ± 20, 1104 ± 75, 1159 ± 135, 550 ± 108, 513 ± 91 and 392 ± 42 μg g^-1 tissue, respectively. Severe contamination by protein or polysaccharides was not found spectrophotometrically. The total RNA fractions were undegraded and could be applied to RT-PCR. Overall, this method drastically reduced the time required for RNA isolation from siliques and dry seeds of Arabidopsis thaliana, and was simple enough to be routinely applied to other tissues.

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Laser microdissection (LMD) is a powerful tool for tissues or single-cell analyses. However, the difficulty of preparing cryosections and total RNA from plants constrains the technology to be unpopular in plant research. Although T7 RNA polymerase-based in vitro transcription protocol has been widely used for amplification from a hint of total RNA, the average length of cDNA becomes one-forth in 2 rounds of amplification. Thus, we employed the SMART protocol for preparing cDNA libraries from about 0.5 ng of total RNA that was extracted from pith of Arabidopsis stem by LMD, and obtained 3,697 Expressed Tag Sequences (ESTs). BLAST search suggested that about 30% of cDNA clones were full-length. As 70% of the ESTs were singletons that indicates the protocol had sufficient quality for further transcriptome analyses. We will also present the comparative study of these protocols when applied to microarray analysis.

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Here we report success of a chloroplast transformation in a poplar (Populus alba) cell. Plastid transformation was performed with poplar leaves by particle bombardment using the chloroplast transforming vector pCB1GFP that carries the coding sequences for a spectinomycin resistance gene (aadA) and a green fluorescence protein gene. From the 40 bombarded leaves, 106 spectinomycin resistant green calli were obtained on selection medium while most leaf sections became bleached. A green fluorescent cell was observed in 17 calli of the 106 calli. The genomic DNA was isolated from 3 of the 17 calli and analyzed by PCR. PCR analysis confirmed that the introduced genes were site-specifically integrated into the plastid genome of the poplar in the all analyzed calli. Regeneration of transformed poplar from calli is in progress. To our knowledge, this is the first report on chloroplast transformation for trees. This work is partly supported by METI.

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We have established a stable genetic transformation method for Hinoki cypress by Agrobacterium--mediated gene transfer system. Embryogenic tissues are co-cultivated with disarmed Agrobacterium tumefaciens strain C58/pMP90 containing a binary vector pBIN19-sgfp, and the infected tissues were screened by both nptII selectable marker and GFP fluorescence. The transformation frequency varied up to 22.5 independent transgenic lines isolated per petri dish that include 250 mg of embryogenic tissues. Eight independent transgenic lines were regenerated through maturation and germination steps of developing somatic embryo and their intensity of GFP fluorescence was observed under a fluorescence microscope. The 35S promoter induced strong GFP fluorescence in embryogenic tissues and roots, but showed quite low intensity in leaves and stems. In searching for constitutive promoters, we are producing transgenic lines expressing a gfp gene under the control of maize ubiquitin promoter.

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Plastid transformation technology has several potential advantages for basic researches and biotechnological applications, including high-level gene expression and gene containment. Glutamine synthetase activity in chloroplasts is responsible for scavenging ammonia produced in the photorespiratory pathway. Indeed, nuclear transformants overexpressing plastidic GS2 showed an improved tolerance to high-intensity light (Kozaki and Takeba, 1996). The nuclear transformants had twice the normal amount of GS2. To achieve higher levels of GS2 protein accumulation in chloroplasts, we have over-expressed rice GS2 gene in chloroplasts. The chloroplast transformants accumulated 40-fold GS2 proteins compared to non-transformed plants in leaves. Plastid-expressed GS2 could provide very high levels (5-7-fold) GS2 activity. Gel chromatography and non-denaturing PAGE analyses demonstrated the formation of catalytically active octamer of GS2 in the transplatomic plants overexpressing GS2.

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Bean bug,Riptortus clavatus, causes a serious damage of soybean production. The insect pest utilizes cysteine proteinases for protein digestion. In contrast, plants contain proteinous inhibitors, cystatins, against endogenous and exogenous cysteine proteinases including insect digestive proteases. In this research, we produced transgenic soybeans overexpressing plant cystatins to confer the insect resistance. We used three plant cystatins, OC1, mCC1 and JTC, from rice, corn, and Job's tears, respectively. Each of the cystatin genes was connected with a seed specific promoter derived from soybean 7S globulin gene or CaMV35S promoter, and nos-terminator, and the resultant cassette was inserted between the green fluorescence protein gene and the hygromycin resistance gene in a pUHG plasmid vector. The plasmids were introduced into soybean embryogenic cells by particle bombardment. Several transgenic plants were obteined by introduction of these constructs and we are now developing fixed lines for transgenes to analyze the resistance to R. clavatus.

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Plant production is limited in acid soils. The main constraint to plant growth on acid soils is the aluminum. The Al ions not only are toxic to root growth but also lead to phosphate deficiency. Certain plant species showing tolerance to these stresses release large amounts of organic acids, especially citric acid, from their roots. Genetic manipulation may be an approach to introduce such traits into plants. In the present study, we generated transgenic Arabidopsis thaliana with repressed cytosolic NADP-isocitrate dehydrogenase (NADP-ICDH) gene involved in citrate catabolism by RNAi technique. NADP-ICDH activities of transformants were repressed to approximately 10% of wild-type plants. These transformants increased citrate content and released 2.5-fold more citrate at the maximum compared to wild-type plants. In addition, transformants showed improved growth on acid soils. These results suggest that repression of NADP-ICDH expression is a useful strategy to enhance the ability of acid soil tolerance of plants.

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Transgenic rice plants expressing a gene encoding rice PHD-finger type homeodomain protein (RNBP97) homologous to alfalfa Alfin1 under the control of CaMV 35S promoter were generated. Ectopic expression of this gene brought several unusual phenomena to the transformants from the beginning of plant regeneration. Some regenerated shoots indicated necrosis and eventually died during in vitro culture. The leaf blade and sheath of some transplanted transformants formed spots similar to the lesion of hypersensitive response. Furthermore, some transformants were headed panicles within one month of cultivation under the long-day condition, and a half of transplanted transformants was sterile. Root growth enhancement observed in transgenic alfalfa was not occurred in the plumules of T1 transgenic plants.

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We have succeeded in disrupting the rice Waxy gene by introducing the hygromycin resistance gene into its intron 1 through homologous recombination. The vector used for the gene targeting carried 6.3- and 6.8-kb Waxy segments for somatic homologous recombination. To examine whether the length of homology affects recombination processes, we are trying to generate the Waxy gene having its entire coding region deleted by employing a vector containing 6.3-kb Waxy promoter and 3.5-kb segment having removed the Waxy coding region. Out of five candidate transgenic T0 plants producing the expected junction fragment for homologous recombination, detected by PCR screening, only two plants showed expected segregation patterns of the Waxy phenotype in both pollens and seeds with iodine staining. We will present molecular analyses of obtained T0 plants and discuss possible implications of gene targeting in rice.

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Transgenes stably integrated into chromosome are often silenced, a phenomena is called gene silencing. Gene silencing has been attributed to several factors, including differences in chromosome position, repeat sequences, and copy number. We transformed Arabidopsis plants with pBI121 (CaMV35S-GUS and NOS-NPTII) using Agrobacterium-mediated transformation. We carefully selected ten lines containing single 'complete' (intact, non-truncated, non-rearranged) copies of the transgene located at different chromosome positions. These lines showed very similar levels of transgene expression, and gene silencing was not observed. In a subsequent study, genetic crosses of isolated lines were performed to generate plants harboring one to eight copies of the transgene. We have been investigating whether gene silencing dependent copy number of the transgene is occur.

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Intervening intron-spacer between sense and antisense sequences forms the most effective dsRNA structures in target gene disruption by RNAi. Previous our research showed that RNAi vector WRI-A with Wx promoter, the 5'UTR sense and antisense Wx sequences, intron-spacer could lead to complete suppression of Wx expression in endosperm, but not in pollen. By contrast, WRI-B carries the substitution mutation at 5'splice site of the intron-spacer could lead to lower degree of Wx suppression endosperm. Further studies examined the effects of various vectors on RNAi as follows; 1) comparison of efficiency of the vectors with another mutation at splice site of intron-spacer, 2) the influence of the dsRNA with different trigger regions on tissue specific suppression of Wx, 3) effect of trigger length of dsRNA on degree of Wx suppression. And results from these experiments will be discussed.

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Previously, we reported binary vectors compatible for Gateway Cloning Technology. They were used for construction of reporter/tag fusion genes. In this paper, we report the Dual Site Gateway Binary Vector System for cloning of two genes. With this system, reporter / tag sequence is independently fused to two genes. This system is very useful for plant research, because the biochemical and cellular analyses of interaction between two genes interested are very important.
In this paper, we report the vector containing new recombination sequences attR3- attR4 in addition to attR1-attR2. Using this vector, two genes were able to be cloned in one vector by LR reaction. We also report application of this system.

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In 2002, a photo-convertible fluorescent protein Kaede was isolated from stony coral, Trachyphyllia geoffroyi, and described. Kaede emits bright green fluorescence, but can change its color efficiently to a bright and stable red fluorescence after exposure to UV or violet light (350-400 nm). The photo-conversion is irreversible. We confirmed that Kaede was expressed correctly and its green-to-red color change was also observed by violet light illumination, in onion, tobacco and Arabidopsis by the particle bombardment method. By using Kaede, we demonstrated that plant mitochondria frequently undergo both fusion and fission. We also developed transgenic Arabidopsis plants that constantly express Kaede targeted to particular organelles. These plants are useful for making further observations of the frequency and manner of fusion of organelles in various organs. We are presently growing T2 plants and investigating the effects of Kaede on their growth.

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