Chloroplasts possess their own genome and the protein synthesis system. The molecular mechanisms for chloroplast protein synthesis have not been fully understood in higher plants. To gain new insights into the mechanism of chloroplast protein synthesis, we have isolated nara (genes necessary for the achievement of RuBisCO accumulation) Arabidopsis mutants which had defects in the chloroplast protein synthesis. In nara12, the translation rate of chloroplast-encoded large subunits of RuBisCO was much lower than that of the wild type. Reduced protein levels were found for not only RuBisCO but also several other photosynthetic proteins. In addition, the processing of chloroplast 23S ribosomal RNA (rRNA) was impaired in this mutant, while that of the other chloroplast rRNAs was slightly affected. These results suggest that NARA12 is involved in the chloroplast protein synthesis via the processing of chloroplast 23S rRNA.

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Dynamin-family proteins are self-assembling GTPases that are involved in membrane fission and fusion events. Although several models for its function have been proposed, the mechanism of dynamin function is not fully understood. Above all, ARC5, a dynamin-like protein, is important to chloroplast division, little is known about the functions of dynamin for division mechanism.
To understand the mechanism of chloroplast division by dynamin, we expressed several point mutants of ARC5 as a green fluorescence protein (GFP) fusion protein in under the control of ARC5 promoter. A mutant of GTPase domain localized as a ring at the division site of the chloroplast. However, the numbers of the chloroplasts in the cell of this plant was comparable to that of arc5 mutant, these results indicate that GTPase activity is essential for dynamin function but not for the localization at the division site. Analysis of other mutant proteins also will be discussed.

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Chloroplasts were originally established in eukaryotes by the endosymbiosis of a cyanobacterium, and chloroplast division apparatus were derived from the ancestral bacterial endosymbionts and the eukaryotic host. Because many of the cell division genes of the cyanobacteria are lost from the plant genome, it is expected that a lot of chloroplast division proteins derived from the eukaryotic host is an unknown. To identify and characterize novel chloroplast division proteins, we screened chloroplast division mutant from activation tagging lines and FOX (full-length cDNA overexpressor) lines in Arabidopsis. As a result of screening from about 30,000 lines, several novel chloroplast division mutants were found and it was thought that one of them was originated from the eukaryotic host. The cause gene encoded unknown function protein, which was predicted to have a chloroplast transit peptide and a coiled-coil motif, one membrane-spanning region. We would discuss the function of the novel chloroplast division protein.

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Chloroplasts are believed to have evolved from an cyanobacterial endosymbiont. In order to establish the permanent endosymbiotic relationship, the host cell has been regulated proliferation of the endosymbionts (chloroplasts) during proliferation of the host cell. Although several components of the chloroplast division apparatus, such as FtsZ and dynamin, have been identified and characterized, the regulatory mechanism of the chloroplast division is still poorly understood.
In unicellular monoplastidic red alga Cyanidioschyzon, the chloroplast division is linked to the cell division cycle of the host cell. By contrast, chloroplasts apparently divide nonsynchronously even in the same cell in higher plants, raising a question of how the timing of the chloroplast division is regulated in cells with multiple chloroplasts.
By investigating several Arabidopsis mutants, we found a novel regulatory mechanism of chloroplast division. We will discuss how the timing of chloroplast division is regulated in cell of higher plants.

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Chloroplast division is driven by both prokaryotic component and eukaryotic component. At present, several factor such as the FtsZ and Dynamin have found, however, more of unidentified factors are required for chloroplast division event. In this study, we isolated a mutant of Arabidopsis with altered chloroplast number and size from the FOX hunting transgenic lines and identified the overexpressed cDNA in this FOX line. This cDNA encoded putative plastid protein possesses three transmembrane domains. This protein is conserved from prokaryotes including cyanobacteria to plants and malarian parasites. We currently are analyzing the localization in chloroplast using GFP fused protein and T-DNA insertion line.

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The catalytic F₁ complex of ATP synthase is the smallest mechanical motor known. The ATPase function is coupled to the stepwise rotation of the γ subunit in a catalytic core formed by three copies of subunits α and β. Tentoxin, a cyclic peptide produced by phytopathogenic fungi of the Alternaria species inactivates the F₁-ATPase in sensitive species at micromolar concentrations, whereas milimolar amounts of the toxin restore and surpass the natural enzyme activity. Although it is known that this inhibition and stimulation is related to the binding of one and two or three molecules of tentoxin, the mechanism of the change of the activity is not known very well. Here we report the detailed molecular mechanism of the regulation revealed by the single molecule analysis.

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The primary stress on photosynthetic organisms is their life-giving sun. To counteract this stress, eukaryotic photosynthesizers have developed methods to recruit light-harvesting carotenoids for photoprotection. During the xanthophyll cycle, violaxanthin is rapidly de-epoxidated to help quench energy flow from the reaction center. While this reaction is efficient in land plants, it is unproductive in the model alga Chlamydomonas reinhardtii. As the earliest branching green algal group, the prasinophytes are of primary interest to the origin of eukaryotic light-adaptation. Our work characterizes the xanthophyll cycle in the model prasinophyte Ostreococcus tauri. Under high-light stress, O. tauri effectively converts up to 50% of its violaxanthin to antheraxanthin to zeaxanthin. Spectroscopic analysis reveals that this conversion correlates to a large increase in non-photochemical quenching, indicative of an effective photoprotective measure. This rapid change is a useful adaptation under changing environmental conditions and is likely a necessary practice for this inter-tidal Ostreococcus species.

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State transition is a mechanism that supports plant survival under various light conditions. Peripheral light-harvesting antennas of PSII (LHCIIs) reversibly migrate between PSII and PSI during state transitions. Recently, we revealed that three LHCIIs are associated with PSI, forming a PSI-LHCI/II supercomplex, in state 2. Notably, CP29 might act as a linker between PSI and major LHCIIs. In this study, we generated CP29-RNAi mutants (b4i) to investigate the specific role of CP29 during state transitions in Chlamydomonas reinhardtii. Sucrose density gradient ultracentrifugation of thylakoids from b4i-8 cells under state 2-promoting conditions revealed that there was no PSI-LHCI/II supercomplex in this mutant. Contrarily, results of both fluorescence and electron flow analyses indicated that b4i-8 could perform state transitions. We conclude that CP29 contributes to the formation of a PSI-LHCI/II supercomplex, but it is not essential for state transitions.

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Under changing light environment, light-harvesting complex II (LHCII) is redistributed between photosystem I (PSI) and photosystem II (PSII) to balance excited levels of each photosystem (state transitions). Recently, a protein complex composed of PSI, LHCIs and LHCIIs has been isolated, which supports the model for attachment of LHCIIs to PSI. However, the mechanism for detachment of LHCIIs from PSII, which should occur simultaneously during state transitions, still remains unclear. Here, we isolated a protein complex composed of PSII and LHCIIs, a so-called PSII-LHCII supercomplex, by using a Chlamydomonas reinhardtii mutant carrying histidine-tagged CP47 through nickel affinity chromatography and determined the structural changes during state transitions. Gel filtration chromatography showed that three different sizes of isolated PSII-LHCII supercomplexes existed. The proportion of each protein complex was changed during the course of state transition. We propose a molecular mechanism for the detachment of LHCIIs in state transitions.

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The photosynthetic electron transfer chain generates proton motive force (pmf) across thylakoid membranes, and the balancing the pmf is important for acclimation to environmental challenges. In this study, a change in the absorption spectra of carotenoid pigments in the thylakoid membranes, the so-called "electrochromic shift (ECS)" was analyzed to gain information on the energy balance in the thylakoid membrane of wild watermelon under drought/high light stresses. Proton flux across thylakoid membranes were estimated from dark-interval relaxation kinetics (DIRK) analysis, and plotted against electron transfer rate (ETR) from photosystem II. The analysis revealed that the slope of proton flux vs. ETR was significantly larger in the leaves under drought than that in the irrigated controls, suggesting the activation of cyclic electron flow around photosystem I under drought stress.

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Chloroplastic NDH (NAD(P)H dehydrogenase) of higher plants is localized in the thylakoid membranes. Recent studies revealed that NDH plays an important role in cyclic electron flow around photosystem I to produce ATP, especially for adapting to environmental changes in C₃ plants and for driving the CO₂-concentrating mechanism in C₄ plants. Although, the 14 subunits of NDH have been identified, "electron input devices" which are responsible for the binding and oxidizing NAD(P)H are still unknown. To reveal the unidentified NDH subunits, we developed a novel bioinformatics strategy by combining a co-expression analysis and a phylogenetic profiling. We found 65 proteins as the candidates for novel NDH subunits by our in silico approach, and measured NDH activity in Arabidopsis lines carrying a T-DNA insertion in these candidate genes. As a result, we found the six novel proteins that were indispensable for the accumulation of NDH. Biochemical characterization of these proteins are discussed.

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The mechanism by which plants regulate channeling of photosynthetically derived electrons into different areas of chloroplast metabolism remains obscure. Possible fates of such electrons include carbon assimilation, nitrogen assimilation and signaling pathways, or return to the plastoquinone pool through cyclic electron flow. In higher plants, these electrons are made accessible to stromal enzymes, or for cyclic electron flow, as reduced ferredoxin (Fd), or NADPH. We investigated how knock-out of an Arabidopsis ferredoxin:NADPH reductase (FNR) isoprotein, and the loss of strong thylakoid binding by the remaining FNR in this mutant, affected channeling of photosynthetic electrons into NADPH and Fd dependent metabolism. We found significant differences in electron channeling in the chloroplasts of mutant and wild type plants, and in addition uncovered evidence that FNR may be involved in stress signaling. Taken together, our results demonstrate the integral role played by FNR isoform and location in the partitioning of photosynthetic reducing power.

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Cyanobacteria possess a hight activity of cyclic electron transport. The several pathways have been proposed, but they are not established. To identify the possible component(s) of the pathway in cyanobacteria, we tried to reconstruct the cyclic electron transport in a cell-free system. Thylakoids with high activities were isolated from Synechocystis 6803 cells by an improved method. In the presence of DCMU, the activity monitored by P700⁺ rereduction was reconsituted by addition of soluble fraction. To identify the component(s), the soluble fraction was fractionated. Ferredoxin was found to be an important component. The activity was successfully reconstituted by addition of Fd and NADPH or NADH. In the case of NADH, rotenone inhibited the rereduction of P700⁺. This suggests that NAD(P)H dehydrogenase complex 1 is the main pathway when NADH is an electron donor. The missing subunits of NDH-1 which act as electron imput device are now under investigation.

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In photosynthesis of higher plants, electrons are transported from photosystem II to photosystem I and finally to NADP+. The electron transport is couple to the proton translocation across the thylakoid membrane. Resulting trans-thylakoid proton gradient contributes to formation of the proton motive force to drive ATP synthesis. In this study, isolation of Arabidopsis thaliana mutants having defects in the photosynthetic electron transport was attempted. Especially, we focused on mutants impaired in the alternative electron transport, e.g. the cyclic electron flow around photosystem I and the Meher-preoxidase reaction. Using chlorophyll fluorescence in low oxygen (2%), we selected 37 mutants from ca. 30,000 F2 plants derived by EMS mutagenesis. In a plant showing severe phenotype in chlorophyll fluorescence, a mutation was found in the gene for fructose-1,6-bisphosphate aldolase (AT4g38970). In this plant, electron donation from the stromal reductants to the intersystem electron carriers was accelerated.

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Chlorophyll fluorescence and absorbance changes at 830 nm were measured at low light in leaves of Arabidopsis thaliana. The level of 830-nm absorption (A₈₃₀) was almost the same as that in darkness, indicating that P700 was in non-oxidized form. However, by illuminating a leaf with a pulse of strong light, A830 did not reach its maximum level. This indicates that there was a population of PS1 centers in which P700 remained non-oxidized and inactive. To investigate this reason, P700 oxidation by the pulse of saturation light was tested under various conditions. Consequently, it was found that reduced inactive P700 was not detected either in the presence of methyl viologen or under far-red light. These results indicate that a lack of electron acceptors in the stroma is responsible for the accumulation of reduced inactive P700 at low light.

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Photosystem I (PSI) cyclic electron transport is essential for photosynthesis and photoprotection. In higher plants, the ferredoxin-dependent pathway is the main route in PSI cyclic electron transport. Overexpression of PGR5, which is essential for this pathway, delayed early chloroplast biogenesis as a result of enhanced activity of PSI cyclic electron transport.
During chloroplast biogenesis, activity of PTOX is essential to activate carotenoid synthesis. PTOX is a homolog of the mitochondrial alternative oxidase (AOX) and has been proposed to serve as the terminal oxidase in chlororespiration. An Arabidopsis mutant, immutans (im), which is defective in PTOX activity, shows a variegated phenotype.
We evaluated an effect of a defect in PSI cyclic electron transport on the im phenotype. In the double mutant im pgr5, the variegated phenotype was partially suppressed. From this result, it is suggested that PSI cyclic electron transport operates in early chloroplast biogenesis.

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The chloroplast NAD(P)H dehydrogenase (NDH) complex functions in PSI cyclic electron transport and chlororespiration. The electron donor binding module is still unclear. An Arabidopsis crr6 mutant was isolated based on the chlorophyll fluorescence. Photosynthetic electron transport and protein blot analyses indicate that the specific defect of the NDH complex in the mutant due to the mutation of CRR6. However, CRR6 protein was stable in other crr mutants. Inconsistently with our previous report, western blot analyses showed that the CRR6 dominantly localized in the stroma fraction and trace amount of it associate with thylakoid membrane. This result suggests that CRR6 transiently interacts with NDH complex, possibly as a subunit of electron donor binding module present in the stroma.

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PGR5 (proton gradient reguleation) was found as an essential factor for the PSI cyclic electron transport from the analysis of the Arabidopsis mutants (Munekage Y. et al.(2002) Cell 110(3):361-371). However, the biochemical property and the actual function of this protein is not known at all. To tackle the function of this protein, we have investigated the exact N-terminal sequence of the mature PGR5 protein, and the localization of the protein in the chloroplasts. In addition, we analyzed the composition of the membrane protein complexes on the thylakoid membrane using the sucrose-density gradient centrifugation to determine which complex in thylakoids is functionally associated with PGR5.

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To avoid photoinhibition, an efficient degradation of D1 protein in the repair cycle of photosystem II is important. FtsH, an ATP-dependent metalloprotease in thylakoid membranes, is believed to predominantly act in this process. We attempt to demonstrate D1 degradation in vivo using Arabidopsis mutants. Despite the availability of the FtsH2 mutant var2, these leaves show variegation and are not good materials for an in vivo biochemical study. To overcome this problem, we incorporated another mutant fug1 that recovers leaf variegation in var2. Under various light conditions, the ability to degrade D1 in the presence of lincomycin was assayed in fug1 and fug1 var2 leaves using immuno-blot against anti-D1 antibodies. These assays showed that the D1 degradation was significantly attenuated in fug1 var2compared with fug1 irrespective of high and low light conditions. These results suggested that FtsH is required for in vivo degradation of the D1 protein.

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FtsH is an ATP-dependent metalloprotease and present as a hetero-complex in thylakoid membranes. FtsH2, encoded in Arabidopsis VAR2 locus, is a major isoform. Lack of FtsH2 results in a typical leaf-variegated phenotype. While we have identified more than 20 var2 alleles, none of the mutations was found in the catalytic center of protease activity (comprised by zinc-binding domain). To test the importance of this domain, we replaced one of the essential histidine residues in the domain with leucine, and over expressed it in var2. We found that this mutation in E. coli FtsH abolishes protease activity in vivo, and that the mutated FtsH2 rescued the variegated phenotype in var2. The result implies that the protein level rather than the protease activity determines leaf variegation. Loss of the protease activity may be mitigated by other isoforms of the FtsH complex. Experiments to clarify these observations are currently underway.

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PsbP, an extrinsic subunit of photosystem II (PSII), is a nuclear-encoded protein that optimizes the water-splitting reaction. In addition to PsbP, higher plants have two PsbP-like (PPL) proteins that show significant sequence similarity to a cyanobacterial PsbP homolog (cyanoP); however, the function of PPLs has not yet been elucidated. In this study, we characterized Arabidopsis mutants lacking either of two PPLs, PPL1 and PPL2. In ppl1 mutant plants, PSII activity was sensitive to high-intensity light and the recovery of photoinhibited PSII activity was delayed. On the other hand, the stoichiometric level and activity of the chloroplast NAD(P)H dehydrogenase (NDH) complex in thylakoids were severely decreased in a ppl2 mutant. These results suggest that during endosymbiosis and subsequent gene transfer to the host nucleus, cyanoP from ancient cyanobacteria evolved into PPL1, PPL2, and PsbP, and each of them has a distinct role in photosynthetic electron transfer.

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We previously developed an algorithm for inferring co-expressed genes using network analysis. Here, we applied the algorithm to a dataset of Arabidopsis DNA microarrays including 1,388 chips. From 22,263 genes, we obtained approximately 300 sets of genes that are highly correlated within individual sets, or co-expressed. The genes in individual sets are functionally related to each other. To systematically infer biological processes associated with individual gene sets, a public database of the co-expressed gene sets was constructed. The database provides information for the individual co-expressed gene sets such as co-expression networks and functional description of genes, and information for the biological processes such as metabolic pathways assignment. Dissimilar to other public co-expression databases that can search genes co-expressed with the query genes (genes-to-genes), our database allows the search for biological processes associated with the co-expressed gene sets (genes-to-processes), which facilitates advanced functional annotations of Arabidopsis genes.

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Integration of transcriptomics and metabolomics enables us to predict the co-regulated genes and metabolites in Arabidopsis. This strategy is useful to identify a set of genes involved in the same metabolic pathway and its transcription factors [1]. In this study, we analyzed the transcripts and metabolites in knockout and over-expression lines of PMG1, PMG2 and PMG3, transcription factors of methionine-derived glucosinolate (MET-GSL) biosynthesis genes, which were predicted by coexpression analysis [1]. In leaves and seeds of a double knockout line pmg1pmg2, MET-GSLs were not detected, and the expression of MET-GSL biosynthesis genes and PMG3 were remarkably repressed in leaves. In seeds of a PMG2 over-expression line, MET-GSL accumulation increased by 10-fold. These results suggest that PMG genes are major transcription factors regulating the MET-GSL biosynthesis genes. [1] PNAS 104, 6478-6483

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We previously reported that PMG1 and PMG2 are transcription factors regulating methionine-derived glucosinolates (MET-GSL) biosynthesis (Hirai et al., 2007). In this study, we analyzed the expression of PMG1, PMG2 and a novel candidate transcription factor, PMG3, in knockout lines of PMG1 and PMG2. Expression of PMG2, and PMG3 was reduced to 30%, and 10%, respectively, in pmg1 in which MET-GSLs were decreased. In pmg1pmg2 in which MET-GSLs were not detected, expression of PMG3 was reduced to 5%. On the other hand, in pmg2 containing comparable amount of MET-GSLs to wild-type, the amount of transcripts of PMG1, and PMG3 was 100%, and 40%, respectively, of those in wild-type. These results suggest that PMG3 is regulated by PMG1 and PMG2 and involves in the regulation of MET-GSL biosynthesis, although PMG1 is a principal regulator.

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Plants respond and adapt to drought, cold and high-salinity stresses in order to survive. We thought that non-coding RNAs have functions in plant abiotic stress responses and have applied Arabidopsis Affymetrix tiling arrays to study the whole genome transcriptome under drought, cold, high-salinity stress and ABA treatment conditions. The tiling array experiments showed that 7,719 novel transcription units (TUs) exist in the Arabidopsis genome. Most of the novel TUs are hypothetical non-coding RNAs and about 90% of them are mapped on the antisense strand of the AGI code genes in the sense-antisense transcripts (SATs). Interestingly, high correlation of the expression ratios (treated/untreated) was observed in the SATs (AGI code genes)/(novel TUs). We confirmed the presence of several stress- or ABA-inducible TUs on the antisense strand by real-time RT-PCR and Northern analyses. Analysis of biogenesis mechanism and the function of the novel stress-responsive antisense RNAs are in progress.

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The phytohormone abscisic acid (ABA) plays an important role for seed dormancy. To explore genome-wide expression patterns and new components involved in ABA signaling in Arabidopsis seeds, comprehensive expression analysis was performed using whole-genome tiling array. In dry seeds, there was no difference in global gene expression between ABA-deficient mutant (aba2) and ABA-over-accumulated mutant (cyp707a1 cyp707a2 cyp707a3 triple mutant). At 24h after imbibition, the transcript levels of many genes in both mutants were drastically changed compared to those of wild type. Transcriptome analysis identified 380 ABA-upregulated and 540 ABA-downregulated AGI code genes at 24 h after imbibition. Moreover, whole-genome transcriptome analysis identified 4,884 non-redundant new transcription units (TUs) in Arabidopsis seeds. Among new TUs, 4,559 TUs probably encode hypothetical non-coding RNAs (ncRNAs). At present, we're proceeding analysis of the functional ncRNAs.

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We have developed a novel method for large scale TSS mapping. The method is composed of a combination of Cap Trapper for preparation of 5' end of full-length cDNA, and Massively Parallel Signature Sequencing (MPSS) for high throughput DNA sequencing. Using this novel method, called CT-MPSS, we have prepared large scale TSS information from Arabidopsis. We have previously identified novel core promoter elements from plant promoters (Yamamoto et al, BMC Genomics 8: 67, 2007; Yamamoto et al, Nucleic Acids Res 35: 6219, 2007), and their relationship to transcriptional characteristics was analyzed using the TSS information. The information of novel core promoter elements and large scale TSS analyses are served by our database (ppdb: Plant Promoter Database, http://ppdb.gene.nagoya-u.ac.jp).

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To understand the molecular mechanisms for pluripotent stem cell formation, we have established an experimental system with the moss Physcomitrella patens, in which a differentiated leaf cell changes to an apical stem cell simultaneously with cell cycle re-entry. It is critical to identify the factors affecting this reprogramming process. We are employing the massively parallel sequencing approach for genome-wide transcriptome analysis. Comprehensive mRNA 3 prime-end and small RNA sequencing were conducted with 454 sequencer. For mRNA 3 prime-end sequencing, we developed the method in which mRNA 3 prime-ends were successfully trapped by improved primer sets. 1.9-million for 3 prime-end sequence (per five samples) and 560,000 for small RNA sequence (per one sample) tags were obtained. Mapping of 3 prime-end sequence tags revealed the high trapping efficiency of 3 prime-ends by this method.

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Plants sense and respond to environmental stimuli, including nutritional deficiency, and biotic and abiotic stresses. The modulation in gene expression patterns is one of the most important processes for adaptation to environmental changes. To develop the method to monitor changes in the composition of nuclear proteins that were induced by environmental stimuli, we attempted nuclear proteome analysis with aerial parts of 5-week-old rice seedling. Purified nuclei were obtained using percoll-density gradient centrifugation and nucleocytoplasmic fraction was extracted with salt. By analyses using a nanoLC/MS system, we identified 182 proteins, which were mostly consisted from histones, ribosomal proteins and RNA helicases. Among these identified proteins, regulatory proteins including transcription factors were estimated to be only ~10%. To improve the coverage of regulatory proteins such as transcription factors, we tried to enrich DNA-binding proteins using heparin- and DNA-affinity chromatography. Results obtained by these methods will also be discussed.

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Light is essential for plant growth. A number of studies have revealed both immediate activation of several signaling pathways and long-term responses of some metabolic pathways. To investigate light responses comprehensively, we performed the proteome analysis using thirteen-day-old dark-grown rice seedlings that were illuminated for 24 hr or not illuminated. Proteins from aerial parts were digested into peptides with trypsin, and the peptides obtained were then analyzed by the use of nano-flow liquid chromatography linked to a mass spectrometer. We identified 868 and 1026 proteins with non-illuminated and illuminated samples, respectively. As our data also indicated that relative amounts of respective proteins could be evaluated by the peak intensities, we performed comparative analysis of identified proteins. We are currently analyzing light-inducible proteins in more detail. We will discuss light-dependent systematic activation of respective metabolic and signaling pathways.

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Cellulose, a major component of the cell walls, is the most abundant biopolymer in plants and serves many uses as industrial materials. However, the cellulose synthesis is so complicated that the mechanisms have been poorly unraveled. By analyzing comprehensive gene coexpression profiles based on publicly available microarray datasets, we identified several candidate genes that functionally associated with the cell wall formation genes.
We experimentally examined functions of the candidate genes. By using inducible RNAi system, we suppressed an expression of a transcription factor (TF) gene coexpressed with genes involved in primary cell wall formation in Arabidopsis T87 cells and Arabidopsis plants. In T87, suppression of the TF revealed lethal phenotypes when grown with dexamethasone. In plants, suppression of the TF caused suppression of root growth and lethal phenotypes. We will report the results of microarray and metabolomics analyses using mass spectrometry, and discuss the function of the TF.

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Plants have adaptive abilities for changing environment by metabolic balance to keep internal homeostasis. To follow this system, we are developing simple methodologies to detect time-dependent changes of metabolites using Arabidopsis T87 cells. Firstly, we performed ¹H-NMR metabolic profiling of cellular metabolites upon treatments with biosynthetic intermediates. Secondly, T87 cells were uniformly labeled with [¹³C₆] glucose and the ¹³C labeled low molecular metabolites were identified with a combination of multi-dimensional NMR methods such as ¹H-¹³C-HSQC, HCCH-TOCSY, and HCACO. Thirdly, we are trying to extract the basic information of metabolic flux with the labeled metabolites by ¹³C -¹³C coupling constants in ¹H-¹³C -HSQC. Additionally, we have examined how the ¹³C metabolic flux could be different in insoluble macromolecule and soluble low molecular metabolites since energy accumulation and consumption in plant cells are essential issues for food and energy sources. We will discuss future perspectives of the ¹³C flow monitoring method.

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We previously proposed a procedure of metabolite annotation to identify mass signals representing metabolites by analyzing LC-FTICR-MS data. Here, we applied the metabolite annotation procedure to Micro-Tom fruit, and constructed the metabolite annotation database, which facilitates a comprehensive characterization of tomato fruit metabolites. Analysis of methanol extract of the fruits at different ripening stages enabled us to provide annotations to 869 metabolites in total, of which 494 appeared to be novel. The number of metabolites was larger in the peel than the flesh at all stages, and increased with ripening. Modifications such as glucosylation and acylation frequently occurred in fruit metabolites. We annotated 70 flavonoids and 93 glycoalkaloids, and confirmed that they undergo various modifications during ripening. The metabolite annotation procedure was further applied to comparative analyses of metabolites in fruits of mutant lines. This work was partly supported by Research and Development Program for New Bio-Industry Initiatives.

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Metabolomics is a powerful high-throughput tool in the functional annotation of genes. We applied gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS)-based techniques to detect changes in the metabolite profile of 345 3-weeks-old independent Arabidopsis lines overexpressing full-length rice cDNAs (Rice-Arabidopsis FOX lines).
Out of these 345 T2-lines analyzed, 234 showed a wild type phenotype and 111 exhibited an altered phenotype under defined growth conditions. The secondary screen of a total of 50 lines confirmed the metabolite change in 26 lines.
Data for one retransformed Arabidopsis line with a metabolic change in accordance with the results obtained in the primary and secondary screen will be presented. The line harbors a rice cDNA of unknown function. The linkage between metabolomic and transcriptomic changes in this line sheds light on the relationship between nitrogen-assimilatory and developmental pathways. The analysis of the corresponding rice overexpressor line will be presented as well.

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Vacuole plays an important role in maintaining the homeostasis of plant cells, that is, the maintenance of the turgor pressure, the accumulation of inorganic ions and metabolites and the degradation of discarded proteins. Until now, however, there are few studies about vacuolar substances analyzed directly and comprehensively. In the former study, we established the isolation method of intact vacuoles from suspension-cultured cells or whole plants and reported the proteomic analysis of the vacuolar membrane proteins. In this study, we analyze the metabolites contained in the vacuolar sap of several plants by using CE-MS and FT-ICR-MS. In consequence, we have detected not only well-known vacuolar substances but also some unexpected substances in the vacuole. We are confirming whether those metabolites actually present in the vacuole, or not.

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We have developed the metabolome analysis system for one vacuole of Chara corallina, including the methods of isolating one vacuole from one internodal cell of Chara corallina and the metabolome analysis using capillary electrophoresis / mass spectrometry (CE-MS). We isolated over 10 μL of the internal fluid of one vacuole from one Chara internodal cell by use of a glass capillary. The vacuolar fluid was directly introduced into CE-MS system without any sample preparation steps. We detected approximately 1,000 peaks from one vacuolar sample on the cation and anion mode of CE-MS analysis. Among them, some peaks were identified as known metabolites, such as amino acids or organic acids, by the verification using both m/z value and migration time of standard compounds. Moreover, we will report the metabolic disturbance in a Chara vacuole caused by various environmental stresses.

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Studies of plant metabolic response against pathogens have focused on a small number of signaling metabolites. Few comprehensive analyses have been performed to obtain further insights into the metabolic regulation in response to pathogen. Here, we performed comprehensive metabolic profiling of Arabidopsis lines that have different levels of virus resistance.
By expressing a resistance gene RCY1 in Columbia, which is susceptible to Cucumber mosaic virus [CMV(Y)], we obtained ER (extreme resistance) line and HR (hypersensitive response) line. We found that ER line demonstrated no difference, whereas HR line demonstrated significant difference between metabolic profiles of mock- and CMV(Y)-inoculated plants at 24 hours after inoculation. Columbia showed the largest difference between mock- and CMV(Y)-inoculation. This indicates that the metabolic change associated with CMV(Y)-inoculation depends on the strength of resistance. In non-inoculated plants, ER line showed a metabolic profile distinct from other two lines, suggesting that ER line constitutively accumulates resistance-related metabolites.

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To elucidate flavonoid biosynthesis in gentian flowers, we attempted molecular cloning of R2R3-MYB genes expressed in flower petals by degenerate PCR technique. Sequence analysis showed that the obtained fragments were classified into 24 groups. Among them, four sequences were abundant and full length cDNA sequences were determined with the RACE technique. The isolated MYB cDNAs were designated as GtMYB2a, GtMYB2b, GtMYB3 and GtMYB4. Phylogenic analysis showed that GtMYB3 and GtMYB4 belong to a group regulating phenylpropanoid and the anthocyanin biosynthetic pathway. The deduced amino acid sequence of the GtMYB4 showed a high homology with maize P and Arabidopsis MYB12. A transient expression assay using gentian petals confirmed that GtMYB4 could activate both promoters of chalcone synthase (GtCHS) and flavone synthase (GtFNSII) genes. Northern blot analysis indicated that GtMYB4 is expressed in early developmental stages of the flower. GtMYB4-expressing transgenic tobacco plants showed increased flavonol and decreased anthocyanin accumulation in flowers.

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We reported previously that anthocyanin biosynthesis in gentian flowers is regulated by temporal and spatial expressions of their biosynthetic structural genes. However, no regulatory factor in gentian plants has been reported yet. In Arabidopsis, petunia and snapdragon, MYB and basic Helix-Loop-Helix (bHLH) have been known as the transcriptional factors regulating anthocyanin biosythesis. We isolated each homologue, GtMYB3 and GtbHLH1, from gentian flower cDNA using degenerated PCR technology. Expression profiles of both genes were similar to the accumulation of anthocynin and the expression of their biosynthetic structural genes during flower development. Transient expression assay showed that GtMYB3 and GtbHLH1 could enhance promoter activities of anthocyanin biosynthetic genes when co-expressed. Yeast two-hybrid system showed the interaction between GtMYB3 and GtbHLH1. In addition, some white-flowered gentian cultivars had aberrant GtMYB3 gene by the insertion of transposable elements. These evidences suggested that both GtMYB3 and GtbHLH1 are involved in anthocyanin biosynthesis in gentian flowers.

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The large variety of over 7000 Flavonoid is derived from modification such as glycosylation, acylation and methylation. In Arabidopsis, the structures of 19 flavonoids suggest that at least 9 family-1 glycosyltransferase (UGT) and 3 acyltransferase are involved in flavonoid modification. However, UGTs are encoded by 107 genes in Arabidopsis. Their huge diversity makes it difficult to determine their physiological functions precisely.
To identify flavonoid UGT comprehensively, we utilized transcriptome coexpression analysis. A UGT gene, UGT5, was found to be correlated with flavonoid biosynthetic genes. UGT5 belongs to flavonoid 3-O-glycosyltransferase family. Compared with flavonoid profile in wild type, flavonol 3-O-pentoside derivatives were lacked in ugt5 mutant. UGT5 recombinant protein converted quercetin to quercetin 3-O-arabinoside. UGT5 recognized flavonol aglycones and UDP-arabinose but not flavonol glycosides and other UDP-sugars. These data show that UGT5 encodes flavonol 3-O-arabinosyltransferase.

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Flavonoid, a huge class of plant secondary metabolites, is generally modified with various sugar moieties. Glycosylation is catalyzed by UDP-glycosyltransferase (UGT), and considered to play important roles not only to increase stability and solubility of flavonoids but also provide hydroxyl residues for further modifications. It is accepted that phylogenetics and biochemical functions (i.e., glycosyl-acceptor specificity and regiospecificity) of flavonoid UGTs are correlated. Here we report the identification and characterization of four flavonoid 7-O-glucuronosyltransferases (F7GAT) and three flavonoid 7-O-glucosyltransferases (F7GlcT) from Lamiales order. The 7GATs specifically utilized UDP-glucuronic acid as their UDP-sugar donor, whereas 7GlcTs utilize UDP-glucose but not UDP-glucuronic acid. The 7GATs form a phylogenetic subclade, apparently apart from the cluster III represented by flavonoid 7GlcTs. This structural diversity suggests that the functional differentiation of UGTs had occurred locally in plant evolution.

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The first step of phenylpropanoid pathway in plants starts from phenylalanine. In plant cells, phenylalanine is converted to courmaric acid via cinnamic acid, then courmaric acid branches to various secondary metabolites. One more important branch point of this pathway is flavanone that is converted to flavone or isoflavone. In this study, we attempted to alter this pathway by using tyrosine ammonia-lyase (TAL), which directly converts tyrosine to coumaric acid instead of phenylalanine, and parsley flavone synthase (FNS). Expressions of the introduced genes were examined by RT-PCR or RNA gel-blot analysis, and the resulted transgenic plants were analyzed by LC-MS and HPLC to investigate profiles of their secondary metabolites. We observed accumulations of specific flavonoids that were dependent on the expression of transgene, TAL or FNS. We thus confirmed the in vivo functions of each trasgene that can alter the biosynthetic pathway of phenylpropanoids in Arabidopsis.

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Norlignans are a class of heartwood substances that accumulate in the heartwood of gymnosperms including Japanese cedar, hinoki cypress, and coast redwood. They are produced as phytoalexines in monocotyledons as well. We have elucidated that coumaryl coumarate is enzymatically transformed to hinokiresinol, but the protein that catalyzes the transformation remains to be elucidated. In this study, we identified the gene encoding the protein.
The protein (named as hinokiresinol syntahse) was purified from Asparagus cell cultures, and the protein contained two distinct polypeptides. The genes were cloned based on the partial amino acid sequences. Each ORF was expressed as recombinant protein in E. coli. Both recombinant proteins showed trans-hinokiresinol synthesizing activity. Surprisingly, the equimolar mixture of the proteins showed cis-hinokiresinol synthesizing activity. Considering that natural and recombinant hinokiresinol synthase are both dimers, it was strongly suggested that geometrical selectivity in hinokiresinol formation depends on the subunit composition of hinokiresinol synthase.

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Plants have two isoprenoid biosynthetic pathways, the cytosolic mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Since the discovery of the MEP pathway, metabolic cross-talk between them has been a great problem. Although many feeding experiments have been reported to demonstrate the existence of the cross-talk, it remains to be solved whether the native cross-talk, not exogenously applied metabolites, compensate for the complete blockage of one isoprenoid biosynthetic pathway. We have previously isolated Arabidopsis mutants in HMG1 and HMG2 encoding HMG-CoA reductase in the MVA pathway. To estimate the contribution of the native cross-talk, we generated hmg1 hmg2 double mutant. However, no double homozygote mutant was obtained. Crossing experiment and microscopic observations suggested that the cause is a defect in the male gametophytogenesis. Our data demonstrate that the native cross-talk from the plastid can not compensate the complete blockage of the MVA pathway at least in the male gametophytegenesis.

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During phosphate (Pi) starvation, galactolipid digalactosyldiacylglycerol (DGDG) and sulfolipid sulfoquinovosyldiacylglycerol (SQDG) increase concomitantly with a decrease in phospholipids. Recently, we reported that auxin is involved in the DGDG accumulation during Pi starvation. SLR/IAA14 is one of the IAA proteins and represses at least two AUXIN RESPONSE FACTOR (ARF) proteins, ARF7 and ARF19. Here, we revealed that DGDG and SQDG accumulation upon Pi starvation was suppressed in the gain-of-function mutant slr and loss-of-function mutant arf7arf19. We also examined the level of gene expression for type B monogalactosyldiacylglycerol synthases (MGD2/3) and sulfoquinovosyldiacylglycerol synthases (SQD1/2) in these mutants. Both expressions were suppressed upon Pi-starved conditions in the mutants. Although SQD1/2 were not suppressed in Pi-sufficient conditions of slr and arf7arf19, MGD2/3 were suppressed in the conditions. Thus, auxin is differently involved in galactolipid and sufolipid syntheses under Pi starvation.

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Monogalactosyldiacylglycerol (MGDG) is not only the most abundant lipid of thylakoid membrane in chloroplast but also has an important role in photosynthesis. In Arabidopsis, MGDG synthases are classified into two types, type A (MGD1) and type B (MGD2 and 3). MGD1 is expressed in photosynthetic tissues, whereas MGD2 and 3 are expressed in nonphotosynthetic tissues such as roots and inflorescences. A knockout mutant of MGD1 shows a disruption of thylakoid membranes and no chlorophyll accumulation, demonstrating that MGD1 has an indispensable role in chloroplast biogenesis.
Since light and cytokinin substantially up-regulate the MGD1 gene expression, we used mutants for light and cytokinin receptors in Arabidopsis and investigated the regulatory mechanism for galactolipid biosynthesis. Lipid analysis indicated that cytokinin signals are important for light-dependent galactolipid accumulation during early seedling growth. The results suggest that the regulation of MGDG synthesis by light and cytokinin is important for chloroplast biogenesis.

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Oxygen evolving photosynthetic organisms, such as plants or cyanobacteria has high amount of glycolipids that constitute the thylakoid membranes more than 90%. This is probably because phosphate is not enough available in nature to build complicated thylakoid membranes by phospholipids. In fact, it is reported that under phosphate limiting condition, content of phosphatidylglycerol (PG), a unique phospholipid in the thylakoid membranes, decrease with concomitant increase of glycolipids in Arabidopsis or Synechococcus sp. PCC7942. Here we identified DGDG synthase gene (dgdA) of Synechocystis sp. PCC6803 and knocked out the gene to analyze functions of DGDG under phosphate limiting condition. The wild-type and dgdA mutant grew similar in optimal medium but the dgdA mutant grew obviously slow under phosphate deprivation. These results suggest that DGDG is required under phosphate limiting condition such as those in natural niches of cyanobacteria.

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Photosynthetic organisms contain C16 fatty acids in the sn-2 positions of glycerolipids, which is largely controlled by the substrate specificity of lysophosphatidic acid acyltransferases (LPAAT). The cyanobacterium Synechocystis sp. PCC6803 contains two LPAAT genes, i.e., sll1848 for C16-acyl-specific LPAAT and sll1752 for C18-acyl-specific LPAAT. A knock-out mutant for sll1848 unusually contains C18-fatty acids in the sn-2 positions of glycerolipids and shows slower photosynthetic growth compared to the wild type. Phosphatidylglycerol (PG) in higher-plant plastids contains palmitate or trans-3-hexadecenoic acids in the sn-2 position. To evaluate the significance of fatty acid composition in the sn-2 position of PG, we introduced a recombinant sll1752 gene into an ats2 mutant that is null for plastid-targeted LPAAT of Arabidopsis thaliana. We will report the effect of gene expression on the fatty acid composition of PG as well as the growth phenotype of the transgenic plants.

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We previously reported that a green alga, Chlamydomonas reinhardtii, induces degradation of sulfoquinovosyl diacylglycerol (SQDG), an acidic sulfolipid in chloroplasts, on sulfur (S)-starvation for yielding an intracellular S-source. We report here that, in contrast to the SQDG degradation, the synthesis of phosphatidylglycerol (PG), the other acidic lipid in chloroplast, is increased to elevate its content up to a level that just compensates for the loss of SQDG. This regulation was proposed to occur through direct sensing of SQDG-loss, but not of S-starvation, because of similar regulation of PG metabolism in an SQDG-deficient mutant under S-replete conditions. Moreover, photosystem I activity of thylakoid membranes isolated from S-starved wild-type cells was decreased in company with partial removal of PG on treatment with phospholipase A₂, which implied that maintaining the total content of these acidic lipids contributes to the photosynthetic integrity under S-starved conditions.

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R3-type single MYB proteins have been shown to act as regulators of trichome and root hair development, but those involved in the regulation of anthocinain biosynsthesis were not known. We identified that AtMYBL2, R3-type single MYB, acts as a repressor of anthocyanin biosynthesis in Arabidopsis. Ectopic expression of AtMYBL2 and the chimeric AtMYBL2 repressor both suppressed accumulation of anthocyanin in rosette leaves, while AtMYBL2 knock-out line prominently enhanced expression of DFR and TT8 genes, with resultant ectopic deposition of anthocyanins. We found that the six amino acids of TLLLFR at the C-terminal end of AtMYBL2 act as repression domain. mybl2 phenotype was complemented by 35S:AtMYBL2, while AtMYBL2 from which the repression domain was deleted failed to complement. Promoter activities of AtMYBL2 were detected ubiquitously in many tissues of aerial parts. We discuss function of AtMYBL2 as a repressor and its functional involvement in the regulation of anthocyanin biosynthesis.

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