Light is essential for plant photomorphogenesis and photosynthesis. Weak light intensity leads to an elongated hypocotyle and pale green cotyledon such as in Arabidopsis. In case of immature leaf of tea, light limitation by covered culture improves greening. While a shading rate of increase leads to enhancement of greening of immature leaf, it also leads to a reduction in yield of tea leaf. Although physiological analyses have been achieved for shading effects, mechanism of greening by covering culture has been remain to be elucidated. The present paper reports the results of not only physiological but also anatomical and molecular study of immature leaf of green tea with covering culture.

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Fruit trees in Rosaceae family, such as apple, have a unique metabolic pathway, that is, Rosaceae trees synthesize sorbitol in source leaves and transport sorbitol to sink organs in addition to sucrose. Sorbitol synthesis leads higher photosynthesis rate comparing with plants, which synthesize sucrose as a sole translocate sugar. Sorbitol is known to be a compatible solute against water and cold stresses and it promote boron movement in plant body. To add sorbitol synthetic ability to non-sorbitol synthetic plants, sorbitol-6-phosphate dehydrogenase (S6PDH) from apple was transformed to Arabidopsis thaliana under the control of source leaf specific promoter, i.e. chlorophyll a/b binding protein (cab) promoter. 6 transformants were obtained. Although one transformant has trace level of sorbitol, other transformants synthesize certain amount of sorbitol. Growth of transformants deteriorated and the plants dead easily. The transformants have small, tick and rough leaves and their flowering time is slightly earlier than wild type.

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Ascorbate (AsA) content in suspension cells of peach (Prunus persica cv. 'Yuuzora') derived from fruit tissue was studied. At day 2 after transfer to new medium, a highest level of 0.4 μM/gFw was observed. AsA content was gradually decreased with the increase in total cell volume, reaching approximately 0.13 μM/gFw at day 18. The ratio of reduced form was 80-90 % which then decreased after day 10. No increase in total cell volume was observed after day 18. The pH of the medium varied lot by lot, although there was a tendency that it was around 4 by day 7, increasing after that, above 5 after day 18. The change in the levels of mRNAs encoding enzymes involving in AsA biosynthesis (L-galactonolactone dehydrogenase, L-galactose dehydrogenase, GDP-mannose pyrophophorylase, GDP-mannose epimerase, aldo-keto reductase) will be presented.

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Genes related to response of various types of stress particularly prone to alternative splicing in higher plants. We have demonstrated that the expression of an Arabidopsis SR protein, atSR45a, is induced by high-light irradiation (Plant Cell Physiol. 48: 1036-1049, 2007). Here we studied the function of atSR45a protein involved in spliceosome. Proteins interacted with atSR45a-2, which was presumed to be a functional form among six types of atSR45a products (atSR45a-1a~e, -2) generated by the alternative splicing event, were screened from Arabidopsis cDNA library by a yeast two-hybrid assay. A key protein involved in the 3^'-splice site recognition, U2AF³⁵b, was isolated by the screening. The interaction between atSR45a-2 and U2AF³⁵b in planta was thus directly examined by BiFC analysis. The YFP fluorescence was observed in the nucleus of onion cells co-expressing atSR45a-2 and U2AF³⁵b. These results suggest that atSR45a serves as a component in spliceosome assembly via interaction with splicing factors.

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The ndhK gene encodes a subunit of the chloroplast NADH dehydrogenase, and is co-transcribed with the adjacent ndhC and J genes on the tobacco chloroplast genome. The ndhK gene product is translated by polycistronic mRNAs but not by monocistronic ones. The ndhK mRNA contains four possible AUG start codons, three of which are within the upstream ndhC coding region. Based on in vitro translation analyses, we report that the major initiation site in tobacco chloroplast ndhK mRNAs is the third AUG codon within the upstream ndhC coding region, ndhK overlapping partly with ndhC. Detailed analysis of translation initiation of the ndhK coding region is reported.

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In eukaryotes, small RNAs play important roles in RNA silencing which are involved in many biological processes. The best studied endogenous small RNAs are microRNAs (miRNAs). In Arabidopsis thaliana, miRNAs are excised from primary miRNA transcripts forming stem-loop structures by the RNase III enzyme DICER-LIKE 1 (DCL1). This enzyme works coordinately with HYL1 and SERRATE. miRNAs are loaded into the RNA-induced silencing complex (RISC) where the complementary mRNAs are cleaved by ARGONAUTE 1, which has the slicer activity. The basic roles of these factors in the biogenesis of miRNA or the regulation of mRNA have been elucidated so far, but it remains incompletely understood how miRNAs regulate the development and physiological condition of plants. Here we studied the phenotype of hyl1 mutant plants which accumulate less miRNAs than wild type plants. Furthermore, we would like to report the possibility of the existence of another factor related to miRNA pathway.

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In various species, proteins that contain dsRNA-binding motifs (dsRBMs) play key roles in the regulation of cellular signaling and gene expression. Arabidopsis thaliana has 16 dsRBM-containing proteins, and we focus on HYL1/DRB-family dsRNA-binding proteins (HYL1/DRB1, DRB2, DRB3, DRB4 and DRB5).
Homozygous drb4-1 mutant plants changed into red-violet color to an extent dependent on their age and anthocyanin accumulation in their leaves had increased. DRB4 cDNA with 35S promoter complemented the phenotype of drb4-1 mutant plants. The results of immunoprecipitation experiments suggested that DRB4 interacts with the Dicer-like protein DCL4 in vivo and functions in the trans-acting siRNA pathway. Now, we are characterizing the components of DRB4 complexes and other DRB complexes.

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Double-stranded RNAs (DsRNAs) that contain a sequence homologous to the promoter sequence can be supplied to plant cells using a plant virus vector, which induce methylation on the promoter and subsequently induce repression of transcription, termed transcriptional gene silencing (TGS). We previously developed a plant virus vector based on the Cucumber mosaic virus (CMV), which is able to efficiently induce RdDM and TGS in plants.
To analyze the induction process of TGS, we cloned various portions of the CaMV 35S promoter sequence into the CMV vector, and inoculated the virus to Nicotiana benthamiana plants containing a single-copy GFP gene expressed under the control of the 35S promoter. We found that the efficiency of the induction of TGS depended on the length of the promoter segment triggering the RdDM, although the siRNAs corresponding to the 35S promoter accumulated to a similar level in TGS-induced and non-TGS induced plants.

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The tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) catalyzes the synthesis of α-linolenate in glycrolipids. A cosuppressed tobacco line was produced by introduction of the sense NtFAD3 transgene under the control of El2 promoter. In this line, the NtFAD3 siRNAs were generated. We introduced an RNAi vector containing an inverted repeat against the part of El2 promoter sequence into this cosuppressed line, and the resultant double transformant showed a recovered phenotype, namely increased α-linolenate content in associated with disappearance of NtFAD3 siRNA. The RNAi construct against the promoter sequence has been considered to cause RNA-directed DNA methylation, which would be followed by the conformational change of chromatin via histone modification. Here, histone modification arround the transgene sequence is investigated by immunoprecipitation of chromatin prepared from the cosuppressed plants and plants recovered from cosuppression.

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Arabidopsis mutant lines generated with ethylmethan sulfonate (EMS) have been selected using reporter genes under the regulation of photosynthesis gene RBCS-3B promoter in order to reveal the mechanisms underlying the organ-specific gene expression. It has been found that gene expression suppressed by exogenous promoters but not by endogenous ones is specifically released in rex2 (relaxed expression of transgenes) line.

DNA methylation status has been analyzed with bisulfite to realize the epigenetic regulation involved in the phenomenon that endogenous and exogenous promoters have the common sequences but the different activities. Methylated cytosine has barely been detected in the exogenous promoter, whereas some methylation has been done in the endogenous one. Furthermore, focusing on chromatin structure and modification of histon proteins, difference in the chromatin structure of exogenous and endogenous promoters has been examined. The mutated region has been narrowed within 50 kbp by genetic analysis, and further subjected to nucleotide sequencing.

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In plant genome, cytosine methylation is found not only at CG sites, but also at CNG and CNN sites. Cytocine DNA methylation at CNG sites is thought to be important especially for transposon silencing. Chromomethylase, which is regarded as a plant specific class of DNA methyltransferase so far, mainly controls CNG methylation. In Arabidopsis, Chromometylase 3 (CMT3) is a main regulator of CNG methylation. To investigate how CNG methylation works in rice genome, which has larger amount of transposons than that of Arabidopsis, we isolated a loss-of-function mutant of OsMET2a, the functional rice ortholog of CMT3. The phenotypes of osmet2a mutants observed suggested the importance of roles of CNG methylation in plant species having transposon-rich genome.

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CTP:phosphorylcholine cytidylyltransferase (CCT, EC 2.7.7.15) is a rate-limiting enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis. In Arabidopsis thaliana, CCT is encoded by CCT1 and CCT2. To understand the physiological importance of the CDP-choline pathway in plants, we have characterized the T-DNA insertion mutants cct1-1 and cct2 as well as the double mutant cct1-1 cct2. Although these mutants grew comparable to the wild type in the vegetative stages, the F2 generation of cct1-1 and the F1 generation of cct1-1 cct2 showed epigenetic homeosis that converted stamens into carpeloids. We herein examined methylation levels of the B-class genes of the ABC model for flower morphogenesis in the wild-type, cct1-1 and cct1-1 cct2 plants. We will discuss possible relationship between B-class gene methylation and epigenetic homeosis.

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The quality control system of the endoplasmic reticulum (ER) ensures that only folded proteins are transported to the down stream organelles. Misfolded proteins accumulated in the ER are degraded by the ERAD (ER-associated degradation), where misfolded proteins are degraded by the ubiquitin-proteasome system in the cytosol. A mutant form of vacuolar carboxypeptidase Y (CPY*) has been used as a model ERAD substrate in yeast cells. Using an Arabidopsis ortholog of yeast CPY (AtCPY), we have constructed a model ERAD substrate in plant cells. When GFP-tagged AtCPY was expressed in Arabidopsis culture cells, it was transported to the vacuole. In contrast, AtCPY*-GFP containing a mutation orthologous to that of CPY* was retained in the ER and degraded in the proteasome-dependent manner. Degradation of AtCPY*-GFP was inhibited by co-expression of a dominant-negative mutant of CDC48 that is necessary for ERAD. We are also constructing a membrane bound ERAD substrate.

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SUMO (Small ubiquitin-related modifier) is a member of the superfamily of ubiquitin-related polypeptides that become covalently attached to various intracellular target proteins as a way to alter their function, location, and/or half-life. Arabidopsis thaliana has 8 SUMO genes (AtSUMO1-8), which are suggested to have individual function. Expression analysis using the GUS reporter gene revealed the tissue specific expression pattern of each SUMO gene. We are now investigating the processing of C terminus, the ability of Sumoylation and the target specificity of each SUMO isoform. Moreover, it was reported that SUMO could convert the protein function through the non-covalent interaction with the target proteins. Since the Gly-Gly motif, which is necessary for the covalent modification, is not conserved in AtSUMO4, 6 and 7, they might function in the protein conformation change through the possible non-covalent interaction with their target proteins.

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Diatoms play an important role as a primary producer on eanth. Recently, whole genome sequences of two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum, were published by Joint Genome Institute (http://www.jgi.doe.gov/ ). We have reported isolation and characterization of PS I complexes from a centric diatom, Chaetoceros gracilis. Here, we show a simpler method to prepare PS I complexes from C. gracilis and T. pseudonana as well. The thylakoids solubilized by n-dodecyl- β -D-maltoside (DDM) was diluted to decrease the DDM concentration and then centrifuged. The resulting pellet was resuspended, and PS I complexes were purified by anion exchange chromatography. This purified PS I complexes had a high electron transfer rate and had all the electron transport components, including menaquinone-4 as A1. We will also discuss characteristics of the fucoxanthin-chlorophyll-binding protein I tightly bound to the PS I core complexes.

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Light-harvesting chlorophyll a/b complex I (LHCI) is antenna complex associated with photosystem I (PSI) core complex. In higher plants, six LHCI distinctive polypeptides are found and four out of six were identified in crystal structure of PSI-LHCI supercomplex. In contrast, Chlamydomonas reinhardtii contains nine distinctive LHCI subunits that were all copurified with PSI-LHCI supercomplex. However, the copy number and arrangement of LHCI in PSI-LHCI supercomplex have not yet been determined. Here we report quantitative estimation of stoichiometry of LHCI subunits in PSI-LHCI supercomplex. The polypeptides that are similar in size were separated by optimized SDS-PAGE systems and were stained with fluorescence dye, FLAMINGO. Intensity of polypeptides was measured by scanning the stained gel and the amount was estimated by calculating the area of the signal, assuming that the signal intensity is proportional to the molecular mass. We will also discuss structure and arrangement of LHCIs in PSI-LHCI supercomplex.

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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 complexes that contain about 13 Chl a/P700. (1) On addition of Chl d with phospholipids (PG), about one-fourth of the added Chl d was bound to the complexes, of which ratio was smaller than that of the exogenously added Chl a, suggesting a low affinity of Chl d to the Chl a-binding sites. (2) The initial rate of the P700 photooxidation was increased by the binding of Chl d, but only with about a half efficiency of the bound Chl a. (3) The Chl d-bound PS1 complexes emitted the fluorescence both from Chl a and d. The excitation spectra showed the excitation energy equilibration between Chl a and d. These results suggest that the bound Chl d acts as an energy sink, transferring its excitation energy to P700.

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All-trans carotenoids have low-lying singlet states with different symmetries and the energy transfer from carotenoid to bacteriochlorophyll is governed by internal conversion and vibrational relaxation. According to theoretical calculation of Tavan and Schulten in shorter-chain carotenoids and our work (Furuichi et. al. 2002), the order of energy states of carotenoid are, from higher energy level, as 1B_u⁺, 3A_g^-, 1B_u^- and 2A_g^- for the number of conjugated double bond n = 11-13. In the present investigation, we focus our attention on detection of those energy states. Especially detection of the 3A_g^- states is rather difficult so we used 30 fs pulse duration and THF as a polar solvent. On the measurement, we could observe stimulated fluorescence from not only optically-active 1B_u⁺ but several low-lying hidden states in the initial stage. According to energy diagram, those stimulated fluorescence from low-lying states are concluded as 1B_u^- and 3A_g^-.

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   Changes in the structure of 15-cis-spheroidene in the T₁ state and intermolecular interaction with peptide binding pocket in the reaction center have been examined by time-resolved Raman spectroscopy at room temperature as a follow-up study of time-resolved EPR spectroscopy [Kakitani et al. Biochemistry 45 (2006) 2053].
   Immediately after triplet excitation, a strong enhancement of the 13-methyl asymmetric-deformation Raman line was seen at 1458 cm^–1, which was ascribed to severe steric interaction between the Car 13-methyl carbon and the peptide C=O oxygen of Gly M178. This steric interaction seems to perturb the initial rotational motion around the cis C15=C15' bond, and to trigger the subsequent rotational motion around the neighboring trans C13=C14 bond. Careful consideration lead us to the idea that the dynamic mechanism through the rotational motion around the central double bond(s) plays the most important role, rather than the static mechanism through the spin–orbit coupling, in the triplet-energy dissipation.

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A series of electron-transfer reaction is started by the charge separation in the reaction center (RC), which must be influenced by the redox potential of the environment. From this viewpoint, we controlled the redox potential of RC, chemically and electrochemically, and examined the changes in the electronic-absorption spectra of RC from Rba. sphaeroides R26.1. In the case of chemical control, we used “sodium ascorbate” as the oxidant and “dithionite” as the reductant. In the case of electrochemical control, we applied voltage to the RC suspension to which mediators, ferrocene and its derivatives, were added. When the voltage was high enough, the RC was oxidized; whereas in low, reduced. Now, we are trying to excite the special-pair bacteriochlorophyll in the RC, and to measure the sub-μs time-resolved absorption spectra.

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In the water oxidizing center of photosystem II, two water molecules are known to cleave into one molecular oxygen and four protons through a light-driven cycle of the S states (S0-S4). However, the definite conclusion has yet to be reached about the number of protons released during individual S-state transitions. In this study, we have estimated the proton release pattern during water oxidation using Fourier transform infrared (FTIR) spectroscopy. The PSII core complexes from the YD-less mutant (D2-Y160F) of Thermosynechoccocus elongatus were suspended in a high-concentration Mes buffer, and flash-induced FTIR difference spectra were recorded. The Mes bands were extracted from the spectra by subtracting the corresponding spectra recorded in deuterated-Mes (D13-Mes). The flash-number dependence of the intensity increase in the Mes signal showed a typical period-four oscillation. Simulation of this oscillation showed that the proton release pattern was consistent with 1:0:1:2 for the S0->S1->S2->S3->S0 transitions.

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The electron transfer reactions of PSII are reversible and the charge-separated states can be partly lost in charge recombination process. The charge recombination reaction is known to re-populate P680^* and to produce delayed fluorescence (DF) in PSII. However, We measured comprehensive decay process of prompt and delayed fluorescence lifetime from 1 ps to 10 sec after the laser excitation in Acaryochloris marina that contain Chl d as the major pigment. We discussed the mechanism of photochemical reaction in PS II of this unique organism.

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Thermo- and acido-phylic primitive red alga, Cyanidioschyzon merolae, is considered to be one of the most primitive eukaryotic oxygenic photoautotrophs. We investigated the biochemical characteristics of photosystem II ( PSII ) complexes in C. merolae. The PSII complexes, retaining high oxygen-evolving activity, were purified as monomeric and dimeric forms. The chlorophyll a specific yield of purified PSII monomer plus dimer was approximately 20% based on the initial solubilaized thylakoid membrane. The proportion of the purified PSII complexes was approximately 1 monomer to 4 dimers. Both of the monomer and dimer were consisted of over 17 subunits, including PsbO, PsbQ, PsbU, PsbV, and Psb30 ( Ycf12 ). The composition of pigments and quinone was the same as that of the PSII complexes from a cyanobacterium, Thermosynechococcus elongatus. These results indicate that the PSII complex in C. merolae is similar to that in cyanobacteria rather than that in green plants.

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PsbY is one of the low molecular mass subunits of oxygen-evolving photosystem II (PSII), with a molecular mass of 4.7 kDa and single trans-membrane helix. Its location, however, has not been identified in the current crystal structure of PSII. Here, we constructed a PsbY-deletion mutant of Thermosynechococcus elongatus, crystallized, and analyzed the crystal structure of the PsbY-deleted PSII dimeric complex. From the difference-Fourier map of wild type-minus-mutant PSII, two strong positive regions were observed in the periphery of PSII dimer which corresponded to an unassigned trans-membrane helix located adjacent to the α- and β-subunits of cytochrome b559 in the 3.7 Å structure reported previously (Kamiya and Shen, 2003, PNAS, 100, 98-103). This helix was also found in the 3.0 Å structure and has been designated helix X2 (Loll et al., 2005, Nature, 438, 1040-1044), but was not present in the 3.5 Å structure (Ferreira et al., 2004, Science, 303, 1831-1838).

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Cl^- is one of the essential cofactors indispensable for photosystem II oxygen evolution. Its location in PSII, however, has not been determined in the current crystal structure due to limited resolution as well as the difficulties in detecting the chloride ion. Here we substituted Cl^- with Br^- in PSII from a thermophilic cyanobacterium Thermosynechococcus vulcanus, and crystallized, analyzed the crystal structure of Br-substituted PSII. Difference-Fourier and anomalous difference-Fourier map between native PSII (Cl-PSII) and Br-substituted PSII revealed two sites of binding of Br^- surrounding the Mn₄Ca-cluster, which were located in each side of the metal cluster. The distances between each of the Br-binding site and Mn atoms are similar with a value of 7.0 Å, and those between Br^- and Ca are also similar with a value of 10.0 Å. These two Br-binding sites may represent the Cl-binding sites in PSII that have important functions in oxygen evolution.

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Lys residues on PsbP responsible for binding with Photosystem II (PSII) remain to be elucidated, although crystal structure of tobacco PsbP has been shown. Previously, we reported using chemical modification of PsbP that the positive charges on Lys residues, especially Lys residues in six domains (Lys11-Lys14, Lys27-Lys38, Lys40, Lys90-Lys96, Lys143-Lys152 and Lys166-Lys174), were important for electrostatic interaction with PSII. In this study, we constructed various spinach PsbP mutants by replacing these Lys with Gly and reconstituted these mutants with PSII to determine the essential lysine residues of PsbP responsible for the binding. The results revealed that Lys residues in Lys11-Lys14, Lys27-Lys38, Lys143-Lys152 and Lys166-Lys174 were essential for the binding. Furthermore, we constructed PsbP mutants by replacing each Lys residue in these domains with Gly and examined their binding abilities. On the basis of these results and crystal structure of PsbP, we will discuss the binding sites of PsbP with PSII.

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We succeeded in isolation of PSII particles retaining high oxygen-evolving activity from a marine diatom, Chaetoceros gracilis (B.B.A. in press). The PSII particles contained 229 Chl a/2 pheophytin. The PSII core complex consisted of CP47, CP43 and D1/D2 core which was prepared by Blue-Native PAGE also contained about 80 Chl a, 14 β-carotene and 3 diadinoxanthin per 2 pheophytin. Thus, the antenna size of the diatom PSII was significantly larger than those of the other organisms. In this study, we attempted to separate CP47, CP43 and D1/D2 core from the PSII particles and analyze pigment compositions in these polypeptides. The diatom PSII particles solubilized with HTG were subjected to DEAE TOYOPEARL and eluted by circulation with buffers containing HTG and various NaCl concentrations. Consequently, we partially purified CP47, CP43, D1/D2 core and will report about pigment analysis of their polypeptides.

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In order to investigate the whole genetic system of legume species, we have been analyzing the genome of a model legume, Lotus japonicus.
Combining clone-by-clone sequencing of large insert clones selected from gene-rich regions with random genome sequencing, genome sequence information covering approximately 90% of the L. japonicus ESTs has been accumulated. The accumulated sequence information was subjected to automatic assignment of protein-coding genes, and as a result, 31000 gene structures could be identified. General features of the gene composition of the L. japonicus genome were surveyed using predicted gene information, and significant increase of gene number compare to Arabidopsis thaliana was observed in several gene families, such as ARF family transcription factor and LysM family receptor-like kinase.
The latest status of the genome 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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As a member of the International SOL Project, we are sequencing the euchromatic region of the chromosome 8, estimated length of which is 17 megabases (Mb). As of Nov. 2007, we finished 91 BACs to Phase 3 generating 9,256,972 bp non-overlapping sequence. We are also accumulating whole genome shotgun sequences derived from a mixture of low-copy BACs in order to use them for filling gaps in the BAC tiling paths. We have already accumulated 1.2 million reads giving a total length of 860 Mb. Gene coverage was assessed using the tomato gene index and 58% of unigenes were shown to be covered by the shotgun sequences. We launched on development of DNA markers using the tomato EST information and mapped over 500 microsatellite markers on an interspecies genetic map. We are currently developing markers polymorphic between tomato cultivars aiming at accelerating map-based cloning and QTL mapping in tomato.

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Plant peroxidases form a numerous multigenic family in higher plants. We have surveyed peroxidase genes in the Populus trichocarpa database and found at least 79 sequences encode complete peroxidase protein with manual annotation. Diversity of peroxidase isoenzymes was investigated from genome structure and amino acid sequences. The coding sequence of the majority of the peroxidase genes are disrupted by three introns. P. trichocarpa PO44 and PO85 were suggested to be an ancestral poplar peroxidase genes by the phylogenetic tree analysis. Within the protein structure of peroxidases, observed changes of highly conserved residue may cause significant change in enzyme kinetics, substrate specificities, and functions. An unique peroxidase isoenzyme from P. alba, CWPO-C, has broad substrate versatility, and Tyr-74 and/or Tyr-177 located on the protein surface were postulated as its oxidastion site. P. trichocarpa PO13, that is ortholog of CWPO-C, was the sole peroxidase possessing both Tyrosine residues in P. trichocarpa.

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Membrane traffic is essential in cellular metabolism and activities of living organs. In plant, transport systems play roles in the acquisition of essential elements as well as in the maintenance of ion homeostasis responding to various environmental stresses. To perform comprehensive analysis of the transporter coding genes in plant species, possible transporter genes classified into 59 families were predicted by using profile and similarity search against the proteome data of Arabidopsis, poplar, rice as well as the UniGene data of 16 plant species. Ortholog prediction by all against all similarity search and phylogenetic profiling of transporter genes were performed to characterize constitution of genes of each family. In this study, we report the results of genome wide comparison in plant transporter genes. Furthermore, these data of possible plant transporter genes were integrated into a relational database with the annotations of predicted domain organization and transmembrane regions.

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After screening of over c.a. 24,000 transgenic Arabidopsis plants randomly expressing the normalized rice full length cDNAs, 8 FOX mutants were isolated that mimic phenotype of over-expression of either iaaM or tms1 (bacterial IAA biosynthesis gene). When cDNAs isolated from these lines were re-transformed into Arabidopsis, transgenic plants showed the original auxin phenotype in T1 generation. These mutants were categorized into three different groups according to the auxin dose-curve response of main root. Among these 8 genes, annotations of two genes show obvious correlation with auxin synthesis (YUCCA homologue) and auxin signal transduction (IAA homologue), respectively. Function of these genes and other rice auxin gene candidates will also be discussed.

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To isolate useful genes from rice by FOX Hunting System, Arabidopsis was transformed using a library of Agrobacterium that has about 13,000 of ruce full-length cDNAs in expression vector. More than 20,000 lines of transgenic plants (rice FOX lines) were screened for high-salinity tolerance by sowing T2 seeds on agar medium containing 150 mM NaCl. As a result, 208 lines were isolated as candidates of high-salinity tolerant lines. Rice cDNA inserted in 181 lines were revealed by gPCR. So far, the isolated 17 cDNAs were introduced in Arabidopsis and 12 retransformed lines showed high-salinity tolerance. R07047 is a line isolated as a high-salinity tolerant line. A rice cDNA for a protein with C2 domain was inserted in R07047 genome. R07047 also showed improved tolerance to osmotic and drought stresses. This work is supported by the Special coordination funds for Promoting Science and Technology.

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To find the function of a gene, systematic analysis of phenotypes is vital. We have proposed a new methodology of mutant phenotypic analysis based on precise three-dimensional (3D) measurement by a laser range finder and micro X-ray computed tomography. However, their measurement systems cannot acquire gene expression data. In this report, we introduce a method to quantify gene expression and location in time series of images towards digital whole mount in situ hybridization(digital WISH). Concretely, we collected two-dimensional image sequence of gene expression using Luciferase tag (LucTag) lines developed in the RIKEN PSC by the ARGUS-50 system. In preliminary data, we found gene expression in the digital image data at shoot apical meristem, root apical meristem, lateral root, and so on. In special, the difference of quantified expression values at the cotyledons and true leaves from the digital image data is discussed.

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Structural analyses of cell are important to bridge molecular functions and phenotype. Recently, various bioimaging technologies are developed and obtained microscopic images are organized as databases. On the other hand, image analyses are depends on human observations and manual measurements in many cases. We are currently developing efficient method for image evaluation of plant cell structures. As a target of analysis, we used confocal images of cytoskeleton and organelles visualized in tobacco BY-2 cells. To evaluate the distribution of images, we tried to utilize clustering analyses. In prior to the clustering, we must develop appropriate distance metrics between images, because standard metrics were lacked in bioimaging analyses. We explored optimizing methods to select the metrics using metadata including annotations. As a result, we developed new clustering algorithm, which can adapt various observation targets. Using this algorithm, we evaluate the cytoskeletal and organellar dynamics during cell cycle progression.

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We have developed a tool, named "RiCES", which is easlily usable by accessing http://hpc.irri.cgiar.org/tool/nias/ces and help research of gene expression mechanism of rice. Researchers who interested in certain gene sets, e.g. drought stress responsive genes, can apply those genes to RiCES, and can obtain cis-element candidates more frequently distributed in promoter region of those genes than others. RiCES can list conserved motifs from promoter region of given gene set, and can evaluates the likelihood of these motifs, as well as those of previously known cis-element motifs. The evaluation is achieved by association rule analysis, a popular data mining method to find hidden relationships. The candidates showing the highest likelihood (specificity) are retained in the final cis-element candidate list. Here we present the current progress of case studies to evaluate accuracy of the tool and to achieve new discovery of cis-elements.

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We classified more than 2,300 ATH1 GeneChips, and found that co-expression linkage tends to intensify with dimensional compression of data matrix using singular value decomposition (SVD). This indicates that the process contributes effectively to the functional prediction of unknown genes. Indeed, the data matrix reconstructed by using up to 40 singular values was sufficient to reproduce the correlations in the fundamental circadian clock and the regulatory relationship of PMG1. The correspondence between singular values and arrays also supported the importance of different tissue types: shoot, root, and stamen (other than leaf) contributed to largest singular values.
The predictive reliability of co-expression relationship depends on the quality and the diversity of dataset selected. So far, little evaluation study has been performed to compare online repositories, while many groups provided co-expressed gene lists based on different dataset and similarity measures. We believe that our evaluation will be helpful for designing transcriptome analysis.

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Mutants having an abnormal photosystem stoichiometry had been effectively isolated by chlorophyll fluorescence induction kinetics from the cyanobacterium Synechocystis sp. PCC 6803. Thus, the kinetics is expected to be a valuable clue to predict function of gene. The change of kinetics could be described as vectors, since the changes are defined by both magnitude and direction. Addition of inhibitors brought about changes on the kinetics by concentration-depending manner, and the pattern of the change in the kinetics varied with different inhibitors. Thus, the length of the vectors represents the extent of the change while the direction of the vectors could be used as an index of the mode of inhibition. When we picked up the mutants with an abnormal photosystem stoichiometry, the phenotypic vectors fell into a certain range of direction. The results suggest that the direction of the phenotypic vectors closely correlate with the function of genes.

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Conservation of gene order in different genomes is a measure of genome evolution. Conventional way of such analysis relies on sequence homology of genes within a selected set of genomes, but the increase in evolutionary distance reduces the reliability of orthology. In this study, we used catalogs of homologous genes that conserved in a set of cyanobacterial genomes estimated by the Gclust server ( http://gclust.c.u-tokyo.ac.jp ) . We analyzed the distance relations of homologous genes between genomes. We previously reported a result of comparison of 16 cyanobacteria. In that result, homologous genes formed neighboring series and formed mosaic structure in global scale. In this presentation, we report the advanced result from the enhanced dataset which contains 25 cyanobacterial genomes .

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Over 1600 genes encoding transcription factors have been identified in the Arabidopsis genome sequence. However, their physiological functions are not yet fully understood. In this study, a small subfamily of single-Myb transcription factor genes, designated as RSM1-RSM4 (RADISLIS-LIKE SANT-MYB 1-4), was characterized. Here, we mainly examined the RSM1 gene, suggesting that this gene plays a role with a close connection to the HLS1 (HOOKLESS 1) gene during the early morphogenesis. The etiolated seedlings overexpressing RSM1 showed several phenotypes similar to those of hls1-1, namely, lack of apical hook with short hypocotyls, and defectiveness in gravitropism. Both RSM1-ox and hls1-1 seedlings were hypersensitive to red light during the early photomorphogenesis, thereby displaying shorter hypocotyls than do the wild type seedlings. These results will be discussed in the context that RSM1 might be implicated in the HLS1-mediated auxin signaling, which is responsible for regulation of the early photomorphogenesis of Arabidopsis thaliana.

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Arabidopsis thaliana has 11 members belonging to the typical type-B ARR (AUTHENTIC RESPONSE REGULATOR) family. Among them, 7 ARRs are implicated as DNA-binding transcription factors in the phosphorelay-mediated cytokinin signal transduction network. To gain insights into the functions of the cytokinin-associated type-B ARRs, here we established an arr1 arr10 arr12 triple loss-of-function mutant, which showed remarkable and severe phenotypes. The observed cytokinin-associated phenotypes of arr1 arr10 arr12 were highly analogous to those reported for certain ahk2 ahk3 ahk4/cre1 triple mutants, which have virtually no cytokinin receptor to propagate the phosphorelay signal transduction. Taken together, it was demonstrated that ARR1, ARR10, and ARR12 together play essential (or general) roles in cytokinin signal transduction. These results will be presented together with the idea that the other type-B ARRs (ARR2, ARR11, ARR14, and ARR18) may play more specific roles spatially and temporally in plants.

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Multiple pathways have been proposed for the biosynthesis of auxin, indole-3-acetic acid (IAA), in plants, and the underlying intermediates are still largely unknown. Indole-3-acetaldoxime (IAOX) has been assumed as a possible intermediate of the Yucca pathway. IAOX has been defied as an intermediate of indole glucosinolate (IG) biosynthesis in Arabidopsis. To distinguish whether IAOX is involved in the Yucca pathway, we developed a new method to analyze IAOX using LC-TOF-MS/MS and D₅-IAOX. As a result, IAOX was detected from wild type seedlings of Arabidopsis, whereas it was not found in the double knockout mutants of Cyp79B2 and Cyp79B3 genes, which encode cytochrome P450 monooxygenases catalyzing the conversion of TRP to IAOX. In addition, we were not able to detect any IAOX from non-crucifer plants that we analyzed. From these results, we concluded that IAOX is involved in the IG biosynthetic pathway in crucifer plants, but not in the Yucca pathway.

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Although phototropism is regulated mainly by photoreceptors, phototropins, several studies suggested the modulation of phototropism by other photoreceptors, phytochromes. In our present study, we isolated the flabby mutant that shows phytochrome-inducible hypocotyl bending in Arabidopsis. The FLABBY gene encodes the ABC-type auxin transporter, PGP19, and its expression is suppressed by the activation of phytochromes. Our results indicate that the phytochromes have at least two effects upon the tropic responses of the hypocotyls in Arabidopsis; the enhancement of hypocotyl bending through the suppression of PGP19, and a PGP19-independent mechanism that induces hypocotyl bending. By the use of an auxin polar-transport assay and DR5:GUS expression analysis, we find that the phytochromes inhibit basipetal auxin-transport and induce the asymmetric distribution of auxin in the hypocotyls. These data suggest that the control of auxin transport by phytochromes is a critical regulatory component of hypocotyl growth in response to light.

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N-1-naphthylphthalamic (NPA) causes the abnormal growth and development of plants by suppressing polar auxin transport. The mechanisms underlying this inhibition, however, have remained elusive. In our current study in Arabidopsis, we show that a defect in the auxin transporter P-glycoprotein 19 (PGP19) suppresses the inhibitory effects of NPA on hypocotyl phototropism and gravitropism, but not on hypocotyl elongation. Expression analysis using the auxin reporter gene DR5:GUS further suggests that NPA inhibits the asymmetric distribution of auxin in a PGP19-dependent manner. These data indicate that PGP19 plays an important role in the mechanism of auxin lateral translocation inhibition by NPA. In addition, we introduce recent studies on the phenotype of pin3pgp19 mutant in this meeting.

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IAA19 is an auxin-responsive gene which mediates tropic responses in hypocotyl and lateral root formation (Tatematsu et al., 2004). For further understanding of IAA19 gene expression, 3kbp promoter and two reporter genes (fluorescent protein and beta-glucuronidase) were fused individually, and introduced into Arabidopsis. In a root of transgenic plants, IAA19:reporter expressed in protoxylem, metaxylem, root cap, lateral root cap. Sporadic expression of IAA19:reporter was observed in central stele along with primary root, and some of them were primordia of lateral root. Luciferase was employed as no-invasive reporter (pIAA19:luc). Time-lapse imaging of bioluminescense revealed that pIAA19:luc expressed in prior to the formation of lateral root. On the other hand, asymmetric expression in lateral root cap was observed when root tip was placed in horizontal position. Further incubation of the root reoriented root tip vertical and the asymmetric expression diminished. We will discuss the advantages of bioluminescence imaging for auxin-responsive gene expression.

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We have previously proposed a new and simple growing system to dissect morphology and elongation by continuous mechanical stimulation on Arabidopsis roots: roots are allowed to grow on dialysis membrane-covered agar plate placed horizontally, resulting in providing continuous mechanical stimulation upon root tip during bending downward. Growth of horizontally grown Arabidopsis thaliana seedlings was found to be almost half as compared to that of vertical control and the reduced elongation of horizontal roots was attributed to enhancement of the ethylene response without that of the ethylene production.
In an effort to understand the effects of continuous mechanical stimulation on Arabidopsis root morphology and elongation, we analyzed hormonal cross-talk especially between ethylene and auxin. The role of auxin in the morphology and elongation will also be discussed based on the DR5::GUS and IAA2::GUS expression studies and the estimation of auxin responsive genes by real-time PCR.

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A plant hormone, ethylene, suppresses 26S proteasome-mediated proteolysis of the EIN3 transcription factor for ethylene signaling in Arabidopsis and allows the accumulation of EIN3. The proteolysis of EIN3 has been suggested to be mediated by SCF^EBF1/2 that contains either of the closely related F-box proteins, EBF1 or EBF2. Here, we show a feedback regulation in ethylene signaling that is caused by direct up-regulation of EBF2 gene by EIN3. Although EBF1 and EBF2 showed a comparable activity for repression of the EIN3-dependent transcription, only the EBF2 promoter was induced by ethylene. Our results revealed that EIN3 directly binds and activates the EBF2 promoter. The ebf2 mutant transformed with the EBF2 gene under the control of a mutated EBF2 promoter in which the EIN3-binding site was disrupted displayed ethylene-hypersensitive phenotype as ebf2 did. These results indicate that ethylene signaling involves a feedback regulation mediated by EBF2 for fine-tuning signaling.

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Immunomodulation is a means to modulate an organism's function by antibody production. Previously, we have succeeded in obtaining GA-deficient phenotypes in arabidopsis and tobacco by expressing single-chain variable fragment (scFv) against bioactive gibberellin (GA), GA4/1. Likewise we have introduced a scFv against GA24/19, biosynthetic precursors of bioactive GAs, into tobacco, which also showed a dwarf phenotype. In arabidopsis, however, the scFv was not well accumulated and its effect was obscure. In this study, we expressed the same scFv as a fusion with GFP. Consequently, transgenic arabidopsis plants accumulated the fusion protein at high level, and showed apparent GA-deficient phenotypes. The phenotype was clear especially in bolting stage, such as late flowering and early completion of the primary shoot growth. These phenotypes were different from those observed for lines expressing anti-GA4/1 scFv. The different mode of immunomodulation, inhibition of GA action and GA biosynthesis, may reflect on the differential phenotypes.

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GAMYB was identified as a positive transcriptional regulator of gibberellin (GA)-responsive genes in barley aleurone. Recently, we showed that the rice GAMYB, OsGAMYB, is also essential for the anther development by using gamyb.
We observed abnormal anthers of gamyb and GA-related mutants, and found that all these mutants showed similar defects in tapetum and exine development. A microarray analysis suggested that the expression of almost GA-responsive genes in anthers is under the control of OsGAMYB. Almost GA-responsive genes contain putative GAMYB binding motifs in promoter regions. We focused on two GA-responsive genes, involved in exine formation, to examine that OsGAMYB really targets GA-responsive genes. OsGAMYB specifically interacted with these putative GAMYB binding motifs in vitro. Furthermore, the mutations to these motifs showed the reduced GUS expression in promoter-GUS lines. Based on these results, we conclude that GA controls these genes expression through the direct regulation by OsGAMYB in anther development.

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Gibberellins (GAs) have many biological roles in higher plants. We performed genetic analysis on rice GA-related mutants, including GA-deficient and -insensitive mutants. The analysis revealed that rice GA-deficient mutations are not transmitted as Mendelian traits to the next generation following the self-pollination of F1 heterozygous plants, although GA-insensitive mutations are transmitted normally. In order to understand these differences in transmission in GA-deficient and GA-insensitive mutants, we examined the effect of GA on microsporogenesis and pollen-tube elongation in rice using new GA-deficient and -insensitive mutants that produce semi-fertile flowers. Phenotypic analysis revealed that the GA-deficient mutant, rpe1, is defective in pollen-tube elongation, resulting in a low fertilization frequency, whereas the GA-insensitive semi-dominant mutant Slr1-d3 is mainly defective in viable pollen production. We also examined the quantitative RT-PCR analysis of GA-biosynthesis and signal-related genes. Based on the observations, we will discuss the ways of transmissions of these GA-related mutations.

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