In higher plants, the profile of gene expression is regulated epigenetically in various developmental events such as flowering and genomic imprinting. However, it is not known whether epigenetic regulation of gene expression occurs in a process forming vascular tissues which is important for plant morphology formation as an individual. To examine the dynamics of chromatin during differentiation of xylem, we use a Zinnia cell culture system, in which isolated mesophylls transdifferentiate into tracheary elements. After adding the inhibitors of DNA methyltransferase and histone deacetylase, transdifferentiation of mesophylls into tracheary elements was inhibited without affecting cell division. The inhibition depended on the concentration of inhibitors and the time when they were added. We will also report the results of microarray analysis of the gene expression in the inhibitor-treated cells using newly developed Zinnia gene chip zinnia-17k.

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Cardiolipin (CL) is widely distributed in various prokaryotes and eukaryotes as a membrane phospholipid. In eukaryotes, it is localized in inner membrane and contact site of mitochondria. CL is suggested to be involved in many mitochondrial functions. To know physiological roles of CL in higher plants, we identified the Arabidopsis gene CLS encoding a CL synthase and isolated mutants in which the CLS gene is disrupted by T-DNA insertion.
We have analyzed knockout mutants of CLS and found that growth of cls/cls mutants during embryonic stage delayed compared to wild type. Promoter-GUS analysis suggested that AtCLS is expressed especially in embryos, young leaves, colmella cells and guard cells. These findings demonstrate that CLS plays a critical role is plant development.

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How distinct cell fates are specified at correct positions within the plant embryo is unknown. To address mechanisms of position-dependent gene activation and cell fate specification, we analyzed the regulatory region of the ATML1 gene, which is already expressed at the one-cell stage and whose expression is later restricted to the outermost, epidermal, cell layer from its inception. A sensitive multiple GFP reporter revealed a modular organization of the ATML1 promoter. Each region contributes to specific spatial and temporal aspects of the overall expression pattern, including position-dependent regulation along the apical-basal axis of the embryo. A 101-bp fragment that conferred all aspects of ATML1 expression contained known binding sites for homeodomain transcription factors and other regulatory sequences. Our results suggest that expression patterns associated with cell fate determination in the plant embryo result from positional signals targeting different regulatory sequences in complex promoters.

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MALE STERILITY1 (MS1) gene of Arabidopsis encodes a nuclear protein with a PHD-finger motif, and is involved in microspore maturation after meiosis. Here we examined the MS1 function. We introduced a chimeric fusion construct of MS1 and a transcriptional repression domain (MS1-SRDX) into wild-type Arabidopsis, and the transgenic plants showed ms1-like semi-sterile phenotype. Since the repression domain has an ability to convert various kinds of DNA-binding transcriptional activators to dominant repressors, this suggested that MS1 functioned as a transcription factor. The PHD motif was required for the MS1 function. Phenotypic analysis of the ms1 mutant and the MS1-SRDX transgenic Arabidopsis indicated that MS1 was involved in pollen exine, pollen cytosol and tapetum development. Next, we searched for downstream genes regulated by MS1 by using dexamethasone-induction system. From these results, we discuss a roll of MS1 in microspore maturation. We also discuss existence of MS1 orthologs in higher plants.

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TEBICHI (TEB) gene of Arabidopsis encodes a homolog of animal MUS308/POLQ proteins which are involved in DNA damage response, and loss-of-function teb mutants show defects in morphology of meristems and organs. teb showed constitutively activated DNA damage responses and an accumulation of cells expressing G2/M-specific cyclinB1;1:GUS in meristems. We show here that this accumulation of cyclinB1;1:GUS-expressing cells in teb was suppressed by a disruption of ATR gene which is involved in DNA replication checkpoint, suggesting teb is defective in DNA replication and activates ATR-dependent checkpoint. However atr enhanced morphological phenotypes of teb. Furthermore, teb atr double mutants frequently developed needle-like leaves indicating teb atr is defective in adaxial-abaxial polarity of leaves. Histochemical analysis of leaf cells and expression analysis of abaxial-specific FILAMENTOUS FLOWER gene showed leaves of teb and teb atr were partially abaxialized, indicating TEB is involved in the establishment of adaxial-abaxial polarity of leaves.

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Leaves are developed from SAM as symmetrical and flat organs. To understand the process of the development of leaves at higher plants, we have analyzed phenotypes of two Arabidopsis thaliana mutant, asymmetric leaves1 (as1) and asymmetric leaves2 (as2). Mutant plants of as1 and as2 generate asymmetrical and downwardly curled leaves with aberrant mid veins and short petioles. Leaves of both mutants can regenerate ectopic shoot on the phytohormone-free medium. Furthermore the class 1 KNOX genes are ectopicaly expressed in leaves of these mutants. These suggest that AS1 and AS2 regulates the expression of class 1 KNOX genes negatively in leaves. To investigate the relationship between true leaf development and repression of class 1 KNOX by AS1 and AS2, we analyzed the phenotypes of the mutants that had various combinations of mutations in class 1 KNOX genes in addition to AS1 gene or AS2 gene.

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To study the shoot formation in orchid, we analyzed the function of Arabidopsis Class I KNOX genes KNAT1 and a leaf gene ASYMMETRIC LEAVES2 (AS2) under the control of Cauliflower Mosaic Virus 35S RNA promoter in Natural Indonesian orchid, Phalaenopsis amabilis. In this study, those two genes were transformed using Agrobacterium tumefaciens LBA 4404. We got 11 lines of KNAT1 transgenic protocorm (developing orchid embryo) that formed multishoots (4-31 shoots developed from one embryo) and 79 lines of AS2 transgenic orchid plants. Detection of transgenic lines were performed by Kanamycin selective medium and PCR. The shoot formation in 4 lines of KNAT1 transgenic plants showed multishoots and alteration of leaf shapes. P35S::AS2 transgenic plants showed smaller leaf blade with abnormal vascular pattern, and dwarf shoots. Histological analysis of protocorm in KNAT1 transgenic plants showed that shoot initially come from some area in the periphery.

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Myrosin cells in Capparales plants are idioblasts that accumulate β-glucosidases, myrosinases, in their vacuoles. Isothiocyanates that work on defense against insects are produced from glucosinolates by myrosinase in damaged tissues. We screened mutants that have abnormal development of myrosin cells in Arabidopsis thaliana , using a transcriptional factor mutant library produced by CRES-T (Chimeric REpressor Silencing Technology), and identified two mutants. These transcriptional factors belong to the same subfamily and their expression patterns are similar to each other. Both transcriptional factors identified are down-regulated in atvam3 mutant that has many myrosin cells compared with wild type. We discuss roles of identified transcriptional factors in differentiation of myrosin cell.

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We report here that functional analysis of the family of TEOSINTE BRANCHED1, CYCLOIDEA and PCF (TCP) transcription factors in Arabidopsis. Expression of the chimeric TCP3 repressor, in which a member of Arabidopsis TCP transcription factors was fused to the EAR-motif repression domain, resulted in formation of ectopic shoots on cotyledons and various defects in organ development. The chimeric TCP3 repressor induced ectopic expression of boundary-specific genes including CUP-SHAPED COTYLEDON (CUC) genes associated with reduction of accumulation of miR164, which cleaves CUC transcripts. By contrast, gain of function of TCP3 suppressed the expression of CUCs and resulted in the fusion of cotyledons and defects in the shoot apical meristem. The TCP3 transcript was accumulated in cotyledons, but not at the cotyledon boundary where CUCs are functioning. Results demonstrate that the TCP3 is a negative regulator of CUCs in cotyledons and controls morphogenesis of shoot organs and formation of shoot meristems.

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Arabidopsis SPL10 is a member of the SBP-box family that encodes a plant-specific transcription factor. SBPs have been shown to bind to AP1 promoter, but their biological function is unknown. To elucidate the function of SPL10, we produced transgenic plants. Expression of the chimeric SPL10 repressor (35S:SPL10-SRDX) caused multiple effects including reduced apical dominance, abnormal inflorescence, short floral organs and shrunk siliques. The silique phenotype was similar to that of ful mutant, and FUL expression was reduced. Expression of AP1, homologous gene of FUL, was not affected in 35S:SPL10-SRDX plants. Although ectopic expression of SPL10 (35S:SPL10) induced no apparent phenotype, overexpression of mSPL10, in which the target sequence of microRNA was mutated, resulted in cauline-like rosette leaves and an accumulation of FUL mRNA. Furthermore, transient assay suggested that SPL10 bind to the FUL promoter. These results indicate that SPL10 directly controls FUL expression and is regulated by microRNA.

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WUSCHEL is known to be a multiple functional gene, which is involved in regulate the stem cell fate of shoot and floral meristems, transition of embryonic to vegetative phase, and phytohormone signaling. WUSCHEL was reported to be an activator of AGAMOUS and a repressor of the ARR genes. These suggest that WUSCHEL has bifunctional transcription factor, which acts as activator or repressor depending on the target gene. But the functional mechanisms of WUSCHEL was not fully analyzed.
The transient assay using 35S-GAL4BD-TATA-LUC reporter gene showed that WUSCHEL had a repression activity. A series of deletion analysis revealed that two regions of WUACHEL gene are necessary for repression activity. One is the EAR-like motif, which previously reported as a repression domain, and the other is novel region that is conserved among WUSCHEL-related homeobox genes. In this report, we discuss the possible function of the repression domains.

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Arabidopsis SHEPHERD (SHD) gene encodes a Hsp90-type molecular chaperon resident in the endoplasmic reticulum. The shd mutant shows a defect in formation of pollen exine. When we joined SHD cDNA to tapetum-specific promoters, SP11p and FBP1p, and introduced them into the shd mutant, only FBP1p:SHD restored the exine structure. This suggested that the the SHD is required for the exine formarion in tapetal cells of tetrad stage, in which FBP1p, but not SP11p, is active. It is supposed that the SHD participates in the secretion of prime exine components from tapetal cells. The shd also shows a defect in pollen tube elongation. It was recovered when the SHD was driven by the pollen-specific LAT52 promoter, whereas not by FBP1p, suggesting that the SHD expression in pollen grains is important for the pollen fertility, and that the correct exine structure is not necessary for the pollen fertility.

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In the self-pollinating flowers like Arabidopsis, coordinated maturation and elongation of floral organs is critically important for the successful pollination. We isolated deco1 mutant, which shows abnormalities in synchronized elongation of stamens and petals (i.e. hastened stamen elongation and insufficient petal elongation) at the flower opening. SEM analysis showed that the cell elongation was enhanced and repressed in the mutant stamens and petals, respectively, in compare to the wild type. The level of endogenous JA was decreased in deco1 petals, that resulted in the block of petal elongation. The DECO1 promoter-GUS analysis revealed that it was expressed specifically in stamen filaments of fully developed but unopened flower buds. These results suggested that DECO1 protein, which is predicted a cytochrome P450 monooxygenase, is involved in the biosynthesis or metabolism of JA in filaments, and that the resultant compound may be important for petal elongation.

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We isolated a dominant mutant, cryptic precocious-1D (crp-1D) which dramatically enhances the precocious-flowering phenotype of 35S::FT. CRP gene was identified by the positional cloning. CRP encodes a large protein which has low similarity to a subunit of the transcriptional mediator complex. To gain insights into its function, we analyzed morphological phenotype of crp-1D mutant in the absence of the 35S::FT transgene. Loss-of-function mutants of crp were obtained from Ds and T-DNA insertion lines and analyzed. crp-1D mutant showed slightly early-flowering phenotype compared with wild type. By contrast, crp loss-of-function mutants showed late-flowering phenotype. These observations suggest that CRP play some role in regulation of flowering. Interestingly, crp loss-of-function mutants also showed pleiotropic phenotype including dwarfism, curled leaf, abnormal shape of floral organs, and sterility. Characterization of the phenotype and analysis of expression patterns will be presented.

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The Arabidopsis ERECTA (ER) gene regulates the shape of organs derived from the shoot apical meristem, and recent genetic studies revealed that ER and two homologues act redundantly in the epidermis differentiation. However, little is known of both upstream regulators and downstream target genes of ER.
To gain new insights into the ER function, we have studied the regulatory mechanism of ER expression. A mutant screen was performed utilizing GUS reporter gene driven by the ER promoter, and identified several mutants with decreased levels of GUS expression. Further analyses revealed that the mRNA accumulation of ER was impaired in one of those mutants, reduced expression of erecta1 (rxe1). Phenotypic analyses of rxe1 mutant implied that in addition to ER, there are more target genes regulated by wild-type RXE1. We will report the molecular characterization of rxe1 mutant.

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The inflorescence of Arabidopsis ecotype Landsberg harboring the erecta mutation shows a corymb-like inflorescence. To understand inflorescence development, we previously screened corymb-like inflorescence mutations, named corymbosa1 (crm1). Cloning of the CRM1 gene revealed that it is allelic to BIG. CRM1/BIG is highly expressed in inflorescence meristems and floral meristems by in situ hybridization and histochemical analysis using transgenic plants. Allelic series of crm1/big mutants, reduced lateral root formation (rlr) were categorized into two classes depending on the defects of plant development. Identification of the molecular defects and Northern blotting suggest that rlr mutants whose phenotypes are changed alternately corresponding with the size of presumed CRM1/BIG protein are hypomorphic alleles. The severities of the corymbose inflorescence in crm1/rlr mutants were correlated with increased levels of PINFORMED1 gene, suggesting that CRM1/BIG controls inflorescence development through auxin transport. Further analyses of the expression pattern of several genes are now under way.

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Short inflorescence stems and curled leaves are the characteristics of acaulis1 (acl1) mutants when grown at 22°C. High temperature is effective to restore the acl1 plant morphology. Even the severe acl1-1 plants, whose rosettes are about 1 cm in diameter and stems are slightly longer than the height of the rosettes, become almost undistinguishable from wild type at 28°C. Growing two acl1 alleles and acl2-1 plants at varied temperature revealed that the sufficient temperature for the restoration is different in each acl mutant. If the common pathway is involved in restoration of acl plant morphologies at high temperature, it is interesting that responses to a certain temperature differ among acl mutants. We found that the size of acl1 plants depends on the concentration of NH₄NO₃ in growth media. acl1-1 plants grown big by NH₄NO₃ supplementation still exhibit curled leaves and short inflorescence stems.

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Streptocarpus (Gesneriaceae) is unique, as some of these species form leaves without shoot apical meristem (SAM). Such processes are analyzed with Streptocarpus rexii, a rosulate species.
To this aim, first, meristematic regions in the seedlings have been identified by incorporating BrdU into tissues and their locations were illustrated by the 3-D program. Simultaneously the 3-D location of groove meristem (GM) was constructed. After merging these two figures, the relationship between the meristematic regions and GM has been visualized.
Subsequently, after isolating homologs for class 1 KNOX-like gene, KNAT1 and MYB-like transcriptional factor, PHANTASTICA, locations of expressions of these genes were identified in the tissues by in situ hybridization. These outcomes suggest the GM is a kind of temporally appeared SAM and seems to move in the tissues. Such similarity and dissimilarity of the leaf formation of this plants will interpreted from the viewpoints of Evo-Devo concept.

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As depicted by the previous speaker, Streptocarpus species display unique morphological patterns, which is the Phyllomorph concept. Molecular basis of such unique morphogenesis was followed by expression patterns of KNOX1 and Phantastica genes. We further explored this analysis to the Gesneriaceae which distribute to the whole world and display unique morphogenesis. One of these characteristics is anisocotyly, which is observed only in the Old World gesneriad.
When we analyzed the expression patterns of KNOX1 gene during the formation of shoot and leaves, it is summarized as follows: morphogenetic potentials of SAM would have been attained in leaves, resulting in unique leaf formation patterns. Furthermore, upon anisocotyly environmental signals such as light play an important role, although we reported previously that cytokinin is a key factor for forming anisocotyly. As it became clear that KNOX1 expression is closely associated with anisocotyly, a hypothetical construction regarding this phenomena will be presented.

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The Golgi apparatus plays a key role in sorting and delivering proteins of various destinations. The Golgi apparatus possesses very unique architecture, which varies depending on the species. However, the relationship between the function and the structure of the Golgi apparatus is not clear, and how the Golgi apparatus maintains its unique architecture is left almost unknown. We are studying the mechanisms of the biogenesis of Golgi apparatus using higher plant, Arabidopsis, as a model system. First, we observed detailed structure and dynamic movement of the Golgi apparatus in living cells under a high-speed and high-sensitivity confocal laser scanning microscope. We are also screening mutant Arabidopsis showing abnormal Golgi morphology. We will report interesting phenotypes of putative mutants as well as the effect of the loss of function of some SNAREs on Golgi.

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The TGN is one of the most important organelles for protein transport at the post-Golgi network, and functions as a sorting spot that directs cargo proteins to a variety of post-Golgi compartments. However, the TGN of plant cells has not been well understood yet. In order to elucidate the structure, function and dynamics of plant TGN, we focused on AtTLg2a/Syp41, the ortholog of Tlg2/syntaxin16 which is localized to the TGN in
yeast and mammalian cells, as a TGN marker. We established the transgenic plants expressing GFP-Syp41/AtTlg2a under the control of the native promoter. The observation under the CLSM (confocal laser scanning microscopy) showed that the TGN is a dot-like BFA (brefeldin A)-sensitive and Wm (wortmannin)-insensitive compartment, which is partially stained with FM4-64. These results suggest that the TGN functions in endocytic pathways in addition to the biosynthetic pathways. We will also discuss dynamics between TGN and the Golgi apparatus.

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We investigate to clarify details of dynamic structure and function of Golgi complex in which locates in the center of membrane traffic. It was found that the secretory glycoprotein α-amlyase I-1 is transport to the plastid through the ER-Golgi system in rice non-secretory cells overexpressing the α-amlyase I-1, suggesting that the Golgi complex has a yet unknown sorting system. In the present communication, we described structural changes in Golgi membranes in the cells overexpressing glycoproteins. EM studies showed that number of the Golgi in transgenic cells was significantly increased in comparison with that in the wild-type. Cell fractionation studies employing a sucrose density gradient centrifugation-floating revealed that the density of the GNTI-associated Golgi membranes in cells overexpressing glycoproteins was markedly increased. It strongly suggests that the components of Golgi membranes are dynamically rearranged for responding to change of the Golgi function.

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A mature tracheary element (TE), a component of vessels and tracheids, is a hollow dead cell and its differentiation process includes a unique cell death program in which vacuole rupture triggers the execution of cell death. To clarify the mechanism of vacuole rupture during TE differentiation, we have analyzed vacuolar membrane dynamics using Arabidopsis cultured cell line expressing GFP-AtRab75 during cell death. Time-lapse imaging revealed that (1) the vacuolar membrane continues to move and fuse dynamically until cell death, (2) at the final stage it ceases moving once, and (3) it dramatically shrinks to disappear. In order to elucidate its molecular mechanisms, we focused on several Rab GTPases and analyzed their function in the vacuolar membrane dynamics during TE differentiation. We also carried out genetic analysis of Arabidopsis mutants defective in the endocytic pathway. Based on our recent results, we will discuss the mechanism of vacuole rupture during TE differentiation.

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Arabidopsis seeds contain a lot of oil bodies, which are surrounded by a lipid monolayer and store lipids mainly triacylglycerols. Oil bodies possess oleosins as major structural proteins and are formed in endoplasmic reticulum during seed maturation. Arabidopsis thaliana has four oleosins in seeds: AtOleS1 to 4. To investigate the role of oleosins in formation of oil body, we isolated each oleosin-deficient mutant of A. thaliana. The mutant plants exhibited wild-type appearance.Electron microscopy revealed that the mutant seeds had larger oil bodies than wild-type seeds. Both AtOleS3 and AtOleS4 are most abundant oleosins in seeds. The mutants, atoles3 and atoles4, developed abnormally larger oil bodies in seeds than the other mutants, atoles2 and atoles1. Our observations suggest that oleosins have an important role in determination of oil body size. We are now investigating the effect of large oil bodies on seed germination and subsequent seedling growth.

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In the cortical region of cell, tubular structures of endoplasmic reticulum (ER) are arranged into meshwork. The contraction of tubular ER in the cortical region has been shown to be suppressed by inhibitors for actin cytoskeleton. We found that tubular structures (ER-tube) were formed in vitro from the vesicles of ER prepared from tobacco cultured cells BY-2, in which GFP-labeled ER (GFP-ER) was expressed, by the addition of GTP, but not by ATP or GDP. On the other hand, the formation of ER-tube was induced in the presence of myosin inhibitor, 2,3-butanedione monoxime, or depolymerizing drugs for actin filaments. However, the SH blockers, such as N-ethylmaleimide or biotin-maleimide, suppressed the formation of ER-tube. Similar phenomenon had been shown in the ER fraction prepared from Xenopus eggs. However, several differences in the formation of ER-tube in vitro were found between plant and animal materials. We will also discuss these points.

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Eucaryotic cells develop several essential membranous components whose morphologies and localizations are regulated by cytoskeletons. In plant cells, development of the large vacuole is indispensable for plant morphogenesis. In this study, we visualized vacuolar membranes and cytoskeletons in Physcomitrella patens by stable expressions of marker genes and analyzed the vacuolar morphogenesis during cell differentiation and growth. In protonemal cells and rhizoids, tubular vacuoles were observed around the cell cortex and cell tips and they flexibly moved. These vacuolar movements were strongly restricted by microtubule inhibitors. Furthermore, the vacuolar membranes were co-localized with microtubules and the vacuole at the cell tips appeared to be pulled by microtubules. These results suggest that the vacuolar structures and distributions are regulated by microtubules in Physcomitrella patens.

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Plant peroxisomes play a critical role in many aspects of growth and development from germination to leaf senescence. In the photosynthetic tissues, peroxisomes contain enzymes that participate in photorespiration, and share the metabolic pathway with chloroplasts and mitochondria. There are some reports on investigating the morphology and motility of peroxisome in root and stem cell (Mano et al, 2002), but few report on those in leaf mesophyll cell. Here we observed localization of peroxisome in Arabidopsis leaf mesophyll cell using transgenic plants stably expressing the fluorescence protein that enable to visualize peroxisomes and cytoskeletal proteins. We investigated then the proper condition regulating the peroxisome localization in detail. Furthermore, we screened a number of the peroxisome unusual positioning mutants (peup) from EMS treated mutant lines. The physiological analyses of peroxisome in wild-type and peup lines show leaf peroxisome having specific mechanism in taking their localization in mesophyll cell.

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The Arabidopsis genome is predicted to encode about 20 proteins homologous to the yeast or mammal peroxisome biogenesis factors (peroxin), but their functions remain unclear. In this study, we generated transgenic plants that were suppressed each peroxin gene expression using RNAi method and analyzed whether these homologues actually function for peroxisome biogenesis or not. The transgenic plants that stably expressed GFP-PTS1 were generated as a parent plant for RNAi plant, and we therefore could observe peroxisomes by GFP fluorescent. In the results, we could classified two groups of Arabidopsis peroxin, one group is that GFP is localized in the cytosol and the other defect on peroxisome morphology. Interestingly, PEX10 suppressed plant phenotype is different from wild type plant whereas other peroxin suppressed plants have same phenotype of wild type plant. Then, we analyzed the phenotype of PEX10 suppressed plant in detail and discuss novel peroxisomal function.

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Ca²⁺ plays a key role as a second messenger in intracellular signaling in stomatal movements. Stomatal movements are regulated by various environmental and endogenous signals, and oscillations in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) are a fundamental component of stomatal closure. [Ca²⁺]_cyt oscillations are mediated by Ca²⁺ flux from extracellular space and internal stores, such as vacuoles and ER. However, little is known about roles of chloroplasts in guard cell signaling. CAS is a Ca²⁺ binding protein that had been previously reported as a Ca²⁺ sensor in plasma membrane. We examined the subcellular localization of CAS and found that CAS is localized to the chloroplast thylakoid membrane. CAS inactivation resulted in significantly reduced [Ca²⁺]_cyt increase and inhibition of stomatal closure induced by external Ca²⁺, while overexpression of CAS promoted stomatal closure. These results provide the first evidence that chloroplasts plays an essential role in the regulation of [Ca²⁺]_cyt oscillations and stomatal closure.

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The Obg and Era-like small GTP binding proteins have been found in various prokaryotic and eukaryotic organisms. In bacteria, it has been shown that they participate in a wide range of cellular processes, including signal transduction, cell proliferation, intracellular trafficking and ribosome biogenesis. However, little is known about the biological role of these GTP binding proteins in eukaryotic cells. Intensive search on gene and protein databases revealed that Arabidopsis has ten Obg-Hflx and eight TrmE-Era Superfamily proteins. The sub-cellular localizations of each protein were determined by transient GFP expression assays using protoplasts or culture cells of Arabidopsis. We found that each proteins target to different organelles including chloroplast (eight), mitochondria (three), nucleus (two) and cytoplasm (two). There was two proteins dual-targeting to chloroplast and mitochondria, also one protein dual-targeting to cytoplasm and nucleus. Sub-cellular localization and T-DNA insertional mutant line analysis provide interesting insights for the evolution of these families.

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A newly isolated Arabidopsis thaliana aberrant chloroplast (abc2 ) mutant shows cotyledon specific albino phenotype. The mutated nuclear gene encoded a novel protein that had a Zn-finger motif. The expression of the ABC2 gene was induced by light irradiation, and was detected at only young plants. Green fluorescence protein and immunoblotting assays indicated that ABC2 protein was chloroplast membrane protein. In the abc2 mutant, the plastids in cotyledon grown under light conditions were abnormal shape and did not develop the thylakoid membrane, but that grown dark conditions was same to that of wild type. ABC2 protein had the reduction activity of insulin, and the oxidative renaturation activity of reduced RNase. These results suggested that ABC2 protein had protein-disulfide isomerase activity, and is a cotyledon-specific chloroplast biogenesis factor.

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Phytosulfokine (PSK), a 5-amino-acid secreted peptide, promotes cellular proliferation, tracheary element differentiation, and somatic embryo formation in vitro. Genes encoding PSK precursors are expressed at considerable level in a variety of tissues throughout plant life cycle, suggesting a role in plant development as well as in vitro culture. The PSK receptor PSKR1 was purified from microsomal fractions of carrot cells by ligand-based affinity chromatography and identified as a member of leucine-rich repeat receptor kinase (LRR-RK). Disruption and overexpression of Arabidopsis ortholog of PSKR1 (AtPSKR1) affects cellular longevity and potential for growth during plant development as well as in vitro culture. We further identified two proteins from the Arabidopsis genome, tentatively designated AtPSKR2 and AtPSKR3, with high sequence identity to AtPSKR1. Here we report the results of a series of experiments including complementation tests, expression patterns, PSK-binding activities of AtPSKR2 and AtPSKR3, and phenotypes of multiple knockout mutants of AtPSKR genes.

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Phytosulfokine (PSK), 5-amino-acid secreted peptide in plants, affects cellular potential for growth via binding to PSKR1, a member of the leucine-rich repeat receptor kinase (LRR-RK) family. PSK interacts with PSKR1 in a highly specific manner. However, it is not known which residues in the PSKR1 extracellular domain constitute the ligand-binding pocket. Here, we identified the PSK-binding domain of carrot PSKR1 (DcPSKR1) by photoaffinity labeling. We cross-linked the photoactivatable PSK derivatives with DcPSKR1, and mapped the cross-linked region by fragmentation. We also established a novel “on-column photoaffinity labeling” methodology that allows to increase the cross-linking efficiency. We purified a labeled DcPSKR1 tryptic fragment and identified a peptide fragment that corresponds to the 15-amino-acid Glu⁵⁰³-Lys⁵¹⁷ region of DcPSKR1 by MALDI-TOF MS. Deletion of Glu⁵⁰³-Lys⁵¹⁷ completely abolishes the ligand-binding activity of DcPSKR1. This region is in the island domain flanked by LRRs, indicating that this domain forms a ligand-binding pocket.

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Like CLV3 gene, some peptides function as a hormone and play important roles for growth and differentiation in plants. The genome-wide protein annotation data from Arabidopsis thaliana and rice (Oryza sativa) has revealed that there are many predicted genes for secreted peptides, which may function as peptide hormones. Here, we have tried a genome-wide survey for plant peptide hormone genes by a bioinformatics approach. Using public databases we analyzed a total of 83508 protein sequences from rice, A.thaliana and Cyanidioschyzon merolae. Based on prediction of secreted protein by the SingalP software and distribution pattern of conserved motifs identified by the MEME software, we identified 197 candidate genes belonging to 22 families with a conserved motif. We are now investigating biological effects of such peptides in planta.

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Although genetic analysis has identified genes involved in seed germination many components still remain to be discovered. We have investigated seed germination of Arabidopsis using chemical genetics. As with mutational analysis, the phenotype caused by small molecule are used as a starting point to identify genes in a particular biological process.
From 10,000 non-annotated chemical library, 5 compounds (E compounds) were identified which inhibit cotyledon greening and expansion. Interestingly ABA or GA signaling mutants showed altered response to E compounds. Consistent with this, genetic screening for E compounds resistant mutants identified new alleles of ABA and GA response mutants. Surprisingly, suppressor screens also identified new alleles of elongated hypocotyl1 locus (hy1). Although hy1 is known photomorphogenesis mutant it is also allele of genome uncoupled2 locus which is involved in plastid retrograde signal. In this presentation, we will discuss possible involvement of E compounds on hormone, light and plastid retrograde signaling.

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Like other winter annual species, germination of Arabidopsis seed is inhibited by high temperature (thermoinhibition). We have found that de novo ABA biosynthesis during imbibition is responsible for thermoinhibition of Arabidopsis seeds. We also found that three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5 and NCED9, were up-regulated by supraoptimal temperatures. To reveal contribution of each NCED gene on thermoinhibition, we selected and produced their T-DNA insertion mutants. The seeds of nced9 mutant showed considerable tolerance to thermoinhibition, but those of nced2 and nced5 showed no apparent phenotypes. The seeds of nced5nced9 double mutant showed higher thermoinhibition tolerance than nced9, but nced2nced9 and nced2nced5 double mutants showed similar germination to nced9 and their parents, respectively. The seeds of nced2nced5nced9 triple mutant showed similar tolerance to nced5nced9. These results suggest that NCED9 and NCED5 genes are unequally redundant and their up-regulation by high temperature is critical for thermoinhibition of Arabidopsis seeds.

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Germination of Arabidopsis seed is delayed as the imbibition temperature fall. To investigate the mechanism of this delay, we selected 10,000 T-DNA insertion lines and isolated four putative mutants which showed rapid seed germination at 5^oC. LTW18-1 was prominent among the four, and the seeds also showed high temperature resistant germination and reduced sensitivity to ABA. The plants were slightly smaller than wild type and had serrated leaves. These phenotypes, except for germination response to temperature, closely resembled to the auxin resistant mutant, ibr5, and the phenotypes of their F₁ indicated that LTW18-1 was allelic to ibr5. Sequencing analysis revealed that IBR5 gene of LTW18-1 had 7 bp deletion in the 2nd exon, which lead frame-shift and pre-mature stop codon. These results suggest that IBR5 works as an ABA signaling factor for germination regulation in a wide range of temperature.

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Auxin regulates various developmental processes, and its homeostasis is rigorously-controlled. In Zinnia elegans cell cultures, isolated mesophyll cells divides and differentiate into tracheary elements (TEs) in the presence of auxin. In this culture system, auxin transport inhibitors such as NPA prevent TE differentiation and high concentrations of auxin overcome the repression of TE differentiation. We found that NPA increased the amounts of NAA-derivatives and decreased free NAA. This free NAA depletion may prevent TE differentiation and cell division.
In this study, using novel IAA-amino acid synthetase inhibitors, we analyzed the auxin metabolism during TE differentiation. As a result, NPA increased the amounts of IAA-amino acid conjugates and decrease of free IAA. Furthermore, treatment of IAA-amino acid synthetase inhibitors on NPA-treated cells decreased the level of IAA conjugates and also recovered the NPA-suppressed cell division. Based these results, we will discuss the auxin metabolism in association with cell division and differentiation.

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Indole-3-butyric acid (IBA) induced vigorous rooting in Saiko (Bupleurum falcatum L.)-cultured roots after about 2 weeks. In medium at the first day of the culture, we found a novel IBA derivative, 4-(3-indolyl)-4-butanolide (IBL).
Synthetic IBL induced rooting on both cultured roots of B. falcatum and cuttings of Vigna radiata L. Wilczek. We histologically compared the effects of IBL and IBA on cultured roots of B. falcatum. IBA induced considerable thickening of the original roots, due to marked cell expansion and division in the cortical tissue. On the other hand, IBL did not show cell expansion, but induced cell division as strongly as IBA. Although auxin suppresses root elongation with unneutral growth of the cotyledons of Raphanus sativus, IBL shows none of those effects. The results suggest that IBL may play a specific role in the rooting process induced by IBA.

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The formation and degradation of IAA-amino acid conjugates (IAA-aa) play an important role in auxin homeostasis. GH3s, early auxin-inducible genes, belong to the acyl-activating enzyme superfamily and encode IAA-aa synthetases. We designed and synthesized N-sulfamolyadenosine (SA) derivatives of auxins as intermediate analog inhibitors of IAA-aa synthetase on the basis of its catalytic mechanism. We previously showed that the SA derivatives are the potent inhibitors of GH3 in an in vitro enzyme assay. In this study, we examined their in vivo activities. In the excised Arabidopsis leaves treated with the exogenous IAA, the endogenous levels of IAA-aa (IAA-Asp, IAA-Glu, and IAA-Gln) greatly increased. When the SA derivatives were applied together with IAA, the increase was significantly repressed. Thus, the chemical approach inhibits all the GH3s involved in IAA-aa synthesis, and the inhibitors are expected to serve as a highly effective tool to study auxin homeostasis.

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Plants produce IAA from the TRP-dependent and -independent pathways. In Arabidopsis, the TRP-dependent pathway involves at least two metabolic routes; the indole glucosinolate and tryptamine pathways. Here, we have developed new isotope labeling systems to assess contribution of the TRP-dependent pathway to IAA biosynthesis in Arabidopsis. The intermediates of the TRP-dependent pathway were efficiently ¹⁵N-labeled by a feeding of [¹⁵N₂]TRP to the seedlings of Arabidopsis trp1-1 mutant. Similarly, the tryptamine pathway was selectively ¹⁵N-labeled using Arabidopsis trp3-1 mutant. Our LC-MS/MS and GC-MS data indicated that IAA is moderately produced from the TRP-dependent pathway, but the tryptamine pathway does not largely contribute to IAA biosynthesis at whole-plant level. We are currently analyzing the contribution of the tryptamine pathway to IAA biosynthesis at organ/tissue levels.

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A rapid decrease of the plant hormone ABA under submergence is thought be a prerequisite for the enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA), a catabolite of ABA, increased with decreasing ABA level after submergence. The oxidation of ABA to PA is catalyzed by ABA 8'-hydroxylase, which is possibly encoded by three genes (OsABA8ox1-3) in rice. The mRNA level of OsABA8ox1 increased dramatically within 1 h after submergence. Treatment of aerobic seedlings with ethylene and ACC rapidly induced the expression of OsABA8ox1. Moreover, pretreatment with 1-MCP partially suppressed induction of OsABA8ox1 expression after submergence. The ABA level was found to be negatively correlated with OsABA8ox1 expression after ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA levels in submerged rice shoots is triggered in part by ethylene-induced expression of OsABA8ox1.

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Plant hormones interact each other and regulate many aspects of plant growth and development. Therefore it is important to analyze all the hormones from the same plant materials. We used to use GC-MS for hormone analysis. However, this method is not necessary suitable for high-throughput and comprehensive hormone analysis. We have recently introduced a LC-ESI-MS/MS system, which does not require derivatization of hormones. Using both GC-MS and LC-MS we will perform comprehensive hormone analysis. We will also report the present situation of the hormone analysis platform of RIKEN PSC.

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Arabidopsis thaliana BOR1 is a borate exporter and is essential for efficient boron(B) translocation under low B conditions. In the root tip cells of P35S:BOR1-GFP plants, BOR1-GFP accumulated in the plasma membrane under B limitation, and was transferred to the endosome and then the vacuole for degradation upon high B supply (Takano et al., 2005).
On the other hand, Arabidopsis thaliana BOR4 was suggested to take part in B export from plant roots under B excess (Miwa et al., this meeting). In the root tip cells of P35S:BOR4-GFP plants, BOR4-GFP still accumulated in the plasma membrane upon high B supply.
To identify domains and motifs important for the different response to B availability, we conducted domain-swapping between BOR1 and BOR4 and also site-directed mutagenesis of potential endocytosis signal motifs in BOR1. We report B-dependent localization of these transporters and discuss the mechanisms of B-dependent trafficking of borate transporters.

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We previously identified a molybdate transporter, MOT1, from Arabidopsis thaliana. Here, we studied roles of MOT2, a MOT1 paralog, in molybdenum (Mo) transport. Transcripts of MOT2 were detected both in shoots and roots, and relative transcription levels of MOT2 were two times higher than that of MOT1. The levels of MOT2 transcript accumulation in plants grown under limited Mo conditions were not significantly different from that in plants grown under sufficient Mo conditions. Mo concentration in shoots of a T-DNA insertion mutant for MOT2 was reduced, and under limited Mo conditions, the growth of mot2 mutant plants was suppressed. The growth was partially restored by supplementing ammonium. Expression of MOT2 increased Mo concentration of yeast cells. Taken together, we suggest that MOT2 encodes a functional Mo transporter required for normal growth under low Mo conditions.

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