In previous study, we reported that expression of NIP5;1, one of NIP family, is strongly induced by boron (B) deficiency in roots and transports B (Takano et, al). NIP6;1 is the most similar gene to NIP5;1 in all nine members of the Arabidopsis NIP family. It is possible that NIP6;1 is also involved in B transport. To verify this hypothesis, we studied Arabidopsis NIP6;1. Transcript accumulation of NIP6;1 was induced by B deficiency, especially in rosette leaves. Expansion of rosette leaves was inhibited only under B deficiency in Arabidopsis T-DNA insertion lines, but not in the wild type. NIP6;1 localized to the plasma membrane and expressed in nodes and vascular bundles of immature young leaves. NIP6;1 facilitated boric acid transport in Xenopus laevis oocytes. Taken together, NIP6;1 is important for B transport in Arabidopsis under low B condition and has distinct roles from NIP5;1.

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Iron acquisition by gramineous plants is characterized by the secretion of phytosiderophores and subsequent uptake of iron as an Fe-phytosiderohore complex. Genes encoding Fe-phytosiderophore transporter have been isolated in maize (ZmYS1) and barley (HvYS1). In contrast to HvYS1, which specifically transports Fe-phytosiderophore, ZmYS1 has broad substrate specificities. Here, we further characterized the ZmYS1 in terms of tissue-specific expression and cellular localization. The expression of ZmYS1 was induced by Fe-deficiency, but unlike HvYS1, the expreesion was found in both the roots and the shoots. Moreover, the expression of ZmYS1 was higher in the shoots than in the roots. In the shoot, the expression was higher in the young leaf showing severe chlorosis than in the old leaf staying green. Unlike barley, maize did not show a distinct diurnal rhythm in both the secretion of phytosiderophore and the expression of ZmYS1. Immunostaining showed that ZmYS1 was localized on the root epidermis.

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Nod-26-like major intrinsic proteins (NIP) in gramineous plants can be divided into three subgroups based on their similarity. Lsi1, which belongs to NIP2 subgroup, has been demonstrating to transport silicon in rice. Here, we compared the permeability of other NIPs to silicic acid by using Xenopus oocytes. Both OsNIP1:1 and OsNIP3:1 did not show transport activity of Si, whereas rice Lsi1 homolog, Lsi6, maize Lsi1 homologs ZmNIP2-1 and 2-2 showed Si transport activity. The substrate specificity of Lsi1 was further characterized by comparing transport activity with three non-charged molecules. Lsi1 showed transport activity for urea and boric acid, but not for glycerol. However the Si transport activity was not or less affected by the presence of equimolar urea or boric acid. The transport activity was inhibited by HgCl₂, but not by low temperature treatment. Immunostaining showed that ZmNIP2-1 was localized at the plasma membrane of epidermal cells.

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Rice is a typical Si accumulator and it requires a large amount of Si for high yield production. We have identified two genes (Lsi1 and Lsi2) encoding Si transporters in rice roots. Here, we characterized the function of Lsi6, a close homolog of Lsi1, in rice. Expression of Lsi6 in Xenopus oocyte also showed transport activity for Si. Unlike Lsi1 and Lsi2, Lsi6 was expressed in both the root and shoot. Immunohistological staining showed that Lsi6 protein was localized at the xylem parenchyma in the leaf sheath and leaf blade. When this gene was knocked out in a T-DNA line, Si uptake by the roots was unaffected, but silicification of leaf abaxial epidermis was frequently observed than wild type. These results suggest that Lsi6 is involved in the xylem unloading and subsequent distribution of Si in the shoot.

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We have isolated a novel gene, OsPI1 from P-deficient rice. Low P induces OsPI1 immediately, therefore, it is suggested that OsPI1 plays an important role in low P adaptation. OsPI1 is thought to belong to TPSI1/Mt4 family, which contains low P inducible non-coding RNAs. Though it is suggested that an Arabidopsis gene of this family related to phosphate-recycling, the detail is still unclear. The purpose of this study is to reveal the function of OsPI1 using OsPI1 knockdown lines.
Seedlings of wild type (WT) and 2 knockdown lines (KD) were hydroponically cultured for 10 d with or without P and used for microarray analysis. Alterations of expression of several genes involved in phosphate-recycling under P-deficiency were smaller in KD than in WT. Furthermore, total P concentration and root/shoot of P-deficient KD was higher and lower than P-deficient WT, respectively. These results suggest that OsPI1 has a critical role in phosphate-recycling.

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The resource of phosphate rock has been depleting, whereas a large amount of organic-P (Po) exists as unavailable form in soil. To liberate inorganic-P (Pi) from Po, plants secret acid phosphatase into the rhizosphere. White lupin has high ability to secret acid phosphatase and grows well under P deficient conditions.
A gene encoding secreted acid phosphatase had been isolated from white lupin and transformed to tobacco plants. The aim of this study is to investigate the ability of P-acquisition by LASAP2 transgenic tobacco. P uptake and growth of LASAP2 line were higher than wild type in sterile medium containing phytate as sole P source. When plants were grown in pots containing soils of long-term experimental field, growth and P accumulation of LASAP2 line were higher than wild type in -P and phytate treatments. These results suggest that overexpression of LASAP2 is effective for P-acquisition from soil Po, including phytate.

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The molecular basis of phloem tissue development and function is largely unknown, although the phloem tissue plays important roles in sugar translocation and long-distance signaling. We have found that a Zinnia HD-Zip gene, ZeHB3, is expressed specifically in immature phloem cells. The overexpression of either ZeHB3 or its Arabidopsis homolog, AtHB5, in Arabidopsis causes the disappearance of the starch granules at the root cap and seedling lethal phenotype. These genes induced ectopic expression of a phloem marker gene, SUC2::GUS, but not other phloem markers, such as AHA3::GUS and APL::GUS. Since the SUC2 encodes a sucrose transporter that is important for the efficient phloem transport, it is possible that the ZeHB3 and AtHB5 are associated with maintenance of phloem function. The downstream analysis of the ZeHB3 and AtHB5 by using microarray analysis and suppressor screening of the overexpressors will also be reported.

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Maintenance of inorganic ion environment is essential for keeping life activities. In plants, however, it is remaining to be resolved how inorganic ions are distributed and accumulated in a leaf. Recently we found that each inorganic ion had a unique gradient within a leaf. For example K⁺ and NO^3- contents in the basal tissue of a leaf was much lower than in a tip. PO₄^3- content of the tissue was constant through a whole leaf. The ion gradients of protoplast isolated from different places of a leaf from the bottom to tip are similar to those of tissues. To discuss mechanisms of the formation of inorganic ion gradients we compared expression levels of known ion transporters in a leaf by using realtime-PCR. To resolve the physiological roles of inorganic ion gradients, we investigated activities of photosynthesis by using 2D-PAM.

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Dynamin and dynamin-related proteins are large GTPases that tubulate and pinch off membranes. In eukaryotes, each member of the dynamin-family is involved in various intra-cellular events, e.g. animal endocytosis, vesicular transport, division of organelles and cytokinesis. The Arabidopsis genome has 16 DRPs grouped into 6 subfamilies (DRP1-6). However, DRPs related to plant endocytosis have not been identified. In previous studies, DRPs in two of these subfamilies, DRP1 and DRP2, have been proposed to have roles in endocytosis. Using Total internal reflection fluorescence microscopy, we observed green fluorescent protein fusions of DRP1A and DRP2B around the plasma membrane in tobacco suspension cells. These fusion proteins were observed as dot-like puncta resembling animal dynamin signals reported previously. The behaviors of GFP-DRP1A and DRP2B puncta have several patterns resembling those reported in animal cells.

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PYK10/BGLU23 is a β-glucosidase that localized in ER bodies. Active PYK10 molecules are involved in a large complex with diameters ranging from 0.65 μm to over 70 μm. Expression levels of some members of jacalin-related lectins (JALs) and GDSL lipase-like proteins (GLLs) are lower in the nai1-1 mutant, which have no ER body, than in Col-0. The analysis of public DNA microarray dataset reveals that some of JALs and GLLs coexpress with PYK10. These results of expression analysis suggest that JALs and GLLs are good candidates of the interacting partners of PYK10. To identify members of the active PYK10 complex, the complex is purified. Some JALs and GLLs are found in the active PYK10 complex. The active PYK10 complex is larger in jal and gll mutants than in Col-0. The difference of subcellular localization of PYK10, JALs and GLLs suggests that their interactions occur when subcellular structure is destroyed.

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Cysteine synthase (CSase) forms cysteine from sulfide and O-acetylserine. In the genome of Arabidopsis, there are nine CSase-like genes (Bsas). Judging from gene expression levels and Km values, it is seemed that Bsas1;1, 2;1 and 2;2 are major CSase and Bsas3;1 is β-cyanoalanine synthase (CASase), which forms β-cyanoalanine from cysteine and cyanide, rather than CSase. In this study, to elucidate the specific role of each Bsas, T-DNA knockout mutants were isolated and analyzed for their gene expressions, enzyme activities and metabolite profiles. In all mutants, expressions of target Bsas genes were repressed, but expression levels of other Bsas were not significantly changed. In bsas1;1, CSase activity was decreased to 54% of wild-type and cysteine content was decreased. In bsas3;1, CASase activity was decreased to 36% of wild-type. These results suggested that Bsas1;1 and Bsas3;1 play the most important role for cysteine biosynthesis and β-cyanoalanine synthesis, respectively.

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Sterols serve as both bulk membrane lipid components and precursors for steroid hormones. In higher plants, cytochrome P450s (CYP710A) catalyze the sterol C-22 desaturase reaction at the last step in the sterol biosynthetic pathway. Homology searches with Arabidopsis CYP710A1 protein sequence led to the identification of EST clones containing sequence stretches derived from two putative CYP710A genes (Pp710A13 and Pp710A14) in the moss, Physcomitrella patens. The deduced primary structures of Pp710A13 and Pp710A14 proteins were 81.2 % identical and contained the unique sequence, which conserved among the sterol C-22 desaturase P450 proteins so far reported, in the helix region distal to the 5^th ligand of the heme. In Arabidopsis Pp710A overexpression lines, stigmasterol (Δ²²-sterol) levels dramatically increased, and enzyme assays using recombinant Pp710A proteins confirmed these C-22 desaturase reactions in vitro. These results indicated that CYP710A family genes are widely conserved as the sterol C-22 desaturase in plant kingdom.

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The D-mannose/L-galactose (D-Man/L-Gal) pathway plays the most important role for ascorbate biosynthesis in higher plants. Many research groups have succeeded in identifying almost all the genes encoding the enzymes in the pathway except the one catalyzing GDP-L-Gal to L-Gal-1P. In this research we set out to characterize and identify the enzyme. Crude extracts prepared from germinating pea seedlings or Arabidopsis leaves showed phosphate-dependent GDP-L-Gal breakdown activities followed by production of GDP and L-Gal-1P, indicating that the reaction was catalyzed by GDP-L-Gal phosphorylase. Because the database analysis indicated that VTC2, a gene isolated from ascorbate-deficient Arabidopsis mutants, had a conserved HIT motif for nucleotide-sugar metabolic enzymes, we suspected that it encoded the enzyme. Three vtc2 alleles showed reduced phosphorylase activities and the recombinant VTC2 protein had GDP-L-Gal phosphorylase activity. These results clearly indicate that VTC2 encodes a novel GDP-L-Gal phosphorylase.

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Caffeine is synthesized from xanthine derivatives through multiple methylation. Three N-methyltransferases, CaXMT, CaMXMT and CaDXMT, were previously identified from coffee plants, and the precursor, xanthosine was successfully converted into caffeine using recombinant enzymes in vitro. To understand the catalytic mechanism of caffeine biosynthesis, we analyzed specific properties of these enzymes. Previous study reported that sequence of a salicylic acid C-methyltransferase (SbSAMT) from Clarkia breweri was similar to that of CaMXMT, and that SbSAMT formed a homo-dimer in vitro. This suggested CaMXMT to form dimer(s), and, since sequences of isolated three N-methyltransferases showed high similarity with each other, they might form hetero-dimer(s). To examine this idea, we used the bimolecular fluorescence complementation, which visualize protein-protein interaction in vivo by YFP reconstitution by two nonfluorescent fragments. Results demonstrated that CaMXMT formed a homo-dimer in cytoplasm, and hetero-dimers with respective CaXMT and CaDXMT. The effects of hetero-dimerization on catalytic activities are under investigation.

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Green leaf volatiles (GLVs) are composed of C6-aldehydes and alcohols. They are formed rapidly after disruption of plant tissues. GLVs are formed from polyunsaturated fatty acids through oxygenation by lipoxygenase (LOX) and subsequent cleavage by fatty acid hydroperoxide lyase (HPL). It has been believed that GLVs are formed from free fatty acids, because LOX prefers free fatty acids and because lipids are degraded rapidly after tissue disruption. In this study, we analyzed compositions of lipids and their hydroperoxides after disruption of leaves of Arabidopsis that has no HPL activity. Unexpectedly, hydroperoxides of free fatty acids could be hardly detected, while, those of the esterified form could be detected. This result might indicate that lipid hydrolysis was nonessential to the formation of GLVs.

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Lithospermum erythrorhizon produces red naphthoquinone pigments called shikonin derivatives in the roots. In the cell cultures of L. erythrorhizon, stimulators of shikonin biosynthesis (Cu²⁺ and MJ) as well as its inhibitors (NH₄⁺ and light) have been identified. Shikonin is biosynthesized from geranyldiphosphate derived via mevalonate pathway and p-hydroxybenzoate via shikimate pathway, whereas the key step of naphthalene ring formation is not clarified yet.
To isolate cDNAs specifically expressed under shikonin production, we applied PCR-select cDNA subtraction technique, where dark-inducible genes were enriched. Out of 240 clones whose expressions were enhanced in the dark, we have selected a polyphenol oxidase-like protein as a candidate gene for the naphthalene-forming enzyme. Time course experiment of the northern blot showed that the gene expression completely paralleled the shikonin production pattern by various regulatory factors. We are preparing the recombinant enzyme to characterize its catalytic function.

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In Nicotinia tabacum, nicotine is a predominant alkaloid which functions as an effective defensive toxin against herbivores. Nicotine is formed by condensation of N-methylpyrrolinium cation and a nicotinic acid derivative, but the enzyme(s) involved in this final condensation step remains elusive. We here report that the previously reported gene A622, which encodes an isoflavone reductase-like protein, and NBB1, which encodes a berberine bridge enzyme-like protein, are both involved in the final condensation steps. Expressions of both genes were tightly regulated by NIC regulatory loci for nicotine biosynthesis. Both genes were specifically expressed in the root, and induced by methyl jasmonate. Suppression of either A622 or NBB1 by RNAi reduced levels of nicotine and related alkaloids in the hairy roots and BY-2 cultured cells. A622-GFP and NBB1-GFP fusion proteins were localized in the plastid and the vacuole, respectively, indicating that final steps of nicotine synthesis may occur in these organelles.

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The white petals of chrysanthemum (Chrysanthemum morifolium Ramat.) are believed to contain a factor that inhibits the accumulation of carotenoids. To find this factor, we performed PCR-Select subtraction screening and obtained a clone expressed differentially in white and yellow petals. The deduced amino acid sequence of the protein (designated CmCCD4a) encoded by the clone was highly homologous to the sequence of carotenoid cleavage dioxygenase. All the white-flowered chrysanthemum cultivars tested showed high levels of CmCCD4a transcript in their petals, whereas most of the yellow-flowered cultivars showed extremely low levels. White petals turned yellow after the RNAi construct of CmCCD4a was introduced. In addition, over-expression of CmCCD4a in yellow petals resulted in reduced level of carotenoid content. These results indicate that in white petals of chrysanthemums, carotenoids are synthesized but are subsequently degraded into colorless compounds, which results in the white color.

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Maltol (3-hydroxy-2-methyl-4-pyrone) is known one of major plant-derived aromatic substance that is widely used for spice and food additive. In order to identify accumulation patterns and physiological functions of maltol and its glycoside in plant, we chose Cercidiphyllum japonicum, a woody plant which is hyper accumulator of maltol, as an explant. Leaves of C. japonicum, i.e. green, yellow, and brown in colour, were collected from late August to early November in 2006 at Toyama Prefecture. They were extracted and purified by appropriate methods and were applied to HPLC analysis. Concentrations of endogenous amino acids and sugars in the samples were also measured. Furthermore, callus culture was established for detailed physiological analysis of maltol and its glycoside in vitro. Callus was initiated from seedling explants on a modified Murashige and Skoog medium containing 2μM TDZ. By using the callus culture system, we discussed stimulating factors for maltol accumulation.

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Characterization of anthocyanin acyl transferases (AATs) using acyl-CoA molecules as acyl-donors have been progressed and the genes encoded in their enzymes have been identified. Recently, the enzyme protein transferring aromatic moieties using 1-O-β-acylglucoses as acyl-donors, acyl glucoside dependent acyl transferase (AGDAT), were purified from petals of Clitoria ternatea and a cDNA encoded its enzyme activity was isolated. We established an anthocyanin-synthesizing cultured-cell line of carrot suspension cultures in which cells produced and accumulated cyanidin 3-[Xyl-(sinapoyl-Glc)-Gal] as a main pigment. In the crude protein extract prepared from the cells, the AGDAT activity was detected using sinapoyl-glucoside as an acyl-donor. In order to synthesize several molecular species of glucosides conjugated with phenylpropanoid derivatives, a recombinant UDP-glucose:phenylpropanoid glucosyltransferase was prepared from E. coli harboring a cDNA isolated from petals of Gomphrena globosa. Using the several species of phenylpropanoid glucosides as acyl-donors, substrate specificity of AGDAT in carrot was characterized.

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Structural changes of chromatin by modification in histone N-tails correlate with transcriptional regulation on development and morphogenesis in eukaryotes.
We are studying genome-wide transcriptional regulation by histone modification under abiotic stress in Arabidopsis. We are using T-DNA tagged kock-out mutants of genes involved in histone modification, such as methylation, acetylation and deacetylation. Drought sensitivity tests of the knock-out mutants for the histone modification-related genes showed that a mutant of histone deacetylase was sensitive to drought stress. Microarray analysis showed that the expression levels of some stress responsive genes were altered in this mutant. The identification of target sites of histone deacetylase and the analysis for enrichment of histone modification in those gene regions are in progress.

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SULTR1;2 high-affinity sulfate transporter facilitates sulfate uptake from the environment. Arabidopsis plants induce SULTR1;2 expression upon sulfur deficiency (-S), which enables the plants to survive under -S condition. To identify the cis-acting element involved in the -S-inducible expression of SULTR1;2, its promoter region was dissected by deletion and base substitution analysis in transgenic Arabidopsis using luciferase as a reporter. The results indicated that -S-responsive expression of SULTR1;2 requires the regions from -371 to -360 and -328 to -323. The -371/-360 region contained a WRKY binding sequence (TGAC), and the -328/-323 region contained a SEF4 motif (AAAAAT), respectively. SEF4 motif is originally reported as a sulfur-responsive element in the promoter sequence of β-subunit gene of β-conglycinin from soybean, suggesting the existence of a common regulatory mechanism inducing -S response between Arabidopsis and soybean.

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Nuclear-encoding sigma factors confer promoter specificity on bacterial-type RNA polymerase in plant chloroplasts. To date, 6 SIG genes encoding sigma factor have been identified in rice and Arabidopsis. To clarify functions of one of the SIG genes, SIG1, we isolated and characterized two allelic mutants of rice SIG1, in which SIG1 is disrupted by insertion of a retrotransposon, Tos17. The mutants impaired in SIG1 were fertile, however, they showed one-third reduction of chlorophyll content in mature leaves. Semi-comprehensive analysis for chloroplast genes revealed that accumulation of psaA operon in mutant heterozygotes reduced to ~30% compared to that in wild type. The reduction of PSI complex resulted in 70% reduction of light-reaction activity. These results demonstrated that SIG1 takes significant roles in maintenance of photosynthetic activity of PSI in matured chloroplasts in leaves by regulating transcription of the genes encoding photosystem I apparatus.

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In plants, the primary enzyme of carbon fixation, RuBisCO (riblose-1,5-bisphosphate carboxylase/oxygenase), is encoded as two subunits, rbcL and rbcS. It is reported that the ML tree based on the amino-acid sequence of RbcL deduced from the nucleotide sequence alignment makes two clusters; R-type (red linage) cluster and G-type (green linage) cluster. G-type RuBisCO coding genes constitute rbcL-rbcS operon, while R-type RuBisCO coding genes constitute rbcL-rbcS-cfxQ operon. It was reported that in Rhodobacter sphaeroides, cfxQ insertional mutant showed to be impaired growth under photoautotrophic conditions.These results predict that cfxQ is necessary for the expression of RuBisCO.
cDNA analysis and complete genome sequence of Cyanidioschyzon merolae 10D indicate that cfxQ is coded both in the plastid genome and in the cell nucleus. In this study, we purpose the function of two cfxQ.

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In plants, some of photosynthetic genes are encoded in the cell nucleus and others are in the plastid genome. In this study we used unicellular red alga Cyanidioschyzon merolae 10D which divides synchronously during the photoperiod and examined the accumulation pattern of transcripts of photo-genes by northern hybridization analysis. The accumulation patterns of the transcripts of photo-genes are categorized into several groups. For example, the amount of transcripts of plastid-coded psaA gradually decreased in the dark and abruptly increased in the light while those of psbA were constant andpsbEFLJ constantly increased through both dark and light conditions. The transcripts of nuclear-encoded psbO decreased abruptly decreased after dark and slightly increased. The variation patterns of the accumulation of transcripts and genes are discussed.

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The genes related to response of various stresses seem to be particularly prone to alternative splicing in higher plants. We demonstrated previously that the transcription of Arabidopsis SR protein, atSR45a, is induced by high-light irradiation. Here we studied the function of atSR45a protein involved in spliceosome assembly. Six types of mRNA variant (atSR45a-1a - e, -2) were generated from the atSR45a pre-mRNA by the alternative splicing event. The expression of atSR45a-1a and atSR45a-2 mRNAs was increased by high-light irradiation. Next, we analyzed the interaction of atSR45a-1a and atSR45a-2 proteins with other splicing factors by a yeast two-hybrid assay. The atSR45a-1a and atSR45a-2 proteins interacted with U1-70K, a key protein involved in the 5'-splice site recognition. In addition, they also interacted with themselves or each other. These results suggest that atSR45a serves as a component in constitutive and/or alternative splicing.

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Expression of the Arabidopsis CGS1 gene that codes for cystathionine γ-synthase (CGS) is feedback-regulated at the step of mRNA degradation in response to S -adenosyl-L-methionine (SAM). This regulation occurs during translation and a short stretch of amino acid sequence (MTO1 region) encoded by the first exon of CGS1 itself is involved in this regulation. Recently, we found that SAM induces translational pausing prior to the mRNA degradation.
CGS1 regulation is reproduced in a cell-free system, and several different intermediates of mRNA degradation accumulate that are truncated at their 5' region. A line of evidence suggests that SAM induces ribosome stacking and the multiple mRNA degradation points correspond to each of the stacked ribosomes. When we analyzed degradation intermediates of mRNAs containing phosphorothioate linkages, which are thought to be resistant to nucleases, intermediates identical to those of unmodified mRNA were detected. This suggests that endonucleolytic cleavages are involved in CGS1 mRNA degradation.

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Here we report a rice recessive and embryonic lethal mutant, club-shaped embryo1 (cle1), which phenotype is appeared after globular stage, and not observed during WT embryogenesis. We identified three loci are responsible for the phenotype. A positional cloning of cle1 indicated that rice Dicer-like1 (OsDCL1) is a prime candidate, because three different mutations were identified in the three alleles. Expression analysis elucidated that CLE1 specifically expressed in the basal region of WT embryo during 4-6 DAP, and the possible target ARF8 is up-regulated. The expression of OSH1, which is a marker of undifferentiated cells of a ventral presumptive shoot apical meristem region, is not restricted in the ventral region, and detected in overall the basal region in cle1 embryo. These results suggest that the loss of function of OsDCL1 might cause retaining the undifferentiated cells in the overall basal region.

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Expression patterns of some small RNAs are regulated at developmental stages or in response to environmental changes. It is quite possible that viral infection alters the expression profile of small RNAs in host plants.
This time we analyzed small RNAs by sequencing small RNAs from Arabidopsis thaliana infected or uninfected with Tobacco mosaic virus.
About 80 % of the total small RNAs in infected plants matched to Arabidopsis genome sequences; about 10 % had the sequence of a part of the viral genome both of positive and negative strand. Of the Arabidopsis-specific sequences, the abundance of some microRNAs was higher in virus-infected plants than in uninfected plants. In addition, small RNAs matching to sequences of tRNA, rRNA and snRNA were found. Curiously, some small RNAs were found specifically in virus-infected plants. It is suggested that the analysis of virus-infected plants is offering us the chance of discovering novel small RNAs.

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We have succeeded to induce RNAi against aadA mRNA that confers spectinomycin resistance in a unicellular green alga C. reinhardtii. More than 80% of aadA mRNA was degenerated by the expression of inverted repeat silencer DNA construct. To disrupt any RNAi related gene, we executed random tag insertion to the RNA induced cell. Sp far about 31,000 tag inserted cells were obtained and screened for its sensitivity change for spectinomycin. 114 showed notable decrecement. Highly likely some of their new phenotypes were result of disruption on the RNAi related genes.
Up- and down-stream genome sequences of a tag were determined by RESDA-PCR method, then based on the inform disrupted gene was deduced by Blast search against whole Chlamydomonas genome. From RNAi response enhanced tag-lines, histone, APG5 genes were identified; while from RNAi response repressed lag lines, protein kinase, ATPase, were identified.

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Small 20- to 25-nt, non-coding RNAs have emerged as important regulators of eukaryotic gene regulation by guiding mRNA degradation, DNA methylation, and modification. In Arabidopsis, about 100,000 small RNAs have been reported. However, their biological roles in the responses to drought, cold and high-salinity stresses have not been well understood.
Recently, 454 sequencing technology has become a powerful tool for the large scale analysis of the small RNAs. We applied the 454 sequencing technology for exploring the small RNAs involved in the responses to drought, cold, high salinity stresses. Large-scale analysis of the small RNAs showed that the expressions of 10 novel miRNAs are regulated by the stresses. Functional analysis of the stress-responsive miRNAs is in progress.

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In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Δ26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. Spa19 and spa23 were photosynthetic revertants and they maintained unusual heteroplasmic chloroplast genomes (PS+ and PS-). Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript. To obtain additional evidence for antisense RNA function in chloroplasts, a segment of atpB antisense RNA was expressed from an ectopic site, which resulted in an elevated accumulation of atpB mRNA. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3'->5' exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed chloroplast genome.

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Unicellular diazotrophic cyanobacteria fix N₂ during the night so as to protect nitrogenase from O₂ that is evolved by photosynthesis. However, N₂-fixation and photosynthesis of many unicellular species occur simultaneously when they were cultured under continuous illumination (CL). Changes in the abundance of nitrogenase (Fe-protein)-containing cells and the activities of nitrogenase and photosynthesis were studied during the acclimation process to CL with Gloeothece sp. 68DGA grown in a continuous culture system. Nitrogenase activity was completely suppressed in the first subjective dark phase upon transferring from 12h light/12h dark cycles (12L/12D) to CL, though about 70% cells expressed nitrogenase. Activities of nitrogenase and photosynthesis became almost constant, and more than 92% cells expressed nitrogenase constitutively after cells were proliferated about 10 generations under CL. Diurnal oscillation of nitrogenase activity recovered from the second dark phase upon transferring from CL to 12L/12D, though about 60% cells still expressed nitrogenase constitutively.

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A previous study on the morphology of zooxanthellae, symbiotic dinoflagellates living inside marine invertebrates, has revealed that two distinct morphologies and motility patterns could be discerned. The motile type has an oval shape while the immotile type has a spherical shape. The model system Symbiodinium microadriaticum CCMP 829 was used to assess the possible implication of different nitrogen sources as inducers of these different motility patterns. S. microadriaticum was cultured in different NO₃^- and NH₄⁺ concentrations and cell density and motility was estimated with a hemocytometer under a microscope. The ratio of motile to total cell number was then calculated. Results showed a proportional relationship between motility and NO₃^- concentration supplemented in the culture medium. These results, which suggest that NO₃^- is capable of inducing zooxanthellae motility, will be discussed in the light of a possible physiological change of zooxanthellae.

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Plastidic dicarboxylate transporters (DiTs) function as the interface between plastidic and cytosolic carbon- and nitrogen-metabolism in plants, because they supply the carbon skeleton 2-oxoglutalate (2OG) to the plastidic GS/GOGAT cycle and export the end product of nitrogen assimilation, glutamate (Glu), from plastids. They are also essential in the photorespiratory pathway. Structurally, it is known that two types of transporters (DiT1 and DiT2) transport 2OG and Glu via malate exchange, respectively. They both have eubacterial origin, although their functions have diversified in the plant kingdom during evolution due to their crucial role in plant metabolism. In addition to these two well-characterized DiTs, Arabidopsis encodes a third DiT (pdct2), the function of which is still unknown (Taniguchi M et al. (2002) PCP, 43: 706- 711). We will report a functional analysis of pdct2.

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Nitrate is a major nitrogen source for plants. We have identified and characterized eight nitrate transport genes (PpNRT2;1-PpNRT2;8) from the moss Physcomitrella patens, which is amenable to homologus recombination. Although its expression level is only 3-6% of total NRT2 expression during steady-state growth with nitrate, PpNRT2;5 is unique among the moss NRT2s in that it is induced quickly when protonemata is transferred from ammomium-containing medium to nitrate- or nitrite-containing medium and that it requires nitrate or nitrite for its expression. PpNRT2;5-disrupted mutant, ΔNRT2;5, grew normally on nitrate-containing medium. However, the activities of nitrate or nitrite uptake of ΔNRT2;5 was lower and was induced slower than in wild-type after transfer of ammonium-grown protonemata to media containing low concentrations of nitrate or nitrite. Our data suggest that PpNRT2;5 has an important role in the initial phase of induction of nitrate/nitrite assimilating activity.

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A facilitated nitrite transport is involved in the nitrite reduction in higher plants. We first cloned cDNA (CsNitr1-L) for a nitrite transporter of chloroplst envelope. CsNitr1-L shows K_m for nitrite of ~0.1 mM being classified as an high-affinity type. Arabidopsis mutants with only one putative nitrite transporter disrupted accumulated nitrite 20-times as much as wild type, showed a phenotype of N deficiency, however, grew with nitrate as sole N source. Nitrite uptake of the mutants was examined by incubating leaf discs with various concentrations of nitrite on illumination. High- affinity nitrite uptake was shown to be lost in the mutants. In contrast, the mutant absorbed nitrite faster than wild type from the media that contained nitrite of unusually high concentration. Accumulated nitrite may induce a low affinity nitrite uptake in Arabidopsis mutants when the plant loses the high affinity one functioning in the uptake of nitrite of physiological concentration.

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The Nitrite Transporter (CsNitr1-S) was first cloned from cucumber. CsNitr1-S is an efflux type nitrite transporter. The CsNitr1-S is located at the plasma membrane and is anticipated to transport nitrite out of the cell at the early stage of germination or de-etiolation. To better understand the function of the CsNitr1-S, transgenic tobacco lines overexpressing and suppressing the nitrite transporter were made by inserting the CsNitr1-S in the sense and antisense directions using Agrobacterium tumefaciens. In this study, nitrate reductase, nitrite reductase, chloroplast development, and intra cellular nitrite concentrations were analysed along the course of seed germination. Results have indicated that CsNitr1-S saves the seed from a toxic effect of nitrite accumulation, which can occur before chloroplast development. The relatedness of the nitrite transporter function with other phenotypes such as drought tolerance will also be discussed.

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Xanthine dehydrogenase (XDH) is a ubiquitous enzyme involved in purine catabolism. Recently, XDH is implicated in the formation of redox-signaling reactive oxygen and nitrogen species such as superoxide and nitric oxide, proposing a possible new role of this classic enzyme. To evaluate the impact of this enzyme on plant growth and development, we generated transgenic plants of Arabidopsis in which XDH expression was downregulated by RNA interference. Knockdown of XDH resulted in a retarded growth phenotype, and significantly impaired fruit development and seed fertility. In addition, XDH-suppressed plants accelerated leaf senescent process as revealed by the decrease in chlorophyll content with a concomitant activation of senescence-associated genes. These results suggest that XDH plays a vital role in plant growth at both vegetative and reproductive phases, and XDH might take part in the control of physiological processes such as leaf senescence.

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Presence of 100-200 ppb ¹⁵N-labled NO₂ in the air almost doubled the growth (biomass, total leaf area), nutrient (C, N, S, P, K, Ca, Mg) uptake and metabolism (free amino acid content) of Nicotiana plumbaginifolia (Takahashi et al., 2005) and Arabidopsis thaliana (Takahashi et al., 2006) as compared with plants grown in absence of atmospheric nitrogen oxides. Mass spectroscopic analyses showed that the contribution of N derived from NO₂ to total plant N is negligible (3-5% or less). The atmospheric NOx seems to act as a multifunctional signal. The present study investigates this plant vitalization signal effect of NO₂ on lettuce (Lactuca sativa), sunflower (Helinthus annuus), cucumber (Cucumis sativus) and pumpkin (Cucurbita moschata) to address its cross-species generality and organ specificity.

Takahashi, M. et al. New Phytol. 168: 149-154 (2005).
Takahashi, M. et al. Cost 859 Workshop, 31 August-2 Septmember 2006, Saint-Etienne, France, (2006).

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Rice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1): these are OsGS1;1, OsGS1;2 and OsGS1;3. To understand an individual role of three GS1 isoenzymes during germination, expression profiles of these genes were analyzed using real-time PCR. Localization of these gene products in situ were also performed.
GS1 protein and its activity were detected following the germination for 3 days. In contrast, GS2 was not detected. Transgenic rice expressing GUS under the control of corresponding GS1 promoter showed that OsGS1;1 genes was active in the shoots. OsGS1;2 and OsGS1;3 genes were also active in the scutellum and the aleurone layers, respectively. Individual function of each GS1 isoenzyme during gerimination will be discussed.

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In cereals, root elongation is stimulated under low nitrogen concentration. Toward isolation of regulatory genes for NH₄⁺ use efficiency, chromosome segment substitution lines developed between Koshihikari and Kasalath were employed to detect QTLs of root elongation under various concentrations of NH₄⁺. Germinated seedlings were hydroponically grown with 5, 50 or 500 μM NH₄⁺ for 7 days, and then maximum length of roots were determined. In particular, Kasalath allele on QTL in long-arm of chromosome 6 greatly promoted the root elongation under all concentrations. This allele increased nitrogen content of whole plants when grown with 50 or 500 μM NH₄⁺ not but with 5 or 1000 μM. These results suggest that the QTL gene could play an important role in controlling of NH₄⁺ use efficiency at a specific range of NH₄⁺ concentration. High-resolution mapping revealed that the target QTL was delimited in a 24.3 kb region.

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Nitrogen utilization is regulated by many factors at various steps. Root elongation is stimulated under low nitrogen concentration in rice. Preliminary study showed that a QTL controlling root length under low NH₄⁺ concentration was detected on the long arm of chromosome 8. A CSSL, SL-225, has a Kasalath segment on a long arm of chromosome 8 in Koshihikari genetic background. To characterize QTL under various NH₄⁺ concentrations, the germinated SL-225 and Koshihikari were hydroponically grown under 5, 25, 50 or 500 μM NH₄⁺ during 9 days. Only under 5 μM NH₄⁺ concentration, root length of SL-225 was significantly stimulated, when compared with that of Koshihikari. This result suggests that this QTL could play an important role in nitrogen utilization under the limited supply of NH₄⁺. Backcrossed progenies, BC₁F₃ derived SL-225, was used for the high resolution mapping. The target QTL was narrowed down to approximately 150 kb-region on chromosome 8.

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In higher plants, some of the genes involved in nitrogen metabolism, such as OsNADH-GOGAT1, are regulated by glutamine (Gln). This suggests that the occurrence of Gln-sensing and -signaling systems. To understand the molecular mechanism of Gln-sensing and -signaling systems, we have focused on ACT domain repeat proteins (ACRs) as a candidate of Gln sensor. Nine genes encoding ACR proteins (OsACR1-9) are found in rice genome. The transcripts of OsACR1 and OsACR5 to OsACR9 were detected. In leaf blades, the accumulation of OsACR9 transcript was maximal at the early stage of development. To analyze the profile of ACR9 protein in rice, an antibody specific to ACR9 was generated. Immunoblot analyses in various organs of rice showed that this protein was accumulated in both leaf blade and panicle during the life-span, with relatively a high accumulation in those organs at the early stage of development. Subcellular/cellular localization of ACR9 will be reported.

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To identify large-scale useful rice gene, we are established rice FOX lines using 13,000 non-redundant rice full length cDNAs to introduce them into Arabidopsis plants.
UV-B radiation can damage plants due to destruct several cell components and reduce gene expressions. To isolate useful rice genes involved in tolerance to UV-B stress, we try to screen UV-B tolerant mutants from rice FOX lines.
We already isolated 50 UV-B tolerant mutant candidates in T2 generation from ca. 3,900 lines by root bending assay screening. Each isolated mutant contained one or two copies of rice cDNA which may encode cell-cycle related genes, stress responsive genes, transcription factors and some unknown function genes. Now we differentiate the isolated mutants based on phenotypes of root growth with or without UV-B irradiation.
This research is supported by the Special coordination funds for Promoting Science and Technology entitled Rapid identification of useful traits using rice full-length cDNAs.

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Ultraviolet B (UV-B) radiation has pleiotropic effects on plant development. There exists two types of trichomes, globular-head ones and sharp-head ones on the surface of cucumber cotyledon. Toluidine blue O stains only sharp-head trichomes. To clarify the effect of UV-B irradiation on the accumulation of polyphenolic compounds in the sharp-head trichomes, we conducted toluidine blue O staining and detection of auto-fluorescence. In connection with the cell division at the basal part of sharp-head trichomes by UV-B irradiation, the area stained by toluidine blue O expanded. In addition, toluidine blue O stained epidermal cell walls round the sharp-head trichomes. The place auto-fluorescence was detected was coincided well with the area stained by toluidine blue O. Also, UV-B irradiation increased lignin content in cucumber cotyledons. Thus, it is possible that UV-B irradiation induces polyphenolic compounds especially stress lignins in and round the sharp-head trichomes on the surface of cucumber cotyledons.

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Rice cultivars vary widely in their UVB sensitivity and this has been correlated with CPD photolyase mutations that alter the structure/function of this photorepair enzyme. Here, in order to test whether CPD photolyase function determines the UVB sensitivity of rice, we constructed transgenic plants bearing the CPD photolyase gene of the UV-resistant rice cultivar Sasanishiki (japonica) in the sense or the antisense orientation using the Sasanishiki, UVB-sensitive Norin 1 (japonica) and Surjamkhi (indica) rice as wild-type plants. The sense transgenic plants had higher photolyase activities than the each wild-type plant, were significantly more resistant to UVB-induced growth damage. Conversely, the antisense transgenic plant had little photolyase activity, was severely damaged by elevated UVB radiation. These results strongly indicate that CPDs are one of principal cause of UVB-induced growth inhibition in rice, and that increasing CPD photolyase activity can significantly produce alleviation of UVB-caused growth inhibition.

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UVB can damage DNA by causing formation of cyclobutane pyrimidine dimer (CPD). Photoreactivation mediated by an enzyme, photolyase, is the major pathway for repairing CPD in plants. Although plant cell has different genome in each nuclei, chloroplast and mitochondria, it is unclear whether CPD photoreactivation is involved in each organelle. We previously reported that the UVB-induced CPD levels on each organelle genome decreased dependently by exposure of blue irradiation. These results suggested that CPD photolyase could function in each organelle in rice. In this study, in order to verify this suggestion, we compared the CPD photorepair capacity in each organelle among two rice strains (Sasanishiki and Surjamkhi) and CPD photolyase transgenic plants. As a result, the CPD photorepair capacity in each organelle was faster in Sasanishiki than that in Surjamkhi, and was not detected in antisense transgenic plant. Thus, CPD photolyase functions in nuclei, chloroplast and mitochondria in rice.

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Cyclobutane pyrimidine dimer (CPD) photolyase is a crucial factor for determining UVB sensitivity in plant. This enzyme binds specifically to CPDs in DNA. By absorbing blue/UVA radiation, the dimer is reversed to monomer pyrimidines and the enzyme is released. Rice CPD photolyase overexpressed in E.coli is known to be a single ca. 55-kDa protein. Up-to-date native class II CPD photolyase has been studied yet. Recently we generated highly UV-resistant Sasanishiki plant bearing the CPD photolyase gene of Sasanishiki in the sense orientation. The content of CPD photolyase in this transgenic plant is extremely higher than that in wild plant. We purified sequentially CPD photolyase from the transgenic plant with several steps of column- and affinity-chromatographies and investigated the nature of the purified enzyme. This paper shows that isoforms of CPD photolyase of two 54- and 56-kDa proteins are involved in rice.

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Thioredoxin (Trx) is a small ubiquitous protein that regulates a number of phenomena via formation or reduction of a disulphide bridge in the target enzyme. Cytosolic thioredoxin has been reported to regulate several membrane proteins that involve in the self-incompatibility and in the innate immunity of plant so far.
To elucidate target proteins of cytosolic thioredoxin in plant membranes comprehensively, we prepared a plasma membrane-enriched fraction from the suspension culture of Arabidopsis thaliana, and solubilized proteins were subjected to the thioredoxin affinity chromatography method. The captured proteins were identified by MALDI-TOF/TOF MS analysis. We present a list of potential targets for thioredoxin including the proteins, which function for the signal transduction. We here discuss the physiological meaning of the redox regulation of the membrane signaling molecules in plant cells based on the results of our thioredoxin affinity chromatography.

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