We have developed a metabolic profiling experimental scheme based on a direct-infusion method on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). The metabolic profiling scheme was designed for metabolome classification and marker metabolite identification, correcting considerable analytical errors intrinsically associated with the high-resolution analyses with FT-ICR MS under unpredictable ion suppression effects in the analytical cell. The scheme consists of 1) optimization of the analysis conditions for reproducible data correction among each MS scan, 2) development of a data analysis tool (Dmass) for automatic mass-error correction, 3) multivariate analyses for metabolome clarification among different samples, 4) identification of marker metabolite candidates through a database (KNApSAcK) search using accurate mass values, and 5) structural analyses for the candidate compounds by MSMS technique. We discuss the application of our experimental scheme for an integrated data analysis of metabolomics and transcriptomics for Arabidopsis suspension cells under different culture conditions.

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Genomic information of tomato is increasing with the progress of tomato genome project. By contrast, metabolic information of tomato is not sufficient to systematically link it to genomic information. Although the metabolic profiling of primary metabolites from tomato fruit has been reported, there has been no report on comprehensive analysis of secondary metabolites. In this study, we analyzed metabolites of tomato (cv. Micro-Tom) fruit comprehensively using LC-FT-MS. LC-FT-MS facilitates measurement of m/z value at sub milli-mass unit. We collected information of each compound, such as retention time, accurate molecular weight, MS/MS data, and UV absorption. More than 500 compounds from tomato fruits were detected, and their chemical information was obtained by comparing our data with open metabolite databases (KNApSAcK, DNP). We also found that the composition of metabolites was different in fruit tissues and maturing stages. Furthermore, many unknown metabolites were detected from tomato fruit.

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Gas chromatography-mass spectrometry (GC-MS) is one of the major analytical instruments in plant metabolomics. The separated compounds through GC are collapsed during the electron impact ionization, and detected as fragments. The compounds are identified by comparing the fragmentation patterns to those of authentic compounds. However, only 10 - 20% of compounds are usually identified in plant materials. In order to compare the metabolite profiles between different samples, we developed a computational tool written in Java that aligns peaks including unknown compounds. Peak detection for each m/z, deconvolution, normalization of retention time, and peak alignment are accomplished. We succeeded to align peaks detected in the GC-time of flight-MS data of multiplicated analysis of a single extraction of Arabidopsis cells. We also successfully compared the metabolite profiles between Arabidopsis and Lotus japonicus.

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In these days of post genome era, metabolome studies attract more attention because the information on metabolites is the key data for so-called systems biology. To analyze the metabolites, mass spectrometry (MS) has been used mainly. However, the MS analysis is not always suitable to detect chemically-diversed metabolites. Therefore, we have been trying to establish NMR-based metabolomics approach. We have conducted the stable isotope labeling of plants, insects and mice, optimization of the extraction solvent for metabolites, building a chemical shift database, and constructing a semi-automtic signal assignment system, SpinAssign. In this talk, we will review these methodologic developments. We will also show the in vivo NMR measurements of Arabidopsis T87 cultured cells treated with ABA coupled with transcriptome analysis, and our attempt to detect metabolite changes in Ds insertion mutants. In addition, the future perspective and the potential of NMR metabolomics will be discussed.

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Silent phenotypes are genetically modified organisms that do not show apparent changes in morphology, yield, or growth rates when compared with parental lines under given physiological conditions. They undergo, however, subtle but systematic metabolic changes that are difficult to detect by targeted metabolite analysis. To analyze silent plant phenotypes, we used a double knock-out mutant of Serat genes (serat2;1 serat2;2); the mutant is known to exhibit relatively reduced amount of O-acetyl-L-serine, cysteine, and glutathione, which play major roles in the sulfur assimilation. In our metabolome analysis, 30 replicates of wild-type roots and their 30 mutants were analyzed using GC-TOF/MS. When multivariate analysis was applied to the non-processed GC/MS files (470 peaks in root; 514 peaks in leaf), the PLS-DA score separated the groups of wild-type clearly from the mutants. Lastly, significance of correlations was assessed considering the number of reaction steps that connect the metabolite pairs in the metabolic network.

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The metabolic profile of rice plant (Oryza sativa, 2 weeks after germination) was compared between wild-type (cv. Nipponbare) and the transgenic rice lines expressing OASA1D, a gene encoding a feedback-insensitive alpha subunit of anthranilate synthase. Profiling by HPLC-PDA revealed that only a limited change in the composition of major aromatic compounds occurred in OASA1D transgenic rice lines, with the exception of a marked increase in the amount of tryptophan. A more detailed survey using HPLC-ESI-Q-MS allowed the detection of a total of 1600 metabolite peaks. Data mining by a combination of correlation analysis, ANOVA and independent component analysis revealed the relatively small but significant changes for about 20 minor metabolites. LC-MS/MS analysis demonstrated that the metabolites that increased in quantity by the expression of OASA1D included such indole compounds as γ-glutamyltryptophan.

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The aim of this study is to identify the functional protein components of rice Golgi complex. In the present study, we attempted to purify cis-Golgi membranes from rice cells expressing GFP-AtSYP31 fusion protein which is known to localize in the cis-Golgi compartment. GFP-AtSYP31 labeled Golgi membranes were succeeded to highly purify by floating membranes in a sucrose density gradient. The specific GFP fluorescence intensity of final cis-Golgi membrane preparation was approximately 20-fold compared with that of crude microsomal membranes. The cis-Golgi membrane proteins were subjected to SDS-PAGE, followed by LC-MS/MS analysis. In addition to the Golgi localized protein such as root hair defective 3, COP-I and -II coatmer proteins, and small GTPase YPT3, Bip, calreticulin and Hsp90 those are well-known as ER membrane proteins were also identified. These results may indicate that there exist a similarity and/or continuance between cis-Golgi and ER membranes.

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In this study, we focus on obtaining the profile of secretory proteins of rice root, and compare the change in the profile among different nutrient stress, to study secretory proteins comprehensively.
After germination, rice were grown in three different culture condition; control, -P and -N for two weeks. Solution was renewed everyday, and the protein sample was collected from the culture solution of the last day. Sample solutions were concentrated. After 2D gel electrophoresis, protein profile was appeared. Many proteins in control treatment indicate that rice secretes proteins constitutively from root even under normal condition. And it was also found that different protein profile was found by -P and -N treatments. Major spots and those spots behaved specifically by the treatments were picked, and the identification of them by MALDI-TOF-MS is being carried forward.

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We used the same sample for simultaneous transcriptome and proteome analysis, and analyzed using KaPPA view. From the transcriptome analysis, when sufficient phosphorus supplied treatment was put as a control, it was observed increased expression of 1) glycolysis related genes, 2) sufolipid and glycolipid synthesizing related genes, stable in 3) TCA cycle composing enzymes genes, and 4) anion channel coding gene, and decreased in 5) amino acids synthesis related genes under phosphorus limited condition (-P). When phosphorus was supplied to -P on one day before the sampling, a decrease in the gene expression was observed in glycolysis related genes. On the other hand, from proteome analysis, though similar tendency was observed in glycolysis related enzymes, the response was reverse in amino acids synthesis related enzymes. And significant increase in anion channel was observed under -P. Thus the fluctuation pattern by phosphorus status was not consistent between transcriptome and proteome.

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Lichens are photoautotrophical symbionts containing algae and can live even in the drought environment. It has been suggested that lichens convert absorbed light energy into thermal energy efficiently in the drought conditions, although the mechanism is still unclear. We measured the lifetime of fluorescence of a lichen, Pertusaria sp. using pico-second time resolved spectrophotometer, and analyzed the process of energy conversion. We obtained following results. (1) The fluorescence of photosystem II decayed much faster in the dried cells. (2) Excitation energy transfer within photosystem II was still observed in the dried cells. (3) The lifetime of photosystem II was increased within 1 min after water-absorption. (4) The blue shift and the shortening of the lifetime were also observed in photosystem I upon dehydration. Similar features were observed in other lichens. We discuss this energy dissipation mechanism from these results.

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Tyrosyl radicals play key roles in the mechanisms of a wide range of enzymes. In the water-oxidizing enzyme, two redox-active tyrosines are present. While the tyrosine that is kinetically competent in electron transfer, TyrZ, may also have a role in the enzyme mechanism, the second tyrosine, TyrD, has a stable radical and is not directly involved in the redox chemistry associated with enzyme function. Despite homologous positions in subunits D1 and D2, TyrZ and TyrD exhibit extremely different kinetics, different redox potentials, and play different functional roles in the enzyme. Thus, they constitute an ideal system for understanding how the protein environment controls the reactivity of tyrosine radicals. In this work, site-directed mutagenesis has been used to modify the hydrogen bond network around TyrD. Then, both the formation of the TyrD radical and its properties have been studied by X-band and high-field EPR. Structural and mechanistic implications will be discussed.

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The local environment of the manganese cluster in photosystem II has been investigated using proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in multiline signals in both the S₀ and the S₂. The 6 pair of lines separated around the position for a free proton was detected. The signals with 2.2-2.3 MHz hyperfine constants disappeared within 3 h of D₂O exchange in both the S₀ and S₂ states. However, the signals with 4.0 MHz hyperfine constants disappeared within 3 and 24 hours in the S₀ and S₂ states, respectively. The different rates were ascribed to oxidation of the liganding Mn ion, leading to a possible model for water oxidation.

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Oxygen evolution in photosytem II (PSII) is performed at the Mn cluster through a light-driven cycle of five intermediates (S₀-S₄). Although Ca²⁺ is known to be an essential cofactor for oxygen evolution, the structural relevance of Ca²⁺ to the Mn cluster remains unclarified. In this study, we have investigated the role of Ca²⁺ in oxygen evolution by detecting FTIR spectra of the Mn cluster in PSII preparations of spinach and the cyanobacterium Thermosynechocuccus elongatus in which Ca²⁺ is replaced with Sr²⁺. In FTIR spectra of S-state transitions, several bands sensitive to Sr²⁺ substitution were found in the symmetric COO^- stretching region, indicating the presence of carboxylate ligands to the Mn cluster affected by Sr²⁺ substitution. The changes were prominent in the S₁->S₂ and S₃->S₀ transitions and similar between spinach and T. elongatus. These results indicate that Ca²⁺ directly interacts with the Mn cluster and is deeply involved in its reactions.

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We examined the effects of replacement of D1-Glu189 with Gln in Synechocystis PCC 6803 on the properties of photosynthetic oxygen evolution. Isolated photosystem II core particles from E189Q mutant evolved oxygen at 70% rate of control wild-type particles. The mutant cores showed normal S₂-state EPR multiline signals. However, the redox potential of mutant Mn-cluster was lower than that of the control one as revealed by thermoluminescence study. Studies by S₂/S₁ FTIR difference spectroscopy revealed that the mutation resulted in the specific changes in IR bands of carboxylate ligands for Mn-cluster and a few carbonyl bands at mid-frequency (1800-1100 cm^-1), and the unique changes in bands ascribed to Mn-cluster core vibrations at low-frequency (650-350 cm^-1). These results indicated that the carboxylate side group of Glu189 ligates a Mn ion. Probably, the carbonyl group of Gln alternatively coordinates the Mn ion to maintain the nearly normal function of Mn-cluster in the mutant.

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Photosynthetic oxygen evolution is catalyzed by a Mn₄Ca cluster which is putatively coordinated by amino acid residues, mostly from the D1 protein of photosystem II. To understand the detailed ligation structure of the Mn₄Ca cluster, we examined the interaction between Mn ions and putative amino acid ligands by means of flash-induced FTIR difference spectroscopy in combination with specific isotope labeling and site-directed mutagenesis using Synechocystis sp. PCC 6803. Histidine-specific ¹⁵N-labeling studies showed the S-state dependent structural coupling between the Mn₄Ca cluster and histidine ligand(s). Alanine-specific ¹³C-labeling studies revealed the unidentate ligation of the D1-Ala344 carboxylate to a redox-active Mn ion. D1-Glu189Gln mutation studies indicated the bidentate ligation of the Glu189 carboxylate to the cluster, while the reported D1-Asp170His mutation studies showed no effect on the FTIR difference spectra. Based on these findings together with recent X-ray crystallographic models, the ligation structure of the Mn₄Ca cluster is discussed.

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Photosynthetic organisms regulate their components and/or reaction mechanisms according to the changes in the environmental temperature so as to keep them in a good condition. We examined changes in the thermosensitivity of photosystem II (PSII) in Synechocystis sp. PCC 6803 grown under different temperatures. Thylakoid membranes and purified PSII complexes were isolated from those cells. Temperature dependencies of the oxygen-evolving activity in thylakoids were different between cells grown under different temperatures, while, those in the purified PSII complexes were the same between those cells. Temperature dependencies of the reaction center activity were the same both in the thylakoids and purified PSII complexes irrespective of the growth temperature. No remarkable difference was observed in protein compositions in thylakoids and purified PSII complexes. These results suggest that one of the major factor for the changes in the thermosensitivity of PSII complexes is the change in the lipid composition of thylakoid membranes.

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Photosystem II (PSII) is a membrane-protein complex containing 14-16 transmembrane and 3 extrinsic subunits with a total molecular mass of 350 kDa, which catalyzes light-induced electron transfer and water-splitting reactions. Crystal structures of PSII have been reported at around 3.5 A resolutions, which are apparently not enough to reveal detailed structures of amino acid side chains as well as that of the Mn cluster. In order to expand the current limit of observable reflections from crystals, we reexamined the preparation and crystallization procedures for PSII from the thermophilic cyanobacterium Thermosynechococcus vulcanus. By modifying the final ion-exchange chromatography step and employing the vapor-diffusion method to grow crystals, we obtained better quality crystals. Furthermore, by optimizing cryogenic conditions for the crystals, we succeeded in obtaining diffraction data of 3.3 A resolution, which are used to reconstruct the structure model of PSII. We will report the current status of crystallization of PSII.

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Phosphatidylglycerol (PG) is the only phospholipid in thylakoid membranes, which are the site of primary processes of photosynthesis. We previously isolated a pgsA mutant of Synechocystis sp. PCC6803 defective in biosynthesis of PG. With the mutant we demonstrated that PG plays important roles in electron transport in photosystem II (PS II).
In this study, we have characterized PSII complex purified from the mutant to investigate the function of PG in PSII complex. The PSII complex of the mutant possessed about 40% oxygen-evolving activity compared to that of wild type. It was also found that extrinsic proteins were dissociated from the PSII core complex of the mutant accompanied with the release of manganese. However, PsbO bound again to the PSII core complex when the mutant cells were incubated with PG. These results demonstrate that PG is required for the binding of extrinsic proteins to sustain functional Mn-cluster in PSII complex.

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Most Chl a in the PS1 RC complexes was removed without any loss of P700 by ether treatment, yielding the antenna-depleted PS1 RC complexes that contain 1 Chl a' and 10-14 Chl a per P700. (1) On addition of Chl a' with phospholipids (PG), 5-10 Chl a' per P700 bound to the antenna-depleted complexes. (2) The P700 photooxidation was enhanced in the Chl a'-reconstituted complexes, suggesting that the bound Chl a' acts as efficient antenna as Chl a remaining in the complexes. (3) When Chl a' was reconstituted with the equal amounts of Chl a, the Chl a' was less bound than Chl a, suggesting the lower affinity of Chl a' than Chl a to the Chl a-binding sites. (4) The more extraction of antenna Chl a resulted in the improper binding of Chl a' to the Chl a-binding sites.

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In a purple bacterium, Rubrivivax gelatinosus, we have shown that a high-potential iron-sulfur protein (HiPIP) is the best electron donor to the RC. Other soluble electron donors, two cytochromes c₈ with different redox midpoint potentials also work as good electron donors to the RC in R. gelatinosus. To clarify the relative contributions of these three electron donors, we have constructed various mutants defective in the genes coding for these proteins. Interestingly, the triple mutant sustained photosynthetic growth ability. Flash-induced kinetic measurements using the intact cells showed photo-oxidation of a cytochrome peaking at 553 nm. The purified cytochrome had an apparent molecular weight of 25 kDa and worked as a good electron donor to the RC in reconstitution experiments. The gene coding for this cytochrome was cloned and sequenced, which has two heme-binding motifs. Mutants lacking this gene were constructed and now analysed to clarify the functions of this new cytochrome.

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The heliobacteria are strictly anaerobic, photosynthetic bacteria that belong to Gram-positive bacteria. They are characterized by the presence of bacteriochlorophyll g as a major photosynthetic pigment and the reaction centers (RCs) of heliobacteria belong to a Fe-S type, like those of green sulfur bacteria and PSI. Fe-S type RCs are able to photoreduce NAD(P)(+) directly via ferredoxin (Fd), although it has not been proven yet in vitro in heliobacteria. We reported that isolation and purification of four ferredoxins, Fd A, Fd B, Fd C, and Fd D, from cells of Heliobacterium Heliophirum fasciatum. The absorption spectra of the purified Fds and g values of cryogenic EPR spectra were typical of those of the 2[4Fe-4S] cluster type. In this study, we investigated NADP(+) photoreduction activity and ESR spectroscopy in the presence of Fds and purified RC from H. fasciatum.

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Green sulfur bacterium Chlorobium tepidum grows phototrophically using various reduced inorganic sulfur compounds such as sulfide and thiosulfate, as well as hydrogen, as ultimate electron donors. The mechanism of thiosulfate oxidation in the oxidizing side of RC is obscure. In this study, we purified thiosalfate-oxydizing multi enzyme from C. tepidum by ammonium sulfate fractionation, anion-exchange chromatography and cation-exchange chromatography. From N-terminal amino acid sequences of subunits of the purified multi enzyme, it was shown that the genes coding subunits of thiosalfate-oxydizing multi enzyme composed a gene cluster in the genome of C. tepidum which is very similar to sox (sulfur oxidation) gene cluster of α-proteobacterium Paracoccus pantotrophus. It is being experimented on whether for the thiosalfate-oxydizing multi enzyme to involve in electron transfer from thiosulfate to RC.

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Excited-state dynamics of all-trans-spheroidene was studied by time-resolved fluorescence up-conversion method. In order to completely remove fluorescent impurities, spheroidene was purified by two series of alternate alumina and silica-gel chromatography. We used TOPAS, whose repetition rate is 1KHz, as our laser source. The laser output was separated to two beams, one worked as gating pulse, the other was frequency doubled by BBO crystal and was used as pumping pulse. The fluorescence and the gating pulse mixed inside the BBO crystal to generate up-conversion signal. Single photon counting system was used to acquire data. The fluorescence kinetics at different wavelengths was different from each other. The lifetimes of different wavelengths were in between 0.18ps to 0.33ps. After doing dispersion correction and intensity calibration, time-resolved fluorescence up-conversion spectra of all-trans-spheroidene was reconstructed. Single value decomposition followed by global fitting will be used to analyse the spectra.

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Energy transfer from carotenoids to chlorophylls competes with internal conversion. Energy diagram of carotenoids has been determined by spectroscopic measurements, and these time constants may follow the energy-gap law. Target carotenoids are lycopene (n = 11), anhydrorhodovibrin (n = 12) and spirilloxanthin (n = 13). The order of internal conversion is expected as 1¹B_u⁺ → 3¹A_g⁻ → 1¹B_u⁻ → 2¹A_g⁻ → 1¹A_g⁻. Each time constant should depend on energy-gap of each step in internal conversion. Time-resolved absorption spectra of these three carotenoids show above four excited states. Spectral pattern and lifetime of each electronic state have determined by singular-value decomposition and global fitting. By the result, the relation with energy-gap law has been examined.

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The internal conversion from 1B_u⁺ down to 2A_g^- facilitates the singlet energy transfer in the channel from 2A_g^- state of carotenoid to Q_y state of bacteriochlorophyll in antenna complex. In-between 1B_u⁺ and 2A_g^- state, spectroscopy such as resonance-Raman excitation profile found 1B_u^-, 3A_g^- state in the carotenoids having the conjugated double-bonding number, n = 9-13. This fact implies the sequential internal conversion from 1B_u⁺ take place through 3A_g^-, 1B_u^- states.
Spheroidene and lycopene were prepared by extraction, twice alumina column, and re-crystallization. They were measured with subpicosecond time-resolved Raman spectroscopy, which accomplished high temporal resolution with the use of two laser pulses for the probing of Raman process. The internal conversion processes of 1B_u⁺→3A_g^-→1B_u^-→2A_g^- were identified by a key stretching Raman line of each excited state. We are going to perform the singular-value decomposition and global fitting and to determine the lifetime of each excited state.

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Submicrosecond time-resolved absorption spectra were recorded for the core (RC-LH1) complexes from purple photosynthetic bacteria, which contain carotenoids(Cars) having different conjugation length (n), Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, Rsp. molischianum, Rps. acidophila 10050 and Rsp. rubrum S1 with and without sodium ascorbate under the argon atmosphere. In the absence of sodium ascorbate, all-trans type T_n ← T₁ absorptions which are reflected in Car composition are ascribable to the LH1 components in core complexes. The lifetimes of the LH1 components give rise to linear relations as functions of 1/(2n + 1); Cars having a longer-conjugated chain tend to decay faster than those having shorter-conjugated chain. On the other hand, with sodium ascorbate, additional 15-cis type T_n ← T₁ absorption was observed. It seems the RC component in core complexes. The lifetimes of RC components decay with the same rate among the set of Cars having different conjugation length.

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Carotenoid (Car) in the reaction center (RC) plays the function of photo-protection (i.e., triplet-energy dissipation). In the RC, a series of electron-transfer reaction is started by the charge separation, and this must influence the redox potential of the environment. From this viewpoint, we determined the lifetime of triplet Car in the RC, and examined if it is affected by the addition of a reducing agent. In the experiment, we excited the special-pair bacteriochlorophyll in RC from Rhodobactor sphaeroides 2.4.1 to the Q_y state, and measured the submicrosecond time-resolved absorption spectra. On increasing of the amount of sodium ascorbate, the signal of triplet Car was increased, and the triplet-state lifetime of Car was decreased systematically. The results show that the decay rate of triplet state is enhanced depending on the amount of triplet state generated. We are planning to examine the case of Rhodospirillum rubrum S1, as well.

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   A green photosynthetic bacterium, Chlorobium limicola, has an antenna complex called ‘chlorosomes’. It absorbs the light energy and transfers its singlet energy to the reaction center. Since chlorosomes contain ‘rod elements’ consisting of higher aggregates of bacteriochlorophyll (BChl) c molecules, we expect that it has a unique excited-state dynamics, because of its cylindrical structure. In this study, we have focused our attention on subpicosecond time-resolved absorption spectroscopy in the near-infrared region.
   Immediately after excitation to the Soret, chlorosomes gave rise to a broad transient absorption which decayed within ∼1 ps. In a higher in vitro aggregate, which exhibited Q_y absorption almost same as chlorosomes, we observed similar excited-state dynamics. In contrast to such higher aggregates, monomeric BChl c in methanol solution exhibited completely-different excited-state dynamics. These results suggest that the former excited-state dynamics may originate from the unique aggregate structure of chlorosomes.

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   Green photosynthetic bacteria have antenna complexes named chlorosomes which absorb light and transport the energy to the photochemical reaction center, where redox energy can be accumulated. The structure of chlorosomes from Chlorobium limicola, i.e. higher-order aggregates of bacteriochlorophyll c (BChl c) isomeric mixture, has been proposed. The structure of the in vitro BChl c aggregates was almost revealed using ¹³C-labeled BChl c and non-labeled BChl c. Reconstitution of chlorosomes is essential to determine the structure of chlorosomes by the same method. Reconstitution can be examined by electronic absorption, circular dichroism spectroscopy, and sucrose density-gradient centrifugation.
   Chlorosome-like aggregates can be formed, in a buffer containing lipids, by injecting a chloroform/methanol extract of chlorosomes (BChl c isomeric mixtures). In addition to this method, several means of preparation of chlorosome-like higher-order BChl c structures have been examined.

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The marine unicellular algae, coccolithophorids produce a calcified biomineral named coccolith. Coccolith has a fine and elaborate ultrastructure of calcium carbonate crystals. In Emiliania huxleyi, a unique acid polysaccharide, coccolith polysaccharide (CP) composed of α-1,3-mannose in the main chain, is located on the coccolith and in coccolith producing vesicles. Therefore, CP has been suggested to be involved in coccolith formation. In order to study on CP biosynthesis, we tried to identify a cDNA of key enzyme in CP biosynthesis from Emiliania EST database and cloned a putative cDNA of GDP-Mannose pyrophosphorylase providing GDP-mannose, a substrate for α-1,3-mannose synthesis. The deduced protein sequence was 382 a.a. (40.9 kDa) in size, and was highly homologous to the known GDP-mannose pyrophosphorylase. This is the first identification of cDNA related to CP synthesis. Regulation of its induction may become a useful tool for the study of the molecular mechanism of coccolith crystallization.

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Most extant cyanobacteria contain almost exclusively C16-fatty acids in the sn-2 positions of glycerolipids, which is under control of lysophosphatidic acid acyltransferases (LPAAT, EC 2.3.1.51). Synechocystis sp. PCC6803 contains sll1848, sll1752 and slr2060 for putative acyltransferase genes and C16 fatty acids in the sn-2 positions of glycerolipids are under control of sll1848. Here, we showed that Δ1848 Δ2060 cells extensively increase C18-fatty acids in the sn-2 positions, and decrease unsaturated C18-fatty acids in the sn-1 positions. Δ1848 Δ2060 cells showed a slow growth at 30 ^oC relative to the wild type and were sensitive to low temperature. sll1752 was upregulated in Δ1848 Δ2060 cells, where stearoyl-dependent LPAAT activity was also increased. Furthermore, LPAAT assays with recombinant proteins and growth complementation of Escherichia coli plsC cells verified that sll1752 encodes stearoyl-specific LPAAT. We conclude that sll1752 is sufficient but not preferable in the photosynthetic growth of this cyanobacterium.

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Sulfoquinovosyl diacylglycerol (SQDG) is present in photosynthetic membranes of most of oxygenic photosynthetic organisms. We here investigated regulation of SQDG metabolism in C. reinhardtii under the sulfur-deprived condition. Cells of C. reinhardtii degraded SQDG concomitantly with removal of sulfur source from the culture. This induction of the degradation was inhibited by actinomycin D or cycloheximide, suggesting that the induction requires de novo synthesis of protein(s) encoded in the nuclear genome. On the other hand, a trace level of SQDG still remained. In line with this, the capacity of SQDG synthesis was kept at the initial level and the mRNA level of the SQD1 gene for SQDG synthesis was increased. This up-regulation of the mRNA level was shown to require de novo protein synthesis through experiments with cycloheximide. These results implied that genes responsible for SQDG synthesis and degradation are simultaneously upregulated in their expression under the sulfur-deprived condition.

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In both chloroplasts of higher plants and cyanobacteria half of the membrane lipid is monogalactosyldiacylglycerol (MGDG). The MGDG synthesis pathway is very different between them. In chloroplasts the MGDG synthesis is one-step reaction catalyzed by MGDG synthase, while in cyanobacteria it is two-step reaction catalyzed by monoglucosyldiacylglycerol (MGlcDG) synthase followed by the epimerization of MGlcDG to generate MGDG. Gloeobacter violaceus PCC 7421 that is known to be branched off at the earliest stage in the cyanobacterial clade have genes very similar to cyanobacterial MGlcDG synthase and plant MGDG synthase (Awai et al., unpublished). We expressed the two genes in Escherichia coli and analyzed the glycosyltransferase activities. The results showed that the both proteins had the activity of MGlcDG synthase while they had no activity of MGDG synthase. We will show more in detail the biochemical and enzymatic properties of the two proteins after purification.

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Monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in thylakoid membranes of chloroplasts. In Arabidopsis, three MGDG synthase genes are encoded on the genome (atMGD1, atMGD2 and atMGD3). MGD1 is localized to the inner envelope membranes of chloroplasts and would mainly contribute to thylakoid membrane construction. In this study, we newly isolated a mgd1 knockout mutant, which showed severe growth defect during seedling development and no chlorophyll accumulation. Lipid analysis revealed that not only MGDG but also digalactosyldiacylglycerol (DGDG) greatly decreased in mgd1, suggesting the necessity of MGD1 in galactolipid biosynthesis. Moreover, MGDG in this mutant possessed no 16:3 fatty acids, which indicates complete defect in prokaryotic MGDG biosynthesis. These data demonstrate that MGD1 is necessary for galactolipid biosynthesis via prokaryotic pathway. Furthermore, MGD1 knockout causes abnormal embryogenesis, suggesting that MGD1 plays an important role in embryo development in addition to seedling growth.

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We previously reported isolation and characterization of an Arabidopsis freezing tolerance 1 (frt1) mutant that showed mature-leaf-specific enhancement of the degree of freezing tolerance due to soluble sugar accumulation. Here, we showed that this mutant shows "restricted sucrose export" phenotypes. Thus, we renamed this mutant rsx1. As reported previously, the rsx1 mutant showed mature-leaf-specific accumulation of transitory starch. We also showed that GUS expression in the rosettes of RSX1promoter:GUS plants occurs in a basipital mode along the leaf vein, suggesting the correlation between RSX1 expression and the expression of source leaf functions. In accordance with this, ¹⁴C-sucrose-feeding experiments revealed that rsx1 plants are restricted in the sucrose export out of mature leaves. We also present some transmission electron micrographs, which strongly supported our hypothesis that RSX1 is a genetic factor involved in the establishment of sucrose translocation pathway in the source leaves of A. thaliana.

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In higher plants, various Z,E-mixed polyisoprenoids, including dolichol and polyprenol, are biosynthesized, showing two different distribution patterns in the chain length, C_50-60 and C_70-120. To understand the physiological functions of Z,E-mixed polyisoprenoids in higher plants, we analyzed abiotic stress response of cis-prenyltransferases (CPTs), which catalyze formation of the structural backbone of Z,E-mixed polyisoprenoids, in Arabidopsis thaliana.
We investigated the expression pattern of nine A. thaliana CPT (AtCPT) genes in response to various abiotic stesses by RT-PCR. AtCPT5 was induced in response to cold treatment, and both AtCPT8 and AtCPT9 were induced in response to high-salinity. AtCPT8 and AtCPT9 expressed in yeast have been shown to catalyze the formation of C_80-100 polyprenyl products in vitro. In contrast, AtCPT5 expressed in Escherichia coli showed novel CPT activity, catalyzing the formation of shorter C_30-35 polyprenyl products. These results suggest the involvement of AtCPT5 and its products in cold stress response in higher plant.

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PHOSPHORYLETHANOLAMINE CYTIDYLYLTRANSFERASE 1 (PECT1) encodes CDP-ethanolamine synthase involved in the CDP-ethanolamine pathway to the biosynthesis of phosphatidylethanolamine (PE) in Arabidopsis thaliana. We herein created pect1-4/pect1-6 F1 plants by a cross between the weak mutant allele pect1-4 and the null allele pect1-6. These plants contained a decreased amount of PE relative to the wild type and showed dwarfism, such as small rosette leaves and a short stem. pect1-4/pect1-6 F1 plants showed reduced cell numbers and limited cell expansion and intercellular space development in rosette leaves and stems, and a short meristematic zone in the root apices. Moreover, stronger fluorescence was observed around meristematic tissues of Pro_PECT1:PECT1-EYFP:Ter_NOS plants. These results suggested the importance of PECT1 in cell proliferation. We also discuss the importance of PECT1 in the development of intercellular spaces and cell expansion, which could be related to excretion of proteins via lipid vesicles.

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We have found two new mutants, cof1-1 and cof1-2 (cuticular defect and organ fusion), in transposon-tagged lines of Arabidopsis, showing same phenotypes of wilty, adhesion in rosette leaves and sterile. By Toluidine-blue test, SEM and TEM, these mutants were detected to have cuticular defects in leaves and stems. These two mutants have a transposon-insertion in same gene, which is the AtWBC11 classified to ABC transporter AtWBC subfamily. AtWBC11 is a highly homologous gene of CER5/AtWBC12, which is reported to be related for cuticular lipid export. Gas-chromatography analysis reveled that the amount of alkanes, the main compound of cuticular wax, extracted from leaves and stems of cof1-1 were remarkably reduced. AtWBC11 protein fused with CFP protein was localized to the plasma membrane of plant cells. These results suggest that COF1/AtWBC11 has an important role in wax transport to construct the cuticle layers for developmental formation.

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In the cyanobacterium Synechocystis sp. PCC 6803, some histidine kinases (Hiks) have been identified as signal sensors. Among them, Hik33 has unique characteristics to respond to several independent stimuli, such as hyperosmotic, salt, cold and oxidative stress. Recently, using yeast two-hybrid screening an accessory protein, Ssl3451, which specifically interacts with the carboxyl-portion of Hik33, has been identified.
In the present study, a truncated form of Hik33, Hik33-c, in which the two transmembrane domains were eliminated, and a full-length Ssl3451 were overexpressed in the cell of Escherichia coli and subsequently both were purified. The purified Hik33-c protein was capable of autophosphorylation in vitro. Interestingly addition of the purified accessory protein to the reaction mixture enhanced the phosphorylation activity of Hik33-c. These results indicated that the accessory protein, Ssl3451, might be involved in regulation of Hik33. This is the first identification of a regulatory component for Hik.

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In addition to the ABC nitrate-nitrite transporter (NrtABCD), Synechococcus elongatus possesses a SulP family permease (LntT) having latent nitrate transport activity. We previously showed that LntT is activated when LntA, a response regulator, is phosphorylated by LntB, a hybrid histidine kinase. Downstream of the lntAB genes is the lntC gene, encoding a hybrid histidine kinase with no autophosphorylation domain. LntC was found to receive the phosphoryl group from LntA in vitro, and was shown to be essential for activation of LntT in vivo. LntC has a C-terminal region of unique amino acid sequence. Expression in S. elongatus cells of a translational fusion of the C-terminal domain of LntC to glutathione S transferase, which mediates dimer formation, led to activation of LntT. These results suggested that dimerization of LntC, which is promoted by phosphorylation of the protein by LntB via LntA, activates LntT in vivo.

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We have shown that the Arabidopsis AtMID1A gene product (AtMid1A) has a Ca²⁺-permeable, stretch-activated channel activity (2004 meeting). AtMid1A and its paralog, AtMid1B, are postulated to act as sensors of mechanical stimuli and participate in generating Ca²⁺ signals. In the last JSPP meeting, we have shown that the atmid1a/b double mutant exhibits a severe growth defect when grown on media containing high concentrations of Mg²⁺, but supplementation of CaCl₂ to the media alleviates this phenotype. We show that MgSO₄ concentrations required for the induction of the growth defect of the atmid1a/b mutant are lowered when the concentration of CaCl₂ in media is lowered. The finding suggests that the growth defect occurs when the ratio of Mg²⁺ to Ca²⁺ is higher. To explain this phenotype, we examine and discuss the following possibilities: (1) Mg²⁺ blocks Ca²⁺ channels other than AtMid1A/B and lowers [Ca²⁺]_cyt ;(2) Mg²⁺ disturbs Ca²⁺ signaling in the cytoplasm.

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The Ca²⁺-permeable, stretch-activated (SA) channel is regarded as a mechanosensor. We have isolated Arabidopsis genes encoding SA channel candidates designated AtMID1A and AtMID1B that can complement the yeast mid1 mutant (2004 JSPP meeting). We examined whether AtMid1A and AtMid1B are involved in touch perception in roots. A significant population of atmid1a and atmida atmid1b roots could not enter the harder medium from the softer medium of the two-phase agar medium, whereas wild-type and atmid1b roots were able to do so. The Ca²⁺ uptake activity of the roots of the AtMID1A-overexpressor was higher than that of wild-type roots. In addition, this activity was inhibited by the SA channel blocker Gd³⁺-, but not by the voltage-gated Ca²⁺ channel blocker verapamil. These results suggest that AtMid1A is an SA channel component required for touch sensing and Ca²⁺ uptake in roots.

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We have collected and completely sequenced 32,127 full-length cDNA clones from japonica rice and homology search was made against NCBI Genbank data. We have focused the existences of known membrane transporter genes in plant and animal. The animal especially developed the channel system (Bcl-2, Connexin, E-ClC, LIC, VIC, etc,.) for rapid transmitting in nerve and muscle tissues and Na⁺ gradient dependent secondary transport system (NSS, SSS, etc,.). On the other hands, the pump system (P-type ATPase, ABC, etc,.) and the secondary transport genes (CPA2, DMT, MFS, MOP, POT, etc,.) using H⁺ as a co-transporter is diverged in plants. We have also compared the numbers of membrane transporter genes in Arabidopsis and rice. Between rice and Arabidopsis, many transporter genes are similar, however, in detail, diversity in the Multi antimicrobial extrusion (MATE), P type ATPase, VIC, monovalent cation: proton antiporter-2 (CPA2), and Carbohydrate transporter (NST, TPT etc,.) were changed.

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In Brassica self-incompatibility, self/non-self recognition is controlled by S-haplotype-specific interaction between the pollen-borne ligand SP11 and its stigmatic receptor-kinase, SRK. The downstream signaling event leading to self-pollen rejection, however, still remains largely unknown. Our recent analyses on the self-compatible B. rapa variant Yellow salson suggested the involvement of MLPK (M-locus protein kinae), a member of receptor like cytoplasmic kinase, in this signaling, but its precise role is still unclear.
Here we report that MLPK is transcribed into two alternative isoforms, MLPKf1 and MLPKf2, by using different transcription initiation sites. Transient expression analyses of Venus fusion proteins in tobacco protoplasts revealed that both MLPKf1 and MLPKf2 is localized to plasma membrane by distinct mechanisms. The expression of mutant form of MLPKf1 that lack myristoylation motif failed to restore the self-incompatibility response in mlpk/mlpk stigmatic cells, suggesting that plasma membrane localization is essential for MLPK to function in self-incompatibility signaling.

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The COP9 signalosome (CSN) is a nuclear protein complex essential for plant and animal development. It has been shown that the N terminus of CSN1 (CSN1N) represses transcription of JNK1 in the JNK1/SAPK signaling pathway. Inhibitory studies suggested that this repression was different from the known proteasome-mediated proteolysis. To further understand this mechanism, interacting proteins (NBPs) directly binding to CSN1N were purified from mammalian cells. SAP130, one of NBPs, is known to be involved in mRNA splicing. SAP130 is a subunit of the SF3b complex that associates with the U2 snRNP complex and the STAGA complex, suggesting that CSN is involved in post-transcriptional regulation.
To characterize this novel function of CSN in the plant kingdom, we have identified the homolog of SAP130 in Arabidopsis (AtSAP130). AtSAP130 was encoded by two genes mapping to chromosome 3 producing identical protein. Here, we will discuss the interaction of CSN1N and AtSAP130.

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The SNF1/AMPK kinases are highly conserved in eukaryotes. Protein kinases of this subfamily exist as catalytically active hetero-trimeric complexes consisting of α, β and γ subunits. Previous reports indicate that interaction between α and γ subunits of SNF1 kinase are strongly in accordance with the deprivation of glucose in the growth medium. Our yeast two-hybrid experiments with the three subunits rice SnRK1 complex convincingly show that the interaction not only between α and γ, but also β and γ subunits is mediated by deprivation of glucose in the growth medium. Some of the experimental evidences also suggest that both dimeric and trimeric forms of protein complexes may exist in vivo .These findings suggest that the regulatory γ subunits of rice SnRK1 may recognize specific signals and interacts with the dimeric complexes comprising of α and β subunits and thus stimulate the catalytic activity of the enzyme complex.

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Using a dynamic method which enabled simultaneous measurement of the cell length and turgidity of Characean internode, a rectilinear correlation was found between them, within 4 sec or so immediately after the step change in outer osmotic pressure. By studying the time dependency of these parameters, the velocity of the water flow V across the cell membrane can be observed instantaneously. Further, the absolute water conductivity Lp can also be estimated from V and the shift in water motive force across the membrane. Then the difference in Lp was accomplished by comparing the effluxing and influxing situations through changing π_e from the holding osmotic pressure of 150 mM mannitol solution to either 200 mM or to 50 mM. There was no significant difference in Lp between them suggesting that the membrane itself is not a rectifier for the water flow.

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Reed is salt tolerant plant, keeps low Na⁺ concentration in shoot under saline condition. It is suggested that reed has the mechanism that translocates Na⁺ back to roots at shoot base (basal part of the shoot). In this study, we evaluated quantitatively ion translocation rate at the shoot base. The shoot base of reed specifically prevents NaCl translocation to shoot. We also visualized intracellular free Na⁺ at the shoot base of reed and the equivalent of rice using specific fluorescent probe. In rice segment, fluorescence was observed whole cells. In reed segments, granule structure was observed in the cell around the vascular bundle, and fluorescence was only observed surface of the granule. The granule was mainly consisted of starch. We suggest that the starch granules produced at the shoot base of reed play an important role in prevention of Na⁺ translocation to shoot.

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Reed keeps Na⁺ concentration in shoot low even under severe saline condition. We suggested that reed specifically suppress Na⁺ translocation from root to shoot at shoot base. We hypothesized that Na⁺-specific transporter or channel in shoot-base cells recover Na⁺ from xylem sap. We cloned a cDNA (pcnhx1) and a part of its genomic clone from reed as a candidate for involvement in this mechanism. pcnhx1 was highly homologous to osnhx1 from rice. Southern analysis suggested that reed has more than two NHX1-like genes. pcnhx1 was expressed in shoot including shoot base and up-regulated by increasing NaCl concentration. We suggest that pcnhx1 is involved in the suppression of Na⁺ translocation to shoot. The function of pcnhx1 will be discussed with results of yeast complementation assays and analysis of antagonism between Na⁺ and other cations at shoot base.

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Synechocystis sp. PCC 6803 contains five putative Na⁺/H⁺ antiporters, two homologous to NhaP from Pseudomonas aeruginosa and three homologous to NapA from Enterococcus hirae. Physiological and functional properties of NapA type antiporters are largely unknown. We examined the isolation and characterization of homologous gene from a halotolerant cyanobacterium Aphanothece halophytica. Two genes encoding the same polypeptide size, designated as ApNapA1-1 and ApNapA1-2, were isolated. ApNapA1-1 exhibited higher homology to Synechocystis's one (SynNapA1) than that to ApNapA1-2. ApNapA1-1, ApNapA1-2, and SynNapA1 complemented the Na⁺- and Li⁺-sensitive phenotypes of E. coli mutant. These three antiporters exhibited strongly pH dependent exchange activities, although the activities of ApNapA1-2 were significantly lower than others. Among them, only ApNapA1-2 complemented the K⁺-uptake deficient E. coli mutant and exhibited K⁺-uptake activity. Structural and functional properties of these antiporters were examined and their results will be presented.

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