CHUP1 protein is essential for chloroplasts to take proper positioning according to light condition. CHUP1 protein contains multiple functional domains, such as 25 amino acid sequence at N-terminus targeting to the chloroplast outer envelop membrane, F-actin binding motif, and prolin-rich region (Oikawa et al, 2003). CHUP1 localization was studied using polyclonal antibody against CHUP1. The result suggests that the hydrophobic sequence at the N-terminus of CHUP1 is important for CHUP1 localization. Next, we studied the relationship between CHUP1 functional domains and recovery of light induced chloroplast relocation in chup1 using constructs defective in each functional domain of the CHUP1 protein. Results showed that CHUP1_1-500aa could partially recover the mutant phenotype of chup1 and the chloroplast positioning was similar to positioning under strong light even under weak light condition, and that C-terminus of CHUP1 is important for chloroplasts to take proper intracellular positioning and may function for G-actin polymerization.

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Plastids contain a eubacterial-type RNA polymerase (PEP) that is responsible for transcription of photosynthesis genes encoded by plastid genome. Analogous to the eubacterial enzyme, PEP is a multi-subunit enzyme composed of a catalytic core complex (a2bbb) and a sigma factor that confers transcription specificity. Arabidopsis contains six plastid sigma factors (AtSig1-AtSig6) encoded by nuclear genome, although the core subunits are encode by plastid genome. Recently, general (AtSig6) and specialized (AtSig2 and AtSig5) sigma factors in plastids have been characterized by analyses of T-DNA tag mutants. To learn more on the functions of AtSig2 and AtSig6, we have invented double mutant plants (sig2sig6). The double mutant exhibited albino cotyledons and true leaves, although the single mutants showed pale green phenotypes. Interestingly, transcripts of psbB operon were seriously defected in the double mutant. These results suggested that AtSig2 and AtSig6 might play a major role in chloroplast transcription

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Morphology and organization of tobacco plastid genome was analyzed by pulse-field gel electrophoresis and observation of individual plastid genomes through a fluorescence microscope. Each single molecule of plastid genome was detected as either a circular or a linear DNA molecule in four forms; monomer, dimmer, trimer and tetramer. Pattern of restriction enzyme fragment showed the presence of linear molecule that has defined ends in internal sequence of inverted repeats. Linear molecule having specific ends were found in plastid DNA isolated from shoots that contain young leaves and meristematic tissues although it were not found in plastid DNA of mature leaves. It seems that linear plastid genome having specific ends are not results of a breakage of circle genome during extraction, but it may have a relation with DNA replication in plastids.

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Chlorophylls are the main light-harvesting pigments that bring photons to reaction centers. It is, however, not yet entirely clear how the light-harvesting complexes are formed and function. The pcb2 mutant is deficient in divinyl protochlorophyllide 8-vinyl reductase and thus accumulates divinyl chlorophylls instead of chlorophylls. To obtain more information about the role of the chlorophylls in photosynthesis, we investigated the photosynthetic activity and the formation of the photosynthetic components in the pcb2 mutant. Chlorophyll fluorescence analysis showed that the photosynthetic electron transport system of photosystem II, especially reaction center, did not function adequately in the pcb2 mutant. Green Gel analysis showed that a component of the photosystem had been disturbed. Electron microscopic analysis showed that the chloroplasts of pcb2 mesophyll cells lacked distinctive grana stacks. We discuss the effect of divinyl chlorophylls on photosynthesis and the formation of grana stacks.

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Most of chloroplast proteins are encoded in the nuclear genome and synthesized in the cytosol as precursors, and then imported into chloroplast through translocon at the outer and the inner envelopes. During protein import under certain conditions, precursors and translocon are irreversibly bound, forming the early-translocation intermediate. By examining the early translocation intermediate, we have discovered that there are three forms of the early-translocation intermediate depending on the GTP/ATP requirement and temperature.
We now start to investigate the interaction between precursors and translocon in the early-translocation intermediate by cross-linking. In brief, the series of a mutant precursor which has single cysteine residues at various positions was constructed and overexpressed in E. coli. These precursors were modified with SH-specific cross-linkers. After forming the different forms of early-translocation intermediate, cross-linking reaction was initiated. The cross-linked products were analyzed by SDS-PAGE. Our current efforts of cross-linking study will be presented at the meeting.

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Higher plants chloroplasts contain at least two RNA polymerases; one is plastid-encoded eubacteria-type RNA polymerase (PEP) and other is nuclear-encoded phage-type RNA polymerase (NEP), which is similar to RNA polymerase of bacteriophage T7. It is proposed that the two RNA polymerases transcribe distinct groups of genes, namely, PEP transcribes photosynthetic genes and NEP transcribes housekeeping genes.
In this study, we tried to down-regulate AtRpoT;3 (corresponding to NEP in Arabidopsis) gene expression using RNA interference (RNAi) technique. As a result, we obtained some RNAi mutant lines with lower AtRpoT;3 expression. The RNAi mutants showed cotyledon-specific albino phenotype, and the most chloroplast transcripts of the mutants were hardly accumulated. We currently are analyzing the expression of chloroplast genes in detail.

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Though cyanobacteria have peptidoglycan layer between inner and outer membranes and 9.genes for its synthesis, this wall structure has not yet been observed in plastids of green plants. We found all homologous genes for the peptidoglycan synthesis pathway in moss Physcomitrella patens. Each disruptant of MurE and PBP genes displayed indistiguishable phenotype whose characters are larger size and decreased number chloroplasts in comparison with those of wild type plants. The chloroplasts morphology of mutants appeared to be similar to those observed in the cells treated with beta-lactam antibiotics. We speculate that moss P. patens harbors peptidoglycan layer between inner and outer envelopes and is indispensable to chloroplast divison.GFP fusion assay revealed that PpMurE and PpPBP are transported to chloroplasts. Furthermore, the complementation test that introduced Anabaena homologous gene into P. patens murE mutant confirmed that Anabaena MurE is functionally identical to those of P. patens.

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The unicellular red alga Cyanidioschyzon merolae 10D has simple cell structure, which contains one nucleus, one mitochondrion and one plastid. C. merolae is considered to be closest to the origin of eukaryotic cells. In order to use this organism as a model cell, it is indispensable to develop reverse-genetical techniques to facilitate functional genomics.
We demonstrated previously that the URA3 mutant (frameshift mutation in URA3 gene, uracil auxotroph) incorporated an exogenous DNA fragment coding for wild type URA3 (Minoda et al. (2004) PCP 45:667-671). However it was hard to distinguish between transformation and reversion mutation. In this study, we performed transformation experiments using modified URA3 gene, which contains two base substitutions unaccompanied by amino acid change. As a result, appearance of uracil prototroph transformant which contained substituted bases, suggests incorporation of exogenous DNA by double-crossover. This result strongly suggests that development of proper marker genes enables gene disruption for reverse genetics.

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Although assimilation of inorganic nitrogen is a critical biochemical process for plant growth, the underlying regulatory mechanism has remained to be resolved. Here, we investigated nitrogen source-dependent gene expressions and its regulation by transcription factor(s) (TFs) in a red alga Cyanidioschyzon merolae. DNA microarray and Northern blot analyses showed that the expression of genes encoding glutamine synthase (GS), nitrate/ammonia transporter (NRT/AMT), nitrate/nitrite reductase (NR/SIRB), and MYB-type TF (CMJ282) were significantly increased in cells grown in culture containing nitrate as a nitrogen source, as compared with cells grown with ammonia. A consensus nucleotide sequence that is similar to GATA-type TF binding motif was found at those promoter regions, suggesting that GATA-type TF(s) contributes to the up-regulated transcriptions. We will also discuss the binding specificity of GATA-type TFs on the consensus nucleotide sequence and its cellular localization.

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The primitive red algae, Cyanidioschyzon, have only a set of organelles, such as nucleus, mitochondrion and chloroplast in the cell. The cell cycle of this organism coordinates with division cycle of chloroplast and mitochondria. However, mechanisms for the regulation of this coordinated cell cycle have not been elucidated. Here, using synchronized cultivation technique, we investigated regulatory mechanisms to initiate organelle replication and that to coordinate organelle cell cycle with that of nucleus.
We identified that replication of chloroplast genome and mitochondrial genome starts before that of nuclear genome by Southern hybridization method. We also identified that a cyclin protein is expressed similar manner with the replication cycle of nuclear genome by Western analysis. These results suggest that the cell cycle of each organelle in Cyanidioschyzon is regulated independently and that as yet known mechanisms might correlate them each other to constitute the coordinated DNA replication and cell division in Cyanidioschyzon.

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Plastids have their own genome and gene expression apparatus derived from endosymbiosis of ancestral cyanobacteria. During subsequent evolution, plastids lost their autonomy and most of function became under nuclear control. In this study, we used the primitive, unicellular red alga Cyanidioschyzon melorae. This alga shows ancestral characteristics on many aspects including plastid genome or transcriptional regulation. Therefore, this organism can be helpful to understand evolution of regulation systems.
Here we used run-on system to understand transcriptional regulation in C. melorae chloroplasts. To examine light-dependent transcription, C. melorae cells were shifted in the dark for 12h, followed by re-illumination for 1h or 6h. Intact chloroplasts were isolated from cells to perform run-on transcription assays. Transcriptional activity was transiently induced after 1 hour light, but declined to the half after 6 hours. This result correlates with microarray analysis, suggesting that gene expression in C. melorae chloroplasts are mainly regulated at transcriptional level.

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The endoplasmic reticulum (ER) has quality control mechanisms that retain misfolded proteins in the ER, and only properly folded proteins are exported from the ER. BiP, Hsp70 in the ER plays a key role in ER quality control. Arabidopsis thaliana has three BiP genes, AtBIP1/2/3. Yeast genetics has identified various dominant negative or temperature sensitive BiP mutants that have mutations in amino acid residues conserved between yeast and Arabidopsis. We constructed a series of AtBiP1 mutants containing these mutations and expressed them in pollen grains and pollen tubes using the pollen-specific LAT52 promoter. Expression of none of the dominant negative AtBiP1 mutants caused defects in pollen germination or tube growth in vitro. However, pollen tubes expressing the mutant AtBiP1 protein did not grow through the style resulting in defective fertilization.

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In contrast to animal cell, the fundamental mechanism to establish cell polarity in plant has been less depicted. Recent studies on several proteins raised the model that plant cell polarity is mainly defined by an ununiform accumulation of plasma membrane proteins under dynamic equilibrium between their endocytosis and membrane expression via vesicle trafficking. To verify this hypothesis, plasma-membrane localized SNAREs, which are essential to membrane fusion in vesicle transport, were used as marker proteins of vesicle trafficking. Transgenic plant lines expressing GFP-fused plasma membrane SNAREs under control of their own promoters have been established in A. thaliana and P. patens. In addition to microscopic observation, new insight by phylogenetic analysis of SNARE family between these organisms will be reported.

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Nuclear matrix is the residual structure after removing nuclear membrane and chromatin from isolated nuclei. Nuclear matrices of animals have three domains, lining of nuclear membrane, intranuclear network of fibrils, and residual nucleoli. Plant nuclear matrix is morphologically similar to those of animals, but at least the major constituent protein of the lining of nuclear membrane is different. Proteins in the internal fibrils have not yet been revealed in plants. Using monoclonal antibodies, we screened cDNA clones that encodes proteins existed in intranuclear network of fibrils of carrot nuclear matrix. One of the cloned cDNAs, 3F5-117had deduced amino acid sequence similar to Arabidopsis thaliana CDC5 (AtCDC5). CDC5 family is required for both cell cycle regulation and pre-mRNA splicing in the nucleus. Existence of a CDC5 homolog in plant nuclear matrix suggested that CDC5 may associate with the intranuclear network of fibrils.

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Transversely oriented cortical microtubule arrays have been proposed to be required for straight cell elongation.
The Arabidopsis thaliana spiral3-1 (spr3-1) mutants are useful for studying interactions between microtubule organization and nucleation. In elongating epidermal cells of spr3-1 roots, cortical microtubules were arranged in shallow left-handed helical arrays, in contrast to wild-type transverse arrays. The SPR3 protein is a homologue of gamma ring protein 84 (Grip84) and spr3-1 mutant has an amino acid substitution mutation in a highly conserved grip motif. In fungi and animal cells, Grip84 is a component of the gamma-tubulin ring complex (γ-TuRC) which shows core nucleating activity of microtubules. Yeast two-hybrid assays indicated that spr3-1 mutation impairs interaction with AtGrip91, another component of γ-TuRC.
Our results indicate that a defect in microtubule nucleation leads to alteration in array organization, possibly by affecting dynamics of cortical microtubules.

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In plant cells, many microtubule-associated proteins (MAPs) control various microtubule (MT) functions. Arabidopsis SPIRAL2 (SPR2) protein is a plant-specific MAP involved in the regulation of cortical MT arrays. SPR2 localizes to the cortical MTs, and the null mutation of SPR2 causes a shallow left-handed helical array in cortical MTs and a right-handed helical growth in longitudinally growing organs such as petiole. SPR2 has HEAT repeat motifs at the N-terminal region and highly conserved region among SPR2 homologs at the C-terminus. In MT binding assay in vitro, MTs directly bound to purified recombinant SPR2 protein independently at the N- and C-terminal regions. Furthermore, we examined the effect of SPR2 on the dynamics of MT in vitro.

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In plants, cortical microtubule (cMT) arrays generally determine the polarity of cell elongation. Many microtubule associate proteins are thought to be involved in formation and maintenance of the array organization. A dominant phs1-1 mutant allele of an Arabidopsis MAP kinase phosphatase-like protein showed compromised cMT organization and stability. Transgenic plants which expressed the functional genomic PHS1 gene fused to GUS and GFP showed strong GUS activity in the elongating region of roots. Moreover, the GFP fluorescence was predominantly detected in cytoplasm. A MAP kinase cascade involving PHS1 may be important for regulation of cMT arrays in the elongation zone of root. To isolate components in the PHS1 phosphorylation pathway, we mutagenized phs1-1 by EMS and isolated many mutants that suppressed the phs1-1 phenotype.

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A group of microtubule-associated proteins called plus-end tracking proteins (+Tips), including End-Binding1 (EB1) family proteins, associates specifically with growing MT plus-ends. +Tips regulation plus-end dynamics and spatial arrangement, directly or indirectly.
Arabidopsis EB1 (AtEB1) tagged with GFP, decorated microtubule plus-ends, in transgenic Arabidopsis plants and in tobacco BY-2 cells. AtEB1 proteins share a conserved calponin homology (CH) domain at the N-terminus, an α-helical C-terminal domain, and a highly polar acidic tail with other homologues. EB1 proteins are thought to stabilize microtubule by forming a plus-end complex in yeast and metazoans, but plant EB1-containg complex is not characterized. When the CH-domain was deleted, the truncated AtEB1 formed a homo-dimer and lost the MT-binding capability. Removal of the acidic tail from EB1 activated microtubule assembly. We propose that AtEB1 takes an autoinhibitory conformation, as seen in animal EB1. We also report our attempt to identify EB1-interacting proteins using LC/MS/MS analysis approach.

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Cva (C-terminal variable acidic amino acid region) of α, β-tubulins consists of the isotype-specific sequence which is extremely rich in acidic amino acids. Peculier modifications as tyrosinylation, polyglutamylation and polyglycinylation on Cva are reported as well as phosphorylation. We analysed β-tubulin isoform spots of IEF by MALDI-TOF MS, and recognized that each IEF spot did not necessarily consist of a single isoform, but might be a mixture of modified isotypes. The advantage of MALDI-TOF MS is that samples less than picomol which are not fully purified could be detected and their sequence could be analysed by PSD or with the use of CPY. Peptides derived from Cva are however, rich in acidic amino acids and may contain phosphate group and so, are relatively difficult to detect. We have examined preparation and detection conditions for Cva peptides and obtained partial information about post-translational modifications of β-tubulin isotypes in synchronized BY2 cells.

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TMBP200 is a homologue of Arabidopsis MOR1/GEM1 in tobacco BY-2 cells. To know functional domains of the polypeptide and a role of TMBP200 in plant cell division, we are attempting to examine subcellular localization and the effects of overexpression of truncated polypeptides of TMBP200 in BY-2 cells. Here, we have established cell lines that express full-length GFP-TMBP200 fusion protein using an inducible expression system. In interphase cells, the GFP fluorescence was observed throughout the cytoplasm and in some cells it was concentrated along cortical microtubule like arrays. In mitotic cells, the GFP fluorescence was localized to the spindle and the phragmoplast. The induced expression of GFP-TMBP200 caused an appearance of multinucleate cells. Currently, we are analyzing mechanisms of multinucleation by expression of GFP-TMBP200.

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In plant cell morphogenesis, it is believed that elongation axes are decided by cellulose microfibril orientation in cell wall and that orientation is controlled by cortical microtubules. Spirogyra is composed of tandem cylindrical cells, and the terminal cells differentiate to be rhizoid of rod or rosette shape. We had indicated that microtubules have an essential role in rhizoid morphogenesis. In the present study, we found that bulging occurred only at the tip region, when oryzalin was added after the start of differentiation. In the presence of DCB, a cellulose biosynthesis inhibitor, cell wall of tip region was burst. These results indicated that control of orientation and synthesis of cellulose at very tip region are essential for rhizoid differentiation. In addition, we tried to observe cellulose microfibrils during differentiation with FE-SEM. It will be discussed how cortical microtubules are involved in cellulose microfibril orientation during rhizoid differentiation in Spirogyra.

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Secondary wall is synthesized only in limited tissues including xylem of stems, anthers and siliques. In this study, we systematically analyzed functions of NAC transcription factors using CRES-T and found that the expression of the chimeric NST1 repressor induced indehiscent anther due to the loss of secondary wall thickenings (SWT) in anther endocecium. Furthermore, the plant expressing NST2 chimeric repressor and the double T-DNA tagged line of NST1 and NST2 also showed the same phenotype. This phenotype of the tagged line was restored by the complementation of NST1 or NST2 expressed by their own promoters. On the other hand, over-expression of NST1 or NST2 induced ectopic SWT in various tissues. The promoter activities of NST1 and NST2 were observed in tissues where SWT occurs. In this report, we will discuss the mechanism that regulates SWT in various tissues and the function of NST3, another gene related to SWT.

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In vitro cell wall-autolysis assays with Zinnia culture system have demonstrated that pectin is one of the most actively degraded substances during tracheary elements (TEs) differentiation. To understand modification of cell walls during TE differentiation, we have isolated and analyzed three Zinnia polygalacturonase (PG) genes, ZePG1-ZePG3. Among these genes, ZePG2 and ZePG3 were specifically up-regulated at 60 h of culture in the TE-inductive medium. We generated a rabbit antibody against the ZePG2 protein, and examined the expression of this protein during TE differentiation. The ZePG2 protein appeared at 48 h and increased rapidly for next 12 h. This analysis also suggested that the ZePG2 protein may be processed during TE differentiation. Analysis of subcellular localization of the ZePG2 protein is underway, and taken together we will discuss the function of polygalacturonases in TE differentiation.

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To investigate phenotypes of a plant that expresses a monoclonal antibody is potentially useful to know in situ functions of its antigen. This method is called immunomodulation. It has been reported that plants expressing monoclonal antibodies against phytohormones show phenotypes similar to those of plants short in the phytohoromones.
We had isolated approximately one hundred of monoclonal antibodies that recognize molecules secreted from differentiating xylem cells by phage display subtraction. In this study, to find extracellular components that have important roles in xylem development, we introduced genes for the isolated antibodies into Arabidopsis plants for immunomodulation. The following three points were devised: 1) a promoter for antibody expression, 2) signals directing intracellular localization of antibodies, 3) negative control for evaluation of the causal linkage between phenotypes and antigen-antibody binding. We will report the results of the experiments.

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Hypergravity increases cell wall rigidity by increasing the molecular size of xyloglucans in dicotyledonous plants. The molecular size of cell wall polysaccharides is determined as a balance between the synthesis and the depolymerization. In the present study, we examined the effects of hypergravity on both processes of xyloglucans. Azuki bean cuttings were incubated with [6-³H]-L-fucose. ³H-Xyloglucans were eluted in the higher molecular mass regions than total xyloglucans detected by the iodine method. Hypergravity did not influence the molecular size of newly deposited xyloglucans. ³H-Xyloglucans obtained from epicotyls grown at 1 g were shifted to the low molecular mass regions during the incubation period, which was inhibited by hypergravity. These results indicate that xyloglucans were deposited into the cell wall as large molecules but they were then demolymerized in the cell wall. Hypergravity-induced inhibition of the depolymerization is involved in the increase in the molecular size of xyloglucans under hypergravity conditions.

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3-Deoxy-D-manno-oct-2-ulosonate (KDO) is a specific component monosaccharide of rhamnogalacturonan II. KDO cytidylyltransferase (CMP-KDO synthetase; CKS) converts KDO into CMP-KDO, which is the immediate precursor of KDO residues in RG-II. Mutant plants lacking CKS activity will be useful to study the physiological role of RG-II and KDO in higher plants. We therefore started this study to isolate and characterize Arabidopsis CKS.
A BLAST search using bacterial or maize CKS sequences as a probe identified an Arabidopsis ortholog, At1g53000. The protein had a putative chloroplast transit peptide and the targeting was confirmed by expressing an GFP fusion protein in protoplasts.
The recombinant protein expressed in bacteria had the CKS activity with a pH optimum of 9.5. Uridine 5'-triphosphate also served as a substrate at an efficiency of 40% to that of CTP. Phenotypic analyses of T-DNA insertion lines and RNAi plants for CKS are now being undertaken.

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We have been surveying plant-proteases that could be used as an effective decomposer of the surplus protein in environment. If plants have SDS resistant proteases, they would show poor profiles in SDS-PAGE. The plant showing the least number of polypeptide bands in tested plants was the white skunk cabbage (Lysichiton camtschatcense). The protease activity of the leaf extract was determined by measuring the digestion of BSA. A higher concentration of the reducing agent was necessary to keep the protease activity in the extract. The protease showed maximum activity in the presence of 0.1% SDS and 0.5% mercaptoethanol, and about 40% of the activity was remained even in 2% SDS. The pH profile with bell shape showed a peak at pH 7.5. The molecular size was estimated as about 28 kDa. It is concluded by the sensitivity to inhibitors that this protease is a cysteine endoprotease.

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The developmental transition in plant leaves, for example greening and senescence, is supported by precise regulation of protein synthesis and degradation. Therefore, investigation of proteases provides important information for plastid maturation. Previously we reported the protease activity in barley leaves. This activity was analyzed by Suc-Leu-Leu-Val-Tyr-MCA. These enzymes required sodium dodecyl sulfate (SDS) at concentrations of 0.1%. The activity with 0.1% SDS showed a good correlation with development and senescence of plastids. The enzyme was purified by the successive chromatography with DE52, Q Sepharose FF anion exchange, Hi Load Superdex 200, and Superdex 200 gel-filtration chromatography. Molecular weight of the enzyme was estimated to be approximately 35 k by SDS-PAGE. Additionally, this enzyme had maximum activity in neutral pH region. We are now under way on complete purification, amino acid sequence, inhibitor study, and substrate specificity to reveal enzymatic properties of this enzyme.

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Leaf senescence is a program to transfer nutrients from senescent leaves to other tissues. The degradation of proteins is proceeded by various proteases. Especially, cysteine proteases play an important role in proteolysis during senescence.
In the previous study, we reported a novel protease having N-succinyl-Leu-Tyr-aminomethylcoumarin degradation activity. This activity dramatically increased in acidic conditions during senescence. To determine the function of this protease, we purified and characterized this enzyme from spinach leaves. It is reported here that a full-length cDNA was isolated from senescent leaves of spinach and exhibited high amino acid sequence homologies to senescence-related cysteine proteases, such as RD21 (Arabidopsis thaliana), DCCP (Dianthus caryophyllus), and CPPIC (Zea mays). We discussed gene expression and protein level of senescence-activated cysteine protease in senescent leaves. A possible role and function of this protease are associated with bulk protein degradation and mobilization during leaf senescence.

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The translation elongation factor-1 (EF-1) contains of four different subunits. They are designated EF-1α, EF-1β, EF-1β' and EF-1γ in plants. EF-1α catalyzes GTP-dependent binding of aa-tRNA to the ribosome, and both EF-1β and EF-1β' catalyzes the GDP/GTP exchange on EF-1α. However, the function of EF-1γ in protein biosynthesis remains unknown. In this study, we analyzed the binding between EF-1γ and other EF-1 subunits to know the subunit composition in plant. In addition, a yeast two-hybrid library was screened to identify novel candidates that interact to EF-1γ.
A yeast two-hybrid assay showed the interaction of EF-1γ with EF-1α, EF-1β and EF-1γ. This result suggests that EF-1γ can form homodimers in the cells. Furthermore, we identified a ribosomal protein L30 as a novel EF-1γ interactor. The interactions between both proteins were also confirmed by other binding assays.

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We have been analyzing the function of a barley protein (P23k) which express abundantly in scutellum of imbibed barley seed. Interestingly, the expression of P23k is also detected in aleuron layer and vascular bundles of ripening seed. These results suggest that P23k is involved in the function of seed. In this study, we examined the intercellular localization of P23k using cell fractionation experiment and immunofluorescence microscopy to know the function of P23k.
The cell fractionation and Western blot analysis revealed that the P23k fraction is nearly identical to the fraction of an endoplasmic reticulum (ER) marker enzyme, cytochrome c reductase. The P23k localization in ER was also confirmed by immunofluorescence microscopy study. These results indicated that P23k is a functional protein localized in ER. In this meeting, we will report the intercellular localization of P23k and discuss the function of P23k in seed.

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Methyl chloride (CH₃Cl) contributes to the destruction of stratospheric ozone. Recent studies clarified that plants are the largest source of CH₃Cl, and a methyl chloride synthase gene, designated as HOL, was identified in Arabidopsis. It could be predicted that HOL also methylates thiocyanate ion (NCS^-). Although the postulated roles of the enzymes include conferring chloride tolerance or production of methyl thiocyanate, which has repellent activities, none of the direct evidences in vivo has been reported so far.
We isolated Arabidopsis T-DNA insertion mutants for HOL and its homologs, HOL-like1 and HOL-like2, and analyzed the responses of the mutants to probable substrates. As a result, the hol mutant solely showed an increased sensitivity to NCS^-, suggesting that HOL is responsible for NCS^- metabolism in vivo. Further analysis of the mutants, enzyme kinetics, environmental responsivity and tissue specificity of the gene expression are underway and will be presented.

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γ-linolenic acid (C18:3 Δ^6,9,12) is widely used as a dietary supplement and for treatment of various medical conditions. It is a component of fatty acids in several plant species. γ-linolenic acid is synthesized by a Δ-6 fatty acid desaturase using linolenic acid (C18:2 Δ^9,12) as substrate. To enable the production of γ-linolenic acid in conventional cereals, we produced transgenic rice plants that express the transcripts of a liverwort Marchanita polymorpha Δ-6 fatty acid desaturase gene MpDES6 under the control of the maize ubiquitin promoter. The transgenic rice plants had 1-2 copies of the transgenes. The MpDES6 gene over-expressed in the transformants. Analysis of leaf, root and seed lipids from transformed plants demonstrated the accumulation of γ-linolenic acid and octadecatetraenoic acid (C18:4 ^6,9,12,15) .

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Stigmasterol, containing a double bond at the C-22 position in the side chain, is one of the major constituents of plant sterols and biosynthesized by the C-22 desaturation of β-sitosterol. However, the gene encoding the C-22 desaturase has not been identified from higher plants. In yeast, cytochrome P450 (CYP61) has been known as the sterol C-22 desaturase, and the CYP710A family has the highest homology (28%) to CYP61 among plant P450s. Thus, we presumed that CYP710A is the plant sterol C-22 desaturase, and investigated the in vitro activity. Recombinant CYP710As expressed in a baculovirus/insect cell system exhibited a notable C-22 desaturase activity with β-sitosterol to produce stigmasterol. As brassicasterol occurs specifically in Brassicaceae family plants and is biosynthesized from 24-epi-campesterol by the C-22 desaturation, we examined the substrate specificity of Arabidopsis CYP710A1~A4. Each CYP710A exhibited the strict substrate specificity for either β-sitosterol or 24-epi-campesterol.

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Fumonisin B1 (FB1) elicits a programmed cell death (PCD) in plants, probably through competitive inhibition of sphingosine-N-acyltransferase. Because this inhibition results in the accumulation of free sphingoid bases in cells, to evaluate the cellular effects of free sphingoid bases involved in the PCD, functional characterization of enzymes that control the intracellular levels of sphingoid bases is very important. Degradation of sphingoid bases proceeds from two specific enzymatic steps. Sphingoid bases are first phosphorylated at the 1-hydroxy group to form sphingoid base 1-phosphate, and then cleaved to a long-chain aldehyde and ethanolamine-phosphate. Sphingosine-1-phosphate lyase (SPL) is a cleavage enzyme responsible for the ultimate step in sphingolipid catabolism. Here we observed that Arabidopsis SPL T-DNA mutants are sensitive to FB1. Mechanisms of the sensitivity will be discussed with several data in this communication.

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Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that plays lipid-mediated signaling involved in abscisic acid (ABA) regulation of guard cell turgor. To evaluate mechanisms of S1P signaling involved in the signal-transduction pathway linking ABA to regulate stomatal switching, functional characterization of enzymes that control the intracellular levels of S1P is very important. Here we communicate the functional characterization of an Arabidopsis enzyme involved in the catabolism of S1P, sphingosine-1-phosphate phosphatase (SPP). In order to evaluate whether a SPP-like gene in Arabidopsis (AtSPP1) encodes a functional SPP, we performed an complementation test using an yeast del1 mutant strain and measured the SPP activity using an HPLC-fluorescence detector system. We will report several results on the expression of the AtSPP1 gene using T-DNA knockout mutants.

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Xylogen is a plant proteoglycan inducing xylem differentiation in vitro. Previous results suggested that xylogen is secreted from differentiating vascular cells, transported to adjacent immature cells, and draws them into the cell fate of vasculature. Here, we report analyses of transport dinamics of xylogen using xylogen-GFP fusion protein.

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In the vesicle transport systems of eukaryotes, COPI (coat protein I) and COPII (coat protein II) work in the transport system between ER and Golgi apparatus. COPII that consists of the small GTP-binding protein, Sar1p and two coat protein complexes, sec23p/24p complex and sec13p/31p complex, work in the transportation from ER to golgi apparatus. In plants, vesicle transport system involves in cell division. In this research, we examine the function of COPII components in development of Arabidopsis thaliana.
In these COPII components, we carry out the analysis of homologs of sec31p, sec13p, sec23p and sec24p that each ATSEC31, ATSEC13, ATSEC23 and ATSEC24. The homology search indicates that there are two homologs for each ATSEC31 and ATSEC13, are seven homologs for ATSEC23 and are three homologs for ATSEC24. In this research, we carry out the expression analysis by promoter GUS and the localization analysis by fluorescence reporters for these homologs.

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Seed storage proteins are synthesized on rER as precursors, are transported to protein storage vacuoles (PSVs), and then are converted into the mature forms. To understand the mechanism of storage protein transport, we examined the detailed phenotypes of Arabidopsis mutant seeds that abnormally accumulate the precursors of the storage proteins. Based on their phenotypes, the mutants were divided into two classes. Class 1 mutants including atvsr1 accumulated a larger amount of pro12S globulins. They secreted storage proteins from the cells. Class 2 mutants accumulated a larger amount of 2S albumins, and had novel structures that accumulate storage proteins. The structures were similar to precursor-accumulating (PAC) vesicles found in maturing pumpkin seeds. PAC vesicles are involved in Golgi-independent transport of storage proteins.

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The green-colonial algae Botryococcus braunii is known to produce large amounts of hydrocarbon. The accumulated hydrocarbon is classified into 3 races A, B, L by their chemical structure. The aim of this study is to characterize hydrocarbon biosynthesis in B.braunii BOT-70 for the establishment of the efficient hydrocarbon production.
We investigate the characteristics of hydrocarbon biosynthesis in BOT-70 by ¹⁴C-tracer experiments. [¹⁴C-methyl]methionine was specifically incorporated into hydrocarbon, differently from race A strain. The obtained results suggest that the accumulated major hydrocarbon is classified as race B that produces botryococcene containing some methyl groups. The hydrocarbon biosynthesis from [¹⁴C-methyl] methionine was highest at the beginning of the growth and then decreased drastically, on the other hand, the hydrocarbon biosynthesis from NaH¹⁴CO₃ was highest at early log phase growth. Botryococcene is supposed to be converted from IPP. We examine whether the origin of IPP is MEV pathway or MEP pathway.

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Nicotine, a major alkaloid in tobacco plants, acts as a defensive compound against insects. When wounding signals are transmitted from leaves to roots, nicotine biosynthesis is activated via jasmonate signal pathway in roots. Nicotine is produced in tobacco roots and transported to leaves for storage, but transport mechanism of nicotine is unknown.
We isolated two transporter genes (MATE1, 2) from tobacco roots. Expression pattern of these genes was very similar to nicotine biosynthetic genes, indicating that NtMATE functions in nicotine biosynthetic cells.
Analysis of nicotine secretion in NtMATE1-overexpressing BY-2 cells and in NtMATE1-suppressed transgenic tobacco plants suggests that NtMATE1 promotes excretion of nicotine from the vacuoles to apoplasts, and consequently facilitates translocation of nicotine from roots to aerial parts.
The functional analysis of NtMATE1 using transgenic tobacco plants is in progress.

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'Kusaie lime' should be regarded as a yellow-fruited form of 'Rangpur lime'. In order to elucidate the mechanism of this alteration, the carotenoid content and composition in flavedo tissue and juice sacs of fruits were analyzed. An unusual accumulation of phytoene was detected in flavedo tissue of 'Rangpur'. But the contents of β-cryptoxanthin, all-trans-violaxanthin and 9-cis-violaxanthin were decreased. Significant differences in total carotenoid were not found. Therefore, it was supposed that biosynthesis downstream from phytoene was suppressed in flavedo of 'Kusaie'. On the other hand, the accumulation of phytoene was not observed in juice sacs of 'Kusaie'. However, total carotenoid of 'Kusaie' was smaller than that of 'Rangpur'. Probably, in juice sacs of 'Kusaie', the amount of carotenoid production may be decreased, or, the resolution of carotenoids may be accelerated. Moreover, the difference of ABA contents between 'Kusaie' and 'Rangpur' will be examined.

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In PAP1-overexpressing mutants, anthocyanins and quercetin-type flavonol glycosides are specifically over-accumulated. Microarray analyses revealed that expressions of 38 genes are higher than in wild type. Acyltransferases (ATs), whose functions are remained undetermined, are up-regulated and expected to be involved in modification of anthocyanins.
Recombinant AT proteins were expressed in Escherichia coli. Malonyltransferase activity was detected with recombinant protein encoded by AT1. The recombinant protein accepted cyanidin 3,5-diglucoside but not cyanidin 3-glucoside as a acyl-acceptor. Coumaroyltransferase activity was detected with recombinant proteins encoded by AT2 and AT3. Although it is not possible to distinguish the transcripts of those 2 genes with microarray analysis, expressions of both genes were confirmed to be higher in pap1-D mutant than in wild type with semi-quantitative RT-PCR. Flavonoids accumulated in knockout mutants of each gene will be analyzed to confirm the functions of those AT genes in planta.

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Blue/violet flowers often contain delphinidin based anthocyanins that contain plural aromatic acyl groups (polyacylanthocyanins). Gentian contains a polyacylanthocyanin, gentiodelphin (delphinidin 3-glucosyl-5, 3^'-biscaffeoylglucoside), that exhibits stable blue color. Yoshida et al. (2000) showed that the color was derived from the intramolecular stacking by the two caffeoyl groups and that the caffeoyl group at 3^' -position mainly contributes to the stacking. The genes encoding anthocyanin acyltransferases have been isolated from gentian, perilla and so on. However, the gene encoding the enzymes that catalyze the transfer of an acyl group to the B-ring has not been isolated. Extensive investigation of gentian anthocyanin 5- acyltransferase (Fujiwara et al., 1999) enabled us to find that this enzyme is 3^' , 5-acyltransferase that acylates both 3^' and 5-glucose. We have also revealed that the acylation at 5-position proceeds to that at 3^' -position. The gene is useful to produce the polyacylated anthocyanin to engineer blue flowers.

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In Perilla frutescens, anthocyanin biosynthetic genes are expressed specifically in red form. The expressions of those genes are induced coordinately by light illumination. So far, two bHLH (MYC-RP and MYC-F3G1), two MYB (MYB-P1 and MYB-C05) and one WD40 (PFWD) factors were isolated and confirmed to be involved in anthocyanin production in red form, however determinant factor of anthocyanin production is still unknown.
The two cDNAs encoding bHLH factors (MYC-RS1 and MYC-RS2) were obtained from red perilla by using PCR-select cDNA subtraction. Between deduced amino acid sequences of MYC-RS1 and MYC-RS2, 7 out of 500 amino acid residues were found to be substituted. Phylogenetic analysis revealed that MYC-RS1 and MYC-RS2 were classified into group IIId, while MYC-RP and MYC-F3G1 were classified into group IIIf of bHLH factors. We introduced MYC-RS into A. thaliana and obtained over-expressing mutants. Metabolites and transcripts in those over-expressing mutants will be analyzed.

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To investigate physiological roles of rutin and flavonol-3-glucosidase (f3g) during cotyledon growth in tartary buckwheat, changes in rutin concentration and f3g activities were measured. The rutin concentration gradually increased just after germination (about 75 mg/g.d.w. in mature cotyledon). Most part of rutin was distributed in epidermis of cotyledon, where proanthocyanidins (condensed tannin) was also distributed. These results suggest that rutin play a role in UV screening. Most part of f3g was distributed in testa, which was stick to cotyledon until 4 DAG. The f3g, which should eluted from testa was also presented on a surface of cotyledon. It indicates that rutin in epidermis would hydrolyzed by f3g on surface of testa. It was recently reported that an anti-fungal compound is formed by oxidation of quercetin (hydrolyzed product of rutin). These results indicate that rutin and f3g may be related to enhancement of the defense system when cotyledon was injured.

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Accumulation of scopolin, a β-glucoside of scopoletin, in Arabidopsis roots indicates existence of the enzyme catalyzing glucosylation of scopoletin.
We selected some of the glycosyltransferase (UGT) genes as the candidates according to the expression levels on the databases and obtained the T-DNA inserted mutants.
The roots of the ugt71c1 (At2g29750) mutants show the severe decrease in the level of scopolin and scopoletin. A high expression level of UGT71C1 is found in the roots of wild-type, whereas a little expression in the shoots.
The UGT71C1 transcript expressed by E. coli expression system exhibits the high activity against the coumarins posessing a 7-hydroxy group.
We concluded that UGT71C1 is the enzyme glucosylating scopoletin in Arabidopsis roots.

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Recent insights into lignin biosynthesis have shown phenylpropanoid ester such as chlorogenic acid (CGA) plays prominent roles in monolignol biosynthesis. However, there is active debate about the biosynthetic pathway for generating phenolic esters. The two different pathways in CGA biosynthesis are known. Generally accepted pathway shown in tobacco and tomato cells used for hydroxycinnamoyl CoA thioester as precursor, and other pathway that used for hydroxycinnamoyl glucose ester is known in sweet potato. We characterized the relationship between both phenylpropanoid pathways involved in CGA biosynthesis in sweet potato cells in suspension culture. Most of enzymes and substrates involved in the glucose ester-derived biosynthesis were detected in vacuole not protoplast fraction, on the other hand, the monolignol biosynthesis used for caffeoyl CoA was detected in cytosol. These results show metabolic channeling by two different phenylpropanoid pathways is probably involved in chlorogenic acid biosynthesis in sweet potato cells.

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Coumarins, which have antimicrobial and antioxidative activities, are often found in the plant kingdom. Enzymes related to coumarin biosynthesis in plants are unclear, although the coumarins are known to be biosynthesized via the phenylpropanoid pathway. The coumarins, scopoletin and its glucoside, scopolin, were detected in Arabidopsis thaliana root. We report here the methyltransferase related to biosynthesis of scopoletin and scopolin in A. thaliana.
We analyzed the coumarins in the T-DNA insertion mutant of caffeoyl CoA O-methyltransferase 1 (CCoAOMT1, At4g34050), which shows the highest expression level of the CCoAOMTs in A. thaliana. The levels of scopoletin and scopolin in the ccoaomt1 roots were ~15% of those in the wild type, suggesting that they were biosynthesized via CCoAOMT in A. thaliana. The levels of esculetin, esculin and cichoriin, which are the coumarins without methylation at 6-hydroxy group, were ~25-fold of those of wild type.

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Lithospermum erythrorhizon produces shikonin derivatives, red naphthoquinone pigments, in the roots. In the cell cultures of L. erythrorhizon, stimulators of shikonin biosynthesis (Cu²⁺, oligogalacturonide, and MJ) as well as its inhibitors (NH₄⁺, 2,4-D, and light) have been identified. Shikonin molecule is formed in vivo from geranyldiphosphate derived via mevalonate pathway and p-hydroxybenzoate via shikimate pathway. However, the biosynthetic reactions responsible for the formation of naphthalene ring are not clarified yet.
Utilizing the dark-inducibility of shikonin synthesis, we applied PCR-select cDNA subtraction technique to L. erythrorhizon cell cultures to isolate cDNAs encoding enzymes involved in the naphthalene frame formation. Out of 240 clones whose expressions were enhanced in the dark, we have found two candidate genes for the naphthalene-forming enzyme, i.e. catechol polyphenol oxidase-like protein and a member of berberine-bridge enzyme family, adding to preciously identified oxidoreductase genes. Characterization of these genes will be presented.

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