Plant cell wall biosynthesis is indispensable to cell elongation and expansion that occur in the morphogenesis. However, the molecular mechanisms of cell wall biosynthesis in the cell growth are poorly understood. To understand the mechanisms, we have been studying seven temperature sensitive elongation-deficient acw1~7 mutants that have altered cell wall structures. Previously, we reported the analysis of acw1 (3), 2, 7 mutants and demonstrated that these ACW genes are required for cellulose biosynthesis. In this study, acw4, 5 and 6 were analyzed in detail.
We analyzed the sugar composition of the mutant cell walls. The results showed that these mutations affect not only cellulose biosynthesis but also non-crystalline polysaccharide biosynthesis. We mapped these mutation loci to chr. 1 (acw4 and 6) and chr. 4 (acw5). Currently, we are progressing the complementation tests for gene isolation.

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In higher plants, APX isoenzymes play a key role in scavenging H₂O₂ and are distributed in at least five distinct cell compartments. Recently, we studied the effects of several stress conditions on the expression of spinach APX isoenzymes. The transcript level of cytosolic APX (cAPX) increased in response to photooxidative stresses, while the levels of other APX isoenzymes were not changed. These results suggest that cAPX may function to control strictly the cytosolic level of H₂O₂ under normal and stress conditions. To clarify the cAPX promoter region for potential binding site of regulatory transcription factors, we constructed chimeric genes consisting of a series of deletion of the cAPX promoter 5' region and GUS gene, and generated the transgenic Arabidopsis plants. The sequence element between -1325 and -860 of cAPX promoter seems to be essential for the high light-induced expression of the GUS gene.

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Alternative splicing in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNAs, one form (tAPX-I) encoding thylakoid-bound APX and three forms (sAPX-I, -II, -III) encoding stromal APX, were regulated by the splicing regulatory cis-element (SRE) (Yoshimura et al., 2002 J. Biol. Chem., 277, 40623-40632). Here, we investigated the possibility that the regulation of alternative splicing by SRE is a general mechanism in higher plants. The truncated 3'-terminal regions of spinach chlAPX gene placed under the control of CaMV 35S promoter were transformed to Arabidopsis which has been found to contain two different genes for chlAPX isoenzymes. All four types of alternatively spliced mRNAs derived from the transgene were detected in the transformed Arabidopsis. The deletion of SRE sequence diminished the expression efficiency of tAPX-I mRNA. These results suggest that SRE regulates the other alternative splicing events.

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We have previously cloned cDNA encoding chloroplastic CuZn-superoxide dismutase (Chl CuZn-SOD) from Spirogyra and its amino acid sequence suggests that the Spirogyra enzyme is a branching point of Chl and cytosolic (Cyt) CuZn-SOD in plants. Here, we have isolated the genomic gene from Spirogyra. The clones (8 kbp) encoding Chl CuZn-SOD were obtained by PCRs. The algal gene sequenced for 3,402 bp contains nine exons and eight introns while plant Chl CuZn-SOD genes eight and seven, respectively. The exon/intron structure of the Spirogyra gene was the same as those of the plant Chl genes except for the first intron. The first intron (191 bp) locates at 9 bp upstream from the cleavage site of Chl transit peptide, which is identical site of the first intron in 5'-UTR of the plant Cyt genes. Thus, the Spirogyra gene seems to represent an ancestor for both types of CuZn-SOD.

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We found an abundant extracellular protein in calli of halophyte Atriplex lentiformis. The deduced sequence of the protein showed high homology to that of germin, which has oxalate oxidase (OXO) and superoxide dismutase (SOD) activities. Therefore, the protein was designated as AlGLP. AlGLP showed SOD activity but not OXO activity. The AlGLP mRNA was strongly expressed in calli and weakly in roots, but not in stems or leaves. The AlGLP expression in the root was inhibited by NaCl or ABA treatment. Phytohormone treatments to the leaves led to the finding that AlGLP transcript level is strongly enhanced by methyl jasmonate. Moreover, wounding or CuSO₄ treatment also induces AlGLP expression. These inductions of AlGLP are inhibited by ABA treatment. In conclusion, we show here that the extracellular SOD is intricately regulated by various environmental stresses.

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We isolated a germin-like protein with Mn-SOD activity (BuGLP) from a moss, Barbula unguiculata. Our purpose in this study is to bring out physiological roles of BuGLP based on its SOD activity.
When NaCl was added into the cells the BuGLP mRNA levels increased, but most of BuGLP protein was released into the medium. On the other hand, the addition of methyl viologen or H₂O₂ decreased the BuGLP mRNA levels dose dependently. The addition of H₂O₂ in the presence of NaCl decreased the BuGLP mRNA levels compared with those in the presence of NaCl alone. This treatment caused the release of BuGLP protein into the medium. These results suggest that H₂O₂ generated from superoxide by BuGLP plays an important role in regulation of BuGLP expression. To clarify the mechanisms that cause these regulations, the promoter region of BuGLP gene was cloned.

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The E.coli gene katE, driven by the promoter of Rubisco small subunit gene of tomato, rbcS3C, was introduced into a tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens-mediated transformation. Catalase activity in transgenic plant was approx. 3-fold higher than that in wild-type plant. Leaf discs from transgenic plants remained green at 24h after treatment with 1 μM paraquat under moderate light intensity, while leaf discs from wild-type plants showed severe bleaching after the same treatment. In addition, ion leakage from transgenic leaf discs was significantly less than that from wild-type leaf discs at 24h after treatment with 1 μM paraquat and 10 mM H₂O₂, respectively, under moderate light intensity. The protect efficiency of the E.coli catalase in the transgenic plant was checked against different oxidative stresses. The transgenic plants showed relatively higher tolerance for paraquat, exogenous H₂O₂ and cold stress than the wild-type plants but not for salt stress

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We found that in Synechocystis PCC 6803 two glutathione peroxidase (GPX)-like proteins utilized NADPH, but not GSH, as an electron donor. To elucidate the physiological function of GPX-like proteins in S. 6803 cells, here we studied the supply of NADPH for GPX-like proteins and the effect of several stresses on the expression of two genes. The t-butyl hydroperoxide (t-BuOOH)-dependent evolutions of oxygen in wild type and mutant cells disrupted by a kan^r cartridge gene indicated that activities of Gpx-1 and Gpx-2 are coupled with NADPH supplied from the photosynthetic electron transport system. By Northern blotting, gpx-1 transcripts were expressed under high light or by the treatment of t-BuOOH and salt, while gpx-2 was up-regulated to high light and salt stress conditions; it was induced within 15 min and then decreased. Interestingly, the transcript level of gpx-2 was not changed in response to the treatment of t-BuOOH.

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Various stress treatments can cause excess active oxygen species (AOS) production. Since generation of AOS is involved in stress-induced injury, enhancement of AOS scavenging activities would confer resistance to stresses to plants. rcd1-2, which is allelic to rcd1-1 (Overmyer et al., 2000), is resistant to paraquat, UV-B and salt stresses because of the enhanced activities of the AOS scavenging enzymes in chloroplasts. On the other hand, rcd1 is more sensitive to ozone fumigation than wild type, and displays programmed cell death after ozone treatment. rcd1 appears to be a mutant that shows stress responses constitutively. Consequently, it accumulates AOS scavenging enzymes without any stresses, and displays programmed cell death even after removal of ozone. RCD1 gene should negatively regulate wide range of stress-related down-stream genes in an unstressed condition.

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Drought is one of the major abiotic stresses affecting plant productivity. Under water deficit conditions, generation and proliferation of reactive oxygen species increases and results in oxidative damage to plant cells. In contrast to this idea established with domesticated or mild environment-adapted plants, wild watermelon, a C3-plant from the Kalahari desert, Botswana, had very unique mechanisms for surviving drought. Under stress conditions, the plants accumulated large amount of a novel compatible solute, citrulline, in the leaves. Citrulline is one of the most efficient hydroxyl radical-scavengers among natural compounds examined so far. Moreover, the plants induced unique set of proteins such as DRIP-1, metallothionein and cytochrome b-561 in response to drought. Some of these factors are thought to be involved in antioxidative defense system of the plants. Molecular analysis of drought-tolerant wild watermelon provides new hints and tools to improve the stress tolerance of crop plants.

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Reactive oxygen species overproduced under various environmental stresses directly or indirectly cause various chemical modifications of plant proteins. In this study, we detected chemical modifications of proteins in stressed cucumber plants (Cucumis sativus L. suyo) by immunoblotting. Antibodies used for analysis are, anti-AGE antibody, anti-carboxymethyllysine (CML) antibody, anti-pentosidine antibody (recognizing glycation-derived modifications, Dojin), anti-malondialdehyde (MDA) antibody, anti-acrolein antibody (recognizing lipid-peroxidation-derived modifications, NOF Life Science). Oxydative modification of proteins was detected by OxyBlot Protein Oxidation kit (Invitrogen). In results, during 8h of drought stresses treatment, increase of modification by MDA was observed both in soluble and membrane protein fractions. Our previous study showed that chemical modification loses stability and function of proteins in vitro, therefore, increased chemical modification of proteins caused under environmental stresses might be involved in the functional loss of stressed plant cells.

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Peroxyacetyl nitrate (PAN), a photochemical oxidant, is one of the most toxic components among common air pollutants and causes severe injury on plant leaves. Petunia leaves are known to show a change from PAN tolerant to sensitive and back to tolerant again, depending on the maturation progress. It has been proven that superoxide accumulates before PAN injury appears, and hydrogen peroxide accumulates in injured leaves. The present study was carried out in order to clarify the role of reactive oxygen species (ROS) and antioxidative enzymes in PAN injury.
PAN injury was restricted under anaerobic conditions, confirming the participation of ROS in the development of the injury. Moreover, POX activity was not only in parallel with the age-dependent rise in tolerance against PAN, but also correlative to the difference in sensitivity between assorted varieties of petunia, suggesting the importance of the scavenging enzymes in the attainment of tolerance against PAN.

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Jasmonic acid (JA) is a cyclopentanone derivative that originates from linolenic acid via an octadecanoid pathway consisting of several enzymatic steps. JA regulates diverse processes such as wound responses, disease responses and pollen maturation in plants. A recent report suggests that a cyclopentenone precursor of JA, 12-oxo-phytodienoic acid (OPDA), can also regulate wound induction of genes. However little is known about the difference in the genes which response to JA and OPDA. We screened JA- and OPDA-responsive genes separately using cDNA macroarray covering about 9000 genes of Arabidopsis. Most of JA- and OPDA-responsive genes are induced by both treatments. However we identified genes which specifically responded to OPDA or JA. Differential effects of JA and OPDA will be discussed in the presentation.

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Jasmonates are widely distributed in the plant kingdom, and modulate wounding responses, disease responses, and anther development. Although various kinds of jasmonates were found in plants, specific roles of each jasmonate are still unclear. Using cDNA macroarray, we identified jasmonic acid (JA)-, methyl jasmonate (MeJA)-, and 12-oxo-phytodienoic acid-responsive genes. JA biosynthesis genes such as LOX2, AOS, AOC, OPR1, and OPR3 are induced by all treatments, however, these genes showed different expression profiles in each treatment. To focus on the MeJA function, we identified jasmonic acid carboxyl methyltransferase (JMT) mutant from a collection of Ds transposon insertion lines made in RIKEN. When jmt was treated by JA, the expression of LOX2 was altered, wheares other JA biosynthesis genes were not changed. In this presentation we will report cDNA macroarray analysis for jmt.

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Most of responses of plant to external stimuli occur via various plant hormones, and it has been indicated that light signal is related to cytokinin. For example, the response to cytokinin in etiolated seedling is imitative of greening response to light. Although there are a lot of individual responses of plant like this example, it is difficult that we image whole system of plant responses to external stimuli.
In this study, we measured the response of 5-day etiolated Arabidopsis to light and cytokinin used by Arabidopsis cDNA macroarray (ca. 9000 locus). In this presentation, we will try to depict the whole response image of these two stimuli. Moreover, considering that light is received by leaf and that cytokinin is mainly produced in root, we will disscuss tissue specificity of light and cytokinin responsive genes.

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Polyamines (PAs) - putrescine, spermidine and spermine (Spm) - are low molecular weight organic cations that are implicated in various physiological and developmental processes in all organisms including plants. Of these, we are interested in Spm action in plants. It was reported that tobacco mosaic virus (TMV)-infected tobacco leaves contain higher levels of Spm in the intercellular spaces. Moreover, Spm application to healthy tobacco leaves induced expression of PR genes. These findings indicate that Spm is closely related with the resistance against plant pathogens. Here we isolated Spm-responsive genes from tobacco using a differential screening method and identified HIN1 gene. Other PAs have no effect on HIN1 induction. The induction was inhibited by antioxidant treatment. These results suggest that Spm induces HIN1, a HR maker gene, via reactive oxygen species-induced signal transduction pathway.

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Ethylene was rapidly produced in sweet potato root tissue in response to infection by black rot fungus (Ceratocystis fimbriata). We have already shown that major sources of ethylene synthesis in the infected tissue may originate from a pathway independent of methionine-ACC pathway.Inhibitors of NADPH oxidase, phospholipase, lipoxygenase, hydroxyl radical and metalions significantly suppressed ethylene synthesis in sweet potato root tissue induced by infection with the fungus. Inhibition by metal ion chelators was restored by the application of cupric ion. These results suggestthat in sweet potato root tissue in response to fungal invasion, unsaturated fatty acids released from membrane phospholipids are hydroperoxidized by lipoxygenase. The resultant hydroperoxy compounds could be degraded in the presence of reactive oxygen species and cupric ion (or copper enzyme), giving rise to ethylene. De novo protein synthesis was required for ethylene synthesis to be induced in the infected tissue.

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ETO1 is a negative regulator of ethylene biosynthesis of Arabidopsis, that interacts with AtACS5. ETO1-transgenic tomato didn't show significant delay of fruit ripening, suggesting lack of sufficient interaction of ETO1 with endogenous ACSs in ripening fruit. ACSs were grouped into three types based on their C-terminal sequences. Y2H experiments showed that ETO1 failed to interact with LE-ACS2 (type 1) and LE-ACS4 (type 3), two major ACS isoforms in developing tomato fruit. On the other hand, auxin-inducible LE-ACS3 (type 2) interacted with ETO1, as well as AtACS5 (type 2). Chimeric construct between LE-ACS2 and LE-ACS3 revealed that the type 2-specific C-terminus is required for the interaction with ETO1. When treated with auxin, ETO1 transgenic seedling produced less amount of ethylene than wild type, despite induction of LE-ACS3 was comparable. These results suggest that ETO1 can regulate ethylene biosynthesis in heterologous plants and the regulation is specific for the type 2 ACSs.

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The ethylene receptors, cytokinin receptors, and phytochromes are composed of N-terminal signal-sensing domain and C-terminal signal-output domain containing histidine kinase or histidine kinase-related kinase. Some bacteria have the genes consisting of the similar domain architecture. Therefore, these plant receptors might be descendants of bacterial progenitor. However, the current phylogenetic analysis using the sequences of kinase domains provides no evidence supporting the monophyletic origins of these receptors. Several evidences suggest that the recombination between the kinase domain and signal-sensing domain has not been rare event. I propose here that these plant receptors represent mosaics of domains with different evolutionary history.

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Based on the hypothesis that phototropism is caused by an unequal distribution of blue light-induced growth inhibitors that inhibit the action of auxin on the illuminated and the shaded sides, not by auxin, some blue light-induced growth inhibitors were isolated and identified from many plant species. As the inhibitors in maize (Zea mays L.), DIMBOA and MBOA were isolated and identified by their spectral analyses. Determination of DIMBOA precursor (DIMBOA-glucoside), DIMBOA and MBOA levels during phototropic curvature and their bioassay indicated that phototropic stimulation induce the conversion of inactive DIMBOA-glucoside to the highest active DIMBOA, which plays an important role in the phototropism of maize coleoptiles, and then metabolizes into MBOA (lower activity), in the illuminated side. Recently, we found that the activity of β-glucosidase, which releases DIMBOA from DIMBOA-glucoside, was enhanced by blue illumination. In this study, we report the role of β-glucosidase on phototropism of maize coleoptiles.

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We demonstrated the search for bioactive substances involved in flower bud formation from Arabidopsis thaliana. A. thaliana grown under short day were transfered to long day or kept under short day, and then, they were extracted with methanol. When compared HPLC chromatograms of each extract, clear decreasing peaks were detected in the long day-treated sample. For identifying them, large amounts of A. thaliana were extracted with methanol. The extract was purified by HPLC,and the isolated sample was determined as monogalactopyranosyl diacylglycerol (MGDG). Whether or not this substance causes flower bud formation of A. thaliana was determined. The substance induced flower bud formation of A. thaliana, which was treated to long day for 1day just before onset of its application. These results suggest MGDG as precursors or substrates of flower bud-forming substances. We will also report two new glycolipids, Arabidopsides A and B from flower forming A. thaliana.

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Tomato is a host plant of Orobanche species, the root parasite weeds. We found that roots of hydroponically-grown tomato produce abundant Orobanche minor seed germination stimulants. Reversed phase HPLC analysis of the root exudates revealed the presence of biologically active fractions, Ly1 (fast moving) and Ly2 (slow moving), the latter being a major fraction. Further HPLC of Ly1 on a phenyl column separated three stimulants Ly1-a, Ly1-b and Ly1-c which seem to be novel strigolactones. The same HPLC of Ly2 gave rise to Ly2-a and Ly2-b, the latter having a major biological activity. Ly2-b constitutes a wide peak with biological activity. GC/MS analysis of the Ly2-b resulted in the identification of four novel strigolactones (compounds A,B,C,D). The compound A had a molecular weight of 342, whereas the compounds B and C, D had a molecular weight of 344. These molecular weights were smaller than those of strigol and orobanchol (346).

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Progesterone is an animal steroid hormone that functions as a corpus luteum hormone, which maintains pregnancy and implantation of fertilized egg. We identified progesterone by using GC-MS in various plants such as Arabidopsis, rice, tomato, pea and apple. Among these tissues, pea shoots contained the highest level of progesterone, about 10 mg/kg fw and rice shoots contained the next highest level of progesterone, about 1.5μg/kg. Thus it was found that progesterone is a ubiquitous constituent and that the level of progesterone is similar to that of other plant hormones. Furthermore, we isolated three rice genes (OsPB1, OsPB2, OsPB3) analogous to human progesterone-binding protein genes. These genes encoded proteins that have a putative steroid-binding domain. OsPB2 and OsPB3 are expressed highly throughout the rice plant ( leaf blade, leaf sheath and spike), suggesting that OsPBs, as well as progesterone, may have physiological functions.

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PSK is a secreted peptide originally found in plant cell culture. This peptide induces the plant cell proliferation and/or differentiation in vitro by means of specific receptors. As a first step of the functional analysis of PSK genes consists of five paralogs, we introduced PSK promotor::GUS constructs in Arabidopsis and analyzed their expression pattern. In Arabidopsis seedlings, GUS expression was observed mainly at vascular bundle in the leaves and stele in roots. PSK should have some functions in these region. We also analyzed their expression pattern in excised leaf disk.. GUS expression was detected around the wound region after 12 h of leaf excision. In the presence of auxin and cytokinin, callus formation was observed only in the GUS positive region, indicating the involvement of PSK in cellular dedifferentiation and restructure of tissue as a wound response.

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Apical dominance is released by excision of apical buds or by auxin transport inhibitors to apical buds or internodes of seedlings. Recently, we demonstrated that application of natural auxin-inhibiting substances, raphanusanin B and 6-methoxy-2-benzoxazolinone (MBOA) not only to apical buds or internodes but also directly to lateral buds of pea (Pisum sativumL.) seedlings released apical dominance, suggesting that lateral bud growth inhibitor(s) were concerned with apical dominance. Therefore, search for the inhibitor(s) led to the isolation of indole-3-aldehyde from the pea seedlings. Endogenous indole-3-aldehyde content was higher in diffusates from explants with apical bud and IAA-treated decapitated explants than in those from decapitated ones. When this compound was applied to the cut surface of etiolated, decapitated plants or directly to the lateral buds, it inhibited outgrowth in both assays. These results suggest that indole-3-aldehyde plays an important role in apical dominance of Pisum sativum.

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Roseobacter denitrificcans has a NO reductase (NOR) homologue that has cytochrome c-oxidase activity. The enzyme is composed of two subunits homologous to NorC and NorB of NOR from other bacterial species. They are encoded in an operon similar to nor gene clusters of Paracoccus denitrificans or Rhodobacter sphaeroides. However, the NOR homologue from R. denitrificans contains copper, while NOR has iron as the non-heme metal in the catalitic center. The purified enzyme has virtually no NOR activity. R. denitrificans also has aa₃-type and cbb₃-type cytochrome oxidases. Inhibition pattern by KCN of the purified NOR homologue and of the membrane fraction differed significantly, indicating that the contribution of NOR homologue to the total cytochrome oxidase activity is small. Results of Northern hybridization and SDS-PAGE showed that the abundance of both mRNA and the protein of NOR homologue increase in light or anaerobic conditions.

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Previous works from our laboratory indicated relatively high hydrogen evolution capacity in the heterocystous cyanobacterium, Anabaena variabilis IAM M58. It has an uptake hydrogenase encoded by hupSL. Bidirectional hydrogenase genes (hox) have also been cloned and sequenced in our laboratory. In this work we report cloning and sequencing of uptake hydrogenase genes hupS and L by PCR strategy. The nucleotide sequence showed high homology to reported sequences of cyanobacteria and bacteria. Deduced amino acid sequences of hupS (897 bases) and hupL (1545 bases) showed 84.6% and 82.2% identities with those of Anabaena sp. strain PCC7120, and 90.6% and 87.3% with Nostoc sp. PCC73102, respectively. However, 99bp intergenic region between hupS and hupL, making hairpin loop structures is significantly shorter than that of Nostoc PCC73102(192bp). These unique characteristics coupled with high hydrogen production capacity make this strain a good candidate for genetic manipulation of hydrogenase genes for improvement of hydrogen production.

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Cyanobacteria have two types of hydrogenase, uptake hydrogenase (Hup) and bidirectional hydrogenase (Hox). We studied distribution of hydrogenase genes and activities among 15 nitrogen-fixing cyanobacteria and found that all of them have hup. Twelve of them also have hox, but 3 of them lack it. Although Nostoc PCC7422 has a hox gene cluster, Hox activity of the cell extracts was found to be very low. We have cloned and sequenced the complete hox gene cluster from the cosmid library. The hox genes in Nostoc PCC7422 are organized as hoxEFUY-ORF-hoxH with only one intervening ORF. While in other strains several ORFs interrupt the hox genes. The strain has hoxE, the observation being the second to the report of Anabaena PCC7120 among 4 nitrogen-fixing cyanobacteria for which hox gene clusters have been sequenced. We have also sequenced hupSL that show high homologies with those of other cyanobacteria.

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Some cyanobacteria produce H₂ as a byproduct of N₂ fixation. We created three hydrogenase mutants from Anabaena PCC 7120 (ΔhupL, ΔhoxH, ΔhupL/ΔhoxH), and showed that the ΔhupL produced H₂ at a rate 4-7 times that of wild-type. The efficiency of light energy conversion to H₂ by the ΔhupL was 1.0-1.6% at not exceeding 50 W/m² (PAR). However, its highest activity lasts for only about 10 hours. It seems that accumulation of combined nitrogen in cells leads to decreases in nitrogenase and consequently H₂ production activities, indicating a need for further improvement.
The active site of nitrogenase contains MoFe₇S₉ to which homocitrate is coordinated. Homocitrate is synthesized by NifV and it was reported in Klebsiella pneumoniae that the nitrogenase of nifV mutant catalyzes the reduction of protons more preferably than N₂ compared with that of wild-type. We have created ΔnifV mutants by gene disruptions from Anabaena PCC7120 wild-type and the ΔhupL.

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Heterocyst is a terminally differentiated cell, which is specialized in dinitrogen fixation. Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 is triggered by deprivation of combined nitrogen in the medium. Although various genes that are upregulated during the heterocyst differentiation have been reported, most studies to date were limited to individual or a small number of genes. Here we report on the genome-wide expression analysis of the response to nitrogen deprivation. Expression pattern of genes upregulated by nitrogen deprivation is classified into three clusters, and it is indicated that extensive changes in gene expression occurred at the eighth hour of nitrogen deprivation. We also noted that many of the upregulated genes were physically clustered to form expressed islands on the chromosome. This suggests that a mechanism of global regulation of gene expression that involves chromosomal structure exists, which is reminiscent of eukaryotic chromatin remodeling.

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Previously we found that the promoter activities of PSI genes were clearly regulated in response to changes in light intensity in Synechocystis sp. PCC 6803. To identify factors involved in the transcriptional regulation of PSI genes, we screened for mutants with aberrant psaA promoter activities by introducing transposon-mutagenized cosmid library into the strain harboring PpsaA-luxAB chimeric construct. To date, 49 mutagenized cosmids covering the half of the whole genome have been assayed, and we isolated 10 transposon-inserted mutants showing high luminescence under low light. Among them, mutants of slr0609, sll1531 and slr0551 actually showed higher psaA transcript levels than the control strain under low light. psaD and psaF genes were also up-regulated, whereas accumulation of psbA transcript was not affected in these mutants. Now, we characterize the phenotype of the specific targeted inactivation mutants of these ORFs.

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In cyanobacteria, several genes that encode phytochrome-like protein have been identified. However their physiological functions are largely unknown. We investigated global changes in mRNA abundance in phytochrome mutants by DNA microarray analysis, which provides cue to identify physiological role of cyanobacterial phytochromes.
The following phytochrome-like genes of Synechocystis sp. PCC6803 were disrupted by insertional inactivation: sll0821, sll1124, sll1473, slr0473, slr1212, slr1393, slr1805 and slr1969. Labeled cDNA probes were synthesized from total RNA of mutant cells grown under continuous light. Transcript of cpcG gene, which encodes phycobilisome rod-core linker polypeptide, was reduced by less than 5% of the wild-type strain in sll1473 mutant. Furthermore distinct patterns of gene expression were observed in other phytochrome mutants to a lesser extent.

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We previously identified a protein that bound specifically to the promoter region of the groESL gene in Synechococcus sp. PCC 7002 and termed it Shl-1. The amino acid sequence contains a Nudix motif, which is specific to a family of Nudix hydrolase that hydrolyzes nucleoside diphosphate derivatives. Shl-1, which was overexpressed in E. coli and purified, hydrolyzed ADP-ribose exclusively among various nucleoside diphosphate derivatives, such as NADH, ATP and GTP. Since free ADP-ribose, a major metabolic product of NAD⁺, is harmful for the cell, Shl-1 might control its level. We disrupted the shl-1 gene in Synechococcus to examine its role as a Nudix hydrolase in vivo and also to clarify whether Shl-1 might regulate the expression of the groESL gene. A homologous gene with undefined functions is present in the genome of Synechocystis sp. PCC 6803. The product of this gene also hydrolyzed ADP-ribose.

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In Rhodobacter sphaeroides f. sp. denitrificans IL106, the DmsS/DmsR two-component signal transduction system regulates the expression of dmsCBA operon encoding DMSO(dimethylsulfoxide)respiration system. DmsS has no putative membrane-spanning hydrophobic amino acid regions, although other sensor histidine kinases generally contain transmembrane regions.
To analyze membrane-binding region of DmsS dmsS-lacZ fusion genes were constructed. Each strain had the LacZ activity, suggesting that each LacZ protein was located in the cytoplasm. The LacZ fusion proteins were recovered in the cytoplasmic and membrane fractions, suggesting that the fusion proteins binded loosely to the membrane from the cytoplasmic side. Although DMSO induces the expression of DmsA protein under anaerobic conditions, no change of dmsS promoter activity was observed by DMSO, suggesting that DMSO is not a transcription signal of dmsS.

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A transcriptional regulation of the Pst system, an ABC Phosphate Transport System, of the Denitrifying Phototroph Rhodobacter sphaeroides f. sp. denitrificans was studied. The Pst system consisted of six genes similar to pstSCAB and phoUB, and its upstream sequence contained a putative regulatory sequence that is analogous to the Pho box involved in phosphate-limitation-induced gene expression in Escherichia coli. Both the amount of the PstS protein and the pstS promoter-driven expression of lacZ activity increased about 2-fold in response to phosphate limitation. Northern blot analysis of the Pst locus showed two different bands, which corresponded to pstS and pstCAB mRNA, suggesting that pstS and pstCAB transcribed by different promoters. The upstream sequence of pstC and phoU had no apparent Pho box, suggesting that the transcriptional regulation of pstCAB and phoUB is not regulated by pho regulon.

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Cyanobacteria contain the carboxysome, which functions as an element of the inorganic-carbon concentrating mechanism (CCM). It has been reported that carboxysome-mutant requires High CO₂. In this study, we investigated whether the increased exogenous Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) may cause the higher photosynthetic affinity for dissolved inorganic carbon (DIC) in High CO₂ requiring mutant.
Carboxysomeless-mutant (CL) was selected as High CO₂requiring mutant. CL has three features as follows: the requisite of High CO₂, the low photosynthetic affinities for DIC, the growth inhibition under 0.5% CO₂. In order to increase the exogenous RuBisCO in CL, 6803psbAII promoter and Chromatium vinosum RuBisCO were selected for constructing the expression vector. In CL transformed expression vector (CL-AX), RuBisCO activity and photosynthetic rate increased about 5 times and 2.5 times respectively. In addition, the recovery of growth inhibition under 0.5% CO₂ was observed in CL-AX.

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I present an approach to compare the genome contents of cyanobacteria, photosynthetic bacteria, non-photosynthetic bacteria, and eukaryotes by whole-genome clustering using the homology group method. Organellar genomes were also included as part of eukaryotic genomes. The clustering was done according to different E-values given by the BLASTP program as a threshold. I found about 70 homology groups that are shared by Arabidopsis and cyanobacteria and/or photosynthetic bacteria but not by non-photosynthetic organisms. The genes in a half of these groups are related to photosynthesis. Many Arabidopsis genes in the remaining half groups possess a putative transit sequence and are candidates for novel photosynthesis-related genes. A phylogenetic relationship was inferred by the presence or absence of homology groups, but this was different from the phylogeny based on 16S rRNA sequences in some important aspects.

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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 phycobilisomes are present in the form of cylindrical elements on the membrane. The 16S rDNA sequence shows that it is branched at the base of the cyanobacterial phylogenetic tree. Gloeobacter could have preservations of the independent generic characters from all other cyanobacteria in the course of evolution. With the objective to obtain basic knowledge on the genetic system of Gloeobacter, we determined the nucleotide sequence of the entire genome. The genome consisted of a circular chromosome of 4,659,019 bp long and no plasmid was detected. As of Dec. 2002, the prediction of all the gene components of this organism is in progress.

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In 2002, the entire genome sequence of thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 was determined. This strain has become a new model organism for genetic engineering and biochemical studies of photosynthesis, because this strain is transformable and protein complexes such as PSII are stable enough for their isolation and crystallization. In this study, we report an improved procedure of transformation that includes antibiotic screening with top agar. Further, we found that T. elongatus BP-1 was naturally transformable with exogenous DNA, and the efficiency was relatively high through a log phase. Based on genome analysis data, tll2230, was presumed to encode type I restriction endonuclease. Disruption of tll2230 enhanced slightly the transformation. We also observed a single-crossover recombination at high frequency in electroporation but hardly any of it in natural transformation.

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Phenylalanine ammonia-lyase (PAL) acts as the key enzyme in regulating metabolic flow from the primary metabolism to phenylpropanoid metabolism, and the genes encoding PAL are regulated by both environmental cues. It has been revealed that the expression of gDcPAL1 gene in suspension-cultured carrot cells is induced by dilution effect, elicitation and UV-B irradiation, and that L1- and L5- boxes in the gDcPAL1 promoter play an important role to the regulation of gDcPAL1 gene expression. We isolated a cDNA for the transcription regulatory factor being highly similar to MYB protein, which binds to L1-box or L5-box using yeast one-hybrid system. We will report the features of DNA-binding activity and specificity of this MYB protein in vivo and in vitro.

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The PAL gene (gDcPAL3) plays the most important role in the induction of anthocyanin synthesis in carrot suspension cultured cells regulated by 2,4-D. Deletion analysis of the gDcPAL3 promoter indicated that the promoter region between -258 and -194 were required to give full activity. This region contained the GCC sequence, which has been known as the cis-element of the ERF family having the plant specific DNA binding domain, AP2/ERF domain. We isolate two AP2/ERF transcription factors, DcERF1, DcERF2, that interact with the GCC sequence of the gDcPAL3 promoter using yeast one-hybrid system from two cDNA libraries, one of which was prepared from carrot cells of the normal line induced for anthocyanin synthesis in the medium lacking 2,4-D, and the other was done from the cells of the mutant line in which anthocyanin was continuously synthesized in the medium containing 2,4-D. Binding activity and expression patterns of them will be reported.

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We analyzed picotee petals composed of colored and white tissues in order to understand activation/inactivation mechanism of the flavonoids biosynthesis in Eustoma grandiflorum. As for the tip-colored variety, flavonoids were accumulated only in the tip tissue and not accumulated in the white tissue throughout the petal growth. As for the base-colored variety, flavonoids were accumulated in the whole tissue in early stage of petal growth and decreased only in the white tissue with the petal growth progress. Furthermore chalcone synthase (CHS) gene transcription was specifically inactivated in the white tissues of opened petals in both varieties. CHS expressions should be responsible for picotee color formation of E. grandiflorum. The flavonoids accumulation profiles suggest that different mechanisms of CHS gene expression might operate between the tip-colored and the base-colored varieties.

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We established a system in which anthocyanin synthesis of regenerated torenia shoots, which derived from the leaf discs in vitro, was induced on the medium containing high concentration of sucrose. In this system, ABA could also induce anthocyanin synthesis on the medium containing low concentration of sucrose. Changes in the amount of endogenous ABA in the regenerated shoots inducing for the anthocyanin synthesis on the medium containing high concentration of sucrose were determined. It is revealed that the amount of endogenous ABA transiently increased just before the induction of anthocyanin synthesis, suggesting that change in the amounts of endogenous ABA may play an important role in the induction of anthocyanin synthesis in regenerated torenia shoots.

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Although a major cue inducing anthocyanin synthesis is light, other factors are necessary to accumulate large amount of anthocyanin. Sugar accumulation enhances anthocyanin synthesis in flower, but there is restricted information about sugar-inducible anthocyanin biosynthesis in vegetative tissues. We developed an anthocyanin induction system by sugar using detached hypocotyls of radish and investigated difference in anthocyanin production between species. Sucrose (175 mM) was the strongest inducer of anthocyanin synthesis in red radish hypocotyls. Glucose and fructose also induced the synthesis, whereas mannose and 3-O-methylglucose did not. Expression of anthocyanin biosynthetic genes (CHS, CHI, F3H, DFR, and ANS) was promoted by sucrose in red radish. The promotion, however, was suppressed in white radish. These results suggest that a portion of signal transduction in anthocyanin induction by sugar may be inactivated in the white radish.

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In higher plants, anthocyanins are the primary red pigments in flower color whereas in most families of the Caryophyllales, betacyanins have replaced anthocyanins as red pigments in flowers and fruits. These two types of red pigment, anthocyanins and betacyanins, never occur together in higher plants. Many kinds of flavonoid compounds other than anthocyanins have been found in Caryophyllales. This suggests that some late stage in anthocyanin biosynthesis might be blocked in Caryophyllales.
We have isolated DFRcDNA from Spinacia Oleraciae by RACE. The cloned spinach DNA contained a full open reading frame of 1026 bp encoding 342 amino acids. The deduced amino acid sequence of spinach DFR was compared with other published DFR amino acid sequences of Arabidopsis, grape, snapdragon, Perilla, rice, carnation, identities of 62-79% being found. The amino acid sequence of spinach DFR exhibited highest identity with those of DFR from carnation, a member of Caryophyllales.

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By carbon-ion irradiation, two independent transparent testa 19 (tt19) mutants were induced. Genetic analysis revealed that tt19 possessed a single recessive mutation on chromosome 5. The characteristics of tt19 mutant are a great reduction of anthocyanin pigments in vegetative parts and of brown pigments in seed coat caused by the oxidation of proanthocyanidins. Map based-cloning enabled us to isolate a single gene, annotated as a glutathione S-transferase-like gene. Disruption of TT19 gene was no effect on the expression of other flavonoid structural genes. Naringenin feeding did not cause anthocyanin pigmentation in tt19, indicating that TT19 could work at any of the anthocyanin biosynthetic steps after the naringenin formation. Vanillin staining illustrated the difference between the wild type and the mutant for the accumulation pattern of proanthocyanidin precursors in immature seed coat. These results suggest that TT19 is involved in relatively last step of both anthocyanin and proanthocyanidin pathway in Arabidopsis.

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Perilla frutescens var. crispa is an interesting model to study the regulation of anthocyanin biosynthesis. Two chemo-varietal forms of Perilla are known, red and green cultivars, which differ in the accumulation of anthocyanins. Only the red cultivar expresses all genes required for the biosynthesis of anthocyanin pigments. Since anthocyanin biosynthesis is highly controlled at the transcriptional level, it is assumed that the change in colour in the two Perilla cultivars is due to the mutation or differential expression of regulatory genes.
A differential diplay reverse transcriptase-mediated (DDRT) PCR-based strategy was employed to identify myb-related genes involved in the regulation of anthocyanin biosynthesis in Perilla. Using this approach we isolated MYBC05, a putative MYB transcription factor which is co-ordinately regulated with the structural genes in a red form-specific manner. The functional analysis of MYBC05 is in progress, and the results in this regard will be presented.

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The non-autonomous Tpn1-related transposons belonging to the CACTA superfamily are the most common spontaneous mutagens in the Japanese morning glory. We have characterized eight spontaneous mutants having Tpn1-related transposons inserted into the ANS gene encording anthocyanidin synthase for anthocyanin biosynthesis. One of them displaying white flowers with variegated red spots and sectors carries 6.4 kb Tpn9 in a unique ANS intron, and the ANS transcripts terminate within Tpn9. The flower variegation is due to somatic excision of Tpn9 from the ANS gene. The seven remaining mutants contain 6.6 kb Tpn8 at the same site of the promoter of the ANS gene, and the Tpn8 insertion suppresses the expression of the ANS gene. The flower pigmentation phenotypes of these mutants vary form snow white to palely colored, and one of them blooms white flowers with marginally pigmented. We will discuss the flower pigmentation of these mutants.

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While the wild type common morning glory (Ipomoea purpurea) displays purple flowers and black seeds, two mutant lines showing white seeds and palely pigmented flowers were found to produce drastically reduced amounts of the transcripts of the IPMYC3 gene, a homologue of the an1 gene for the bHLH transcriptional factor that regulates flower and seed pigmentation in petunia. The genomic sequences revealed that both mutants carry two insertions of transposable elements into the IPMYC3 gene which comprises 8 exons. In one mutant, two copies of Tip100 belonging to the Ac/Ds family are inserted into the second intron and the seventh exon. The other mutant contains two different Mu-related transposable elements in the second exon and the fifth intron. Moreover, the latter mutant appears to carry an additional mutation for flower pigmentation.

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Hydrangea flower color varies very easily responding to the environmental conditions. To clarify the in vivo mechanism of the blue color development of hydrangea sepals we measured the vacuolar pH and analyzed the organic and inorganic components of colored cells. The mixture of blue and colorless protoplasts, prepared from the blue sepal by the reported procedure, was separated by micro-manipulation to give pure blue protoplasts. By using a micro ODS column (0.3 mm φ) and micro-HPLC system we could quantitatively analyzed the composition of anthocyanin and co-pigments in a single blue cell. We also succeeded in measurement of Al-content by using 50 protoplasts. Delphinidin 3-sambubioside (2) was isolated from blue sepals with major anthocyanin, delphinidin 3-glucoside (1). 2 with 5-caffeoyl quinic acid 3 and Al³⁺ in acetate buffer (pH 4) gave bluer solution than the mixture of 1, 3 and Al³⁺.

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