In a virescent mutant of rice, leaves in seedling is initially yellow with less chlorophyll, and then gradually becomes more greenish as the mutant grows. The mutable virescent plant, pyl-v (pale yellow leaves variegated) showed yellow leaves with green sectors, and the variegation was caused by recurrent somatic mutation from the recessive pyl-v allele to the green Pyl-r (Pyl-revertant) allele. We showed that the pyl-v allele is caused by the disruption of a nuclear-coded chloroplast protease gene, OsClpP5 due to an insertion of a novel DNA transposon, nDart1. We showed that OsClpP5 can be localized in chloroplasts and that the defects in grana stacks of chloroplasts of yellow leaves in seedlings do not recover even after the leaves become green. Phenotypic features in pyl-v mutant with regard to the expression of OsClpP5 gene itself and environmental factors affecting the OsClpP5 expression will be discussed.

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To uncover functions of a large population of rice genes efficiently, we are employing Full-length cDNA Over-eXpresser gene (FOX) hunting system. Producing FOX-rice lines, we have observed phenotypes of over 25,000 independent FOX-rice calli. Among them, two calli showed a green color on a 2,4-D-containing medium, on which wild-type rice calli are ivory yellow. In the two calli, OsGLK1 gene, which encodes a GARP-transcription factor, was ectopically overexpressed. After transfer of wild-type calli to the regeneration medium, greening of calli and an increase in the OsGLK1 mRNA level occurred synchronously. Transmission electron microscopy showed that chloroplasts with grana stacks were differentiated in the OsGLK1-FOX calli. However, in darkness, both the OsGLK1-FOX calli and plants did not show the green color and the differentiation of grana stacks. These data strongly suggest that OsGLK1 is a key regulator of chloroplast development under the control of light and phytohormones.

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Chloroplast has developed the sophisticated translation system using only 30 tRNAs that correspond to all 20 amino acids. Researches of sigma factors of eubacteria-type plastid encoded RNA polymerase (PEP) suggest that transcription of some tRNAs including tRNA-Glu, which is essential for tetrapyrrole synthesis, is regulated by SIG2 and SIG6 during chloroplast development. In this study, we investigated the extended dynamism of chloroplasts tRNA expression in response to environmental condition. In the dark condition photosynthetic mRNAs were surely decreased, whereas chloroplast tRNAs were accumulated inversely. This phenomenon was observed even in SIG2 or NEP deficient mutants. Similar accumulation was occurred by change of N/C balance in medium or by addition of chloroplast translation inhibitors. Overexpression of the plastid glutamyl-tRNA synthetase, however, did not affect the accumulation. Based on these results, we will discuss physiological meaning and molecular mechanism of the unique dynamism.

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Cytokinin plays a crucial role for maintenance of shoot meristem function. Cytokinin is first synthesized as a nucleotide form, and then converted to the bioactive free-base form. Although the activating step is important to regulate cytokinin activity, the genetic and biochemical nature has not been identified. We report that the LOG (LONELY GUY) gene, whose mutants confer defects in the maintenance of shoot meristems, encodes a novel cytokinin activating enzyme in rice. LOG has phosphoribohydrolase activity, which directly converts a cytokinin nucleotide to the free-base form with the release of a ribose 5'-monophosphate. Our findings revise the long-held idea of multi-step reactions, cytokinin nucleotide can be directly activated to the free-base form by LOG. We will show you data biochemically and genetically characterizing the LOG and its homologs.

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Cytokinins play a crucial role in various aspects of plant growth and development. The spatial and temporal distribution of bioactive cytokinin levels is strictly controlled in plant development events. Recently, LONELY GUY (LOG) was identified from rice as a novel cytokinin-activating enzyme that works in the final step of bioactive cytokinin synthesis. In Arabidopsis genome, 9 genes (AtLOG1- AtLOG9) are predicted as homologs of rice LOG. To elucidate the functions of Arabidopsis LOG family genes, we characterized the activities of AtLOGs proteins and the expression patterns of AtLOGs genes. Several AtLOGs proteins have the activities in the final step of bioactive cytokinin synthesis as well as rice LOG. Analysis of GUS::AtLOGs fusion genes revealed that each AtLOGs genes show the different expression patterns. Possible role of the AtLOGs genes in plant growth and development will be discussed.

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CYP735A1 and CYP735A2 catalyze the hydroxylation of isopentenyladenine-type cytokinins (CKs) to trans-Zeatin (tZ)-type CKs. To clarify the physiological roles of the CK hydroxylation, we analyzed insertional mutants and overexpressor of the CK hydroxylase genes. cyp735a1 and cyp735a2 did not show any visible phenotype but double mutant, cyp735a1cyp735a2, displayed phenotypes only in the aerial part such as reduced size and increased rosette branches. In cyp735a1cyp735a2, amount of tZ-type CKs was lower than 3 % of that in WT and amount of iP-type CKs was twice of that in WT. Partial complementation of the mutant phenotypes by the application of tZ indicated that mutant phenotype was caused by the decreased amount of tZ-type CKs. Transgenic overexpressors of the hydroxylase genes had increased amount of tZ-type CKs but displayed no visible phenotype. We will also show the results from gene expression analysis of the double mutant using DNA microarray chips.

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In A. thaliana, His-Asp phosphoerelay is involved in both the cytokinin- and ethylene-dependent signal transduction pathways, in which certain His-kinases particularly act as cytokinin-receptors or ethylene-receptors. Among 11 His-kinase-related proteins, 8 of them are directly implicated in either cytokinin or ethylene signaling. But, the remaining three HKs are less characterized. Here we clarified an interesting aspect of physiological functions of the least characterized one, named AHK5 (also known as CKI2). Three independent ahk5 T-DNA insertion mutant lines were established in the hope of revealing a common phenotype for all of them. The results suggested that AHK5 His-kinase regulates root elongation through an ETR1-dependently integrated abscisic acid (ABA) and ethylene signaling pathway. This new function of AHK5 will be discussed in the context of canonical signaling pathways of ABA and/or ethylene, such as those during germination and/or triple responses.

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Brassinosteroids (BRs) are thought to be biosynthesized from campesterol via campestanol in plants. Recently, we have characterized that Arabidopsis CYP90C1 and CYP90D1 are redundant C-23 hydroxylases. Analyses of their substrate specificities revealed a novel shortcut in BR biosynthesis, which allows direct conversion of early 22-hydroxylated intermediates to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol via C-23 hydroxylation. Here, we report biochemical characterization of this novel shortcut, focusing on 5α-reductase (DET2) and C-3 oxidase. These recombinant enzymes were heterogenously expressed, and their activities were measured in vitro. DET2 catalyzed 5α-reduction of not only (24R)-ergost-4-en-3-one (4-en-3-one) but also 22-hydroxy-4-en-3-one and 22,23-dihydroxy-4-en-3-one. The substrate specificity of C-3 oxidase showed that (22S)-22-hydroxycampesterol is a better substrate than (22R,23R)-22,23-hydroxycampesterol, whereas campesterol is not metabolized. Thus, the shortcut is likely a main route of BR biosynthetic pathway.

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We tried to perform the chemical genetics screening by Brz (brassinazole) that was synthesized as the first specific inhibitor of brassinosteroid biosynthesis. Target of Brz220 is the cytochrome P450 enzyme encoded by DWF4. In order to analyze in detail the mechanisms of brassinosteroid signal transduction, we screened for mutants that showed longer hypocotyls than wild type when grown with Brz220 in the dark, and designated bil mutants (Brz-insensitive-long hypocotyl). We identified a semidominant mutant, bil5, from fast neutron-treated lines. Hypocotyl elongation of these plants on Brz medium was at least twice that of the wild type. Adult bil5 plants showed pale green and thin leaves, and thin and shortened inflorescences. The root elongation of bil5 was also inhibited. The pale-green leaves of bil5 were in contrast to the dark-green leaves of brassinosteroid-deficient mutants, and chloroplast gene expression was lower in bil5 than in wild type.

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Brassinosteroids (BRs) are steroid hormones of plants that regulate plant growth and development. BRs are perceived by a cell surface receptor kinase BRI1, then various components is predicted to be involved to transduce the BR signal in the cell. To identify additional components involved in BR signaling, we have analyzed various dwarf and semidwarf mutants from Arabidopsis mutant, which is insensitive to the BR synthetic inhibitor Brz. Brz causes deetiolation and dwarf phenotypes similar to those of BR-deficient mutants. We had tried to screen mutants that showed to etiolate on medium containing Brz. Here we identify a bil4 (Brz-insensitive-long hypocotyls 4) mutant from activation tagging line. bil4 mutant showed slender dwarf phenotype which small and twisted leaves ,and short and thin inflorescences.

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Brz (brassinazole) was synthesized as the first specific inhibitor for brassinosteroid biosynthesis. In the dark germination, Brz-treated plant expressed chloroplastic genes that are expressed as very low level in dark-grown cotyledon without Brz. When the dark-germinated plant with Brz was transferred to the light conditions, chloroplast development was accelerated as compared to the condition without Brz. In the light, Brz treated adult plant showed high-green leaves as compared to leaves without Brz. So we tried to screen for a mutant that showed pale green phenotype with Brz in the light, and isolated bpg2 (Brz-insensitive-pale green2) mutant. A T-DNA insertion site of bpg2 mutant was identified in an ORF of novel gene. TargetP program predicted that BPG2 localized to chloroplast. Expression of bpg2 gene was induced by light, and was highest in leaves. These results might suggest that BPG2 has an important role for regulation of chloroplast differentiation.

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Progesterone is an animal steroid hormone that functions as a corpus luteum hormone. Recently, we identified progesterone from Arabidopsis, rice, tomato and pea, suggesting that progesterone is a ubiquitous constituent of plants. To clarify the biological function of progesterone in plants, Arabidopsis was germinated and grown in the presence or absence of progesterone under the various light conditions. Arabidopsis hypocotyl and petiole showed elongation in some conditions. These functions might be similar to brassinolide functions. Analysis of possible function of progesterone for another plant organ is in progress. In addition, to clarify the molecular mechanism of progesterone bioactivity for plant, we analyzed candidate genes related to progesterone by microarray analysis.

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A callus can be considered as one of the phenotypes of the plant cells. There may be factors that induce and/or maintain the dedifferentiated state. To identify such factors, we performed comparative analyses of the gene expression profiles by DNA microarray between Arabidopsis seedlings and three callus lines, and found that a gene for the plant specific transcription factor of AP2/ERF family was upregulated in callus. Transgenic plants that express the gene under the control of 35SCaMV promoter showed morphology alteration. Transformants with severe phenotype formed callus in the shoot, hypocotyl and root. These callus generated from the transformants grew and maintained dedifferentiation state in the phytohormone-free medium. The seedlings of T2 generation can dedifferentiate into callus under the lower concentration of auxin, in which condition wild type seedlings do not form callus. We discuss the possible function of the transcription factor in the regulation of callus formation.*

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Although accumulation of sequential analyses of cell number and size has revealed many aspects of mechanisms of leaf expansion, it was difficult to understand details of how leaf develops since cell proliferations and cell expansions occur simultaneously during leaf expansion. To overcome the difficulty, we developed a computer simulation of behavior of palisade tissue.
Fundamental information constituting the simulator is cell locations and volumes. We introduced cell expansion potential and cell proliferation potential depending on the location of the objective cell. Since pushing force of each cell can be presumed as the same, shape of a given cell was determined by arrangement of surrounding cells. Migration distance of the cell was determined by sum of expansion of cells, which located at basal part compared to the objected cell. In the presentation, we will introduce the simulation model and calculated results from several parameter sets.

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We studied salt homeostasis in mangrove. Ion contents were measured in various organs of several mangrove species distributed in Iriomote Island, resulting that seawater-derived sodium was detected in every vegetative organ of shoots. Sodium content of propagules was however significantly lower than that of other vegetative organs in all the species assayed suggesting a sodium filtration between these organs. Concentration gradient of sodium between propagules and other organs was highest in Avicennia marina. Moreover as Na⁺/K⁺ ratio was lower than 1 only in A. marina, this species was expected to selectively filtrate sodium. We hypothesize that xylem sap needs to pass through the parenchyma for the selective ionic filtration. A placenta to which vascular bundles from a flower stalk connected adjoined a hypocotyle at each parenchyma. Further analysis using a fluorescent sodium indicator showed organ-specific localization of sodium, concluding that A. marina filtrated sodium selectively and effectively in placenta cells.

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Petal color of morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from reddish-purple to blue during blooming. The color change is caused by an unusual vacuolar pH increase of the epidermal colored cells. During flower opening process the volume of colored cell increased two- to three-fold. To clarify the mechanism of this phenomenon we analyzed the ion concentration of the colored cells during flower opening. After measurements of the cell number and volume of the colored protoplasts cations and anions of the cell sap were analyzed using capillary electrophoresis. The major cation was K⁺ and its concentration was 50 mM at -24 h. The K⁺ concentration once decreased but turned to increase reaching 40 mM at the full opening. The major anion in the cells was Cl^- and PO₄^3-. The concentration of both ions decreased towards the full opening. The relationships between petal color development and cell enlargement are discussed.

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We screened a rice full-length cDNA library for novel cation transporters involved in the salt-tolerance. As a result, the Shaker family K⁺ channel OsKAT1 was found to be a suppressor of the salt-sensitive phenotype of mutant yeast. OsKAT1 also suppressed a K⁺ transport-defective phenotype of another mutant, indicating the function of OsKAT1 in K⁺ uptake. Under salt stress conditions, cellular Na⁺ was decreased in OsKAT1-expressing yeast following the increase of K⁺. The improvement of salt-tolerance and the increase of cellular K⁺ were observed also in rice cells overexpressing OsKAT1, suggesting OsKAT1 may participate in maintenance of cytosolic cation homeostasis during salt stress and protect the cells from Na⁺. The expression of OsKat1 was restricted to internode and rachis of wild type plant while other Shaker family genes were expressed in various organs. OsKAT1 may be involved in salt-tolerance of rice plant cooperatively with other K⁺ channels.

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The ionic transport systems have important roles on 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 intracellular behavior of chloride ions, a counter ion for sodium ions, under the high salt condition.
We have been investigating about the two chloride channel genes in rice. We found three cDNA clones (AK066375, AK101523, and AK119459) for the novel rice chloride channel genes other than OsCLC-1 and OsCLC-2 in the database. They had 55.0% to 60.7 % identity of amino acid sequence with OsCLC-1. Although OsCLC-1 and OsCLC-2 were expressed in most tissues, two novel clones expressed in a specific organ. We found the expression level of novel CLC genes increased in the clc-1 mutant line, but not in the clc-2 mutant line.

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When plants suffered wounding, they generate electrical signals. Characeae is a suitable material for this study because of simple morphology. Using Chara corallina, I developed a system to analyze the response. Specimens composed of two tandem internodal cells were prepared. When one cell (victim cell) was kiled by cutting, another cell (receptor cell) generated two kinds of resposnes: rapid depolarization and long-lasting depolarization. My previous study showed that the latter resposne is induced by potassium released from vicitim cell. In the present study, analysis was carried out with special interests in the rapid response. I hyothesized that loss of turgor pressure of the victim cell upon killing may induce the rapid response. When 1 M methanol was added to the external medium of the victim cell, the recepor cell generated a rapid spike similar to that generarated upon cutting the victim cell.

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Plants generates receptor potential which is explained as an activation of mechanosensitive Ca²⁺ channels, followed by an activation of Cl^-.
What triggers the activation of Ca²⁺ channels may be a change of membrane tension or membrane stretching as follows. Membrane potential, E_m, significantly changed during hypertonic treatment (plasmolysis) and the following hypotonic treatment (deplasmolysis). By contrast, the deplasmolyzed cell did not show any change in E_m upon the second hypertonic treatment. This may suggest that membrane streching triggers changes of E_m, because membrane stretching should occur during plasmomolysis and deplasmolysis but not during the second hypotonic treatment. Effect of membrane stretching upon activation of Ca²⁺ channel was also studied. Significant increase in cytoplasmic level of Ca²⁺ occurred during the plasmolysis and during the deplasmolysis, but did not during the second hypotonic treatment. We discussed the mechanism of receptor potential in relation with the membrane stretching.

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We have isolated the gene encoding K⁺ channel from tobacco cells (Nicotiana tabacum cv. SR1). The gene products rescued the E. coli mutant defective in K⁺ uptake transport, but did not restore the growth of the K⁺-uptake-deficient yeast strain. Likewise, we could not find the K⁺ current when the gene was expressed in Xenopus laeivs oocytes. To determine the location of the K⁺ channel expression at the subcellular levels, the immunoblotting detection has been performed. A single band corresponding to the deduced molecular mass was seen in the tonoplast fraction, suggesting that the channel is located in the tonoplast. Thus we used plant or yeast expression system to measure the K⁺ channel current in the vacuole membrane. Compared with the currents of the non-transformed cells, the cells expressing the gene gave the K⁺ channel-mediated K⁺ currents. The K⁺ transport activity was dependent on the change of pH.

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Phytic acid, inositol hexakisphosphate, IP6, a storage compound of phosphorus in seeds, is accumulated in protein storage vacuole during seed maturation. The synthesis and physiological function of IP6 are still unclear in higher plants. We succeeded in inducing synthesis of phytate in suspension-cultured Arabidopsis cells by Pi supplementation. It has been proposed that IP6 is finally synthesized by addition of a last phosphate group to inositol pentakisphosphate by one of inositol phosphate kinase ; IPK1. To confirm the subcellular distribution of AtIPK1, we expressed the GFP fusion protein transiently in Arabidopsis cultured cells. We observed fluorescence of GFP at the cytosol of cultured cells. It suggests that IP6s may be synthesized at the cytosol in Arabidopsis cultured cells, and then transported into the vacuole for storage.

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Proteins and lipids are delivered from the trans-Golgi network (TGN) to the cell exterior by secretory vesicles. However, little is known about the behavior of the vesicles. Secretory Carrier Membrane Protein (SCAMP) has four transmenbrane domains and various domains involved in the intracellular trafficking. We have been analyzing about the localization and the function of SCAMP in plant cells. Confocal laser scanning microscopy (CLSM) and TEM observation by high-pressure freezing technique revealed that SCAMP in tobacco BY-2 cells was localized in the plasma membrane (PM), TGN and novel vesicle cluster, designated SCAMP-containing vesicle cluster (SVC). By TEM, four-dimensional CLSM and total internal reflection microscopy, we showed that the SVC existed away from TGN. Moreover, we observed that the SVC fused with PM and cell plate. From these results, we conclude that the SVC is involved in the secretory pathway from the TGN to the cell exterior.

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The number of SNARE genes in Arabidopsis genome is much greater than that in any other model organisms. Among them, in addition, many SNARE molecules localize predominantly on the plasma membrane (PM) compared with other intracellular compartments. To elucidate physiological function of each PM-localized SNARE, we analyzed spacio-temporal expression profiling of 9 PM-resident Qa-SNAREs in Arabidopsis roots. One of them, SYP123 showed the specific expression pattern exclusively in the trichoblasts with elongating root hairs. Loss-, or gain-of-function analysis of SYP123 depicted morphological abnormality, or elongation defect in root hairs. These results indicate that SYP123 may function in the elongation step of root hair, not in the bulging phase, and that, furthermore, this Qa-SNARE may control the orientation of polarized vesicular transport by its precise localization on PM of root hair region.

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Arabidopsis thaliana has two different types of Rab5-related GTPases (conventional-type Ara7 and Rha1 and plant-unique-type Ara6). We have found that only one guanine nucleotide exchange factor (GEF), AtVps9a, can activate all Rab5 members in vitro. In the atvps9a-1 mutant, whose GEF activity is completely lost, embryogenesis is arrested at the torpedo stage and cells hypertrophied and aligned irregularly. GFP-Ara7 and Ara6-GFP diffused in cytosol like GDP-fixed form of Rab5s, suggesting that both types are not activated in atvps9a-1. In the leaky allele, atvps9a-2, elongation of the primary root was severely affected. Overexpression of GTP-fixed form of Ara7 suppressed the atvps9a-2 mutation, but that of Ara6 had no apparent effects. These results indicate that the two types of plant Rab5 members are functionally differentiated, even though they are regulated by the same activator, AtVps9a.

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Rab5 GTPase is known to regulate not only the endosomal fusion but also signaling through endosomes.The Rab GTPases are activated by specific guanine nucleotide exchange factors (GEFs), which accelerates the exchange of GDP to GTP. The Vps9 domain, the catalytic core for the activation of Rab5, is conserved in all Rab5 GEFs. We have already demonstrated that the AtVps9a can activate all Arabidopsis Rab5 members (Ara6,Ara7 and Rha1). To examine the activation mechanism of Rab5s in detail, we performed mutational analysis of AtVps9a based on the crystal structure of AtVps9a/Ara7 complex. We introduced several amino acid changes in Vps9 domain and revealed that conserved Tyr (225) and Asp (185) in the Vps9 domain are essential for the GEF activity. We also demonstrated that truncated AtVps9a shows specific increase in the GEF activity toward Ara6. We are now examining the effect of these mutations on plant development using transgenic Arabidopsis.

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We describe here the establishment of a novel gain of function system named as Fox hunting system (Full-length cDNA over-expresser gene-hunting system) for random over-expression of a normalized Arabidopsis full-length cDNA library. Full-length cDNAs from Arabidopsis were sequenced and c.a. 10,000 non-redundant clones were randomly expressed in Arabidopsis plants. We observed 1,487 morphological mutant candidates while growing 15,547 T1 transformants. One of the most frequently observed phenotypes was that of pale green leaves and stems. When 115 such T2 lines were observed 59 of these lines showed the pale green phenotype in dominant or semi-dominant fashion. One of such pale green line showed early flowering and tall phenotypes. Full-length cDNA causing the phenotype encode small protein (95 A.a.) having signature of secretion proteins. Usefulness of the FOX-hunting system to elucidate the function of unknown genes will be discussed.

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F-box protein acts as a receptor for target protein of ubiquitination in SCF complex. By database analysis, we found 568 F-box protein genes in Arabidopsis genome, however functions of most of the F-box proteins are still unclear. To carry out large-scale functional analysis we have cloned ORFs of F-box protein genes in recombination type cloning vector. We first employed RT-PCR cloning using total RNAs from various tissues to clone novel splicing variant as well as predicted ORFs. As to ORFs that was not cloned by the RT-PCR, we amplified the ORFs using the full-length cDNAs. All cloned PCR fragment were sequenced. This cloning resulted in finding lots of unpredicted transcripts that were candidates for splicing variants. We characterized the splicing variants by the analysis of their sequences, structures and expression patterns. We present here the results of the ORF cloning, characterization and expression study of cloned ORFs.

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In order to investigate the whole genetic system of legume species, we initiated a large-scale genome analysis of a model legume, Lotus japonicus.
Combining clone-by-clone sequencing of large insert clones from gene-rich regions with random genome sequencing, genome sequence information covering approximately 90% of the L. japonicus ESTs has been accumulated. By generating PCR-based markers from the accumulated genome sequences, positional information on 80 % of the sequenced clones has been obtained. The sequence data, predicted gene information and mapping information are available through the web database, http://www.kazusa.or.jp/lotus/. In order to establish the basis for transfer of knowledge on L. japonicus to other legumes, comparative genome analysis between L. japonicus and other legumes is currently in progress.
The latest status of the genome sequencing project and the features of the L. japonicus genome appearing from the functional annotation and comparative genome analysis will be presented.

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Model legume Lotus japonicus has been used for symbiotic nitrogen fixation (SNF) study. Lotus has LORE1, endogenous active Ty3-gypsy retrotransposon, and attempt to establish gene tagging system using LORE1 is undergoing. Here, we report the identification of another active retrotoransposon, LORE2 (Lotus Retrotransposon 2).
Responsible genes of two independent SNF mutants, isolated from Ac/Ds tagging population, were found to be interrupted by endogenous Ty3-gypsy retrotransposons. They were very similar to each other and designated as LORE2. Number of cognate LORE2 copies in Gifu was estimated as around twenty.
LORE2 transcripts were detected in all tissues investigated, however, up-regulation of transcription in calli, well-known phenomenon for other active retrotransposons in plant, was not seen for LORE2. It was assumed that LORE2 transposition in the two SNF mutants had happened in intact plant during sequence of screening for SNF mutants from the population.

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Ion transporters play central roles in facilitating acquisition of nutrients in plants; however little is known about their regulation at the molecular levels. In Arabidopsis, 1968 genes are predicted to encode transcription factors. To investigate transcriptional regulation of nutrient uptake processes, we performed a large-scale RT-PCR-based screening of transcription factor mutant collection, focusing on starvation responses of the transcripts of major nutrient uptake transporters in Arabidopsis roots. All available candidates of transcription factor mutants were collected from RIKEN transposon-tag collection, and homozygous insertion lines representing the mutants of 265 transcription factors were established. SULTR1;2 sulfate transporter and NRT2;1 nitrate transporter, playing important roles in acquisition of sulfate and nitrate, respectively, were used as profilers in RT-PCR screening. From this screening, we isolated several primary mutant candidates whose induction of SULTR1;2 on sulfur deficiency or repression of NRT2;1 on sucrose deficiency were significantly altered from the wild-type responses.

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Gene coexpression analysis that depicts gene-to-gene correlations became prevalent in plant transcriptome research. Methods of network analysis have been developed to detect connected groups of genes from a large network. However, within the group, it was difficult to exclude the possibility that the group was also highly connected with genes on the outside of the group. To extract genes with dense within-group connectivity and sparse outside connectivity, we developed a novel algorithm by introducing quantitative index "network specificity". To identify genes specifically coexpressed with a query gene, the first procedure was to extract a group of genes that have high network specificity to a query gene. The second procedure was to calculate network specificity of the group to estimate within-group coexpression quantitatively. By applying this algorithm to genome-wide analysis of Arabidopsis gene coexpression, we identified a number of coexpression groups.

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Plant vacuole is a enormous organelle that accumulates a variety of solutes such as nutrients, proteins, second metabolites, and toxic ions. Vacuole not only conserves solutes but also contributes to dynamic events, such as cytosolic pH control, Ca2+ signaling and stomatal movement. These functions are supported by a great number of tonoplast proteins and their interactions. In this study, we investigated protein complexes of vacuolar membrane comprehensively.
We prepared vacuolar membrane from suspension-cultured cell of Arabidopsis thaliana. Solubilized membranes were subjected to 2D- blue native- 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) under blue native-PAGE. We also screened protein complexes in the vacuolar membrane by co-immunoprecipitation method using the antibody library.

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Recent advances in the studies with phloem sap have suggested that it might contain molecules involved in signal transduction. On a search for proteins and peptides that might function as signaling molecules, proteome analysis was performed with rice phloem sap of high purity that was obtained by the insect laser methods. Proteins and peptides in the sap were separated into several fractions, using gel electrophoresis, ion-exchange chromatography, and ultrafiltration techniques, and then subjected to the analysis with ion trap mass spectrometer equipped with nano-flow liquid chromatography. We identified more than 70 proteins including putative zinc finger-type transcription factors as well as metabolic enzymes and stress-responsive proteins that had been reported as phloem proteins. Based on our results, we will discuss possible mechanisms underlying signal transduction mediated by long-distance transport system. We also report proteins and peptides in xylem sap.

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Phylogenetic profiling is used to find lineage-specific, conserved proteins in a set of sequenced genomes, and thus helps functional annotation of genomic data. I have developed a clustering software called Gclust, which uses the output of all-against-all BLASTP results, and outputs protein clusters, analogous to the COG of NCBI. In contrast with the COG, one can customize the dataset to infer clusters in the Gclust. In addition, datasets including both prokaryotes and eukaryotes can be processed. Such clusters are used for phylogenetic profiling. The Gclust server (http://gclust.c.u-tokyo.ac.jp/) was designed to provide all biologists with the ease in phylogenetic profiling. One can find easily all protein clusters that are conserved in a selected set of organisms. I will present some examples, such as the chloroplast proteins of endosymbiont origin, and the conserved proteins in nitrogen-fixing symbiotic bacteria.

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Comparative genomics based on synteny relies on sequence homology of genes within a selected set of genomes, but the increase in evolutionary distance reduces the estimation of orthology. In this study, we use homolog groups instead of orthologs. We also estimate correlations of gene locations between genomes. We obtained 16 cyanobacterial datasets from the Gclust server (http://gclust.c.u-tokyo.ac.jp) developed by N. Sato. As a result, significant correlation of gene location was found within a window of fifty genes. Based on such correlations, the cyanobacteria are classified into marine group, fresh water group and Anabaena family. In addition, the results of hierarchical clustering using these data are consistent basically with molecular phylogeny. These results show that the application of our method is not restricted to cyanobacteria.

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Distribution profiles of short sequences along a promoter region has been comprehensively analyzed in silico, and hundreds of short sequences have been detected as putative promoter constituents. Based on their localization profiles, the identified sequences were classified into three groups, including TATA box and REG groups. Identified sequences of the TATA box group are consistent with the ones reported in previous studies. REG includes functionally proved cis elements, and candidates of novel cis-regulatory elements. Comparison of LDSS-positive sequences from plant and mammalian genomes will be introduced. This method does not require any biological information such as microarray data, and can detect minor elements as well.

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Heliobacteria are strict anaerobic primitive photosynthetic bacteria that have a homodimeric type I reaction center (RC) complex. Their RCs are essentially analogous to the heterodimeric photosystem (PS) I reaction center. The redox components in the electron transfer chain of Heliobacterial RCs are not fully clear yet. We studied the flash-induced ESR signal in the isolated RC core complex of Heliobacterium modesticaldum, which is depleted of iron sulfur centers FA/FB. We detected the transient ESR signals different from those reported in the membrane preparations of this bacterium. The results suggested that the electron transfer pathways are somewhat different between PS I and the Heliobacterial RC.

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Most Chl a in the PS1RC complexes was removed without any loss of P700 by ether treatment, yielding the antenna-depleted PS1RC complexes that contain 12-14 Chl a per P700. (1) On addition of Phe a, Zn-Chl a and Cu-Chl a with phospholipids (PG), about one-third of the added Zn-Chl a was bound, similarly with Chl a, to the complexes. Phe a bound a half less than Chl a. Cu-Chl a bound exponentially with increasing its amounts added. (2) The initial rate of the P700 photooxidation increased by the number of the Zn-Chl a bound, while it was not enhanced by the bound Phe a or the bound Cu-Chl a. (3) These results suggest that the bound Zn-Chl a acts as efficient antenna as Chl a, while Phe a and Cu-Chl a could not bind to the Chl a-binding sites in the complexes, resulting in no recovery of antenna activities.

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Genes encoding subunits of photosystem I (PSI genes) in the cyanobacterium Synechocystis sp. PCC 6803 are actively transcribed under low-light conditions, whereas their transcription is coordinately and rapidly down-regulated upon the shift to high-light conditions. To identify the molecular mechanism, we searched for common light-responsive elements in the promoter region of PSI genes. First, by the deletion analysis of the psaAB and psaD promoters, we found that these promoters possessed a common light-responsive element located just upstream of the basal promoter region. These AT-rich upstream sequences enhanced the basal promoter activity under low-light conditions and their activity was transiently suppressed upon the shift to high-light conditions. Successive analysis of the other PSI promoters revealed that their light response was also achieved by AT-rich sequences located at the (-70 to -46) region. Thus, AT-rich upstream elements are responsible for the coordinated high-light response of PSI genes dispersed throughout Synechocystis genome.

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We previously reported that regulation of the suf operon by SufR, which is an Fe-S cluster binding redox sensor. In Escherichia coli, the suf operon is regulated by another transcription factor IscR which carries Fe-S cluster. IscR homologues are found in many cyanobacteria, but critical cysteine residues are not conserved. Here, we report the role of iscR homolog (slr0846) in Synechocystis sp. PCC 6803. DNA microarray analysis of a slr0846 disruptant showed marked decrease in expression of psaAB. Consistently, the mutants exhibit lower PSI/PSII ratio and decreased chlorophyll content. Growth of the mutants was retarded under normal light conditions and severely suppressed under high light conditions. These results suggested that the Slr0846 is involved in regulation of PSI transcript levels. To detect direct interaction of Slr0846 with the psaAB promoter, we are now doing gel shift assays. We also report a suppression mutation that recovers expression of psaAB.

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The biosynthesis of iron-sulfur (Fe-S) clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of Fe-S clusters. Here, we report biochemical properties of HCF101 from Arabidopsis which is involved in biogenesis of [4Fe-4S] proteins including photosystem I (Lezhneva, et al. Plant J. 37, 174-85). We were able to succeed in expressing and purifying HCF101 recombinant protein from E. coli cells. UV/vis absorption spectrum analysis indicated that the purified protein possessed a certain Fe-S cluster-like cofactors, which was sensitive to metal chelation by EDTA or reduction by dithionite. The results of gel filtration analysis of the purified protein and chloroplast stroma fraction suggested that HCF101 existed as a dimer. In addition to the results above, we would like to present the latest data of biochemical studies including an assay of Fe-S cluster transfer from HCF101 to apo-substrate.

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Photosystem I complex consists of 14 subunits and tightly binds LHCI to form PSI-LHCI supercomplex. It is believed that multiple steps are included in assembling a mature PSI-LHCI supercomplex from a number of subunit and cofactor components and would be assisted by several assembly factors. However, biochemical evidence is not sufficient to propose a reliable assembly mechanism. In a previous report, we identified an assembly intermediate in the green algae Chlamydomonas reinhardtii by pulse-chase protein labeling experiments. In this study, we characterized in more detail the assembly intermediate using immunochemical approach. We found that the assembly intermediate shows photosystem I activity but specifically lacks two peripheral subunits located in close proximity to LHCI. The size of the assembly intermediate was much smaller because LHCIs were easily dissociated from PSI-LHCI supercomplex during a mild detergent solubilization. Therefore, we suppose a working hypothesis of the assembly mechanism in PSI-LHCI supercomplex.

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Phycobilisome is a main light-harvesting complex in cyanobacteria and red algae. It has been suggested that phycobilisome transfers energy not only to PSII but also to PSI, although the molecular mechanism has not been elucidated. Previously, we reported that two rod-core linker proteins (CpcG1 and CpcG2) form distinct supramolecular complexes of phycobilisome in Synechocystis sp. PCC 6803. In contrast to the classic model, CpcG2-associated phycobilisome lacked core components and was suggested to serve as PSI antenna. We also showed that CpcG2 was predominantly associated with the isolated thylakoids, while CpcG1 was not tightly associated. Here, we solubilized and fractionated photosystems by glycerol density gradient centrifugation. The results suggest that CpcG2 is directly associated with PSI trimer. Specific interaction between CpcG2 and the thylakoid are also studied under various conditions. We will discuss the specific interaction of the CpcG2-phycobilisome and a possible mechanism of energy transfer to PSI.

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The chloroplastic NAD(P)H dehydrogenase (NDH) plays a role in cyclic electron flow around photosystem I to produce ATP, especially for adapting to environmental changes. While NDH contains 11 subunits that are homologous to the subunits of prokaryotic respiratory complex I (EC 1.6.5.3) and 3 subunits that are unique in chloroplastic and cyanobacterial NDH, subunits that serve as electron input device are not yet unidentified. To isolate the unidentified subunits, we performed the phylogenetic profiling and selected 37 Arabidopsis proteins as potential candidates for NDH subunits. Further characterization of T-DNA insertion mutants indicated that knockout of two ndh-gene-candidates actually impaired the NDH-dependent increase in chlorophyll fluorescence and drastically decreased the amounts of NDH complex. We discuss the possibility of two proteins as the novel NDH subunits.

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The NAD(P)H dehydrogenase (NDH) complex mediates photosystem I cyclic and chlororespiratory electron transport. Eleven plastid-encoded and three nuclear-encoded subunits have been identified so far, but the entire subunit composition is still unknown. The Arabidopsis crr3 mutant was isolated based on its lack in NDH activity using chlorophyll fluorescence imaging. The CRR3 gene (At2g01590) encodes an unknown protein containing a putative plastid-targeting signal and a transmembrane domain. CRR3 localize to thylakoids, and is essential for the accumulation of the NDH complex. NDH complex is also required for stabilizing CRR3. These results suggest that CRR3 interacts with the NDH complex. In contrast to the other NDH subunit, CRR3 is not conserved in cyanobacteria. We propose that CRR3 is a novel subunit of the NDH complex, which is specific to chloroplasts.

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Photosystem I (PSI) cyclic electron transport is essential for photosynthesis and photoprotection. In higher plants, the ferredoxin (Fd) -dependent pathway is the main route in PSI cyclic electron transport. Although a small thylakoid protein, PGR5 (PROTON GRADIENT REGULATION 5), is essential for this pathway, the fuction of the PGR5 protein is still unclear and there are numerous debates on the rate of electron transport in vivo and its regulation.
We evaluated Fd-dependent PQ reduction in ruptured chloroplasts by using a Arabidopsis mutant, pgr5. We detected high activity of PGR5-dependent cyclic electron transport even in the presence of linear electron transport. In the wild type, a competition between NADP⁺ photoreduction and PGR5-dependent cyclic electron transport was observed. From these results, it is suggested that the rate of PGR5-dependent cyclic electron transport is high enough to explain the mutant phenotype and the redox state of NADP⁺ regulates the activity of this pathway.

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Photosynthetic energy conversion in plants involves the formation of proton gradient across thylakoid membranes, but the mechanisms for balancing the membrane potential has been poorly elucidated. We found that drought stress induces selective decomposition of the ε subunit in the CFoCF₁ ATP synthase. Thylakoid membranes from the stressed leaves showed reduced efficiencies in proton gradient formation and energy coupling, but addition of the recombinant ε subunit significantly suppressed their leaky property. We conclude that the selective decomposition of the ε subunit induces partial uncoupling of the thylakoid membranes under drought, and hence contributes to the excess energy dissipation in chloroplasts.

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In higher plants under drought/strong light stress, limited entry of CO₂ into the leaf causes the suppression of photosynthetic carbon fixation, and the electron transport chain is prone to be over-reduced, leading to the oxidative damages in photosynthetic apparatus. To elucidate the regulation of photochemistry under such conditions, we analyzed the change in the proteome of the membrane fraction of wild watermelon leaves suffering the stress. Two-dimentional electrophoresis and Western blot revealed that there were multiple spots for the chloroplast Rieske protein, a subunit of the cytochrome b₆f complex, with very similar molecular weights but with different isoelectric points. Furthermore, some new spots with more acidic pI appeared under the stress, but disappeared upon resuming irrigation. These results suggested the involvement of post-translational modification of Rieske protein in the regulation of electron transport under excess light stress.

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In the purple bacterium Rhodovulum sulfidophilum, spheroidene monooxygenase (CrtA) converts spheroidene (yellow) to spheroidenone (red). Purpose of this study is to establish the whole cell biosensor system for environmental monitoring with low-cost using the color change evoked by CrtA in R. sulfidophilum.
An arsenite or dimethyl sulphide (DMS) sensor has a CrtA gene under regulation of the ars operator/promoter region and ArsR from E. coli or under regulation of the intrinsic DMS dehydrogenase gene promoter. Using the sensor strain, 0.6-6 μg/l arsenite or 3-30 μM DMS could be detected without adding any color-forming reagent. An increase in a molar ratio of spheroidenone and a shift in the color coordinate from a yellow to a red hue were observed, as the analyte concentration was raised. These results show that the genetically engineered R. sulfidophilum cells can be used to monitor quality of water environments by the naked eyes.

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