We obtained time-series data of the transcriptome and metabolome of sulfur-starved Arabidopsis. By analyzing the data using batch-learning self-organizing mapping, we found that known glucosinolate (GSL) biosynthesis genes were coexpressed under sulfur deficiency. Based on the assumption that unknown genes coexpressed with known GSL biosynthesis genes are also involved in GSL biosynthesis, we found a candidate gene encoding a transcription factor regulating GSL biosynthesis genes. We also conducted coexpression analysis using publicly available 1388 microarray data of AtGenExpress, and found that this gene is coexpressed with the genes involved in Met-derived GSL (MET-GSL). To confirm the predicted function as a transcription factor regulating MET-GSL biosynthesis genes, we analyzed the transcriptome and GSL profile of a knockout mutant and overexpressing T87 cell culture lines of this gene. The results showed that this gene is actually a positive regulator specific to MET-GSL biosynthesis genes but lesser for Trp-derived GSL biosynthesis genes.

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Integration of transcriptomics (DNA-microarray) and metabolomics (infusion-FTMS) enabled us to predict the co-regulated genes and metabolites in Arabidopsis under sulfur deficiency. In particular, this strategy is useful to identify the gene group involved in the same metabolic pathway. In this study, our integrated analysis was applied to identification of the side-chain-elongation enzyme (ELONG) of methionine-derived glucosinolates (MET-GSLs). The candidate genes, which are thought to catalyze the three steps in MET-GSLs ELONG reactions, were successfully predicted based on co-expression and co-accumulation patterns with known GLS biosynthesis genes and MET-GSLs. These candidate genes are homologs of the gene coding for leucine biosynthesis enzymes. In the MET-GSLs ELONG gene knock out Arabidopsis mutants, the levels of elongated MET-GSLs markedly decreased. These results suggest that these candidate genes are committed in the biosynthesis of elongated MET-GSLs.

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Rice Sekiguchi lesion (sl) mutant spontaneously induces a run-away type cell death on its leaves without an infection of pathogens. Recently, a gene responsible for the mutation has been isolated by map-based cloning and assigned as a putative cytochrome P450 (CYP71P1) based on the homologies with other plant CYPs. In this study, the metabolic profile of sl mutant was analyzed to estimate the function of CYP71P1 gene. The metabolites extracted from three lines of rice suspension cell cultures, wild type (WT), sl mutant (SL), and CYP71P over-expresser under sl mutant background (OX) were served for the metabolic profiling analysis using LC-ESI-MS (Shimadzu LCMS-2010). The analysis of the metabolic profile data indicated that level of tryptamine was increased while that of serotonine was decreased in sl mutant cells. The same phenotype was also observed in the leaf tissue of sl mutant suggesting that the function of CYP71P1 is tryptamine 5-hydroxylase.

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We propose a concept of "metabolite annotation", and report an application of metabolite annotation procedure to analyze plant metabolome data obtained by LC/FT-ICR-MS. We tentatively define "metabolite annotation" as a series of processes including clustering of MS peaks derived from single metabolites, and providing metabolite peaks with chemical information such as molecular formulas, MS/MS fragmentation patterns, and UV/visible absorption patterns. We used data of tomato fruit samples obtained by LC/FT-ICR-MS to establish metabolite annotation procedure. Combination of accurate m/z values and relative intensity of isotopic ions allowed us to cluster peaks and narrow down the candidate molecular formulas. We currently provide annotation to more than 300 metabolite peaks. As a result of tomato analysis, we conclude that metabolite annotation is informative for structural elucidation, and that metabolite annotation allows coherent comparison of metabolite peaks in numbers of LC/FT-ICR-MS data. Additionally, a comprehensive metabolite annotation facilitates a prediction of metabolic pathway.

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We present a novel informatics system for automatic high-throughput processing of metabolic profiling data obtained from liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry (LC/FT-ICR-MS). The data processing system is implemented with tools that facilitate 1) normalization of m/z value fluctuations in each single scan using a regression analysis of m/z values of internal mass calibrants, 2) organizing corrected m/z values from a large number of scans in a single m/z value-matrix, and 3) elimination of noise peaks and detection of statistically significant peaks. In addition, we report an application of the system to metabolome data obtained from tomato fruit samples. Peaks selected by the system are annotated with chemical information including putative molecular formulas, MS/MS fragmentation patterns, and UV/VIS absorption patterns. Our data processing system provides firm basis for metabolite annotation procedure, and allows coherent analysis of numbers of LC/FT-ICR-MS data.

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To integrate metabolomic data generated by analytical instruments with transcriptomic data obtained by microarray experiments, and to facilitate identification of gene functions and metabolic regulations, we developed a web-based analytical tool, KaPPA-View (kpv.kazusa.or.jp/kappa-view/), which represents profile changes of transcriptome and metabolome as color changes of symbols for genes and metabolites on pathway maps. Here we report the new version of the tool, KaPPA-View2. Availability of a lot of gene expression data in public enables us to investigate regulatory features of gene expressions by calculating indices such as Pearson's correlation coefficients. KaPPA-View2 can represent inter-gene and inter-metabolite relationships as smooth curves overlay on pathway maps. Gene expression correlation data provided from ATTED-II (www.atted.bio.titech.ac.jp) and user-uploaded data are available to draw relationship curves. This function facilitates us to elucidate functional differences between paralogous genes.

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Plants release a certain amount of photosynthate as Rhizodeposition. Though individual compounds have been surveyed so far, total of the root exudates is not figured out. We have established aseptic cultivation system of rice to obtain root exudates without microbial contamination. The results of global analyses of root exudates by Gas Chromatography - Mass Spectrometry (GC-MS) are reported.
Root exudates was collected from rice (Oryza sativa cv. Nipponbare) grown under +P / -P conditions on hydroponic culture. Sterility of the system was confirmed by 1 / 10 SCD medium. Root exudates were eluted by methanol from freeze dried sample, and then analyzed by GC-MS. About 90 peaks were detected and a half of which were identified by AMDIS software. Under P deficiency, decreased exudation of sugars and increased amines and fatty acids were indicated by PCA. Further analysis with UPLC-MS, expanded for secondary metabolites is in progress.

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The cinnamate-monolignol pathway, a biosynthetic route with one of the highest metabolic flows in plants, produces a wide variety of phenylpropanoid compounds. The pathway enzymes are often encoded by multiple genes and accept several substrates leading to the formation of a complex metabolic grid. A major challenge is due to the difficulties in understanding whether or not dynamic metabolic flows and physiological substrate combinations may be deducible through in vitro enzyme studies with putative substrate compounds. The extremely high sensitivity and resolution of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR/MS) warrant accurate MS measurements, and its performance has been developed as a unique metabolomics tool. Here, using the FT-ICR/MS metabolomics approach, we simultaneously studied sequential reactions using recombinant proteins of 4CL (4-coumarate:CoA ligase), HCT (hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyltransferase), and a cytochrome P450 with multiple substrates and discuss the potential of the FT-ICR/MS metabolomics for high-throughput screens for enzyme reactions.

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Flavonoid is a class of major plant secondary metabolites with C6-C3-C6 skeleton derived from phenylpropanoid-acetate pathway. More than 6000 flavonoids are reported from various plants. Flavonoid serves as not only pigments but antioxidant, anti-inflammatory agents. Structural analysis is the key, because structural classification reveals, physico-chemical properties, modification patterns, biosynthetic pathways, and pathway evolution in plants. Structure-species information provides evolutionary relationships. In this context we developed hierarchically classified flavonoid structure database related with pathway chart. In the database, almost 6000 flavonoids are included and classified with 12 digit ID system by its core structure, hydroxylation patterns, and substitution patterns. We also developed viewer system to display structure-species relationship related with pathway chart. Software system is available at http://www.metabolome.jp/. We plan to add other phenylpropanoids and plant secondary metabolites to the viewer system in the future.

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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 transcription factor (TF) genes that were coexpressed with cell wall formation genes.
We experimentally examined functions of the TFs using Arabidopsis cultured cell T87. Overexpression of one of the TF genes, tightly coexpressed with genes involved in secondary cell wall formation, caused phenotype change, which was confirmed by Fourier transform infrared spectrophotometer analysis. RNAi-induction of other TFs that coexpressed with genes involved in primary cell wall formation using glucocorticoid-inducible transcription system revealed lethal phenotypes when grown on dexamethasone. With a focus on these TFs, we will also report microarray analyses and metabolomic analyses using mass spectrometry.

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Plant isoprenoid pathways produce various metabolites including important industrial materials such as rubber. To investigate regulatory mechanisms of the pathways, we analyzed co-expression networks with correlation coefficients that were calculated from the 771 microarray data of AtGenExpress. According to the outcome networks, we selected 12 genes as candidates for further analysis. Full-length cDNA (RAFL) clones for the genes were obtained from RIKEN and overexpressed them under 35S CaMV promoter in the T87 Arabidopsis suspension-cultured cells. The transgenic cell lines were analyzed with DNA microarray and non-targeted metabolite profiling using GC/MS, UPLC-Q-TOF/MS and FT-ICR/MS. We preliminarily found that two candidate genes actually affected gene expression of MEP and MVA pathway enzymes, and that several unidentified peaks in GC/MS distinguished the overexpression lines from control. Currently we are working on identifying the metabolite peaks influenced as well as analyzing transgenic cell lines for the other candidate genes.

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In previous study, we reported that one of the isopentenyltransferase in Arabidopsis, AtIPT3, is a key determinant of nitrogen-dependent cytokinin biosynthesis. AtIPT3 is an enzyme that catalyzes the initial step in the biosynthesis of cytokinin, and the gene expression is induced by nitrate. In rice, seven genes of isopentenyltransgerase (OsIPT1-5, 7-8) were identified as the genes involved in the cytokinin biosynthesis. The intracellular localization of each gene products is observed in chloroplast, mitochondrion, or cytosol, respectively. To identify the key determinant of nitrogen-dependent cytokinin biosynthesis in rice, we analyzed gene expression of OsIPT in the nitrogen-treated rice root and shoot. In the results, we found that some OsIPT genes are induced in root and/or shoot both by nitrate and ammonium ion. It is different from the result of AtIPT3, which is induced only by nitrate. It suggests that there are different mechanisms of nitrogen-induction from OsIPTs and AtIPT3.

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Plants uptake and assimilate nitrate and ammonium, and then biosynthesize various compounds involving nitrogen, including amino acids, protein and nucleotides. Nitrogen supply not only directly induces expression of nitrogen-responsive genes but also elevates the levels of assimilated products, triggering the secondary responses. To comprehensively investigate alterations in metabolite contents and gene expression pattern, we performed metabolic profiling and microarray analyses, using rice seedlings that were treated with 10 mM ammonium nitrate for different periods. Results of the analyses revealed different alterations in shoots and roots, and also allow us to identify both transcription factors that were induced rapidly and transcription factors that were induced in parallel with an increase of glutamine. Such transcription factors were members of the AP2-EREBP, the MYB, the NAC, the WRKY, or the zinc-finger family. We are currently attempting to identify the target genes of the transcription factors.

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We previously reported an increase of free amino acids and enhancement of nitrogen assimilation in transgenic Arabidopsis expressing maize Dof1, a transcriptional activator for expression of the genes encoding enzymes involved in carbon skeleton production. Here, we show ammonia-dependency of the Dof1 effects. While the increase in amino acid contents was observed neither in shoots nor in roots of the Dof1 transgenic Arabidopsis grown on the medium without ammonia, a significant increase in amino acid contents was specifically detected in shoots of the transgenic Arabidopsis grown on the medium with ammonia. Furthermore, reduction in malate and fumarate contents and activation of AtPEPC1 and AtPK1 were also observed only in shoots of the transgenic Arabidopsis grown on the medium with ammonia, suggesting that the ammonia-dependent Dof1 effects are specific to shoots. The gene expression profiling for further characterization of the Dof1 transgenic Arabidopsis is currently in progress.

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Citrulline is an efficient hydroxyl radical scavenger which accumulates in the leaves of wild watermelon under drought. Previously, we found that the activities of four key enzymes in citrulline metabolic pathway are modulated under drought. In this study, we focused on the regulatory mechanisms for acetylglutamate kinase (AGK) activity, which is up-regulated under drought. Enzymatic assays using leaf crude extracts showed that AGK from unstressed leaves was strongly feedback-regulated by arginine. In contrast, AGK from stressed leaves was insensitive to arginine inhibition, implying that PII protein, a regulator of carbon and nitrogen status, is involved in this regulation. Moreover, addition of PII protein to crude extracts from unstressed leaves relieved the inhibitory effect of arginine on AGK activity. Furthermore, immunoblot analysis revealed that abundance of PII protein increased in wild watermelon leaves under drought. These results suggest that PII protein plays pivotal roles in citrulline accumulation in wild watermelon.

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Rubisco is the key enzyme responsible for photosynthesis and its amount is increased with increasing N supply. In this study, changes in the mRNA levels of rbcS nuclear multigene family (genes coding for the small subunit of Rubisco) in response to N supply were examined in rice. Rice plants were grown hydroponically with different levels of N supply and the mRNA levels in tenth leaf blade were determined. There are five rbcS genes (designated as OsRBCS1 to OsRBCS5) and the mRNAs of OsRBCS2 to OsRBCS5 were highly accumulated irrespective of N supply, The maximum levels of these mRNAs were increased with high N supply. The maximum mRNA levels of OsRBCS2 and OsRBCS4 were decreased with low N supply, whereas these of OsRBCS3 and OsRBCS5 were increased. Therefore, it is concluded that the mRNA levels of rbcS multigene family were differentially regulated in response to N supply in rice.

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Ribulose 1,5-bisphosphate carboxylase oxygenase ( RuBisCO ) catalyzes both carboxylation and oxygenation reactions of RuBP. The specificity for carboxylation relative to oxygenation is represented by S_rel (S_rel = Vmax(CO₂) Km(O₂) / Vmax(O₂) Km(CO₂) ). We expect that the creation of RuBisCO with high S_rel enables plants to acquire high photosynthetic efficiency.
RuBisCO of red alga Galdieria partita shows the highest S_rel of 238 among RuBisCOs examined so far. Galdieria RuBisCO has a unique structure involved in high S_rel. This structure is a hydrogen bond called the latch structure between the main chain oxygen of Val332 and the amino group of Gln386. No latch structure can be seen in plant and cyanobacterial RuBisCOs.
To clarify the relationship between S_rel and the latch structure, we introduced the latch structure into cyanobacterial RuBisCO known to show low S_rel . Mutant RuBisCO was likely to show higher S_rel than that of wild type.

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Chloroplastic carbonic anhydrase in the marine diatom Phaeodactylum tricornutum, PtCA1, is assumed to balance Ci species in the chloroplast. PtCA1 gene (ptca1) is a typical CO₂-responsive gene whose expression is repressed under elevated [CO₂]. In this study, mechanisms of regulation of the ptca1 promoter, Pptca1 was investigated. The core-regulatory region of Pptca1 (downstream -70bp relative to the transcription-start site) comprises of three putative cis-elements (CRE1, P300-binding site, and CRE2). Deletions or substitutions of these elements were carried out, fused to uidA-reporter gene, and introduced into P. tricornutum. Legions in CRE1 and p300bs caused a lack of ptca1 repression even in high CO₂. Moreover, treatment of cells with a cAMP analogue or a cAMP-phosphodiesterase inhibitor efficiently repressed the endogenous Pptca1 even in air, but these repressive effects were disappeared by deleting CRE1 from the Pptca1. The function of cAMP for CO₂ sensing mechanism in marine diatoms will be discussed.

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The β-type carbonic anhydrase (PtCA1) localizes as clustered particles at the surface of the girdle lamellae in the marine diatom Phaeodactylum tricornutum . Although, physiological roles of PtCA1 and its particle formation are not known, GFP analysis of N- or C-terminus truncation of the mature PtCA1 suggested that 263-282 region of C-terminus might be crucial for the particle formation of PtCA1. Hydrophobic Cluster Analysis for C-terminus (253-282) region of PtCA1 suggested that hydrophobic amino acids, M, L, I, L, and L could form a hydrophobic cluster on one face of the putative C-terminus α-helix. The hydrophilic residue, glutamic acid, was substituted for each or all of cluster-forming hydrophobic residues, M, L, I, L, and L in PtCA1-GFP fusion. As a result, the expressed GFP fusions dispersed with the stroma area without forming particles, indicating that these cluster-forming-residues play an essential role for the particle formation of PtCA1.

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In CO₂-limiting stress conditions, aquatic photosynthetic organisms induce active uptake systems for inorganic carbon (Ci) that enable the accumulation of Ci within the cell (Carbon-Concentrating Mechanism: CCM). In a green alga, Chlamydomonas reinhardtii, the regulatory factor CCM1 (CIA5) is indispensable for induction of the CCM. The CCM1 has two putative zinc-finger domains in its N-terminal region. Considering the fact that the mutant cia5, which has a H54Y point mutation in CCM1, does not induce the CCM under the Ci-limiting stress conditions, the putative zinc-fingers were considered to be indispensable for the function of CCM1. In order to analysis the zinc-binding activity of the putative zinc-binding site, the N-terminal region of the CCM1 were fused with GST-tag and subjected to atomic absorption analysis. We discuss about the importance of the zinc binding for the induction of CCM.

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Acclimation to CO₂-limiting stress is achieved by monitoring of the environmental changes through putative CO₂-signal transduction pathways. Our focus is to understand how the changes in CO₂ levels are sensed and the roles of low-CO₂ responsive genes. Using cDNA array, we identified a gene, named as LciB, encoding a 48-kDa soluble protein, showed rapid induction during the acclimation process in low-CO₂ conditions. Induction of the LciB mRNA was impaired in a mutant, ccm1, that cannot acclimate to low-CO₂ conditions and LciB complemented a mutant, pmp1, lacking a part of CO₂-transport activity. We generated RNAi strains that decrease the abundance of the LCIB protein and carried out a photosynthetic characterization. We also examined the subcellular localization of LCIB protein by immuno-histochemistry. Based on the above observation, it is possible that LCIB protein is associated with putative CO₂-transports under low-CO₂ conditions.

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The most abundant unicellular calcifying alga, Emiliania huxleyi (Coccolithophrales, Haptophycea), is known to form a huge bloom in the ocean. Therefore, the alga plays a significant role in the global carbon cycle. To know physiological factors for bloom formation, it is essential to reveal the initial metabolism of photosynthesis. Using a ¹⁴C-radiotracer technique, we analyzed primary metabolites in photosynthesis. The labelling pattern was typical for C₃ photosynthesis but the operation of beta-carboxylation was also active to produce significant amount of amino acids such as Ala, Asp and Glu. By the homology search of β-carboxylation enzymes in the Emiliania EST library, partial sequences highly homologous to phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PYC) were found. Northern blotting analysis revealed that the PYC transcript increased under illumination whereas the PEPCK transcript was suppressed. These results indicated that PYC is a responsible enzyme for active anaplerosis during photosynthesis in this alga.

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One of rice cultivars, Oryza glaberrima has been thought as unfavorable environment resistance. However, the physiological adaptation of Oryza glaberrima to water stresses is not understood. The physiological response of Oryza glaberrima to flash floods was studied.
This experiment was conducted in Guinea from May to June in 2006. Saligbeli (Oryza glaberrima), Ballawe and IR 49830-7-1-2-2 (Oryza sativa) were used. The seedlings at 12 days old were submerged completely for 7 days at 1 m water depth.
Saligbeli and Ballawe elongated the leaf sheath after submergence, which resulted in the significant higher shoot elongation than that of IR 49830-7-1-2-2. Before submergence, the ratio of the root to the shoot of Saligbeli was higher than that of other cultivars, but the ratio became the same level ratio of other cultivars during submergence, suggesting that Saligbeli maintains the upper part growth by changing the distribution of dry matter weight during submergence.

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Carotenoids (Cars) are group of pigments complementary to bacteriochlorophyll (BChl), which play important functions of light-harvesting, which include capturing light energy by Car followed by transferring its singlet energy to bacteriochloropyll. In this paper, subpicosecond time resolved absorption spectra of carotenoids having 9-11 conjugated double bonds were recorded in the visible region, upon excitation at different vibronic excited states, 1B_u⁺(v = 0,1,2). The spectral data matrices were analyzed by singular-value decomposition (SVD) followed by global fitting by the use of a sequential model. SVD was used to extract all significant components and the decay time constants determined by global fitting. The lifetimes (τ) of the 1B_u⁺, 1B_u^- and 2A_g^- states shortened with the number of conjugated double bonds (n) among neurosporene (9), spheroidene (10) and lycopene (11). The linear dependence of lnτ as a function of 1/(2n+1) was explained in term of the Englman-Jortner energy-gap law.

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Carotenoids transfer the light energy to chlorophylls in plants by the use of singlet excited states. Vibration spectroscopy of deuterium-substituted substances can be a powerful tool to investigate the excitation and relaxation processes. β-carotene in natural abundance and 6 different types of deuterium-substituted β-carotene (12-d, 12,12'-d₂, 14-d, 14,14'-d₂, 15-d, 15,15'-d₂) were synthesized, purified, and ¹H NMR ¹H-¹H COSY, ¹H-¹H NOESY spectra were recorded.
   The assignment shows high-field-shift of ¹H signal adjacent to deuterium. Though the signals of di-substituted species were simple, mono-substitution made the signals complicatedly split and overlapped. These results showed the expected deuterium substitution was performed.
   It is expected the frequency and coupling parameter among vibronic modes should change. We are trying to measure 1) ground state Raman spectroscopy to identify the spectral patterns 2) sub-picosecond stimulated Raman spectroscopy to find out the isotope effect on relaxation processes in the excited states.

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The triplet-state absorption spectra were recorded for carotenoids (Cars), whose number of conjugated double bonds (n) being in the region of 9–13, to determine the dependence on n of the T₁ lifetimes (τ), and reached to the following conclusions: (1) A substantial shift of the linear dependence to shorter lifetimes, on going from solution to the LH2 complex, was ascribed to the twisting of the Car conjugated chain. (2) A substantial decrease in the slope of the linear dependence, on going from the reconstituted LH1 to the LH1 component of the RC-LH1 complex, was ascribed to the minor-component Car forming a leak channel of triplet energy. (3) The loss of conjugation length-dependence, on going from the isolated RC to the RC component of the RC-LH1 complex, was ascribed to a triplet-energy reservoir consisting of bacteriochlorophylls in the RC component.

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A marine purple photosynthetic bacterium, Rhodovulum sulfidophilum, synthesizes a membrane-bound cyatochrome c, in addition to the water-soluble cytochrome c₂, as a possible electron donor to the photochemical reaction center. The gene coding for this cytochrome c was cloned and analyzed. The deduced molecular weight was 50,528. Its C-terminal heme-containing region showed the highest sequence identity to the water-soluble cytochromes c₂. Phylogenetic analyses suggested that this membrane-bound cytochrome c has evolved from cytochromes c₂. Mutants lacking this cytochrome or cytochrome c₂ showed the same growth rate as the wild type. However, a double mutant lacking both cytochromes c showed no growth under photosynthetic conditions. It was concluded that either the membrane-bound cytochrome c or the water-soluble cytochrome c₂ work as a physiological electron carrier in the photosynthetic electron transfer pathway of R. sulfidophilum.

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Filamentous anoxygenic phototrophic bacteria (formerly called green non-sulfur bacteria) are positioned in the most deeply branched lineage among phototrophic bacteria in the 16S rRNA phylogenetic tree. They have a quinone-type reaction center complex which is similar to that of purple photosynthetic bacteria. However, other components working in the photosynthetic electron transfer pathway of the filamentous bacteria have not been fully clarified. In this study we purified and characterized the candidate protein, auracyanin, from Roseiflexus castenholzii. The purified auracyanin showed a typical absorption spectrum of a blue-copper protein and had a midpoint potential of 236mV. We are also trying to purify the possible quinol oxidoreductase in the filamentous anoxygenic phototroph.

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All-trans carotenoids have low-lying singlet states with different symmetries. The energy transfer from carotenoid (Car) to bacteriochlorophyll (BChl) is governed by internal conversion and vibrational relaxation. In the present investigation, we focus our attention on the vibrational relaxation. The 1B_u⁺ state plays the most important role in Car-to-BChl singlet-energy transfer, in both shorter-chain and longer-chain Cars. We measured the time-resolved absorption spectrum of spirilloxanthin (the number of conjugated double bonds, n = 13), having the shortest lifetime in the 1B_u⁺ state, by the use of approximately 40 fs pulses. In the initial time evolution, two peaks appeared that is to be assigned to the 1B_u⁺(1) and 1B_u⁺(0) states; the former decayed much faster than the latter. The lifetime of vibrational relaxation is substantially longer than that of internal conversion, so that the Car-to-BChl singlet-energy transfer and the internal conversion can take place competitively from plural 1B_u⁺ vibronic levels.

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In order to understand the important functions of carotenoids in photosynthesis systems, their excited-state energies and lifetimes should be determined correctly. Because the time-resolved fluorescence up-conversion spectroscopy is free from stimulated emission and transient absorption, it is very suitable to achieve that. For the sake of obtaining the up-conversion signal of the 1B_u⁺(0)→1A_g^-(0) transition and minimize the effect of vibrational relaxation, spheroidene was excited upon the 1B_u⁺(1)←1A_g^-(0) transition. We have recently succeeded in obtaining time-resolved fluorescence data by the use of 1 KHz, 100 fs pulses. SVD followed by global fitting was used to analysis the time-resolved fluorescence up-conversion spectra, we found that two dynamic processes were present in the spectra. We assigned the faster and slower processes to the relaxation dynamics of 1B_u⁺ and 1B_u^- states, respectively. The lifetimes of 1B_u⁺ and 1B_u^- states were determined to be 0.04 ps and 0.30 ps, respectively.

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The possible mechanism of dissipation of triplet energy in reaction center (RC) bound carotenoid has been established by a pair of investigations which proposed that the series of configurational changes of carotenoid could facilitate the triplet-energy dissipation. The sub-microsecond time-resolved Raman spectra of spheroidene bound to the reduced RC of Rb. sphaeroides 2.4.1 were recorded at room temperature. The OD800 nm=100 cm^-1 sample with 100 mM ascorbate acid were pumped at 590 nm and probed at 532 nm respectively. After spectral analysis we observed a series of time-dependent spectral changes which could be attributed to the triplet-state configurational changes of the RC bound carotenoid. As strengthened by our preliminary study of time-resolved EPR spectroscopy (Yoshinori Kakitani, et. al., Biochemistry 2006, 45, 2053-2062), this investigation provided us the definitive evidence of the triplet-state configurational changes of the RC bound carotenoid functioned as the dissipation of triplet-energy under physiological temperature.

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We analyzed energy transfer and electron transfer process in a Synecocystis PCC6803 mutant that has an inactivated pgsa gene, which functions as one of the terminal enzymes in the biosynthesis of a lipid phosphatidylglycerol (PG). 1) The depletion of PG from the medium increased the fractions of un-assembled PS II reaction center (RC) complex lacking CP43 or CP47. 2) The amount of PS I RC decreased. 3) The de novo synthesis of PS II proteins continued but the newly formed proteins were not incorporated into the PS II and PS I RC complexes. 4) We analyzed 77K fluorescence lifetime to know the energy transfer. The results indicated the decreased intensity of 694 nm band from CP47, the increases in the intensity and the lifetime of the 683 nm band, which was assigned to allophycocyanin fluorescence, while the lifetime of phycocyanin fluorescence was not affected indicating the proper assembly of phycobilisomes.

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The chlorophyll dimer P680 traps the light energy and its redox potential (E'₀ > +1.2 V) fixes the driving force available for water oxidation. In this study, to know the effect of a ligand of D1 side of P₆₈₀ (P_D1) to its redox potential in PSII, we analyzed PSII from the thermophilic cyanobacterium Thermosynechococcus elongatus in which a normal His ligand of P_D1 was substituted for either Ala or Gln by site-directed mutagenesis.
Both mutant cells grew photo-autotrophically. However, the amounts of phycobilliproteins in the mutants were smaller than in the WT*. The energy transfer from phycocyanin to PSII via allophycocyanin in both mutants was not efficient as that in WT*. Purified PSII core complexes were fully active in oxygen-evolution. FTIR spectra shows the stractural changes of P₆₈₀. Flash-induced absorption changes and themperature shifts in themoluminescence spectra suggested possible changes in the redox potential of P₆₈₀ ⁺/ P₆₈₀.

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The redox-active tyrosine Y_D (D2-Tyr160) in photosystem II (PSII) serves as an accessory electron donor to P680. When oxidized, Y_D releases a proton and becomes a neutral radical Y_D^·. The X-ray structures of the PSII core complex showed that D2-His189 and D2-Gln164 are located near Y_D forming a H-bond network, which should play an important role in proton-coupled electron transfer. In this study, we have studied the H-bond structure of Y_D using Fourier transform infrared (FTIR) spectroscopy. Light-induced Y_D^·/Y_D FTIR difference spectra were obtained with PSII core complexes from Thermosynechococcus elongatus. The 1252 cm^-1 band was assigned to the coupled mode of CO stretching and COH bending vibrations by labeling of Tyr residues with [4-¹³C]Tyr and measurement in D₂O. From quantum chemical analysis of this CO/COH band, it is concluded that Y_D is H-bonded with both D2-His189 and D2-Gln164.

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Bicarbonate is known to be a ligand to the non-heme iron located between Q_A and Q_B in PSII. It has also been proposed that bicarbonate is a ligand to the Mn cluster and controls O₂-evolving reactions. However, details of its structural relationship remain unclarified. In this study, structural coupling of bicarbonate with the Mn cluster was studied using Fourier transform infrared (FTIR) spectroscopy. Flash-induced FTIR difference spectra of S-state cycle were recorded using PSII in the presence of H¹²CO₃^- or H¹³CO₃^-. In ¹²C -minus-¹³C double difference spectra, bicarbonate bands were observed at the 1st flash but no bands were detected at the 2nd-4th flashes. These bicarbonate bands were identical to those in the non-heme iron spectrum measured with Mn-depleted PSII. Thus, no bicarbonate band arising from the S-state cycle was detected in FTIR spectra. This result strongly suggests that bicarbonate is not a ligand to the Mn cluster.

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Tiller of rice plants is corresponding to a lateral branch in dicots and its number is recognized as an important agricultural trait. It is well known that increment of tiller number is significantly repressed when rice plants are cultivated under dense planting. However, mechanism of the repression has been unclear yet. Based on results obtained from other plants, responses against dense planting are induced by high FR/R ratio in ambient light quality, which is monitored by phyB.
To clarify function of phytochromes in monitoring of dense planting, we examined tiller numbers of several phytochrome mutants grown either under sparse or dense planting. Not only Nipponbare but also all mutants examined (phyA, phyB, phyC, phyAphyC, phyBphyC) showed normal responses against dense planting. It is noteworthy that absence of phyB give no effect to monitoring of dense planting, suggesting that phyB is dispensable for these responses in rice plants.

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Under low-light conditions, chloroplasts accumulate in the light to capture weak light efficiently (accumulation response), while under high-light conditions, they escape from the light to avoid their photodamage (avoidance response). In a seed plant Arabidopsis thaliana, the accumulation response is mediated by two blue light receptors, termed phototropins (phot1 and phot2) that act redundantly, and the avoidance response is mediated by phot2 alone. We recently isolated a mutant, kac1, which is deficient in chloroplast photorelocation movement. Map-based cloning revealed that KAC1 gene encodes a novel protein specific to plants. KAC1 transcripts are detected in leaves, stems, flowers and roots. KAC1 proteins are enriched in soluble fraction and are detectable also in microsomal fractions. The amount and the localization pattern of KAC1 proteins do not change in mutants, phot1phot2, jac1 and chup1, which are deficient in chloroplast photorelocation movement, comparing to those of wild type plants.

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Phytochromes regulate various physiological light responses in plants. In Arabidopsis thaliana, the over-expression of N-terminal domain of phytochrome B linked to GFP-GUS, rescued some phyB phenotypes. To reveal the function of rice phyB molecule, transgenic rice (phyB-2) plants over-expressing OsPhyBN450 and OsPhyBN651, fused to the GFP-GUS, were examined. Rice phyB mutant showed some altered photomorphogenesis such as longer coleoptile and pale colored first leaf under red light, and increased angle of second leaf blade under blue light. Under red light, those phenotypes were partially rescued by over-expression of OsPhyBN651, but not by OsPhyBN450. Under blue light, plants with OsPhyBN450 exhibited a similar phenotype to the wild type while plants with OsPhyBN651 exhibited a drastically stronger phenotype than wild type. These results indicate that the signaling mechanism of rice phyB molecule differs from that of A. thaliana , and distinct phyB domains may be required for various physiological responses in rice.

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The bending of a plant toward the direction of intense light is called phototropism. This directional growth response is caused by the plant growth regulating substances. In this aspect, Bruinsma-Hasegawa hypothesis (1990) stated that the gradient of growth-inhibiting substances (phototropism-regulating substances) in the illuminated side is a key factor of bending during phototropic curvature. Although some phototropism-regulating substances were isolated from several plant species, the molecular mechanism underlying this phenomenon is still largely remained. The symbolic phototropism-regulating substances, cis- and trans-raphanusanins were isolated from radish hypocotyls. To understand the role of raphanusanins in phototropism and the responsible genes of growth inhibition, differential display was performed between the raphanusanin applied and control hypocotyls. We could isolate some candidate genes. Almost all positive clones were related to biotic and abiotic stress. The detailed roles and functional impacts of these genes in the growth inhibition will be presented.

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Regulatory mechanisms of stress responses in rice largely remain unclear. Using rice microarray, we identified many abiotic stress-responsive genes. Among them, a gene for a bHLH transcription factor down-regulated by drought stress has been studied. The bHLH protein showed a high sequence homology with Arabidopsis PIF1, driving us to name the transcription factor OsPIF1. Transgenic rice plants overexpressing OsPIF1 and dominant loss-of-function rice mutants with the chimera repressor changed internode elongation, suggesting that OsPIF1 functions as an important growth regulatory factor in response to drought stress in rice. Here, we clarified that OsPIF1 is highly expressed in a node using promoter-GUS analysis. We also found a candidate of target genes of OsPIF1 using microarray analysis. The candidate gene encodes an ethylene biosynthesis enzyme. The gene expression was strongly detected in the node and reduced by drought stress. Currently, we are trying to identify target genes of OsPIF1.

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Phytohormone abscisic acid (ABA) inhibits blue light-dependent stomatal opening and enhances the stomatal closure under drought stress by unknown mechanisms. Here we show that nitric oxide (NO), a second messenger of ABA signaling, specifically inhibits blue light-dependent stomatal opening. Blue light-dependent stomatal opening and H⁺ pumping were inhibited by a NO donor SNP. All of these inhibitions were restored by c-PTIO, a specific NO scavenger. Importantly, c-PTIO partially blocked the inhibitions of blue light-dependent stomatal opening and H⁺ pumping by ABA. Furthermore, SNP inhibited blue light-dependent phosphorylation of the plasma membrane H⁺-ATPase and a subsequent binding of a 14-3-3 protein. However, SNP had no significant effect on the phosphorylation of phototropins, blue light receptors of stomatal opening. These results suggest that NO is responsible for the inhibition of blue light-dependent stomatal opening by ABA through the intercept of blue light signaling between phototropins and the H⁺-ATPase.

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Establishment of an effective supplementary lighting procedure was necessary to improve the quality of hydroponically grown red-leaf lettuce without increasing the production cost. The effect of light quality in supplementary lighting on the flavonoids (anthocyanins and flavonols) concentration and the expression of those biosynthesis related genes was investigated in leaves of the lettuce. Flavonols concentration increased in the immature leaves than those grown under the other light source, such as sole UV-B, blue and red. Grown under the simultaneous light with UV-B and blue. At this time, one of the key enzymes, flavonol synthase (FLS) expression in the immature leaves was almost coordinately regulated with the level of flavonols. It is notable that FLS expression in the immature was especially increased under the UV-B and blue simultaneous lighting with the immature leaves. The Inoue Enryo Memorial Foundation for Promoting Sciences supports a part of this work at 2006 fiscal year.

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Red light irradiation at the beginning of dark period (RB) or blue light irradiation at the end of dark period (BE) at a low level promoted the biomass production in spinach (Hanyu and Shoji, 2000). We supposed this promotion of growth was related to the leaf area enlargement, and investigated how RB or BE affects leaf expansion. We grew spinach plants under white light at 220 µmol m^-2 s^-1 for 12 h with RB or BE at 30 µmol m^-2 s^-1 for 0.5 h. RB or BE promoted the biomass production accompanied by the increase in leaf area. Microscopic analysis of the leaf transverse sections revealed that RB increased the leaf cell size, while BE did not affected it. These results suggested that RB accelerated leaf expansion by the increase in leaf cell size and BE accelerated leaf expansion in the increase of leaf cell number.

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Plants have a energy dissipating mechanism called qE-quenching (qE) induced by pH of trans-thylakoid membranes under excess light conditions. It has been suggested that zeaxanthin formed by de-epoxidation of violaxanthin (xanthopyll cycle) is involved in qE in higher plants. However, Chamydomonas reinhardtii has a decreased level of qE and does not express PsbS protein that is essential for qE in higher plants. Therefore, it is inferred that the mechanism of qE could differ among plant species. In this study, we performed PAM fluorescence measurement and HPLC analysis using a mutant npq1 lor1 that lacks violaxanthin de-epoxidase and does not accumulate zeaxanthin. The results indicated that npq1 lor1 and wild type showed similar levels of qE. Therefore, it is suggested that the small level of qE obserbed in Chlamydomonas is independent of xanthophyll cycle.

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Suspension-cultured cells of azuki bean do not grow in a liquid medium containing mannose (Man) but callus cells proliferate actively on agar medium converting Man to sucrose (Suc). In the present study, we examined the effects of Man on cell-growth and sugar composition in azuki-bean seedlings aseptically grown under the light/dark conditions.
Plant-seedlings were grown for 7 days on agar media containing MS salts supplemented with or without 90 mM Suc or Man. Man strongly inhibited the length, fresh weight and the cell-wall dry weight of roots, but it increased the levels of Suc, Glc and Fru of cells under both conditions. Man also inhibited cell-wall synthesis and cell-growth in shoots. Abundance of Suc in cotyledons implied an importance of its translocation to shoots.
We conclude that Man inhibits cell-wall synthesis and hence inhibiting cell-growth in the plants,unlike in callus cultures.

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The role of plant purple acid phosphatases (PAPs) has ever been proposed for the degradation of organophosphates, when plants are under phosphate starvation. Nevertheless, phosphate deprivation did not induce some PAPs in tobacco cells. In the transgenic tobacco cells overexpressing the PAP (NtPAP12), not only wall regeneration but also cellulose deposition was accelerated on the surface of tobacco protoplasts. Since PAPs are members of a group of nonspecific phoshomonoesterase, containing protein phosphatases, calcineurin (type 2B) and protein phosphatases type 1, the tobacco PAP could probably function as the type of protein phosphatase. We have identified some potential substrates for NtPAP12 in suspension-cultured tobacco cells by phosphoproteome.

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In the previous presentation, we suggested the potential substrate related to wall proteins for the tobacco purple acid phosphatase (NtPAP12). In order to examine whether they are really physiological substrates in the tobacco cells, those enzyme activities in the transgenic cells overexpressing NtPAP12 were compared to those in the wild-type cells. Those activities in some wall enzymes were enhanced in the transgenic cells, on the other hand, some wall enzymes were suppressed in the transgenic cells. In this presentation, we would suppose a possible dephosphorylation-action of a wall enzyme by the purple acid phosphatase, which related to the phenotype in the transgenic cells.

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We suggest that purple acid phosphatases (PAPs) have a physiological role relating to β-glucan deposition in suspension-cultured tobacco cells. To study the function of PAP in an intact plant, we expressed tobacco PAP (NtPAP12) constitutively in Arabidopsisi thaliana. In this work, we analyzed the phenotypes between the transgenic lines, T-DNA knock-out lines of AtPAP10 and the wild-type. Since AtPAP10 has the highest homology to NtPAP12 among Arabidopsis PAP genes, we suppose that roles of AtPAP10 correspond to NtPAP12. The cells of rosette leaves in the transgenic lines were smaller in diameter than those in the wild-type and the knock-out line. We will analyze compositions and quantities of wall polysaccharides in the three lines.

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Xylan is the main hemicellulose in secondary xylem wall of angiosperm and has β-1, 4 bound main chain. An endo-β-1, 4-xylanese gene, which was originated in barley, was introduced into poplar with 35S CaMV promoter. Several lines of trasngenic poplar were obtained and the xylanase activities of the lines were much higher than that of the wild type. The result of methylation analysis showed that the amount of xylan was significantly less than that in wild type. Not so much as xylan, the transgenic poplar contained less lignin than the wild type. Moreover, fluorescent microscopic observation suggested that the lignification of interfascicular xylem in the trasgenic would be later than that in the wild.

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The overexpression of xyloglucanase increased the length of stem, accompaning by a decreased elasticity (Young s modulus) in the growth zone, an increased elasticity in mature tissue, whereas cellulose deposition was enhanced in both regions. Thus, degradation of the xyloglucan network can be a useful strategy for accelerating growth and cellulose content in trees. The phenotype of leaf in the transgenic poplars was changed to sun leaf, which showed smaller size and thicker than that of the wild type. The leaf also showed high photosynthesis rate, high CO2 conductance and low responding speed of stomata. In the grafting test, the accumulation of cellulose was accelerated in the stems of the transgenic poplars with the leaves of the wild type. These results suggest that the activation for sink function affects the source function.

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