In the oil palm, plants regenerated from tissue culture frequently show morphological abnormality called "mantled", in which stamens change into carpels, and the epigenetic change possibly occurs in the class B gene. Here we examined methylation of the oil palm EgDEF1, EgGLO1 and EgGLO2 genes in the wild-type plant. Promoters or 5' UTRs of them were not methylated. From exon 4 to intron 6 of EgGLO1, both CG and non-CG sites were highly methylated. In the corresponding region of EgGLO2, only CG sites were highly methylated. Their sequences were highly homologous to each other, but only EgGLO1 had a microsatellite-like sequence. These suggest that the microsatellite-like sequence is involved in the non-CG methylation of EgGLO1. This study was supported by "Molecular genetic studies of the oil palm (Elaeis guineensis) for improvement of the oil production" in '03 from New Energy and Industrial Technology Development Organization (NEDO) in Japan.

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Pyridine nucleotides not only act as mediators of redox reactions but also have important roles in signaling pathways and in gene regulation. The turnover of NAD(P) is performed by the pyridine nucleotide cycle (PNC). Diversity in PNC has been found in species and organs. In the present study, we examined details of the PNC in higher plants by investigating the metabolism of ¹⁴C-labelled compounds, by determining the enzyme activities of several plant materials, and by bioinformatic analyses. In plants, the specific PNC which consist of seven enzymes (PNCVII) is proposed. Proposed cycle is NAD → nicotinamide mononucleotide → nicotinamide riboside → nicotinamide → nicotinic acid → nicotic acid mononucleotide → NaAD → NAD. In addition, the cycle including nicotinic acid riboside is found in some plant species. Nicotinic acid, an intermediate of the cycle, is released for the synthesis of trigonelline and/or nicotinic acid glucoside.

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In the Japanese morning glory (Ipomoea nil), several responsible genes for unstable mutations were isolated, and all transposable elements inserted in these genes belong to En/Spm (CACTA)-related element, Tpn1 family. All Tpns identified were non-autonomous elements that truncated transposase gene and captured host gene sequences. The mRNAs for transposase proteins TNPδ and TNPα are expressed by alternative splicing from autonomous Tpn as other En/Spm-related elements, and they mediate the transposition of non-autonomous elements. TNPδ has TNPD/TNP2 domains conserved in other En/Spm transposons. On the other hands, TNPα includes C-terminal sequence showing a homology to cysteine protease domain. This protease domain is also conserved in Rim2 transposon from rice and CACTA-related transposons from Medicago, but not En/Spm from maize and Tam1 from Antirrhinum, suggesting that Tpn have a different transposition mechanism.

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Soybean is important crop since soybean seeds contain a large amount of protein and lipid, and useful as functional foods and energy source. However transformation efficiency of soybean is lower compared to rice and tobacco. Therefore, the technology of transgenic soybean was not so common. Transformation using Agrobacterium tumefaciens has advantage to introduce the low copy transgene stability. We aim to establish the soybean transformation using Cotyledon node method with A.tumefaciens to produce the transgenic soybean containing high amount of nicotianamine (NA) in their seeds. NA has the ability to suppress high pressure. We have established the condition to get the regenerates after many trial and errors. We are improving transformation efficiency more by examination the condition as germination status, hygromycin concentration, light intensity and temperature.

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Gene targeting (GT) is an effective tool for functional genomics. We have developed an efficient and generally applicable homologous recombination (HR)-dependent GT procedure in rice, which comprises, (1) a large-scale Agrobacterium -mediated transformation, (2) a strong positive-negative selection, and (3) an efficient PCR screening to detect junction fragments generated by HR. To avoid any potential undesirable side effects, caused by manipulating recombination and/or repair system(s), for elucidating the function of a target gene, we employ wild-type rice cultivars for GT. Because independent GT events by HR should generate an identical genomic structure with a previously designed sequence alteration(s), the experimental demonstration of the capability for the reproducible isolation of recombinants with the anticipated gene structure would be very important. At present, we have reproducibly succeeded in generating rice GT plants of eight different genes (Waxy, Adh1, Adh2, OsDDM1a, OsDDM1bOsMET1a, OsMET1b, OsMET1a, OsMET1b, and OsDRM1a).

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Gene targeting (GT) by homologous recombination (HR) in higher plants is thought to be difficult because its targeting frequency is too low to be detected. We have developed an efficient HR-promoted GT procedure in rice by a large-scale Agrobacterium-mediated transformation with a strong positive-negative selection (PNS) and subsequent PCR screening and reproducibly succeeded in generating transgenic rice plants having one of eight different genes modified. In addition, we detected randomly integrated and truncated T-DNA segments containing our positive marker without intact negative markers in surviving calli with PNS. We also found both expected true gene targeting and one-sided invasion events. To look into the recombination crossover points, we performed GT with a vector having several point mutations in homologous regions and determined their sequences in a few targeted plants obtained. These results are discussed with regard to molecular processes of homologous and nonhomologous recombination.

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We were establishing lines for genome wide functional analysis of Arabidopsis genes by employing gain-of-function techniques, activation tagging and FOX hunting system. However the isolation of important genes, that cause severe phenotypes like embryonic lethal, is difficult from these lines for constitutive 35S promoter. And many transcription factors should be included in these important genes, because they play pivotal role for regulation of various biological functions in plants. We are, therefore, establishing novel lines, and the feature of these lines is the functional induction of transcription factors transformed in plants by employing glucocorticoid-mediated transcriptional induction system. And the important knowledge might be acquired by comparison between these lines and CRES-T lines, that are established by employing gene silencing system using dominant repressor, since they shows loss-of-function and gain-of-function phenotypes, respectably. In this report, we describe about the detail of these lines and discuss the usefulness as genome resource.

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Among 2,090 chloroplast proteins identified by computational prediction in Arabidopsis, we collected 3,416 tag-lines disrupted by transposon or T-DNA that encode 1,374 chloroplast proteins. From this collection, we have not only isolated mutants with visible phenotype (apg mutants), but also collected homo-lines without clear phenotypes. By screening of 702 of RIKEN Ds tag-lines, we identified 578 homozygotes with no obvious phenotype, 59 mutants showing abnormal seedling and 62 mutants of which homozygotes were not obtained. Among 43 genes that caused abnormal phenotype, 20 genes existed several alleles with similar phenotypes, suggesting their important role for chloroplast development. 59 mutants that could not obtained homozygotes are valuable resource to understand the roles of plastid development in plant embryogenesis. For the functional analysis of the nuclear-encoded chloroplast proteins that affect abiotic stress response, we isolated several candidates for stress-related chloroplast proteins by screening of 578 homozygotes. We will discuss their possible functions.

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Fluorescence Lifetime Imaging Microscopy (FLIM) is a technique to visualize the fluorescence lifetime in cells. The fluorescence lifetime is the time of fluorophore to remain at the excitation state before returning to the ground state. FLIM can be used to measure FRET, pH, ion concentration, amount of reactive oxygen species, etc. I applied FLIM to analyze the organellar environment. Various GFP lines targeted to the organelles were used to examine the difference in the lifetime depending on the organelles. In addition, the effect of pH change over the lifetime was examined. These data will be a basis of future research on the intracellular dynamics during development or under various environmental stresses.

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A brown alga P. littoralis is unique to that so mach as 7 group II introns are located in the mitochondrial genome. Four group IIB introns in the LSU rRNA gene and three group IIA introns in the cox1. We analyzed the arrangement of group II introns for 8 P. littoralis specimens collected at world wide habitats. The first intron located in the LSU rRNA gene of a specimen collected in France and the fourth intron of a specimen harvested in Japan had a exceptionally long evolutionary distance against the cognate introns found in P. littoralis specimens. In addition to that these introns harbored an intact or nearly intact tripartite ORF in their arm-IV loop. Based on the result of molecular phylogenetic analyses, we concluded that these introns are the result of recent reinvasion of cognate introns.

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Students who received high school education guided by the new national curriculum standards, so called the "cram-free education", entered universities in the spring of 2006. Anticipating the arrival of students with new educational background, faculty members of Division of Biology in The University of Tokyo, who take charge of introductory education to Life Sciences, have developed three new experiments and refined the current eight experiments for the course. Additionally, in accordance with the start of an interfaculty program of the university, the members have also developed a DVD resource that would facilitate course instructions and enhance educational achievements.

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Leaves with structural variegation have partly whitish division not because of lack of chlorophyll but because of diffused reflection due to air spaces between epidermis cells and palisade cells. Ecological meaning and developmental processes of structural variegation have not been known yet.
We chose Schismatoglottis calyptrata which grows in tropical rainforest floor and Saxifraga stolonifera which distributes in Japan as model cases to underatand ecological meaning and developmental processes of structural variegation, because these species have both strain with/without variegation in leaves. Although the efficiency of carbon fixation in variegated leaves seems to be less than that of normalleaves, curiously, they were reported to coexist under same condition.
Here we show that the structural variegation decreases the photosynthetic efficiency. We also analyzed differences of quality and quantity of chlorophyll between variegated and normal leaves. Furthermore, what stage in development and why do structural variegation appear are also reported.

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Photosynthesis is affected by the conductance of CO₂ from the atmosphere to the site of carboxylation. The location of chloroplasts in the mesophyll cells may affect the length of the diffusion pathway for CO₂. Using different light treatments to induce chloroplast movements we studied the effect of chloroplast location on a number of photosynthetic parameters in Arabidopsis thaliana. In addition, we examined these parameters in mutants which do not show a chloroplast avoidance response under high light conditions. Furthermore, we investigated the internal conductance after externally applying an inhibitor of chloroplast movement. Following gas-exchange measurements we sampled the leaves for anatomical measurements to quantify the length of the diffusion pathways. Our results indicate that chloroplast movement affects internal conductance for CO₂, and this in turn exerts a small effect on the photosynthetic capacity in A. thaliana.

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To identify useful genes of rice, we generated rice FOX (full-length cDNA overexpressor) lines that rice full-length cDNAs were over-expressed in Arabidopsis plants. In this study, we have used imaging of chlorophyll fluorescence to screen photosynthesis-related mutants. So far, we screened about 6,000 lines and isolated 23 candidates with altered chlorophyll fluorescence parameters. Isolated mutants could be classified into several categories depending on these parameters. Moreover, chlorophyll fluorescence was also measured after high-light treatment (1,000 μE, 1 h) to isolate high-light tolerant mutants. We isolated 2 lines that showed tolerance to high light from about 2,500 lines. In these mutants, various rice cDNAs were transformed into Arabidopsis such as cDNAs encoding enzymes that have a chloroplast transit peptide, transcriptional-related proteins and unknown proteins. This research is supported by the Special coordination funds for Promoting Science and Technology entitled Rapid identification of useful traits using rice full-length cDNAs.

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During C₄ photosynthesis, CO₂ is first fixed into C₄ acid by phosphoenolpyuvate carboxylase in the mesophyll cell. The C₄ acid is transported to bundle sheath cells (BSC), where it is decarboxylated, released CO₂ being refixed by Rubisco. Since this CO₂ concentrating mechanism decreases the Rubisco oxygenase activity, C₄ photosynthesis can be advantageous under water stress or high temperature. However, C₄ metabolism requires additional ATP compared to C₃ metabolism. It is assumed that additional ATP can be produced by cyclic electron transport around photosystem I which can increase ΔpH across the thylakoid membrane. In Arabidopsis, two cyclic pathways, PGR5-dependent pathway and NDH-dependent pathway, have been shown to be functional. To elucidate the role of the two cyclic pathways during C₄ photosynthesis, we investigated localization of PGR5 and NDH in leaf tissues of Flaveria C₃ and C₄ species. Results suggested that both cyclic pathways are up-regulated in C₄ species, especially in BSC.

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We tested the hypothesis that ferredoxin (Fd) limits the activity of CEF-PSI in vivo and that the relief of this limitation promotes the non-photochemical quenching (NPQ) of Chl fluorescence. In transplastomic tobacco expressing Fd from Arabidopsis in its chloroplasts, the minimum yield of Chl fluorescence (Fo) was higher than in the wild type. Fo was suppressed to the wild-type level upon illumination with far-red light, implying that the transfer of electrons by Fd-quinone oxidoreductase from the chloroplast stroma to plastoquinone was enhanced in transplastomic plants. The activity of CEF-PSI became higher in transplastomic than in wild-type plants under conditions limiting photosynthetic linear electron flow. Similarly, NPQ of Chl fluorescence was enhanced in transplastomic plants. On the other hand, pool sizes of the pigments of the xanthophyll cycle and the amounts of PsbS protein were the same in all plants. All these results supported the hypothesis strongly.

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In C3 photosynthesis plants, [CO₂] in the chloroplasts (Cc) is often lower than half the ambient [CO₂]. Such reduction of Cc is attributed to CO₂ gas diffusion conductance from stomatal cavities to the chloroplasts (gm). Some recent studies have suggested that plasma-membrane-type aquaporin contents have positive relationships with gm using transgenic plants. Drought stress generally decreases gm. However, it is uncertain that aquaporin contents explain the change in gm. Tobacco (Nicotiana tabacum) plants were intermittently irrigated (stressed) while some were constantly irrigated (control). gm in stressed plants was about one-third that in control. gm had a positive relationship against area-based leaf nitrogen content in both treatments. A significant level of PIP1-type aquaporin was not found, and PIP2-type aquaporin content was slightly lager in stressed plants than in control. These results suggest that the reduction of gm in response to drought is independent of leaf aquaporin contents.

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Cyanobacteria modify their photosynthetic apparatus to mitigate their oxidative stress under high-light condition. Sll1961, a putative transcription factor involved in the response to high-light, was previously identified. The sll1961 mutant could not regulate photosystem stoichiometry under high-light condition. Although Sll1961 have a certain homology with a transcriptional regulator, the actual role of Sll1961 in the process of high-light acclimation is totally unknown. We picked up some candidates for its targets through the microarray analysis of the sll1961 mutant. The level of transcripts of slr0364, slr0366, and slr2057 were reduced in the sll1961 mutant. The differences in photosystem stoichiometry of the mutants of those genes from that of the wild type were less than that of the sll1961 mutant but some of the differences were significant. It implies that Sll1961 is involved in the modulation of photosystem stoichiometry through the transcriptional regulation of several genes during the acclimation to high-light.

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We have previously reported that the large subunit of Rubisco (LSU) is degraded by ROS in cucumber leaves under chilling-light conditions. In present study, we investigated what are causal factors of LSU degradation by ROS. When leaves of eight species having different chilling-sensitivity were chilled under illumination, LSU was degraded only in cucumber and bean. In cucumber and bean, LSU degradation followed PsaB degradation. Furthermore, the rapid increase of thermal dissipation, the decrease of activities of antioxidant enzymes (AOE) and the increase of inactivated Rubisco without binding of anything (E-form Rubisco) were observed in cucumber and bean. On the other hand, the slow increase of thermal dissipation, the maintenance of AOE activities and the decrease of E-form Rubisco were observed in wheat and arabidopsis. These results suggest that the combination of inactivation of AOE, PsaB degradation and the increase of E-form Rubisco can cause LSU degradation under chilling-light conditions.

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Chlamydomonas raudensis (UWO241) is a psychrophilic green alga found in the permanently ice-covered Lake Bonny, Antarctica. Because of the thick ice cap, which only blue-green light can pass through, the light environment in the lake is not disturbed throughout a year. It has been reported that C. raudensis is deficient in state transition, one of the plant photoacclimation mechanisms, due probably to the stable light environment. In this study, we investigated the differences in photoacclimation mechanisms between C. raudensis and model organism C. reinhardtii. We carried out the biochemical and spectroscopical analyses. The obtained results indicate that the psychrophile has a unique photoacclimation mode other than state transition.

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Any visible injury can not be observed when the whole rice seedlings (Akitakomachi) are exposed to chilling conditions around 10°C, except the leaf-rolling, likely a result of water deficit caused by root inactivation. We, however, observed the severe injury that often led to leaf death after such treatments, only when the roots were not exposed to low temperature. We compared chlorophyll fluorescence parameters, in order to help define the mechanism of this chilling injury. The rice seedlings of the 3rd leaf stage were treated in low air and high root temperature (10°C/25°C) with the 12h-light (650 µmol·m^-2·s^-1) and 12h-dark period. A big change occurred between the first and second light period. This led to a big and rapid decrease in the photosynthetic capacity. The mechanism of this chilling injury is discussed in terms of the function of photosynthesis, water supply from roots and visible injury.

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Tyrosine Z(Y_Z) radical has been trapped by illumination at -20 C in Ca-depleted spinach PS II. ELDOR (Electron Electron Double Resonance) was observed at 4.2 K. The observed time profile showed 20 MHz oscillation that corresponds to the distance of 14 A between Mn₄ spin center and Y_Z radical by a point dipole approximation. According to X-ray analysis of cyanobacterial crystal the distances between manganese are about 3 A and the size of cluster is too large for a point dipole approximation.
We have fitted the experimental profile by spin projection and the positions of X-ray analysis for the assumed valence states of each manganese. In the S2-state EXAFS study suggests Mn(III)Mn(IV)₃. The best fitted configuration by was found to show that the manganese 1 position takes valence 3, which is closest to Y_Z. The method was applied to ELDOR for Y_D-Mn-cluster in the S2 and S0-state.

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PsbM is one of the low-molecular mass subunits of PSII with an approximate molecular mass of 4 kDa and a single trans-membrane helix, and is conserved from cyanobacteria to higher plants. PsbM is located in the central part of PSII dimer in the crystal structure, thus may function in joining the two PSII monomers to form the dimer. In order to study the role of PsbM, we purified PSII from a PsbM-deletion mutant of Thermosynechococcus vulcanus, and crystallized it. We collected the diffraction data to a 4.2 Å resolution, and solved the structure of the mutant PSII. Difference Fourier map between the wild type and mutant PSII indicated that the electron density corresponding to PsbM was lacking in the mutant. The ratio of monomer to dimer was increased in the mutant than that in the wile type, indicating that PsbM is indeed required for the stabilization of PSII dimer.

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Although the central part of photosystem II (PSII) is highly conserved from prokaryotic cyanobacteria to eukaryotes, there are some differences in the extrinsic proteins involved in oxygen evolution among different organisms. The crystal structure of PSII from cyanobacteria has been reported; however, no reports have been published on the structure of any eukaryotic PSII. Red alga is one of the eukaryotic algae closely related to cyanobacteria, but its PSII differs from that of cyanobacteria in that the former contains a 20 kDa protein, the fourth extrinsic protein. In order to elucidate the structure of red algal PSII, we initiated the crystallization trials with PSII from a red alga Cyanidium caldarium. In order to do so, we established a procedure for the large-scale purification of PSII from the red alga, which yielded PSII dimer with a high oxygen-evolving activity. We will report the results of our purification procedures and crystallization trials.

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Lipids are one of the important constituents of membrane protein complexes, and 14 lipid molecules have been identified in the crystal structure of photosystem II (PSII) from a thermophilic cyanobacterium. In order to study the roles of lipids in PSII, we treated PSII dimer complexes from cyanobacteria and higher plants with phospholipase A₂ (PLA₂) and lipase. PLA₂- and lipase-treatments of PSII from a thermophilic cyanobacterium Thermosynechococcus vulcanus decreased the oxygen-evolving activity by 40% and 16%, respectively. The same treatments of spinach PSII, however, decreased the activities by more than 80% in both cases. Analysis of lipid contents indicated that PLA₂- and lipase-treatments decreased the contents of PG and MGDG to a significantly higher degree in spinach PSII than in T. vulcanus PSII. Thus, the association of lipids in spinach PSII is significantly weaker than that in the thermophilic cyanobacterium, which leads to a significantly increased instability of the spinach PSII.

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PsbZ is a membrane protein of ~6.5 kDa found in Photosystem II (PSII) complexes from cyanobacteria to higher plants. The two helices of PsbZ are located on the perimeter of the dimer near CP43 and PsbK in the cyanobacterial PSII structure. In tobacco and Chlamydomonas reinhardtii, PsbZ is necessary for the stability of the PSII-LHCII super complex and mediates nonphotochemical quenching of chlorophyll fluorescence. However, in cyanobacteria the function is still not clear. In this work, we deleted psbZ gene from Thermosynechococcus elongatus BP-1 and analyzed the function of PsbZ. No difference was observed in the growth under the normal condition and the oxygen evoluving activities in cells, thylakoids, the purified PSII complexes between the wild type and the mutant. Electrophoretic profile of the mutant revealed that PsbZ and PsbK were lost in the purified PSII complexes. It was concluded that PsbZ is important for the stable binding of PsbK.

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PsbP is an extrinsic protein that is associated with photosystem II (PSII) core at the luminal side in higher plants and green algae. In our laboratory, tobacco plant in which PsbP was downregulated by RNAi was produced (ΔPsbP). In ΔPsbP tobacco, Fv/Fm value was severely decreased, whereas most of PSII subunits were accumulated. In addition, the amount of photosystem I was markedly decreased, while the amount of the subunits of NDH and Cyt b₆/f complex were increased. These observations were not found in Chlamydomonas mutant lacking PsbP and suggest that knockdown of PsbP could cause the large effect on the property of the photosynthetic electron transfer in higher plants. In this report, further analysis on ΔPsbP plant using the chlorophyll fluorescence and thermoluminescence will be described. Effects of the PsbP knockdown on the gene expression and on the protein-complex formation in the thylakoid membranes will be also reported.

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PsbP, an extrinsic subunit of photosystem II (PSII), has been reported to be present only in plants and green algae. However, recent genomic and proteomic studies have demonstrated the presence of PsbP homolog in cyanobacteria (CyanoP), whereas CyanoP was reported to be rarely associated with PSII. This suggests that the function of CyanoP and PsbP are quite different, and PsbP should have developed as an important factor of PSII regulation during evolution of photosynthetic oxygenic organisms. Consistent with this suggestion, it has been reported that higher plants still have PsbP-like (PPL) proteins, which show higher homology with CyanoP than PsbP. In Arabidopsis thaliana, there are two PPL proteins and we named these proteins as PPL1 and PPL2. Characterization of ppl1 and ppl2, mutants lacking PPL1 and PPL2 proteins, respectively, is on-going to elucidate their functions.

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Diatoms are unicellular algae found throughout the world^,s ocean and freshwater, and one of the most important algae in aquatic ecosystems and the global carbon cycles. Photosynthesis by marine diatoms is predicted to be comparable to that of all terrestrial rain forests. In spite of their significance, little is known about Photosystem 2 (PS2) in diatoms. Isolation of diatom PS2 have been blocked at the level of obtaining thylakoid membranes that are capable of oxygen evolution, because it is difficult to break silica-based rigid cell-wall. In this study, we found that diatom cells were readily disrupted by freeze-thaw, and succeeded in preparation of thylakoid membranes retaining high oxygen-evolving activity. Furthermore, diatom PSII particles were prepared by centrifugation of Triton X-100 extracts of the thylakoid membranes. The diatom PSII particles contained five extrinsic proteins, PsbO, PsbQ^,, PsbV, hypothetical protein and PsbU. We will report about various properties of the diatom PS2.

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Photosystem II (PSII) contains different extrinsic proteins among different organisms. Recently, Enami et al. succeeded in the preparation of PSII particles having high oxygen-evolving activity from a marine diatom Chaetoceros gracilis and found that the diatom PSII particles contained five extrinsic proteins, PsbO, PsbQ', PsbV, hypothetical protein and PsbU. In this study, we cloned and sequenced the genes encoding these five extrinsic proteins. Full-lengths of psbO, psbQ', hypothetical protein, and psbU cDNAs were isolated by degenerate RT-PCR and RLM-RACE. On the other hand, DNA fragment containing psbV gene was isolated using a combination of degenerate RT-PCR and inverse PCR. The results show that the former genes are encoded in the nuclear genome. After sequencing the genes, we found that all of the five extrinsic proteins have typical features for thylakoid transfer peptides. This suggests that the hypothetical protein is also one of the luminal extrinsic proteins functioning in oxygen evolution.

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Acaryochloris marina is a unique prokaryote in which Chl d is a major pigment. The Chl a content of A. marina is variable, but it is always present. The primary electron donor of A. marina PS I was assigned to Chl d by Hu et al. In contrast, the primary electron donor of PS II is still under debate. Delayed fluorescence of PS II in the ns range was found only in Chl a region in intact cells. To identify the primary electron donor, we isolated the PS II particle from A. marina by a combination of two kinds of chromatography and sucrose density gradient centrifugation. The purified PS II preparation contained Chl d, Chl a, Pheo a, and α-carotene. Furthermore, PS II complexes retained electron transfer activity. Based on these observations, the assignment of the pigment and its role of PS II RC will be discussed.

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Phytochrome signaling involves a complex network of processes with branches taking place in distinct cellular and subcellular compartments. For long time, phytochtomes were considered cytoplasmic proteins. However, it is now known that photoconversion of phyA and phyB to their active forms triggers nuclear translocation. For phyB, nuclear translocation is necessary and sufficient to control seedling deetiolation. However, for phyA, the subcellular sites for the control of deetiolation responses remained to be investigated. We have produced two new types of GFP-fusion transgenic plants expressing phyA-GFP with a nuclear localization signal (APAGL) or with a nuclear export signal (APAGE) under the PHYA promoter and in the phyA mutant background. The predicted subcellular distribution and protein accumulation levels comparable to the Wt-phyA were corroborated. Selected lines are being used to dissect effects of altered subcellular compartmentalization on phyA signaling. Results on the physiological and cell-biological characterization of these lines will be discussed.

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Light is the most important environmental factor that controls leaf shape and structure. When plants are grown under a high intensity of light, thick leaves, referred to as sun leaves, are developed. In sun leaves, palisade cells are greatly elongated in the adaxial/abaxial direction.
In Arabidopsis thaliana, three classes of photoreceptors, phytochrome, cryptochrome, and phototropin, are known. However, it remains unknown whether these photoreceptors are involved in the sun-leaf development. To investigate the effects of these photoreceptors, we examined sun-leaf formation in photoreceptor deficient mutants, phyB, cry1cry2, and phot1phot2. In the phot1phot2 mutant, the polar cell elongation was dramatically reduced. By contrast, elongation of palisade cells was observed normally in the cry1cry2 and phyB mutants. We further confirmed that blue but not red light effectively induced the elongation. These results suggest that phototropin regulates the polar cell elongation in palisade tissue in response to high intensity of light.

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AppA is a novel flavin-based blue-light photoreceptor controlling photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides. AppA is based on two domains: one is N-terminal flavin-binding domain named BLUF, and the other is C-terminal region where a transcriptional repressor PpsR could associate. Previous spectroscopic analyses have indicated that W104 is necessary for light-induced beta-sheet structural changes in AppA. To get more insight into the role of the amino acid, we biochemically characterized W104A mutant protein, and physiologically analyzed the mutant strain that harbors the W104A mutation on its genome. Obtained results indicated that W104A mutation causes loss of sensitivity of AppA to blue-light for controlling its in vivo and in vitro activity. Together with recent-solved structures of several BLUF proteins, mechanism of photocycle reaction of AppA will be discussed.

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We discovered two novel flavoproteins in the coenocytic alga, Vaucheria frigida (Xanthophyceae, Heterokontophyta, Stramenopile). They have each one bZIP- and one LOV domain. They are probably photoreceptors for the blue light-induced branching, a photo(cyto)morphogenesis, as the response is completely inhibited when their mRNAs are knocked out by RNA interference (PCP meeting at Niigata 2005). One of these bZIP-LOV proteins is suggested to be involved in the sex-organ development. We found several orthologs in mRNA of Fucus distichus (Phaeophyta) and in the genome of the marine diatom, Thalassiosira pseudonana (Ambrust et al. 2004). We thus name the bZIP-LOV photoreceptor AUREOCHROME. AUREOCHROME seems to be the common and intrinsic blue light photoreceptor of Stramenopile. Instead, no PHOTOTROPIN has been found in Stramenopiles, We are intending to isolate AUREOCHROMEs from other members of the Stramenopiles and the related algal groups whose chloroplasts are red algal symbiotic origin, such as Haptophytes and Cryptophytes.

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LOV domains form subfamily of a PAS domain in the Light-Oxygen-Voltage sensing proteins. One of the major LOV proteins, phototropin (phot) has two LOV domains named LOV1 and LOV2. Despite the almost identical crystal structures, LOV1 and LOV2 have distinct functions. LOV2 plays significant roles in the photoregulation, however, LOV1 doesn't. One of the important roles of LOV1 is thought to be a dimerization site. Oligomeric structures, therefore, of LOV domains in Arabidopsis phot and their light-induced changes were studied by using a chemical cross-linking technique. Arabidopsis has two phots, phot1 and phot2, a broad-range and a strong light sensor, respectively. LOV1 of both phot1 and phot2 forms a dimer. However, oligomeric structures of LOV2 differ between phot1 and phot2. Phot2-LOV2 exists as a monomer and showed light-induced changes in the molecular size. Phot1-LOV2, while, has multiple oligomeric forms, such as, monomer, dimer and tetramer that showed no light-induced alteration.

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Phytochromes are well-known photoreceptors mediating photomorphogenetic responses in plants. Plant phytochromes and bacteriophytochromes covalently bind a linear tetrapyrrole as a chromophore, and show red/far-red reversible photoconversion. PixJ1 is a phytochrome-like photoreceptor necessary for positive phototaxis in a unicellular cyanobacterium Synechocystis sp. PCC 6803. We have already reported that PixJ1 protein covalently bound a linear tetrapyrrole phycocyanobilin (PCB) and showed a novel blue (λ_Amax= 435 nm)/green (λ_Amax= 535 nm) reversible photoconversion. In this study, the interconvertible photoreactions of PixJ1 were investigated by light-induced UV-Vis difference spectroscopy at low temperature. The photoreactions of a PCB-bound cyanobacterial phytochrome, Cph1, were also studied. For the photoconversion of Cph1 Pr to Pfr, an intermediate lumi-R was observed at 133 K, while the photoconversion of PixJ1 from Pb to Pg showed a lumi-R-like intermediate below 77 K. Above 250 K, Pg form was obtained. We will discuss the possible photoreactions of PixJ1.

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It has been reported that there is the crosstalk between phytochrome B (phyB) and phytochrome C (phyC) in Arabidopsis. However, we found that the crosstalk between phyB and phyC in rice (Oryza sativa ) is quite different from that in Arabidopsis. In rice phyB mutants, phyC protein levels were greatly reduced in the seedlings grown in the dark, but not in the light. Intriguingly, phyC protein exhibited higher light-labile in WT than in phyB mutants. All these results indicate the involvement of phyB in regulating phyC levels in rice. On the other hand, phyC-mediated responses were not distinguishable in rice phyB-deficiency mutants, suggesting the involvement of phyB in phyC-regulated photomorphorgenesis. To access the molecular mechanisms of crosstalk between phyB and phyC in rice, several transgenic lines were produced and analyzed. Implications of interactions between phyB and phyC on the versatility of plant light sensing and signaling mechanisms will be discussed.

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Cyanobacteria harbor many putative GAF-containing photoreceptors that may bind a linear tetrapyrrole as a chromophore (cyanobacteriochrome). Previously, we reported novel properties of a tetrapyrrole-binding GAF domain of TePixJ of Thermosynechococcus elongatus BP-1 that was expressed in Synechocystis. Purified protein showed reversible photoconversion between the 433nm and 531nm-absorbing forms. Molecular mass of the chromophore was identical to phycocyanobilin (PCB) but spectral properties were slightly different. Here, we report detailed denaturation analysis of TePixJ and Synechocystis phytochrome Cph1. After denaturation with acidic urea, both chromophores showed photoconversion without protein, however, the spectra of TePixJ chromophore were clearly different from those of Cph1 PCB. Global analysis of time-resolved spectral changes revealed several distinct intermediates in both pathways of photoconversion in TePixJ. These results suggested that the tetrapyrrole of TePixJ isomerizes between the ring C and D upon photoconversion like phytochrome but configuration in the other positions may be stereochemically distinct from each other.

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Phytochromes are red and far-red light receptors in plants. The GAF domain of phytochromes is essential for chromophore holding. A distinct but related subgroup of the GAF domain is abundantly present only in cyanobacteria (denoted cyanobacteriochrome). Recently, our group reported that one of cyanobacteriochrome GAF domains (TePixJ) bound phycocyanobilin (PCB) and showed blue-green photoreversibility. In this study, we focused on the PixJ homolog (AnPixJ) in Anabaena sp. PCC 7120 whose GAF domain is distinct from that of TePixJ.
We expressed the second GAF domain of AnPixJ as a His-tagged protein (H₆AnPixJ-GAF2) in PCB-producing Escherichia coli and purified it. H₆AnPixJ-GAF2 showed photoreversibility between green-absorbing form (543 nm) and red-absorbing form (648 nm). Acidic denaturation analysis suggested that the chromophore photoreaction of H₆AnPixJ-GAF2 is essentially the same as that of orthodox phytochrome. From these, possible light-sensing mechanism of AnPixJ will be discussed. Finally, we will discuss about the perspective of diverse cyanobacteriochromes.

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In plants, phytochromes play roles in various photoresponses as red-farred light photoreceptor. Recently, novel photoreceptors which have a phytochrome-like GAF domain were found in cyanobacteria and named "Cyanobacteriochrome". CcaS is a putative cyanobacteriochrome which controls light-dependent expression of phycobilisome protein (CpcG2). CcaS is a His kinase protein which contains the putative cyanobacteriochrome GAF domain and flavin-binding LOV-like domain.
In this study, we prepared His-tagged CcaS-GAF from Synechocystis 6803 and phycocyanobilin (PCB)-producing Escherichia coli. Both proteins showed reversible photoconversion between green-absorbing and red-absorbing forms with difference spectra very similar to each other. This suggests that CcaS-GAF binds PCB in Synechocystis 6803. Then, His-tagged CcaS without transmembrane region was purified from PCB-producing E. coli. The absorption spectra showed that CcaS bound both PCB and flavin, suggestive of a composite photoreceptor. Autophosphorylation activity was detected. Effects of light illumination and phosphotransfer from CcaS to a cognate response-regulator will be presented.

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We have recently shown that the nitrogen of atmospheric nitrogen dioxide (NO₂) taken up byplants is incorporated into organic nitro-compounds. We investigated nitration of protein tyrosine from Arabidopsis thaliana fumigated with NO₂. The extracted proteins were separated by the two-dimensional PAGE, and analyzed by Western blot using anti-nitrotyrosine (NT) antibody. Seven protein spots were detected, and all of them were identified, using the MALDY-TOF MS and peptide mass finger printing (PMF) using MS-Fit program, to be subunits of oxygen-evolving complex (OEC) of photosystem II; PsbP and PsbO. Some protein spots different from PsbP and PsbO reacted with the antibody when the proteins extracted from the isolated chloroplast were separated and identified nitrated as described above. However, the signal intensity of them was weaker than that of PsbP and PsbO. The present results strongly suggest that NO₂ fumigation-mediated nitration of tyrosine residues occurs in a protein-specific manner.

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8-Oxo-guanine(8-oxo-G), an oxidized form of guanine by attack of active oxygen species are one of the major sources for spontaneous mutations in nucleic acids. To prevent the mutagenic consequences of 8-oxo-G, organisms have developed cellular defense mechanisms by the sanitation of nucleotide pools and base excision repair pathway. We have reported previously that AtNUDX1 protein hydrolyzes 8-oxo-(d)GTP and completely suppresses increased frequency of spontaneous mutations in the E. coli mutT ^- strain. In this study, we studied the physiological function of AtNUDX1. AtNUDX1 suppressed transcriptional errors caused by 8-oxo-GTP in E. coli mutT ^- strain. The level of 8-oxo-G in AtNUDX1-knockout plants was significantly increased compared with that in the wild-type plants under normal and stressful conditions, indicating that AtNUDX1 play an important role in the sanitization of nucleotide pools. We are progressing toward to analyze the localizations of AtNUDX1 and the enzymes involved in the base excision repair pathway.

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Galactinol synthase (GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides (RFOs). Recently, we have reported that the transcription of GolS1 and GolS2 is induced by treatment with H₂O₂, as is the transcription of HsfA2 (Plant J., 2006). Here we investigated the physiological roles of galactinol and/or RFOs as novel intracellular antioxidants. In leaves of the HsfA2-overexpressing transgenic Arabidopsis plants, the transcript levels of GolS1, 2 and 4 and raffinose synthase (RS) 2 were highly induced and thus levels of galactinol and raffinose increased. In the wild-type plants, the transcript levels of GolS1-4 and 8 and RS2 were induced and the levels of galactinol and raffinose were increased in response to oxidative stress. Galactinol and RFOs effectively protected salicylate from attack by hydroxyl radicals in vitro. Furthermore, GolS-overexpressing plants had increased levels of galactinol and raffinose in the leaves and showed increased tolerance to oxidative stress.

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A decrease in catalase activity is a key factor increasing salt stress-induced oxidative injury in rice seelings. Catalase activity is inhibited by salicylic acid (SA). NaCl treatment increased SA content in rice seedlings. On the contrary, in Echinochloa crus-galli seedlings which are tolerant to salinity, SA content did not increased. Rice and E. crus-galli seedlings were treated with NaCl after SA application. No significant difference in growth and Φ_PSII was observed between SA treated and non-treated rice seedlings under salt stress. On the contrary, SA pre-treatment enhanced the reduction of growth and Φ_PSII in E. crus-galli seedlings. Moreover, in E. crus-galli seedlings, SA + NaCl treatment caused a decrease in catalase activity and an increase in superoxide disumutase. It was suggested that SA accumulation is involved in the change in antioxidative enzyme activity, resulting in the induction of oxidative injury.

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Lipid peroxide-derived carbonyls (aldehydes and ketones) are reactive species. Like reactive oxygen species, they work as signal molecules at low concentrations and damage various cellular components at high concentrations. In order to elucidate the regulation and physiological effects of carbonyls in leaves, we here established a comprehensive analysis of carbonyls. Leaves of Arabidopsis thaliana were extracted with acetonitrile. The extract was derivatized with 2,4-dinitrophenylhydrazine, and separated on a reversed-phase HPLC column. More than 50 species of carbonyls were detected, among which aldehydes and ketones of carbon chain length 3-10 with or without C=C double bond, were identified by comparison of their retention time with standard compounds. Three ecotypes Col-0, Nos, and Ler, showed similar profiles of carbonyls except that Col-0 showed lower 3Z-hexenal and 2E-hexenal levels, corresponding well with the lack of hydroperoxide lyase (HPL) activity in Col-0. This result imply the HPL-independent production of aldehydes in leaves.

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Under environmental stresses, excessive light in chloroplasts result in oxidization of cell components by reactive oxygen species. Aldehydes and ketones derived from lipid peroxides inhibit the Calvin cycle in the chloroplasts. On the other hand, the over-expression of aldehyde scavenging enzymes in plants enhance the tolerance to environmental stresses. Thus, aldehydes and ketones are involved in environmental stresses. To examine what kinds of aldehydes and ketones are produced in chloroplasts, extract from spinach chloroplasts was derivatized with 2,4-dinitrophenylhydrazine and analyzed with HPLC. According to comparison with standard compounds, 8 kinds of saturated aldehydes such as butyraldehyde, n-hexanal and n-nonanal, 3 kinds of α,β-unsaturated aldehydes such as 2E-hexenal and 2E-nonenal and at least 9 kinds of unidentified aldehydes and ketones were detected. The most abundant compounds were 12-oxophytodienoic acid and acetone (35 μmol/mg Chl, 15 μmol/mg Chl, respectivly).

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It is well known that ozone induces the loss of chlorophylls (Chls) in plants. However, the mechanism for ozone-induced Chl degradation is still unknown. We attempted the search for ozone-dependent Chl degradation paths in both in vivo in leaves and in vitro , using HPLC and TOF-MS.

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Functional genes related to the Cd tolerance/accumulation were identified in rice using a recently developed genome-wide gene screening procedure, Full-length cDNA Over-eXpressor gene (FOX) hunting system. For screening of Cd tolerance related genes, the T0 FOX-rice calli cultured on a Cd-containing medium were digitally analyzed for their growth rates. For screening of Cd accumulation related genes, Cd contents in the T2 FOX-rice seedlings were quantified after a few days culture with Cd. In addition to Cd measurement, Cu, Fe, Mn and Zn contents were also measured in the same seedlings. So far, we have analyzed more than 10,000 FOX-rice calli and 500 FOX-rice seedlings, and obtained about 100 Cd tolerance-enhanced calli and 130 Cd/the other metal content-changed seedlings. Those contained some known genes related to Cd-stress responsive one, such as phytochelatin, kinases, transporters and anti-oxidant function, and many possibly functional unknown genes.

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We have been working on phytoremediation, which is a method to clean soil and water of pollutants such as heavy metals using plants. Many researches have been carried out to enhance the ability of tolerance and accumulation of heavy metals by a gene manipulation technique, however, few have succeeded. We employed T-DNA tagged mutants to understand the mechanism of the heavy metal tolerance and accumulation ability of plants. In this study, to identify heavy metals tolerance determinants, we screened approximately 40 hyper sensitive mutants of cadmium (Cd) from a T-DNA tagged population which involved more than 13000 lines in Arabidopsis Col-0 gl-1 background. These mutants showed higher sensitivity to Cd than wild type under the condition of 50 μM Cd. TAIL-PCR revealed the location of the introduced T-DNA in mutant genome.

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