FtsH protease is an ATP dependent protease whose function is to degrade damaged proteins. The major physiological substrate of this protease is D1 protein that is one of the main subunit of photosystem II. Knockout mutants of this protease displays variegated phenotype in Arabidopsis, thus physiological function of this protease is maintenance of chloroplast proteins. Previously, we constructed over-expression system of FtsH protease using E.coli-pET vector system. This system produced recombinant protein as inclusion body, however these were possible to be activated by urea mediated refolding. The refolded proteases showed only ATPase activity. Protease activity has not been detected, though we tried almost all of the conditions which have been reported. In this study, we calculated affinity between FtsH protease and D1 protein by means of molecular dynamics simulation, and analyzed unfoldase activity using bovine serum albumin as a substrate.

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Protein ubiquitination and degradation are key processes in the development of eukaryotes and their various physiological responses. Skp1 forms SCF E3 ligase with Cullin1, F-box protein, and RBX1 to catalyze protein ubiquitination in co-operation with enzymes E1 and E2. In Arabidopsis thaliana, there are 21 Skp1-like (ASK) genes. ASK20 has two translational products, ASK20A and ASK20B, due to variation in splicing. These deduced proteins have four helix domains at their N-termini, which bind with F-box motif and are conserved in all ASK proteins, while they also have functionally unknown regions at their C-termini, which are absent in other ASK proteins. In this study, we performed several fundamental characterizations of ASK20 to understand its function. In addition, possible functions of ASK20A and ASK20B will be discussed.
This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

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Small ubiquitin-related modifier (SUMO) is covalently attached to many cellular targets via a three-step enzyme pathway. While the ubiquitination leads to protein degradation, the SUMO modification regulates protein-protein interactions, as well as localization and stability of target proteins. In Arabidopsis, eight SUMO genes are encoded and four of them (AtSUMO1, 2, 3, and 5) are found in the EST database. In order to reveal the functional specificity of each SUMO gene, we analyzed their expression patterns. By the RT-PCR analysis, constitutive expression of AtSUMO1 and 2 was observed, while AtSUMO3 and 5 showed tissue-specific expression. We confirmed the expression of AtSUMO4 in root tissue. Further analysis using the promoter-GUS genes is currently in progress. Furthermore, Arabidopsis has two isoforms, AtSAE1a and b, for the E1 enzyme. We are also analyzing the expression patterns of these genes, respectively.

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Proteomic analysis with isolated vacuoles from Arabidopsis leaves detected a major protein that belongs to GDSL-lipase family. We designated it as VML (Vacuolar Major Lipase). Arabidopsis has more than 100 GDSL-lipase family proteins. However, the physiological roles are not fully understood. To analyze the nature of the protein, subcellular fractionation was carried out. The result revealed that VML was concentrated in the 100,000-g-pellet fraction. Further fractionation by linear sucrose-density-gradient centrifugation concentrated VML in the fraction that did not overlap marker proteins for vacuolar membranes, ER, or Golgi complex. VML was detected as large oligomers by treatment with various concentrations of cross-linker. These findings suggest that VML forms a large complex within the vacuoles. We obtained VML-deficient mutant and analyzed it to elucidate the physiological function.

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COP9 signalosome (CSN) is an 8-subunit nuclear protein complex. Null mutations for CSN results in lethality, indicating its essential role in development. To date, enzymatic activity has been detected in CSN to deconjugate Rub1 from E3 ubiquitin-ligases, which then modulates activity of ubiquitin-proteasome degradation pathway.
Functional dissection of mammalian CSN revealed that N-terminal portion of CSN1 (CSN1N) represses activated signals of the SAPK/JNK1 signaling pathway. CSN1N also reduces accumulation of JNK1, leading to suppression of c-Jun phosphorylation. To analyze this regulation, we have isolated proteins directly binding to CSN1N (NBPs). NBPs cover a novel spectrum of proteins, including SAP130, DDX15/hPrp43/mDEAH9, CFIm68, which are components of transcription/RNA processing complexes.
We have isolated the Arabidopsis NBP orthologs and its knockout plants. Detailed biochemical analyses on CSN-NBP interactions together with in planta research should reveal this novel mechanism of CSN. Here we discuss the complex interaction between CSN, COP/DET/FUS, and their interacting partners.

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Signal peptide peptidase (SPP) was identified as a homologue of presenilin, catalyzes intramembrane proteolysis of some signal peptides cleaved from a preprotein. Searching the Arabidopsis database, we found an orthologue of human SPP (AtSPP) and five homologues (AtSPPL1-AtSPPL5). In situ hybridization showed that AtSPPs were highly expressed in the shoot meristem and the epidermis of radicle during germination. At the reproductive stage in shoot apex, AtSPP was expressed in the inflorescence meristem, whearas the expression of homologues took place in the lower part of rib zone, where cell division is activated to form vascular bundle. Subcellular localization of AtSPPs were investigated by transporting a GFP fused protein in Deep cells. We detected GFP-AtSPP on the ER membrane and both GFP-AtSPPL2 and GFP-AtSPPL3 in the endosomes. These results suggest that AtSPP and AtSPPLs are functionally different from one another, having differential roles in plant physiology.

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Chlamydomonas sp. strain W80 is characteristically different from known Chlamydomonas types because of having tolerance to salt and cadmium. In this study, cystein protease from the cells of C. sp strain W80 (W80CP) was purified and characterized. W80CP was purified by five steps of chromatography using hydrophobic, ion-exchange and gel-filtration. This procedure resulted in a purification of 3090-fold with a yield of 2.6%. Its activity was optimal at pH 8.0. The K_m value for Boc-LRR-MCA was estimated to be 0.44 μM. W80CP was not inhibited by PMSF and EDTA and also not activated by cations, but fairly inhibited by cystein protease inhibitors, leupeptin and N-ethylmaleimide, suggesting that this enzyme is cystein protease. Molecular weight of W80CP was determined to be approximately 102 k. Analysis of SDS-PAGE of W80CP suggests that W80CP is dimer. We are now studying amino acid sequence of this enzyme and cloning of the gene.

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Arogenate dehydratase (ADT) catalyzes the last step of phenylalanine (Phe) biosynthesis in plants. A gene,OsPDTH, that is rice homolog of bacterial prephenate dehydratase, was identified from rice mutant (MTR1) as a mutation point. This mutation is responsible for resistance to 5-metyltryptophan and high levels of Phe accumulation. We characterized OsPDTH using a wheat embryo cell-free system, and clarified its function as ADT. In vitro chloroplast import assay revealed that the OsPDTH protein translocates into chloroplasts. Kinetic analysis revealed that OsPDTH showed high substrate affinity to arogenate over ten times more than that to prephenate. ADT activity of OsPDTH was feedback-inhibited by Phe, and the mutant enzyme showed resistance to Phe. We concluded that high-level accumulation of Phe in the MTR1 is ascribable to the insensitivity of the mutant ADT to Phe.

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The moss Pogonatum inflexum contained proteins exhibiting SOD activity and comigration with a tracking dye in 7.5% native-PAGE. We purified the protein (SOD-like A) and characterized its molecular properties. SOD-like A indicated a subunit molecular mass of 15 kDa. In 13% native-PAGE, the cell extract of P. inflexum showed the presence of a minor SOD-like B with faster migration than SOD-like A. SOD-like A and B were sensitive to cyanide and H₂O₂, suggesting the presence of copper as a prosthetic metal. However, immunoblotting analysis indicated that both proteins differ with a typical chloroplastic or cytosolic CuZn-SOD. The molecular masses of SOD-like A and B were estimated by Furgason plot analysis to be 40 kDa and 10 kDa, respectively. Thus, SOD-like A is a homodimmer with somewhat higher molecular mass than those of typical plant CuZn-SODs of 32 kDa. Therefore, it is likely that P. inflexum contains novel types of SOD.

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This research aims to reveal the function of isoamylase (ISA) and pullulanase (PUL) in rice endosperm. We have already established that ISA1-ISA2 hetero- and ISA1 homo-complexes co-exist in rice endosperm while there is only ISA1-ISA2 hetero-complex in potato tuber. To suppress the OsISA1 and OsISA2 by the RNAi technology, the calli of Kinmaze and rice PUL mutant were transformed.. The ISA1 suppressed lines lost the ISA activity in the endosperm and displayed the phytoglycogen-storage phenotype. The ISA2 suppressed lines had only the ISA1 homo-complex activity but no substantial phenotypic changes were observed, suggesting a considerable overlap in functions between ISA1 homo-complex and ISA1-ISA2 hetero-complex and PUL. These results sharply contrast with the previous studies showing that the reduction of ISA2 expression results in sugary phenotype in Arabidopsis leaves and potato tuber. This suggests that the function of a considerable overlap in functions between ISA1-homo and ISA1-ISA2 hetero-complexes and PUL.

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Previously purified maize acetylcholinesterase (AChE) was 88kDa dimmers consisting of 42-44kDa polypeptides. The full-length cDNA is 1471 nucleotides encoding 394 amino acid residues. The maize AChE homologs were widely distributed in plants but not in animals. The homologs in monocot shared high homology (92-64%) with maize AChE, whereas dicot showed relatively low homology against maize AChE (57-53%). Thus, we presume that there is difference of the primary structure or enzymatic function between monocot and dicot. In this study, purified AChE from siratro was 125kDa trimmers consisting of 41-42kDa polypeptides. The full-length cDNA is 1441 nucleotides encoding 384 amino acid residues. The homolog against siratro AChE shared 90% identity with soybean, while maize AChE showed low homology (45%). From the results, the primary structure and subunit of siratro AChE were clearly different from maize AChE. Meanwhile, the enzymatic properties of siratro AChE was nearly similar that of maize AChE.

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Recent studies clarified that plants are the largest source of CH₃Cl, and methyl chloride synthase gene, designated as HOL, was identified in Arabidopsis. Although it is predicted that HOL methylates Cl^-, Br^-, I^-, NCS^- and HS^-, its physiological significances are still unkown.
We analyzed recombinant proteins of HOL (AtHOL1) and its homologs, AtHOL2 and AtHOL3, and demonstrated that each protein catalyzes S-adenosylmethionine-dependent methylation of Cl^-, Br^-, I^-, NCS^- and HS^-. AtHOL1 and AtHOL2 showed the highest activity with NCS^-. Analyses of the growth responses of the Arabidopsis T-DNA insertion mutants (hol1, hol2, hol3) to the substrates showed that hol1 solely increased sensitivity to NCS^-. We showed that CH₃SCN was released from homogenized Arabidopsis seedlings but not from hol1. It is known that NCS^- is produced by hydrolysis of a glucosinolate and CH₃SCN has repellent activities. These results implies that AtHOL1 is involved in the metabolism of a glucosinolate and wounding responses.

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Disaccharide glycoside-specific glycosidases (diglycosidases) specifically hydrolyze the β-glycosidic bond between disaccharide and aglycons. Diglycosidases belong to glycosidase family 1, in which various monoglucosidases (β-glucosidases) are also classified. Disaccharide glycosides are found in diverse plants, suggesting the wide distribution of diglycosidases in the plant kingdom. Diglycosidases show over 50% amino acid identities with β-glucosidases, indicating that diglycosidases have evolved from β-glucosidases. To elucidate structural basis of alteration in their glycone specificities, we carried out X-ray crystallographic analysis of β-primeverosidase (PD, a diglycosidase from Camellia sinensis). The 3-D structure of PD complexed with a substrate analog inhibitor was determined at 1.8 Å resolution. The conformation of most catalytic residues was conserved between PD and β-glucosidases. Interestingly, Ser473 and Gln477 in PD were found to form the hydrogen bonds with the second xylose of primeverose. Thus, diglycosidases evolved from monooglucosidases as the consequence of slight structural change in the substrate binding pocket.

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During phosphate starvation, membrane galactolipid digalactosyldiacylglycerol (DGDG) accumulates concomitantly with a decrease in phospholipids. Recently, we revealed that auxin is involved in this process. Auxin signaling is activated by degradation of IAA proteins that bind directly AUXIN RESPONSE FACTOR (ARF) and thus inhibit transcription of ARF-regulated auxin responsive genes. SLR is one of the IAA proteins, and ARF7 and ARF19 are known to interact with SLR. Here, we revealed that DGDG accumulation upon phosphate starvation was suppressed in the gain-of-function mutant slr and loss-of-function mutant arf7arf19. During phosphate starvation, DGDG ratio of total polar lipids was increased up to 35% although its ratio was about 20% under phosphate-sufficient conditions in Arabidopsis shoot. In slr and arf7arf19, however, the DGDG increase by phosphate starvation was suppressed to 25% and 30%, respectively. These results indicate the involvement of the auxin signaling proteins in substitution of phospholipids to galactolipids during phosphate starvation.

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Phosphatidate (PA) phosphatase (PAP) is the enzyme which yeilds diacylglycerol (DAG) by dephosphorylating phosphatidic acid (PA), an end-product of Kennedy pathway with which glycerolipid metabolism commonly shares. Although homologues for mammalian membrane-associated PAP were reported in Arabidopsis,there is no evidence as to the chloroplast-localized or soluble type of PAP despite their importance in lipid metabolism.
Here,we detected a novel type of membrane-associated PAP in cyanobacteria and its five homologues in Arabidopsis that were distinct from mammalian-type PAP. Results of analyses showed that three out of five were chloroplastic PAP. Meanwhile, recent identification of soluble PAP in Yeast (Han et al,2006) suggests two homologues in Arabidopsis. Therefore, Arabidopsis has at least two additional PAP families. Identification and characterization on these novel PAPs will be presented.

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Sphingoid base 1-phosphates are resolved into ethanolamine-phosphate and C16-fatty aldehyde catalyzed by sphingosine phosphate lyase (SPL). In this study, to clarify the physiological role of SPL in Arabidopsis, we performed gene expression analysis of an Arabidopsis SPL gene, AtSPL1, and functional analysis of spl mutants. AtSPL1 transcripts were slightly decreased by low humidity stress. To assess the functional consequence of the loss of function of AtSPL1, we characterized two independent Arabidopsis mutants (atspl1-1 and atspl1-2) with T-DNA insertions in the AtSPL1 gene. The phenotype of atspl1-1 and atspl1-2 was indistinguishable from wild-type plants under normal growth conditions. Next, we compared the rate of water loss in excised leaves from wild-type and mutant plants. The mutant plants showed slower water loss than wild-type plants. Futhermore, we found that stomatal closing in the two lines was more sensitive to ABA than in the wild type.

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Glucosylceramides (GlcCers) are major glycolipids in plasma membranes and tonoplasts. The genome-wide studies on a glycosyltransferase gene family in animal, plant, fungal, and bacterial origin have revealed that plant GlcCers may be synthesized by glucosylceramide synthase (GCS) (UDP-glucose:ceramide glucosyltransferase). However, the genes for plant GCS were poorly reported; only from cotton. During the initial stages of our studies, even when an Arabidopsis cDNA homologous to a cotton GCS gene was expressed in Saccharomyces cerevisiae, such transformants failed to synthesize GlcCers, which might provide some clue to the mechanism of the glucosylation to ceramides. To overcome the difficulty of the functional analysis of Arabidopsis GCS genes, we currently constructed mutants lacking GlcCers in yeast Kluyveromyces lactis. Here we will discuss the function of glycosyltransferase genes in Arabidopsis.

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Anthocyanins are major constituents of flower color and their biosynthesis pathway leading to anthocyanidin 3-glucosides is well conserved among higher plants. The glucosylation was catalyzed by anthocyanidin 3-glucosyltransferase (A3GT). Anthocyanidin 3-glucosides are usually further glucosylated with other glucosyltransferases. However, in rose, a single enzyme (anthocyanin 5, 3-glucosyltransferase) glucosylates 5-position of an anthocyanidin followed by 3-position (Ogata et al., 2005). We isolated a rose genomic clone encoding a protein (RhA3GT) belonging to the A3GT family (46-57% identity). RhA3GT showed A3GT activity in vitro. The expression level of this gene in rose petals is below detection level in northern analysis. A rose cDNA, encoding a protein with 42-44% identity to A3GTs, was also isolated. The protein (RhF3GT) showed glucolyltransferase activity to flavonols but not to anthocyanidins in vitro. Transgenic rose plants habouring an RNAi construct of RhF3GT gene exhibited white or paler flower color due to decrease of anthocyanins.

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S-adenosylmethionin:Anthocyanin methyltransferase (AMT) catalyzes methylation of anthocyanins and contributes to colour variation of anthocyanins and flower colour. Torenia AMT (ThAMT) gene was isolated with using a petunia AMT gene (WO2003-062428). ThAMT was revealed to encode anthocyanin 3', 5'-methyltransferase activity by its expression in E.coli. Rose and many other flowers do not produce substantial amount of methylated anthocyanins. Expression of genes of ThATM and a flavonoid 3', 5'-hydroxylase in roses resulted in malvidin, petunidin and peonidin production and modified flower colour. AMT gene is a useful molecular tool to engineer and diversify flower colour.

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We have previously identified the gene encoding 1-O-acylglucose:anthocyanin 3'-O-glucoside-6'''-O-acyltransferase (3'AT; CtAT1) which is responsible for the biosynthesis of ternatin C3 from ternatin C5 in blue butterfly pea flowers. This enzyme, consisted of heteromeric 31- and 24-kDa subunits, is a serine carboxypeptidase-like acyltransferase containing three potential N-glycosylation sites. Recombinant CtAT1 protein was expressed in Sf9 cells in a baculovirus expression vector. LC/MS analysis of the recombinant-enzymatic reaction mixtures, in which various ternatins were reacted as substrates, indicated that CtAT1 had antocyanin 5'-O-glucoside-acyltransferase (5'AT) and anthocyanin 3'-O-(4-O-glucosyl-(6-O-p-coumaroyl))glucoside-acyltransferase (3'AT2) activities as well as first 3'AT activity. To investigate the subcellular localization of CtAT1, fusion proteins of CtAT1-green fluorescent protein (GFP) were engineered and stably expressed in tobocco BY-2 cells. Expression of CtAT1-GFP fusion protein showed that CtAT1 localized to the vacuolar lumen. These results suggested that CtAT1 catalysed the acylation involved in polyacylated anthocyanin biosynthesis in the vacuole.

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We prepared transgenic plants that express a chimeric repressor for single-MYB domain protein and attempted to identify those involved in flavonoid biosynthesis by isolating seeds with different color from wild-type. We found that the expression of the chimeric repressor for AtMYBL2 resulted in pale-color seeds. Interestingly, the ectopic expression of AtMYBL2 (35S:AtMYBL2) also induced light color in the seeds. The contents of anthocyanin in leaves both of 35S::AtMYBL2 and 35S::AtMYBL2SRDX transgenic plants were much lower than those of wild type plants. Transient analyses revealed that 6 amino acids at the carboxy-terminal region of AtMYBL2 sufficiently confer the repression activity. Expression of the trancated AtMYBL2 from which a C-termini was remomved, and homozygous AtMYBL2 T-DNA tagged line exhibited an increase of anthocyanin contents in rosette leaves. These data suggests that AtMYBL2 is a negative regulator for flavonoid biosynthesis.

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Lithospermum erythrorhizon produces shikonin derivatives, naphthoquinone-type secondary metabolites, in the roots. Shikonin is formed by the coupling of two key intermediates, geranyl diphosphate (GPP) derived from mevalonate pathway and p-hydroxybenzonate via shikimate pathway. It is proved that GPP is provided from mevalonate pathway and the GPP synthase is localized in cytosol as a sole exception in higher plants characterized thus far. From EST information we cloned a homolog of FPP synthase (LeFPPS) as a candidate of GPP synthase of cytosolic localization. Recombinant LeFPPS purified via His-tag demonstrated FPPS activity from GPP and IPP as substrates but not GPPS and FPPS activity from DMAPP and IPP. However, the correlated expression of LeFPPS with shikonin accumulation suggested that the in vivo function of LeFPPS is GPPS. Indeed, the addition of cell-free-extract of L.erythrorhizon converted the FPPS activity to strong GPPS activity, suggesting LeFPPS functions as a catalytic subunit of GPPS in vivo.

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To study physiological roles of anthocyanins in the sprouts of common (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum), we performed structural identification of them, and then investigated varietal differences of its concentrations using 19 cultivars. Buckwheat sprouts contained 0.67- 1.17 mg / g.d.w. of anthocyanins. They were mono- or di- glycosylated forms of cyanidin. Tartary buckwheat sprouts contained 0.29 - 0.60 mg / g.d.w. of anthocyanins. Their structures were same as that found in common buckwheat sprouts except for absence of a galactopyranoside. The mutant line, which contained high amount of anthocyanins (50.0 mg / g.d.w.), can accumulate it even grown in the darkness (27.7 mg / g.d.w.) whereas cultivars cannot. Anthocyanins were distributed in the epidermis of cotyledons and hypocotyls. It indicates that anthocyanin in buckwheat sprout would have a role of UV screen.

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Growth of suspension-cultured Catharanthus roseus cells ceased during phosphate starvation, but the cells grew again upon addition of Pi even after long-term starvation. The metabolic fate of [³³P]Pi was studied in Pi-starved cells. Immediately after administration, the most heavily labelled organic compounds are nucleotides, followed by sugar phosphates. The RNA, protein and free nucleotide content decreased gradually during Pi-starvation; however, these compounds, especially nucleoside triphosphates and UDP-glucose, increased markedly after addition of Pi. The transcript level of phosphate transporter and the activities of acid phosphatase, 5^'- ,3^'- nucleotidase and adenosine nucleosidase were all reduced by the addition of Pi. In contrast, the activities of adenine phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamidase were markedly increased in the Pi-fed cells. Little or no increase was observed in adenosine kinase. In the light of these results we discuss the possible involvement of net nucleotide synthesis in the initial metabolic events of recovery from Pi-deficiency.

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Tea (Camellia sinensis ) and Camellia japonica belong to Theaceae. C. sinensis is one of the caffeine-containing plants, on the other hand, caffeine is not accumulated in C. japonica. The aim of this study is to clarify the reason why C. japonica is not purine alkaloid-producing species.
The caffeine biosynthetic pathway contains three S-adenosylmethionine(SAM)-dependent methylation steps. Caffeine synthase(TCS) , which is investigated by our group , is the SAM-dependent N-methyltransferase. TCS is involved in the last two steps(N-1 and N-3 methylation) of caffeine biosynthesis in tea leaves. First of all, we could show the presence of caffeine synthase homologous gene in C. japonica genome. The transcripts could be detected by RT-PCR, but not by northern blot analysis. We isolated two types of TCS homologous gene from C. japonica leaves by RACE. The identity of amino acid sequence between TCS homologous genes and TCS1 was 90% .

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Rice is important crop and used as a model plant for genome research. In order to isolate genes useful for molecular breeding of rice, we started large-scale analysis of rice genes using FOX Hunting System (full-length cDNA over-expressor gene hunting system) and Arabidopsis.
Wild type Arabidopsis accumulates proanthocyanindins and seed color is brown. Mutants of Arabidopsis in anthocyanin biosynthesis, transport of anthocyanin or regulation of these genes have pale brown or yellow seeds.
A plant with yellow seeds and the other with pale brown were isolated from FOX lines. Anthocyanin levels in leaves or stems and proanthocyanidin levels in seeds of these lines were lower than those of wild type. On the other hand, mRNA levels of genes involved in anthocyanin and proanthocyanidin biosynthesis were not lower significantly when they were compared to wild type. Analysis of both introduced genes and involvement of genes in anthocyanin accumulation was in progress.

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Plants with 'Purple leaf' that anthocyanin appears constitutively in the leaf are very popular and has been used in the arranged cut flowers and the gardening plants. 'Purple leaf' plant has been obtained as a bud mutation from various plants. But it is not known in the petunia that is one of the most often-used bedding plants. The OSB2 gene has been cloned from the Purple leaf locus of rice and codes the transcription factor of the bHLH type. When having introduced the OSB2 gene with the CaMV 35S promoter into petunia var. 'Purple Wave', anthocyanin accumulated only in the leaf edge part of the petunia. When the transgenic petunia crossed with the other petunias transformed with the gene of another transcription factor of the myb type, some of the offspring accumulated a lot of anthocyanin in the leaf and the flower. These results will be discussed.

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Two types of red pigment, anthocyanins and betacyanins, never occur together in the same plants. Although anthocyanins are widely distributed in higher plants, betacyanins are characteristic constituents of most species of Caryophyllales.
We have cloned cDNAs of DOPA dioxygenase (DOD) which is known as a key enzyme in betalain biosynthesis from red cell cultures of Phytolacca americana. Phytolacca DOD (PaDOD) had a conserved motif found in DODs of betacyanin synthesizing plants. Expression patterns of PaDOD in cell cultures and in various organs of Phytolacca plants were investigated by semi-quantitative RT-PCR. In red cell cultures, the highest expression of PaDOD was observed just before betacyanin accumulation. In Phytolacca plants, the transcripts of PaDOD were found not only in betalain-containing cells but also in green or white cells. Further studies on the function of DOD in Phytolacca plants are in progress.

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The preprophase band (PPB) of plants is a band of microtubules that underlies the plasma membrane in prophase cells. The PPB plays an important role in the determination of the site of division site of plant cells. Clathrin-coated vesicles (CCV) as well as structurally related non-coated vesicles (NCV) were frequently observed at the PPB. To understand the role of the vesicles and actin in PPB development, we have quantitatively analyzed the effects of cytochalasin D on the distribution and the morphology of these vesicles and of cytoskeletal structure in high-pressure frozen onion epidermal cells by electron tomography. The average size of the NCVs decreased by treatment with cytochalasin D, suggesting a role of actin in vesicle fusion. Cytochalasin D also affected MT organization in the PPB. In particular, the PPBs become wider, due to greater lateral separation of the MTs, and simultaneously the length of the average MT decreases.

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Vacuoles are the compartment of amino acids in yeast as well as in plant cells. Several transport systems operate in the Saccharomyces cerevisiae vacuole. Concanamycin A, an inhibitor of the vacuolar H⁺-ATPase, inhibited uptake of amino acids but not of 2-Deoxy-D-glucose by Schizosaccharomyces pombe cells, suggesting involvement of vacuole in amino acid uptake by cells. We found that S. pombe fnx1 (SPBC21D10.04c) and fnx2 (SPBC3E7.06c) genes are phylogenetically related to S. cerevisiae VBA2 (YBR293w) gene encoding vacuolar basic amino acid transporter. GFP-fused Fnx1p and Fnx2p localized exclusively to the vacuolar membrane of S. pombe. Uptake of lysine, asparagine or isoleucine was impaired by Δfnx1 mutant cells whereas uptake of lysine or isoleucine impaired by Δfnx2 mutant. These results suggest that Fnx1p and Fnx2p are involved in vacuolar transport by S. pombe with a moderate specificity for amino acids.

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We have developed the in vitro chloroplastic protein import system free from the radio-labeled proteins with use of recombinant precursors. The import efficiency of this system is improved from the previous reports, thus we are able to handle the newly imported protein in chemical amounts. We are currently developing the application of this in vitro chloroplastic protein import system. We will present our current effort on the development of the application.

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In the vesicle transport systems of eukaryotes, 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 transport from ER to golgi apparatus. 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 ATSEC31s, ATSEC13s, ATSEC23s and ATSEC24s. The homology search indicates that there are two homologs for Sec31p (ATSEC31), two homologs for Sec13p (ATSEC13), seven homologs for Sec23p (ATSEC23) and three homologs for Sec24p (ATSEC24). In this research, we carry out the expression analysis and the intracellular localization analysis. In this poster, we report about the intracellular locatization analysis for ATSEC31, ATSEC13 and ATSEC23. Additionally, we examin whether ATSEC31 and ATSEC13 interact each other. In this examination, the fluorescence of YFP was detected, suggest that ATSEC31 interacts with ATSEC13.

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We previously showed that AtATG6, an Arabidopsis homologue of ATG6, restored autophagy and vacuolar sorting of CPY protein in yeast atg6 mutant. In Arabidopsis cells, AtAtg6 and phosphatidylinositol 3-kinase (AtVps34) colocalized with AtAtg8, a marker protein of the pre-autophagosomal structure (PAS). Here we further show that GFP-FYVE proteins, which interact with phosphatidylinositol 3-phosphate, colocalized with AtAtg6 and AtAtg8 on the punctate structure, which may represent the PAS of plant cells. We propose that synthesis of phosphatidlylinositol-3 phosphate by phosphatidylinositol 3-kinase at the PAS is an important process in autophagosome formation. We have also found that atg6 was male sterile because ATG6-deleted pollen failed to germinate. Transmission electron microscope and environmental scanning electron microscope showed that atg6 mutant pollen grains were normal in appearance. All these results suggested that AtAtg6 is essential for pollen germination but not for early pollen development.

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Arabidopsis zig mutant exhibits abnormal shoot gravitropism and morphological defects in stem and leaves. The ZIG gene encodes a Qb-SNARE VTI11 that plays roles in vesicular trafficking between trans-Golgi network and vacuoles.
To understand the molecular basis of vesicle transport involved in physiological function of higher plants, we performed a screening for the second mutation that can suppress defects of zig-1. zig suppressor 3(zip3) partially suppresses abnormalities in gravitropism and morphology observed in zig-1. The cytological analysis revealed that the zip3-1 partially restored the dynamics of amyroplasts and vacuolar membrane in the gravity-sensing endodermal cells.
Based on the map position, a nonsense mutation was found in AtVPS35b gene in the zip3 mutant. The complementation analysis identified ZIP3 as AtVPS35b encoding one of three Arabidopsis genes homologous to yeast VPS35. The Vps35p is a subunit of retromer involved in retrograde transport from prevacuolar compartment to TGN in yeast.

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In pollen, the membrane structure is largely reconstructed throughout the pollen development and pollen tube elongation. Particularly, large amount of cell wall and plasma membrane (PM) components are selectively transported to the tip region of pollen tube during the pollen tube elongation process. In order to search SNARE (soluble N-ethyl-maleimide sensitive factor attachment protein receptor) molecules which are involved in the pollen maturation or pollen tube elongation, we generated transgenic Arabidopsis expressing GFP-fused PM Qa-SNARE molecules under the control of their own promoters, and found that totally four SNARE molecules (SYP112, SYP124, SYP125 and SYP131) were specifically expressed in the pollen. We also report the subcellular localization of these SNAREs in pollen.

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Proteins synthesized in the endoplasmic reticulum (ER) are correctly folded before translocation. If protein folding is incorrect, genes for ER-resident chaperones such as BiP are induced. This phenomenon is known as the ER stress response or the unfolded protein response (UPR). Studying molecular mechanism of the ER stress response in Arabidopsis, we identified a bZIP transcription factor, AtbZIP60, induced by an ER stress inducer tunicamycin. AtbZIP60 has a putative transmembrane domain in its C-terminal region, suggesting proteolysis triggers its activation. Immunoblot analysis indicated that AtbZIP60 protein was cleaved by tunicamycin and DTT treatment. Subcellular fractionation showed that full-length and cleaved AtbZIP60 localized to the ER and the nucleus, respectively, supporting the above hypothesis. In order to investigate the physiological role of AtbZIP60, detection of cleaved AtbZIP60 under normal growth condition will be tested.

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Seed storage proteins are synthesized on rER as precursors, are transported to protein storage vacuoles, and then are converted into the mature forms. We have isolated vacuolar sorting mutants for storage proteins. atvsr1 mutant secretes the storage protein precursors to the extracellular space 1). mag2 mutant accumulates the precursors in the novel structures derived from rER within cells 2). Different accumulation manners of the precursors in atvsr1 and mag2 indicate that AtVSR1 and MAG2 function in the different step of the storage protein transport from each other. We generated a double mutant of atvsr1 and mag2. The double mutant germinates and grows normally. Electron microscopy revealed that the double mutant accumulated the storage protein precursors in both intracellular and extracellular spaces of seeds.
1) Shimada et al. Proc. Natl. Acad. Sci. USA, 100, 16095 (2003)
2) Li et al. Plant Cell, (2006) in press

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We have performed to estimate phloem flow velocity with the Positron Emitting Tracer Imaging System (PETIS), a noninvasive measurement device. ¹¹CO₂ was supplied to leaves of rice plants and the translocations of ¹¹C-photoassimilates were monitored using PETIS. We have developed an analytical program to estimate the phloem flow velocity from PETIS data by determining the arrival time of ¹¹C-photoassimilates to the basal part of shoot. This method was applied to a PETIS experiment testing plant response to cadmium toxicity. As a result, the phloem flow velocities in the cadmium-treated plants were estimated to be slower than those of control plants.

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We have developed prototype of the multi-element imaging system for plant study using CdTe semiconductor detector, which has high-energy resolution. The feasibility of this system for gamma-ray emission imaging of radioactive multi-nuclide tracer was examined by imaging experiment with a plant. The distribution of the two sample tracers, technetium and thallium, fed to a tobacco plant was successfully visualized for each nuclide simultaneously. The presented imaging method will yield plant molecular imaging, which visualizes dynamics of some competitive elements in intact plant, non-invasively and quantitatively.

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Iron-fortified rice will contribute to ameliorate the iron deficiency anemia which is a serious problem of insufficient nourishment in Asia. We have already expressed ferritin gene in rice seeds using an endosperm-specific promoter and obtained transgenic rice seeds whose iron concentrations were increased. However, higher levels of ferritin expression in rice seeds hardly increased iron concentration. In this report, we aimed to further increase iron concentration in rice seeds by enhancing iron absorption and transport ability by introducing the barley HvNAS1, HvNAAT-A,-B and IDS3 genes encoding biosynthetic genes of mugineic acid family phytosiderophores. First, we investigated iron concentrations of transgenic rice seeds with introduced HvNAS1alone, HvNAS1 and HvNAAT-A,-B, or IDS3 gene alone. Second, we introduced soybean ferritin gene, SoyferH2 connected to two endosperm-specific promoters, along with HvNAS1, HvNAAT-A,-B and IDS3 genes, using a marker-free vector. We will report ferritin expression and iron concentrations of T₂ seeds.

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Sedum species, which possess the CAM metabolism, are widely utilized for extensive green roofs as they are tolerant to drought environments. Especially, naturalized species, Sedum mexicanum and S. acre, are most intensively used. In this study we compared transpiration rates of three Sedum species that naturally habitat in Japan, S. makinoi, S. bulbiferum and S. polytrichoides subsp. yabeanum var. setouchiense to four naturalized species, S. mexicanum, S. acre, S. lineare and S. sarmentosum to evaluate their adaptability to roof environments.
Transpiration rates of S. makinoi and S. polytrichoides subsp. yabeanum var. setouchiense were very slow and comparable to four naturalized species, indicating their adaptability to roof environment in terms of water use efficiency. Contrary, the transpiration rate of S. bulbiferum was approximately 10-fold higher than those of other species. This suggests that S. bulbiferum shows relatively low adaptability to roof environments and higher evaporative cooling ability of buildings.

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Phytoremediation is a good technique to clean or remediate Cd-polluted soils. In order to practicalize this technique, it is essential to control long-distance transport of Cd in vascular tissue. In this study, we tried to investigate proteins in the phloem sap to elucidate the mechanism of long-distance Cd transport in the sieve tubes.
Oilseed rape plants (Brassica napus L.) were grown hydroponically for about three months and treated with 30μM Cd for 48 hours. Phloem sap was collected from these plants after Cd treatment. Effects of Cd on sugars and amino acids concentration in the phloem sap were not observed. Additionally, difference was not found in protein concentration in the sap (protein concentration in the phloem sap was approximately 0.25 mg mL^-1). 2D-PAGE analysis revealed that several proteins in the phloem sap responded to Cd treatment. We tried to identify these proteins by PMF analysis.

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Boron (B) is an essential elements for higher plants. Recently, AtNIP5;1, a member of Major Intirinsic Protein family, was identified as a boric acid channel responsible for efficient root uptake of B in Arabidopsis (Takano et al., 2006). To reveal the molecular mechanisms of B transport in rice, we analyzed OsNIP3;1, a rice gene most similar to AtNIP5;1.
Accumulation of OsNIP3;1 transcripts was induced by several fold upon exposure to B limitation in roots. GFP-OsNIP3;1 fusion protein mainly localized to the plasma membrane in tobacco BY2 cells. Tissue specificity of OsNIP3;1 was investigated with transgenic plants carrying Promoter:GUS fusion gene. In roots, GUS staining was observed both in steles and outer cortex cells. In leaf blades, GUS staining was observed in cells around vesicular bundles. These data suggest that OsNIP3;1 is an B- inducible channel involved in both root uptake and distribution of B in rice plants.

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Aquaporins are membrane proteins that facilitate membrane transport of water and other small molecules in various organisms. Arabidopsis has 35 isoforms of aquaporins, which are classified into four subfamilies (PIPs, TIPs, NIPs, and SIPs) . In this study, we focused on four NIPs (NIP1;1, 1;2, 5;1, and 6;1) that have not been studied extensively yet, and investigated their water permeability, subcellular localization, and tissue-specific accumulation.
A stopped-flow spectrophotometric assay of the membrane vesicles from yeast cells harboring NIPs revealed that the four members had only low water permeability. The transient expression of NIP1;1, 1;2, and 6;1 linked with green fluorescent protein in Arabidopsis cultured cells showed their localization on the ER membrane. In addition, we performed promoter-β-glucuronidase (GUS) analysis to examine tissue-specific accumulation.

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Boron (B) is essential for plants but toxic when present in excess. Arabidopsis thaliana BOR1 is a borate exporter for efficient B translocation under B limitation (Takano et al. 2002). Using P35S:BOR1-GFP plants, we have shown that BOR1 is regulated by B-dependent endocytosis and degradation (Takano et al. 2005).
Here we report localization and B-dependent regulation of BOR1-GFP expressed under control of the BOR1 promoter. BOR1-GFP was localized to steler cells in mature portion of roots and also to various cells in root tip under B limitation. In root tip, BOR1-GFP was preferentially localized on the plasma membrane of the proximal side of the epidermal cells. These results suggest that BOR1 functions for B transport into xylem in mature portion for root-to-shoot B translocation while it is for radial B transport towards inner portions in root tip.

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Arabidopsis MCA1 and MCA2 genes (formerly AtMID1A and AtMID1B, respectively) complement the lethality of the yeast mid1 mutant defective in a putative stretch-activated channel component (2004 JSPP meeting). By the two-phase agar method we developed, we have found that the root of an mca1-null mutant is incapable of sensing the hardness of agar (2006 JSPP meeting). Here, we performed the functional characterization of Mca1 and Mca2 in yeast. Mca1 was found to be localized in the plasma membrane as an integral membrane protein. Since yeast Mid1 must cooperate with yeast Cch1 to function as a high-affinity Ca²⁺ channel, Mid1 alone cannot complement the lethality of the mid1 cch1 double mutant. By contrast, Mca1 and Mca2 complemented individually the lethality of the double mutant and enhanced Ca²⁺ accumulation. The results suggest that Mca1 and Mca2 function as a Ca²⁺-permeable channel component whose action mechanism is different from that of Mid1.

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The high salt concentration in soil is one of the important factors for growth and yield of crops. Rice is not so tolerant to salt, but have the mechanism for the exclusion of salt from a cytoplasm. The genes for salt tolerance were selected by rice FOX (Full-length cDNA overexpressor gene) hunting system and microarray technique using a slide glass spotted 44k oligonucleotides encoding rice genes (Agilent Technologies, Inc.) We selected genes concerned with salt tolerance by FOX hunting, such as the genes for zinc finger containing proteins. Many genes were selected as a gene functioning salt tolerance by microarray technique. The genes selected by FOX hunting did not appeared in the genes result from the microarray experiment. However, the genes selected by both methods were categorized in the groups having a similar function.

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Plants are exposed to a large number of environmental stresses such as dehydration, cold and high salinity. Plants respond to these stresses at the physiological and biochemical levels. Moreover, many genes including enzymes for metabolism and sugar and proline transporters are induced by the stresses. In this study, we analyzed ERD6 and its homologues encoding putative sugar transporters. ERD6 was isolated from a cDNA library prepared from Arabidopsis plants exposed to dehydration stress for 1 h. Expression of ERD6 and its homologues in response to dehydration, cold and high salinity stresses was analyzed by northern blot hybridization. The expression levels of these genes were increased by these stresses. We observed histochemical expression patterns of ERD6 and its homologues in Arabidopsis plants using a GUS reporter gene and detected different expression patterns between ERD6 and its homologues. Currently, we are analyzing phenotypes of knock out mutants of these genes.

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Leaf temperature provides an indicator of the transpiration of plants, because evaporation of water causes leaf cooling. With a high throughput leaf thermal imaging CO₂ screen, we isolated two allelic Arabidopsis mutants (high leaf temperature 1; ht1-1 and ht1-2) altered in their ability to control stomatal movements in response to CO₂. The strong allele, ht1-2 exhibits a dramatically impaired CO₂ response but functional reactions to blue light and ABA, suggesting a specific role for HT1 in stomatal CO₂ signalling. HT1 encodes a protein kinase expressed mainly in guard cells. Phosphorylation assays demonstrate that the activity of the HT1 protein carrying the ht1-1 or ht1-2 mutation is greatly impaired or abolished, respectively. Furthermore, dominant negative HT1:kw transgenic plants which lack HT1 kinase activity show a disrupted CO₂ response. Further analysis suggested that the HT1 is a Raf-like protein kinase functioning as CO₂ signalling molecule in cytosol.

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In higher plant, MAPKs are suggested to be concerned in cell proliferation and pathogenetic responses. Although there are about 20 MAPK homologs in Arabidopsis thaliana, many are unknown in function.
     Chlamydomonas reinhardtii, a unicellular alga, is most suitable model organism for analysis of MAPK systems in plant, because there are much available EST's and genomic information. We have already analyzed some MAPK homologs; CrMPK1,2. Because genomic information (Chlre3) had been available recently, we have searched MAPK homologs in Chlamydomonas. These analyses suggested that there were four TEY-type and four TDY-type MAPKs, including two plant-specific TDY-type MAPKs expressed by pathogenetic infection in higher plant, and MAPK homologs similar to ones found in pathogenetic flagellate. These results suggest that MAPKs in Chlamydomonas are evolutionally and functionally interesting.
     We are now analyzing the expression of each MAPK by RT-PCR.

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