We have used imaging of chlorophyll fluorescence to screen the T-DNA-tagged Arabidopsis mutants. To isolate mutants defective in high light response, plants were exposed to two types of high light stress; short-term and log-term irradiation. To observe short-term response, transient fluorescence kinetics was monitored during short irradiation (12 sec). For long-term treatment, plants were exposed to high light for 24 hr and then chlorophyll fluorescence was measured. To date, we screened 8,000 seedlings and isolated nine candidates showing different fluorescence kinetics from that of WT. Six out of nine mutants had defects in response to short-term irradiation. The rest of the mutants showed lower fluorescence intensity than WT only after 24 hr of high light. PAM fluorescence measurements indicate that several mutants differ in photosynthetic parameters (qP, qN, ETR, Fv/Fm) from WT, indicating that various types of mutants can be isolated by this screening system.

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Excess light exposure leads to inhibition of photosynthetic electron transport and photooxidative stress in plants. In this study we characterised photooxidative stress response of the knockout mutant of A. thaliana in signal recognition particle (cpSRP43) named chaos, which has permanently reduced photosystem II antenna. Chlorophyll fluorescence parameter and oxygen evolution data indicated that photosynthesis was more strongly inhibited by excess light (2,000 μmol/m²/s) illumination in chaos compared to WT. However, when returned to low light conditions chaos recovered faster from photoinhibition. ATP/ADP ratio was higher in chaos than in WT in low light-acclimated condition. When plants were subjected to photooxidative stress conditions (650 μmol/m²/s, 4 °C), chaos exhibited better photosynthetic performance and survival compared to that observed for WT. It is concluded that a lack of functional cpSRP43 protein leads to a higher energy conservation and to constitutive tolerance to photooxidative stresses.

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Singlet oxygen, generated by photodynamic actions, is a strong oxidant that potentially damages various molecules of biological importance. We investigated the action in vivo of singlet oxygen in the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen by the presence of rose bengal resulted in inhibition of the repair of the photodamaged PSII and not in acceleration of direct damage to PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, that of the D1 protein. Northern blot analysis indicated that the accumulation of psbA mRNAs for the D1 protein was unaffected by the production of singlet oxygen, suggesting that the primary target of singlet oxygen might be translation.

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Glycinebetaine (betaine) is one of the compatible solutes and is accumulated in a large number of organisms in response to stress conditions. It has been demonstrated in transgene systems that betaine enhances tolerance against various kinds of stress. Synechococcus sp. PCC7942 which has been transformed with the codA gene retains the ability to accumulate betaine when choline is exogenously supplied as the substrate. We found that the transformation enhanced the tolerance of cell against cold stress via the increased ability to repair the photodamaged photosystem II. However, at normal growth temperature, the betaine synthesized in vivo only slightly enhanced the tolerance to photoinhibition. This observation may suggest that the repair of photodamaged photosystem II is sufficiently fast under normal growth conditions and is not enhanced further by betaine. We are now characterizing the effect of betaine on photoinhibition under salt stress.

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At the last meeting, we reported that hypertonic treatments of a terrestrial, highly drought-tolerant cyanobacterium, Nostoc commune, can mimic drying processes in this cyanobacterium. We also reported that N. commune actively (not passively) deactivates photosynthetic systems at an early stage of drying.
In order to further study how N. commune changes photochemical reaction center complexes in various stages of desiccation, we observed the effects of light and high temperatures under various osmotic conditions. In N. commune, the PSII activity was not decreased by light, and its temperature sensitivity shifted to a higher value by 5^oC. On the contrary, in Synechocystis sp. PCC6803, which has no desiccation tolerance, the PSII activity was further inhibited by light, and the temperature sensitivity was not changed under hypertonic conditions.
We will also discuss on effects of liquid cultivation on drying stress in various strains of N. commune.

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Freezing tolerance of cyanobacteria, Nostoc commune and Synechocystis sp. PCC6803, and the effects of alcohol were studied by measuring their photosynthetic activities. Because freezing stress usually causes water stress simultaneously, the effects of freezing stress were compared with those of drying stress. N. commune is terrestrial and is highly tolerant to drying stress, while Synechocystis is not. The following results were obtained. 1) N. Commune can survive freezing stress even at temperatures as low as -78^oC, while Synechocystis could not when it was frozen at -20^oC. 2) Decreases in fluorescence emission from photosystem (PS) II and photochemical activities of PSI and PSII showed that water loss from the N. commune cells was not so large as in the case of air drying. 3) Addition of 5% methanol to the Synechocystis solution protected the cyanobacterium from freezing damage. The effects of alcohol will be discussed.

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In the present study we investigated the effect of the Calvin cycle inhibition on photoinactivation of PSII. When Chlamydomonas reinhardtii (wild type) cells were exposed to light, glycolaldehyde (GA), which inhibits the Calvin cycle, stimulated the photoinactivation. The effect of GA on the photoinactivation was similar to that of chloramphenicol. The photoinactivation in a Rubisco missense mutant (10-6C) was faster than that in wild-type. However, it was not further accelerated by GA and chloramphenicol. After photoinactivation treatment, PSII activity in wild-type cells gradually recovered under low light. However, no significant recovery was observed when the Calvin cycle is inhibited. The inhibition of the Calvin cycle suppressed the synthesis of proteins, which are encoded in chloroplastic genome. These observations suggest that the level of photoinactivation is enhanced by the inhibition of the repair system when the Calvin cycle is inhibited by high temperature.

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We have recently found in Arabidopsis thaliana a novel, oxidative stress-induced enzyme NADPH:2-alkenal α,β-hydrogenase (ALH) that catalyzes the α,β-hydrogenation of 2-alkenals, the lipid peroxide-derived endogenous toxins. We made ALH-overproducing transgenic tobaccos to verify the in vivo function of ALH. Three homozygous transgenic lines, whose leaves contained >20 times higher ALH activity than in the non-transgenics, showed higher tolerance against 4-hydroxynonenal (HNE), the major 2-alkenal, which inactivated PSII and caused necrosis, when administered to the leaves. The transgenics also showed higher tolerance against methylviologen (MV)-light treatment. The MV tolerance was solely attributable to the HNE detoxication by ALH, because the tolerances against HNE and against MV were correlated each other, while the levels of major known antioxidant enzymes, e.g. SOD, APX and catalase, in transgenic and control plants were the same. Thus, the lipid peroxide-derived 2-alkenals are injuring molecules in photooxidative stress. ALH protect cells by detoxifying them.

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Chloroplast NAD(P)H dehydrogenase complex (NDH) reduces the plastoquinone pool nonphotochemically, which functions in cyclic electron flow around photosystem I. In order to clarify the physiological role of NDH, we isolated nuclear mutants, crr2 (chlororespiratory reduction 2-1, 2-2) affected in the chloroplastic NDH activity in Arabidopsis by using chlorophyll fluorescence imaging system. Accumulation of NDH complex is specifically impaired in crr2 alleles. Consequently, crr2 lacks the NDH activity drastically, although the photosynthetic electron transport is unaffected. CRR2 encodes a member of AtPCMP family which is related with the PPR proteins predicted to function in the post-transcriptional regulation of organellar gene expression. Northern analysis revealed that crr2 has a defect in the intergenic processing of chloroplast RNA between rps7 and ndhB. We conclude that CRR2-dependent intergenic processing is essential for the ndhB translation and the formation of NDH complex.

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Cyclic electron flow was first discovered based on its coupling with ATP synthesis approximately 50 years ago. However, it has been still matter of debate whether it contributes to photosynthesis. Two redundant pathways including FQR and NDH function in this electron flow. In contrast to the main pathway, FQR, which is essential for ΔpH generation to induce NPQ, NDH possibly functions compensatory to prevent the over-reduction of the stroma. To clarify the physiological function of cyclic electron flow around PSI, we created a double mutant between pgr5 lacking the FQR activity and crr2 affected in the ndhB expression. crr2 pgr5 was completely defective in cyclic activity and cannot grow even under low light conditions. In crr2 pgr5, electron transport was drastically restricted and PSI was highly reduced because of the stromal over-reduction. We conclude that cyclic electron flow is essential for the photosynthesis to maintain the proper ATP/NADPH production ratio.

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To monitor over-reduction in stroma induced by photo-oxidative stress, we employed a simple non-destructive method to monitor redox state around photosystem I before and after high-light illumination based on the level of P700 oxidation by far-red light (FR). Oxidation level of P700 induced by FR illumination decreased during daytime and recovered at night. The recovery in the dark of once decreased oxidation level of P700 proceeded slowly with a half recovery time of about 5 hours. This depression of was probably due to cyclic electron donation to P700 or charge recombination within PSI. In either case, stromal over-reduction induced by sunlight was the direct cause, since the depression of P700 oxidation under FR was reproduced by illumination with saturating white light to dark-adapted leaves. Possible effects on photosynthetic activity of over-reduction induced by high-light and their physiological relevance is discussed.

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In order to reveal the molecular mechanisms of unusual pigmentation patterns of flowering peach, gene expression of pink-coloured flowers and variegated ones were investigated by RTPCR Southern-hybridization. The CHS gene was strongly expressed in both of pink-coloured flower buds and variegated ones. But the DFR gene and the AS gene were strongly expressed only in pink-coloured flower buds. Therefore variegation considered to be caused by a transcription factor regulating the expression of structural genes in the later part of anthocyanin synthesis. Complementation of pigment synthesis was attempted by introducing five genes encoding Antirrhinum transcription factors into variegated flowers by particle bombardment. Red spots were reverted in petals by Rosea1 one of MYB-like transcription factors. Two MYB-like clones expressed only in pink-coloured flower buds were obtained by RTPCR cloning as a candidate responsible for the variegation.

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Molecular analysis of the FWA gene revealed that all fwa mutants are gain-of-function epigenetic alleles of a homeodomain-containing putative transcription factor (Soppe W. et al. 2000). Ectopic expression of the FWA gene is associated with hypomethylation of the 5' direct repeats of the gene, and induces a late flowering phenotype. The heritable epigenetic late flowering trait is also found in the repeatedly selfed ddm1 (decrease in DNA methylation) mutant.
To understand epigenetic character of the FWA gene, we investigated parent-of-origin-dependent gene expression in the endosperm. The result indicated that the expression was observed only in the maternal allele, whereas the paternal allele was silenced. Furthermore, silencing of the paternal allele was not maintained in met1 (DNA methyltransferase1) or fwa-1 mutants. Based on these findings, we concluded that the FWA gene is a novel imprinted gene expressed in the endosperm.

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Closterium peracerosum-strigosum-littorale complex have two sexes, mating-type plus (mt⁺) and minus (mt^-) cells, and sexual reproduction in this species was easily induced in the laboratory. Two sex pheromones (PR-IP Inducer and PR-IP) were isolated and characterized. In addition, two sex pheromones, sexual cell division-inducing pheromone (SCD-IP-minus and SCD-IP-plus), were detected and evaluated.
SCD-IP-minus was released from mt^- cells and induced the SCD of mt⁺ cells and vice versa. Partially purified SCD-IP-minus showed quite similar characters to PR-IP Inducer, which induced the production of PR-IP in mt⁺ cells. Moreover, recombinant PR-IP Inducer produced in yeast induced the SCD of mt⁺ cells as well as the production of PR-IP in mt⁺ cells. The SCD could be induced by rather shorter period of treatment with the pheromone than the latter. It was strongly indicated that PR-IP Inducer exerted two different biological activities although their inducing mechanisms seemed to be different.

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As the first step to understand the comprehensive mechanism of sexual reproduction of Closterium, cDNA library was constructed from cells that were in the various processes of sexual reproduction and a total of 1,190 5'-end ESTs were established. The 604 ESTs of them were resulted in 174 redundant groups, thus the total number of unique sequences in EST set was 760. Similarity search against the public non-redundant protein database indicated that 370 groups showed no significant matches. Among them, homologues of previously known sex pheromones were detected. Using those non-redundant and other unidentified ESTs, cDNA microarrays containing 3072 cDNAs were prepared. As a first attempt, cDNAs prepared from cells in early stage of mating culture and in non-mating culture were used as probes. As a result, several genes expressed specifically in a mating culture were identified. These included genes encoding sex pheromones, their homologues, and a putative receptor-like protein kinase.

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Nitrogen (N) deprivation of Chlamydomonas reinhardtii cells triggers not only an adaptation program to acclimate to N starvation, but also a gamete program which permits them to mate to form zygotes. We searched genes from the Chlamydomonas cDNA macroarrays, whose expression is greatly enhanced under -N conditions, and isolated 19 novel genes, named Nsg (N-starved gametogenesis). In this study we analyzed their expression patterns during synchronized gametogenesis.
Synchronized vegetative cells in the early G₁ phase were incubated in -N medium. Gametic differentiation occurred between 2 and 4 h, and over 95% cells became gametes within 5 h incubation. Northern blot analyses showed that Nsg genes are classified into 3 temporal classes; early (1-2 h, 1 gene), middle (3-4 h, 15 genes) and late (6-7 h, 3 genes). Early and late genes appeared to be related to the adaptation program, while middle genes to be related to the gamete program.

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The dioecious plant, Silene latifolia has chromosomal distal end specific satellite DNA (Sl-distal-satDNA). Eleven BAC clones containing this satellite DNA were isolated from a partial BAC library of S. latifolia. They were digested with six restriction enzymes and hybridized with the Sl-distal-satDNA as a probe. This Southern blot demonstrated that they contain the Sl-distal-satDNA showing typical ladder patterns of repeated DNA. A phylogenetic tree was constructed using the neighbor-joining algorithm to investigate pair-wise similarities between individual repeat units of the Sl-distal-satDNA. The repeat units derived from eleven BAC clones formed four clusters named as SacI, KpnI, E and F family, respectively. Multi-colored FISH with the SacI family and the KpnI family probes demonstrated that the KpnI family is localized at only one end of chromosome 7. These results suggest that there is nucleotide diversity among individual families of the Sl-distal-satDNA.

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In the liverwort M. polymorpha, a specific Y chromosome is present in the male, and an X chromosome is found only in the female plants. This X/Y exclusiveness strongly suggests the presence of sex-determining factors on the sex chromosomes. The liverwort Y chromosome has an approximately 4-Mb region (YR1) which has accumulated its own repetitive sequences. The other part of the Y chromosome is designated as YR2 which contains genes unique to the Y chromosome. We have obtained draft sequences of 25 representative PAC clones and a 470-kb contig in YR1. Forty-five PAC clones were selected from two contigs (5.94 Mb) in YR2 and sequenced. Most genes identified in YR1 are present in multicopy. Contrastingly genes present in YR2 are mostly single-copied. Gene structure evolution of the Y chromosome and its genes in M. polymorpha as well as difference in gene structure between the X and Y chromosomes are discussed.

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Arabidopsis ABI3 gene is known as a transcriptional factor which expresses during plant embryogenesis from very early stage, but the mechanism for regulating the gene expression is still unclear. In our laboratory, C-ABI3, a carrot homologue of ABI3 had been isolated and showed that the expression was observed during somatic and zygotic embryogenesis from the early stage. In this study, we analyzed the mechanism which regulates C-ABI3 expression. At first, C-ABI3 promoter region was isolated, and a cis-regulatory element which relates to regulation of embryo-specific gene expression was identified and named as Carrot Embryonic Element 1 (CEE1). CEE1 regulated embryo-specific gene expression in carrot somatic embryos and Arabidopsis zygotic embryos. Moreover, the existence of some nuclear proteins which interact to CEE1 was suggested by EMSA data. By using yeast-one-hybrid system, we isolated seven CEE1 binding factors (CEE1BFs). All of the CEE1BFs genes expressed in carrot somatic and zygotic embryos.

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ABI3, LEC1 and FUS3 are thought to be major transcriptional activators expressed specifically during seed formation. Deficient mutants of the seeds show the reduction of storage proteins, which are classified into late embryogenesis abundant (LEA) proteins. We had identified embryogenic cell proteins (ECPs) from carrot, and isolated the corresponding genes (DcECP31, DcECP63). These genes are regulated by carrot ABI3 homolog, C-ABI3, and expressed in the lately stage of seed development. However, in carrot, FUS3 gene homolog has not isolated yet, and there are no deficient mutants of C-ABI3, C-LEC1. We had already isolated Arabidopsis homolog to DcECP31 and DcECP63 (AtECP31 and AtECP63) and are examining the expression pattern. Using deficient mutants and transgenic plants, we clarified that factors controlling expression of AtECP31 were different from those of AtECP63.

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Somatic embryogenesis is unique character of plant cells. To understand the molecular mechanism of somatic embryogenesis, we tried to isolate novel embryogenesis-related genes. In carrot, Embryogenic Cell (EC), can be maintained in the medium with auxin. Only after the depletion of auxin, EC starts the embryogenesis. We speculated that some genes expressed during the early stage after auxin depletion would be essential for somatic embryogensis. We report here one cDNA 18B-H10 which showed specific expression in embryogenic cells by auxin depletion.
18B-H10 cDNA had 1523bp nucleotides, and encoded a polypeptide of 443 amino acids. Homology search analysis showed that 18B-H10 was most homologous to carrot EP1. 18B-H10 was preferentially expressed in EC 8hrs after auxin depletion, then the expression decreased. Carrot seedling and Non-Embryogenic Cell (NC) which loses regeneration ability showed low expression of 18B-H10. Further characterization of cell specific expression and physiological roles of 18B-H10 are on going.

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We have developed direct somatic embryo-forming system in which carrot hypocotyl explants are treated with 2,4-D for brief time and then cultured in 2,4-D-free medium. We think that the transition from somatic cells to cells which have embryo-forming function in the earliest events during somatic embryogenesis is firstly promoted by treating with 2,4-D. Thus we search for genes expressed specifically in 2,4-D-treated hypocotyl explants. To find these genes, we prepare three samples as following for differential display method; (1)hypocotyl explants treated with 2,4-D for 48hrs, (2)hypocotyl explants treated with 2,4-D for 24hrs and then cultured in 2,4-D-free medium for 24hrs, (3)hypocotyl explants cultured in 2,4-D-free medium for 48hrs. We obtain three genes, No.130, No.151 and No.209 which are specifically expressed in 2,4-D-treated samples by RT-PCR analysis. In addition, these genes were expressed in cell clusters and blobular embryos, but expressed hardly in the heart- and torpedo-shaped embryos and plantlets.

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In carrot somatic embryogenesis, 2,4-D promotes the transition from organized somatic cells to cells that have embryogenic function. However, 2,4-D has well known to inhibit development of embryogenic cell clusters to embryos. We examined in what manner the development of cell clusters to embryos was inhibited by 2,4-D in this study.
We observed with SEM that the membrane on superficial cells of cell clusters treated with 2,4-D was disrupted. The genes that were specifically expressed in cell clusters treated with 2,4-D were searched by subtraction method. Consequently we found a gene that had high homology with tomato polygalacturonase. Actually, we confirmed that high polygalacturonase activity was present in cell wall fraction of cell clusters treated with 2,4-D. We suppose that cell wall polygalacturonase induced with 2,4-D degrades pectin polysaccharides consisted of cell wall, resulting cessation of subsequent development of cell clusters by inhibition of cell elongation and adhesion between cells.

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Suspension-cultured Bruguiera sexangulacells were grown in NaCl solutions as high as 200 mM (Kura-Hotta et al. 2001). When cell were cultured in 150 mM NaCl increased respiration was observed and this was accompanied by a decrease in fructose-6-P (F6P) and increase in fructose-1,6-bisP, indicating activation of ATP-dependent phosphofructokinase (PFK) and/or pyrophosphate: F6P phosphotransferase (PFP). Partially purified activities of both PFK and PFP were stimulated by NaCl (in vitro). The apparent Km value of PFK for F6P was 1.1 mM. In the presence of 150 mM NaCl the Vmax/Km value increased more than 2-fold. Higher concentrations of ATP inhibited the PFK activity, but NaCl completely counteracted this inhibition. The mangrove PFP activity required fructose-2,6-bisP. Increases in the activities of these key glycolytic enzymes by salt seems to be one of the mechanisms facilitating salt tolerance of mangrove cells. Kura-Hotta et al. Plant Cell Environ. 24:1105 (2001)

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We have identified N6D medium with 10% glucose promoted starch accumulation in rice callus. During this condition, AGPase large subunit gene (accession D50317, AGPL) was increased in proportion to the starch accumulation. To clarify the starch-accumulation mechanisms in callus, we firstly analyzed transcriptional responses of starch and sugar metabolic genes to sugar in the medium. Nine of starch and nine of sugar metabolic gene expressions were analyzed by Quantitative RT-PCR. According to expression pattern, these genes were divided into six groups: 1) increased or 2) decreased in proportion to starch contents, 3) just induced or 4) suppressed by sugar contents, 5) ubiquitous, and 6) very low. There was no gene, which shows the expression pattern such as AGPL. These results suggested AGPL is key enzyme of starch accumulation in callus. We will investigate the regulation factor for expression of AGPL gene in this condition.

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Storage α-polyglucan in cyanobacteria is glycogen, which is regarded as the primitive form of starch (amylopectin) in plants. To elucidate the mechanism of amylopectin biosynthesis in plants, we isolated mutants of Synechococcus PCC 7942, affected in genes encoding glycogen synthase, glycogen branching enzyme (BE) and isoamylase (ISA). In addition to the single mutants, double mutants for BE and ISA were also obtained.
Analysis by capillary electrophoresis showed that glycogen of wild type Synechococcus, after debranching treatment, had the maximum chain length of DP 6. In BE mutant, the proportion of the peak chains of DP 6 was significantly decreased, while that of longer chains (≥10) was inversely increased. In ISA mutant, the amount of very short chains (DP 2 and 3) increased, but chains with length around 8 decreased. In BE/ISA double mutant, the cumulative effects in the corresponding single mutants were observed.

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A novel ethanol hypersensitive mutant, gek1 was isolated from Arabidopsis. The gek1 mutants cannot germinate nor grow in the presence of 0.03% ethanol while they grow normally in the absence of ethanol. The GEK1 gene encodes a novel protein without any known motifs. Interestingly, similarity searches showed that only plants and archaea have the GEK1-related genes.
In order to address the GEK1 function, we tried to see the effect of the overexpression of GEK1 in Arabidopsis, E. coli, S. cerevisiae, and S. pombe. In Arabidopsis, GEK1 overexpression not only recovered the ethanol hypersensitive phenotype but also conferred ethanol tolerance, while GEK1 co-suppression enhanced ethanol sensitivity. On the other hand, the ethanol tolerance of E. coli and yeasts was not affected by GEK1 expression. These results imply that GEK1 protein is involved in a novel ethanol metabolic pathway that is specific to plants and probably to archaea.

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We aim to establish a simple comprehensive analytical method for sugar phosphates to know the phosphate metabolism in plants. For this purpose, we applied anion exchange separation and electrochemical detection (HPAE-PAD) method for Arabidopsis. It is necessary to inactivate enzymes immediately after sampling for applying the chromatography. We tried to examine the extraction by hydrochloric acid, TCA, microwave, ultra filtration or heating to inactivate enzymes. The extraction by hydrochloric acid and TCA caused anion overloading in anion exchange column, and it was difficult to determine sugar phosphates. Changes in metabolites levels were observed during the sample homogenization using ultra filtration or heating. Heating or microwave application after the lyophilization of frozen sample made better detection of sugar phosphates. Sugar phosphates in Arabidopsis which was grown in different Pi concentration media, were determined by this extraction method.

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Glycerol-3-phosphate acyltransferase (EC 2.3.1. 15; GPAT) catalyzes the committed step of the biosynthesis of glycerolipids in eubacteria and eukaryotes. Eukaryotes contain multiple genes for GPAT in their genome, which copes with the idea of symbiotic evolution of eukaryotic cells. GPAT activity localized in three cellular compartments in plant cells, i.e., ER, plastids and mitochondria, and the evolutionary relationship between the gene for plastid-targeted GPATs (ATS1) and that for cyanobacterial GPAT has attracted our major interest. However, our current knowledge of the gene for cyanobacterial GPAT is scarce. We herein conducted a comprehensive ProScan search for a conserved sequence motif of acyltransferases (Lewin et al. (1999) Biochemistry 38: 5764-5771) in the genome database of Synechocystis sp. PCC6803 and identified a putative acyltransferase gene that is probably a potent candidate for the ancestral gene for ATS1.

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Monogalactosyldiacylglycerol (MGDG) is a typical lipid in oxygen evolving photosynthetic organisms and occupies over half of the lipids in thylakoid membrane of chloroplast and cyanobacteria. MGDG is, therefore, considered as an essential factor for photosynthetic apparatus. In fact, from crystal structure analysis, MGDG is found to associate with reaction center protein of PSI. Although genes for MGDG synthesis have been isolated from higher plants, no one could find the ortholog from the Cyanobacteria genome of which has been sequenced.
Recently, we reported that there are two pathways for MGDG synthesis (UDP-Glc and UDP-Gal dependent) in cyanobacteria and the activities are conserved between Synechocysitis PCC6803 and Anabaena PCC7120. We assumed that the genes for these activities were highly conserved in both cyanobacteria and identified the UDP-Glc dependent glycolipid synthase gene by comparative genomic analysis. In this presentation, we will report the strategy for the identification and discuss knock-out mutant of the gene.

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1,2-Diacylglyceryl-3-O-carboxyhydroxymethylcholine (DGCC) is a common lipid constituent of Haptophyceae. We have investigated to characterize the biosynthetic pathway of DGCC in Pavlova sp. (Haptophyceae). Feeding experiments using ¹⁴C-labeled compounds demonstrated that a precursor of DGCC was choline via ethanolamine, suggesting that the trimethylammonium group of polar group was derived from choline. The polar group of 1,2-diacylglyceryl-O-4'-(N,N,N-trimethyl)homoserine (DGTS), which was present in ferns, mosses and green algae was reported to be synthesized from methionine as C₄ backbone. The formation of the trimethylammonium group in DGTS was suggested to be responsible for the three steps of the lipid-linked N-methylation in Rhodobacter sphaeroides. Our results indicated that the biosynthetic pathway of polar group of DGCC was different from that of DGTS. To elucidate the origin of the carboxyl group in polar head group of DGCC, feeding experiments using ¹³C-labeled compound and the following NMR analysis are now in progress.

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Plants are known to have two pathways (prokaryotic and eukaryotic) for galactolipids synthesis each of which has characteristic fatty acid composition for diacylglycerol backbone. Among both, eukaryotic pathway is likely more important for galactolipid synthesis since almost all plants have that pathway. By pulse chase analysis, phosphatidylcholine (PC) has been considered to be an intermediate of eukaryotic galactolipids. Actually, under phosphate-starved condition, characteristic digalactosyldiacylglycerol (DGDG) is accumulated by eukaryotic pathway, and fatty acid composition of the DGDG is very similar to that of PC on outer envelope membrane.
In this study, we focused on PC-specific phospholipase C (PC-PLC). By using EST database, we found that there are homologs of bacterial PC-PLC only in plants. Thus, these homologs may contribute to plant-specific lipid metabolism. There are six homologs in Arabidopsis and we found that expression of only one gene (PC-PLC3) responds to phosphate starvation.

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The green microalga Botryococcus braunii produces large amounts of liquid hydrocarbons and is classified into three races, A, B, and L depending on the types of the hydrocarbons. The B race produces two types of triterpenoid hydrocarbons, methylated squalenes and botryococcens, which are promising as renewable fuels. In order to understand isoprenoid biosynthesis by the B race of B.braunii, the incorporation of ¹³C labelled glucose into tetramethylsqualene and C₃₀botryococcene was studied by quantitative ¹³C NMR spectroscopic analyses. The resulting labelling patterns showed that the isoprene units of those triterpenoids are derived from the methylerythritol phophate (MEP) pathway. We also isolated and characterized a gene for 1-deoxy-D-xylulose 5-phophate synthase (DXS) from this alga which catalyzes the first step in the MEP pathway.

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PnC401 which was isolated from Pharbitis nil cv. Violet showed specific expression during the flower inductive darkness. PnC401 and AtC401, a homologue in Arabidopsis thaliana, showed circadian regulated expression. By the reporter analyses using AtC401 promoter::luciferase transgenic Arabidopsis plants, we showed 146 bp fragment was sufficient to regulate the rhythmic expression of that AtC401. To investigate the regulation mechanism in detail, we determined the transcriptional initiation site by primer extension experiment and found 73 bp of downstream sequence in the 146 bp fragment. The AtC401 promoter fragments in which the downstream (-174/+2) from the initiation site was deleted could not drive the expression of luciferase reporter, and the fragment fused to 47 bp minimum fragment of CaMV35S promoter did not show rhythmic expression of the reporter. These results suggest that the rhythmic expression of AtC401 is regulated by the element(s) located in the downstream sequence from the transcriptional initiation site.

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We had isolated PnCRY1 (Pharbitis nil Cryptochrome 1) whose transcripts transiently increased during the 16-h flower inductive darkness in leaves from an absolute short-day (SD) plant P. nil cv Violet, using fluorescent differential display (FDD) method. PnCRY1 encoded a putative polypeptide of 675 amino acids with a molecular mass of 76 kD. The deduced amino acid sequence of PnCRY1 showed 79% and 73% amino acid identity with that of tomato and Arabidopsis, respectively. PnCRY1 mRNA accumulation showed diurnal fluctuation in both SD and long day (LD) conditions. This oscillation showed circadian rhythm during the extended dark period (DD), but damped under continuous light condition (LL). We wish to show the results of its expression analysis in various conditions (different photoperiodic condition, effect of night break treatment, organ specificity, difference of cultivars, etc), and discuss on the participation of PnCRY1 in the photoperiodic induction of flowering in P. nil.

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Circadian clock components, LHY, CCA1, TOC1 and GI, are thought to compose a negative feed-back loop to control their expression reciprocally in Arabidopsis. Loss-of-function of both LHY and CCA1 causes acceleration of flowering under short days (SD). GI expression peaks in the morning in the lhy cca1 double mutants, although it normally does in the evening in wild-type. GI gene is required for the early flowering phenotype of lhy cca1 under SD. These results suggest that the early flowering phenotype of lhy cca1 may be caused by mis-expression of GI. It is reported that CO, a downstream factor of GI, promotes flowering through two floral activators, FT and SOC1. To understand molecular mechanisms how clock components control flowering, we have generated multiple mutants on circadian rhythms and flowering time regulation. Here we report genetic analysis of interactions among clock components, LHY and CCA1, and floral inducers, CO, FT and SOC1.

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In many organisms, a wide variety of biological processes are controlled by circadian rhythms. Two Myb-related genes, LHY and CCA1, have been shown to be closely associated with clock function in Arabidopsis. Lack of LHY or CCA1 shortens the period of the rhythm in the expression of clock-controlled genes under continuous conditions, and also accelerates flowering under short days. lhy cca1 double mutant plants flower much earlier than the progenitor single mutants and lose free-running rhythms in constant light. Loss-of-function of a blue light receptor gene, FHA/CRY2 or a floral inducer gene, CO, causes late flowering phenotype and decreased expression of a floral activator gene, FT, under long days. To understand molecular mechanisms on photoperiodic control of flowering, we have tested genetic interactions among LHY and CCA1, CO and FHA/CRY2. Our results suggest that both CO and FHA/CRY2 are downstream factors of LHY and CCA1 to control flowering.

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Circadian rhythms are driven by endogenous biological clocks that regulate many processes in a wide variety of organisms. At least 4 genes, LHY, CCA1, TOC1 and GI, have been shown to be closely associated with clock function in Arabidopsis. Loss-of-function of either LHY or CCA1 shortens the period of the rhythm in the expression of clock-controlled genes under continuous light, and also accelerates flowering under short days (SD). To identify more mutations which affect circadian rhythms, we have screened EMS-mutagenised lhy-12 seeds for plants which flowered earlier than the lhy-12 in SD. We found that two mutations, 38elf-1 and 70elf-1, in a similar way to the cca1, enhanced 1) early flowering phenotype in SD and 2) shift in the phase of expression of GI, of the lhy-12 mutant. Characterization of these enhancer mutations is underway. They may define further cca1 loss-of-function alleles or genes required for CCA1 regulation.

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