Water channel proteins, termed aquaporin, belong membrane proteins and involved water movement across membrane. We have previously reported that plant's stem conducts light toward root. In addition, we confirmed that several aquaporin gene expression levels in Arabidopsis root were altered by far-red light irradiation. These observations suggest axial light conduction might modulate water transport activity through regulation of aquaporin level in root. To elucidate whether water transport activity was controlled by light, we monitored water dynamics in Arabidopsis root under light-dark cycles using ¹H-NMR imaging. Under light condition, water content in root was decreased compare to that in dark. Moreover, autonomous rhythm of water dynamics were observed under continuous light and dark conditions, suggesting both light signal and circadian clock might modulate water transport in root. Here, we will present about correlation between aquaporin gene expression level and water transport activity in root under light-dark cycles.

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In higher plants, vacuoles increase their volume according to cell enlargement and become large vacuoles that occupy most of the cell volume. The large vacuole plays an essential role in cell elongation. However, little is known about the dynamics of vacuolar structures in the biogenesis of large vacuole. To observe the vacuolar dynamics in living cells, we have already established a transgenic BY-2 cell-line expressing a GFP-AtVam3p fusion protein (BY-GV), and have developed 3-D reconstruction software, named REANT.
In this study, we tried to clarify the morphological changes of vacuoles during the regeneration of large vacuoles in the BY-2 miniprotoplasts. The 3-D reconstruction and morphometrical analyses were performed using the BY-GV and REANT system. We found that the complicated network of vacuoles was developed during the regeneration of large vacuoles. Also, an increase of vacuolar surface area was occured before the increase of vacuolar volume.

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Plant vacuole is a multifunctional organelle which storage solutes and proteins, digests them, control cytosolic pH and ions, fill large volume of cell space and others. Vacuole also contributes to dynamic events, such as Ca²⁺ signaling and stomatal movement. These functions were supported by a great number of tonoplast proteins and their interactions. In this study, we searched protein complex of vacuolar membrane comprehensively.
We prepared vacuolar membrane from suspension-cultured cell of Arabidopsis thaliana. Solubilized membranes were subjected to 2D- BN- SDS- PAGE to separate complexes of membrane protein. We detected approximately 20 candidates for hetero-oligomeric protein complex and about 20 candidates for homo-oligomeric protein. Using antibody library for vacuolar-membrane transporters of about 40, we analyzed apparent molecular masses of protein (complex) that contains target antigen under BN-PAGE. We also tried immunoprecipitation of the solubilized vacuolar membrane using the antibody library to pull down protein complexes of transporters.

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The genome secquence of rice, which is one of the main crops of the world and important for survival of human kind in the future, was completed in 2002, though the database is in a draft stage. In the genomic sequence of rice, we have identified 36 different MIP-encoding genes and 2 pseudogenes. We examined and described all the MIPs in the genome sequence of rice. Twelve different genes belong to PIP subfamily and the number is as similar as Arabidopsis and Zea. As low similarities were found between 3 PIP2s and the other PIP2s, the 3 PIP2s might be categorized into a new PIP subgroup after functional analysis. Rice-Corn ortholog analysis revealed two new MIP-encoding genes in the genomic sequence of corn. Comparison of them with Arabidopsis thaliana and Zea mays resulted useful information on structure and function of several subfamiles of MIPs.

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Internodal cells of a brackish water charophyte Lamprothamnium succinctum regulate their turgor pressure upon hypotonic treatments within 1 h. During the regulation, vacuolar KCl is released to regulate the turgor pressure. Cytoplasmic concentrations of K⁺, Na⁺ and Cl^- after hypotonic turgor regulation were analyzed to know effects of efflux of vacuolar KCl through the cytoplasm. They were measured with an ion chromatography using cells whose vacuoles were replaced with artificial cell saps free from those inorganic ions. Immediately after the end of hypotonic turgor regulation, cytoplasmic concentration of K⁺ decreased and then gradually increased. Cytoplasmic concentration of Cl^- transiently decreased. On the contrary, cytoplasmic concentration Na⁺ increased transiently. The same tendencies were observed in response to a stronger hypotonic stress. Cytoplasmic concentrations of K⁺, Na⁺ and Cl^- were regulated against the transient changes induced by hypotonic treatments.

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To elucidate mechanism(s) of aluminum (Al)-induced growth inhibition, the effects of Al on sucrose uptake and water uptake were investigated in tobacco cells. Cells were treated without (control) or with Al in a simple calcium solution containing 3% sucrose (pH 4.5-5.0). Control cells exhibited sucrose and water uptake continuously, which was inhibited by Al immediately. PCMBS and CCCP also inhibited sucrose uptake in control cells, indicating that Al inhibits H⁺-dependent sucrose transporter. The inhibition of sucrose uptake by Al was reversible in Al-free nutrient medium, but became gradually irreversible after 9-h exposure, together with increases in respiration inhibition, ATP depletion, reactive oxygen species production and loss of growth capability. These results suggest that Al inhibits sucrose uptake, which is immediately linked to water uptake inhibition and cell elongation inhibition, then, after prolonged sucrose starvation, decreases in mitochondrial functions lead to irreversible cell death process.

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In order to elucidate a possible role of cytosolic free calcium ion ([Ca²⁺]_cyt) in signal transduction pathway in response to aluminum (Al) accumulated on cell surface, relationship between [Ca²⁺]_cyt status and Al toxicity symptoms was investigated in tobacco cells expressing recombinant aequorin. Compared to control cells, Al-treated cells exhibited two types of transient increase in [Ca²⁺]_cyt :the first one immediately after a start of Al exposure which depended on both Al ion and a decrease in pH by Al exposure, and the second one from 3-h Al exposure which was triggered by mechanical stress (pipetting) and was closely related to a loss of growth capability. These results suggest that the accumulation of Al on cell surface sensitize cells to mechanical stress which causes an increase in [Ca²⁺]_cyt.

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To elucidate physiological roles of the TPC1 family, putative voltage-gated Ca²⁺ channels, we generated and characterized transgenic rice plants and cultured cells overexpressing OsTPC1 mRNA, as well as a retrotransposon-insertional knockout mutant. The overexpressing plants showed reduced growth and abnormal greening of roots. Growth of Ostpc1 seedlings was comparable to the control on agar plates, while significantly reduced in adult plants. These results suggest that OsTPC1 functions as a Ca²⁺-permeable channel involved in the regulation of growth and development. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of [Ca²⁺]_cyt rise in response to a fungal elicitor, while did not affect hypoosmotic shock-induced [Ca²⁺]_cyt increase. Cosuppression of NtTPC1s also caused suppression of elicitor-induced PCD and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca²⁺ mobilization induced by pathogenic elicitors, and have crucial roles in various stress-induced signal transduction pathways.

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Potassium ion (K⁺) is one of a fundamental mineral nutrients for plant growth and development. It is also involved in cell elongation and cell division of tobacco BY-2 cells. Therefore, in this study, we compared the role of K⁺ and K⁺ channels between cell elongation and cell division. At first, when we examined K⁺ channel gene expression between cell elongation and cell division, gene expression of an inward K⁺ channel gene, NKT1, was induced both in cell elongation and cell division while an outward K⁺ channel gene, TORK1, was induced in the condition of cell division. Treatment of tetraetylammonium, which is known to inhibit outward K⁺ channels, reduced cell division but accelerated cell elongation. Furthermore, cell osmotic pressures measured by plasmolysis method was lower during cell division than during cell elongation. These results suggest that reduction of cell osmotic pressure by activation of outward K⁺ channel activity promotes cell division.

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The ionic transport systems function to maintain the ionic homeostasis in the cells under the salt stress. Sodium ions were removed from the cytoplasm by Na⁺/H⁺ antiporters localized on tonoplasts and plasma membranes. We hardly know about intercellular behavior of chloride ions, a counter ion for Na⁺, under the high salt condition. It were reported that AtCLC-a and -c play a role in controlling the intracellular nitrate status in Arabidopsis. However, other functions were not known. To assess the function of rice chloride channel genes OsCLC-1 and -2, we isolated loss of function mutants from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17. We found mutants of the OsCLC-1 or -2 were defects in the growth and development at the vegetative stage. So, OsCLC-1 and -2 are important for growth of rice.

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The ion transporters in the cytoplasmic and organelle membranes play many important roles of the ionic homeostasis in the cytoplasm. We have restricted knowledge about the plant ionic transporters. So, we tried to analyze the function of ionic transporters using loss-of-function mutation of rice, that is mutant panel. We isolated the mutants inserted a retrotransposon, Tos17, in chloride channel genes(OsCLC-1 and -2) and the vacuolar Na⁺/H⁺ antiporter gene(OsNHX1) from the panel of O. sativa, cv Nipponbare. The mutant of OsCLC-2 was sensitive to NaCl treatment in the growth. All mutants isolated need the high concentration of some nutrients in various patterns, especially in very young stage.

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Plant growth is tightly linked to development of vacuole. Because vacuole occupies most (>95%) of cell space. Two H ⁺ pumps, vacuolar H ⁺ -ATPase and vacuolar H ⁺-pyrophosphatase (V-PPase) contribute to acidification of vacuolar space. They support development of vacuole energetically. V-PPase is a plant specific H ⁺ pump of which substrate is inorganic pyrophosphate.
To investigate physiological function of V-PPase, we isolated two mutants of Arabidopsis thaliana of which V-PPase gene (VHP1) was inserted by T-DNA from mutant library. Both mutants grew as well as wild type when they were cultivated in medium containing carbon source. The mutants, however, shows defect in growth in autotrophic condition. Size of mutant plants was about half of that of wild type. Severe growth-defect was observed when light limiting condition. These results indicate that V-PPase is not essential for survival of plant cell but extremely important for natural growth of plant.

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SULTR1;1 and SULTR1;2 encode high-affinity sulfate transporters co-localizing at epidermis and cortex of Arabidopsis roots during sulfur-starvation (-S). The double mutant, sultr1;1 sultr1;2, was impaired of high-affinity sulfate uptake activity, causing severe growth defects.The wild-type plants showed significantly higher level of sulfate uptake as compared with the sum of uptake rates independently measured for sultr1;1 and sultr1;2 single mutants, suggesting that co-expression of SULTR1;1 and SULTR1;2 on -S may synergistically facilitate the acquisition of sulfate. Co-expression of SULTR1;1 and SULTR1;2 also resulted in synergistically increasing sulfate transport activities in yeast; however this positive regulatory effect was repressed by the supply of organic sulfur. Physical interaction between SULTR1;1 and SULTR1;2 was demonstrated by co-purification of their complex. SULTR1;1-SULTR1;2 complex was stably formed both under +S and -S conditions and functioned as a regulatory unit of sulfate transport, optimizing sulfate uptake capacity in response to the change in sulfur conditions.

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We demonstrate that SULTR2;1/SULTR3;5 and SULTR4;1/SULTR4;2 are essential components that regulate the root-to-shoot transport of sulfate. SULTR2;1 and SULTR3;5 were both expressed in xylem parenchyma and pericycle cells in roots. In a yeast expressing system, SULTR3;5 accelerated sulfate uptake when co-expressed with SULTR2;1. In planta, the root-to-shoot transport of sulfate was restricted in the knockouts suggesting that SULTR2;1/SULTR3;5 provide maximum capacity of sulfate uptake. SULTR4 facilitates efflux of sulfate from the vacuoles and regulates internal distribution of sulfate. GFP fusion protein of SULTR4 was accumulated in the tonoplast and was localized in the pericycle and xylem parenchyma cells of roots and hypocotyls. Vacuoles isolated from the sultr4;1 sultr4;2 mutant showed excess accumulation of sulfate, which was substantially decreased by overexpression of SULTR4;1-GFP. In seedlings, supplied sulfate was retained in the root of the sultr4;1 sultr4;2 mutant, whereas decreased accumulation of sulfate was found in the root of SULTR4;1-GFP overexpressing plants.

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We have isolated Arabidopsis hma1 mutant by monitoring chlorophyll fluorescence. HMA1 is expected to be involved in the transport of Zn/Co/Pb/Cd. The hma1 showed a growth defect in the presence of 100 μM ZnSO₄ and FeSO₄-depleted medium. Moreover, the growth defect caused by excess Zn was restored by the addition of excess FeSO₄. The photosynthetic parameters of hma1 did not show significant difference from that of WT under normal air condition. In CO₂ free air, however, the hma1 showed slightly delayed NPQ, suggesting that limitation of alternative electron transport might result in the decrease of NPQ due to insufficient pH gradient. Even under O₂ free condition, the NPQ induction of hma1 was slower than that of WT, indicating that water-water cycle is not involved in the defects in hma1. These results suggest that Fe deficiency caused by excess Zn may lead to alteration of alternative electron transport in hma1.

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Recently, many genes involved in photoperception, signal transduction, and pilus biogenesis, which are responsible for cellular motility and phototaxis, have been identified in a unicellular motile cyanobacterium Synechocystis sp. PCC 6803. Meanwhile, a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, whose genome sequence was determined, also shows motility and phototaxis. Here, we report functional analysis of genes involved in motility and phototaxis by using T. vulcanus strain RKN which is closely related to T. elongatus. T. vulcanus forms slightly spreading colonies on agar plates under normal culture conditions. In the course of our study on glycosyltransferase, we found a certain strain of the glycosyltransferase mutants showed enhanced motility with negative phototaxis toward white light. Moreover, addition of glucanase to the agar plate significantly enhanced motility. We disrupted several genes involved in motility and phototaxis on the genetic background of the motile glycosyltransferase mutant. We will report phenotype of those disruptants.

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Many cyanobacteria show phototactic motility by employing pilus structures. Synechocystis sp. PCC 6803 has been a model organism for molecular analysis of genes involved in such photoresponses. Previously, we isolated SyPixJ1 protein that serves as a photoreceptor for positive phototaxis and showed the reversible photoconversion between 435 nm and 535 nm forms (Yoshihara et al. 2004 PCP). However, purification was not sufficient to eliminate any other pigments from the preparations because it is expressed in membranes. Here, we expressed in Synechocystis a chromophore-binding domain of TePixJ from a thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1. The purified TePixJ_GAF with a His-tag showed the same blue-green reversible photoconversion as SyPixJ1. By contrast, TePixJ_GAF did not show spontaneous dark reversion. These results clearly demonstrated that the unique photoreversible cyanobacteriochrome serves as a photoreceptor for phototactic motility widely in cyanobacteria.

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Phytochromes are well-known photoreceptors mediating photomorphogenesis in plants. Recently, distinct homologs of plant phytochromes have been found in several bacteria. These proteins covalently bind a linear tetrapyrrole as chromophore, and show red/fared reversible photoconversion. pixJ1 was found as one of the genes necessary for positive phototaxis in a unicellular cyanobacterium Synechocystis sp. PCC 6803. PixJ1 has a similar amino acid sequence to chromophore-binding regions of phytochromes. His-PixJ1 protein purified from Synechocystis cells covalently bound an unknown linear tetrapyrrole and showed the novel blue (λ_Amax=435 nm)/green (λ_Amax=535 nm) reversible photoconversion. In this study, the assemblies of the chromophore-binding domain of PixJ1 with several linear tetrapyrroles and their absorption spectral properties were studied. Assembly with phycocyanobilin (PCB) well simulated the blue/green photoconversion, although PCB-bound cyanobacterial phytochrome, Cph1, is known to show a red/far-red reversible photoconversion. We will discuss the possible involvements of PCB to the blue/green photoconversion of PixJ1.

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We have examined photoreceptor of phototaxis in thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. Action spectra of the wild type cells indicated the involvement of two or more photoreceptors for the phenomenon(Kondou,et al.2001). At least one of them should be phytochrome-like protein, because of the observation of the red and far-red light reversible manner in the phototaxis.
Thus we examined the phototactic activity in disruptants of phytochrome-like genes. The action spectrum of the phototaxis in the disruptant of tlr1215 gene was measured and was compared with that in wild type. The activity of phototaxis in the disruptant of this gene decreased significantly at the red region (570nm and 640nm), indicated that the product of the tlr1215 gene was a red light receptor for phototaxis. Zinc-stain analysis of the recombinant apoprotein of Tlr1215 with biliverdin indicated that the chromophore binding occurred in vitro. Furthermore, we will discuss the role of other phytochrome-like genes.

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Slr1694 is a BLUF (sensor of blue-light using FAD) protein and a blue-light photoreceptor in the
cyanobacterium Synechocystis sp. PCC6803. Illumination of Slr1694 induced a signaling
light-state characterized with a red-shift in the UV-visible absorption of flavin, and formation of the
bands from flavin and apo-protein in the light-minus-dark Fourier-transform infrared (FTIR)
difference spectrum. The FTIR bands were classifiable into three groups according to their decay
rates (t_1/2 = 1.5, ~2.4 and 4.5 min) and the effects of deuteration. The C4=O stretching bands of a
flavin isoalloxazine ring had the highest decay rate and were most effected by deuteration, which
corresponded to the decay properties of the absorption red-shift. The result indicated that the
hydrogen bonding at C4=O is responsible for the UV-visible red-shift. A photocycle mechanism
involving the proton transfer was proposed.

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AppA is a novel blue-light receptor controlling photosynthesis gene expression in purple bacteria. Light-induced FTIR spectrum of the AppA showed simple features, which were composed of two sets of derivative-shaped sharp bands at 1709(-)/1695(+) and 1632(+)/1619(-) cm^-1. We reconstituted the BLUF domain from free FAD and an apo-protein isotopically labeled with ¹³C, and compared the spectra with those for uniformly ¹⁵N-, ¹³C-labeled or unlabeled sample. It was shown that the light-induced FTIR spectrum of the unlabeled BLUF domain of AppA was predominantly composed of multiple apo-protein bands, while a C(4)=O stretching of an isoalloxazine ring was the only band exclusively assigned to FAD. The results showed that relatively large structural changes occur in the protein backbone of the BLUF domain of AppA upon illumination. These changes will be discussed in relation to the mechanistic role of the BLUF domain in the process of blue-light perception by AppA.

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BLUF is the FAD binding blue-light sensory domain that is widely distributed among the genomes of photosynthetic bacteria, cyanobacteria and Euglena. Tll0078 protein of thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 has the BLUF domain. The absorption spectrum of this protein showed 10 nm red shift upon the light illumination at room temperature. We studied the photocycle of purified Tll0078 protein expressed in E.coli. The fluorescence decay of the Tll0078 protein showed three exponential decay components with a lifetime of 120 ps (95 %, relative amplitude), 710 ps (4 %) and 4.56 ns (1%). The transient absorption change showed the formation of 10 nm red shifted form at 10 ns after the laser excitation at room temperature. These results indicate the extremely fast primary photoconversion process of BLUF domain at room temperature.

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Recently, BLUF proteins were found as flavin-binding blue light sensors in some bacteria and eukaryotes. BLUF domain shows red-shifted absorption spectrum when illuminated by blue light. The conserved Tyr-21 of AppA was a critical amino acid residue for this photocycle in Rhodobacter sphaeroides. Previously, we reported that BLUF protein Tll0078 of a cyanobacterium Thermosynechococcus elongates BP-1 showed such photocycle. Here, we generated the site-directed mutated Tll0078 at the conserved Tyr-8. Tll0078_Y8A protein, where Tyr-8 was replaced with Ala, was overexpressed and purified from Escherichia coli. Purified Tll0078_Y8A bound flavin, but did not show red-shifted absorption spectrum upon illumination. On the other hand, the absorbance around 370 ~ 470 nm bleached when illuminated for longer time and slowly recovered when in the dark. This may suggest light dependent reduction of flavin. We will discuss about mechanism of photocycle and structure of near flavin of BLUF domain.

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Cell death in plants caused by ozone is promoted by ethylene and salicylic acid, while reduced by jasmonate (JA). Using ozone-sensitive jasmonate-semi-insensitive Arabidopsis mutant oji1, it has been suggested that JA suppresses cell death by inhibition of ozone-induced ethylene production. On the other hand, ascorbate (AsA) protects cells from direct oxidation by removing active oxygen species. In wild type Ws-2 and oji1, ascorbate contents increased during ozone exposure in spite of visible injury, while sulfur dioxide gas exposure decreased ascorbate contents. From these, it is inferred that sulfur dioxide causes cell death by direct oxidation while ozone induces both programmed cell death and antioxidative function in surviving cells. Treatment with methyl jasmonate suppressed the increase in ascorbate contents in wild type. This result suggests that JA-mediated signaling pathway may possibly participate in regulation of ascorbate contents during ozone stress.

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Plants are exposed to various environmental stressors associated with generation of reactive oxygen species (ROS). To remove ROS, plants synthesize antioxidant substances such as ascorbate (ASA). The leaf injury under ozone exposure in the ASA-deficient Arabidopsis mutant (vtc1) has been explained by the ROS generation. However, recent studies showed that pathogen-infected vtc1 accumulates higher levels of salicylic acid (SA) glucoside, indicating not only the ROS generation but also phytohormone-pathways are involved in the processes leading to the leaf injury. In the present study, we focused on two phytohormones, SA and ethylene. Accumulation of these phytohormones was higher in vtc1 than in Col-0 under ozone exposure. The transcript levels of PR-1 and PR-4 were also higher in vtc1. These results indicate that the leaf injury in vtc1 arises not only from the ROS generation but also from the accumulation of SA and/or ethylene. Analyses of double mutants vtc1/sid2, vtc1/ein2 are underway.

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Four novel and single-recessive O₃-tolerant mutants, toz (tolerant to ozone) 1, toz2, toz3 and toz4, were isolated from 60,000 EMS-treated seeds of Arabidopsis (Cvi-0). Observation of visible injury as well as ion-leakage analysis demonstrated clear differences between mutants and Cvi-0. Induction of AtGST3, which is regard as a marker for generation of reactive oxygen species (ROS), in mutants was lower than in Cvi-0, suggesting that generation of ROS in mutants was low-level. Moreover, two of them (toz2 and toz3) showed characteristic gene expression patterns induced by O₃ and used for further analysis. In toz3, O₃-induced expression of AtVSP1, a jasmonate-inducible gene, was remarkable, and the MeJA-induced inhibition of root elongation was higher than the other mutants and Cvi-0. Thus, O₃-tolerance in toz3 might be occurred increasing JA-sensitivity because JA signaling modulates O₃-induced leaf injury. The toz3 showed constitutive expression of PR-1, indicating that TOZ3 may regulate O₃-sensitivity through SA-mediated pathway.

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We previously reported that transgenic Arabidopsis plants overexpressing Zinnia cytosolic glutathione reductase (GR) show retarded root formation and reduced number of root meristematic cells [Henmi et al. (2004) PCP suppl. s45]. This suggests that GR expression affects the rate of root formation. Using the GR-overexpressing and the T-DNA inserted cytosolic GR-knockout plants, we here verified the role of the glutathione redox state on the rate of root formation. On growth media containing GSSG, the rate of root formation of the knockout plants did not respond to GSSG, while that of wild-type plants was accelerated or suppressed by the various concentrations of GSSG. The response to GSSG was amplified in the GR-overexpressing plants, compared to wild-type plants. Considering these together with the short-root phenotype of the knockout plants, it is suggested that the glutathione redox state controlled via GR activity regulates the rate of root formation.

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It is generally considered that the antioxidant GSH scavenges reactive oxygen species generated during chilling stress, resulting in prolonging the life in plants. We, however, show a paradoxical action of GSH, hastening effect on senescence, in Eustoma grandiflorum, a plant that is evergreen without chilling. Application of GSH induced plant senescence even without chilling. Buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, completely prevented the chilling-induced senescence without decreasing the plant growth rate. BSO-mediated inhibition of the chilling-induced senescence was abolished by GSH but not by other thiols. The application of GSH at various concentrations to a plant with chilling increased endogenous GSH and promoted senescence in a dose-dependent manner. These suggest that GSH essentially and specifically regulates plant senescence, resulting in shortening the life span of E. grandiflorum.

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We recently showed that the mitochondria elicit the signal in response to respiration and regulate the photosynthesis gene's expression, in a unicellular green alga, Chlamydomonas reinhardtii. However, it is uncertain whether the target genes of this signaling is restricted to photosynthesis genes or not. To answer this question, we searched for the genes whose expressions are regulated in response to mitochondrial respiration, with the aid of Chlamydomonas cDNA macroarray, on which 10000 cDNAs are spotted. We found that the mRNA levels of ca. 600 genes changed when acetate, a respiration substrate was added to Chlmamydomonas cells. Among them, ca. 150 genes were regulated by respiration, because their responsiveness was disturbed by a respiration inhibitor, antimycin A. Characterization of these genes and physiological significance of the mitochondrial signal will be discussed.

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Rapid generation of reactive oxygen species (ROS) is an important component of plant defense responses against pathogens. ROS production is inhibited by diphenylene iodonium (DPI), suggesting that NADPH oxidase-like enzyme mediates generation of ^.O₂^-. Plant NADPH oxidase homologs (rboh; respiratory burst oxidase homolog) possess hydrophilic N-terminal domain containing two Ca²⁺ binding EF-hand motifs. However, regulatory mechanisms and physiological functions of rboh are still largely unknown. For functional analyses, heterologous expression system was applied. AtrbohD was transiently transfected to human embryonic kidney (HEK) 293T cells, and ROS generation was detected by chemiluminescence. Treatment with a Ca²⁺ ionophore induced rapid and transient production of ^.O₂^-. This activity was completely inhibited by DPI, suggesting that rboh is directly activated by binding of Ca²⁺ to its EF-hands to show NADPH oxidase activity.

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We characterized nucleotide pyrophosphatase (OsNPP1) that hydrolyzes ADPglucose linked to the starch synthesis. We further succeeded to identify and isolate a novel cDNA clone encoding NPP isoform that has high homology (61%) with OsNPP1. This gene was designated as OsNPP2. We produced the transgenic rice cells transformed with Ubi::OsNPP2 and NPP2 was purified from the OsNPP2 overexpressed cells. The molecular size of purified enzyme was estimated to be approximately 70 kDa, and the binding of NPP2 to ConA-Sepharose column indicated that this enzyme is glycoprotein same as NPP1. The NPP1 enzyme catalyzed the hydrolytic cleavage of various nucleotide-sugar. By contrast, NPP2 did not hydrolyze the nucleotide-sugars although the enzyme was able to react the nucleotide diphosphate, such as ADP and UDP. These results strongly suggested that the physiological function of OsNPP2 is distinguishable from that of OsNPP1. The morphological phenotype of OsNPP2 overexpression rice plant also will be discussed.

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We investigate nucleotide pyrophosphatase (NPP1) that hydrolyzes ADPglucose to G1P and AMP. Previously, we reported the purification and cDNA cloning of NPP1, and the evidence that NPP1 occurs in the plastid strongly suggested that NPP1 is a novel factor involved in regulation of starch biosynthesis. Here, we produced and characterized a series of transgenic rice plants which overexpressed NPP1 gene to clarify the physiological role of the enzyme. It was found to decrease starch content and to increase soluble sugar contents in the transgenic rice plants. Furthermore, activities of some starch metabolism related enzyme dramatically increased in the transgenic plants. These results supported the conclusion that NPP1 operates on regulation of starch biosynthesis. Interestingly, the transgenic rice plants exhibited growth stimulation in post-germination stage and increase of plant height in heading stage. Based on these results, we will discuss physiological role of the enzyme.

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We investigated whether α-amylaseI-1 is involved in the degradation of starch in rice leaf chloroplasts. We generated a series of α-amylaseI-1 co-suppressed and overexpressed transgenic rice. In the co-suppression lines, the seed germination and seedling growth delayed. However, the retardation effects for growth were overcome by supplementation of sugar. Interestingly, significant increase of starch accumulation in the young leaf tissues was observed under sugar-supplemented condition. In contrast, starch contents in leaves were reduced in the overexpression lines. In immunocytochmical analysis with specific anti-α-amylase I-1 antiserum, immuno-gold particles deposited at chloroplasts and extracellular space in young leaf cells. In addition, mature protein species of α-amylaseI-1 bearing oligosaccharide side chain were detected in the isolated chloroplasts. We further examined expression and targeting of α-amylaseI-1 fused with GFP in re-differentiated green cells, indicating that the fluorescence of expressed fusion protein co-localized with the chlorophyll autofluorescence in the transgenic cells.

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Rice α-amylases were known to be encoded by multigene, and 20 isoforms were identified and characterized in the germinating seeds and tissue culture. α-Amylase I-1 (RAmy1A) was a glycoprotein bearing N-linked carbohydrate chain, that was actively synthesized and secreted from the secretory tissues. However, immunocytochemical analyses with specific anti-α-amylase I-1 antibodies revealed that α-amylase I-1 locates in the amyloplasts or chloroplasts of living cells of rice. To determine the intracellular localization of different α-amylase isoforms, we investigate the expression and targeting of enzyme/GFP fused protein in onion epidermal cells. The fluorescence-labeled α-amylase I-1 was found to be co-localized with the plastid, whereas the targeting ratios of the other isoforms into plastid were different from that of α-amylase I-1. We will discuss a possible mechanism for plastid targeting of α-amylase I-1 and difference of physiological function of each α-amylase isoform.

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The chloroplastic NADP-malate dehydrogenase (MDH) is engaged in indirect export of excess reducing equivalents from the stroma. Using transgenic rice overproducing sorghum MDH (19- and 39-fold increase in activity), we investigated the activation of foreign MDH and the effects of MDH overproduction on photosynthesis. The steady state level of MDH activation under growth light conditions was above 80 % in sorghum, while it was 40 % in both transgenic and non-transgenic rice. However, MDH was rapidly inactivated after transfer to darkness in both sorghum and rice, while its activation by illumination proceeded much slower in transgenic rice. The MDH activity reached maximum within two min in sorghum and non-transgenic rice, while in transgenic rice it continued to increase even after 60 min, probably due to short supply of the reduced thioredoxin. Dependences of CO₂ assimilation rate on light intensity and Ci remained unchanged by overproduction of MDH.

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Diplotaxis includes C3-C4 intermediates as well as C3 species. D. muralis (Dm, 2n=42) is thought to be originated from natural hybridization between D. tenuifolia (Dt, 2n=22; C3-C4) and D. viminea (Dv, 2n=20; C3). We investigated leaf anatomical and photosynthetic traits of these species to elucidate the genetic background of Dm. The BSCs of Dt contained centripetally arranged many chloroplasts and mitochondria, whereas those of Dv did few organelles. The BSCs of Dm showed intermediate traits between Dt and Dv. In Dt, GDC P-protein was localized in the BSCs, whereas in Dv it occurred mainly in the mesophyll cells. In Dm, GDC occurred in both cell types, but more densely in the BSCs. Correspondingly, Dm showed intermediate values of CO₂ compensation point between Dt and Dv. These data support the hypothesis that Dm was created by natural hybridization between the C3-C4 intermediate and the C3 species.

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To clarify the contributions of fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) to the increase in the photosynthetic rate, we generated transgenic tobacco plants expressing cyanobacterial FBPase-II (TpF) or Chlamydomonas SBPase in chloroplasts (TpS). In TpF-11 plants with 2.3-fold higher FBPase activity, final dry matter and photosynthetic activity were 1.30- and 1.15-fold higher, respectively, than those in wild-type plants. However, TpF-9 plants with 1.7-fold higher FBPase activity did not show these phenotypes. On the other hand, TpS-11 and TpS-10 with 1.6- and 4.3-fold higher SBPase activity, respectively, showed the increased photosynthetic activity and growth rate, but TpS-2 plants with 1.3-fold higher SBPase activity did not show these phenotypes. These data suggest that both FBPase and SBPase are the limiting factors for the RuBP regeneration in the Calvin cycle and that FBPase contributes to partitioning of fixed carbon for RuBP regeneration or starch synthesis.

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Under the poor availability of inorganic carbon (CO₂, HCO₃^-, etc. :Ci) for photosynthesis, Chlamydomonas or cyanobacteria induce the active uptake systems for Ci within the cell (Carbon-concentrating mechanism :CCM). In Chlamydomonas, the regulatory factor CCM1 is indispensable to induce the CCM. In order to detect the CCM1 protein, anti-CCM1 antibody was generated using His-tagged CCM1. A protein band with a higher M.W. was detected in western blot analysis. Considering with the fact that the deduced M.W. of CCM1 was 70,073, some modifications of CCM1 were suggested. The expression of CCM1 was constitutive and independent of CO₂ concentration. Moreover, CCM1 has five Cys residues conserved in the zinc-finger domain. The strains expressing the modified CCM1 with D60H, D60C, D60V were created respectively. Since all the strains grew as in the case of wild type under the CO₂-limiting condition, it was suggested the Asp-60 is replaceable in the function of CCM1.

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A green alga Chlamydomonas reinhardtii acclimates to low CO₂ condition by induction of carbon concentrating mechanism (CCM). The mechanisms of CO₂ sensing and regulation of CCM are poorly understood. To understand the regulation mechanism of response against CO₂ condition, we employed an open gas exchange system as a method of rapid characterization of CO₂ uptake of cells. Light dependent CO₂ uptake was observed, which was consistent with previous studies analyzed by silicone oil filtering centrifugation method. In wild type, low and high CO₂-adapted cells showed CO₂ uptake activities of 53.4 and 8.8 μmol CO₂/mg Chl h, respectively. A Ccm1 deficient mutant showed 8.8 μmol CO₂/mg Chl h of activity in both low and high CO₂-adapted cells. We will report about the nature of CO₂ uptake in regulatory mutants of CCM and also characterize effects of photosynthetic inhibitors on CO₂ uptake of Chlamydomonas cells with this system.

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Fruit sweetness correlates with sugar accumulation into fruits during their development. Sugar content in tomato fruits is known to increase just as during salt- or drought-stress. These effects are interpreted as the result of water withdrawal from the fruit and transfer to the plant, thereby raising the concentration of sugars in the fruits. Another possible factor is the induction of free sugar accumulation in the leaves under abiotic-stress, with consequent movement of sugars to the fruits. We gave oxidative-stress to 4-months-old tomato and melon plants by supplying the hydrogen peroxide solution to the soil. This treatment caused an increase of sucrose-phosphate-synthase activity and subsequent free sugar content in the leaves. It also caused an increase in free sugar content of the fruits. Furthermore, the production levels of the fruits were slightly increased after supplying the hydrogen peroxide solution, although the oversupply of hydrogen peroxide prevents plant growth.

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  Concerning the structure of chlorosomes, two kinds of models have been proposed; one is ‘the monomer-based stacking’ and the other is ‘the dimer-based stacking’. We tried to determine the structures of BChl c artificial aggregates and chlorosomes by the use of solid-state NMR, electronic-absorption and circular dichroism spectroscopies.
  In the case of BChl c artificial aggregate, we measured the 2D spin-diffusion spectra which can detect through-space ¹³C–¹³C correlations. The through-space correlations experimentally determined were compared with those simulated by intermolecular distances in the models. The structure of BChl c artificial aggregates could be explained not by the monomer-based stacking but by the dimer-based stacking; in which the macrocycles are stacked like a stair case. This structure realizes the red-shift of the Q_y absorption as observed spectroscopically.
  In chlorosomes, the CD spectral pattern was simulated only by the left-handed cylindrical arrangement of dimers.

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It is reported that IscS (a homolog of NifS) and IscU protein in Azotobacter vinelandii and Escherichia coli are involved in assembly of iron sulfur (FeS) proteins. It is thought that mobilizing sulfur from free cystein is incorporated into apo-FeS protein to form FeS cluster by IscS and that IscU play a role as scaffold protein. IscS and IscU homolog were also reported from oxygenic photosyntetic bacteria, cyanobacteria Synechosystis PCC 6803. Whole genome sequence of Green sulfur Bacterium Chlorobium tepidum revealed that the putative iscS iscU are present on the genome. Phylogenetic analysis indicated that the iscS of C. tepidum is classified with different from that of E. coli and cyanobacteria. To investigate the function of iscS and iscU of C. tepidum, we over-expressed this genes in E. coli. The biochemical function of purified proteins from E. coli will be reported.

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The reaction centers of heliobacteria belong to a Fe-S type, like those of green sulfur bacteria and PSΙ. Four Ferredoxins, Fd A, Fd B, Fd C, and Fd D, were extracted from heliobacterium Heliophilum fasciatum cells, and purified by ammonium sulfate fractionation, anion-exchange chromatography and hydrophobic interaction chromatography chromatographies under strictly anaerobic conditions. The absorption spectra of the purified Fds were typical of those of the 2[4Fe-4S] cluster type Fds, with absorption peaks at about 385 and 285 nm and with a shoulder at about 300 nm. Cryogenic EPR measurements showed that the purified Fds were dithionite reducible at pH8.0 and those spectral features were typical 4Fe-4S type iron-sulfur cluster. Although A₃₈₅/A₂₈₀ ratio of Fd B and Fd C were not changed significantly up to 24h under the air at 4°C, those of Fd A and Fd D were decrease by 50% and 20% respectively.

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Heliobacteria are strictly anaerobic, nitrogen fixing photosynthetic bacteria. On the basis of 16S rRNA sequence analysis, heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Heliobacteria synthesize a unique photopigment, bacteriochlorophyll (BChl) g, which has an interesting structural similarity to chlorophyll (Chl) a from oxygenic phototrophs. BChl g contains an ethylidene side group at C8 of ring II that, in the presence of oxygen and light, isomerizes to produce an ethyl group, giving rise to a ring structure that is the same as in Chl a. A heliobacterium Heliobacillus mobilis contains a major cluster of photosynthesis genes which include genes involved in BChl and carotenoid biosynthesis and structural gene of reaction center apoprotein. We are investigating photosynthesis gene cluster from another species of heliobacteria, Heliophium fasciatum, and will discuss the comparison study of the photosynthesis gene cluster between heliobacteria.

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The RCs of purple bacteria and FAPs are similar to each other, and seemed to have derived from a common ancestral protein. The puf genes form one operon puf B-A-L-M-C in purple bacteria and R.castenholzii (FAP), while form two operons, pufB-A-C and pufL-M, in another FAP, Chloroflexus aurantiacus. This feature of R.castenholzii seemed to reflect that of the common ancestor. To obtain more information of photosynthetic genes of the common ancestor, we studied the transcriptional organization of the puf operon in R.castenholzii. In northern hybridization experiments, small amounts of 3.5kb (pufBALMC) and 2kb (pufBALM) mRNAs, and a large amount of 0.3kb (pufBA) mRNA were detected as in the case of purple bacteria. 5'end of these mRNAs were detected at the 140 base upstream of the pufB. The putative promoter sequences similar to that of Cfl.aurantiacus were detected at the corresponding position.

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The pst operon of Rhodobacter sphaeroides f. sp. denitrificans, which encodes the phosphate-specific transport system, is composed of six genes, pstS, pstC, pstA, pstB, phoU and phoB. Northern blot analysis of the Pst locus showed two different bands, which corresponded to pstS and pstCAB mRNA. Although a phosphate-regulated promoter (Pho box) located upstream of the pstS gene, the upstream sequence of pstC and phoU had no apparent Pho box. The pstC and phoU promoter-driven expression of lacZ activities were reduced to the back ground level in the response to the phosphate limitation. The DNA-binding activity of PhoB was not observed when the organism was grown in the phosphate-limited medium except for the pstS promoter, suggesting that the transcriptional regulation of pstCAB and phoUB is not regulated by pho regulon.

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Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing the reduction of rotochlorophyllide D-ring to form chlorophyllide. DPOR consists of two separable components, L-protein and NB-protein, which are structurally related to Fe-protein and MoFe-protein of nitrogenase, respectively. Here we show structural models for L-protein and NB-protein from Rhodobacter capsulatus based on analyses of molecular mass and Fe-S centers. The elution profiles of the two components in gel filtration chromatography indicated that L and NB-protein are a homodimer [(BchL)₂] and a heterotetramer [(BchN)₂(BchB)₂], respectively. L-protein semi-purified with 6xHN affinity tag showed an EPR signal with g-values of 1.95 and 1.86, suggesting that L-protein carries a [4Fe-4S] cluster similar to Fe-protein. In contrast, semi-purified NB-protein showed complex EPR signals with g-values of 2.03, 1.94 and 1.92, which suggest the presence of [4Fe-4S] cluster(s) different from MoFe-protein. Structural similarity between DPOR and nitrogenase will be discussed.

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Chlorophyll a and bacteriochlorophyll a biosynthesis pathways are identical except that the latter has two additional steps. The terminal step of these synthesis pathways is esterification with long-chain alcohol. This reaction is catalyzed by ChlG in chlorophyll synthesis and BchG in bacteriochlorophyll synthesis. A recent study, however, suggested that the mutant of cyanobacteria expressing BchF, a specific enzyme in bacteriochlorophyll synthesis, accumulates 3-hydroxyethylchlorophyllide a with a long-chain alcohol. In this study, we constructed mutants lacking bchG and bchXYZ to analyze the substrate specificity of BchG and ChlG in vivo. These mutants accumulated intermediate pigments, metabolites of bacteriochlorophyllide a, chlorophyllide a and 3-hydroxyethylchlorophyllide a, respectively. Results on introduction of chlG of Synechocysts sp.PCC6803 into these mutants will be discussed.

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Heterocyst is a terminally differentiated cell of cyanobacteria which is specialized in nitrogen fixation. Upon limitation of combined nitrogen in the medium, heterocysts are formed with a semi-regular spacing of 10-15 cells. Heterocyst differentiation involves a complex series of coordinated cellular process to develop an efficient environment for nitrogen fixation. To understand how heterocyst differentiation is regulated, it is crucial to identify genes related to heterocyst differentiation. We analyzed a pattern of gene expression after nitrogen deprivation using oligo DNA microarray in Anabaena PCC 7120 and a hetR mutant strain, DR884a. The HetR protein is a key regulator in the regulation of heterocyst differentiation. Disruption of hetR blocks the earliest stage of heterocyst differentiation and overexpression of hetR leads to ectopic formation of heterocysts even under condition replete with combined nitrogen. We try to identify genes related to heterocyst differentiation by comparing gene expression patterns in these two strains.

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We showed that the inactivation of the htpG gene from the cyanobacterium Synechococcus sp. PCC 7942 causes loss in both basal and acquired thermo-tolerances, indicating an indispensable role of the HtpG protein for the survival under high-temperature stress.
We observed the reduction of phycocyanin content in the htpG mutant grown under normal conditions. The phycobilisome purified from the mutant by sucrose density gradient ultra-centrifugation is lighter than that from the wild type. Phycobilisomes are composed of phycobiliproteins and linker polypeptides. In contrast to phycobiliproteins, linker polypeptides were prone to aggregation when dissociated from the phycobilisome complex. We found that the level of the 30-kDa linker polypeptide is reduced in the mutant's phycobilisome and that the purified HtpG can prevent the in vitro aggregation of the linker polypeptides at normal and high temperatures. Studies on an interaction between HtpG and a linker polypeptide are in progress.

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