Among a variety of putative transposable elements found in the liverwort Y chromosome, one class contains a DNA methyltransferase (DNMT) domain associated with the polyprotein typical for retrotransposons, and was designated as DNMT-containing repetitive element (DRE). To date, no retrotransposon associated with DNA methyltransferase has been reported. DRE has gag-pol genes of Ty3-gypsy type and long terminal repeats (LTRs), but the 3'-LTR is located between another pair of direct repeat. The DNMT domain shows the highest similarity to that of the mammalian DNA methyltransferase 3A (DNMT3a), which is required for imprinting of germ cells. Furthermore, each copy of DRE appears to have incorporated a reverse-transcribed product of different mRNA. Approximately 150 copies of DRE are present in both the male and female genomes, but the mechanism of its transposition is currently unclear.

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Students who received high school education guided by the new national curriculum standards, so called "cram-free education," entered universities in the spring of 2006. Anticipating the arrival of students with new educational background, members of Biology division, who take charge of "the Introductory Course in Laboratory Experiments" in the field of life science, newly developed three experimental materials for the course and also carefully refined the old contents. In this meeting, we report on the two new experiments, "Separation of photosynthesis-related proteins by SDS-PAGE", using cyanobacterium and "Diversity of plant reproduction processes", using green algae, ferns, and seed plants, and one refined experiment, "Structure of vascular bundle in angiosperms". We will also exhibit a new textbook for the experiments accompanying DVD as a new educational tool that has been developed in order to improve the course instructions.

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A miniature cultivar Micro-Tom has attracted much attention as a model of tomato (Solanum lycopersicum) because it has small size (10-20 cm in height), a short life cycle (70-90 days), and grows well in ordinary laboratory spaces. We have constructed full-length cDNA libraries from Micro-Tom and generated ESTs from the clones as a genomic resource. From the ESTs, non-redundant cDNA clones were selected and used to obtain the full-length cDNA sequences. At present, we generated 57,271 ESTs (including Acc. BW684914-BW692959, DB678295-DB727670) and 1673 full-length sequences (Acc AK224591-AK224910, AB211518-AB211526). We have analyzed the ESTs and full-length sequences by using BLAST and InterPro programs. Additionally, to elucidate intron-exon structures of the tomato genome, we compare the full-length sequences with genomic sequences provided from the international tomato genome sequencing project. All the information is available from a database KaFTom (http://www.pgb.kazusa.or.jp/kaftom/).

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Fagopyrum esculenthum is one of the several crop species possessing strong allelopathic properties. In our previous study we had identified eight allelochemicals in buckwheat and analyzed by microarray analysis two important compounds such as rutin and gallic acid.The gene expressions of 20 days old A. thaliana plants were analyzed using Affymetrix GeneChips ATH1. The results showed 168 and 55 genes with higher expression after 6 hours of exposure to gallic acid and rutin, respectively. However, only 14 genes were found common for both compounds. The study revealed some genes which are important in regulating plant responses to stress. Induced genes fell into different functional categories mainly, metabolism; cell rescue, defence and virulence; cellular communication/signal transduction mechanism and transcription. This study may lead to a better understanding of the allelochemicals mode of action which in the future could be used in biological control of weeds.

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Little is known about how gene expression between orthologous genes of two species is quantitatively conserved among plant species. To address quantitative gene expression conservation during plant evolution, we compared quantitative gene expression data of two model plant species, Arabidopsis and tomato, which were obtained from DNA array analyses of leaf and root of two species. With FASTA search, we selected 4,274 putative orthologous gene pairs from the GeneChip sequence data for the species, and found that a strong conservation (< E-100) was seen in 19% of gene pairs, although some gene pairs exhibited low conservation. Quantitative expression levels in Gene Onthology categories such as "electron transport or energy pathways" were well conserved. To know a tendency of quantitative expression profile differences at the metabolic pathway level, we used plant metabolic pathway maps of KaPPA-View, and found that genes in "Sterol biosynthesis" were apt to express highly in tomato.

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Recent genomic analyses revealed that plants contain a large number of genes that are predicted to be involved in the ubiquitin/26S proteasome pathway. For example, nearly 700 F-box proteins have been annotated in Arabidopsis. These observations strongly indicate that ubiquitination contributes to various biological processes in plants. Indeed, genetic analyses have shown that the ubiquitin/26S proteasome pathway components regulate embryogenesis, hormonal responses, circadian rhythms, floral homeosis, photomorphogenesis, trichome differentiation, senescence, and pathogen defense. However, only a few target proteins for ubiquitination were so far identified.

Here, we present a novel method for identifying ubiquitinated proteins from plants. Ubiquitinated proteins were enriched by an affinity column that utilizes ubiquitin-binding domains isolated from Arabidopsis. After trypsin digestion, peptides were separated and analyzed by 2DLC-MS/MS. 2D separation was performed online with a strong cation-exchange chromatography followed by a reverse-phase chromatography.

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The two-dimensional gel electrophoresis has been used as a powerful tool to separate proteins, and it is recently used as good resolution method for the subsequent mass spectrometric analysis. Generally, pretreatment of samples for two-dimensional electrophoresis involves solubilization, denaturation and reduction to completely break up the interactions among proteins, and removal of all interfering compounds to ensure efficient separation. One of the problems in the pretreatment is that large amount of proteins is lost, because some proteins would be aggregate and difficult to resolubilize them. Here, we show the method to get reproducibly clear images of the two-dimensional gel electrophoresis without complicated pretreatment.

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Peroxisomes are organelles, ubiquitously found in eukaryotic cells, in which a variety of oxidative metabolic reactions are compartmentalized. Peroxisomal proteins are synthesized in the cytosol, and the function after their post-translational transport into peroxisomes. Most of peroxisomal proteins have been shown to contain one of two peroxisomal targeting signals (PTSs) within their amino acid sequences. Using the PTS sequences, we predicted that 286 peroxisomal genes are present in Arabidopsis genome. Of these, functions of only 29 proteins have been demonstrated. This result suggests that peroxisomes may have divers functions than those we know at present. To explore unidentified peroxisomal functions, we analyzed proteins in soybean peroxisomes by combination of 2-dimensional gel electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The peroxisomal proteins were obtained and were then identified by peptide mass fingerprinting analysis using a soybean EST database.

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To identify the mechanism of GA signal transduction in rice basal part, a proteomic approach was used. Growth of rice seedlings were stimulated when treated with 5 μM GA₃ for 24 h to extract proteins. The difference in protein expression pattern between control and treatment was displayed by 2D-DIGE. The expression of five proteins were increased and one protein was decreased. Identification of these proteins by mass spectrometry analysed that they were elongation factor 1 beta, 21 kDa polypeptide, abscisic acid, stress and ripening 5 (ASR5) and fructose-diphosphate aldolase. After quantification of the relative expression levels of these proteins, ASR5 was significantly up-regulated by GA₃. However, ASR5 did not change at mRNA level. Polyclonal antibody was raised against ASR5 that was analyzed for subcellular distribution and ABA response. The results indicate that ASR5 is distributed in nucleus and cytoplasm, and responded to both ABA and GA.

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Photosystem II complexes are comprised of ~20 subunit proteins. Many of those subunit proteins are the hydrophobic membrane proteins; e.g., the grand average hydropathicity (GRAVY) of D1 and D2 proteins exceed 0.3. Similarly, other complexes in photosynthetic electron transport chain also consist of hydrophobic membrane proteins as well as small numbers of hydrophilic extrinsic subunit proteins. The expression level of these proteins under variable environment will be well investigated with two-dimensional gel-electrophoresis using iso-electric focusing for the first dimension (2D-gel). However, such kind of hydrophobic membrane proteins are not separated reliably on the conventional 2D-gel. We have developed a novel 2D-gel system to analyze hydrophobic proteins. For the iso-electric focusing, agarose was used as a supporting matrix and n-dodecyl-β-D-maltopyranoside was used as a surfactant. Using this system, all known subunit proteins in photosystem II complexes larger than ~10 kDa were reproducibly and clearly separated on the 2D-gel.

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Plant metabolomics has various potential applications; for example improvement for the useful products. To achieve this purpose, we have been trying to develop methodologies to describe the metabolic network analysis of atomic redistribution process using uniformly stable isotope labeled plant cells measured by hetero-nuclear NMR.
Previously, we showed the atomic redistribution analysis of carbon source [¹³C₆] glucose using simple closed system (Arabidopsis T87 cells). At this time, we will show detailed strategies of signal assignments by a combination of multi-dimensional NMR methods such as ¹H-¹³C HSQC, HCCH-TOCSY, HCCH-COSY, HCACO. Introduction of these correlation spectra exhibits overcoming of signals overlapping and unambiguous signal assignments. By use of these assigned NMR signals, we monitored the time-dependent changes of the signal intensities at atomic level in order to follow the metabolic network at atomic level.

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Technical improvement and optimization of analytical conditions for mass spectrometry enabled to detect many metabolites with high-throughput and high-resolution. However, using a single system like GC-MS can not lead a comprehensive analysis against all metabolites that have diverse chemical nature. Therefore, RIKEN metabolomics research group uses various analytical systems, such as GC-TOF/MS, LC-TOF/MS, and CE-TOF/MS, for detecting as many metabolites as possible. In these instruments, we use CE-TOF/MS system. CE-TOF/MS can perform the high resolution analysis for ionic compounds quantitatively. First, we have analyzed standard compounds using three methods to detect cations, anions, and nucleotides, and then identified 129 standard compounds based on their migration times and their detected m/z values. In analyses of Arabidopsis extracts, we could quantify 48 known metabolites and detect 3332 unknown peaks. We now analyze other standard compounds to increase the number of quantifiable metabolites. Moreover, we perform metabolic profiling analysis toward the Arabidopsis mutants.

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The NMR-based metabomics approach has much potential for not only basic science but also for applied science in plant systems. Firstly, 1D-¹H-NMR metabolic fingerprinting can be used for characterization of global metabolic changes such as RIKEN Arabidopsis disruptome collection and core-collections of rice and wheat strains. Identification of changed metabolites would be performed by multi-dimensional NMR coupled with stable isotope labeling of plants and by use of our original Java software SpinAssign and original standard database. In addition to this, the ¹³C labeling can be used in metabolic flux analysis of plant cells at atomic level. Furthermore, we have observed nicely resolved ¹H-¹³C cross peaks of non-extracted, insoluble small and macromolecules by magic-angle-spinning NMR experiments. Finally, the ¹³C-labeled plant biomass can be also used as nutritional resources for animal systems. We will show how the ¹³C labeled plants can be metabolized by animal organs using the NMR technologies.

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[Background] In spinach, integrated relationship among metabolites and nitrogen (N) nutrition is still ambiguous. We examine the influence of different nitrogen source under hydroponics conditions on spinach and focused to the profile of metabolite by using GC-MS. [Method] In Experiment 1, N concentration of nutrient solution was supplied by NO₃ at the rate of 1, 2 and 4 mmol L^-1, respectively. In Experiment 2, NO₃ and NH₄ were applied with the ratio of 10:0, 5:5 and 3:7, respectively. The extraction media (Methanol: Chloroform: Water =33: 16: 23 ) was added to lyophilized leaf disks. After TMS derivatization, each sample was injected into GC-MS. [Results] Principal component analysis shows that components reflected the amount of nitrogen amount and form applied. From the loading analysis, amino acids correspond to the nitrogen amount and form, and sugars and organic acids correspond to the amount of nitrate applied.

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The legume crops have lower nitrogen use efficiency (dry matter productivity per the amount of absorbed nitrogen) than the cereal crops. It would be due to, in legume crops, low photosynthetic rate per unit nitrogen of the leaves and the consumption of photosynthate for protein synthesis of the seeds. However, it is not clear about the detail of metabolic mechanism that caused mentioned above physiological phenomenon in the leaves and the seeds. In this study, diurnal changes of metabolic profiling in seeds in rice and soybean were compared in order to get the basic knowledge of the difference of metabolic mechanism between the cereal and legume crops. As a result, an amino acid and organic acid of soybean seeds increased than those of rice. It was suggested that larger carbon skeletons supply to an amino acid in seeds of soybean.

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[Objective] Catharanthus roseus produces about 130 terpenoid indole alkaloids (TIAs), including valuable medical substances. To date, many genetic, molecular biological, and biochemical studies on TIAs biosynthetic pathway were conducted, however, precise and comprehensive analysis of whole metabolism in C. roseus cell has not been conducted yet. In this study, metabolic profiling of methyl jasmonate (MeJA)-treated C. roseus shoot and cultured cell was obtained in order to analyze metabolic alteration when TIAs biosynthesis is activated. [Method] Treatment of MeJA was performed by transferring shoot or suspension cultured cell to the medium added with MeJA. Time-course sampling was conducted and the metabolites were extracted and purified from these samples. Sugars, amino acids and organic acids were analyzed by GC/MS. Sugar phosphates, nucleotides and CoA compounds were analyzed by CE/MS. And TIAs were determined by UPLC/MS. Obtained metabolic profiles were applied to multivariate analysis.

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We carried out comparative analyses of both metabolites and transcripts using Arabidopsis plants under low-temperature and drought stress conditions and transgenic plants overexpressing DREB1A (35S:DREB1A) and DREB2A (35S:DREB2ACA). Metabolites analyses were performed using GC-TOF-MS, CE-ESI-MS and LC-IT-MS. Identified metabolites were statistically compared by principal component analysis. Then, we selected candidates of critical factors under low-temperature and drought conditions. Transcripts analyses were performed using an Agilent Arabidopsis2 oligo array. We found that genes encoding some of the metabolic key enzymes were induced under low-temperature and/or drought conditions and, a clear correlation was observed between induction of the genes and increments of synthesizing sugars in the plants. In addition, DREB1A was related to the regulation of starch degradation and raffinose synthesis. Using GFP fusion proteins we found that a raffinose synthase encoded by a low-temperature inducible gene was located in chloroplast suggesting that raffinose was accumulated in chloroplast under low-temperature condition.

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Previously, we constructed Dual Site Gateway Binary Vector for cloning two genes on separate position. In this paper we report the new Gateway Binary Vector (R4 pGWB) to connect two DNA fragment using a part of Multisite Gateway system. R4 pGWB has attR4-CmR-ccdB-attR2 acceptor site and by the LR reaction using L4-promoter-R1, L1-cDNA-L2 and R4 pGWB, we can make promoter::cDNA construct easily. We also made R4 pGWB series for tag/reporter fusion at C-terminal of cDNA. These new vectors were named Hybrid Gateway Binary Vector system and could be a powerful tool for promoter swapping experiments.
In the report we show the promoter analysis of the gene that expressed in guard cell, as example of application of this system.

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 In plants, RNA silencing induced by highly transcribed sense transgenes makes it difficult to express the transgene at high level. SDE1 of Arabidopsis thaliana is an RNA-dependent RNA polymerase that synthesizes an anti-sense RNA from an RNA template and plays an essential role in the transgene-induced RNA silencing. To obtain transgenic lines that express transgene at high level, we produced Arabidopsis transformant in which expression of sde1 is reduced.
 We cloned a 1500-bp DNA fragment including the first intron of Arabidopsis sde1 and ligated its 5'-1100-bp fragment inversely at the 3'-end, to produce an intron-spliced double-stranded RNA. The construct was introduced via Agrobacterium and the expression of the sde1 gene in the transformed Arabidopsis was examined by RT-PCR using primers specific to sde1. The results indicated decrease in the level of sde1 mRNA in the tranformant, suggesting the possibility of suppression of RNA silencing.

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In vivo recombinational cloning in yeast is the efficient method for the cloning of multiple DNA fragments and large DNA fragments exceeding 10 kbp. Still, this cloning method has been limited to experiments with yeast vectors because many cloning vectors lack yeast replication origins. Therefore, we developed a new system to apply the yeast-recombinational cloning to T-DNA binary vectors, such as pCAMBIA. A newly constructed helper plasmid pSU7 and pSU23 allows the in vivo conversion of plant vectors into vectors replicable in yeast. The DNA fragments to be cloned are PCR-amplified with the addition of homologous sequences to a vector. Cotransformation of the helper plasmid into yeast with the vector and PCR-amplified DNA fragments, results in the conversion of the vector into a vector replicable in yeast and simultaneously allows cloning of the DNA fragments into the same vector.

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Woody plants have gotten a lot of attention as CO₂ absorption sources and an alternative energy resource. Our goal is to develop new woody plant species with high growth performance and tolerance to environmental stresses by genetical modification. One of our aims is to improve functions of Eucalyptus root system, for example the acquisition of nutrition and tolerance to stresses. But one year or more is required for getting transgenic Eucalyptus and analysis of their function. Therefore we focused on the utilization of transformed hairy roots which can be generated in a relatively short term. In this study, we developed a hairy-root transformation system that made the function analysis possible in one to two months. With this system, we are now evaluating the effectiveness and impact of promoters and genes in Eucalyptus.

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The genes for building up chloroplasts express in leaves of plants by perception of light. To know the underlying mechanism, we the selected mutants ces102 and ces103 (callus expression of RBCS) of Arabidopsis by activation tagging on the criterion of expression of RBCS-3B in calli. The gene of At3g05490 was activated in ces102, and the same phenotype was reproduced by ectopic expression of this gene, leading to the conclusion that the CES102 is the At3g05490 product. Fluorescence of CES102-sGFP was observed in endoplasmic reticulum by transient expression. As for ces103, both At3g59860 and At3g59870 were activated and the greening phenotype was reproduced by over-expression of any of these genes. While expression of RBCS-3B in the At3g59860-expressing calli was almost compatible with that in ces103, the ectopic expression of At3g59870 resulted in elevation of RBCS-3B transcripts These results indicate that CES103 would be At3g59860.

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To investigate the feasibility for using the key enzymes of formaldehyde (HCHO) fixation from methylotrophs to generate the functional ornamentals which have enhanced HCHO detoxification capacity, we introduced the fused HPS/PHI gene from Mycobacterium gastri MB19 into ornamentals, petunia and pelargonium. Western blot analyses showed that the fused HPS/PHI was successfully expressed in the two ornamental plants. The two transgenic plants displayed better resistance to HCHO than the wild-type plants when they were grown on MS medium with HCHO or exposure to gaseous HCHO. Our measurements indicated that the HCHO assimilation ability of the transgenic lines was increased 30-40% than the wild-type plants. The transgenic ornamental plants also showed a 15-40% increase in their efficiency to take up exogenous HCHO as compared with wild-type. The results suggest that the fused HPS/PHI can be applied to ornamentals to produce functional plants for phytoremediation of HCHO pollution.

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Crops that were conferred useful traits by genetic engineering have potential to increase crop yields, improve agricultural practices, or add nutritional quality to products. Despite broadly expanding of genetically modified crops over the world, risk of undesirable diffusion of antibiotics resistant genes to ecosystem still exists. To avoid this problem, development of selection marker derived from native plant gene would be favorable for plant transformation. We are now intending to develop a novel technique, with the aid of self-cloning application. As the candidate genes for native selection marker, we focused plant transcription factors that response to abiotic stress. Because some of the defective mutants show tolerance to abiotic stress, we attempt to isolate transgenic Arabidopsis plants that shows tolerance to various stress, such as salt and drought, and for herbicide exposure by CRES-T system. Molecular approaches and tools for the development of self-cloning in Arabidopsis will be discussed.

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Feral transgenic oilseed rape (Brassica napus) has been detected in central Japan. In this study, we investigated the distribution of transgenic oilseed rape and occurrence of hybridization of transgenic B. napus with feral populations of its closely related species (B. rapa and B. juncea) in the west of Japan in 2005. The progenies of 50 B. napus, 82 B. rapa and 283 B. juncea maternal plants from 95 sampling sites in seven port areas were screened for herbicide-resistance. Transgenic herbicide-resistant seeds were detected from 12 B. napus maternal plants growing at seven sampling sites in two port areas. A portion of the progeny from two transgenic B. napus plants had two kinds of herbicide-resistance transgenes. Therefore, two types of transgenic B. napus plants are likely to have outcrossed with each other. As found in the previous study, no transgenic seeds were detected from B. rapa or B. juncea.

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The cDNA for glutathione-dependent formaldehyde (HCHO) dehydrogenase (FALDH) from Arabidopsis and the fused gene for 3-hexulose-6-P synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) from a methylotrophic bacterium, Mycobacterium gastri MB19, were over-expressed in tobacco, respectively under the control of tomato rbcS-3C promoter. The FALDH and the fused HPS/PHI were expressed in the cytoplasm and chloroplasts, respectively in the transgenic tobacco. Our assay indicated that overexpression of the FALDH gene and the fused HPS/PHI gene led to an increase in FALDH and HPS/PHI activities in the two transgenic tobacco plants, respectively. The transgenic tobacco overexpressing fused HPS/PHI (HPS/PHI tobacco) showed higher tolerance to HCHO than the transgenic tobacco over-expressing FALDH (FALDH tobacco) when they were grown on MS medium with HCHO or treated with gaseous HCHO. Our measurement also suggested that the HPS/PHI tobacco had stronger ability for HCHO detoxification than FALDH tobacco.

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Since April 2002, National BioResource Project (NBRP) has promoted life science research through preservation and distribution of biological materials of model organisms. Plant resource projects on Arabidopsis, rice, wheat, barley, legume, morning glory, chrysanthemum, and algae have been supported by the NBRP. The RIKEN BioResource Center (BRC) Experimental Plant Division preserves and distributes seeds, cultured cells and cDNA clones of Arabidopsis and some other model plants through the NBRP. Current NBRP will terminate by the end of next March, and the 2nd term of NBRP is expected to start from April 2007. Here, we will present the current status and future plans of plant resource project in RIKEN BRC.

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Tomato (Solanum lycopersicum) has been an excellent model system for analysis of plant mechanism, which cannot easily be studied in Arabidopsis or rice, such as fruit physiology and secondary metabolites accumulation. Mutational approaches have been highly instrumental for the study of the genetic and molecular bases of traits. To obtain information on the genetic mechanism of fruit ripening, we constructed mutation libraries induced by irradiation with gamma-ray and accelerated heavy-ion. We used a determinate miniature cultivar Micro-Tom, which is suitable for large mutagenesis because of its small size and rapid life cycle.
We found a mutant whose leaf color was pale. The mutant also showed pale surface color of the fruit, but there no significant differences between the mutant and WT in the content of carotenoid and expression level of the genes involved in carotenoid biosynthesis.

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Real-time monitoring of gene expression using a bioluminescence reporter strain is one of the most powerful methods for the analysis of gene expression in living cells with a high sensitivity, a broad dynamic range, and a high time-resolution. The method allows easy isolation of a large number of mutants affecting the expressions of any genes in the genome. However, there were many problems in previous systems. We have developed a high-throughput real-time bioluminescence monitoring system, which consists of two types of high-throughput bioluminescence monitoring apparatus and the bioluminescence-analyzing program RAP. The system enables the automatic monitoring and analysis of bioluminescence from 4,800 individual samples in one assay under uniform light conditions.
Here, we will introduce the real-time bioluminescence monitoring system and the cloning and analysis the circadian clock genes in cyanobacteria and Arabidopsis.

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A series of cDNA clones encoding luciferases with four different colors was obtained from the bioluminescent click beetle, Pyrophorus plagiophalam. The click beetle genes have been developed as a reporter vector system and have become available commercially. The multi-color luciferases provide an ideal dual-reporter system because the two reporter enzymes are highly similar and because both signals are generated by D-luciferin treatment. To investigate the usefulness of multi-color luciferases as reporter genes in higher plants, we assayed the transient expression of luciferase genes by microprojectile bombardment. Although their expression levels were relatively low, luminescence from green and red luciferases were separable under the CCD camera equipped with interference filters. Results of time-course experiments and the inducible promoter assay suggest that the multi-color luciferase system is applicable to plant cells. We will present examples of the use of green and red luciferases to monitor regulated gene expression in higher plants.

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We have developed an imaging system and software for analyzing plant growth and development. To define specific developmental changes, a comprehensive analysis of visible phenotypes required a large-scale image database including more than a thousand growth images per plant that was acquired every minute. We also developed a software platform to observe serial images quickly, measuring growth parameters automatically.
We applied this method for early development of phytochrome-deficient mutants of rice, and analyzed the effects of mutation on growth. The results promptly showed us that the effects of light on either photoinhibiton of extension growth or photoinhibiton of revolving movement of coleoptiles are different according to their growth stages. These two photoresponses were not always simultaneously induced.
We would like to discuss potential opportunities of image processing techniques and statistical techniques to provide a framework for the analysis of future plant growth and development data.

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We develop a graphics tool for observing growth process of rice roots. The tool has two major functions for monitoring images of rice roots:
(1) Semi-automatic analysis of spatiotemporal growth characteristics of rice roots
(2) Effective visualization of the analyzed characteristics
In the semi-automatic analysis, the spatiotemporal image processing technique is used for analyzing the growth characteristics, such as length, area, and inclination angle. Results of the analysis can be visualized effectively using the proposed tool. For instance, the tool can compare the graphs that represent characteristics of large amounts of samples. This has been achieved by making the graphs translucent and overlapping them. The observation procedure of the growth process is conducted effectively by combining the functions prepared in the proposed tool.

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The superior performance of hybrids (heterosis) is generally evaluated with traits such as yield and biomass that are directly related to agricultural importance; however, the dependence of these characters on multiple genetic factors and their interaction with the environment have obscured the common molecular mechanisms underlying heterosis. Here, by using a newly developed system for time-lapse monitoring of the growth of rice seedlings, we have characterized parameters in the early growth of F₁ hybrids between japonica and indica varieties of Oryza sativa L, especially with respect to the effect of chromatin modification. Inhibition of histone deacetylases induced apparently similar growth retardation in both inbreds and hybrids independent of their relative growth rates. However, detailed characterization of growth parameters by regression showed that the decrease in the growth rate could be divided into components that showed distinctive responses to TSA treatment between inbreds and hybrids.

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Red clover is an important forage legume widely cultivated in most temperate regions. However, its usefulness is limited due to its short perenniality. Perenniality is determined by interactions among various factors, such as biotic abiotic stress and flowering characteristics, which are controlled by QTLs. Growth monitoring of red clover individuals during plant growth period should be efficient for revealing and splitting up the factors those contribute to perenniality. However, traditional growth analysis required sampling of plants, and it was impossible to chase individual growth habit of the field plants by nondestructive inspection. In this presentation, we reported the result of growth monitoring of red clover individuals on the field level using Field Imaging System and QTL detection with the obtained data. Field Imaging System was a measuring device of field plants, composed with personal computer, digital camera and software for picture filming and instrumentation.

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Most of plastidic proteins are nuclear encoded and are imported into plastids after synthesized in the cytoplasm. Majority of them are synthesized as precursors with plastidic localizing signals, transit peptides, at their amino termini. These proteins are thought to translocate through Toc and Tic complexes in an energy-dependent manner. Toc and Tic complexes are the translocons embedded in both the outer and inner envelope membranes, respectively. Once proteins are imported into plastids, some of them are further targeted to thylakoid membrane. Protein localization systems to thylakoid membrane are different from the Toc-Tic pathway, rather resembled to the bacterial protein secretion systems. There handful of Toc- and Tic-mutants are isolated in Arabidopsis, many of which exhibit pale-green as phenotype. Therefore, protein import through envelope membranes is important for the development of plastids. Current knowledge on protein import into plastids will be discussed at the symposium.

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The assembly of photosystem II (PSII) complex has been studied, especially in rapid turnover of photodamaged D1 protein in photoinhibition. However, there is still much to be learned about molecular mechanism and pathway of the PSII assembly. We have analyzed structure and function of the PSII subunits by using Synechocystis sp. PCC 6803 and the thermophilic cyanobacteria, Thermosysnechococcus elongatus BP-1 and T. vulcanus. We have prepared various PSII complexes lacking PSII subunits such as PsbX, PsbI, PsbK, PsbTc, PsbH, PsbM, and PsbZ. PsbI, PsbK, PsbTc, PsbH, and PsbM are important for the stability of dimeric PSII complexes. Deletion of psbTc resulted in loss of PsbM and deletion of psbH resulted in loss of PsbX in the PSII complex. We are now studying crystal structure of psbTc and psbM mutants. We recently found a gene involved in destabilization of PSII. We will discuss assembly and stabilization of PSII complex of cyanobacteria.

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Photosystem I (PSI) complex in photosynthetic electron transport plays roles in light-harvesting and photochemical reaction. The components and reactions of the complex have been extensively studied. However, little is know on the assembly process. The reasons why little is know on the assembly process are; (1) assembly process is difficult to study in vitro, and (2) constituent components are efficiently and rapidly integrated into a functional complex in vivo. Here we will focus on the chloroplast-encoded Ycf3 and Ycf4 essential for the PSI assembly. Of great interest is that Ycf4 is a component of a large complex containing PsaF, Cop2 as well as Ycf4. In addition, several site-directed mutants of ycf4 genes were generated to get some insight into the assembly process. We have also identified and characterized an assembly intermediate by refined biochemical characterization and pulse-chase protein labeling. We will propose an assembly model for PSI complex.

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Chlorophyll molecules are major components of thylakoid membranes and play important roles in photosynthesis. It is well know that chlorophyll accumulation is closely related to thylakoid formation. In etiolated seedlings, chlorophyll is absent and thylakoid membranes are not form. Development of thylakoid membranes are accompanied by chlorophyll accumulation during greening. On the contrary, degradation of chlorophyll molecules and thylakoid membranes is observed during senescence. However, we do not know whether chlorophyll metabolism regulates thylakoid formation or formatin of thylakoid membranes regulate chlorophyll metabolism.
In order to clarify the roles of chlorophyll metabolism in thylakoid formation, we examined the mutants of chlorophyll metabolism. These experiments suggest that chlorophyll metabolism regulates the thylakoid formation. I will discuss the mechanisms in this symposium.

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Monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in thylakoid membrane of chloroplasts. It constitutes for almost 50% of the total thylakoid lipids. MGDG is synthesized by MGDG synthase in chloroplast envelope. Since MGDG is so abundant in chloroplast and a second major galactolipid, digalactosyldiacylglycerol is synthesized from MGDG, biosynthesis of MGDG is one of the most important steps to construct chloroplast.
In Arabidopsis, three genes for MGDG synthase are encoded in the genome. Among them, MGD1 is localized in inner envelope and mainly contributes development of thylakoid membrane. Recently, we isolated a T-DNA tag mutant of the MGD1 gene. Unexpectedly, homozygous mutants showed severe defects in not only greening process but also early stage of embryo development. MGDG content of the mutant was less than 10% of wild type. Defect of MGD1 gene also resulted in large deformation of the plastid structure, demonstrating the importance in proper chloroplast development.

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To determine essential nuclear-genes involved in chloroplast development, we systematically analyzed albino or pale green Arabidopsis Ds-tagged knock-out mutants which was produced by using a two-component transposon system based on the Ac/Ds element of maize as mutagen. We have isolated about 40 mutants with albino or pale green (apg) phenotypes. Identified APG genes have sequence homology with house keeping proteins involved in photosynthesis, translation, transcription, translocation and so on. In this meeting, we present phenotypes of five apg mutants (apg3, apg4, apg5, apg11 and apg12), and discuss functions of the APG genes in chloroplast development.
To shed light on the regulation of plastid proteins during plastid differentiation, we also isolated intact chloroplasts, etioplasts and chromoplasts of wild type plants and apg mutant plastids by Nycodenz gradient centrifugation in Arabidopsis and tomato. We then analyzed plastid proteomic profiles in these different plastids.

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A histidine kinase (Hik) and a response regulator (Rre) consist a two-component system, which performs signal recognition and transduction pathway. In general, cyanobacteria contain more numbers of two-component systems per unit of genome size than other bacteria.
We identified functions of several Hiks and Rres in the cyanobacterium Synechocystis sp. PCC 6803. Among them, Hik33 is one of the most interesting Hiks, which might respond to several stimuli, such as cold, salt, hyperosmotic and oxidative stress, and transduces the stimuli to at least two Rres depending on the stimuli perceived. Moreover, Hik33 associated with a novel small protein, Ssl3451, which enhanced the in vitro activity of phosphorylation of Hik33 up to ten-fold. A disruptant of the ssl3451 gene indicated that it might also function in vivo as a regulator of Hik33. Hik33 and Ssl3451 are conserved in all the cyanobacterial genome, indicating that cyanobacteria develop unique features of two-component systems.

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Phototrophic organisms respond to photooxidative stresses as well as normal stresses. In this talk, I summarize transcriptional regulation that responds to the various photooxidative stresses. Based on microarray analyses and functional characterization, we identified three photooxidative stress-responsive regulons, i.e. Slr1738(PrxR)–sll1621(prxA), Sll0088–slr0074, and Slr1245–sll1161. Of these, Slr1738 is a Zn and Fe-binding regulator that recognizes redox changes. Sll0088 coordinates FeS cluster to recognize redox changes. We also identified LexA regulation in Synechocystis. Generally, LexA is the single strand DNA-activated protease and regulates gene expression. But we found that LexA lost protease activity at least in Synechocystis. Finally, we recently identified IscR homolog in Synechocystis and many cyanobacteria. It is shown that IscR homolog is essential for expression of PSI genes. Based on these findings, we will discuss general view of responses to various photooxidative stresses.

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Cyanobacteria constitute a remarkable group of prokaryotes, not only having an oxygen-producing photosynthetic system but also showing unique conservation of molecular chaperone gene structures. To reveal the features of cyanobacterial stress proteins, we have studied major molecular chaperone family, Hsp70/DnaK, Hsp40/DnaJ and Hsp90/HtpG.
In the cyanobacterium Synechococcus elongatus PCC 7942 genome, three dnaK and four dnaJ homologs were identified. We analyzed their expression profile upon various stresses. Then we found that a conserved sequence, located in the upstream of several hsp genes exclusively in cyanobacteria, was indispensable for the expression of stress responding dnaK gene.
Moreover, we carried out yeast two-hybrid screening to explore the function of Hsps and found out several specific interactions. Those were between DnaJ and RNase E, which is an essential endoribonuclease, and HtpG and HemE, which is a metabolic enzyme of tetrapyrrole biosynthetic pathway. These findings suggest novel functions of Hsps in cyanobacteria.

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Oxidative stress inhibits the synthesis de novo of proteins that are required for the repair of photosystem II in the elongation step of translation. To investigate in vitro the mechanisms whereby ROS inhibit translational elongation, we prepared an in vitro translation system from Synechocystis sp. PCC 6803. The in vitro translation of the D1 protein was arrested in the presence of H₂O₂. However, addition of the reduced form of elongation factor G (EF-G), which is known to be particularly sensitive to ROS, was able to rescue the arrest of translation. Furthermore, overexpression of EF-G in Synechococcus sp. PCC 7942 increased the tolerance of cells to H₂O₂. Thus, EF-G might be a primary target of inhibition by ROS in the translational machinery. Interaction of EF-G with thioredoxin suggests that the redox state of EF-G might regulate the translational machinery.

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Cyanobacteria thrive under various salinity conditions. Here, the recent data on salt-stress responses in a halotolerant cyanobacterium Aphanothece halophytica will be presened. A. halophytica can grow at high salinity up to 2.0 M NaCl. This organism synthesizes betaine from glycine by two N-methyltransferase enzymes which were induced upon salt stress. Betaine transporter of A. halophytica is unique. It is the Na⁺-betaine symporter and its affinity for betaine was high. A. halophytica has NhaP- and NapA-type Na⁺/H⁺ antiporters. Substrate specificity of ApNhaP1 was different from that of ApNapA1. Two ApNapA1 antiporters, ApNapA1-1 and ApNapA1-2, also have different ion specificity. It was shown that Ca²⁺/H⁺ antiporter plays an important role for salt tolerance in cyanobacterium. Based on these data, the molecular mechanisms of responses on salt stress in cyanobacteria will be discussed.

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The diversity of organisms arises through adaptation to the environment. Thus, how organisms have evolved and diversified may be understood by studying the diversity of organism's defense mechanisms against environmental stresses.
In this presentation, I will discuss about cyanobacterial responses to environmental changes such as temperature and nutrient availability, by focusing on functions of molecular chaperones. We have shown that molecular chaperones play important roles in acquisition of tolerance to various stresses. Through these studies, we started to think that light-harvesting phycobilisomes may be one of the major targets for molecular chaperones. Intriguingly, HtpG, a homolog of Hsp90, from E. coli, the model heterotrophic organism, did not protect components of phycobilisomes at high temperatures, although its cyanobacterial counterpart did. The results may indicate that cyanobacterial HtpG has evolved to interact with phycobilisomes in order to sustain photoautotrophic life.

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We have investigated the gene expression profile using a DNA microarray when the cells of Anabaena sp. PCC 7120 were exposed to certain stress conditions. Nitrogen depleted conditions caused an induction of "Nif gene islands". Those gene expression are mediated by a transcription factor, NrrA which is under the control of global nitrogen-responsive transcription factor, NtcA. NrrA regulates HetR which is a master transcription factor for nitrogen metabolic genes. A terrestrial cyanobacterium, Nostoc sp. HK-01 shows a high drought stress tolerance. We have found that a sigma factor, SigI, plays a key role in the cellular response to desiccation stress. It was also found that the genes involved in treharose metabolism were induced by desiccation stress. As for salt stress, a transcription factor, OrrA, was found to be important to regulate gene expression in response to high salt concentration. However, the regulatory mechanism by OrrA has not yet been elucidated.

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A phragmoplast, a cytokinetic apparatus in plant cells, builds a cell plate during cytokinesis. Formation of new microtubules at the outer margin of the phragmoplast is essential for expansion of the cell plate. We successfully observed microtubule dynamics of the outer margin of the phragmoplasts in living tobacco BY-2 cells expressing either GFP-α-tubulin, a microtubule marker, or GFP-EB1, a microtubule plus end marker. New microtubules initiated at the side of phragmoplast microtubules, elongated outside of the microtubule mass of the phragmoplast towards an equitorial plane and finally incorporated into outermost margin of the phragmoplast.

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Electron tomography has emerged as an important tool for the study of three-dimensional ultrastructure, ranging from biological nanosystems to reconstructions of whole cells. It is ideally suited for studying membrane systems.
The preprophase band (PPB) is a ring-like band of microtubules in plants that underlies the plasma membrane in G2 and prophase cells. The PPB plays an important role in the determination of the division site. Recent studies using live imaging experiments raise the question about the classical hypothesis that the Golgi derived exocytosis in the PPB involves in the marking of the division site. However, any quantitative analysis at electron microscopical level
has not yet done.
In the present study, we have investigated the mechanism of PPB membrane modifications in epidermal cells of cryofixed onion cotyledons by analyzing the endocytic and secretory structures associated with the PPB formation in electron tomograms.

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Post-Golgi organelles, such as the trans Golgi network, endosomes, and vacuoles are mutually connected by a vesicular transport system. This inter-organelle traffic seems to be strictly regulated temporally and spatially, and disordered post-Golgi traffic affects the various cell function. Recent molecular genetic study also revealed that the post-Golgi network participates in the various plant function of higher order, such as response to the environment, morphogenesis, and transport and perception of the phytohormone. We are studying the dynamics and function of the post-Golgi network especially focusing on the endosomes. Combination of the high-speed and high-sensitivity imaging system, genetic analysis, and biochemical study enabled us to unveil the dynamic and essential functions of post-Golgi organelles.

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