We analyzed the responses of cyclic electron flow around PSI (CEF-PSI) to the light environments, using a model plant Arabidopsis thaliana. After the plants were grown under low-light conditions (LL) for 3-weeks, they were transferred to high-light conditions (HL). Both photosynthetic linear electron flow (LEF) and the electron flux in PSI were evaluated from both the quantum yields of PSII (QY(PSII)) and PSI (QY(PSI)), respectively. The activity of CEF-PSI was estimated as QY(PSI) - QY(PSI). After the transition from LL to HL conditions (24, 48, 96 h), these above parameters were measured. On the transitions, Fv/Fm decreased from 0.82 to 0.78. Chl fluorescence parameters, both qL and NPQ, increased. [P700+]/[P700]total also increased. Further, the activity of CEF-PSI increased. The values of both qL and [P700+]/[P700]total also increased. Thse results inidicated that the redox level of PQ regulated the activity of CEF-PSI.

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We characterized the response of cyclic electron flow around PSI (CEF-PSI) to low-nitrogen nutrient conditions using rice plants. Rice plants were hydroponically cultured with the two level nitrogen concentration (MN, 1 mM NH4NO3; LN, 0.3 mM) under high (HL) and low light (LL) intensities. Both Photosynthetic linear electron flow (LEF) and the electron flux in PSI were evaluated from both the quantum yields of PSII (QY(PSII)) and PSI (QY(PSI)), respectively. The ratio of QY(PSI) to QY(PSII) in both HL- and LL-plants increased in response to LN-conditions. These results indicated that the photosynthesis-limited conditions at LN induced the activity of CEF-PSI. We could not detect any difference of QY(PSII) at lower light intensities in all rice plants. However, QY(PSI) in LN-plants were larger than those in MN-plants. These lowered QY(PSI) were activated by the pre-illumination to their leaves. These results showed that low-nitrogen nutrients activated PSI and its enhancement contributed to the activation of CEF-PSI. Activation mechanism of CEF-PSI will be discussed.

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We characterized cyclic electron flow around PSI (CEF-PSI) using the intact leaves of rice which PSII was photoinhibited, to elucidate the effect of photosynthetic linear electron flow (LEF), estimated from QY(PSII), on CEF-PSI. Rice leaves were exposed to high intensity of light under photosynthesis-limited conditions. With the light treatments, maximum quantum yield of PSII and net photosynthesis rate decreased. NPQ of Chl fluorescence was suppressed by the light treatments. On the other hand, qL, the Chl fluorescence parameter for the redox level of PQ, increased. The ratio of P700+ to total P700 also increased. These results indicate that photoinhibition of PSII induced the increase in oxidation of photosynthesis electron transport system, especially PQ. Different from LEF activity, the relative electron flux in PSI was not affected by PSII photoinhibition. Furthrmore, the relationship between QY(PSI) and QY(PSII) with the slope below 1 was kept even after the photoinhibition. However, the activity of CEF-PSI increased, because LEF activity decreased. The molecular mechanism for the regulation of CEF-PSI activity and its physiological significance will be discussed.

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Photosynthesis is one of the most important determinants of crop productivity. To identify photosynthesis-related genes in rice, we generated Arabidopsis lines that rice full-length cDNAs were expressed. We have used imaging of chlorophyll fluorescence to screen photosynthesis-related mutants. Overexpression of two rice cDNAs (OsLFNR1, OsLFNR2) lead to altered chlorophyll fluorescence both in Arabidopsis and rice. Chlorophyll fluorescence analysis indicated that OsLFNR2 overexpression plant was impaired in linear electron transport and Fd-dependent PSI cyclic electron flow. OsLFNR1 overexpression plants showed growth defect. However, the growth defect was recovered when the plants were grown in nutrient rich medium. When OsLFNR1 overexpression plants were grown in nitrogen limited conditions, they exhibited long roots compared to WT. This suggests that OsLFNR1 overexpression plants have defect in nitrogen assimilation. Expression of OsLFNR genes result in suppression of endogenous FNR genes both in Arabidopsis and rice. From these results we propose that two isoforms of FNR control the balance between the demand for nitrogen assimilation and photosynthetic activity.

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Arabidopsis has three PsbQ-Like (PQL) proteins in addition to the PsbQ subunit of the oxygen-evolving complex (OEC) of photosystem II. Recent proteomic and in silico co-expression studies suggested that the two PQL proteins, PQL1 and PQL2, might have a function in the chloroplast NAD(P)H dehydrogenase (NDH) complex. In this study, we report the characteristics of Arabidopsis mutants lacking either of two PQLs, pql1 and pql2. The NDH activity estimated by chlorophyll fluorescence analysis was severely decreased in pql1 and pql2 mutants, indicating that both PQLs are required for the NDH activity. In thylakoid membranes of the wild-type plants, PQLs were tightly associated with the NDH complex and protected from protease treatments, while unassembled PQLs were unstable in the mutants lacking known NDH subunits, NdhB or PsbP-Like protein 2 (PPL2). Defect of PQL1 caused drastic reduction of the known NDH subunits in pql1 mutant, while the NDH subunits were partially stable in pql2 mutant, indicating that the functional role and the binding site in the NDH complex would be differed between PQL1 and PQL2.

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Chloroplast NAD(P)H dehydrogenase (NDH) is a homolog of complex I in mitochondria and bacteria. NDH mediates the backflow of electrons from stroma reductants to plastoquinone in cyclic electron transport around photosystem I (CET). Recently, the biochemical analysis indicated that NDH forms a supercomplex with PSI in thylakoid membranes. However, in contrast to the elucidation of fine structure of complex I, the entire subunit composition and electron input site of NDH are still unclear. Here we report the identification and characterization of a novel protein required for NDH activity. Its homologs were also found in cyanobacteria but not in Chlamydomonas, which lacks NDH. The protein localizes to thylakoid membranes but has no transmembrane and any known motifs. The Arabidopsis knockout mutant did not show a transient increase in Fo of Chl fluorescence after turning off of actinic light, indicating the impairment of NDH activity in vivo. This protein co-migrated with NDH-PSI supercomplex on BN-PAGE. However, the lack of the protein did not affect the stabilities of the supercomplex and other NDH subunits.

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Type I chaperonins are large double-ring assemblies present in bacteria (GroEL), mitochondria and chloroplasts (Cpn60) and they are involved in the efficient protein folding. In E. coli, GroEL consists of 14 identical subunits and 85 substrates exhibit an obligate dependence on GroEL for folding, suggesting a broad range of GroEL substrates. In contrast, chloroplasts comprise two different Cpn60 isoforms, α and β. Two Cpn60α and four Cpn60β subunits were found in Arabidopsis chloroplasts and they were assembled into mixed Cpn60α7β7 oligomer. While cpn60α and cpn60β1β2 mutants are lethal, cpn60β4 was only defective in NDH activity. Consistent with the phenotype of cpn60β4, an NDH subunit, NdhH, was detected in the chaperonin complex containing Cpn60β4 as a substrate by mass analysis. Cpn60β4 has an additional C-terminal extension compared with other 3 Cpn60β subunits and this extension was shown to be critical for the specific function of Cpn60β4 on the NDH assembly. These facts imply that Cpn60β4 has acquired some structure features during the evolution to accomplish the folding of the specific substrate NdhH.

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Light reactions of photosynthetic consist of linear and photosystem I (PSI) cyclic electron transport. The light reactions have been generally explained by linear electron transport, and the physiological function of PSI cyclic electron transport was unknown for a long time. Recently, it was clarified that linear electron transport cannot supply enough ATP required for CO₂ fixation and extra ATP is produced by PSI cyclic electron transport. In higher plants PSI cyclic electron transport consists of two pathways. The main route depends on PGR5 protein, while chloroplast NAD(P)H dehydrogenase (NDH) is involved in the minor pathway. The PGR5-dependent pathway is sensitive Antimycin A, but its target is still unclear. We discovered that Arabidopsis transformed with the Pinus thunbergii PGR5 gene showed Antimycin A-resistance. We also determined the amino acid that determines the resistance. The results suggest that Antimycin-A binds to PGR5 or protein closely localized to PGR5 in thylakoids.

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Photosystem I (PSI) cyclic electron transport is essential for photosynthesis and photoprotection. In angiosperm, the PGR5 (PROTON GRADIENT REGULATION 5)-dependent pathway is the main route of electrons in cyclic electron transport and contributes to ATP synthesis.
Previously, we reported that overaccumulation of PGR5 accelerated the rate of PSI cyclic electron transport, resulting in delayed chloroplast development. Enhanced PSI cyclic activity may cause higher reduction of the plastoquinone (PQ) pool, disturbing chloroplast development. The Arabidopsis immutans mutant, which is defective in plastid terminal oxidase (PTOX), exhibits leaf variegation, probably because of a lack of oxidized PQ supply to phytoene desaturase involved in carotenoid biosynthesis during chloroplast development. These results imply the link of the pheonotypes between PSI cyclic electron transport and PTOX. Here, we discuss the function of these alternative electron transport pathways, which were clarified from our genetic analysis.

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Peroxisomes are organelles present in most of eukaryotic cells and participate in the metabolism of fatty acids and other metabolites. Unlike chloroplasts or mitochondria, peroxisome proteins are synthesized in the cytosol and imported across their membranes. Thus there must be a peroxisome-specific protein transport system which is classified as peroxisome membrane protein transport and matrix protein import. Pex16 is a peroxisomal membrane protein that has been shown to regulate peroxisome biogenesis and membrane morphology. Pex7 is a soluble import receptor that recognizes peroxisomal targeting signal type 2 (PTS2)-containing proteins in the cytosol and transports them into peroxisomes across peroxisome membranes. Using transgenic plants overexpressing green fluorescence protein (GFP) tagged PEX7 and PEX16, we analyzed protein complexes of PEX7 or PEX16 proteins. By combination of co-immunoprecipitation technique and Orbitrap MS analysis, we identified 31 and 6 candidate proteins as binding partners of Pex7 and Pex16, respectively. Functional analysis of each candidate protein including the localization is under investigation for understanding peroxisomal biogenesis.

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Peroxisomes are one of the membrane-bounded organelles, and ubiquitously found in eukaryotic cells. Peroxisomes play vital metabolic function in all eukaryotes. However, the metabolisms are different depending on each organism. In higher plants, peroxisomes are responsible for β-oxidation of fatty acids, photorespiration and synthesis of the plant hormone. Hence, lack of peroxisomal enzymes usually results in remarkable phenotypes such as embryonic lethality or dwarfism under normal condition. peup1(Peroxisome unusual positioning1) in this study, shows different morphology and subcellular localization of peroxisomes with wild-type peroxisomes. In peup1 mutant, the number of peroxisomes seems to increase than wild-type. Additionally, catalases which are major peroxisomal proteins, are accumulated excessively. Notably, the specific activities of catalases in peup1 mutants is as half as these of catalases in wild-type. These results indicate that PEUP1 control the quality and quantity of the peroxisomes.

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Peroxisomes are single membrane-bound organelles that are ubiquitously found in eukaryotic cells and have pivotal roles in β-oxidation of fatty acids and degeneration of hydrogen peroxide. Additionally, peroxisomes in higher plants have specialized functions, such as biosynthesis of phytohormones and photorespiration. Since unlike mitochondria and chloroplasts, peroxisomes does not have own genome, the enzymes participated in peroxisomal functions are encoded in nuclear genome and transported into peroxisomes after protein synthesis in the cytosol. The functions of peroxisomes are plastically controlled under environmental changes. However, precise machineries of differentiation of peroxisomes have not been clarified. To identify the factors regulating peroxisome biogenesis, we have screened the aberrant peroxisome morphology (apm) mutants, in which peroxisomes display abnormal phenotypes, elongated or enlarged peroxisomes. Some mutants show a defect in peroxisomal protein transport. In this presentation, we will report the recent work on the machinery of peroxisomal protein transport in higher plants.

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The Golgi apparatus plays an essential role in protein sorting and transport in eukaryotic cells. In animal and plant cells, the Golgi apparatus consists of several flattened membrane sacs called cisternae, which form a stack. In mammalian cells, Golgi stacks are centralized by microtubules and fuse together to form a huge single ribbon-like structure. In contrast, individual Golgi stacks are distributed widely in the cytoplasm and move along actin filaments in plant cells. The plant Golgi apparatus has been also known to keep its morphology and function throughout mitosis, suggesting that the plant Golgi apparatus proliferates in the late stages of mitosis. To examine the dynamics of the plant Golgi apparatus, we visualized Golgi stacks in plant cells by using GFP, mRFP and photoconvertible fluorescent protein, Dendra2. The observation by confocal laser scanning microscopy revealed that individual Golgi apparatus communicated with each other. In this meeting, we will discuss the biogenesis of the plant Golgi apparatus during mitosis.

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In all eucaryotic cells, the post-Golgi organelles, such as the trans-Golgi network (TGN), endosomes, vacuoles and the plasma membrane, are connected by vesicular traffic. This complex network plays a critical role in several higher-order functions. 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 SYP43, 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-SYP43 under the control of the native promoter. The observation by CLSM (confocal laser scanning microscopy) showed that the There are two types of the TGN; One mainly locates beside the Golgi apparatus and behaves together, the other is independent from the Golgi apparatus. We will also discuss dynamic movement between TGN and the Golgi apparatus.

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The trans-Golgi network (TGN) is known to be a major sorting organelle. However, recent work has suggested that the TGN is also involved in the endocytic pathway in plant cells. To clarify whether the TGN acts in endocytosis, it is first necessary to visualize the endocytic route in plants. Using a Leu-rich repeat transmembrane receptor kinase, FLAGELLIN SENSITIVE2 (FLS2), as an endocytosis marker, we have succeeded in visualization of endocytosis process in Nicotiana benthamiana. In order to investigate the proteins that function in endocytosis through the TGN, we focused on RABs, small GTPases regulating targeting and/or tethering of transport vesicles. Rab11 is the first candidate because it is localized on recycling endosomes and the TGN. In addition, Rab5 is known to be involved in endocytosis in mammalian and plant cells. We present how Rab11 and Rab5 mediate endocytic transport of FLS2.

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It is well known that pavemant cells show their jigsaw puzzle-like shapes in leafe epidermis of dicotyledonous plants. However, intracellular dynamics during the cell morphogenesis are largely unknown. We found the interesting localization of mRFP-tagged sialyl transferases (ST-mRFP), which label the cytosolic side of trans-Golgi membrane. ST-mRFP fluorescence was observed in the pavement cell border, whereas it was also in trans-Golgi bodies as previously reported. In guard cells, ST-mRFP strongly labeled the both ends of the guard cells. We confirmed that ST-mRFP localized in apoplast by observation of the plasmolyzed cells and dual observation with plasma membranes by vital FM4-64 staining. The observation in various leaf growth stages showed that the apoplastic localization become pronounced during cell maturation. Also, our image analysis techniques quantitatively revealed that the fluorescence intensity has strong correlation with the curvature of the cell border, suggesting that ST-mRFP tended to localize at the curved region.

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Recent progress on microscopy techniques allows us to provide tremendous amounts of microscopy data that can easily surpass the volume of omics data. We therefore believe that there will be a global trend toward the development of microscopy image databases and data mining technologies. We have obtained serial optical sections of various fluorescent-labeled structures in Arabidopsis guard cells, and made the image sets public as LIPS (Live Images of Plant Stomata) database. In this study, we performed localization analysis using statistical approaches. We visualized and analyzed the intracellular distribution of the structures by the averaged intensity projection (AIP) image obtained from 100-120 images. These colocalizations were quantitatively evaluated by the intensity correlation coefficient among the AIP images. From a series of the studies, it has been suggested that microtubules and mitochondria mainly localize in the proximal side, whereas actin microfilaments and endosomes localize in the distal side. Also, actin microfilaments and endoplasmic reticulum were observed in the connection of the guard cells.

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The nuclear envelope, which separates cytoplasm and nucleoplasm in eukaryotes, is lined with nuclear lamina from nucleoplasmic side. It has been shown that the animal nuclear lamina has a mesh like-structure composed of a lamin protein, an intermediate filament, and contributes to maintenance of nuclear envelope structure and diverse nuclear functions. However, plants have no homologues of lamin and few homologues of nuclear envelope proteins in their genomes. This suggests that plants have evolved their own nuclear architecture. We aim to reveal the molecular basis of nuclear structure in higher plants. We generated transgenic Arabidopsis plants that stably expressed nucleus-targeted GFP to visualize nuclei. To screen the mutants having abnormal shapes of nuclei, the transformants were treated with EMS. We found that one of isolated mutants, which has spherical and smaller nuclei, had a base substitution mutation in LINC1 gene that encodes a coiled-coil protein localized at nuclear periphery for regulating nuclear morphology. The other mutants we isolated are now being investigated.

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Plant lipids have an important role in plant life processes, such as membrane component, energy source in seeds, signal molecular in stress response, and anti-fungal/bacterial compounds in defense response. A specific organelle, oil body, stores plant lipids. Oil bodies are accumulated in seeds, anthors and stressed leaves. Leaf-oil bodies are located in cytosol, in contrast to plastoglobules in plastid. Leaf-oil bodies are increased in the number by senescence and various stresses including fungal infection. Colletotrichum higginsianum is a hemibiotrophic fungal pathogen and causes anthracnose disease on Arabidopsis thaliana and other crucifer hosts. Interestingly, Colletotrichum higginsianum infection induced a leaf-oil-body related protein around the fungal lesion. The leaf-oil-body related protein might contribute to defense response against Colletotrichum higginsianum infection. We suppose that leaf-oil bodies and the leaf-oil-body related protein inhibit fungal spread.

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The endoplasmic reticulum (ER) body is an ER-derived organelle that is specific to Brassicales plants. In Arabidopsis thaliana, a transcription factor, NAI1, regulates the expression of PYK10, a beta-glucosidase of major ER body component, and NAI2, a novel ER body protein for ER body formation (1,2). We found that the co-expression of NAI2 and PYK10 is sufficient for ER body formation in onion (Allium cepa), a non Brassicales. These findings indicate that NAI2 and PYK10 is sufficient for the ER body formation. NAI1 regulates the expression of membrane of ER body 1 (MEB1) and MEB2. MEB1 and MEB2 are integral membrane proteins that localize to the ER body membrane. Depletion of NAI2 alters localization of MEB1 and MEB2, which become diffusely distributed within the ER network. The artificially induced ER bodies accumulate MEB2 in onion cells. These findings indicate that ER body components, NAI2 and PYK10, regulate ER body formation and localization of MEB2.
(1) Matsushima et al., (2004) Plant Cell. 16, 1536-1549.
(2) Yamada et al., (2008) Plant Cell. 20, 2529-2540.

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Chloroplasts are partially mobilized to the vacuole via RCBs by autophagy. We have analyzed factors affecting the RCB-creation in Arabidopsis excised leaves. We suggested that the sugar contents in leaves were the main factors affecting the RCB-creation, and the presence of mechanism specifically controlling the RCB-creation in autophagy. In this study, we analyzed the RCB-creation in leaves of starch-less mutants (pgm, adg1) and starch-excess mutants (sex1, mex1). In continuous illumination, the RCB-creation was accelerated in leaves of starch-less mutants. There were more starch content and lower creation of RCBs in starch-excess mutants than wild-types. In the condition of 14-hour photoperiod, we investigated the RCB-creation from highly expanding to senescent stages. The maximum number of RCBs in each stage was significantly higher in starch-less mutants and lower in starch-excess mutants compared to wild-types. These results indicate that the starch metabolism have a critical role in the RCB-creation.

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Sugars are the prime carbon and energy source and an important signal for growth and development in plants. To elucidate molecular mechanisms of sugar response in plants, we identified evolutionarily conserved and glucose responsive nuclear proteins by proteome analysis of rice nuclear proteins and investigation of the sugar responsiveness of the corresponding genes. There were two WD40 proteins (NuGWD1 and 2) among selected proteins. We confirmed that these transcripts were induced by glucose and sucrose in both rice and Arabidopsis. We isolated T-DNA insertion lines for AtNuGWD1. In the siliques of atnugwd1 heterozygous Arabidopsis, aborted ovules were observed, suggesting either lethality of atnugwd1 homozygous embryo or abnormal female gametogenesis. The result of GST pull down assay using OsNuGWD1 protein revealed that OsNuGWD1 bound a complex containing ribosomal proteins. Homologs of NuGWD1 and 2 are evolutionarily conserved from yeast to human. Taken together with previous reports on function of yeast homologs, these results imply that glucose elevates levels of NuGWD1 and 2, which promote 18S rRNA biogenesis and assembly of 40S small ribosomal subunit in plants.

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A candidate for a gene encoding MGDG synthase was identified from the green sulfur bacterium, Chlorobaculum tepidum (formerly called Chlorobium tepidum), through a newly developed computer-added gene discovery system. This gene, mgdA, encodes a 49-kDa protein that belongs to a beta-glycosyltranferase family. The primary structure of MgdA shows no significant similarity to any MGDG synthases identified so far from other organisms. MgdA expressed in E. coli showed MGDG synthase activity in vitro by use of UDP-galactose, but not UDP-glucose, as a substrate. mgdA is essential for this bacterium; only heterozygous mgdA mutant could be isolated. We performed complementation analysis of Arabidopsis MGDG synthase (MGD1) mutant, mgd1-2, by heterologous expression of MgdA that contains the chloroplast transit peptide of MGD1 at its N-terminus. The complementing line showed almost normal levels of MGDG, although mgd1-2 mutation causes about 98% reduction in MGDG content. From these observations, we conclude that MdgA is a novel MGDG synthase, which has been established and conserved in Chlorobi phylum. The physiological function of MgdA will be discussed.

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Cardiolipin (CL) is widely distributed in various prokaryotes and mitochondria of eukaryotes as a membrane phospholipid. We identified the Arabidopsis gene CLS encoding a CL synthase as the first gene from the multicellular organisms. We also isolated mutants in which the CLS gene is disrupted by T-DNA insertion and found some abnormalities in the cls/cls mutants. The abnormalities of cls-2/cls-2 plant were severer than those of cls-1/cls-1 plant. In vivo labelling experiments in which the seedlings were incubated with [³³P] Pi for 17 hours revealed that the incorporation of radioactivity into CL in the total phospholipid labelling was higher in the cls-1/cls-1 than in the cls-2/cls-2, and that the extent of the CL labelling was correlated with the phenotypes. We also measured the labelling of CL in the mutants in which CLS expression can be induced by exogenously added estrogen. By the induction of CLS expression with estrogen, the extent of CL labelling in the mutant increased and concomitantly the growth of roots was recovered. These findings demonstrate that CLS can control the level of CL and the cls mutants are nice tools to investigate the physiological roles of CL.

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Sterols are isoprenoid-type lipids that are biosynthesized via the cytosolic mevalonate pathway. Through the study of several sterol biosynthetic genes, we have identified that sterols play important roles on cell elongation, senescence, male gametophyte development and chloroplast differentiation. To understand total physiological function of sterols, we have comprehensively collected and characterized Arabidopsis mutants lacking sterol biosynthetic genes.
Mutants on 19 loci were obtained as homozygote genotype. The sterol accumulation and profiles of these 19 mutants were quantitatively analyzed. The gene expression profiles of these mutants were examined by microarray analyses and the data were analyzed statistically. The results of sterol analyses and transcriptomics suggest that the magnitude of influence of sterol deficiency is compared with that of brassinosteroid deficiency. Although the alteration of sterol composition also affects gene expression profiles, the magnitude of influence was less than that that of sterol and brassinosteroid deficiency.

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About 120 glucosinolates (GSLs) are predominantly synthesized in the plant family of the Brassicaceae. GSLs are sulfur-containing secondary metabolites and their breakdown products confer e.g. resistance to herbivores and pathogens or exhibit anticarcinogenic properties in humans.
Synthesis of GSLs is tightly connected with the synthesis of amino acids in the primary metabolism, mainly methionine, phenylalanine and tryptophane.
These amino acids form the skeleton of the GSLs. Side-chain elongation, core GSL biosynthesis and side-chain modification lead to the final products of aliphatic, aromatic and indole GSLs.
Integration of transriptomic and metabolomic data of S-deprived Arabidopsis plants led to the identification of many genes in GSL biosynthesis. Intriguingly, this approach also uncovered changes of genes in primary amino acid metabolism.
We aim to unveil the impact of the primary amino acid metabolism in glucosinolate biosynthesis by characterizing candidate genes identified by coexpression analysis and metabolic profiling. Furthermore, we want to elucidate missing steps in GSL biosynthesis by this approach. Our latest results will be presented.

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Camptothecin is produced in the hairy roots but not in cell suspension culture of Ophiorrhiza pumila. The hairy root specific metabolites were profiled by non-target analysis by using infusion FT-ICR-MS. Furthermore, the gene expression of two enzymes, triptophane decarboxylase (TDC) and secologanin synthase (SLS), involved in the biosynthetic pathway of indole alkaloids were modified by RNAi silencing. The metabolic changes in these hairy roots were analyzed by LC-FT-ICR-MS. From the exact mass of the ion peaks that changed correlatively with the suppressed gene expression, candidates of intermediates were predicted.

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Legumes show a pool of different triterpenoid saponins, however, most of their biosynthesis pathways are yet unknown. Previously, licorice (Glycyrriza spp.) β-amyrin 11-oxidase CYP88D6, which catalyzes the conversion of β-amyrin to 11-oxo-β-amyrin in the glycyrrhizin pathway, was identified using a yeast heterologous expression system. Similarly, CYP-A21 (tentative name) was identified as a second important P450 that is responsible for the oxidations at C-30 of 11-oxo-β-amyrin to produce glycyrrhetinic acid. In this work, using the Medicago truncatula EST database, 7 homologs of CYP-A21 and 2 homologs of CYP88D6 were identified and analyzed for their potential β-amyrin oxidizing activity using a yeast expression system. One of the CYP-A21 homologs (75%amino acid identity to CYP-A21) was able to catalyze three oxidation steps at C-30 of P450-amyrin to produce 11-deoxoglycyrrhetinic acid. Furthermore, when the activity of this CYP-A21 homolog was tested in combination with CYP88D6, GC-MS results showed their ability to produce glycyrrhetinic acid. The combination of various legume P450s are now in progress in order to generate new β-amyrin derivatives.

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Artemisia annua produces an anti-malarial sesquiterpenoid, artemisinin. A cytochrome P450 monooxygenase CYP71AV1 was identified from A. annua. The CYP71AV1 is a multifunctional enzyme capable of oxidizing amorpha-4,11-diene at the C12 position to generate artemisinic acid via artemisinic alcohol and artemisinic aldehyde in artemisinin biosynthetic pathway.
In this study, we revealed that CYP71AV1 homologous genes (more than 94% identity at amino acid level) were expressed in artemisinin non-producing species; A. afra and A. absinthium, inconsistent with the absence of amorpha-4,11-diene and transcripts for amorpha-4,11-diene synthase (ADS).
To analyze amorpha-4,11-diene oxidizing activity of these CYP71AV1 homologs, ADS and each of CYP71AV1 homologs were coexpressed in yeast, and the ethyl acetate extracts of the cultures were analyze by GC-MS. Putative sesquiterpene alcohol, with retention time different from artemisinic alcohol (C12 hydroxylated form), was detected from yeast expressing ADS and each of CYP71AV1 homologs. These results indicate that these homologs have amorpha-4,11-diene oxidizing activity with different regiospecificity from CYP71AV1.

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The diversity of biosynthesis of pyridine nucleotides and related secondary metabolites in plants was investigated using more than 40 species. In contrast to animals, the de novo pathway of NAD synthesis in plants is initiated from aspartate, and nicotinamide produced by the catabolism of NAD and was converted to nicotinic acid, and then reutilised the synthesis of NAD. In contrast to other organisms, the seven-member pyridine nucleotide cycle is operative in plants. In addition, bypasses catalysed by newly discovered nicotinic acid riboside kinase and/or nicotinamide deaminase appear to be functional in some plant species. All plants investigated produced trigonelline (TG) or nicotinic acid glucoside (NaG) as secondary metabolites derived from nicotinic acid. TG is formed in a wide variety of plant species. Large quantity of TG is accumulated in seeds of coffee and Leguminosae plants. NaG formation was found in limited species including Brassicaceae plants, such as Arabidopsis and canola. Organ specific formation of TG and NaG was also found in some species, for example, TG is formed in potato leaves, but NaG is formed in tubers. Possible roles of TG and NaG will be discussed.

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Lysine decarboxylase (LDC) is the key enzyme involved in the first step of quinolizidine alkaloids (QAs) biosynthesis. We have cloned the lysine/ornithine decarboxylase (L/ODC) from alkaloid-containing cultivar of L. angustifolius by using PCR-select-subtraction and 5'/3'-RACE techniques. The purified recombinant protein expressed in E. coli exhibited decarboxylase activities towards both L-ornithine and L-lysine with similar K_m value. The decarboxylase activities toward both substrates were competitively inhibited by DFMO, which is a specific inhibitor of ODC. We also characterized L/ODC genes from Sophora flavescens and Echinosophora koreensis which produce QAs. Kinetic study of these two purified recombinant L/ODCs showed the decarboxylase activity toward both substrates with similar K_m as same as L/ODC from L. angustifolius. The comparison of the catalytic efficiency (k _cat /K_m) of these three L/ODCs revealed that the preference for L-ornithine over L-lysine is only 0.5-1.5 times. This is the first report on an L/ODC presumably involved in QAs biosynthesis having almost equal decarboxylase activities towards both L-ornithine and L-lysine from plants.

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Anthocyanins and betacyanins, two types of red pigment, have never been found to occur together in plants. Although anthocyanins are widely distributed in higher plants, betacyanins have replaced anthocyanins in the Caryophyllales. The accumulation of flavonols in the Caryophyllales suggests that the step(s) of anthocyanin biosynthesis from dihydroflavonols to anthocyanins could be blocked in the Caryophyllales. So far, anthoacyanidin synthase (ANS) cDNA was isolated from plants of the Caryophyllales and an enzyme activity assay showed that the Caryophyllales possess functional ANS. However, the expression profile revealed that ANS are not expressed in most tissues and organs except the seeds in Spinacia oleracea, suggesting the transcriptional regulation might cause the lack of anthocyanin in the Caryophyllales. Therefore, we are trying to reveal the transcription mechanics of ANS from two aspects of their regulatory elements, cis and trans.
We first analyzed the expression of Caryophyllales ANS in Arabidopsis transformed with ANS promoter::GUS constructs. Furthermore, we have tried to find transcription factors which activte Spinacia ANS promoter.

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The two types of red pigment, anthocyanins and betacyanins, never coexist in the same plant. While the anthocyanin biosynthetic genes have been cloned and analyzed, the betacyanin biosynthetic pathway is still poorly understood. We carried out the biochemical analysis of two Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) that may be involved in betalain biosynthesis. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. The site-directed mutagenesis experiments suggested the contribution of a conserved motif to full DOD activity and the presence of another essential element(s) for dioxygenase activity. The chimeric proteins of PaDOD1 and PaDOD2 were constructed and analyzed to identify the essential regions for DOD activity. Multiple essential regions for DOPA dioxygenase activity were found in PaDOD1. The evolutionary mechanism of DOD genes is discussed using phylogenetic analysis.

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Multiple copies of flavonoid biosynthetic genes with different patterns of expression in several tissues and induction following environmental stimuli were identified in Legume. We have identified that gene duplication had occurred in the transcription factors regulating proanthocyanidin biosynthesis in model legume Lotus japonicus. Three copies of a homologue of Arabidopsis TRANSPARENT TESTA2 (TT2), which is an MYB transcription factor, were present in the Lotus japonicus genome. Transient assays suggest that three LjTT2s are different in their transcriptional activities. Chimera constructs exchanging N-terminal R2R3-MYB domain and C-terminal region of each LjTT2 revealed that R2R3-MYB domains play an important role in the activity of LjTT2. Furthermore detailed analysis using the site-directed mutagenesis indicated that three amino acid residues in R2R3-MYB domain contribute to the transcriptional activities of LjTT2s.

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Condensed tannins (CTs) are polymers composed of flavane-3,4-diols, intermediates of anthocyanin biosynthesis, and their derivatives flavane-3-ols. CTs are known for the anti-microbial and anti-herbivorous activities, and health-promoting benefits as food compositions. To clarify the biosynthetic mechanism of CTs, we have been investigating the viridicaulis1 (vic1) and vic2 mutants of Lotus japonicus, which are deficient in both CT and anthocyanin . We reported that the VIC1 gene encodes a bHLH protein at the JSPP meeting last year. In this study, we predict the vic2 gene by positional cloning and found a nonsense mutation at the 354th base in an orthologous gene of Arabidopsis thaliana TTG1 encoding a WD40 repeat protein, which results in the deletion of the WD40-repeat domains. Anthocyanin and CT accumulation in the vic2 mutant were complemented by the wild-type allele. Real-time PCR analysis showed reduced level of the transcripts encoding dihydroflavonol-4-reductase (DFR) and anthocyanidin synthase (ANS) in vic1 and vic2 mutants and the recovery to the wile-type level in the complementation tests, which suggests that VIC1 and VIC2 are regulatory factors of DFR and ANS.

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Flavonoid is one of the major groups of second metabolites in plants. Many Arabidopsis mutants with defects of the flavonoid accumulation, which are called transparent testa mutants from white color of their seed coats, have been reported. Analyses of these mutants revealed the major pathway of flavonoid synthesis. However, the mechanism of the final step of the incorporation of flavonoids into vacuoles is not known.
To elucidate the relationship between the flavonoid accumulation and vacuolar sorting system, we screened a mutant seeds with white seed coats from the library of gfs (green-fluorescent seeds) mutants, which have a defect in vacuolar protein sorting (1). The isolated mutant gfs9 had pale-brown seed coat and was defective in the transport of seed storage proteins into protein storage vacuoles. Our findings suggest that GFS9 is a novel protein involved in both the flavonoid accumulation and the vesicle transport.
(1) Fuji et al. (2008) Plant Cell, 19, 320-332.

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Calystegia soldanella (Convolvulaceae), Vitex rotundifolia (Verbenaceae) and Lathyrus japonicus (Leguminosae) are originally growing in coast, but they are also native to inland Lake Biwa. The seashore is a harsh environment for plants to inhabit due to various stresses, e.g. sea water and strong UV radiation. Flavonoids are one of the most effective anti-stress substances that are synthesized in plants. In this survey, the flavonoid compounds of C. soldanella, V. rotundifolia and L. japonicus were qualitatively and quantitatively compared with those of inland Lake Biwa populations. In coastal C. soldanella, the content of quercetin glycosides having ortho-dihydroxyl groups in B-ring were from ca. 4.5 to 10.5 fold than those of kaempferol glycosides having mono-hydroxyl group in B-ring. On the other hand, inland populations were from 0.8 to 1.8 fold. The flavonoids of V. rotundifolia were qualitatively and quantitatively the same with both sites. In contrast, those of L. japonicus were qualitatively different.

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5-O-methylcyanidin 3-glucoside was isorated from Egeria densa. When detached leaves were incubated in sucrose solution under the light, both anthocyanin synthesis and chlorophyll degradation were induced simultaneously. This system may be regarded as one of model system to elucidate the mechanism of "autumnal coloration." When detached etiolated leaves were incubated in 0.6 M sucrose solution, all the cells were plasmolyzed, in some of which the protoplasts were divided into nearly equal halves within a cell. Anthocyanin was synthesized only in nucleated halves. This result indicated that nucleus were required for anthocyanin formation. When detached leaves were irradiated partially with UV-light and incubated in distilled water, anthocyanin was synthesized in whole leaves. These results suggested the some signal factor(s) inducing for anthocyanin formation might transmit in a leaf. When detached leaves were incubated with sectioned stems in distilled water, anthocyanin synthesis was induced, suggesting that some signal molecule(s) was released from sectioned stems in water acting for detached leaves. We are going to identify and purify the signal molecules from sectioned stems.

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Veronica persica develops small bluish flowers. We analyzed flavonoids in the blue petals by HPLC, TOF-MS, and NMR, and determined its major anthocyanin and flavone to be delphinidin 3-O-(2-O-(6-O-coumaroyl)-glucosyl)-6-O-coumaroyl-glucoside-5-O-glucoside and apigenin 7-O-(2-O-glucuronosyl)-glucuronide, respectively. This flavone glucuronide caused a dose-dependent bathochromic shift toward a blue hue with the anthocyanin pigment, showing an intermolecular co-pigment effect. The significant co-pigmentation prompted us to explore genes responsible for these flavonoids. Based on the structural similarity to Lamiales flavonoid 7-O-glucuronosyltranferases, we isolated a cDNA encoding a UDP-sugar-dependent glycosyltransferase (UGT88D8). UGT88D8 catalyzed the 7-O-glucuronosylation of flavones. Moreover, based on the structural similarity to sugar-sugar glycosyltransferases, we also identified UGT94F1 as an anothocyanin 3-O-glucoside-2''-O-glucosyltransferase. Considering the remarkable expressions of UGT88D8 and UGT94F1 genes in petals, they are involved in the blue flower coloration of V. persica by catalyzing the co-pigment and pigment biosyntheses.

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In red petals of carnation, the main pigment was identified as 3, 5-di-O-(β-glucopyranosyl) pelargonidin 6''-O-4, 6'''-O-1-cyclic malate. Anthocyanins are known to be glycosylated by uridine diphosphate (UDP)-glucose dependent glycosyltransferase. However, some of the reactions, including 5-O-glucosylation in carnations, have not yet been elucidated. Here, we assessed to detect that sugar donor candidates in protein-free extracts from carnation petals prepared using 50% ethanol. The presence of candidate donors was tested with a crude protein extract from carnation petals using cyanidin 3-glucoside as the acceptor. After the enzyme reaction with the candidate in the crude protein extract, an additional peak was observed in a high-performance liquid chromatography chromatogram. The retention time of the peak and its ultraviolet-visible spectrum corresponded to that of cyanidin 3,5-diglucoside. This work demonstrated the presence of anthocyanin glucosyltransferase activity at 5 position using a donor that was apparently different from UDP-glucose. This research was partially supported by the Research and Development Program for New Bio-industry Initiatives.

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Glycosylation of anthocyan facilitates the solubility and stability of the molecule in the vacuole, and also is related to variation of flower color. Therefore, the anthocyan glycosyltransferase (AnGT), which transfer glycosyl residues to anthocyan, are essential as a factor affecting flower coloration. In this study, we used flowers of the common coral tree (Erythrina crista-galli L.), which is indigenous from southeastern Brazil to northern Argentina and has large, nontubular, brilliant crimson flowers from June to October. The flower contains cyanidin 3-O-sophoroside as the major anthocyanin in the petal, pelargonidin 3-O-glucoside as the major in the calyx, and cyanidin 3-O-glucoside as the major in the peduncle. Although anthocyanidin 3-O-glucosyltransferase (An3GT) has been studied in detail to date, there have been no reports of changes in its properties with flower organ differentiation. In this study, we report the isolation and purification of AnGT from each flower organs. As the result, we confirmed the differences of enzyme's properties and substrate specificities in AnGTs from each flower organs.

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Classical microsurgical experiments have been suggested the presence of some signal molecules communicating between shoot apical meristem (SAM) and organ primordium. Recently, molecular genetic approach reveled many key molecules responsible for the SAM function. But it is not enough to understand the timing and pathway of signal transduction. To elucidate the followed mechanism, it is indispensable to disrupt specific signaling pathways by microsurgical technique and observe developmental effect using expression marker lines. In tomato, laser ablation system is established, but it is limited because of the shortage of expression maker lines. In this study, we tried to establish UV laser ablation system in SAM of Arabidopsis. Now we are succeeded in single cell ablation in the SAM of GFP maker lines (FILAMENTUS FLOWER promoter::GFP and CLAVATA3 promoter::GFP). Here, we will discuss the ablation effect on organ development.

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Leaf venation pattern is determined when the provascular prepro/procambium is formed during the leaf primordium development. However, the molecular mechanisms as to how undifferentiated cells acquire the cell fate to be the prepro/procambium and how polarized provascular development is established are still largely unknown. To understand the mechanisms of leaf-venation patterning, we have isolated and analyzed Arabidopsis mutants defective in formation of leaf veins. NO VEIN (NOV) and VASCULAR HYPERPLASIA (VAH) genes were necessary for formation of the normal leaf-venation pattern. NOV and VAH are required for formation of the normal prepro/procambial pattern in leaves. It has recently been shown that NOV mediates auxin-dependent cell specification and patterning in the Arabidopsis embryo, shoot and root (Tsugeki et al., Plant Cell, 2009). Further analyses on roles of NOV and VAH in vascular development will be presented.

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The NAC transcription factors, VND6 and SND1/NST3, have been shown to be master regulators for differentiation of metaxylem vessel cells and fiber cells, respectively. However, how these processes are regulated by VND6 and SND1 is unclear. To investigate downstream genes regulated by VND6 or SND1, we established novel in vitro transgenic systems using Arabidopsis suspension cultured cells in which VND6 or SND1 was overexpressed. We confirmed that the in vitro culture systems could reflect processes of xylem cell differentiation in planta. Then, we performed microarray experiments with the suspension cultured cells we had established. We identified a number of downstream genes. Based on these results, we will discuss the gene regulation mechanism of xylem cell differentiation.

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Cell-to-cell peptide-signaling mechanisms like animal systems have been considered in functioning in diverse ranges of plant development. Recent advances in analyses of CLV3/ESR-related (CLE) peptides have revealed some of their functions in modulating cell proliferation and differentiation, but most of CLE peptide functions remain unknown. Here we applied an Arabidopsis pollen tube culture to examine the effects of 27 synthetic CLE peptides on pollen tube growth. Pollen tubes grew longer in the presence of certain types of CLE peptides. CLE peptide-receptor genes were supposed to be expressed in pollen tubes or pollen grains when the pollen tube growth differently responded to each CLE peptide. We next chose receptor-like kinase genes from those similar to the known CLE receptor genes, based on published gene expression data of pollen. The corresponding T-DNA insertion mutants are now being subjected to the pollen tube culture with the synthetic CLE peptides, which showed the promoting activities on wild type pollen tube growth. This approach would contribute to identify new CLE peptide-insensitive mutants, in which receptor-like kinase genes are impaired.

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When roots encounter localized source of nitrate, lateral root growth is stimulated. Studies with Arabidopsis indicate a MADS-box transcription factor, ANR1, is a key regulator of this mechanism. We have been characterizing AGAMOUS-LIKE 21 (AGL21) which belongs to this gene family. However, unlike ANR1, AGL21 was suggested to function when supply nitrogen was limited. The agl21 mutants had reduced lateral root growth compared to the wild-type plants when nitrate concentrations were 0.03 - 0.1 mM or when 0.1 mM glutamine was substituted for nitrate as an alternative nitrogen source. The overexpression of AGL21 was able to restore the growth of lateral roots in the agl21 mutant both in the presence and absence of nitrate. Consistent with AGL21's role in regulating lateral root growth, the AGL21 promoter activity was present specifically in the root tips of the primary and lateral roots. In Arabidopsis, AGL16, AGL17, and AGL21 are highly homologous to ANR1. An agl17 agl21 anr1 triple mutant was constructed to investigate the individual roles of ANR1 homologues whose transcripts were mainly found in roots. The results will be discussed with the analysis of promoter-GFP plants.

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CLE genes are the family of genes encoding signaling peptides in their C terminal regions homologous to CLAVATA3 (CLV3). CLE genes are thought to regulate the size of meristems and their differentiation. Previous findings suggested that CLE1-7 are expressed in the root. The purpose of this study is to clarify the roles of CLE1-7 in the root development in Arabidopsis.
Expression of CLE1, 3, and 7 was induced by low NO3- in the medium, and was repressed by high NO3-. CLE1, 2, 3, 4, and 7 was expressed in pericycle cells. Lateral root lengths of CLE1-7 overexpressors were shorter than that of the wild-type plants. CLE1-7 was suggested to act as repressors of lateral root elongation.
The clv1 mutants having mutations in CLV1, the receptor of CLV3, had longer lateral roots than the wild-type plants. Microscopic analysis of CLV1-GFP revealed that CLV1 was expressed in the phloem companion cells of root. These results suggested that the CLV1 act as a receptor of CLE1-7 signaling peptides. We will also show the results from the analysis of the root length of CLE3 overexpressor in clv1 mutants, and discuss about the relationships between CLE3 and CLV1.

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Tyrosine sulfation is a posttranslational modification found in peptides and proteins synthesized by the secretory pathway in most eukaryotes. In plants, this modification is critical for the biological activities of a subset of peptide hormones such as PSK and PSY1. To investigate the mechanisms of tyrosine sulfation in plants, we purified TPST activity from microsomal fractions of Arabidopsis cells and identified a 62-kD protein that specifically interacts with the sulfation motif of PSY1 precursor peptide. AtTPST is expressed throughout the plant body, and the highest levels of expression are in the root apical meristem (RAM). A loss-of-function mutant of Arabidopsis displayed a marked dwarf phenotype accompanied by stunted roots, pale green leaves, reduction in higher order veins and early senescence. Interestingly, root growth defect in tpst mutant was not recovered by the addition of PSK and PSY1 in the medium, suggesting that as-yet-unknown sulfated peptide is required for normal root growth. We are searching for this peptide by in silico gene screening approach.

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Root cap has several important physiological functions, such as protection of the root meristem and gravity sensing. In Arabidopsis thaliana, root cap cells are generated by coordinated cell division and differentiation of two types of stem cells with distinct cell lineage. After initial differentiation, root cap cells rapidly go through a maturation process and slough off within several days. We isolated a dominant Arabidopsis mutant, urp7-D, in which stem cells that give rise to the root cap were lost and root cap-like character was given in the epidermis. In urp7-D, a NAC domain transcription factor, SOMBRERO (SMB) was ectopically expressed throughout the root. Consistently, loss-of-function smb mutants showed enhanced cell division of the root cap stem cells, as well as delayed root cap differentiation. Expression of SMB initiates in the daughter cells produced by the division of the root cap stem cells, and then gradually increases as they mature. These results indicate that SMB is a key regulator of root cap differentiation and maturation.

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In vascular plants, lateral root (LR) formation is critical for the development of the root architecture. To get insight into the mechanisms of LR formation, we identified the fewer roots (fwr) mutant with a decreased number of LRs in Arabidopsis. The fwr mutation is a single recessive and strongly reduces the frequency of LR formation. The fwr mutant was less sensitive to exogenous auxins that induced LR formation. Fine mapping and sequence analysis revealed that the fwr has a missense mutation in the GNOM gene, encoding an ARF-GEF that functions in vesicle trafficking. This was confirmed by the allelism test between the fwr and GNOM T-DNA insertion mutant. GNOM is required for the establishment of the apical-basal axis during embryogenesis and acts during auxin-mediated growth and development by affecting the intracellular location of PIN1, an auxin efflux carrier. Although several types of gnom mutant alleles with embryonic or seedling lethality are known, few mutants that are specifically defective in LR formation have been reported. We will discuss the role of GNOM in LR formation, based on phenotypic analysis of the fwr mutant.

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