To elucidate the physiological functions of glutathione in plants, we tried to identify glutathionylated proteins in the suspension-cultured Arabidopsis cell [PCP 44: 655]. One of the proteins proved chloroplastic fructose 1, 6-bisphosphate aldolase (FBA), which had four cysteine residues. To examine the target cysteine residue of glutathionylation and its relation to FBA activity, we generated four recombinant mutant FBAs in which one of the four cysteine residues was replaced with alanine. GSH inhibited the activities of wild-type and three mutant FBAs, but had no influence on that of the remaining mutant FBA. This indicates that FBA activity was regulated by the binding of GSH to the specific cysteine residue. On the other hand, GSSG did not inhibit the activities of wild-type and four mutant FBAs, implying that the GSH-dependent regulation of FBA activity is not a simple redox regulation of the cysteine residue. Further analysis is now in progress.

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In chloroplasts, Fd forms an electron transfer complex with redox enzymes, such as Fd:NADP⁺ oxidoreductase(FNR), sulfite reductase, nitrite reductase and glutamate synthase. Fd therefore influences redox metabolism through the formation of intermolecular electron transfer complexes, and these are the subject of our research.
Two maize leaf FNR isozymes;L-FNR1 and L-FNR2, have previously been described and we have also identified a new L-FNR. Chloroplast fractionation revealed L-FNR1 was located at the thylakoid membrane, new L-FNR in the stroma and L-FNR2 in both fractions. Using Fd-affinity chromatography with wild type and mutant Fd-resin columns we found interaction of different FNR isoenzymes strongly depend on specific Fd amino acid residues. Therefore, despite very high sequence homology, these L-FNR isoenzymes vary dramatically in location and Fd interaction.

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In C4 plants, PEPC, which is expressed in the cytosol of mesophyll cell, plays a key role in CO₂ fixation. PEPC is activated by phosphorylation of the conserved Ser residue near N-terminus in response to light. Recently, we first cloned a specific protein kinase (PEPC-PK) from Flaveria trinervia, a C4 plant. Furthermore, we showed that recombinant PEPC-PK was redox-regulated. Here, we report two experimental results relating to the mechanism of redox-regulation. (1) Inactivated PEPC-PK by oxidation could be recovered with cytosolic thioredoxin system (1 μM Trx, 100 μM NADPH, 50 nM thioredoxin reductase). Trx-h, a cytosol-type Trx, activated PEPC-PK more effectively than chloroplast-type Trx-f and -m. (2) Among six Cys residues, a pairwise replacement of Cys53 and Cys250 to Ala abolished the sensitivity to oxidants and reductants. These results suggested that PEPC-PK activity was redox-regulated via thioredoxin through an intramolecular thiol/disulfide formation between Cys53 and Cys250.

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Our previous research with purified PEPC-PK suggested a possible redox regulation via thioredoxin. Through our several experiments of the oxidative-stress treatment in plants, we demonstrate the occurrence of this regulation in vivo. When maize plant was exposed to a cold stress under light, the degree of phosphorylation of PEPC was abruptly decreased. And when detached maize leaves were treated either with DCMU or diamide, the phosphorylation state changed similarly. Thus PEPC-PK activity seemed to be somehow inhibited under the stress. Succeeding experiments with cell extracts from stressed leaves revealed that the PEPC-PK activity could be recovered by incubation with dithiothreitol. Although previous studies suggested a rapid turn-over rate of PEPC-PK, our data showed that the PEPC-PK activity is not lost by degradation but redox-regulated in a reversible manner at least under oxidative stress conditions. Possible significance of redox regulation in the cytosol will be discussed.

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In higher plants, light and cytokinin co-operatively regulate MGDG synthase (MGD) (Yamaryo et al, PCP, 2003). Although light induces accumulation of the mRNA and subsequent increase in MGDG, MGDG does not accumulate by far-red light (700 nm≤) whereas red light (600 nm≤) can. It indicates that MGDG synthesis requires red light (600-700 nm). MGD is also known to require some reducing agents to preserve its activity so that MGD may require the reducing equivalent generated by photosynthesis. In this report, we analyzed the effect of reducing agents and thioredoxin on MGD activity with recombinant MGD (rMGD) expressed in E. coli. The SH residues of the purified rMGD were oxidized and the effects of reducing agents were analyzed. Consequently, we found that thioredoxin effectively reduced the disulfide bond in MGD. We also analyzed the effect of membrane lipids on MGD activity, and found that PA enhanced rMGD activity most effectively.

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To clarify the sugar-signaling pathway of higher plants, the ghs1 (glucose hyper-sensitive 1) mutant of Arabidopsis was isolated and characterized. The ghs1 mutant had an increased sensitivity to sugar, showing a dramatic inhibition of chlorophyll synthesis, accumulation of anthocyanin, and developmental arrest of leaves when grown on medium containing more than 5% glucose. The ghs1 mutant is a single recessive loss-of-function mutation caused by a T-DNA insertion in the GHS1 gene, which encodes the plastid 30S ribosomal protein S21. The mutant showed 1) reduction in the translation product but not the transcript for plastid-encoded rbcL, 2) reduction in photosynthetic activity monitored with pulse-amplitude modulated fluorometry, 3) impaired chloroplast development. These results indicate that the deficiency of such chloroplast functions as photosynthetic activity observed in the ghs1 mutant is caused by impaired plastid protein synthesis associated with loss of ribosomal S21 protein. Relationships between the GHS1 gene and sugar-signaling are discussed.

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We have developed a practical method for the comprehensive analysis of sugar phosphates by high performance anion exchange chromatography with pulsed amperometric detection coupled with a titanium oxide column as a trap-column. Using this system, levels of inorganic phosphate, sugar phosphates and a nucleotide phosphate from Arabidopsis grown at three different Pi concentrations in nutrient media, were investigated. These compounds were also measured in the Pi-related mutants (pho1 and pho2). Glc 1-P, Glc 6-P, Frc 6-P, Gal 1-P and Man 1-P increased in proportion to increases in the in vivo level of Pi in wild type plants. In contrast levels of Suc 6-P and UDPG decreased as the in vivo Pi levels increased. Except for Suc 6-P and UDPG, the responses to Pi level in the mutants were similar in wild type plants. However Suc 6-P and UDPG responded differently.

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In higher plants, a variety of Z,E-mixed polyisoprenoids have been detected. However, the physiological roles of these compounds and the mechanism of chain length determination of cis-prenyltransferases (CPTs) which catalyze the formation of Z,E-mixed polyisoprenoids have been hardly elucidated. In order to understand the catalytic mechanisms of CPTs and the physiological roles of Z,E-mixed polyisoprenoids in higher plants, we cloned and characterized genes encoding CPTs in Arabidopsis thaliana.
In Arabidopsis genomic sequences, we found 9 genes that have high homology to CPT genes. By RT-PCR analysis, the expression pattern of the genes in various tissues was shown. According to the expression pattern, 7 cDNAs were isolated. Expression of these cDNAs in yeast rer2 strain, which is deficient in CPT activity, suppressed temperature-sensitive phenotype of the mutant. Moreover, the crude extract from these yeast strains harboring each cDNA from Arabidopsis showed actual prenyltransferase activity, catalyzing the formation of C₅₀-C₁₀₀ polyprenyl diphosphates.

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Labyrinthulids are fungoid protists in the kingdom Chromista. In the coastal ecosystem, they are important decomposers. Labyrinthulids produce polyunsaturated fatty acid, docosahexaenoic acid (DHA). The biosynthetic pathway of the DHA has been investigated, but few cytological studies on the DHA biosynthesis have been conducted. We observed lipid bodies stained with Nile Red and found that the cells accumulate lipid bodies during the cell development from the zoospore to the vegetative cell. It is suggested that the electron microscopy using freeze-substitution technique can reveal lipid bodies containing 1,3-dipalmitoyl-2-DHA-triacylglycerol. Using this technique, we found more lipid bodies containing DHA in the vegetative cells than in the zoospores. Analysis of fatty acid content and composition demonstrated that the zoospores contain lower level of fatty acid than those in the vegetative cells. On the other hand, the ratio of DHA in the fatty acid was higher in the zoospores than in the vegetative cells.

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Arabidopsis contains two isogenes for CTP:phosphorylcholine cytidylyltransferase (CCT), AtCCT1 and AtCCT2, whose contribution to the enhanced biosynthesis of phosphatidylcholine (PC) at low temperature remains to be genetically evaluated. We previously reported that T-DNA-tagged disruption of AtCCT2 scarcely affected the accumulation of PC at low temperature, despite the selective enhanement of AtCCT2 expression in wild type at low temperature.
Here we revealed that AtCCT1 contributes to the enhancement of CCT activity at low temperature without significant enhancement of expression levels in this disruptant. We examined the distribution of CCT activity and AtCCT1 protein in differentially centrifugated fractions of rosette homogenates and found that enhancement of the CCT activity in the 150 k x g pellet fraction at low temperature accompanies little increase of AtCCT1 protein. These results suggested that both of the two CCT isogenes of Arabidopsis contribute to the activation of PC biosynthesis at low temperatures by different mechanisms.

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We investigated the role of a phospholipid, phosphatidylglycerol, in the PSI complex through characterization of a disruptant of Synechocystis sp. PCC6803 as to the cdsA gene responsible for the PG synthesis. The mutant showed decreased levels of PsaA and PsaB that construct the PSI core with a decrease in the PG content. Besides, the quantitative decrease in the PSI complex was shown to be caused by a drop of the population of the trimeric form, but not of the monomeric form. Thus, it was demonstrated that PG is responsible for the maintenance of the level of the PSI complex, and also for the trimeric form of the PSI complex.

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Two types of red pigments, anthocyanins and betacyanins, never occur together in the same plant. Nevertheless of the lack of anthocyanin, other flavonoids, especially major flavonols are widely found in the Caryophyllales. This suggests that the step(s) of anthocyanin biosynthesis from dihydroflavonols to anthocyanins could be blocked in the Caryophyllales.
We have isolated cDNA of DFR (dihydroflavonol 4-reductase) from Spinacia oleracea and Phytolacca americana, plants of the Caryophyllales. These DFR showed high degree of homology (62-82% identity) with DFRs of anthocyanin-producing plants. A putative NADPH binding region, which is likely part of the co-factor binding site, was located at the N-terminal in both of DFRs.
Both of recombinant DFRs expressed in E. coli converted dihydroquercetin to leucocyanidin. This indicates functional DFR is expressed in Spinacia oleracea and Phytolacca americana, which are the Caryophyllales.

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It is assumed that betacyanin was synthesized via betanidin since betanidin glucosylating enzymes were reported in early studies. However, we detected the glucosyltransferase activity for cyclo-DOPA that is the precursor of betanidin in petals of four o'clock (Mirabilis jalapa L.) which suggested the existence of betacyanin biosynthetic pathway via cyclo-DOPA glucosylation. Here, we succeeded that isolation and characterization of the cDNA encoding for cyclo-DOPA glucosyltransferase.
Thirteen cDNAs encoding UDP-glucose: flavonoid 3-O-glucosyltransferase homolog were isolated by PCR using the cDNAs synthesized from petals of four o'clock as a template. All of them were introduced into in E. coli to produce proteins encoded in the cDNAs and cyclo-DOPA glucosyltransferase activity was measured to the crude extracts prepared from the E. colis, indicating that one clone cDNA encodes cyclo-DOPA glucosyltranseferase assay. Northern hybridization analysis revealed the corelation between expression of transcripts corresponding to the cDNA and contents of betacyanin in four o'clock.

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In previous research, we found that one possible mechanism to be variegated flower color phenotypes is caused by transposition of transposable element dTdic1 in Dianthus caryphyllus. dTdic1 was an Ac/Ds-like transposable element, and more than 20 copies of dTdic1 family were found in the carnation genome. The cyanic color of most flowers comes from anthocyanins, while betacyanins produce the color in flowers of Centrosparmae excluding Dianthus. Mirabilis nyctaginaceae is one of flower, which produces betacyanins, and it has many flowers mutable with variegated flower color phenotypes, which might be caused by transposable element. It is thought that transposable elements play a very important role in altering the genome during evolution, but it is unknown in how it has spread. We identified a transposable element, Tnm1, which belongs to an Ac/Ds superfamily, but it was different from dTdic1 structurally. We report about a relation between the insertions of Tnm1and flower color.

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MADS-box gene was postulated to be related to anthocyanin biosynthesis. As a MADS-box gene regulated proanthocyanidin biosynthesis in Arabidopsis. We tried to isolate MADS-box gene from red root of sweet potato. Degenerate primer was designed based on amino acid sequence of MADS domain of MADS-box genes, then 3'-RACE was performed. A new MADS-box gene was isolated, which was named IbMADS1. The expression of IbMADS1 was analyzed by northern blot analysis. The maximum transcript level was observed in floral buds, pink roots beginning color and red roots fully colored. A lower expression was found in developing tubers. The transcript was not detectable in flowers at anthesis, buds, leaves, stems and white roots. The expression of anthocyanin biosynthetic gene, CHS, CHI, F3H, DFR, ANS and UFGT, were also analyzed. We will report the possibility that IbMADS1 is related to anthocyanin biosynthesis base on our results.

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The PAP1 gene, which encodes an MYB transcriptional factor, up-regulates the flavonoid biosynthetic gene expression. In this research, we integrated the studies of metabolomics and transcriptomics with wild-type, pap1-D mutant and PAP1-overexpressed transgenic plant, to elucidate a detailed anthocyanin accumulation mechanism.
By metabolome analysis using HPLC-PDA-MS and FT-MS, it was confirmed that PAP1-overexpressing lines highly accumulated 11 cyanidin derivatives in the leaves. In addition, by the transcriptome analysis using Affymetrix GeneChip, 38 up-regulated genes were identified as those encoding glycosyltransferase, acyltransferase and transporter, which are expected to be involved in flavonoid biosynthesis.
These results showed that the PAP1 gene is a transcriptional factor which regulates specifically the anthocyanin accumulation. Our findings suggest that a functional genomics approach is useful for the complete identification of genes for anthocyanin accumulation in Arabidopsis.

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The pap1-D, activation tagging mutant over-expressing Myb-like transcription factor PAP1, specifically over-accumulates anthocyanins. Genes upregulated in pap1-D were expected to be involved in biosynthesis and sequestration of anthocyanins. Microarray analysis revealed that genes upregulated in pap1-D included those encoding enzymes involved in anthocyanin biosynthetic pathway, transcription factors such as Myb and WRKY, and glycosyltransferases (GTs), acyltransferases (ATs) and glutathione-S-transferases (GSTs) whose function were remained undetermined. Those GTs, ATs and GSTs were expected to be involved in modification of aglycons and sequestration of anthocyanin.
Out of those candidate genes, genes encoding GTs were cloned, and recombinant proteins were expressed in E. coli. Enzymatical properties of those recombinant GTs were analyzed. Metabolomic analysis of A. thaliana mutants that defective in each gene was also performed. Roles that each gene performed in planta will be discussed.

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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 without adding the high concentration of sucrose. Determination of endogenous ABA in the regenerated shoots producing anthocyanin in the presence of high concentration of sucrose revealed that the amount of endogenous ABA transiently increased just before the induction of anthocyanin synthesis. These results suggested that change in the amounts of endogenous ABA might play an important role in the induction of anthocyanin synthesis in the regenerated torenia shoots. In the present study, cDNA subtraction was performed to isolate the genes up-regulated in torenia regenerated shoots producing anthocyanin. Several genes which were especially expressed in the shoots producing anthocyanin were found by this screening method.

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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 and nuclear localization of this MYB protein in suspension-cultured carrot cells.

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It has been known that the genes for the enzymes involved in anthocyanin synthetic pathway express in specifically differentiated tissues and organs. 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. Previously, we isolated two AP2/ERF transcription factors, DcERF1, DcERF2, that interact with the GCC sequence of the gDcPAL3 promoter. However, the function of these genes was unknown. Transactivation functions of DcERF1, DcERF2 using carrot suspension cultured cells will be reported.

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The environmental factors which control the growth of Microcystis developing massively in eutrophic water-bodies have been investigated such as light and temperature, but effect of potassium on the growth have not been evaluated.
The generation time of Microcystis was 22-32 hrs at 0-2.5 mM KCl whereas they could not grow at 5-40 mM KCl. At 10 mM NaCl, the growth rate was almost the same as that in the control cells although they were unable to grow at 5 mM K₂SO₄, indicating that the inhibition of growth is responsible for potassium, but not for chloride. The apparent Km(NaHCO₃) values in photosynthesis of Microcystis cells were 56 μM without KCl and 279 μM with KCl(40 mM) and no significant difference in Vmax between them was observed. It is concluded from these observations that inhibitory effect of potassium on photosynthesis in Microcystis leads to the suppression of Microcystis growth.

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APX isoforms localized in chloroplasts of higher plants are rapidly inactivated by attack of hydrogen peroxide if the second substrate ascorbate is depleted. This is the reason why chloroplasts are damaged under oxidative stresses. We studied the molecular mechanism of the H₂O₂-induced inactivation of chloroplastic APXs. We found chloroplastic APXs have amino acid sequences inserted in C-terminal half domain, which is not found in those of H₂O₂-tolerant APX isoforms. When this region was inserted into the corresponding site of a Galdieria APX, an H₂O₂-tolerant APX, it showed rapid inactivation by addition of H₂O₂ under depletion of ascorbate. This indicate that interaction between the catalytic site and this region is responsible for the inactivation of APX by H₂O₂. Furthermore, we found that the inactivation of the chloroplastic APX associated with covalent binding of the heme located at the catalytic site to the apoprotein.

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When Chlamydomonas cells are exposed to CO₂-limiting stress conditions, they induce a set of genes for a carbon-concentrating mechanism (CCM) under the CO₂-limiting conditions. To elucidate molecular mechanisms of CO₂-signal transduction pathways, we have isolated regulatory mutants of the low-CO₂ inducible gene Cah1, which encodes a periplasmic carbonic anhydrase. Using arylsulfatase reporter gene driven by the Cah1-promoter, a regulatory mutant of Cah1 was isolated and named as C44. C44 showed a moderate high-CO₂ requiring phenotype and partially defective in the CCM-induction. The C44-mutation was caused by a single-tag insertion. The corresponding gene, LciR1 encodes a protein containing a single Myb-like DNA binding domain in its N-terminus region. Expression of the LciR1 was also induced by lowering CO₂, and regulated by Ccm1 gene which controls carbon concentrating mechanism in Chlamydomonas.

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Photosystem II (PSII) is particularly sensitive to oxidative stress. We investigated the effects in vivo of reactive oxygen species (ROS) in the photodamage to PSII in Chlamydomonas reinhardtii. Increases in intracellular concentrations of ROS, caused by the presence of ROS-generating reagents, such as methyl viologen, hydrogen peroxide, and rose bengal, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the presence of these reagents. These observations suggest that ROS act primarily by inhibiting the repair of photodamaged PSII and not by accelerating damage to PSII in Chlamydomonas.

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Glycinebetaine (betaine) is one of the compatible solutes and is accumulated in various organisms in response to stress conditions. Synechococcus sp. PCC7942 that had been transformed with the codA gene for choline oxidase retained the ability to synthesize betaine in the presence of choline and enhanced tolerance against cold and salt stress. We have already demonstrated that betaine enhanced the repair of photosystem II (PSII) during photoinhibition with salt stress although neither salt stress nor betaine affected the photodamage to PSII. A further investigation of the effects of betaine on photoinhibition of PSII in Synechococcus cells showed that salt stress inhibited the degradation and synthesis de novo of D1 and caused the slow repair of PSII. Betaine alleviated both kinds of inhibition, resulting in accelerated repair of PSII. We will also discuss about the effect of salt stress and betaine on transcription of the psbA gene.

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The research of simultaneous measurements of CO₂ exchange and growth have been continued to evaluate the impact of photosynthesis on biomass. Plants of radish, cv White Cherrish with big storage root were grown for 6 days under 4 different conditions (1: ambient CO₂ of ca. 380 ppm and 23 molE/m²⁺/day, 2: elevated CO₂ of ca. 750 ppm and 23 molE/m²⁺/day, 3: ambient CO₂ of ca. 380 ppm and 15 molE/m²⁺/day, 4:elevated CO₂ of ca. 750 ppm and 15 molE/m²⁺/day) and the rates of CO₂ exchange were monitored continuously during whole periods of growth analysis. The results of growth analysis of RGR, NAR, LAR, LWR, SLA, leaf area, rates of photosynthesis and respiration, and water use efficiencies under 4 different conditions will be discussed comparing the results with radish cv. Kosena with small storage root which was presented last year.

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Cyanobacteria possess a CO₂ concentrating mechanism (CCM) that allows cells to perform efficient CO₂ fixation. The CCM is considered to consist of two basic steps, accumulation of intracellular HCO₃^-, which is mediated by multiple active uptake systems for CO₂ and HCO₃^-, and CO₂ fixation in the Rubisco-containing micro-compartment known as carboxysome. Because of the presence of a carbonic anhydrase in the carboxysome, it is supposed that the carboxysomal carbonic anhydrase (CCA) catalyses rapid conversion of HCO₃^- to CO₂, resulting in a high local CO₂ concentration at the site of fixation. In this study, we detected high CO₂ fixation activity in partially-purified carboxysome preparations from the wild-type cells, confirming that carboxysome is the site of CO₂ fixation. By contrast, carboxysome fraction of a CCA-less mutant showed much lower CO₂ fixation activity, verifying that CCA is important for efficient CO₂ fixation.

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Transcription of the rbcLS operon, encoding RuBisCO, is activated under low-CO₂ conditions in the cyanobacterium Synechococcus sp. PCC 7942. We found three potential CbbR recognition sites in the upstream region of rbcL, two of which were shown to be essential for the low-CO₂ responsiveness of the promoter. Two CbbR homologues (CmpR and RbcR) have been identified in Synechococcus. The expression level of the rbcLS operon in a cmpR-deficient mutant is higher than that in the wild-type strain, implying that RbcR is likely the regulator of the rbcLS operon. Since no rbcR-null mutant was obtained, we constructed an overexpression strain of Synechococcus, in which the rbcR gene is expressed from a nitrogen-regulated promoter. This strain showed a 2-fold increase in the expression level of rbcLS under the induction conditions. These results suggested involvement of RbcR in activation of the rbcLS operon in Synechococcus.

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Aquatic photosynthetic-microorganisms including diatoms possess inorganic CO₂-concentrating mechanism (CCM), when grown in air levels CO₂. Carbonic anhydrase (CA) is known to be a key component in CCM. We have previously demonstrated that β-type CA (PtCA) is induced upon CO₂ limitation and that light stimulates PtCA induction in the marine diatom Phaeodactylum tricornutum.
In order to elucidate the molecular mechanisms of light stimulation in PtCA expression, the function of PtCA promoter region was analyzed in relation to light conditions. Several length of upstream region of PtCA gene were isolated and were fused to GUS gene. The pattern of GUS expression sharply agreed with that observed in PtCA expression and responsibility for CO₂ was well conserved with -1292/-70 regions. Responsibility of these truncated promoter regions for light stimulation effect on PtCA expression is reported.

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In the genus Flaveria, there are various photosynthetic types, C3, C3-C4 intermediate and C4 (NADP-ME type) plants with highly close genetic backgrounds. To isolate cDNAs for novel proteins involved in C4 photosynthesis, we carried out differential screening for F. trinervia cDNA library with either of the two 32P-labeled cDNAs as probes, which were prepared from mRNAs of C3 type F. pringlei and C4 type F. trinervia. Besides Ppc, Ppdk and Me, 56 clones were identified and classified into 29 genes. Among them 19 genes were highly homologous to known genes and other 10 genes were novel. These genes were expressed much higher in leaves of light period than in leaves of dark period and in other organs, suggesting their functions in light leaves. Here we will show detailed expression analyses for selected genes, e.g. their tissue specificity and leaf stage dependency, and discuss possibilities of their involvement in C4 photosynthesis.

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To clarify biochemical and molecular mechanisms of shift of optimum temperature for photosynthesis, we grew spinach (Spinacia oleracea) at 15C or 30C and analyzed various photosynthetic properties.
Temperature optima of RuBP carboxylation, assessed by the gas exchange techniques, were 18C and 24C in the leaves grown at 15C and 30C, respectively, although temperature dependence of carboxylation has been believed to be unaffected by growth temperature. Results of the Two-dimensional gel electrophoresis of Rubisco revealed significant differences in the isoelectric point in Rubisco small subunits depending on the growth temperature. It is likely that, in spinach grown at the lower temperature, expression of isozyme(s) of Rubisco small subunits having greater activity at the lower temperature, or some post-translational modification of Rubisco small subunits contributes to the downward shift in the optimum temperature for photosynthesis.

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Decrease in photosynthetic activity of sweet potato source-sink model plants placed under sink-limited conditions was mainly due to the binding of inhibitor to Rubisco. This inhibitor was suggested to be a sugar phosphate by analysis using HPLC.
The phenols contained largely in leaf extract from sweet potato disturbed purification of inhibitor using activated Rubisco. Moreover, pectins contained largely in the extract adsorbed strongly the inhibitor. Therefore, purification of the inhibitor and analysis of the inhibitor by NMR was prevented.
In order to obtain the inhibitor in high purity, it is under examination about the removal method of an impure ingredient.

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The production of reactive oxygen species (ROS) in chloroplasts is stimulated during leaf senescence and under stress conditions.It is suggested that ROS act as a trigger of protein degradation.Actually,direct degradation of the large subunit of Rubisco by ROS has previously been shown in vitro.However,it is unknown whether ROS cause the fragmentation of Rubsco-LSU in vivo.In this study,we examined whether the fragmentation of the LSU by ROS occurs in vivo. When leaf discs of a chilling sensitive plant ,cucumber, were incubated in the light at 4^oC,five fragments of the LSU were time-dependently appeared.The fragmentation was inhibited by the ROS scavengers,n-propyl gallate and tiron.While FeSO₄ stimulated the fragmentation,iron specific chelator,deferoxamine,prevented it.Furthermore,when the purified cucumber Rubisco was exposed to the hydroxyl radical-generating system,the fragments produced were identical to those of the light-chilling discs in molecular weights and isoelectric points. These results suggest that the direct fragmentation of LSU by ROS occurs in vivo.

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There are little information about the effect of nitrogen nutrition on the photosynthetic proteins after full expansion to senescence. The purpose of this study is to examine how the supply of nitrogen after full expansion influences major photosynthetic proteins in the 8th leaves of rice. Rice plants were grown under different levels of nitrogen (1 mM N and 4 mM N) at the stage of full expansion of the 8th leaves or at the 7th day after full expansion. In spite of the treatments, the ratio of Rubisco-N to total leaf N contents was greater in the leaves with 4 mM N. However, the respective ratios of LHCII, CF₁ and Chl to total leaf N contents were little different between them. Thus, Rubisco was more largely affected by the N supply even after the stage of full expansion in rice.

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The feathered (fe) mutants in the Japanese morning glory have strongly crumpled or rolled leaves, and flowers with deeply split or tube-like petals folding back at their tips. The fe mutant phenotypes suggest that FE is involved in establishment of organ polarity. For example, number of stomata in abaxial side decreased in fe mutants. FE gene was isolated from a germinal revertant using Simplified Transposon Display (STD) method which allows visualization and isolation of transposon-flanking sequences from high copy-number lines. FE gene has a high homology with KANADI1 (KAN1) gene in Arabidopsis. We suppose that FE expresses on abaxial side of lateral organs, and specify the abaxial identity as KAN. Gentic and molecular analyses showed that the strong fe phenotype is caused by an additional mutation in fe background.

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We cloned a KNOX class1 gene in Physcomitrella patens by RT-PCR and named PpKNOX1 (PpKN1). Champagne and Ashton (2001) reported two KNOX class1 genes, MKN2 and MKN4, and a 267-bp fragment (MKN5). MKN2 was identical to PpKN1. We cloned flanking regions of MKN5. We inserted GUS reporter gene at the end of PpKN1 and MKN5 coding sequence. The transgenic mosses showed GUS activity specifically in egg cells and young sporophytes. Arabidopsis thaliana expressing PpKN1 cDNA under the control of cauliflower mosaic virus 35S promoter showed lobed leaves, which is similar to those in A. thaliana expressing other KNOX class1 genes. These data suggest that PpKN1 and MKN5 functions in meristem formation and maintainance of the sporophyte, which do not form shoot but exists transiently to form seta and sporangium. However, PpKN1 disruptants formed fairly normal sporophytes. We are now producing PpKN1 MKN5 double disruptant and triple disruptants.

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Cell elongation is essential for plant morphogenesis. Recently, several genes related to the process have been identified using molecular genetics. However, the comprehensive information of gene expression and regulation during the process are poorly understood. In this study, we examined oligomicroarray analysis to get comprehensive information about the gene expression profiles during cell elongation in A. thaliana.
For microarray experiments, RNA samples were prepared from wild type and cellulose deficient mutants and were used as templates for Cy3- or Cy5-labeled cRNA synthesis. We used the Agilent Arabidopsis 2 Oligo Microarray and analyzed the data using computer software 'Luminator' (Rosetta Biosoftware). We have already collected several gene expression profiles in the hypocotyls, the rosettes and the stems. We are currently analyzing the data to identify the genes whose expression is induced during cell elongation in the organs.

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Plant leaves have not only two-dimensional dimensions but also a thickness. A shape and/or a size of mesophyll and/or epidermal cells, and a layer number of mesophyll cells can determine leaf thickness. It has been previously reported that the number of mesophyll layers is affected by growth conditions. However, the molecular mechanisms of the control of the layer number are still unknown. In order to reveal the mechanisms for leaf-thickness control, we developed new equipment for measurement of leaf thickness. In the course of the screening of mutants of the leaf-thickness regulation, we isolated an Arabidopsis N374 mutant that possesses the increased number of the mesophyll layers from T-DNA tagged lines. N374 mutation not only increased number of the mesophyll layers but also decreased the cell size and a leaf area. The latest results of analyses of N374 will be presented.

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As the only member of C-terminal binding protein (CtBP) family in Arabidopsis thaliana, ANGUSTIFOLIA (AN) has not only D-isomer-specific 2-hydroxy acid dehydrogenase (D2-HDH) motif which is conserved among CtBPs, but also LXCXE motif, PEST motif, cell-cycle-specific phosphorylation site and nuclear localization signal.* We carried out the comparative analysis of AN protein with authentic CtBP/BARS family, focusing on the above-mentioned AN-specific motifs which are not found in CtBPs. AN did not show ability of interaction with the C-terminal region of E1a. Moreover, AN-specific C-terminal region was found to be required for self-association. Although AN has LXCXE motif, AN did not interact with Rb in yeast two-hybrid system. Various types of mutated ANgene were constructed and the ability of complementation of the an mutation was examined for each mutated AN. Our data strongly suggest that AN has evolved different molecular function from CtBPs.

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Morphogenesis of leaf blade is dependent on the determinate and two-dimensional cell proliferation. We found that this process is subdivided into two phases based on the properties of direction of cell proliferation in Arabidopsis thaliana. During Phase I, cell proliferation in a leaf primordium occurs along its longitudinal axis, and during Phase II this restriction is released so that cell proliferation occurs two-dimensionally. angustifolia3 (an3) mutant of A. thaliana is defective in the maintenance of cell division activity during Phase II and develops narrower leaves than wild type. Cloning of AN3 gene revealed that it encodes a putative transcription coactivator. We searched for AN3-interacting proteins and identified putative transcription factors by yeast two-hybrid system. To test if genes for these transcription factors are coordinately expressed with AN3, examination of their tissue-specific expression patterns is in progress. Phenotypes of overexpression of AN3 and AN3 interacting proteins will also be reported.

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Stipule is a leaf-like appendage of leaf. To understand molecular mechanisms of the stipule development, mutants (#1148 and #216) of model legume Lotus japonicus were analyzed. Ternately divided compound leaf of wild-type plant in L. japonicus usually accompany two stipules, while leaves in the mutants lack the stipules and have more than three narrow or needle-like leaflets. Observations of transverse sections of the needle-like leaflets revealed that cells with adaxial identity are absent from the mutants, suggesting morphological similarity with phan mutant in Antirrhinum majus. We isolated two PHAN/AS1/RS2 homologs from L. japonicus genome. Results of analysis of the nucleotide sequences and the expression of the homologs will be shown, and whether either of homolog is a mutated gene in the mutants will be discussed.

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To understand developmental process of leaves, we have analyzed ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) genes. AS1 and AS2 are involved in the establishment of the leaf venation systems, formation of symmetrical and flat lamina and regulation of expression of class 1 knox genes in leaves to maintain the developmental state of cells. Transcripts for AS1 were accumulated in the central region in leaf primordia, and transcripts for AS2 were entirely found in SAM and leaf primordia, and predominantly detected in the epidermal layer in leaf primordia. In this study we show that overexpressor of AS2 generated upwardly curled leaves, while as1 and as2 mutants generated downwardly curled leaves. The results suggested that AS2 might be involved in regulation of cell proliferation of adaxial side of leaf primordia in the presence of AS1. We further show the results of characterization of the AS1 and AS2 proteins.

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Recessive mutations in ASYMMETRIC LEAVES2 (AS2) display asymmetric leaf-shape, altered venation pattern, higher potency of regeneration and ectopic expression of class1-KNOX in leaves of arabidopsis. AS2 is a plant-specific novel protein that belongs to novel AS2/LOB-family. To investigate the function of AS2 protein, we constructed and analyzed 35S:AS2-GR transgenic arabidopsis. Transiently expressed AS2-GFP-GR fusion proteins localized in nuclei of BY-2 cells dependently on Dex. High amount of AS2-GR fusion proteins were accumulated in 35S:AS2-GR transgenic arabidopsis independently from Dex. 35S:AS2-GR transgenic arabidopsis exhibited hyponastic leaves or growth-arrest when they were treated with 10 nM or 100 nM Dex, respectively. Finally, 35S:AS2-GR transgenic plants could exhibit flat and symmetric leaves dependently on Dex even in as2-1 background. Thus we concluded that AS2-GR fusion protein was functional and that AS2 was nuclear protein. Recently, we are analyzing the effects of chemical compounds on the phenotype of 35S:AS2-GR transgenic and as2 mutant plants.

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To identify genes expressed in the procambium and/or vasculature, we have been creating enhancer-trap lines in Arabidopsis and screened for the lines expressing a reporter gene in the vasculature. The enhancer-trap T-DNA includes GFP as a reporter gene and pBluescript II for the plasmid-rescue method to isolate genomic DNA adjacent to the inserted T-DNA. So far, we have screened about 20,000 T1 transformants and obtained 346 lines in which GFP fluorescence was detected in the procambium and/or vasculature of rosette leaves (105 lines), roots (209, out of which 10 are specific to the root apex), shoots and roots (16), and shoots except rosette leaves (16). Besides further screening T1 transformants, we are currently isolating genomic DNA adjacent to the T-DNA insertion sites by TAIL-PCR and plasmid-rescue procedures to identify genes responsible for the vasculature-specific expression.

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To dissect vascular pattern formation genetically, we have isolated and analyzed van mutants of arabidopsis that are characterized by fragmented minor veins. Here we report the map-based cloning of the VAN3 gene.
Fine chromosome mapping of the van3 mutation localized it at the position of 30 cM of chromosome V. Sequencing of this region revealed that a single base substitution, G to A, exists in one of the predicted CDS of the van3 genome. We introduced the wild-type genome fragment containing this candidate CDS into the plants heterozygous for the van3 mutation. Phenotypic and genotypic analyses of the resultant T2 progenies confirmed that this fragment can rescue the vascular defect of the van3 mutant, leading to the conclusion that the candidate CDS corresponds to the VAN3 gene. The predicted product of the VAN3 gene shares sequence similarity with Arf GTPase-activating protein involved in the vesicle transport process.

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We have cloned the VAN3 gene as a causal gene of van3 mutant, defective in leaf vascular continuity. VAN3 encodes a putative novel type ArfGAP protein in Arabidopsis thaliana. Here we report ArfGAP activity of VAN3, and its subcellular localization.
First, we determined ArfGAP activity of VAN3 by an in vitro GTP/GDP exchange assay. VAN3 protein, which was prepared from E.coli havoring VAN3, stimulated hydrolysis of GTP to GDP binding to yeast Arf1p. Next, we determined the subcellular localization of VAN3 using a fusion protein of VAN3 and Venus. VAN3-Venus expressed under the control of 35S promoter showed fluorescent dots moving around in the cytoplasm. Double labeling experiments using VAN3-Venus and FM4-64, revealed high colocalization of FM4-64 staining vesicles and VAN3-Venus staining dots, suggesting VAN3 locates in the endosomal compartment. Efforts are underway to reveal colocalization of GNOM ArfGEF and VAN3 ArfGAP, and cell specificity of VAN3 gene expression.

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To dissect vascular pattern formation, we have isolated Arabidopsis van3 mutant showing the fragmented minor veins. For the aim to unveil the molecular mechanisms of the VAN3, we performed yeast two hybrid screening. ADL1A, a GTPase dynamin, was isolated as a VAN3 interacting protein. From the analysis of ADL1A::ADL1A-GFP transgenic plants, ADL1A can be co-localized with the endosome marker FM4-64. ADL1A expression was observed in the provascular cells of the torpedo-stage embryos, trichomes, and stigmatic papillae. adl1a mutants produced unbranched trichomes, abnormal isotropically expanded papillae cells, and discontinued veins. Furthermore, adl1a adl1e double mutant showed embryo lethality without producing the procambium cells. These results suggested that the ADL1A would widely regulate the single cell differentiation step, and in some cases ADL1A activity could be regulated by the VAN3 GAP.

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We evaluated the degree of chromatin condensation around the HSP18.2, H4, PCNA, V-ATPase and ADH gene promoters in Arabidopsis plants and suspension cells. The DNase I sensitivities were at a similar level between these genes and among leaves and suspensions irrespective of their expression states, however the local DNase I hyper-sensitive sites (HS sites) were found at the every promoter except the ADH gene at the same position in leaves as well as in suspension cells. The nucleosome positioning around the HS site was changed by heat activation in HSP18.2 promoter. Furthermore, at the ADH promoter the HS site is found in suspension cells but not in leaves, and the ADH gene is induced by ABA in suspensions but not in leaves. Taken together, it is suggested that these HS sites are involved in gene regulation by controlling the accessibility of transcription factors to the promoter.

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Some of upstream ORFs (uORFs) in 5′UTR are known to repress translation of downstream major ORF. In these cases, ribosome reinitiates translation at ATG of downstream ORF after translation termination of uORF. It was reported that the ribosomal protein L24 (RPL24) is involved in translational reinitiation of polycistronic mRNA. Here we report that an Arabidopsis mutant, in which an RPL24 gene was deleted, has unique phenotypes in gynoecium development. Similar gynoecium defects have been shown in ett and mp mutants. Because ETT and MP have uORFs, we examined whether their uORFs affect the translation efficiency of downstream ORF by mesophyll protoplast transient expression assay with reporter genes placed at downstream of the 5′UTR. The results showed that their uORFs repress the expression of downstream ORFs. We hypothesize that the gynoecium phenotypes shown in L24 mutant were caused by decrease of reinitiation efficiency of ETT and/or MP translation.

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7SL RNA is an RNA component of Signal Recognition Particle (SRP). It is thought that 7SL RNA was derived from ancient tRNA, therefore transcribed by RNA polymerase III. In plant 7SL RNA genes have USE (Upstream Element) and TATA sequences as transcription regulatory elements at upstream of transcribed region, and don't have internal promoters like as tRNA or 5S rRNA genes. In mammal 7SL RNA have propeller secondary structure at 5' region of RNA, and contributing auto-regulation of transcription. In this study, we introduced point mutation into Arabidopsis 7SL RNA genes and mutated genes were used as templetes for in vitro transcription assay with tobacco nuclear extract, From this assay, it is strongly suggested that a cis-regulatory element located in transcribed region and RNA was destrabilized when a mutation introduced as dissolving propeller motif which was also located in 5' region of Arabidopsis 7SL RNA.

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