Phytic acid (inositol hexakisphosphate ; IP6), a storage compound of phosphorus, is accumulated in protein storage vacuole during seed maturation. Recently IP6 was reported to be in crystal of TIR1, auxin receptor of Arabidopsis, suggesting that IP6 might be related to auxin signaling. The synthetic mechanism of IP6 is, however, still unclear in higher plants. To confirm the subcellular distribution of enzymes for inositol phosphate synthesis, we expressed the GFP fusion proteins transiently in Arabidopsis cultured cells. We observed fluorescence of all GFP-tagged enzymes in the cytosolic space. This suggests that IP6 may be synthesized in the cytosol. In various plants it is revealed that IP6 is localized in vacuole and endoplasmic reticulum. To clarify IP6 localization in Arabidopsis cells, we isolated the vacuoles and protoplasts and determined the IP6 concentration in them. We also analyzed interactions among enzymes for inositol phosphate synthesis.

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We investigate function of nucleotide pyrophosphatase/phosphodiesterase (NPP) gene family in rice (Oryza sativa L.). Six genes of NPP homologues are existed and located on chromosomes 3 (NPP3), 8 (NPP1), 9 (NPP6) and 12 (NPP2, 4, 5). Recombinant proteins for NPP1, 2 and 6 were expressed and purified from transgenic rice cells. Computer-assisted analyses showed that the deduced amino-acid sequences contain numerous N-glycosylation sites and cleavable hydrophobic signal sequences. All NPP1, 2 and 6 proteins actually conatined N-linked oligosaccharide chains recognized by Con A and Endo-H. However, confocal laser scanning microscopy of rice cells expressing NPP1-GFP, NPP2-GFP and NPP6-GFP revealed that they similarly localize in the chroloplast compartment. The results obtained suggest that the NPP family is synthesized and transported from ER-Golgi system to plastids, and possibly involved in the control of plastidial levels of nucleotides linked to glycosylation.

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Rice α-amylase I-1 (AmyI-1) is a secretory glycoprotein bearing a typical N-linked oligosaccharide chain. However, it was found that α-amylase is involved in degradation of plastidial starch in living cells. Furthermore, the traffic of ER-to-Golgi was shown to be necessary for targeting AmyI-1-GFP into plastid in onion epidermal cell system, indicating that possible existence of a traffic route of AmyI-1 from the endomembrane system to plastids.
To clarify the plastidial targeting route of AmyI-1, simultaneous expression of trans-Golgi and plastid markers, ST-mRFP and WxTP-GFP, in onion cells was performed. In the cells expressing fluorescent trans-Golgi and plastid markers, they frequently co-localized when co-expressed with AmyI-1. Three-dimensional time-lapse imaging in onion cells and electron microscopy of high-pressure frozen/freeze-substituted rice cells demonstrated that contact and subsequent absorption of the Golgi-derived membrane vesicles with cargo into plastids occur in the cells.

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Most peroxisomal matrix proteins are synthesized in cytosol with a targeting signal PTS1 at C-terminus or PTS2 at N-terminus. They are recognized by receptors, Pex5p and Pex7p, respectively, and imported into peroxisomes by binding to the docking factor, Pex14p, on peroxisomal membrane. Catalase, peroxisomal enzyme, has neither typical PTS1 nor PTS2. Peroxisomal import system of catalase is unknown. The study using pumpkin catalase, Cat1, suggested that internal PTS1 like sequence 'QKL' is functional for targeting, but the interaction between Cat1 and Pex5p was distinct from typical PTS1. Then we analyzed subcellular localization of mRFP-Cat1 fusion protein in Pex5p-defficient Arabidopsis generated by RNAi. Inhibition of Cat1 import into peroxisome in Pex5p-deficient Arabidopsis suggests that Pex5p is involved in catalase import. By the analysis using Arabidopsis mutants, we revealed that Pex14p, Pex13p, Pex12p and Pex10p which are necessary for PTS1 and PTS2 import, are also necessary for catalase import.

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Plastids are surrounded by two membrane layers, designated as the outer and inner envelope membranes, which have various transport and metabolic activities. We have identified and characterized a novel class of inner envelope membrane proteins, designated as Cor413 inner envelope membrane group (Cor413im). Previously, we showed that Cor413im proteins are integral membrane protein and targeted to the chloroplast envelope both in vitro and in vivo. Cor413im proteins are predicted to have multiple transmembrane segments. However, the targeting mechanism of polytopic envelope membrane proteins remains to be characterized. In this study, we investigated the targeting mechanism of Cor413im to the chloroplast envelope by sucrose density gradient centrifugation and sodium carbonate extraction. We will discuss the possible targeting mechanism of Cor413im to the chloroplast envelope membrane.

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Many of the genes for membrane proteins are encoded in the genome of land plants. Although most of these gene products are predicted to function as solute transporters, only a relatively small number of plant membrane proteins have been functionally characterized to date, mostly because of the experimental difficulties associated with their biochemical analysis. Here we report an improvement of proteoliposome reconstituting system for analyzing plant transporters by modifying wheat germ cell-free translation system. We established optimized conditions for the reconstitution system with Arabidopsis thaliana phosphoenolpyruvate/phosphate translocator 1 (AtPPT1) as a model transporter. A high activity of AtPPT1 was achieved by synthesis of the protein in the presence of Brij35 and liposomes. We also determined the substrate specificities of three putative rice PPT homologs with this system. The cell-free proteoliposome reconstitution system provides a valuable tool for functional analysis of transporter proteins. Nozawa et al. PCP, 48, 2007

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11S seed storage protein (globulin) families are produced as precursor in the endoplasmic reticulum (ER), then translocated to storage vacuole and processed into mature form. The family shares conserved bi-cupin structure, and are assembled into trimer form within ER. Amino acid sequence and physical structure of 11S globulins are putative determinants in translocation from ER to storage vacuole (Maruyama et al., 2006). Furthermore, glutelins (11S globulin of rice) show about 40% homology to glycinin (11S globulin of soybean), and are assembled into trimer with glycinin and transported to PB-II in rice seed. It remains unclear whether rice glutelin can be self-assembled or requires chaperons for folding and assembly. We demonstrated by analysis on sucrose density gradient and Blue-Native PAGE that in vivo trimer formation of proglutelin in immature seed and newly synthesized proglutelin can be self-assembled into trimer in vitro.
*Maruyama et al. (2006) Plant cell, 18, 1253

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Regulation of stomatal aperture relies on turgor changes within a pair of guard cells. The main solutes involved in the osmoregulation are K⁺, Cl^- and malate^2-. Through a high-throughput leaf thermal imaging screen, we isolated the Arabidopsis mutant cdi3 (carbon dioxide insensitive 3) that overaccumulates these osmoregulatory ions in guard cells, leading to CO₂ insensitivity in plant gas exchange regulation. The CDI3 protein is a distant homologue of bacterial and fungal C4-dicarboxylate transporters, and is localized specifically to the plasma membrane of guard cells. Our results identify CDI3 as a regulator of guard cell ion balance essential for stomatal movement. Arabidopsis CDI3 homologues, which are expressed in different tissues, but not in guard cells, complemented the cdi3 phenotypes when expressed ectopically in guard cells. Thus CDI3 family members have an evolutionarily conserved function in distinct Arabidopsis tissues.

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Arsenic is toxic to organisms. Arsenite (As(III)) and arsenate (As(V)) are the two main chemical forms of arsenic. As(III) and As(V) are taken up by bacteria, yeast and human via aquaglyceroporins and phosphate transporters, respectively. Plants also take up As(V) via phosphate transporters. However, the molecule(s) which mediates As(III) uptake in plants have not been identified.
We expected the loss-of-function mutant of As(III) transporter would be tolerant to As(III), as the As(III) could not enter roots. We searched As(III) tolerant mutant using EMS mutagenized Arabidopsis thaliana seeds and obtained 3 lines. We also analyzed tolerance of T-DNA mutants of NIPs, which is a homologue of aquaglyceroporins. Among the lines tested, only nip1;1 showed tolerant to As(III). We therefore sequenced the genomic region of NIP1;1 of As(III) tolerant mutants. All three mutants had a mutation in NIP1;1. These data indicate that NIP1;1 is involved in the As(III) uptake.

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Arsenic intake from contaminated crops is a threat for human heath. It is estimated that more than tens millions of people are exposed to risk of chronic toxicity and carcinogenesis. In paddy field, As is mainly present in the form of arsenite (As(III)). However, the molecular mechanism responsible for As(III) uptake is unknown in rice. In the present study, we report that both Si influx transporter (Lsi1) and efflux transporter (Lsi2) are involved in As uptake.
A short-term uptake experiment showed that both lsi1 and lsi2 mutants defective in Si uptake accumulated less As in the shoots. Addition of Si significantly decreased the As accumulation in the wild-type rice, but not in the mutants. When Lsi1 was expressed in the yeast or Xenopus oocytes, an increased transport activity for As(III), but not for As(V) was observed. These results suggest that Lsi1 and Lsi2 are involved in As(III) uptake.

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Maize is able to accumulate Si at a high concentration in the shoots, but the transporters involved have not been identified. Here, we isolated and characterized three maize silicon transporter genes (ZmLsi1, ZmLsi2 and ZmLsi6), which are homologues, respectively, of rice silicon transporters (Lsi1, Lsi2 and Lsi6). Each gene shared more than 80% identity between maize and rice. Both ZmLsi1 and ZmLsi6 showed Si-influx transport activity, while ZmLsi2 showed efflux activity when they are expressed in Xenopus oocyte. ZmLsi1and ZmLsi2were expressed mainly in the roots, while ZmLsi6 was in the shoot. Immunostaining showed that ZmLsi1 was localized at the plasma membrane of root epidermal cells, while ZmLsi2 was in the endodermal cells, and ZmLsi6 was in the leaf xylem parenchyma cells. These results suggest that ZmLsi1 and ZmLsi2 are involved in the uptake of Si from the roots and ZmLsi6 is involved in the Si distribution in leaves.

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Silicon is a beneficial element for plant growth, which helps plant to overcome various stresses such as pest, pathogen, drought and heavy-metal toxicity. Our goal is to understand the silicon uptake mechanism in barley by physiological and molecular biological approaches. Kinetics analysis indicated that the concentrations in symplast and xylem were always higher than that in the external solution and saturated at higher concentrations, suggesting that the radical transport of silicon from the external solution and the xylem loading are actively mediated by transporter(s). HvLsi1 and HvLsi2 were then isolated as homologous genes to rice silicon transporters. Both genes were specifically expressed in the roots. HvLsi1 showed silicon influx transporter activity in Xenopus oocytes. Immunostaing showed that HvLsi1 is localized on the distal side of outer cortex cells, which is slightly different from the localization of Lsi1 in rice. Characterizations of HvLsi2 will be reported as well.

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Protoporphyrinogen oxidase (Protox) catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX in chlorophyll and heme synthesis. Protox is encoded by the HemY gene in eukaryotes, and by the HemG gene in Escherichia coli. Since homologues for neither HemY nor HemG were found in the genomes of most cyanobacteria, it has been suggested that these organisms have a yet-unidentified type of Protox. Interestingly, most cyanobacteria seem resistant to diphenyl-ether herbicides which are specific inhibitors of HemY-type Protox. To identify a cyanobacterial gene encoding Protox, we first introduced the Arabidopsis HemY gene into Synechocystis sp. PCC6803 and then mutagenized the cells by transposon tagging. Subsequently, we screened the tagged lines for mutants that were sensitive to diphenyl-ether herbicides. We isolated several cell lines sensitive to diphenyl-ether herbicides and identified a single gene that was tagged in these cell lines. We found that this gene was essential for Protox activity in Synechocystis.

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3,8-Divinyl-chlorophyllide reductase reduces the vinyl group on the pyrrole ring B of tetrapyrrole molecules. Previously, we identified a divinyl reductase gene (DVR) from Arabidopsis thaliana by the means of genetics. However, DVR homologs were not found in most of the cyanobacterial group except five Synechococcus species. In this study, we identified cyanobacterial DVR by the combination of a bioinformatics approach and reverse genetics. We developed a bioinformatics tool to determine the species specific genes. By this tool, we found two genes were present only in the genomes which contain no Arabidopsis DVR homologs. We disrupted one of the two genes (slr1923) in Synechocystis sp. PCC6803, and found that the mutant cells accumulated divinyl chlorophyll instead of monovinyl chlorophyll. slr1923 was homologous to the gene encoding the β subunit of F420 reducing hydrogenase in organism. We concluded that slr1923 encodes DVR in Synechocystis sp. PCC6803.

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Chlorophyllide a oxygenase (CAO) catalyzes the conversion of chlorophyll a to chlorophyll b. The amount of chlorophyll b is regulated by the protein level of CAO. The CAO protein consists of three domains, namely A, B, and C domains. Among these domains, only the A domain is necessary for the regulation of the CAO protein level. Previously, we found that chlorophyll b triggers the degradation of CAO. In order to understand the regulatory mechanism of CAO, we mutagenized a transgenic plant of Arabidopsis thaliana that overexpressed the fusion protein of GFP and the A domain. We isolated 73 mutants that were impaired in the regulatory mechanism of CAO by the observation with laser-scanning confocal microscope. We classified these mutants into 11 groups according to the accumulation patterns of the GFP-CAO fusion protein. We will report the phenotypes of the mutants and will discuss the possible regulatory mechanism of CAO.

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Chlorophylls and their derivatives have a photooxidative activity under the light. Thus, in higher plants, chlorophyll biosynthesis is properly regulated at several biological steps such as transcription, translation and enzyme activation. Previous study in Arabidopsis showed that chlorophyll biosynthesis is strictly regulated at the transcriptional levels in response to light and growth conditions. To unravel the regulatory mechanism for chlorophyll biosynthetic genes, we focused on the chlorophyll accumulation in Arabidopsis roots. We found that cytokinin plays an important role in root greening. By contrast, auxin signals were indicated to have a negative effect on chlorophyll accumulation in the root. Transcriptome analysis showed that these hormonal signals are involved in the regulation of co-expression of chlorophyll biosynthetic genes. These hormonal effects on root greening were, however, completely abolished in hy5 mutant, suggesting that HY5 is a point of convergence between light and cytokinin/auxin signaling pathways in the root.

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Etioplasts accumulate chlorophyll (Chl) precursors, protochlorophyllides (Pchlides), that form prolamellar body (PLB) as ternary complex with NADPH and POR. Previously, Reinbothe's group proposed that PLB is formed with two POR homologs, PORA and PORB, which respectively bind to Pchlide b and Pchlide a (the LHPP model). However, this model is still controversial, because other studies failed to detect presumably accumulated Pchlide b.
We previously reported that the kinetic properties of POR from a cyanobacterium Gloeobacter violaceus were similar to those of PORs from higher plants rather than a cyanobacterium Synechocystis sp. PCC6803. In this study, we introduced the Gloeobacter POR gene into the Arabidopsis PORA knockdown mutant, and showed that 1) Gloeobacter POR was fully functional in chloroplast biogenesis, and 2) it could form PLB. Given that cyanobacteria do not synthesize Chl b, our results do not support the LHPP model. Evolutionary implications of the enzyme will also be discussed.

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A crystal structure of the catalytic component, NB-protein, of light-independent protochlorophyllide (Pchlide) reductase from Rhodobacter capsulatus has been solved at 2.3 Å resolution. The structure contains two copies each of homologus BchN and BchB subunits, and a [4Fe-4S] cluster in the interface. A surprise of the structure is direct coordination of BchB-Asp36 to the cluster, instead of BchB-Cys95 anticipated to coordinate the cluster based on the sequence similarity. The structure in the presence or absence of Pchlide has revealed the displacement of C-terminal helix of BchB when accommodating Pchlide. Intriguingly, the orientation of bound Pchlide is mainly provided by hydrophobic interaction, keeping the reduction site of ring-D portion away from the [4Fe-4S] cluster. These structures would have important implications for the mechanism of stereospecific Pchlide reduction, and comparison with the evolutionary related nitrogenase, which catalyzes the reductive formation of ammonia from dinitrogen, illustrates a functional framework of nitrogenase-like enzymes.

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Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing D-ring reduction to form chlorophyllide. Three subunits of DPOR, BchL, BchN and BchB, show significant similarity to those of three nitrogenase subunits, NifH, NifD and NifK, respectively. In the previous study, we have confirmed the nitrogenase-like features of DPOR by the reconstitution of purified components. A crystal structure of NB-protein showed that the [4Fe-4S] cluster is coordinated by three Cys residues of BchN (Cys26, Cys51, Cys112) and Asp residue of BchB (Asp36). To address the functional significance of these residues in the cluster coordination and the Pchlide reduction, we have constructed a series of strep-tagged NB-protein mutants of the Cys and Asp residues (C26A/S, C51A/S, C112A/S, D36C/A/S, C95S/A). The activity of the mutant NB-proteins was evaluated in vivo by the complementation of the bchN- or bchB-disrupted mutants of R. capsulatus and in vitro by biochemical analysis of the purified NB-protein.

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Dark-operative protochlorophyllide (Pchlide) reductase (DPOR) is a nitrogenase-like enzyme catalyzing the D-ring reduction of Pchlide in the penultimate step of chlorophyll biosynthesis in most oxygenic photosynthetic organisms. DPOR is the determinant enzyme for greening in the dark of the photosynthetic organisms. How the oxygen-sensitive enzyme operates in oxygenic photosynthetic cells remains unsolved. Here we report the functional expression of the cyanobacterial DPOR catalytic component, NB-protein (ChlN-ChlB), in E. coli. The chlN and chlB genes from the cyanobacterium Leptolyngbya boryana (formerly Plectonema boryanum) were artificially connected to form a small operon, chlN-chlB, in the overexpression plasmid. ChlN protein with Sterp-tag was co-purified with ChlB by an affinity column from the soluble fraction of E. coli. The addition of thus purified NB-protein to a crude extract of the LPOR-lacking mutant of L. boryana obviously stimulated the DPOR activity, indicating that an active form of NB-protein was expressed in E. coli.

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The photosynthetic green sulfur bacterium Chlorobium (Chl.) tepidum containes chlorophyll (Chl) a_PD as well as bacteriochlorophyll (BChl) a_P and BChl c_F. Chl a_PD serves as a primary acceptor as well as accessory pigments in the reaction center. This pigment has the same chlorin π-system as Chl a_P found in oxygenic photosynthetic organisms except for a long hydrocarbon chain on the C-17 position, that is, the former is esterified with Δ2,6-phytadienol group instead of phytol group. It is known that a precursor of phytol, geranylgeraniol is stepwisely reduced by geranylgeranyl reductase. However, the reaction process to produce Δ2,6-phytadienyl ester is not still revealed. In the genome of Chl. tepidum, two paralogs of geranylgeranyl reductase gene have been found and presumed to be involved in the hydrogenation process of geranylgeraniol. In this study, the mutants lacking these genes were constructed and analyzed to clarify the biosynthetic pathway of Chl a_PD.

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We identified the carotenoids in the cyanobacterium, Thermosynechococcus elongatus BP-1. β-Carotene (51%, mol% of total carotenoids), β-cryptoxanthin (trace) and zeaxanthin (8%) were found. Further, caloxanthin (6%) and nostoxanthin (14%) were present. Myxol glycoside (5%) and OH-myxol glycoside (15%) were typical carotenoid glycosides in cyanobacteria. Although ketocarotenoids, such as echinone and ketomyxol glycoside, were also typical carotenoids in cyanobacteria, they were absent. Glycoside moiety of myxol glycoside is under study. Among the carotenogenesis genes, only crtE is functionally confirmed. From homology search, presence of crtB, crtP, crtQ and crtR was suggested. β-Carotene ketolase, crtO and crtW, were absent. A homologous gene crtG of β-carotene 2-hydroxylase, which produces nostoxanthin from zeaxanthin, was found, and its function is under study. The biosynthetic pathway of carotenoids will be discussed. (Takaichi, Mochimaru (2007) Carotenoids and carotenogenesis in cyanobacteria: unique ketocarotenoids and carotneoid glycosides, Cell. Mol. Life Sci. 64: 2607-2619)

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A coccolithopholid Emiliania huxleyi is a unicellular marine alga and requires selenium (Se) for its growth. In E. huxleyi, we previously reported six selenoproteins (EhSEP1~6) and among them, EhSEP2, is a novel protein disulfide isomerase containing Se. These phenomena suggest that E. huxleyi may possess unique selenoproteins. In this study, we characterized EhSEP1 to get new knowledge about physiological function of Se. After purification of EhSEP1, we determined partial amino acid sequences and cloned a full-length cDNA for EhSEP1. The deduced amino acid sequence of EhSEP1 had strong similarity to those of thioredoxin reductases (TR), which are well-characterized selenoproteins. The crude extracts of E. huxleyi exhibited significant TR activity, when Se was supplied into the culture media. However, levels of the EhSEP1 mRNA were constant despite amount of supply of Se. These results suggest that the translation of EhSEP1 might be responded to the levels of available Se.

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The coccolithophorid Emiliania huxleyi, a marine unicellular calcifying alga, produces mineralized scales called coccoliths by intracellular calcification. The process of calcification was not fully understood due to the lack of the information of proteins associated with the calcification.
We found that the calcification was promoted under phosphate-depleted conditions and low-temperature conditions and a protein which has an apparent molecular mass of 32-kDa was specifically synthesized under the conditions. We purified the protein and determined its N-terminal sequence. From the searching expressed-sequence-tag (EST) database, we found the sequence in the protein which has a highly conserved FK506-binding protein domain (FKBP), an EF-hand motif and an endoplasmic reticulum (ER) retention sequence. Because the deduced molecular mass of the protein was 20.4 kDa, the protein might be post-translationally modified in vivo. Mechanisms of the calcification may be elucidated by analyses of the functions of the protein.

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Sec-pathway is known to one of the main translocation pathway of some proteins in bacteria and plants. It is constituted of essential components SecA and SecYE translocon. SecA operates to translocate precursor proteins into thylakoid lumen in plants. Normally most of the organisms encode only one secA gene in its genome.
While primitive red alga Cyanidioschyzon merolae encodes two secA genes, one in the nucleus and the other in the plastid genome. Phylogenetic analysis showed that two secA genes grouped into different clusters. The purpose of this study is to clarify the differences of two SecAs. Previous study demonstrated that these two secA genes were transcribed. Two SecA proteins were purified to investigate ATPase activities and translocation assays. Now we attempt to isolate the chloroplasts and thylakoid membranes of C. merolae. Moreover substrates for each SecA proteins are searched by Pull-down assay.

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The presence of high ADP hydrolysis activity in Mimosa pudica was previously reported. However, biochemical characteristics of the enzyme were not studied in detail. In the present study, we purified ADP hydrolysis enzyme (ADPase) from Mimosa plant by sequential column chromatography. The purified ADPase was detected as smear bands on SDS-PAGE gels with relative molecular masses of 67 kDa, indicating that some isoforms of ADPase resulted from the post-translational modification such as glycosylation. The determined partial amino acid sequences of the peptides showed similarity with other plant apyrases. Inhibition of NTP/NDP hydrolysis activity with triflupromazine (TFP) indicates that Mimosa ADPase belongs to the ecto-apyrase group. The ratio of the rate of ATPase/ADPase of apyrases from the other plants range from 0.7 to 1.5, whereas the ratio of the Mimosa ADPase is more than 4. Thus, Mimosa ADPase is a novel enzyme. I plan to elucidate a physiologic functions in detail.

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Rapid petiolar bending and leaf closing of Mimosa pudica L. occurs in response to mechanical, thermal, electrical, and chemical stimuli. We have reported that phosphorylation/dephosphorylation cycles at tyrosine residue play a role in this seismonastic movement. However, protein tyrosine phosphatase (PTP) in plants is less well-characterized than those in animals. In the present study, we have isolated and characterized cDNA clones (MpPTP1 and MpPTP2) encoding tyrosine phosphatase of Mimosa plant. For characterization, the biochemical properties of these clones, GST-fused MpPTPs were expressed in E. coli, and its phosphatase activity was measured. Both clones exhibited a tyrosine-specific phosphatase activity. Analysis of expression profiles of these clones reveals that MpPTP1 was specifically expressed in root. We further examined the effect of PTP-inhibitor, phenylarsine oxide (PAO), on development. Unusual root growth was observed by incubating seedlings in PAO-containing media. Thus, we consider that MpPTP1 affects the root development negatively in mimosa pudica.

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In mammalian brown adipose tissue, uncoupling protein 1 (UCP1), triggers a proton leak and converts the energy into heat. Although the recent finding of plant UCPs in non-thermogenic tissues has precluded their involvement in thermogenesis, there are few studies of them in thermogenic tissues. Therefore, we analyzed two cloned UCP cDNAs, SfUCPa and SfUCPb, isolated from the thermogenic spadix of skunk cabbage. We show that SfUCPA, not SfUCPB, is the major uncoupling protein and that it is integrated into the inner mitochondrial membrane. Using spadix mitochondria, we examined the uncoupling activity of SfUCPA. Although SfUCPa transcripts were constitutively expressed in various tissues irrespective of thermogenic stages, SfUCPA protein existed only in the thermogenic tissue or stage. Quantitative immunoblot analysis revealed that SfUCPA was an abundant protein in spadix mitochondria. Our results suggest that SfUCPA may be involved in the heat production of skunk cabbage.

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AtPCaP1 is localized to plasma membrane via N-myrisotylation and association with PI(3,5)P₂. The protein is composed of 225 amino acid residues and rich in Glu and Lys. No enzymatic functional motif was found in AtPCaP1. The physiological function is unclear. In the present study, we prepared the recombinant AtPCaP1 to determine its cation-binding properties. By CD spectrum and DSC (differential scanning calorimeter) analyses, AtPCaP1 has been revealed to have unique properties: namely, the binding of divalent cations and the high protein concentrations decreased the thermal denaturation temperature. In other words, these conditions decreased heat stability of AtPCaP1. Furthermore, AtPCaP1 specifically changed higher-order structure by binding of Cu²⁺. Interestingly, it binds 8 Cu²⁺ ions per molecule with K_d of 10 μM even though AtPCaP1 has no His or Cys, We will report more detailed information and discuss its physiological role.

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Recently, we found novel cation-binding proteins in Arabidopsis thaliana. One of these proteins, AtPCaP2 consists of 168 amino acid residues and contains N-myristoylation signal and repetitive unique motifs (VEExK). To estimate its physiological role we characterized molecular properties of AtPCaP2. Quantitative RT-PCR revealed that AtPCaP2 was predominantly expressed in roots. The promoter::GUS expression analysis confirmed the root specific expression. The mRNA level was increased more than three-fold when plantlets were treated with bacterial elicitor peptide, heavy metals such as Fe²⁺ and Mn²⁺, low temperature, drought, abscisic acid and osmotic stresses. When a AtPCaP2::GFP fusion protein was expressed in Arabidopsis cells, green fluorescence was clearly observed on the plasma membrane. Further analyses revealed that AtPCaP2 was N-myristoylated at the N-terminal part and has capacity to bind specific phosphatidylinositol phosphates. These properties suggest a possibility that AtPCaP2 is involved in responses to a variety of physiological stresses.

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Our biochemical analyses of AtHOL1, which is involved in methyl halide emissions in Arabidopsis, and its isoforms designated AtHOL2 and AtHOL3 showed that the three recombinant AtHOL proteins could catalyze the S-adenosyl-L-methionine-dependent methylation of Cl^-, Br^-, I^-, NCS^-, and HS^-. We were interested in the results indicating that Cl^- methyltransferase activities of three recombinant proteins were very low whereas the NCS^- methyltransferase activity of AtHOL1 was high. Analyses of the AtHOL1-disrupted mutant showed that AtHOL1 methylated NCS^- produced in homogenized Arabidopsis tissues and CH₃SCN was synthesized. Our results implied that NCS^-was derived from indolyl-glucosinolates and S-adenosyl-L-methionine was rate determining for CH₃SCN synthesis in homogenized tissues. Glucosinolate are defensive compound and when tissues are damaged, they are hydrolyzed by myrosinase. It was reported that indolyl-glucosinolate accumulated by the treatments of methyl jasmonate or elicitors from plant pathogens. The possible physiological roles of AtHOL1 will be discussed.

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Nudix hydrolases are widely distributed among eukaryotes, bacteria, archaea, and viruses. They are divided into subfamilies based on their major substrates: ribo- and deoxy-nucleoside triphophates, ADP-ribose, NADH, CoA, and dinucleoside polyphosphates. Previously, we have demonstrated the molecular characterization of cytosolic Nudix hydrolases (J. Biol. Chem. 2005 280: 25277-83, Plant Cell Physiol. 2007, 48: 1438-49). Here we report the comprehensive analysis of chloroplastic AtNUDXs (AtNUDX19~24, 26, and 27). Among the chloroplastic AtNUDXs, AtNUDX19 and 23 showed NAD(P)H and FAD pyrophosphatase activities, respectively. AtNUDX26 and 27 acted on diadenosine polyphosphates. These results suggest that AtNUDX19 and 23 are pertinent to the regulation of diverse metabolic pathways, such as photosynthesis, redox reactions, and light sensing via hydrolyzation of NAD(P)H and FAD in chloroplasts. We are progressing toward to analyze their physiological functions using knock-out and overexpressing mutants.

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Radioisotope tracer studies indicate that the biosynthesis pathway proceeds via D-galacturonate (D-GalUA) as the intermediate in Euglena cells. To clarify the Euglena pathway in detail, we have purified D-GalUA reductase and L-galactono-1,4-lactone (L-GalL) dehydrogenase, and cloned a cDNA encoding aldonolactonase (ALase). The purified D-GalUA reductase utilized NADPH as an electrondonor with Km values of 3.79 ± 0.5 mM and 4.67 ± 0.6 mM for D-GalUA and D-glucuronate, respectively. The recombinant ALase catalyzed L-galactonate and L-gulonate with the apparent Km values 1.55 ± 0.3 mM and 4.55 ± 0.23 mM, respectively. On the other hand, L-GalL dehydrogenase purified from Euglena mitochondria showed much higher affinity for L-GalL (Km=0.21 mM) rather than L-GulL (Km=113.9 mM). Furthermore, introduction of double-strand RNA for the ALase to Euglena cells resulted in the cell growth arrest. These results clearly demonstrated that D-GalUA pathway is the major route for ascorbate biosynthesis in Euglena.

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Acylamino Acid-Releasing Enzyme (AARE) is protease conserved widely among microorganism, mammal and plant. AARE catalyzes the N-terminal hydrolysis of N^α-acylpeptide, which releases N^α-acylated amino acids. It was found in vitro that AARE degraded oxidized and glycated protein. We analyzed plant AARE function in the degradation of oxidized protein using transgenic Arabidopsis thaliana.
Arabidopsis overexpressing AARE was generated using 35SCaMV-promoter and AARE silencing Arabidopsis was also created using RNAi. AARE expressions were confirmed by western blot and measuring AARE activity. Oxidative stress tolerance and levels of oxidized proteins were compared between wild-type (Col-0) and transgenic plant.
AARE silencing plants are more sensitive to oxidative stresses than wild-type one. The oxidized protein levels increased as AARE activity decreased.

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Prenyltransferases occur widely in prokaryotes and eukaryotes, among which membrane-bound type enzymes are involved in the biosynthesis of various quinines, e.g. plastoquinone and in the formation of prenylated aromatic secondary metabolites like planylated flavonoids in plants. However, due to the difficulty in the biochemical handling almost nothing is known about the enzymatic function of those membrane-bound prenyltransferases. In this study, we used LePGT1, which accepted p-hydroxybenzoic acid (PHB) and geranyl diphosphate (GPP) as substrates, as a model of such membrane-bound prenyltransferases and the crucial amino acids were identified by site-directed mutagenesis. Moreover, we generated the 3D structure of substrate binding pocket by molecular modeling, which provided a reasonable structure to explain the biochemical data obtained by the characterization of mutant enzymes. The domain determining the substrate specificity of LePGT1 to GPP will be also discussed using this model.

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It is known that plants grown under high light conditions have larger amount of xanthophyll cycle pigments compared to those of the plants grown under low light conditions. However, little is known about the regulation mechanism of biosynthesis of xanthophylls in green leaves. In recent years, many photosynthesis related genes have been reported to be regulated by chloroplast redox signals. In this study, we examined the regulatory effect of redox-state of the photosynthetic electron transport chain on the xanthophyll biosynthesis.
The photoautotrophically cultured tobacco cells grown under high light conditions did not exhibit an increase in xanthophyll cycle pigments pool size when DCMU was added to the culture medium. In contrast, lowering the CO₂ concentration in the atmosphere of culture flasks induced an increase in the xanthophyll cycle pool under low light condition. Our results suggest that chloroplast-derived redox signaling regulate the xanthophyll biosynthesis.

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The first step of chlorophyll biosynthesis is catalyzed by Mg-chelatase composed by subunits, CHLI, CHLD and CHLH. Mg-chelatase requires ATP hydrolysis attributed by CHLI. Arabidopsis possesses two CHLI isoforms, CHLI1 and CHLI2, showing similar expression profiles, but CHLI2 is suggested to have limited function on Mg-chelatase complex. Previously, we showed that Arabidopsis CHLI1 is active ATPase and a target of chloroplast thioredoxin (Trx). Here, we revealed that CHLI2 shows ATPase activity with lower V_max and higher K_{m ATP} values than those of CHLI1. We further confirmed Trx-dependent reduction of S-S bond of CHLI2 and thiol-modulation of ATPase activity. By crossing of homozygous CHLI1 (cs) and CHLI2 (CSHL_GT13937) mutants, we could select pale-green plants in F2 progenies, which segregated into three different types showing cs like pale-green, almost albino, and intermediate plants with ratio of 1:0.8:2. PCR analysis confirmed that CHLI2 mutation is semidominant, suggesting CHLI2 certainly functions in Mg-chelatase complex.

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Chlorophyllide a oxygenase (CAO) is a rieske-type monooxygenase that catalyzes the conversion of chlorophyll a to chlorophyll b. CAO protein consists of three domains, namely, A, B and C domains. The A domain alone is involved in the regulatory mechanism of the CAO protein level. Previously, we reported that both accumulation of chlorophyll b and presence of Clp protease are necessary for the activity of CAO degradation. In this study, we demonstrated that a sequence of 10 amino acid residues which locates in the middle of the A domain is essential for the regulation of the CAO protein level. Furthermore, addition of this sequence to GFP significantly reduced the accumulation of GFP in transgenic plants, indicating that this sequence may is a tag to induce degradation of chloroplast proteins. Possible degradation mechanisms that involve the recognition of the sequence tag by Clp protease will be discussed.

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Chlorophyll b reductase catalyzes the conversion of chlorophyll b to chlorophyll a. In this study, we investigated the functions of two chlorophyll b reductase isoforms, namely, AtNYC1 and AtNOL, in Arabidopsis thaliana. The AtNYC1 gene was predominantly expressed during leaf senescence, while the AtNOL gene was constitutively expressed in leaves. An AtNYC1 knock-out mutant showed the stay-green phenotype during leaf senescence. Degradation of chlorophyll b, carotenoids, and LHCII was markedly retarded in the mutant. In contrast, the AtNOL knock-out mutant didn't show an obvious stay-green phenotype. These results indicate that AtNYC1 and AtNOL have distinct and independent physiological functions in Arabidopsis thaliana. Furthermore, we examined the in-vivo activity of AtNOL and found that the enzyme was able to convert the chlorophyll b molecules bound to LHCII.

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Coproporphyrinogen III oxidase (CPO) catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX in heme biosynthesis that is shared with the chlorophyll biosynthesis in photosynthetic organisms. There are two structurally unrelated CPOs; oxygen-dependent (HemF) and oxygen-independent (HemN) oxidases. The cyanobacterium Synechocystis sp. PCC 6803 has one hemF-like gene, sll1185, and two hemN-like genes, sll1876 and sll1917. To address how the different types of CPO operate in the environments under oxygen-fluctuated environments, three mutants lacking one of the three genes were isolated. The Δsll1185 mutant failed to grow under aerobic conditions with anomalous accumulation of coproporphyrin III. The Δsll1876 mutant grew much slower than wild-type under micro-oxic conditions and the coproporphyrin III accumulation was induced by 5-aminolevulinate feeding. In contrast, Δsll1917 grew as well as wild-type. These results suggested that Sll1185 and Sll1876 operate as the dominant CPO under aerobic and micro-oxic conditions, respectively.

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A mutant (slr1923^M) of Synechocystis sp. PCC6803 has been created through inactivation of the gene slr1923 and was characterized. The chlorophyll of the mutant is replaced from 3-vinylchlorophyll (normal Chl) to 3,8-divinyl chlorophyll. We have concluded that Slr1923 is related with chlorophyll metabolism and the mutant failed to carry out the reduction of 8-vinyl group of 3,8-divinyl(proto)chlorophyll(ide). In the present study, characterization of reaction centers of the mutant was carried out. Absorption spectra of both the isolated photosystems showed that Soret band peak position shifted to longer wavelength significantly. Compared with wild type, the mutant PSII showed a relatively higher fluorescence peak of 685 nm band at 77K. P700 difference spectra indicated that Soret peak is red shifted which is the same characteristics of divinyl-Chl a absorption spectra. These data confirmed that PSI primary electron donor (P700) is also composed of divinyl-Chl a. Other features will also be reported.

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Recently, we isolated the purple photosynthetic bacterium, Rhodopseudomonas sp. Rits which possessed BChl a intermediates with non/di/tetrahydrogeranylgeranyl groups as well as phytyl group at the C-17 propionate [1]. In carotenoid biosynthesis, this bacterium mainly accumulated some intermediates of spirilloxanthin including rhodopin and 3,4-didehydrorhodopin. We observed that these intermediary pigments located in peripheral antennas as well as RC-LH1 core complex [2]. Although we have demonstrated that the composition of these pigments was dependent on irradiated light intensity, the effect of incubation period on the pigment compositions has not been analyzed yet. In this study, we measured the quantitative changes of intermediates of BChl a and carotenoids in the Rits strain during cultivation. We will discuss about the relationship between pigment compositions and the types of antenna proteins in this bacterium.
[1] T. Mizoguchi et al. (2006) FEBS lett. 580: 6644-6648.
[2] J. Harada et al. (2007) Photosynth. Res. in press.

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In the conventional ultra-fast up-conversion fluorescence measurement, fluorescence emitted from the opposite side of the laser excitation is detected. Only a transparent sample could be measured by this type of system. For the noninvasive in vivo measurement, we developed a new reflection-type system, in which the fluorescence emitted at the same side as the laser excitation can be detected. We measured the rise and decay of fluorescence from the green alga inside dried lichen. Lichens are drought tolerant, and have a mechanism to convert the excess light energy into heat to prevent the photoinhibition. We directly measured the time constant of the fluorescence lifetime of dry lichen to be 20 ps. No fluorescence rise corresponding to the dacay was observed suggesting that the most of the excitation energy is quenched within 20 ps.The reflection type system enables us to perform noninvasive ultra-fast measurements of opaque samples like leaves in vivo.

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State transitions are regulated by redox state of cytochrome b₆f complex; when the complex is reduced (state-2), a part of light harvesting complex II (LHCII) migrates from PSII to PSI, forming PSI-LHCI/II supercomplex. Here we improved conditions to induce cells to state-2 more completely by illuminating plastocyanin-deficient mutant or by dark incubation of wild type in anaerobic condition in the presence of glucose/glucoseoxidase. The thylakoid membranes isolated from these cells were solubilized and fractionated by sucrose density gradient ultracentrifugation. More PSI-LHCI/II supercomplex was obtained in A-3' band. It is noted that two thirds of CP26 remained associated with the supercomplex although only a trace amount was recovered in A-3' in the previous report. This result may be derived from the fact that the cells were induced thoroughly to state-2. We will report relative abundance of PSI-LHCI and LHCII by dissociating these two complexes in the presence of NaCl.

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Photosystem I (PSI) complex consists of 15 subunits and a number of cofactors, and forms PSI-LHCI supercomplex. In higher plants, PSI particles retaining all these subunits could be isolated while most PSI particles purified from Chlamydomonas lost PsaN, PsaO and PsaP. Chlamydomonas contains the psaN and psaO genes and a psaP homologue. We generated antibodies against PsaN, PsaO and PsaP to address localization of these subunits in Chlamydomonas PSI complex. These three subunits were easily dissociated from PSI complex when separated on sucrose density gradient. However, PsaN and PsaO were reduced in PSI deficient mutants. When thylakoidal polypeptides were chemically cross-linked, the cross-linked products of PsaN/PsaF and PsaO/PsaL were observed. In addition, PsaN was associated with PSI-LHCI/II supercomplex that is separated only from the state2 thylakoids. Thus the PsaN binding affinity to PSI changes depending on the state transitions.

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We generated Synechocystis sp. PCC6803 strains, which express His-tagged PsaF and PsaJ subunits, for simple and rapid purification of PSI complex. In the generated strains, F-His and J-His, the C-terminus of PsaF and the N-terminus of PsaJ were tagged with 6 histidine residues. Growth profile of F-His and J-His cells under photoautotrophic condition was similar to that of wild type. No difference in photosynthetic activity was also observed among wild-type, F-His and J-His cells. PSI complexes could be simply purified from the F-His and J-His cells by solubilization of thylakoid membranes with dodecylmaltoside followed by a Ni²⁺ affinity column chromatography. The purified PSI complexes were separated to monomer and trimer by ultracentrifugation with glycerol density gradient. Blue-Native PAGE and SDS-PAGE analyses of the purified PSI complexes indicated the existence of two distinct monomers having different subunit composition.

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Acaryochloris marina is a unique cyanobacterium that produces chlorophyll (Chl) d as a major photosynthetic pigment. Recently, the complete genome sequence of A. marina MBIC 11017 was determined, and the PsaI was not found in the genome. The crystal analysis of PS I showed that PsaI is localized in the attaching site of individual monomers. We identified a new subunit and plural PsaK subunits in the PS I complexes of A. marina. We presumed that this new subunit might replace the function of PsaI in A. marina. In PS I, the redox potential of the primary electron donor (P740) and localization of Chl a are still controversial. We estimated a redox potential of P740 and found that it was inconsistent with previous report (Hu et al. 1998). Based on these observations, the assignment of the pigment and its role of PS I will be discussed.

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Acaryochloris marina is a unique cyanobacterium that possesses chlorophyll d as a major photosynthetic pigment. The photosystem (PS) I and II of A. marina have been isolated and well characterized. The primary donor of PS I was assigned to Chl d/Chl d' heterodimer. The redox potential of the primary donor was reported as +335 mV (vs. SHE), which is lower by approximately 100 mV than those of the primary donor of photosystem I in other photosynthetic organisms; however this reported value has not yet been consistent with other experimental results (Benjamin et al, 2007; Telfer et al 2007). In the present study, we have tried to measure the redox potential of the primary donor of A. marina PS I with a spectroelectrochemical technique. We will compare the redox potentials of the primary donor of PS I in the various organisms including A. marina.

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A thermo- and acido-philic primitive red alga, Cyanidioschyzon merolae, is a unicellular eukaryote. Total genome sequence have been reported. Concerning the genes encoding subunits of the photosytem I (PS I) complex, genome of C. merolae has both eukaryotic and cyanobacterial type genes. From this view point, the PS I seems to have a chimeric feature of cyanobacterial and eukaryotic PS I.
PS I complexes were highly purified from C. mearolae and analyzed precisely. All of the subunits predicted from the genome sequence were detected, confirming that all of the corresponding genes were actually expressed in C. merolae. The purifiled PS I complex was found to be a monomer associated with three kinds of proteins homologous to light-harvesting chlorophyll a/b proteins . The electron carrier A1 may not be a phylloquinone. These results indicate that the PS I complex has a mixed feature of cyanobacterial and eukaryotic PS I complexes.

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We have introduced functional membrane protein complex into a silica mesoporous material (SMM) that has the nano-scale pores of honeycomb-like hexagonal cylindrical structure in its inside. The photosynthetic reaction center (RC) complex (129 kDa) and light-harvesting LH2-complex (137 kDa) of a thermophilic purple photosynthetic bacterium Thermochromatium tepidum were shown to be introduced into SMM (ref 1). In this study, we introduced the trimer of photosystem I (PSI) reaction center complex (1068 kDa) purified from a thermophilic photosynthetic cyanobacterium Thermosynechococcus (T.) elongatus into SMM. Adsorption to SMM increased the heat stability of P700 photochemical activity and protein structure. The PSI-SMM conjugate can be a new material for the solar energy conversion.
Oda et al.(2005) :J. Phys. Chem. B 110 (3),1114-1120

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