Methods for analyzing the distribution of mineral elements were applied to the tissues in the shoot apex of the Pharbitis nil., a short-day-plant. The fluorescent probes of Mag-fluo-4 AM and Fluo-3 AM were used for visualizing the distribution of Mg and Ca, respectively. Results showed that there was a difference between the distribution patterns of these elements. Magnesium was highly accumulated at the top of the shoot apex, whereas Ca distribution was rather homogeneous throughout the shoot apex. After the 16-hour's dark period, there appeared the different distribution pattern of Mg, suggesting the specific role of Mg to flower induction. The results shown by the method of the fluorescence probes was supported by SEM-EDS analysis where freeze-dried sections of the shoot apex were applied.

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The sequence comparison revealed PEONY (PN), an Ipomoea nil MADS box gene, is most likely to be a counterpart of snapdragon class C gene, PLENA, not FARINELLI. To investigate the functions of PN in vivo, transgenic petunias constitutively expressing PN were generated. PN overexpressors (PNox) showed homeotic changes that are similar to blind(a petunia class A mutant) indicating that PN acts as a class C gene. In order to understand the relationship between floral organ identity and flower coloration, the effects of disordered floral organ identity on the transcription of anthocyanin biosynthetic structural genes were investigated in Pnox. ANTHOCYANIN1 (AN1) and ANTHOCYANIN2 (AN2), transcriptional activators of the structural genes, were drastically down-regulated in Pnox. Subsequently DIHYDROFLAVONOL 4-REDUCTASE was transcriptionally suppressed, whereas CHALCONE SYNTHASE-A remained unchanged. Further analysis of petunia homeotic mutants is underway. The relationship of floral organ identity and anthocyanins biosynthetic pathway will be discussed.

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We report the isolation, sequence, and gene expression patterns of RMD1, a novel member of the STMADS11 subfamily from rice. Northern blot analysis revealed that expression pattern of RMD1 is different from that of other members of STMADS11 subfamily reported so far. All the members of STMADS11 subfamily has been reported that their expression is detected in vegetative tissues, but RMD1 is mainly expressed during flower development and embryogenesis. In situ hybridization analysis revealed that RMD1 expression is localized in developing stamen primordia and outermost cell layer in embryos. Over/ectopic expression of RMD1 in transgenic rice plants induced abnormal reproductive growth similar to transgenic tobacco plants expressing STMADS16. These results are discussed in terms of a possible role for RMD1 in promoting similar molecular function to STMADS11-like proteins although their expression patterns quite differ.

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We have studied effects of high temperature stress on reproductive development in barley plants. Exposed to high temperature (30°C day/25°C night) for five days at the panicle differentiation stage, the following pollen development was completely aborted. In this study, we found that the abortion was due to the defect of early development of tapetum cells. In addition, expression levels of histone H3 and H4 family genes didn't increase in the abortive panicles, whose levels particularly increased under normal condition. Expression levels of housekeeping genes such as a 60S ribosomal protein gene also decreased. In order to identify whether such a high temperature generally affected vegetative growth and somatic cell division, we measured each growth curve of two types of culture cells and young shoots at 30°C. The results indicate that the high temperature specifically affects the early development of tapetum cells.

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The 6b gene of Agrobacterium tumefaciens is tumorigenic in certain plants. We have shown that the gene induces morphological abnormality and phenylpropanoid metabolism in tobacco. To study further these processes, the 6b gene from AKE10 stain was placed under the control of dexamethazone-inducible promoter and introduced to tobacco. Upon induction, the transgenic seedlings showed;
(1) Developmental abnormalities including high growth rate of whole seedlings, altered morphology of SAM, and multiple outgrowths in cotyledons in which vascular tissues are densely formed.
(2) High accumulation of a number of phenolics,including chlorogenic acid, which could be partly due to increased transcripts of phenylalanine ammonia lyase gene.
(3) Increased sensitivities of growth inhibition to auxin transport inhibitors. This could reflect altered intracellular auxin levels and/or signal transduciton process (under investigation).
From these results we speculate that the 6b gene may induce phenylpropanoid biosynthesis pathway, promoting vascular development in close association with tumorigenic outgrowth.

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The ascorbate content declined rapidly in broccoli (Brassica oleracea L. var. italica) florets during postharvest senescence. In this study, ascorbate peroxidase (APX), ascorbate oxidase (ASO), L-galactono-1,4-lactone dehydrogenase (GLDH), ascorbate free radical reductase (AFRR), dehydroascorbate reductase (DHAR) and glutathione reductase (GSH) were investigated in enzyme activities and gene expression after harvest and treatments with plant hormones. It is observed that mRNA levels of chloroplastic APX (BO-sAPX and BO-tbAPX) decreased greatly within 12 h after harvest in broccoli florets. In addition, as a result of the suppression of other chloroplastic genes involved in ascorbate synthesis (BO-AFRR 1 and BO-DHAR), active oxygen species accumulation and ascorbate degradation may have been accelerated in chloroplasts. We also found that the mRNA expression of GLDH and chloroplastic genes involved in ascorbate metabolism was suppressed by the treatments with methyl jasmonate and ABA.

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Ascorbate functions as an antioxidant to be oxidized to monodehydroascorbate (MDA) radical. Its regeneration is essential to avoid damages by oxidative stress. In plant, MDA reductase (MDAR) containing FAD catalyzes the reduction of MDA by ping-pong mechanism using NAD(P)H. The first step of this reaction is the reduction of FAD by NAD(P)H, which is inhibited by thiol reagents. Cytosolic MDAR has two Cys residues at the conserved positions, but it has not been clarified which of Cys residues participates in its activity. Mutated MDARs whose Cys residues were substituted to Ala or Ser were designated C69A, C69S, C198A and C198S respectively. The C69S showed 32% of the activity of the native enzyme and lability of binding with FAD. The K_m values for NAD(P)H of mutants were comparable to those of the native enzyme. Now we are analyzing the rapid reaction between NADH and FAD on these enzymes.

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Phosholipase A2(PLA2) hydrolyzes glycerophospholipids to produce free fatty acids and lysophospholipids. In animals, secreted PLA2s(sPLA2) have been recognized as a large super family of distinct enzymes that play a central role in diverse cellular processes. In plants, little is known about the biological functions of sPLA2, although there are some reports of purification and cDNA cloning of sPLA2. To investigate biological function of sPLA2 in tobacco, Nicotiana tabacum, we attempted to isolate cDNAs for sPLA2 from tobacco. Initially, we performed RT-PCR with primes selected on highly conserved regions of sPLA cDNAs using total RNA extracted from tobacco flower. Next, we performed 5'- and 3'-RACE using gene-specific primers derived from the respective partial sequences. The cDNAs of sPLA2 from tobacco encoded 145 and 159 amino acids, respectively. The deduced amino acid sequences were shown to have a putative signal peptide. At present, we have studied gene expression of sPLA2 in tobacco.

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Fatty acids are peroxidized by lipoxygenase and subsequently cleaved by hydroperoxide lyase (HPL), which leads to the formation of short-chain aldehydes. Two different types of HPL, which can use 13- and 9-hydroperoxy derivatives of C18 polyunsaturated fatty acids as substrates, are known. So far, nobody cloned monocotyledonous HPL. In this study, the full-length cDNA from barley seedlings coding for HPL was cloned and expressed in Escherichia coli and characterized. Barley HPL has 50.1% similarity with Arabidopsis HPL in amino acid level. The enzyme was specific 13-hydroperoxy linoleic and linolenic acids and did not use the 9-hydroperoxy isomers as substrates. The expression analysis of barley HPL gene is being carried out under various treatments.

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Among a number of potential ferredoxin reductase genes in Arabidopsis genome, we have identified AtMFDR to correspond to a homologue of adrenodoxin reductase (mitochondrial ferredoxin reductase), and AtMFDX1 and AtMFDX2 as the genes for ferredoxins similar to adrenodoxin (mitochondrial ferredoxin). The deduced primary structures of AtMFDR and AtMFDXs contained the signature sequences involved in the electron transfer, and the recombinant AtMFDX1 and AtMFDR proteins were fully active to reconstitute the electron-transfer activity with NAD(P)H as the electron donor. Protein-gel blot analysis indicated constant accumulation of AtMFDR protein in leaf, stem, and flower. Subcellular fractionation analysis demonstrated membrane association of AtMFDR protein, and immunohistochemical studies revealed specific structure for the AtMFDR localization. Detailed analysis is in progress to clarify the membrane structure where the proteins exert their physiological functions.

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DAD1 is a gene essential for anther dehiscence in Arabidopsis. Recently, it has been reported that DAD1 encodes a phospholipase A1 involved in jasmonate synthesis. In tomato EST database, we found several DAD1-like lipase (DLL) gene homologs. One of them, namely, LeDLL1, shows high and transient expression during seed germination, when formation of jasmonate is not so remarkable. Thus, we expected that LeDLL1 is involved in the other events, such as fat mobilization during seed germination. In this study, we explored expression profile of LeDLL1. Enzymatic characters of the recombinant LeDLL1 expressed in E. coli was also examined. These analyses showed that expression profile of LeDLL1 was highly coincided with the timing of fat mobilization during germination of tomato seeds. Recombinant LeDLL1 showed activity against neutral fats. Accordingly, it might be conceivable to expect that LeDLL1 is involved in fat mobilization. Further studies are now underway to reveal the function.

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Chlorophyll degradation is involved in crimson foliage, aging of leaf and fruit ripening, as well as ordinary chlorophyll turnover. In Arabidopsis thaliana, two isozyme genes, chlorophyllase 1 (CLH1) and chlorophyllase 2 (CLH2) are isolated, that catalyze the first reaction of the degradation, and RCC reduction enzyme gene (RCCR) that catalyzes later stage of degradation is also isolated. In this study, we analyzed the expression mainly on CLH1 during aging and development of Arabidopsis.
CLH1 mRNA remarkably accumulated in several hours after treatment with methyl jasmonate. In contrast, gibberellin and cytokinin had no effect.
Analysis of a transformant containing GUS fusion gene with CLH1 promoter indicated that CLH1 was expressed in green tissues such as leaf, sepal and silique.
The analysises of the expression of CLH2 and RCCR will be reported.

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Citrulline ia accumulated up to 0.3 M in wild watermelon leaves and functions as a hydroxyl radical scavenger under drought condition.However, the mechanism of the accumulation is unclear.
Then, we analyzed glutamate acetyltransferase (GAT) which catalyzes the first and fifth steps of citrulline biosythesis simultaneously.
The purified GAT enzyme was composed of two different kinds of peptides, of about 28 kDa.The N-terminal sequence of each peptide was homologous to the 37th to 49th and the 249th to 283rd residues of the GAT homolog from Arabidopsis thaliana.In yeast and Bucillus, a single chain precursor protein of GAT is self-cleaved leading to form the active heterotetrameric molecule.We are now analyzing the regulation in catalysis of this enzyme.

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Bisphenol A (BPA) is a potential endocrine disrupter, used as a stabilizer for many types of plastics. It has recently been a cause for anxiety because of the environmental release of BPA. It has been demonstrated that BPA is metabolized to BPA-ο-β-D-glucopyranoside (BPAG) in the tobacco seedlings (Plant Cell Physiol. 2002, 43: 1036-1042). This reaction may be catalyzed by BPA specific glucosyltransferase (BPAGTase). In this study, we identified BPAGTase activity in cell-free extract of both tobacco suspension cultured cells (Nicotiana tabacum cv. BY-2) and seedlings (Nicotiana tabacum cv. Xanthi NC). Km of BPAGTase for BPA was 82μM. This value was equivalent to those of other plant glucosyltransferases. Specific activity in the leaves was 4 fold higher than that in the roots. Our results indicated that tobacco plants have BPAGTase to metabolize to BPA and leaf tissue may be mainly responsible for the β-glucosylation of BPA.

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The interaction network of double-stranded RNA (dsRNA) and dsRNA-binding proteins (dsRBPs) is a critical component of various biological processes including gene silencing. We identified seven genes that encode putative dsRBPs, HYL1, ADB2, ADB3, ADB4, CAF, RTL1 and CPL1, in the genome of Arabidopsis thaliana. All seven putative dsRBPs have two conserved dsRNA-binding motifs (dsRBMs) consisting of about 70 amino acid residues, similar to prototypical dsRBMs, such as those of human PKR. Using bacterially produced recombinant proteins, and Northwestern and Far-Westernanalysis, we characterized in vitro dsRNA binding activity and protein-protein interactions for these seven putative dsRBPs. By Northwestern analysis, we demonstrated that HYL1, ADB2, ADB3, ADB4 and CAF bound the 500-bp dsRNA specifically, whereas RTL1 and CPL1 did not. Results of Far-Western analysis indicate that these dsRBPs specifically interact to each other via their dsRBMs.

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Tulip flower oscillation, opening and closing of petals is repeated at least two weeks from the first flowering and then petals fell down. We could produce this petal oscillation in the dark by changing the temperature accordingly. During oscillation, opposite petals apertures were proportional to water content transferred via the stem from medium, the amount was almost three times at 20C compared to at 5C. Ruthenium red, a Ca²⁺ channel blocker and BAPTA, a Ca²⁺ chelator inhibited petal opening and water movement at 20C almost by 80 and 90% respectively. But they had no any effect on petal closing and water movement at 5C. Phosphorylation of a 31 kDa membrane protein at serine and/or threonine site at mid-temperature by membrane associated CDPK, and its dephosphorylation at 5C suggested its possible involvement in the tulip petal oscillation.

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Three species of Symplocarpus, S. foetidus, S. nipponicus and S. nabekuraensis, are distributed in Japan. Except for S. foetidus, however, detailed measurements of heat-production and genes that confer thermogenic metabolism have been poorly investigated. In the present study, we have compared the levels of heat-production and gene expressions of mitochondrial uncoupling proteins (UCPs) and alternative oxidase (AOX), both of which act as an important heat generator, in S. foetidus and S. nipponicus. Continuous records of the spadix temperatures revealed that S. foetidus maintains a body temperature of around 20^oC even when the ambient temperature drops below freezing, while S. nipponicus does not show any significant increases in its body temperature. Interestingly, both S. foetidus and S. nipponicus accumulate high levels of UCPb and AOX transcripts in the spadix. These results suggest that expression of UCP and AOX genes does not fully explain the capacity of heat-production in the Symplocarpus family.

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The spadix of skunk cabbage, Symplocarpus foetidus, maintains an internal temperature of around 20 ^oC even when the ambient air temperature drops to freezing. In long-term measurements of the spadix temperatures, we found that distinct complex oscillations occur in time series of the spadix temperature. To clarify the relation between the observed temperature oscillation and the mechanism of thermoregulation, detailed analysis of changes in the spadix temperatures was performed using dynamical system theory. It was revealed that the oscillation showed chaotic behavior, and the number of factors that control such a complex oscillation were predicted to be two. Furthermore, it was shown that the oscillatory period of the spadix temperature was independent of the weight of the spadix. Based on these results, it was concluded that the thermoregulation of skunk cabbage could be modeled by an analogy to a damping oscillation without any parameters of the weight of the spadix.

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Cold-shock induced RNA binding protein family in Anabaena sp. PCC 7120 has an RNA recognition motif (RRM). In vitro RNA binding assay showed that the affinity to homopolymers of RbpA1 containing C-terminal glycine-rich domain was: poly(U) > poly(G) > poly(A) > poly(C), and the same result was obtained with RbpD lacking glycine-rich domain. Affinity of RbpG containing C-terminal long region was: poly(C) > poly(U) > poly(A) > poly(G). The R83E point mutant of RbpA1RRM84, an 84 amino acids protein containing RbpA1 RRM, was shown to lack affinity to poly(G). But in the present using full-length RbpA1 or RbpD constructs, point mutation of Arg, did not affect binding affinity. Though RbpA1 protein and RbpA1RRM84 protein showed the same tendency of binding to poly(A), poly(C) and poly(G), binding affinity was stronger in RbpA1. This suggests that the C-terminal region of RbpA1 is important in increasing binding affinity.

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A cold-regulated wheat cDNA, WCSP1, was identified and found to contain an N-terminal cold shock domain (CSD) with two consensus RNA binding motifs, and a glycine-rich region which is interspersed with three C-terminal Cys-X₂-Cys-X₄-His-X₄-Cys (CCHC) zinc fingers (J.Biol.Chem 277,35248). RNA and protein blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively. WCSP1 induction was cold-specific, because neither abscisic acid treatment, drought, salinity nor heat stress induced WCSP1 expression. Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers. Structural and expression similarities to E. coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation. EST database searching revealed that the CSD occurs widespread among diverse plant genera and is highly conserved (Plant Physiol. 131, in press). A complete CSD protein family of Arabidopsis was found to display a differential response to low temperature treatments.

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Protein phosphorylation and dephosphorylation are implicated in the development of freezing tolerance during cold acclimation in plants. We previously showed that freezing tolarance of moss Physcomitrella patens protonema cells increased by treatment with ABA, cold. We isolated the PARK gene encoding protein Ser/Thr kinase induced by ABA and cold. The PARK kinase domain was similar to S-domain receptor like protein kinase, but it did not possess the extracellular domain.
The GST-PARK fusion protein phosphorylated histon IIIS and myelin basic protein and that the kinase itself underwent rapid autophosphorylation. When the PARK-GFP fusion gene was introduced into plant cells, the fluorescence signal was observed in the plasma membrane. Gene knock-out plants of PARK had reduced survival rates after freezing as compared to the wild type plants. These results indicate that PARK might regulate signal transduction processes of ABA- or cold-induced freezing tolerance.

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Here we report the isolation and characterization of a novel bZIP-encoding cDNA from rice which belongs to the lip19 subfamily. The cDNA showed highest identity (60.9% and 86% at DNA and amino acid sequence levels, respectively) to OBF1 from maize, thus named OsOBF1 (Oryza sativa OBF1). The gene product, OsOBF1, localizes in nuclei and binds to the hexamer sequence 5'-ACGTCA-3' but is unable to bind the ocs sequence, differing from the DNA binding specificity of maize OBF1. In an in vitro system OsOBF1 not only takes a homodimer form but also a heterodimer form with LIP19. OsOBF1 transcripts were abundantly detected in young mature leaf blades, whereas lip19 ones were more accumulated in senescing leaf blades. Based on those data, we will discuss a function of OsOBF1 and a possible role in transcriptional regulation of a heterodimer of OsOBF1 and LIP19 in growth development and stress response in rice plants.

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A rapid synthesis and accumulation of small heat shock proteins (sHSPs) upon heat-shock is one of the important cellular responses in heat stressed higher plants. However, function of plant sHSP remains unclear at present. In the present study, we have shown that ER-sHSP confers thermal protection of other proteins in vitro.
Tomato endoplasmic reticulum-located sHSP (ER-sHSP) cDNA (LeHSP21.4) was successfully cloned. Northern-blot analysis revealed the heat-inducible character of LeHSP21.4 mRNA. To use the recombinant LeHSP21.4, the molecular chaperone function of ER-sHSP was confirmed in vitro. The recombinant LeHSP21.4 was able to enhance the renaturation of chemically denatured lactate dehydrogenase (LDH) and citrate synthase (CS). The protein prevented the thermal aggregation and inactivation of CS at 45^oC. Most of proteins of E. coli cell extracts containing the ER-sHSP were protected from heat-denatured precipitation, whereas extracts from cells not expressing the protein were very heat-sensitive at these conditions.

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When organisms subjected to heat stress, patterns of gene expression change in a cell. Exclusive synthesis of heat shock proteins (HSPs) mRNAs occurs and these HSP mRNAs are efficiently translated while translation of existing normal cellular mRNAs is suppressed. It has been reported the 5' untranslated region (5'-UTR) of mRNA of HSP gene plays an important role in the efficient translation during heat stress. We report possibility that the expression of Arabidopsis thaliana HSP gene, HSP18.2, was also regulated at translational level under heat stress condition. We show translation was not suppressed during heat stress when the 5'-UTR sequence of HSP18.2 was inserted at upstream of initiation codon of β-glucuronidase gene in A. thaliana T87 protoplasts, Nicotiana tabacum BY-2 protoplasts and stable transgenic BY-2 clones. We also show that the secondary structure of 5'-UTR of HSP18.2 was unchanged by heat treatment in vitro.

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The psychrophilic bacterium, Colwellia maris, grows at low temperatures (5-15^oC). Although groESL and dnaK genes of this bacterium are expressed at 20^oC, their promoter regions are similar to the heat shock promoter of E. coli. To reveal specific mechanisms of heat shock response in psychrophiles, we cloned the rpoH gene and investigated its expression.
Using mixed oligonucleotides primers designed from conserved amino acid sequences in several RpoH, a partial fragment of the rpoH gene was obtained. Its deduced amino acid sequence showed 64% identity to the corresponding region of E.coli RpoH. The RpoH box, which is specific to RpoH, was identified in this DNA fragment. Northern blot analysis showed that the level of rpoH transcript reached the maximum level when treated at 20^oC and that the rate of degradation was much slower at 20^oC than at 10^oC. These observations suggest that rpoH mRNA stability contribute to RpoH regulatrion.

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Previously, we showed that heat shock strongly enhanced accumulation of transcripts of the htpG and groEL genes in Synechocystis sp. PCC 6803 in the light, but heat induction was much lower in the dark. DCMU and DBMIB differentially affected transcript accumulation of these genes, suggesting that the redox poise of the plastoquinone pool is critical for the HSP gene expression.
In the present study, we examined the effect of light on the heat shock response in detail. Light did not affect stability of the HSP mRNA significantly. The level of the groEL and hsp17 mRNAs did not decrease in the dark for 3 hours after a heat shock. Heat shock strongly enhanced accumulation of the GroEL protein in the light. The acquired thermo-tolerance was greater in the light than in the dark. These results indicate that cyanobacteria have evolved a unique regulatory mechanism by light to induce heat shock genes.

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In the Synechocystis sp. PCC 6803 genome, an ORF encoding a HrcA homolog is present and the DNA element CIRCE has been identified around the transcriptional start site of the two groEL genes, groEL1 and groEL2, suggesting that the groEL genes are controlled by the CIRCE/HrcA system. The HrcA repressor interacts with CIRCE and controls eubacterial dnaK and/or groEL operons.
We inactivated the hrcA gene in Synechocystis sp. PCC 6803. In the hrcA mutant, the groEL genes were derepressed at 30^oC. But their expressions were not fully derepressed, suggesting that another level of heat shock control exists.
The inactivation of the hrcA gene did not affect the expression of other heat shock genes such as clpB, htpG, dnaK, hspA at a normal growth temperature. However, some change in the heat shock response of these genes was brought about by the mutation.

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HSP90/HtpG constitutes one of the most abundant and conserved heat-shock protein (HSP) families. Compared with HSP90 in eukaryotes, little is known about a role for the HSP90 homologues in prokaryotes due to the lack of a mutant that shows a clear phenotype under stress conditions.
Previously, we showed that the inactivation of the htpG gene from the cyanobacterium Synechococcus sp. PCC 7942 causes loss in thermotolerance, indicating an indispensable role of HtpG for the survival under heat stress.
In the present study, we over-expressed the cyanobacterial htpG gene in E. coli and purified the HtpG protein. HtpG forms a homo-dimer and prevented the irreversible aggregation of a model substrate such as citrate synthase by heat. On the other hand, a truncated form of HtpG lacking the C-terminal 124 amino acids could not form an oligomer, indicating that the region is important for the oligomer formation.

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Previously, we reported that the constitutive expression of HspA, a small heat-shock protein (HSP) increased thermal resistance of PSII and protected phycocyanin from heat-induced photo-bleaching in Synechococcus sp. PCC 7942. These results indicate that a small HSP plays a role in the protection of the light harvesting phycobilisome and the electron transport system under heat stress.
In the present study, we examined whether molecular chaperones such as small HSP and GroEL may interact with phycobiliproteins. We purified phycobilisome from the mutant by the sucrose density gradient ultracentrifugation method, resulting in the separation of the heavy and light fractions containing phycobiliproteins. The heavy fraction contained purified phycobilisomes. The light fraction contained GroEL and HspA as well as phycobiliproteins. The dissociation of phycobilisome may have produced an intermediate complex which interacts with these molecular chaperones. In vitro experiments are in progress to show the physical interaction between molecular chaperones and phycobiliproteins.

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The photosynthetic antenna complex is composed of the core and peripheral antenna. While size and composition of the core antenna are not influenced by the amount of light, those of the peripheral antenna could vary under the different light conditions. Details for this light acclimation have not been well understood.
We first isolated a complex of the photosystemII and the LHCII from a green alga C.reinhardtii grown under the normal conditions. The obtained complex was examined by SDS-PAGE. Bands corresponding to the antenna subunits were then determined by microsequencing. Seven subunits of LHCI and three of LHCII were identified together with information from the EST library. Composition of the subunits which constitutes for the peripheral antenna was further investigated by using the membranes prepared under different light conditions. Acclimation of the light harvesting antenna to environmental light will be discussed.

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Higher plants change their photosynthetic antenna size upon acclimation to different light environments. In our previous studies, we revealed that expression of chlorophyllide a oxygenase (CAO) controls antenna size. Transgenic Arabidopsis plants overexpressing CAO have large antenna size and increased amounts of LHC proteins. To understand the roles of CAO proteins in light acclimation, we investigated the effects of alterations in CAO expression in the transgenic plants and ch1-1 mutant whose CAO gene is deleted. (1) The degree of thylakoid membrane stacking in the transgenic plants was same as that in the wild-type plants. ch1-1 lacks most of LHC proteins, but showed clear membrane stacking. (2) The de-epoxydation rate of xanthophyll-cycle pigments were higher in ch1-1 and lower in the CAO-overexpressing plants than that in the wild-type plants. Based on the results, we discuss the mechanism of light acclimation in higher plants.

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Chlorophyllide a oxygenase (CAO) synthesizes chlorophyll b from chlorophyll a and is encoded by nuclear genomes in higher plants. The enzyme regulates the formation of light harvesting complex (LHC) by controlling the synthesis of chlorophyll b. It is important to determine the localization of CAO in chroloplasts to understand the mechanism of LHC formation.
Comparison of various CAO amino acid sequences and computer prediction analysis showed that Arabidopsis CAO is composed of four domains, i.e., transit peptide (36 residues), A domain (134), B domain (30) and C domain (336). In this study, GFP (green fluorescence protein) fused with various CAO domain(s) were expressed transiently in pea leaves or stably in Arabidopsis transformants. The localization of CAO domain(s)-GFP was investigated by fluorescence microscopy. The results showed the different localization patterns of these proteins, suggested that the C domain has an important role in localization of CAO in chloroplasts.

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Photoautotrophic cells provide a unique system for photosynthetic research. We have studied the high light adaptation of photoautotrophic cell culture of Petunia hybrida. The cells grown under high light condition (HL cells) had larger total pool size of xanthophyll cycle pigments, that is total contents of violaxanthin, antheraxanthin and zeaxanthin, and higher violaxanthin ratio which can be converted by zeaxanthin after photoinhibitory treatment in comparison with low light grown cells (LL cells). The distribution of the xanthophyll cycle pigments among photosynthetic pigment-protein complexes in the thylakoid was examined after dark adaptation and after photoinhibitory treatment. The fraction containing major LHCII and minor LHCII induced more zeaxanthin after photoinhibitory treatment in both cells. LHCII proteins in HL cells had more xanthophyll molecules per polypeptide and higher zeaxanthin even after dark adaptation, suggesting that the LHCII proteins in HL cell had extra zeaxanthin.

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Photoinhibition of photosynthesis was studied in Vicia faba leaf discs at chilling (4oC), room (25oC) and high (40oC) temperature under 2000 *mole quanta m-2s-1. Quantum efficiency of PS II (FV/FM) showed a linear time dependent decrease at room temperature. The decrease was further enhanced at chilling temperature and maximum at high temperature.
The far-red (FR) light (13.5W.m-2) induced *I/I0 at 830nm (P700+FR) and FR+SP (saturating-pulse) induced P700+TOT in vivo, decreased with photoinhibition. High temperature treatment accelerated the decrease in P700+FR and P700+TOT in darkness. High light accelerated the decrease in P700+FR but caused no significant change in the P700+TOT compared to that in darkness at room temperature, suggesting that P700 pool could not be fully oxidised during photoinhibition at room temperature. However, chilling temperature caused severe damage (*80%) to P700+TOT. This study shows a characteristic difference in the temperature dependent changes in PS I activity under photoinhibitory condition.

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The effects of coldness on Saintpaulia leaf vary depending on the modes of cooling. When cooled rapidly to 5C, Saintpaulia leaf lost variable chlorophyll fluorescence (Fv) and was led to irreversible injuries of chlorosis, but when cooled to 5C at the rate of 1C/min or slower, the leaf was free from this injury and showed normal photosynthetic oxygen evolution at room temperature. This slowly cooled leaf was readily injured if recovered to room temperature soon after cooled to 5C and exposed to sudden chilling, while if incubated at 5C for10 min or longer, the slowly chilled leaf was found to be tolerant to sudden cooling, indicating that this plant is not cold labile natively and that the conversion to cold hardy leaf was achieved within 10 min. It was assumed that the sudden cooling cause destabilization of cells, whereas this reaction was inactivated during the incubation at 5C.

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We have shown that in the C₄ plant maize, CO₂ assimilation does not start in the absence of O₂, but after photosynthesis starts once, CO₂ assimilation is able to proceed even under anaerobic conditions. In order to understand a mechanism why O₂ is required to start CO₂ assimilation, detailed analysis under various O₂ concentrations was done.
In 2% O₂, CO₂ assimilation and transpiration rates at the steady state after dark-adaptation were 80% compared with those in 21% O₂. ΦPS2 and ΦPS1 were 70% and 100%, respectively. In O₂ lower than 2%, these parameters were further lowered. In 0% O₂, CO₂ assimilation rate and ΦPS1 were 15% and 40%, respectively, compared with those in 21% O₂. ΦPS2 and transpiration were close to zero. These results suggest that O₂ is required to start water-water cycle, and it produces essential ATP for CO₂ assimilation cycle and opening of the guard cell.

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Ascorbate peroxidase (APX) isoenzymes are known to play an important role in scavenging H₂O₂ in higher plants. APX utilizes ascorbate (AsA) as a specific electron-donor in the scavenging reaction. The object of this report is to elucidate the reaction mechanism of APX by the X-ray structure analysis and the single-crystal microspectroscopy. The crystals of APX were soaked in the reservoir solution containing 10 μM H₂O₂ or a mixture of H₂O₂ and 100 mM AsA and frozen rapidly at 100 K. Diffraction data were collected at SPring-8. The side-chain of Arg, one of the catalytic residues, was found to be shifted toward the ferryl-oxygen in the crystal soaked in H₂O₂. The electron density of the crystal soaked in H₂O₂ and AsA showed the location and orientation of AsA. The structural features and absorption spectra provided invaluable information for the reaction mechanism of APX.

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We elucidated the inactivation mechanism of ascorbate (Asc) peroxidase (APX) in chloroplasts. In the presence of nigericin, illumination of intact chloroplasts decreased electron flux in PSII (Je(PSII)), with the uptake of O₂. APX was simultaneously inactivated. These results show that inactivation of APX suppressed the consumption of electron from PSII for the regeneration of Asc. On the other hand, in the absence of nigericin, both the decrease in Je(PSII) and O₂ uptake were suppressed. This indicates that non-photochemical quenching and cyclic electron flow within PSII protect WWC activity by preventing H₂O₂ production in its cycle. Furthermore, we found that APX reaction rate in chloroplasts depended on H₂O₂ production rate and that Asc regeneration did not limit H₂O₂ scavenging. These results indicate that even in the presence of Asc, H₂O₂ produced in WWC decomposes APX intermediates, that is, functioning of WWC is dangerous for APX.

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Tetrapyrrole compounds, such as chlorophylls and hemes, are synthesized in many enzymatic steps. For regulation of tetrapyrrole metabolic pathway, it is generally considered that several specific isoforms catalyzing particular enzymatic steps control the flow of tetrapyrrole intermediates by differential regulation of gene expression depending on environmental and developmental factors. However, coordination of such regulatory steps and orchestration of total tetrapyrrole metabolic pathway are still poorly known. In this study, we developed miniarray system, which enables to monitor the specific gene expression profiling of total tetrapyrrole metabolic pathway. Gene specific regions of total 35 Arabidopsis genes corresponding to tetrapyrrole metabolism were amplified by RT-PCR, and used as probes for the miniarray system. After confirmation of the specificity of these probes by genomic Southern hybridization, all probes were spotted onto nylon membranes together with light- and phytohormone-responsive genes as control. Results of gene expression profiling with this miniarray system will be presented.

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Most phototrophic organisms including cyanobacteria synthesize chlorophyll by the two different protochlorophyllide (Pchlide) reductases; one is light-independent reductase (DPOR) and the other is light-dependent reductase (LPOR). We report here differentiation of the two Pchlide reductases in response to the environmental oxygen concentration in the cyanobacterium Plectonema boryanum.
A DPOR-less mutant (YFC2), an LPOR-less mutant (YFP12) and the wild type of P. boryanum were cultivated photoautotrophically bubbling with mixture gas (N₂-2%CO₂) containing various amount of oxygen (0-21%) under high light conditions. While YFC2 grew as well as the wild type in all examined conditions, YFP12 grew only under the oxygen concentrations less than 5% with the maximal growth rate (ca 62% of the wild type) at 0% O₂. This result indicated that LPOR is essential for growth under the conditions with oxygen more than 5%. An evolutionary aspect of chlorophyll biosynthesis will be also discussed.

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NADPH-protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide a. Three differentially regulated POR isoforms have isolated from Arabidopsis thaliana. To elucidate the physiological function of each POR isoform, we screened POR mutants in T-DNA tagged populations. Isolation of porB mutant has previously presented at 2001th JSPP meeting, and here we present the isolation and analysis of T-DNA knockout porC mutants. Two independent porC mutants were obtained after screening. No detectable PORC was accumulated in two mutants, indicating both mutants are null mutation of PORC. Like porB, porC seedlings grown under white light showed comparable Chl accumulation and photosynthetic activity to those of wild type. However, only porB showed higher sensitivity to the far-red block of greening response. These results suggest that PORB and PORC are functionally redundant under normal growth conditions. However, under limited environmental or developmental conditions, expression of particular POR isoforms is important for normal chloroplast development.

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Chlorella, when heterotrophically cultivated in the dark in the presence of 33 mmol/L of Zn, starts to proliferate after a long lag phase of about one or two months. We found that under these conditions, Zn-Chl a is gradually accumulated during the stationary phase. (1) Whereas only a small amount of Chlide a was detected during long lag phase, Chls start to be synthesized as the cells start to grow. (2) Pheo a and a small amount of Chl b were first accumulated during the early log phase. (3) Zn-Chl a was gradually accumulated and finally reached to about 10% of Chl a in the late stationary phase. (4) Low-temperature fluorescence of Chl a shows that F685 was prominent in the early log phase, while F725 became prominent in the late stationary phase. (5) These results suggest that Zn-Chl a is incorporated and act as antenna in Chl-proteins.

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In dicotyledonous plants, the apical region of the embryo acquires bilateral symmetry in addition to previous radial symmetry as two cotyledons develop at the opposite position. Mutations in the PIN1 (PIN-FORMED1) and PID (PINOID), cause comparable defects in seedlings, as cotyledons fusion and modified cotyledon number. The pin1 pid double mutantion severely suppressed the cotyledon development. In these mutants, we analyzed expression patterns of several marker genes. The results suggest that PIN1 is involved in establishment of bilateral pattern, that PID is involved in maintaining bilateral symmetry, and that PIN1 and PID coordinately promote cotyledon development through the regulation of CUC genes expression. To understand auxin distribution, we also analyzed expression pattern of YUCCA, which encodes the enzyme involved in IAA biosynthesis. Depending on these results, we want to discuss that auxin distribution through PIN1 and PID is involved in cotyledon development and cotyledon separation with bilateral symmetry.

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CUC1 (CUP-SHAPED COTYLEDON1) and CUC2, redundantly involved in SAM formation and organ separation during embryogenesis, encode NAC domain proteins respectively. Each cuc1 or cuc2 single mutant results in no obvious phenotype, but cuc1 cuc2 double mutant has a cup-shaped cotyledon and lacks an embryonic SAM. To identify genes, involved in these processes together with CUC2, we isolated several enhancers of cuc2 exhibiting a cup-shaped cotyledon phenotype. While more than half of enhancers are novel cuc1 alleles, some have mutations in a NAC box gene (CUC3). We are investigating the role of CUC3 in SAM formation and organ separation.

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The shoot apical meristem (SAM) is responsible for the morphogenetic events in plants. In order to understand the mechanisms underlie the SAM formation and organization, we tried to isolate two genes named SHOOT ORGANIZATION1 (SHO1) and SHOOTLESS2 (SHL2) from seedling lethal and embryo lethal mutants, sho1 and shl2, respectively. Defects in sho1 mutant include abnormal leaf shape, short plastochron, and irregular phyllotaxis, and those defects are supposed to be caused by flattened shape of sho1 SAM. shl2 lacks SAM during embryogenesis. Recently, weak alleles of shl2 were isolated and those showed quite similar phenotypes to sho1. This indicates that SHO1 and SHL2 could act in the same pathway in the SAM formation or the organization. Map based cloning of these genes revealed that both genes encode proteins related to RNAi interference (RNAi) pathway. This result suggests that genes related to RNAi mechanisms regulate the shoot formation and organization in rice.

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Plant leaves develop from a shoot apical meristem along three axes (adaxial-abaxial, proximal-distal, medial-lateral axes). The asymmetric leaves2 (as2) mutants generate leaf lobes and leaflet-like structures in bilaterally asymmetric manner. To understand the molecular mechanisms behind the leaf development, we have isolated the AS2 gene. AS2 encodes a novel protein involved in a gene family with 42 members in Arabidopsis. The N-teminal region of AS2 which contains C-motif (Cx2Cx6Cx3C) and leucine-zipper-like structure is conserved in all proteins encoded by this family. We designated it AS2 domain. The as1 mutant shows the phenotypes similar to those of as2. AS1 encodes a MYB related protein. Using immunoprecipitation experiments, we showed that AS1 and AS2 interact in vitro. Expression analysis showed that AS1 and AS2 are expressed in overlapping regions of the leaf primordia. Our results suggest that AS1 and AS2 forms protein complex, which may regulate an early stage of leaf development.

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Building of the plant architecture is achieved by the highly organized growth and differentiation of cells. Arabidopsis mutant, asymmetric leaves2 (as2), exhibited ectopic leaf lobe and altered leaf venation pattern in asymmetric manner. Shoot regeneration potency from leaves of as2 mutant is higher than that of wild type. Leaf is generated and differentiated from totipotent shoot apical meristem (SAM). Furthermore class1-KNOX genes which encode SAM-related homeobox proteins were expressed ectopically in developed leaves of as2 mutant. Thus AS2 may be involved in spatially and/or temporally proper cell differentiation and also in direct or indirect repression of class1-KNOX. AS2 gene encodes a protein that belonged to a novel family (named AS2 family). AS2 interacted with Myb-like transcription factor ASYMMETRIC LEAVES1. To further understand how AS2 regulates the differentiation of cells and the expression of class1-KNOX, we carried out screening of another factors that interacted with AS2 physically and/or genetically.

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The eye (embryo yellow) mutant that has defects in the maturation stage of the embryogenesis was isolated from a screening of T-DNA tagged Arabidopsis lines. The germinated plants of the eye mutant were dwarf and formed many adventitious leaves in the shoot apex. The EYE gene was predicted to encode a protein with a putative coiled-coil region near the amino terminus. Phenotypes of the eye mutant were reversed by expression of the truncated EYE protein that includes this region. This result indicates that this region plays a critical role in a normal development of the plant body supported by the EYE protein. The predicted EYE protein was homologous to Cog7 that is a subunit of a mammalian Golgi-localized COG complex. Phenotypes of the eye mutant might be caused by defects in the function of the Golgi apparatus.

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We isolated a rice orthologous gene to Arabidopsis SCR(OsSCR) and investigated its expression in root and around the shoot apical region. OsSCR was specifically expressed in the endodermis layer and QC cells of root in the rice plant. Interestingly, OsSCR was also expressed at high level in almost cells of young leaf primordia (P1 and P2) except cells, which would form vascular tissues. As progress of the leaf development, the OsSCR expression was localized to the specific cell file, which would develop stomata at the later stage, then the expression was localized into the guard mother cells (GMC) and subsidiary cells. OsSCR expression was down-regulated in GMC when it was divided symmetrically. The expression pattern of OsSCR suggests that OsSCR is involved in the asymmetric cell division not only of the endodermis/cortex initial cells in root but also in the process of stomata development.

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Axillary buds developing at the axils of leaves produce additional shoot systems, lateral branches. The rice TB1 gene (OsTB1) was first identified based on its sequence similarity with maize TEOSINTE BRANCHED 1 (TB1), which is involved in lateral branching in maize. Both genes encode putative transcription factors carrying a basic helix-loop-helix type of DNA-binding motif, named the TCP domain. Transgenic rice plants overexpressing OsTB1 exhibited markedly reduced lateral branching without the propagation of axillary buds. We also demonstrated that a rice strain carrying a classical marker mutation, fine culm 1 (fc1), contains the loss-of-function mutation of OsTB1 and exhibits enhanced lateral branching. Expression of OsTB1 was observed throughout the axillary bud, as well as the basal part of the shoot apical meristem, vascular tissues in the pith and the lamina joint. Taking these data together, we concluded that OsTB1 functions as a negative regulator for lateral branching in rice.

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