Current methods for determining gene function simply cannot keep pace with the rapid accumulation of new sequence information. In the case of Synechocystis PCC 6803 genome, sequence information is now available for the estimated 3200 genes residing in its 3.6 million nucleotide genome, yet definitive function has been assigned to less than two thousands of these. The classical approach to assigning each gene its function is to analyze the phenotypes of mutants. Chlorophyll fluorescence kinetics is excellent phenotype in the sense that changes in the fluorescence reflect numerous kinds of mutations in the genome. We showed possibility to systematically predict function of genes by the analysis of the fluorescence kinetics of mutants. The data are available on our web-based database and can be analyzed quantitatively. We will discuss what kind of genes is suitable for such analysis and what is not.

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Anabaena, a kind of cyanobacteria, can grow heterotrophically under dark conditions by sugar addition as well as autotrophycally with light illumination. Early studies by electron microscopy revealed that some cyanobacteria show changes in morphology of thylakoid membrane and composition of pigment-protein complexes in cases of heterotrophic incubation. Microscopic fluorescence spectral analysis, which provides us fluorescence spectra of thylakoid membrane with high spatial resolution, will help us understand changes in properties and morphology of thylakoid membrane. Our two-photon excitation line-scanning fluorescence spectral microscopy can record fluorescence spectra with nearly diffraction-limited spatial resolution and wavelength resolution of 2 nm. We studied how thylakoid membrane of anabaena depends on nutritional environment. We have confirmed durability of Anabaena thylakoid membrane after obtaining a few tens of z-sections of spectral imaging. We have acquired highly accurate fluorescence spectra and intensity distribution of heterotrophically incubated anabaena cells, which exhibit characteristic features in comparison to autotrophic ones.

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Cyanobacteria are oxygenic photosynthetic bacteria, many of which have ability to fix nitrogen. Filamentous cyanobacteria of the genus Nostoc produce differentiated cells such as heterocysts (specialized cells for nitrogen fixation) and hormogonia (transient motile filements which function as infective units), and many of them are known to establish symbiotic association with various eukaryotic hosts, including plants, algae and fangi.
Molecular-phylogenetic analyses of free-living and symbiotic Nostoc species suggest that symbiotic association may have evoloved polyphyletically within the clade. Co-culturing experiments using a bryophyte host indicate that their ability of hormogonia formation may not correspond to plant-infection efficiency.
Continued molecular-biological and physiological study of Nostoc species will provide a clue to understand mechanisms and evolution of the cyanobacterium-plant symbioses.

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Phosphorylation is one of the most frequent and significant modifications that regulate a large variety of proteins in plants. However, a comprehensive overview of experimentally verified phosphorylation events in plants has been lacking, while this is now available for the other organisms. Here we present large-scale phosphoproteome analyses of Arabidopsis and rice employing powerful MS-based technologies combined with an improved version of the phosphopeptide enrichment methods ¹⁾.
Using un-fractionated whole cell lysates of Arabidopsis and rice cells, we identified 3,937 and 5,510 highly confident phosphorylation sites, respectively, which are the largest data sets in plants to date. To our surprise, the proportions of phosphotyrosine among the phospho-residues in Arabidopsis and rice are estimated to be 4.2% and 2.8%, respectively, which are strikingly close to that in humans. These results clearly indicate that tyrosine phosphorylation events play an important role in plants.
1) Mol Syst Biol. 2008;4:193. Epub 2008 May 6.

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Vacuole plays an important role in maintaining the homeostasis of plant cells, that is, the maintenance of the turgor pressure, the accumulation of inorganic ions and metabolites and the degradation of discarded proteins. These functions of the vacuole predict that the vacuole include many kind of substances. We have analyzed contents of intact vacuoles directly and comprehensively by using CE-MS and FT-ICR-MS. There are organic phosphate compounds in the vacuole irrespective that the vacuole includes phosphatase. The proteomic analysis of vacuolar membrane and vacuolar sap was also conducted. Relationship between proteins and metabolites in the vacuole is discussed. We have overexpressed the unknown tonoplast proteins of Arabidopsis suspension-cultured cells whose function are unknown. We will report the comparative analysis of the vacuolar metabolites of the wild-type and transgenic cells.

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Vacuole of plant cells is a storage organelle of various metabolites. However, an actual diversity of metabolites in a vacuole has not been clarified. In this study, we performed a comprehensive annotation of metabolites in the isolated vacuoles from suspension cultured cell of Arabidopsis (DEEP stain) using LC-FTICR-MS, to elucidate basic metabolite profile of vacuole. A metabolite profile of the whole cell fraction was also analyzed. From the isolated vacuoles and the whole cells, 736 and 876 metabolites were detected in positive mode, respectively, and 713 and 989 metabolites were detected in negative mode, respectively. The whole cell extract contained a larger number of metabolites in a m/z range smaller than 500 Da. Interestingly, several metabolites in nucleic acid metabolism and amino acid metabolism were detected exclusively in the isolated vacuole or the whole cell. This work was partly supported by CREST, JST.

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It has been known that metabolite concentrations are different among not only each cell in organism but also organelles in a cell. However, because metabolite amounts in a single cell and organelle are too small to detect, direct determination of actual conditions of metabolites in a cell is quite difficult. To overcome this problem, we have utilized a giant internodal cell of Chara corallina for a comprehensive metabolomic analysis in a single cell. For metabolomic analysis, we have used CE-MS (capillary electrophoresis / mass spectrometer) which is useful for the separation and detection of ionic compounds. In this study, we identified 55 metabolites in a single cell. Additionally, we conducted metabolomic analysis of a single vacuole and cytoplasm easily isolated from a Chara internodal cell. Metabolomic fluctuations in a cell and organelles under different light conditions were examined.

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In order to investigate an inter-tissue diversity of plant secondary metabolisms, the metabolic profiles of 32 tissues of Arabidopsis thaliana (Col-0 ecotype) were acquired by using the LC-Q-TOF/MS method with considering growth conditions of the public transcriptome data such as AtGenExpress Development. The obtained data matrix (called AtMetExpress Development LC/MS) contained 2522 peaks with annotations of about 100 metabolites. A detected metabolite (m/z 138, Rt 2.06 min) specifically accumulated in flower buds and apical meristem was identified to be tyramine from the annotation data A comparison of the accumulation profiles with that of gene expression data revealed that one homolog of putative aromatic amino acid decarboxylase gene is likely to be responsible for the tyramine biosynthsis in A. thaliana due to those identical accumulation and expression patterns.

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We established a platform to analyze a wide range of plant lipids based on liquid chromatography mass spectrometry, and utilized for analysis of lipidome of Arabidopsis T-DNA insertion lines to identify new lipid metabolic genes. To narrow down the new genes involved in the lipid metabolism, we analyzed transcriptional network of genes involved in the lipid metabolism in Arabidopsis, and we prioritized a previously-uncharacterized gene (UGP3) highly coexpressed with known glycoglycerolipid biosynthetic genes. Profiling of leaf lipids in the knockout mutants of this candidate gene revealed that no sulfolipid was accumulated in these mutants, indicating the participation of UGP3 in sulfolipid biosynthesis. Recombinant UGP3 was able to catalyze the formation of UDP-glucose from glucose-1-phosphate and UTP. A transient assay using fluorescence-fusion proteins indicated chloroplastic localization of UGP3. These results indicated that UGP3 was chloroplastic UDP-glucose pyrophosphorylase involved in the biosynthesis of UDP-glucose, the precursor of UDP-sulfoquinovose for sulfolipid biosynthesis.

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Understanding which metabolites accumulate in arrhythmic plants will help us to elucidate the mechanisms regulating plant growth and development by the circadian clock system. To this end, we performed comprehensive metabolite phenotyping of two arrhythmic mutants: Arabidopsis pseudo-response regulator (PRR9, 7, and 5) triple mutant and CCA1-overexpression. Our approach, which is based on gas chromatography-mass spectrometry, demonstrated that the triple mutant exhibits large alteration in primary metabolism. The triple mutant is characterized by a drastic increase in the metabolite levels of tricarboxylic acid (TCA)-cycle intermediates, osmolytes and several antioxidants. The results from both light/dark cycle and continuous light suggest that the linkage between the circadian clock and the TCA-cycle is very robust. Our integrated approach revealed that PRR9/7/5 negatively regulates the chlorophyll, carotenoid-ABA, and vitamin E biosynthetic pathways, and highlights them as novel outputs of the circadian clock.

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Rice plants have three homologous genes for cytosolic GS (GS1). In GS1 family, OsGS1;1 has the critical role for normal growth and grain filling in rice. However, in terms of regulatory relationships among nitrogen assimilation, growth, and changes of metabolic networks, the function of OsGS1;1 is not completely understood. To address this issue, metabolite and transcript profiling was conducted using OsGS1;1-knockout mutant. The metabolite profiles of the mutants showed the imbalance of sugars and amino acids in leaf blade, whereas those in roots displayed unique pattern. Metabolite-correlation analysis could capture the newly appeared metabolic networks in the mutant triggered from incomplete assimilation of ammonia. The integrated analysis using the results from metabolite correlations and microarrays can be revealed the distinct and important role of OsGS1;1 in both shoots and roots, in which it maintains the appropriate levels of metabolites for normal growth under continuous ammonium supply.

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Tomato is a climacteric fruit whose ripening is induced by ethylene. Remarkable metabolic changes in tomato fruits occur during ripening, however, it is unclear which metabolites are affected by ethylene, and how ethylene controls metabolic pathways of these metabolites. To confirm metabolites responding to ethylene comprehensively, we investigated metabolic profiling of ripening-mutant tomato fruits. Mutant tomato fruits inhibiting ethylene production or perception (nor, rin and Nr) and their wild type (cv. Rutgers) were grown in the same condition and harvested at mature green stage and red-ripe stage of wild type. Primary metabolites were analyzed by GC-MS after TMS-derivatization, and approximately 100 metabolites were identified and compared. Secondary metabolites were analyzed by UPLC-TOFMS and their metabolic profiles were compared. The metabolite annotation data, consisting of about 800 metabolites, were used for prediction of structures of secondary metabolites. These comprehensive analyses enabled to discriminate the metabolite which is affected by ethylene.

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Zinc finger protein genes are known to have diverse functions in plants, such as transcription and cytoskeleton organization. In this study, we found that an Arabidopsis zinc finger protein gene was involved in regulating sugar metabolisms. We constructed an inducible RNAi construct of the gene and introduced into Arabidopsis suspension-cultured cells. Suppression of the gene resulted in no proliferation of the cells. Metabolites of the transgenic cells were analyzed by GC-TOF-MS, and the results were subjected to network analysis. The network suggested a change of amounts of myo-inositol, which is one of the precursors in cell wall synthesis. Fourier transform infrared spectrophotometer analysis supported the changes in cell walls. Overexpression of the gene in Arabidopsis suspension-cultured cells resulted in an increase of myo-inositol, which rescued the cell growth that was suppressed when myo-inositol was omitted from the medium. These results suggest that the gene is involved in myo-inositol synthesis.

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Accurate mass measurement using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR/MS) allows prediction of elemental compositions of each analyte. We have powered our FT-ICR/MS-based metabolomics scheme by implementing an analytical function for reconstituting metabolic activities on the van Krevelen diagram, which is constructed by plotting atomic ratios of each elemental composition. In this report, we discuss the possibility of this analytical tool for gene function analysis. Practically, metabolome data from wild-type plants and T-DNA insertion mutant lines were converted to elemental compositions with ion intensity information, and were superimposed on the van Krevelen diagram. Analysis of a cytochrome P450 and an acyltransferase genes using this scheme allowed us to speculate metabolic activities related to these genes of interest. These novel enzyme functions were elucidated by enzyme assay with recombinant proteins, demonstrating the effectiveness of our new approach.

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Rumex obtusifolius L. (Polygonaceae) is one of the most formidable weeds in infesting fields, arable lands and pastures through the world. In the early 20 century, the occurrence of R. obtusifolius in Japan was first reported in the northern island, whereas today it can be found in almost all areas.
Rumex plants contain high concentrations of oxalate. Excess uptake of this substance is toxic to animals. In plants, oxalate plays an important role as a source of hydrogen peroxide, which may act as defense molecules. Furthermore, some plants secrete oxalate to the rhizosphere, which could detoxify metallic ions.
In this investigation, we analyzed the oxalate metabolism in R. obtusifolius. Metabolite analysis by CE-MS showed that higher level of oxalate and amino acids were accumulated in R. obtusifolius. The analysis of R. obtusifolius grown in the dark indicated that citrate in stems play an important role in oxalate accumulation in leaves.

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The trap of Aldrovanda vesiculosa, an aquatic insectivorous plant, consists of a pair of lobes (trap-lobes). There is a central region in trap-lobes that is composed of three cell layers and has some sensory hairs. Bending of the sensory hairs cause the decrease of turgor in inner cells and then trap-lobes close.
In the former report, we showed that various Ca²⁺ channel blockers had no effect on the movement, but EGTA inhibited the closing although it took longer than 2 hours. We also found that Tetrandorine, and Tetracaine inhibited the closing.
In the present study, we found, using a conventional microelectrode method, that the excitability of cells in the central region under Tetrandrine and Tetracaine treatment was not affected. But, PAO completely diminished the action potential. .
We further analyze the effect of these inhibitors on the sensory hairs. The role of Ca²⁺ signaling in the closing is discussed.

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LKP2 protein contains 3 functional domains: the LOV, F-box, and kelch repeat domains. The LOV domain is involved in the blue light perception and protein-protein interaction. The F-box motif is found in F-box proteins and the kelch repeat is a protein-protein interaction module. Arabidopsis plants overexpressing LKP2 cDNA exhibited the elongated hypocotyl under light conditions, delayed flowering time under long-day conditions, and arrhythmia under continuous light or dark conditions. We used DNA microarray analysis to identify the genes with altered expression levels in the LKP2 overexpressor. Microarray analysis revealed that 0.35% of the total genes in the LKP2 overexpressor were upregulated by more than 2-folds, compared to the expression levels in the wild type. The upregulated genes included some plant hormone-responsive and stress-responsive genes. In order to confirm whether LKP2 functions in stress responses, we assessed the expression levels of stress-responsive genes and the stress responses in the LKP2 overexpressor.

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Roots show hydrotropism in response to gradient in moisture or water potential. We recently isolated ahydrotropic mutant of Arabidopsis, miz1 and identified the gene responsible for its phenotype. miz1 roots display not only ahydrotropic growth but also a reduced phototropism. To explore the regulatory mechanism related to these phenotypes, we examined whether MIZ1 transcription and MIZ1 protein localization are modulated by moisture gradient or light stimulation. MIZ1 transcription was observed at root cap and vascular bundle, when seedlings were grown under continuous light. Under the same conditions, MIZ1 protein was localized not only at root cap and vascular bundle but also at cortial cells where MIZ1 gene was not expressed. While these expression patterns were unchanged upon hydrostimulation, MIZ1 expression diminished only at the root cap cells when seedlings were grown in the dark. These results suggest that MIZ1 at the root cap regulate cross-talking of hydrostimulation and light stimulation.

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Transcriptional repressors were recently proposed to play a key role in modulating the plant response and tolerance to abiotic stresses. A subset of these transcriptional repressors belongs to the C₂H₂-type zinc-finger gene family and contains an EAR repressor domain. To characterize the role of this type of proteins in the stress response and tolerance, we analyzed the function of Arabidopsis genes encoding two different C₂H₂-type zinc finger proteins (AZF1, AZF2). We generated transgenic Arabidopsis plants harboring an AZF1/AZF2-GFP construct driven by AZF1- and AZF2-promoter. The AZF1- and AZF2-GFP proteins were detected in roots under normal growth conditions. Microarray analyses revealed that expression of many common genes was decreased in plants overexpressing AZF1 and AZF2 driven by a dexamethasone (DEX)-inducible promoter. Currently, we are performing transient gene expression assays using Arabidopsis protoplasts to identify downstream target genes of AZF1 and AZF2.

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The OsNAC5 gene is a member of the stress-responsive NAC transcription factor gene family in rice. There are many homologous genes to OsNAC5 or OsNAC6 in monocot crops, respectively. We analyzed function of the OsNAC5 gene homologous to OsNAC6. Expression of OsNAC5 was induced by high salinity, and the OsNAC5 protein was localized in nucleus. Transactivation experiments using a rice protoplast system showed that OsNAC5 had a transcriptional activation domain in the C-terminal region. We generated the transgenic rice plants overexpressing OsNAC5, and found that transgenic plants showed enhanced tolerance against high-salinity. Microarray analysis of the transgenic rice plants overexpressing OsNAC5 revealed that many stress-responsive genes including LEA proteins were up-regulated. Transactivation experiments showed that OsNAC5 enhanced the expression of the LEA promoter. These data indicate that OsNAC5 is one of important transcription factors controlling the expression of many stress-responsive genes including LEA proteins.

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DREB2s are transcription factors that interact with a cis-acting DRE sequence and activate expression of downstream genes involved in abiotic stress response and tolerance in Arabidopsis thaliana. There are five DREB2 homologues in rice and they are called OsDREB2 genes. Among them, OsDREB2B is closely related to other DREB2-type genes in monocots. OsDREB2B protein exhibited higher transactivation activity than other OsDREB2s in transient transactivation assays. OsDREB2B produced two forms of transcripts, and the functional transcript of OsDREB2B was accumulated by low temperature, high temperature, drought and high salinity stresses in rice seedlings. Some drought- or high-temperature-stress inducible genes were induced in transgenic Arabidopsis expressing OsDREB2B ectopically. These data suggested that OsDREB2B functioned as a DREB2-type transcription factor. We are now analyzing the target genes of OsDREB2B using transgenic rice.

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The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and concerns to various physiological processes in the bacterial, plant and animal cells. Transgenic rice plants expressing a molecular chaperone DnaK from a halotolerant cyanobacterium Aphanothece halophytica exhibited the enhanced activities for Calvin-cycle enzymes, and showed faster growth and higher seed yield compared with the wild-type rice under normal growth conditions. Transgenic rice plants also showed enhanced tolerance for high temperature and salt stress compared with the wild-type rice seedlings. High-temperature treatment during the reproductive stage decreased total dry weight of seeds in both transgenic and wild-type rice, but more severely in the wild-type. Similar results were obtained for salt stress during the reproductive stage. These results suggest a relation of increased folding activity with enhanced stress tolerance, increased seed yield, and total plant biomass.

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Beta vulgaris accumulates large amounts of sucrose, betaine, and raffinose in tap roots. Regulation of betaine synthesis, transport, ansd accumulation is important, but its mechanisms are poorly understood. Here, we examined the the changes of accumulations of osmoprotectants. One month old Beta vulgaris accumulates betaine 2 - 5 micro-mol/gFW even under the control conditions. Upon salt stress, the levels of CMO and betaine increased significantly in all the leaves, stems, and roots. The levels of Na+ also increased upon the salt stress. A betaine/praline transporter gene was isolated from sugar beet. The changes of accumulation of sucrose, cations, genes for betaine synthesis and transport under these conditions will be presented.

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Proline accumulates under osmotic stress and acts as an osmolyte in plant. Proline biosynthesis occurs via two pathways from either glutamate or ornithine, proline biosynthesis from glutamate, in which Δ¹-pyrroline-5-carboxylate synthetase (P5CS) is the key enzyme, appears to be the predominant pathway under stress conditions. Investigating the gene expression of P5CS in rice, we attempted to overexpress the gene expression of P5CS in rice. In the transgenic rice overexpressing P5CS mRNA, Pro content was increased six fold higher than the wildtype. We treated the rice cells with salts (NaCl or CaCl₂). After 17 hours of 200mM NaCl treatment, the viability of the transgenic cell was higher than the wildtype moreover, after 6 hours of 400mM NaCl treatment significant difference was detected. Similar results were detected on CaCl₂ treatment. In addition, the growth of their callus on the solid medium containing 100mM NaCl was the significant difference.

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Recent research has underscored the importance of nicotinamide adenine dinucleotide (NAD) biosynthesis. In Arabidopsis, genes involved in the NAD biosynthetic pathway have not been fully determined. Previously, we identified the gene encoding NAD synthetase (NADS), which acts in the final step of NAD biosynthesis. Transcriptional analysis revealed that guard cells (GCs) have relatively high levels of NAD biosynthetic gene expression. The NAD level in the epidermis including GCs was transiently decreased by abscisic acid (ABA), whereas nicotinate mononucleotide adenyltransferase (NMNAT) activity was increase by ABA; however, the increase was significantly delayed compared to the decrease in NAD. The activity of NADS was in a steady state during NAD fluctuation. Stomatal movement and drought tolerance were correlated to the activity of NMNAT. We proposed that NAD biosynthetic activity is involved in regulation of stomatal aperture, possibly via the regulation of NMNAT activity.

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In plants, most of genes involved in metabolism belong to multigene families. It was reported that there are 272 cytochrome P450 genes and 107 family glycosyltransferase genes in Arabidopsis. Their huge diversity makes it difficult to determine their physiological functions precisely.
To identify flavonoid UDP-dependent glycosyltransferase (UGT) comprehensively, we utilized transcriptome coexpression analysis using known flavonoid related genes as queries. A UGT gene, UGT3, was found to be highly correlated with anthocyanin biosynthetic genes. UGT3 belongs to the flavonoid-UGT subfamily which can glycosylate a hydroxyl group of sugar moiety linked to the flavonoid aglycone. The anthocyanin profiles of ugt3 Ds-transposon inserted mutants are different from that of wild type when grown in high sucrose condition. UGT3 recombinant protein could catalyze the xylosylation of cyanidin 3-O-glucoside. UGT3 recognized UDP-xylose but not other UDP-sugars. These data show that UGT3 encodes a novel anthocyanin O-xylosyltransferase.

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Plants are exposed to many kinds of environmental stresses. To overcome these stresses, plants respond with biochemical and physiological changes, for example, expressing genes involved in metabolism and transport. Especially, sugars accumulating during environmental stresses are thought to play important roles in stress tolerance. In this study, we analyzed a stress-inducible monosaccharide transporter gene ERD6 (ERD6A) and its homolog ERD6B. ERD6 was originally isolated from a cDNA library from Arabidopsis plants that were exposed to dehydration stress for 1 h. We analyzed stress-responsive and tissue-specific expression patterns of the ERD6A and ERD6B genes. We also examined subcellular localization and transport activity of the ERD6A and ERD6B proteins. These results suggested that ERD6A and ERD6B might differentially contribute to acquisition of abiotic-stress response possibly by sugar storage or remobilization.

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Glycyrrhizin is the major triterpene saponin produced by Glycyrrhiza glabra, and stored in the roots and stolons. We examined a transport mechanism of glycyrrhizin by using cultured G. glabra cells, because they cannot synthesize glycyrrhizin de novo. We showed that the G. glabra cells were able to uptake glycyrrhizin from the medium. Treatment with several inhibitors suggested that glycyrrhizin uptake depended on ATP level. Anion blockers, pH gradient destroyers, and glycyrrhetinic acid inhibited glycyrrhizin uptake. To clarify the molecular mechanism of glycyrrhizin transport, membrane vesicles of the G. glabra cells were fractionated using a discontinuous sucrose gradient. The tonoplast-enriched vesicles exhibited ATP-dependent glycyrrhizin uptake activity. Further characterization of glycyrrhizin transport using various inhibitors revealed that sodium orthovanadate, a P-type ATPase inhibitor, and glycyrrhetinic acid strongly inhibited glycyrrhizin transport.

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Boron (B) is essential for plant growth but toxic when present in excess. Arabidopsis thaliana BOR1 is a plasma membrane borate exporter and is essential for efficient B translocation from roots to shoots under B limitation. In transgenic plants expressing BOR1-GFP under control of the BOR1 promoter, BOR1-GFP was localized preferentially in the plasma membrane of proximal side of various root cells under B limitation, and was transferred to vacuole for degradation upon high-B supply. In contrast, GFP tagged BOR4, a BOR1 homolog suggested to be involved in B exclusion from root cells, was accumulated stably in the plasma membrane of distal side in epidermal cells irrespective of B conditions. Analysis of BOR1/BOR4 chimera and mutant proteins revealed that sorting motifs embedded in a probable cytoplasmic-loop region of BOR1 but not of BOR4 are involved in the endocytic and polar trafficking.

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Boron (B) is essential but excess B is toxic in higher plants. In Arabidopsis, a B efflux transporter, BOR1, which localized preferentially in the plasma membrane of proximal side of root cells, functions for efficient translocation of B from the soil under B limitation condition. However, under enough B condition, the function of BOR1 is considered to be not needed or detrimental. Indeed, the accumulation of BOR1 is rapidly decreased by the ressupply of high levels of B. BOR1 is transferred from the plasma membrane to the vacuole via endocytic pathway. However, it is unknown which intracellular factors are involved in the machinery. We will report a novel factor involved in the machinery and discuss how plant cells effectively regulate B uptake.

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The zig-1 mutant of Arabidopsis lacking Qb-SNARE VTI11 shows abnormal shoot gravitropism and morphological defects. VTI11 functions in vesicular trafficking between trans-Golgi network and vacuoles. To understand the molecular basis of vesicle transport involved in physiological function of higher plants, we have analyzed zig suppressor(zip) mutants that can suppress defects of zig-1.
The zig suppresor4 is recessive mutant that partially suppresses defects of zig-1. Based on the map position, a mutation was found in splicing acceptor site of the gene encoding homolog of adaptor protein complex-3(AP-3) μ3 subunit. AP complexes have been known as coat proteins of transport vesicles in yeast and animal cells.
T-DNA insertion mutants of this gene also suppressed defects zig-1 as well as zip4. Finally, we identified ZIP4 as AP-3μ3 by complementation analysis. These results indicate that loss of function mutation of AP-3 μ3 acts as suppressor of zig-1.

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In higher plants, vesicular traffic plays important roles in plant functions and development. Rab GTPases, key regulators of vesicle transport, have been widely conserved among eukaryotes. Among these Rabs, the Rab5 group, which consists of RHA1, ARA7 and ARA6 in Arabidopsis thaliana, regulates endocytosis.
To investigate their roles in plant development, we analyzed T-DNA tagged knockout Arabidopsis lines. Although each single KO had no obvious phenotype, the rha1-/- ara7-/- double mutant was never segregated. Cross-pollination analysis revealed that the double KO was male gamete lethal. Structural analysis of the rha1-ara7- pollen by light and electron microscopy showed abnormal callose depositions within the pollen grain, indicating that ARA7 and RHA1 might have important function during the pollen development.

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In Arabidopsis thaliana, 57 Rab GTPases are encoded in its genome, 26 of which are classified into Rab11 group (RabA1-RabA6). On the other hand, yeast and animals possess a few Rab11 genes. This implies that Rab11 has evolved in a unique way in plants. However, precise function of plant Rab11 remains largely unknown. To reveal the function of plant Rab11, we analyzed subcellular localization of 9 members belonging to RabA1 subgroup (RabA1a-RabA1i). We generated transgenic plants expressing GFP/Venus-tagged RabA1s, which are driven by their own promoters. Detailed observation of GFP-RabA1e revealed that it localizes on dynamically moving dotty compartments, which partially overlap with Syp42 and Syp43-labelled TGN. We also found that RabA1e-compartments accumulate in the tip region of growing root hairs. These results suggest that RabA1e functions in secretion from the TGN to the PM for the tip growth of root hairs.

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In animals, RAB5 GTPase is known as a molecular switch regulating various aspects of endosomal functions including membrane fusion, motility, and alteration of lipid compositions. RAB5 fulfill these diverse functions through the interactions with effector molecules. In Arabidopsis thaliana, there are two conventional type of RAB5 members, ARA7 and RHA1. The ara7 rha1 double mutant shows gametophytic lethality, which indicates redundancy in their functions and essentiality. Signals delivered from these plant RAB5s should be also mediated via effector molecules to induce downstream reactions, but no such effectors have been identified thus far.
We looked for effectors of A. thaliana RAB5s by the yeast two-hybrid method. We found that one of the candidates is translocated in a cell depending on the nucleotide state of ARA7. In this meeting, we will report the biochemical and cytological properties of these effector candidates.

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RAB5 is conserved in a broad range of eukaryotic organisms including plants. In addition to the conventional type of RAB5 homologs, land plants conserve plant-specific RAB5 homologs with unique structure (ARA6 in Arabidopsis thaliana). Our previous studies provided a new insight that land plants have established an original and possibly more complex mechanism for endocytotic regulation using ARA6, which should be required for the plant-specific way of living. In order to elucidate the molecular basis of such system, ARA6 effectors were determined. We have identified seven putative plant-unique RAB5 effectors(PUFs), among which PUF3 exhibits very interesting behavior. PUF3 localizes on ARA6 positive endosomes, which is regulated by the nucleotide state of ARA6. We have also found that PUF3 binds preferentially to PI4P via PH domain. In this meeting, the cell biological and physiological significance of PUF3-ARA6 interaction will be discussed.

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Mammalian Rab5 GTPase is known to regulate not only endosomal fusion but also signaling through endosomes. Rab GTPases are activated by specific guanine-nucleotide exchange factors (GEFs), which accelerate the exchange of GDP for GTP. Vps9 domain, a catalytic core for activation of Rab5, is conserved in all Rab5 GEFs identified thus far. We have already demonstrated that the Arabidopsis VPS9a can activate all of three Arabidopsis Rab5 members (ARA6, ARA7, and RHA1). VPS9a comprises the conserved Vps9 domain at N-terminus and C-terminal region with no similarity to known domains. We found that truncation of the C-terminal region of VPS9a confers increased GEF activity toward ARA6. Detailed functional analysis of the C-terminal region of VPS9a is now underway.

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The production of phytoalexin momilactones, plant defense-related diterpenoid compounds, is induced in rice upon recognition of pathogenic invasion. The momilactone biosynthetic genes (OsCPS4, OsKSL4, CYP99A2, CYP99A3, and OsMAS) are clustered on chromosome 4 and are coexpressed after elicitation that mimics pathogen attack.
To elucidate regulatory mechanisms for the coordinate expression of the genes, we first performed promoter analysis of OsKSL4, and identified that a bZIP-type transcription factor is involved in the OsKSL4 gene expression. OsTGAP, which was screened by microarray analysis under elicitation, was identified as a bZIP-type transcription factor that directly regulates the elicitor-inducible expression of OsKSL4. We also found that the expression of five clustered genes was hardly detected in the ostgap mutant, and was strongly enhanced in the OsTGAP overexpressing lines. These results indicated that the OsTGAP is a master transcription factor regulating expression of the momilactone biosynthetic gene cluster.

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Bacterial blight (Xanthomonas oryzae) is one of the most destructive pathogens to rice in south-west area of Japan and South East Asia. The rice cultivar Nipponbare shows the high field resistance against the disease. We inoculated bacterial blight pathogens to Nipponbare mutants which were induced by the insertion of the endogenous retrotransposon Tos17, and have screened mutants which lacked the field resistance. One of the mutants, XC20, had tightly related to a Tos17 insertion in the ninth chromosome. A translational region of about 430 bp (xc20) were found near the Tos17 insertion. This XC20 protein had a sequence similarity to calmodulin binding protein ZmSAUR2 of maize and pathogenesis induced protein upa5 of pepper. Because the field resistance against the disease was recovered in the transgenic XC20 mutants in which native xc20 gene were introduced, we concluded the xc20 was related to the field resistance against bacterial blight disease.

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To investigate whether the kinase activity of Pti1a is required for the regulation of defense signaling, we made amino acid substitution (T233A) of Pti1a which is a major phosphorylation site by Pti1a-interacting kinase1 (Pik1). Pti1a^T233A complements ospti1a mutant phenotypes including cell death induction, PR1b gene expression and broad resistance to rice blast. Furthermore, 35S:Pti1a^T233A transgenic lines exhibited the enhanced susceptibility against compatible race of bacterial blight. These results indicated that the phosphorylation of Thr-233 of Pti1a is important for induction of basal resistance. To investigate the Pti1a regulatory system, we characterized Pik1 which functions upstream of Pti1a. The Pik1 expression was observed in peripheral region of HR after inoculation of Magnaporthe grisea. Pik1 was transiently phosphorylated 10 minutes after treatment of H₂O₂, suggesting that Pik1 functions at the early stage of ROS signaling in disease resistance.

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MAPK cascades play an important role in defense signaling after recognition of elicitor signals. In rice, several MAPKs are strongly activated by elicitor treatment but the downstream responses are remained elusive. We analyzed the defense responses regulated by an elicitor-responsive MAPKK, OsMKK4. Using conditional gain-of-function transgenic system, we demonstrated that OsMKK4 induces activation of OsMPK3 and OsMPK6, expression of multiple defense-related genes and cell death but not ROS production. Analysis of genes regulated by OsMKK4-OsMPK3/OsMPK6 MAPK cascade revealed that this MAPK cascade tightly regulates phytoalexin and lignin accumulation. Using osmpk6 mutant, we confirmed that OsMPK6 functions for the induction of cell death and phytoalexin accumulation. We are analyzing transcription factors which are regulated by OsMKK4-OsMPK3/OsMPK6 MAPK cascade. We will compare the expression pattern or expression level between in osmpk6 mutant and in the wild type to identify regulators of cell death and phytoalexin accumulation.

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Rice blast is one of the most widespread and destructive diseases of rice. The panicle blast occurring after heading stage causes decreased yield and reduced quality of brown rice. Pb1 is a panicle-blast resistance gene, which was identified as a major quantitative gene. The blast resistance by Pb1 is effective during adult stages, and has not experienced breakdown for more than 20 years. We isolated Pb1 by map-based cloning to investigate the mechanisms underlying its characteristic traits of resistance. In 26kb of Pb1 region, five open reading frames were predicted but gene expression was detected for only P15 and P18. Complementation experiments showed that only P15 conferred blast resistance to rice. Pb1 encoded a protein of CC–NBS–LRR-family R protein of 1,296 amino acids. Expression analysis showed that the strength of blast resistance roughly paralleled with the expression levels of Pb1 at respective growth stages.

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Pb1 (Panicle Blast1) is a so-called field resistance gene for a fungal pathogen Magnaporthe grisea. The resistance by Pb1 is durable, of broad spectrum for fungal races, and effective especially during reproductive stages. Recently, Pb1 was isolated and encoded a Coiled Coil-Nucleotide Binding-Leucine Rich Repeat (CC-NB-LRR) protein. MLA, a CC-NB-LRR-type R protein of barley for Bluminia graminis, has been reported to translocate to the nucleus and interacts with a WRKY transcription factor (TF) after pathogen recognition. We tested the interaction of Pb1 with WRKY TFs of rice by using yeast two-hybrid, revealing that Pb1 specifically interacts with WRKY45 that plays a crucial role in plant-activator-induced resistance and salicylic-acid-mediated defense signaling in rice. While MLA-interacting WRKY TF is a transcriptional repressor, WRKY45 is a transcriptional activator; therefore the molecular mechanisms involved in the MLA- and Pb1-mediated signaling pathways are presumably different.

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Phospholipase D (PLD) plays an important role in plant biotic and abiotic stress responses. To study the function of PLD in rice, we made knockdown (KD) plants by introduction of RNAi constructs specific to each PLD gene. The OsPLDβ1- and OsPLDδ2-KD plants were found to exhibit the increased disease resistance to infection of a major fungal pathogen of rice, pyricularia grisea. The up-regulation of defense related genes such as PR-protein genes was observed in the OsPLDβ1-KD plants but not in OsPLDδ2-KD ones. On the other hand, the up-regulation of WRKY family transcription factor genes was observed in both OsPLDβ1- and OsPLDδ2-KD plants. These results suggested that OsPLDβ1 and OsPLDδ2play a negative role in induction of defense reactions through the partly different signal transduction pathways.

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Plant causes immune response after perception of flagellin which is component of bacterial flagella filament. Previously FLS2 was identified as flg22 receptor in Arabidopsis thariana. Flagellin recognition system in rice, however, is still not known. Therefore we examined the flagellin perception system in rice. Rice recognizes two domains, flg22 and C-terminal region in flagellin. We attempted to identify receptor for flg22 in rice and identified OsFLS2, ortholog of FLS2. When OsFLS2 was overexpressed in rice cells, ability of flg22 perception was increased. OsFLS2 could complement A. tharianaΔFLS2 mutant, suggesting that OsFLS2 is functional for flg22 perception. To identify the receptor for C-terminal of flagellin, microarray analysis was performed. Among the identified gene, we concluded that STR1 and STR2 are candidate for the receptor. When both genes were overexpressed in rice, flagellin perception ability was increased. Moreover, STR1 and STR2 deficient mutants lost the ability of C-terminal flagellin perception.

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Plants have evolved PAMP- and effector-triggered innate immunity to fight against pathogen attacks. Rac GTPase, RACK1, RAR1, SGT1 and Hsp90 are characterized as important components for plant defense signaling. However the relationship and molecular functions of these components are largely unknown. Here, we characterized a novel OsRac1 interacting protein, Sti1, which is a well-known component of steroid receptor complex in animal, as a key regulator for disease resistance. We found that suppression of Sti1 dampened resistance, while overexpression of Sti1 enhanced resistance in rice. Interestingly, Stil interacted with Hsp90 and plant PAMP receptor homologs (RLKs). Our results extend the existing tripartite interaction of RAR1, SGT1 and Hsp90 to a protein complex network, termed as "defensome", including Sti1, Rac1, and RLKs. Thus, we hypothesize Stil may function as an adaptor linking PAMP receptors and molecular switch (Rac1) together and contribute to PAMP-triggered innate immunity in rice.

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OsRac1 functions as a key regulator of defense signaling in rice. So far, we isolated OsRac1 interacting proteins by affinity chromatography and yeast two-hybrid screen. But, these results only indicated in vitro and direct interaction of these proteins with OsRac1. Therefore, we tried to isolate the native protein complexes. The aim of this study is to reveal the existence of OsRac1 complex in vivo, and study how the composition of OsRac1 complex is altered by elicitor treatment in suspension cell cultures.
Proteins extracted from rice transgenic cultured cells of constitutively active OsRac1 (CA-Rac) and dominant negative-OsRac1 (DN-Rac) were applied to gel filtration. Proteins fractionated by gel filtration were probed for proteins involved in innate immunity by western blotting. So, we found that the CA-Rac complex was composed of a higher molecular weight complex than DN-Rac complex and the OsRac1 complex was composed of a larger complex after elicitor treatment.

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Most resistance (R) genes encode nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins, which act as intracellular receptors for pathogen signals. It is largely unknown how NBS-LRR resistance proteins transduce pathogen signals to their downstream effectors and thereby execute innate immunity in plants. We have previously found that the small GTPase OsRac1 plays critical roles in R gene-mediated defense response in rice. Therefore, we searched for OsRac1-interacting molecules and found that OsRac1 directly interacted with Pit, Pib, Pi9 and Xa1 which are NBS-LRR-type R proteins for rice blast and rice bacterial blight. An expression of dominant negative form of OsRac1 suppressed Pit-mediated defense response against rice blast fungus. An active form of Pit activated the OsRac1 activity in rice protoplasts. These results suggest that OsRac1 forms a complex with R proteins and acts as a downstream effector of R proteins, thereby contributing defense signaling in rice.

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Because of its difficulties with the observation, little is known about the molecular mechanism of angiosperm fertilization. Previously we showed that in Torenia, pollen tube attractant is secreted from the synergid cells. Using this in vitro pollen tube guidance assay, a molecule, which induced competence to the pollen tube to respond to the attractant, was uncovered and named AMOR (activation molecule for response capability). AMOR was heat-stable and was collected in a fraction of arabinogalactan proteins (AGPs). To understand its role for the induction of competence, we investigated strategies to purify sufficient amount of AMOR to characterize its molecular nature, and active fractions obtained from column purification were analyzed further. We also found that AGPs from vegetative tissues of other plant species had some AMOR-like activity. We are now investigating whether AMOR belongs to AGPs and whether sugar chains in the AMOR fraction contribute to AMOR activity.

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The phytohormone ethylene influences numerous plant growth and developmental processes. The first rate-limiting step in ethylene biosynthesis is catalyzed by ACC synthase (ACS), which is encoded by a multigene family that is regulated at both the level of transcription and protein stability. Using a yeast two-hybrid screen, we identified Arabidopsis orthologs of DnaJ (ATJ2/ATJ3) as proteins that interacted directly with ACS5. The ATJ3 interaction was confirmed in vitro and in planta and found to be specific for type-2 ACS proteins. Disruption of the two closely related ATJ2 and ATJ3 genes blocks the stabilization of ACS5 protein that occurs following treatment of etiolated seedlings with cytokinin and light. The atj2/atj3 double mutants were altered in other aspects of plant growth, including flowering time and fertility. These studies reveal a novel role for DnaJ and add to our understanding of the regulation of protein turnover in plants.

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ACC synthase (ACS), a rate-limiting enzyme of ethylene biosynthesis, is regulated not only transcriptionally but also post-translationally. We previously reported that LeACS2, a wound-inducible ACS in tomato, is immediately phosphorylated after translation and almost all LeACS2 molecules act as phosphorylated form and that the half-life of dephosphorylated LeACS2 is shorter than that of phosphorylated LeACS2. These evidences suggest that dephosphorylation step defines the rate of ACS turnover. Among Arabidopsis mutants which overproduce ethylene, we focused on rcn1 which disrupts a regulatory subunit of protein phosphatase 2A. Auxin treatment induced the expression of AtACS4, followed by ethylene production in etiolated Arabidopsis seedlings. Although the expression levels of AtACS4 mRNA were equivalent between wild-type and rcn1, ACC contents and ethylene production in rcn1 were higher than those in wild-type. Theses results suggest that the elevated level of ethylene production in rcn1 results from the alternation of AtACS4 turnover.

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