In Cyanobacteria, the amount of photosystem (PS) I complex decreases under high-light conditions. Although the decrease of PSI content is mainly achieved by the coordinate down-regulation of promoter activities of PSI genes, the mechanism by which PSI promoter activities are modulated is totally unknown. In this study, we investigated promoter architecture of psaAB encoding reaction center subunits of PSI. To identify cis-regulatory elements, various fragments of psaAB upstream region were fused to luxAB reporter gene, and luminescence levels from each construct were compared. It was found that psaAB has two light-responsive promoters (P1, P2), and each promoter possesses positive and negative cis-elements that do not confer the light-response. DNA mobility shift assay showed that each promoter binds different proteins. Now we are trying to identify light-responsive elements by introducing base substitutions to both promoter regions.

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Pirin is a well-conserved protein among various organisms. It is involved in the process of apoptosis in human and tomato, whereas roles of its bacterial counterparts are totally unknown. We found that sll1773 encoding a Pirin homolog and ssl3389 encoding a putative transcriptional regulator are co-transcribed under stressed conditions in a cyanobacterium Synechocystis sp. PCC 6803. We named sll1773 and ssl3389, pirA and pirB, respectively. slr1871 encoding a LysR family transcriptional regulator located immediately upstream of pirAB in the divergent direction. Expression of pirAB was derepressed by disruption of slr1871 under non-stressed conditions, indicating that this gene works as a repressor of pirAB. Therefore, we named slr1871, pirR. Further investigation on the relationship between pirAB and pirR revealed that complicated interaction was observed between three genes in response to the changes in environmental conditions.

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Gloeobacter violaceus PCC 7421 is a unicellular cyanobacterium containing distinctive characteristics. This strain lacks thylakoids, and the light-harvesting and photosynthetic apparatuses are associated with the cytoplasmic membrane. The 16S rDNA sequence shows that it is branched at the base of the cyanobacterial phylogenetic tree. These features support the idea that Gloeobacter holds ancestral characters for oxygenic photosynthesis. To reveal the genetic background responsible for characteristics of Gloeobacter and evolution of oxygenic photosynthesis, we determined the nucleotide sequence of the entire genome. The genome consisted of a circular chromosome of 4,659,019 bp long with an average GC content of 62%. The genome comprises 4430 potential protein-encoding genes. Forty-one percent of the genes showed sequence similarity to genes of known function. Comparison of the assigned gene components with those of other cyanobacteria has unveiled distinctive features that reflect the phylogenetic distance between them.

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We investigated the kinetics and function of the redox-components in reaction center of Hb. modesticuldum at cryogenic temperature by ESR spectroscopy. The core complexes of Hb. modesticuldum were illuminated at 230 K and cooled down to 14 K under illumination. We detected assymmetrical ESR signal with g = 2.0060 and 12 G line-width. This signal was assigned to the menasemiquinone radical analogous to A₁^- in PS I and proved that menasemiquinone exists in the core complexes. Under the same condition we also observed A/E/A/E (A: absorption E: emission) spin polarized signals by transient ESR at early times after flash. On the other hand, E/A/E pattern of polarized signals was observed in the dark frozen samples. We will discuss these results in comparison with PS I.

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The reaction center (RC) complex of green sulfur bacteria consists of four subunits, PscA (core homodimer), PscB (F_A/F_B protein), PscC (cytochrome c_Z) and PscD subunit (a 17-kDa polypeptides of unknown function). To ascertain the function of PscD, we constructed a mutant lacking the PscD subunit in Chlorobium tepidum. This is the first-time achievement of the direct mutagenesis in the RC complex of green sulfur bacteria.
Kinetics of the fluorescence showed that the bacteriochlorophyll a fluorescence in the mutant decayed slower compared to that in the wild type. This result suggested that the efficiency of energy transfer from the FMO protein to the RC decreased in the mutant because of the lack of PscD. Our interpretation could be supported by the previous study of the three-dimensional imaging analysis of the Chlorobium RC complex which speculated the direct contact of PscD with the FMO protein.

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Photosynthetic Reaction center(RC) was purified from an acidophilic purple bacterium Acidiphilium rubrum that uses Zn-bacteriochlorophyll a as a major pigment. We measured femtosecond fluorescence kinetics by fluorescence upconversion to determine the electron transfer time from Zn-BChl dimer(P) to BPhe(H) in the purified RC. Accesary Zn-BChl(B) was excited by Ti-Safire laser at 815 nm of 180 fs width. The rise and decay of fluorescence from P were 160 fs and 3.3ps, respectively. The former represents the energy transfer from B to P, and the latter the electron transfer from P to H. The two time constants are almost equal to those in RC of R.sphaeroides that has typical Mg-BChl a type RC. Angles of dipole moment between B and P estimated by fluorescence depolarization was 32 degree. Energy transfer from LH1 to RC was estimated to be 42 ps by the fl photon counting.

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Previously, we cloned a repressor, SPB, for pufoperon under aerobic or semiaerobic light condition from Rhodobacter sphaeroides. In this study, we identified a novel protein, SIP which interacts with SPB, and cloned the corresponding gene. Amino acid sequence showed SIP had 35% identity to SPB and contained a DNA binding domain. Previously we suggested SPB included a region homologous to the leucine-zipper motif and it might contribute to the formation of a dimer. Immunoblot analysis showed SIP protein was expressed constitutively under all conditions tested, although SPB-SIP interaction took place under aerobic condition. Thus, our present results suggest SIP and SPB might form a heterodimer and bind to puf promoter. Furthermore, these results indicate SIP-SPB interaction is not regulated by SIP or SPB expression but by modification of SIP or SPB and do not contradict our previous hypothesis that transcription of puf operon is regulated by phosphorylation/dephosphorylation of SPB.

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We have been investigating the transport mechanism of berberine alkaloid using Coptis japonica (Ranunculaceae), as a model plant. In C. japonica, berberine is biosynthesized in root, but is highly accumulated in the rhizome as the main alkaloid, suggesting that berberine is translocated from the root into the rhizome. We have recently reported that an MDR type ABC protein, CjMDR1, functions as a berberine influx transporter in the rhizome. To further clarify the physiological function of CjMDR1 in planta, we produced transgenic C. japonica plants with modified expression of Cjmdr1. In fact, the introduction of 35S promoter drived Cjmdr1 sense construct produced a transgenic plant (Sense-2) with reduced expression of Cjmdr1. Since this transgenic C. japonica plant showed stable reduction of Cjmdr1 expression, we are now characterizing the effect of Cjmdr1 expression on the accumulation of berberine as well as the expression of biosynthetic genes.

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Nicotine, a major alkaloid in tobacco plants, is produced in tobacco roots and transported to the vacuole of leaves for storage. A low nicotine mutant (nic1nic2) is defective in nicotine biosynthesis and accumulation very low levels of nicotine. By differential display technique, we found that two highly homologous genes (NtMATE1, NtMATE2) are expressed at very low levels in nic1nic2 compared to wild type. Function of MATE-type transporters is largely unknown in plants. We speculate that NtMATE1/2 participate in nicotine transport, because expression of these genes is coordinately regulated with bona fide nicotine biosynthesis genes. We are currently investigating the intercellular localization of NtMATE1/2 and their nicotine transport activity using NtMATE1-expressing cultured BY-2 cells. We also plan to analyze accumulation of nicotine in the NtMATE1-suppressed and -overexpressed transgenic tobacco plants.

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Tobacco has served as a useful material in plant physiology and biochemistry. Despite these experimental advantages, molecular and genomic tools have not been well established in this plant. Here, we report construction of partially normalized cDNA library from root and leaf tissue of a diploid tobacco species, Nicotiana sylvestris, and preparation of cDNA micro-array chips. Single-pass sequences were obtained from a total of approximately 13,000 cDNA clones and were assembled into approximately 6,500 non-redundant cDNA groups. Database searches on nuclear sequences of Nicotiana species in Genbank revealed that these cDNA groups represent approximately 3,500 unique tobacco genes. The ESTs are used to prepare cDNA micro-array chips. We are analyzing gene expression patterns in relation to nicotine biosynthesis using these chips.

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Purine alkaloids producing species accumulate caffeine (1,3,7-trimethylxanthine) or theobromine (3,7-dimethylxanthine) as a major compound. Caffeine biosynthetic pathway contains three S-adenosylmethionine(SAM)-dependent methylation steps. The available data support the operation of 7-methylxanthine to caffeine via theobromine by a bifunctional caffeine synthase which catalayses the last two steps (N-1- and N-3 methylation) in caffeine-accumulated species. In this study, we focused on two N-methyltransferases. One was caffeine synthase (TCS1) from Camellia sinensis and the other was theobromine synthase (PCS1) from Camellia ptilophylla, which was theobromine-accumulated species. TCS1 was 91% identical to PCS1. We produced the recombinant enzyme introduced to the artificial modification in amino acid sequence with E. coli expression system to clarify the region related to the substrate specificity. The residues from 148 to 287 out of 369 amino acids in TCS1 determined the substrate specificity, in particular, position 221 arginine was contributed to the specificity.

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Anthocyanin is widely distributed in higher plants and in tissue cell it is accumulated in central vacuoles. However, detailed mechanism of compartmentalization of anthocyanin is still unclear. Using red radish (Raphanus sativus cv. Pekin mizudaikon), which accumulates anthocyanins in taproots, we have studied on the transport system of anthocyanins from the cytoplasm to vacuoles.
First, we tried to determine the structure of anthocyanins in red radish. Anthocyanins were extracted with 0.5% TFA- 50% CH₃CNaq. from cambium and parenchyma tissues of taproots. The HPLC analysis revealed that the composition of anthocyanins was different between the cambium and parenchyma tissues. We isolated seven anthocyanins and four of them were new pigments. All of them had a pelargonidin nucleus, and contained feruloyl, p-coumaroyl and/or malonyl residues. Major anthocyanins in the cambium were higher glucosylated and acylated than those in parenchyma. We will discuss on the structural characteristics and physiological significance of taproots anthocyanin.

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Flower pigments, anthocyanins, are localized in vacuoles of colored epidermal cells. The concentration of pigments in the vacuole is ca. 10^-2 M, and the anthocyanins are stabilized to develop beautiful colors by various molecular stackings. We are studying the mechanisms of in vivo flower color development. To investigate molecular stackings of anthocyanins in vacuoles, we measured circular dichroism (CD) of living petals and colored cells. The pigment of blue flower of Nemophila menziesii was a metalloanthocyanin composed with anthocyanins, co-pigments, and metal ions, nemophilin. The complex composed from Mg²⁺ showed purple color, but that of Mg²⁺ and Fe³⁺ showed blue color. Visible absorption spectra and CD of the fresh blue petal and colored protoplasts were measured. Both spectra were the same as the complex composed with Mg²⁺ and Fe³⁺. The CD showed the characteristic exciton-type negative Cotton effect in the visible region, indicating the chiral molecular stacking of anthocyanidin chromophores.

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Ternatins, polyacylated anthocyanins accumulated in blue petal of butterfly pea, are delphinidin 3-(6''-malonyl)glucoside-3',5'-diglucoside (ternatin C5) derivatives. Independently purified 3'- and 5'-glucosyltransferase (3'GT and 5'GT) were characterized to be similar in biochemical properties. We isolated the cDNA, CtBGT1, based on the internal amino acid sequences of the purified 3'GT. The CtBGT1 encodes a polypeptide of 447 amino acids with a molecular mass of 48,649. The sequence indicates that CtBGT1 belongs to the UF3GT family. Surprisingly, the CtBGT1 sequence shows only 8% identity to the functionally related gentian 3'GT (AB076697). The recombinant CtBGT1 expressed in E. coli was found to catalyze the sequential glucosylations at 3'- and 5'-OH groups of delphinidin, resulting in the formation of ternatin C5. Although CtBGT1 express in mauve line petals accumulating delphinidin 3-(6''-malonyl)glucoside, it contains a stop codon in its ORF. These results proposed that novel UDP-glucose:anthocyanin 3',5'-glucosyltransferase encoded by CtBGT1 is involved in ternatin biosynthesis.

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In order to modify the blue flower color in cultivated gentian, we first attempted to introduce an antisense construct targeted for chs. Successful production of transgenic plants with loss of the flower color enable us to promote genetic modification in gentians. Here we applied RNAi method to reduce the chs expression. A binary vector with chs inverted repeat under the control of the gentian chs promoter was constructed and used to study potential utility of RNAi in gentian. Compared with antisense strategy, RNAi-inducing construct changed the flower color more frequently, but its suppression was not enough and in most cases caused faint blue flowers. When the individuals with flower colors ranging from white to faint blue were analyzed, the relationship between accumulation of anthocyanin and suppression of flower color was observed and northern analyses indicated the flower color-dependent reduction of the endogenous chs.

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We describe here the genes relating to formation of white and pink color flowers in the cultivated genitans. Expression analyses of the flavonoid biosynthesis-related genes showed that one of the two white-colored cultivars was deficient in the gene for ANS and this was confirmed by complementation in transient expression assay by particle bombardment. The other one was considered to be derived from the mutation in the regulatory trans-acting factor(s) for flavonoid biosynthesis by the expression analysis of the petals with the spontaneous pigmentation under cold-stressed conditions. Namely, the accumulations of the CHS,F3H,F3'H and ANS transcripts were induced and those of CHI and F3'5'H were reduced. Furthermore, sequence analysis of the genomic F3'5'H gene in pink-colored cultivar revealed the pink mutation was caused by an insertion in the gene itself. Details on this insertion are now being studied.

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We analyzed genes expressing in carnation petals to understand the metabolism in the petals and obtain the genes encoding 2',4,4',6'-tetrahydroxy chalcone 2'-glucosyltransferase (THC2'GT). THC2'GT stabilizes the yellow tetrahydroxychalcone and thus the gene encoding THC2'GT should be a useful molecular tool to engineer yellow flowers.
EST analysis of 2,600 clones, sequencing of 3,700 clones of a subtracted cDNA library (petals-leaves) and screening of a petal cDNA library with previously cloned glucosyltransferase cDNAs yielded more than 30 kinds of putative glucosyltransferase cDNAs. Arabidopsis is known to have about 100 glucosyltransferase homologues in the genome. Carnation may have more than Arabidopsis. Functional analysis of these cDNAs is interesting and in progress in our laboratory.
(A part of this study was supported by the Bio-oriented Technology Research Advancement Institution, Japan.)

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We cloned 35 glucosyltransferase (GT) cDNAs from the yellow petals of carnation (Dianthus caryophyllus cv. light cream candle), and expressed them in E.coli. Four clones (T170, T128, CGT93, S12A2) of them encode the UDP-glucose: 2',4,4',6'-tetrahydroxychalcone 2'-glucosyltransferase (THC2'GT) activity that is recognized as critical to yellow flower coloration of carnation, cyclamen and periwinkle. Overexpression of T170 and T128 in transgenic petunia plants successfully generated THC 2'-glucoside (THC 2'-G) in the petals. Their flower color did not change probably due to the low levels of THC 2'-G accumulation.
We also cloned T170 homologous genes from cyclamen (Cyclamen persicum) and periwinkle (Catharanthus roseus). Their recombinant proteins synthesized in E.coli exhibited THC2'GT activity.
(A part of this study was supported by the Bio-oriented Technology Research Advancement Institution, Japan.)

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A green alga Chlamydomonas reinhardtii has a comparable light-harvesting machineries to vascular plants including chlorophyll a/b-binding outer antennae which is composed of the major and minor complexes, and chlorophyll a-binding core antennae. However, it shows very high growth rates under high light conditions (2000μE/m²/s ), where most of vascular plants can not survive. This is probably due to the effective NPQ induction observed in the high-light acclimated cells since the levels of photoinhibition and NPQ are well correlated with the growth light intensities.In this study, we study the mechanism for the high NPQ induction in the high-light acclimated C. reinhardtii. Physiological properties of the wild-type algal cells grown under high light conditions were firstly examined. The outer antenna fractions were then isolated and biochemically characterized. The obtained results will be discussed in relation to the algal NPQ mechanism and the structure of the high-light acclimated LHCII.

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The light-harvesting complexes for PSII are generally divided into three categories; The core antennae (CP47/CP43), the minor antennae (monomeric LHCIIs), and the major antennae (trimeric LHCIIs). While the minor antennae have been suggested to have some role on quenching excessive light energy rather than harvesting it because of its high content of the xanthophyll cycle pigments, the Arabidopsis antisense plants of either CP26 or CP29 were reported to show no phenotype. Therefore, it is still controversial. In this study, we suppressed expression of one of the minor antennae CP29 in C. reinhardtii by RNAi technology. Transformants of the RNAi construct was first confirmed to have very low level of Lhcb4 mRNA as well as CP29 protein. Then, effects of CP29 suppression on the physiology are examined. Based on the obtained results, the role of CP29 on the algal physiology will be discussed.

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Photosynthetic pigments are bound to their specific proteins to form pigment-protein complexes that enable the efficient energy transfer between different pigments. Light-harvesting apparatus can be divided into the core and peripheral antenna. Core antenna is composed of chlorophyll a-protein complexes and their pigment composition and organization do not change under any environmental conditions. Peripheral antenna is composed of light-harvesting chlorophyll a/b-protein complexes (LHC) in green plants. However, the mechanism concerning the preferential incorporation of chlorophyll b into LHC have not been revealed.
In order to elucidate the mechanism, we introduced prochlorothrix CAO into Arabidopsis thaliana and investigated the pigment compositions of the complexes. The core antenna of chlorophyll a-protein complexes became chlorophyll a/b-protein complexes in the transgenic plants. We will report the photosynthetic performance of the transgenic plants and discuss the regulation of chlorophyll distribution and the photosynthetic performance of the transgenic plants.

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Chlorophyll a and b are synthesized by at least 17 different enzymes in land plants. Among the genes encoding these enzymes, only that for divinyl-protochlorophyllide reductase has not been identified. On the other hand, Chlorophylls are degraded by at least 7 different enzymes, among which three have not been identified. In order to identify these genes, we developed an HPLC-based method to screen for Arabidopsis mutants that have defects in chlorophyll metabolism. We grew approximately 2000 EMS-mutagenized M2 seedling on vermiculite for 3 weeks and analyzed their pigment compositions by HPLC. Then, we transferred the plants in darkness for 4 days and compared their pigment compositions to those before dark treatment.
We isolated several mutants that accumulate or lack some intermediate molecules of the chlorophyll metabolism, including one that accumulate divinyl-chlorophylls.

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Oxygenation of pheophorbide a is a key step in chlorophyll breakdown. Several biochemical studies have implicated that this step was catalyzed by an iron-containing and ferredoxin-dependent monooxygenase, pheophorbide a oxygenase (PaO). It has been proposed that inhibition of its activity leads to the "stay-green" phenotype. We searched the Arabidopsis genome for a possible PaO encoding gene and identified three such genes,Tic55,ACD1 and ACD1-like. We produced transgenic Arabidopsis plants which expressed antisense RNA as a method to inhibit the expression of these genes. After they were kept under darkness for 5 days and again illuminated, the leaves of the antisense ACD1 plants (AsACD1) were bleached. The rate of decrease in chlorophyll a was not influenced and pheophorbide a was accumulated in senesced AsACD1 leaves. These results demonstrated that ACD1 encodes PaO, and its inhibition led to photooxidative destruction of the cell instead of the "stay-green" phenotype.

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Chlorophyll a is synthesized from glutamic acid by many steps. Most of the genes and enzymes responsible for chlorophyll biosynthesis have been identified and well characterized. However, the gene for divinyl protochlorophyllide 8-vinyl reductase (DVP reductase), which converts divinyl protochlorophyllide a to monovinyl protochlorophyllide a, has not been identified.
In order to identify the gene for DVP reducatase, we isolated the mutant of Arabidopsis thaliana in which monovinyl chlorophylls are replaced by divinyl chlorohylls. This mutant is supposed to have a mutation in the gene for DVP reductase.
The mutants can grow photoautotrophically, but the growth rate was slow compared to wild type Columbia. The mutant has high divinyl chlorophyll a/b ratio and exhibits pale green phenotype. The PAM experiments showed that maximum photosynthetic electron transfer rates and Fv/Fm ratios were decreased in the mutant.

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Cyclic electron flow around PSΙ is essential to induce of NPQ by recycling electrons from reduced ferredoxin or NADPH to plastoquinone. PSΙ cyclic electron flow consists of two redundant pathways depending on FQR and NDH activities. Arabidopsis mutants lacking FQR activity, pgr5, and lacking NDH activity, crr mutants, were isolated and characterized. To screen mutants affected in alternative electron flow functioning with these pathways, we isolated mutants displaying the high chlorophyll fluorescence under CO₂-free, 5%-O₂ condition. In CE46-48 mutant, NPQ induction and ΦPSΙΙ were lowered than in the wild type under CO₂-free, 5%-O₂ condition. While NPQ induction was impaired during the induction phase of photosynthesis, it was rather normal during steady steate. CE46-48 may be defective in the alternative electron flow that are independent from FQR or NDH. We are investigating the impact on the activity of O₂ reduction by PSΙ.

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Chloroplast gene expression has been shown to be regulated at transcriptional level and also at various post-transcriptional steps, including RNA stabilization, RNA processing, RNA splicing and translation. We isolated Arabidopsis thaliana mutants pgr3 (proton gradient regulation 3) .The accumulation of Cytocrome b₆f complex was affected in pgr3-1 and pgr3-2 but not in pgr3-3, while that of the NDH complex was in pgr3-1 and pgr3-3 but not in pgr3-2. PGR3 contains of 27 PPR (Pentatrico-Peptide Repeat) motifs. PPR motifs are present in proteins functioning in the post-transcriptional regulation of organellar gene expression.Actually,pgr3 defects impaired the stabilization of petL operon RNA and possibly also translation of petL and one of ndh genes. We determined the localization of PGR3 in the thylakoid membrane.These results suggest that PGR3 is involved in different steps of chloroplast RNA maturation for distinct target RNAs in the thylakoid membrane.

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It is not clear at the molecular level how cyanobacteria acclimate the photosynthetic antenna, phycobilisomes, to variable light conditions. Recently we showed that in Synechocystis the transcript level of a rod-core linker gene, cpcG2, was regulated by a unique two-component system comprising a phytochrome-like histidine kinase and OmpR-type response regulator. Synechocystis has two paralogous cpcG genes (cpcG1 and cpcG2) in the genome. We created their disruptants, and analyzed assembly of the phycobilisome through sucrose density gradient ultracentrifugation. We found that the phycobilisome in the cpcG2 mutant almost fully assembled, while in the cpcG1 mutant phycobiliproteins partially assembled to form several subcomplexes. In the double mutant assembly was more suppressed than in the single cpcG1 mutant. These results suggest that CpcG2 plays an auxiliary but regulatory role in the assembly of phycobilisome. Further investigation will be done to elucidate the role of CpcG2 in acclimation to light conditions.

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We reported that three polypeptides in the thylakoid membranes of chloroplasts from green spores of the fern Osmunda japonica decreased during spore germination. The 24-kDa protein was tightly bound with thylakoid membranes. We have attempted to determine a gene of the 24-kDa protein by PCR using the information of amino-terminal sequences of proteolytic products. A computer search of the protein database revealed similar features between the 24-kDa protein and ELIPs (early light-induced proteins) homologs of Tortula ruralis and Onoclea sensibilis.

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Regulation of photosynthetic electron transport was studied in intact leaves on the basis of the low-wave phenomenon, i.e. the transient drop of yield of modulated chlorophyll fluorescence shortly after application of a pulse of saturating light. Measurements of chlorophyll fluorescence, CO₂ uptake and absorption at 830 nm of leaves showed that low-waves of chlorophyll fluorescence are induced by increased non-photochemical quenching as a result of induction of cyclic electron flow around photosystem I (PSI). It was suggested from observations of low-waves that cyclic PSI plays a role in the regulation of the rate of photosynthetic electron transport by increasing thermal dissipation of excess excitation energy in photosystem II. PSI cyclic flow, however, was not induced under the conditions where incident light intensity was so high as to fully saturate photosynthesis (e.g. at 1,100 μmol photon m^-2s^-1 in normal air).

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The OsRac1, a small GTPase, functions as a regulator of the production of reactive oxygen species in rice defense response. To know the OsRac1 gene expression pattern, we introduced OsRac1promoter::GFP into rice. The OsRac1::GFP expression was observed in vascular tissue of rachillae, lemma, palea, collar, lateral root primordia, tiller bud and mesocotyle. To examine the reactive oxygen production and the occurrence of cell death in these organs, the DAB and the Evans blue staining in roots were performed. A part of the epidermis and the cortex of parent root and the primordia of lateral root were stained by DAB and the basal region of lateral root in parent root was stained by Evans blue. The lateral root development was inhibited by the treatment with DPI, an inhibitor of NADPH oxidase. These results suggest that the reactive oxygen production by OsRac1 would be required for the root development.

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To elucidate the mechanisms of establishment and maintenance of shoot apical meristem (SAM), we analyzed the function of SHOOTLESS2 (SHL2) and SHOOT ORGANIZATION1 (SHO1) genes in rice. shl2 and sho1 mutants are defective in the formation of functional SAM during embryogenesis. shl2 mutant is defective in the formation of SAM. The SAM in sho1 is flat shaped and has fewer indeterminate cells than wild type. The formation of this defective SAM is thought to cause the abnormal morphologies of leaves and much shorter plastochron in sho1.
Positional clonings of SHL2 and SHO1 genes revealed that both genes encode the components of the machinery of RNA interference (RNAi). Recently, it has been reported that RNAi related mechanism is used to regulate the development through the production of endogenous small RNA called microRNA (miRNA). In this presentation, we will report the roles of SHL2 and SHO1 in the production of miRNA.

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Dicotyledonous plants acquire the bilateral symmetry in addition to the previous radial symmetry at the transition stage as two cotyledon primordia are developing from the apical region of the embryo. Our previous studies show that PIN-FORMED1 (PIN1), which encodes a putative auxin efflux carrier, is essential for the establishment of the bilateral symmetry and that pinoid (pid) enhances the abnormality of pin1 mutation. To isolate new genes involved in the establishment of bilateral symmetry, we screened for mutant loci causing the defects of symmetrical cotyledon formation in the background of pid mutation. We identified three loci except for PIN1 gene and found a point mutation of gene involved in the energy metabolism in one of three mutants. We will present the molecular analyses on these mutants and discuss on the relationship among the loci, auxin, and bilateral symmetry.

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In Arabidopsis, root epidermal cells differentiate either into hair cells or into hairless cells. One of the R3-type Myb gene, CAPRICE (CPC) is a positive regulator of the hair-cell differentiation and CPC is preferentially transcribed in hairless cells. By the analyses of the CPC promoter, we identified the cis-acting region which is responsible for the hairless-cell-specific transcription. This region contains two putative Myb protein binding sites (pMBS). When the second pMBS was mutated, hairless-cell specific transcription was not observed. R2R3-type Myb protein, WEREWOLF (WER) is considered to be a positive regulator of the hairless-cell differentiation. We showed that WER could bind to the pMBS of the CPC promoter by yeast one-hybrid analysis and gel shift assay. We will discuss about the regulatory system of the CPC transcription and the epidermal-cell differentiation.

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Intercellular communication is a crucial process to build the multicellular organism such as higher plant. Cell-to-cell movement of macromolecule has been thought to play an important role in this communication.
CPC:GFP fusion protein can move from cell-to-cell in epidermal cells in Arabidopsis root. Two motifs in CPC, one in the N-terminal region and the other in the Myb domain, are required for cell-to-cell movement. Amino acid substitution experiment indicated that both W76 and M78 are critical for cell-to-cell movement of CPC.
To elucidate the molecular machinery for cell-to-cell movement of CPC protein, we isolated the CPC-interacting proteins by yeast two-hybrid screening. One clone encodes the novel protein which has a D/E/K rich domain. This clone did not interact with bait containing W76A mutation, suggesting that this protein was involved in cell-to-cell movement of CPC.

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CAPRICE (CPC) encodes a small protein with R3 MYB motif and regulates epidermal cell differentiation in Arabidopsis. We identified three CPC-like genes (CPL-1, 2, and -3) in the Arabidopsis genome. Overexpressers of these three genes showed a reduction of the number of trichomes and an increase in the number of root hairs, which is similar to the effect of 35S::CPC. Analyses of promoter-GUS and in situ hybridization indicated that CPL2 and CPL3 were preferentially expressed in stomata guard cells, whereas CPL1 was expressed in trichomes and root epidermal cells.
WERWOLF (WER) encodes R2-R3 type Myb protein, repressing the root-hair formation. To understand the difference between activation and repression by Myb transcription factors in plants, we made the chimera construct of CPC and WER and transformed then into wer-1 or cpc-2. The results showed CPC R3 and WER R3 motifs were not interchangeable with each other.

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Efficient induction of gain-of-function mutations is essential for functional assignment of genes whose loss-of-function mutations do not cause obvious phenotypic defects. In order to identify genes responsible for root morphogenesis, we are establishing a new activation tagging system that utilizes a yeast transcriptional activator GAL4 and its recognition sequence (UAS). In this system, a T-DNA containing five copies of 17-mer UAS is randomly inserted into the genome of GAL4:VP16-expressing Arabidopsis lines established by Jim Haseloff and coworkers. The simple transcriptional activation by GAL4:VP16-UAS interaction is expected to efficiently induce ectopic expression of tagged genes in a pattern defined by the GAL4:VP16 expression. Since the host lines also harbor a GFP reporter placed under the UAS, plants defective in root patterning can be easily screened in the primary transformants based on altered GFP expression. TAIL-PCR analysis of isolated mutants has identified several genes to which no biological functions have been assigned.

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Recent genetic analyses of the symbiotic processes in legume revealed that several steps are common for rhizobial nodulation and arbuscular mycorrhization. A symbiotic mutant of Lotus japonicus, Ljsym71, is the non-nodulating mutant. Nod factor could not induce calcium spiking and internal hyphae of arbuscular fungi could not penetrate into cortex. We have named the causative factor as TRINITY (TRI), after the fact of a tripartite relationship among Legume-Rhizobia-Arbuscular mycorrhiza symbioses. We constructed BAC/TAC contig around TRIlocus. Genetic markers within the contig were developed and used for delimiting the locus to approx. 155kb. Among 4 candidate genes, we have identified TRI, which is a novel gene and does not include any domain with known function. We have isolated 14 alleles of tri induced by EMS and somaclonal mutation. The latter mutation showed 100b-2kb deletion in TRI locus, indicated that the region is a hot spot of heterozygous deletion.

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We have isolated the TRINITY (TRI) gene from a symbiotic mutant Ljsym71 that controls the symbioses of rhizobia and mycorrhiza. The predicted amino acid sequence has revealed that TRI has a plastid-transit peptide and four transmembrane domains, and is homologous to potassium transport proteins. TRI is presumed to have a critical role in the signal transduction pathway because symbiotic responses of Ljsym71 are completely repressed in the early stage of nodulation. Southern hybridization analysis has shown the existence of a TRI homologue, and we have successfully isolated this gene, Sister of TRINITY (SOT). Because SOT also has a plastid-transit peptide and was assumed to present with TRI, the relationship of symbiosis and interaction with TRI are expected. TRI homologs are widely distributed in leguminous and non-leguminous plants. This fact indicates that the function of TRI plays an important role in not only symbiosis, but also general intracellular events.

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Legume plants establish symbiosis with rhizobia and arbuscular mycorrhizal fungi in root. In this study, we have analyzed Ljsym82, a symbiotic mutant of Lotus japonicus acc. Gifu. When rhizobia were infected with Ljsym82, this mutant formed many bumps, but failed to develop mature nodules. Infection thread growth was arrested in the root hair. When arbuscular mycorrhizal fungi were infected, their internal hyphae could invade in cortical cells, but early senescence of arbuscules was induced. By mapping analysis, it was suggested that this gene was located at the linkage group 2. After the locus was narrowed to roughly 20 kbp, Mutation point was identified by sequence analysis of candidate genes. Putative protein consisted of 518 amino acid residues. In this mutant, nonsense mutation was occurred at 107th amino acid. This gene did not possess any known domain.

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Infection threads are important for symbiotic nitrogen fixation of legumes such that the structure is a pathway of rhizobia infected from rhizosphere to plant cells. With the aim of identifying the factors involved in infection thread development molecular-genetically, we have isolated and analyzed symbiotic mutants of Lotus japonicus that show defect in the development. We have identified 5 complementation groups of B-129 background, and carried out fine mapping using MG-20 as a crossing partner. Of these, Ljsym74 and Ljsym80 were mapped in LG1, Ljsym82 and Ljsym84 in LG2, and Ljsym79 in LG5. Mapping of Ljsym74 locus was further investigated using B-303 as a crossing partner, because the locus was initially mapped at the co-segregated 0cM region between B-129 and MG-20 due to the translocation event. By phenotype analysis, those mutants were categorized in two aspects of infection thread behavior; point of abortion and structure.

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Glutamate decarboxylase (GAD) is an enzyme that catalyzes the conversion of glutamate to γ-aminobutyric acid (GABA). We previously reported that rice has two distinct isoforms of GAD (i.e., OsGAD1 and OsGAD2). The former carries a C-terminal calmodulin-binding domain (CaMBD) that is common in counterparts from dicotyledonous plants, but the latter does not (Akama et al., 2001).
In order to explore a role of the C-terminal peptide of these two GAD isoforms more in detail, the genes that lack the C-terminal were introduced in E.coli expression vector pET32a to thus purify recombinant GAD proteins (i.e., OsGAD1Δand OsGAD2Δ) with a nickel chelate affinity chromatography. An in vitro enzyme analysis with these recombinant proteins as well as wild-type GAD proteins indicated that OsGAD1Δ possesses a high and constant activity, irrespective of the presence of Ca²⁺/CaM and OsGAD2Δ displays five times higher activity than wild-type OsGAD2 protein.

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Enzymes responsible for catabolism of glutathione are not clear in higher plants. In mammals and microbes, γ-glutamyltransferase (GGT) catalyzes the transfer or the hydrolysis of γ-glutamyl moiety of γ-glutamyl-compounds. In several plants, GGT activities were detected both in the soluble fraction (s-GGT) and in the fractions solubilized from the pellet with NaCl (b-GGT). We examined the localization of GGTs in radish plants and concluded that s-GGT is present in the soluble fraction and b-GGT is localized to the cell wall. 4 isozymes (2 s-GGTs and 2 b-GGTs) were purified from radish cotyledons. Enzymatic properties were similar among s-GGTs and as so among b-GGTs, but differed between s-GGTs and b-GGTs. We cloned 2 radish GGT cDNAs and analysed their expression. The transgenic tobacco plants overexpressing these genes were produced. GGT activities were detected in the bound fraction and properties of the recombinant enzymes were similar to those of radish b-GGTs.

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It has been demonstrated that bisphenol A (BPA), an endocrine disrupter with estrogenic properties, is metabolized to BPA-ο- β-D-glucopyranoside in the tobacco seedlings (Plant Cell Physiol. 43: 1036-1042, 2002). This reaction may be catalyzed by BPA specific glucosyltransferase (BGT). In this study, we isolated 3 types of cDNAs encoding BGT by RT-PCR method with RNA of tobacco suspension cultured cells (Nicotiana tabacum L. cv. BY-2). Nucleotide sequence of the cDNAs were completely identical to those of NtGT1a , NtGT10a and NtSAGT (DDBJ Ac. No.AB052557, U32643, AF190634), respectively. Extracts of E. coli cells harboring these cDNAs had BGT activity, indicating that they encode BGT. Transcripts for NtGT10a and NtSAGT were high amount in both 4 weeks-old tobacco (Nicotiana tabacum L. cv. Xanthi NC ) leaves and root. But transcript for NtGT1a was only detected in the leaves. Our results indicate that BGT constitutes a multi-gene family.

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ADP-glucose is glucose donor for srarch synthesis. We identified and purified the enzyme that hydrolyzes ADP-glucose to AMP and G1P from the shoot tissues of germinating rice seeds. The molecular size of purified enzyme was estimated to be 285 kDa polypeptide composed of 70 kDa subunits. The NPP cDNA clone was isolated by using the amino acid sequence information determined by Edman sequencing and nESI-MS/MS analysis. The substrate specificity and the amino acid sequence of the enzyme revealed that it belongs to a widely distributed group of plant nucleotide pyrophosphatase (NPP; EC 3.6.1.9). Immunocytochemical analysis using electron microscopy with anti-NPP antibodies showed that NPP localizes in chloroplasts and cell wall in green leaves. Furthermore, the transient expression analysis of NPP-GFP fusion protein supported that it was transported to the plastid. These results strong suggested that NPP is involved in the regulation of starch biosynthesis.

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Isoamylase (ISA) is known to play an essential role in amylopectin biosynthesis. Recently, Hussain et al. (2003) characterized three ISA isoforms from potato tuber, and showed that all higher plants have three distinct ISA isoforms encoded by three gene families.
cDNAs of OsISA1, OsISA2 and OsISA3 ORF were identified by RT-PCR. Their predicted amino acid sequences showed high homologies to the corresponding ISA families of higher plants. OsISA1 was mainly expressed in endosperm, while OsISA3 was mainly expressed in leaves. OsISA2 was considered to be inactive on its own, but present in both tissue. The ISA preparation purified from rice endosperm contained only a single OsISA1 band, whereas that from potato tuber contained ISA1 and ISA2 bands. These results suggest that OsISA1 is a homo-oligomeric enzyme whereas potato ISA is a multimeric enzyme composed of ISA1 and ISA2.

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A diaphorase protein purified from Bacillus subtilis was found to be the yumC gene product from N-terminal amino acid sequencing. YumC is a homo-dimer of 94kDa with one molecule of FAD per subunit. YumC supports diaphorase activity with higher affinity to NADPH than NADH, low NADPH oxidase activity and ferredoxin oxidoreductase activity. YumC shows high amino acid sequence identity to the novel type FNR from green sulfur bacterium Chlorobium tepidum. A BLAST search with YumC as a query sequence revealed that there are more than 30 genes in prokaryotes coding for similar proteins variously annotated as thoredoxin reductase, NAD(P)H oxidase, etc. These genes are present notably in Gram-positive bacteria except for clostridia, and less frequently in Archaea and proteobacteria. We propose that YumC together with C. tepidum FNR constitute a new group of FNR, which should be added to already established plant type, bacteria type, and mitochondria type FNR groups.

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Protein modification caused by polyunsaturated fatty acid peroxidation products was examined by Western blotting using monoclonal antibodies raised against major lipd peroxidation products. Reaction mixture contained model protein (BSA or Rubisco), C18 unsaturated fatty acid methyl ester (methyl oleate (18:1), methyl linoleate (18:2) or methyl linolenate (18:3)) in the presence of a metal-catalyzed oxidation system (Fe(III)/ascorbate/O₂). In results, the most effective protein modifier was methyl linolenate which caused protein adducts derived from peroxidation products such as malondialdehyde (OHCCH₂CHO), acrolein (CH₂:CHCHO) and crotonaldehyde (CH₃CH:CHCHO). The monoclonal antibodies reacted with several proteins in cucumber leaves, and internal amino acid sequences analysis showed that they were chloroplast-localizing proteins. Therefore chloroplasts, which contain most abundant linolenate as a membrane component among plant organellar, may be a major site occurring protein modification by polyunsaturated fatty acid peroxidation.

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Fatty acid alpha-oxygenase, which is one of the oxylipin metabolism enzymes, produces fatty acid alpha-hydroperoxide by insertion of one oxygen molecule to the alpha-position of free fatty acids. It is known that the fatty acid alpha-oxygenases in dicotyledonous plants such as Arabidopsis and tobacco are induced by pathogen infection and oxidative stress. However, the physiological function of fatty acid alpha-oxygenase is still unclear. In this study, in order to elucidate the physiological function of fatty acid alpha-oxygenase, the expression profile of the enzyme was investigated with rice. Although the fatty acid alpha-oxygenases in dicotyledonous plants were mainly induced via the salicylic acid pathway, that in rice was markedly increased only by the jasmonic acid pathway but not by the salicylic acid pathway. In rice seedlings of 9 days after germination, fatty acid alpha-oxygenase was induced by oxidative stress caused by methyl viologen and by heavy metal-ion stress.

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Hemolysin-like proteins (HLP) exist not only in pathogenic but also in non-pathogenic bacteria. Although these proteins seem to have a physiological function other than hemolysis, it has not been clarified. In a previous study, we purified HLP from Synechocystis sp. PCC6803 and showed that it underwent a large conformational change from 90 kDa to >200 kDa by heat treatment in the presence of SDS. In the present study, we found that when the HLP was heated in the absence of SDS and then supplemented with calcium ion, the mobility returned to 90 kDa. This fact suggests that the release/binding of calcium ion induces the conformational change of the protein. In immuno-electron microscopy, abundance of HLP molecules existed in the surface layer. We expect that the conformational change of the protein is related to the motility of the bacterium. We will discuss this hypothesis in the presentation.

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We previously found that selenium is essential for growth of coccolithophorids (Haptophyte algae). Selenium is also an essential element for animals as a constituent of selenoproteins. In the present study, we identified the selenoprotein in a coccolithophorid, Emiliania huxleyi, and tried to elucidate the physiological function of selenium in this alga. Six kinds of selenoproteins (SEP1 to 6) were found in E. huxleyi cells. We analyzed the cDNA-sequence of SEP2 by RT-PCR using degenerate primers. A BLAST analysis of the deduced amino acid sequence revealed homology to protein disulfide isomerase (PDI), especially around the thioredoxin domain. SEP2 cDNA contained in frame TGA codon, that codes selenocysteine, at the position corresponding to cysteine residue at the reaction center of PDI in other organisms. These results suggest that, in a coccolithophorid, selenium may be specially participated in folding and assembling pathways of proteins as a component of PDI.

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