In Arabidopsis, Lec1, Lec2, Fus3 and ABI3 have been cloned as transcription factors to control seed development. Especially, Fus3 and ABI3 play important roles during mid- and late embryogenesis. Although it is believed that these transcription factors control the gene expression during seed development coordinately, it has not been clearly understood its molecular mechanisms. We have been conducting DNA microarray analysis to examine gene expression controlled by these transcription factors. When the transgenic Arabidopsis containing FUS3 gene with glucocorticoid inducing promoter was treated with DEX and ABA, expression levels of several hundreds of genes were changed to compare those of vector only control transgenic plants. It was observed that genes such as involved in accumulation of storage proteins or lipids and ABA synthesis, various transporters and transcription factors responsible for developmental control were upregulated.

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SPK specifically phosphorylates sucrose sythase, that catalyzes the reaction of an initial step in the starch biosynthesis, for its activation. Antisense SPK transformants lack the ability to accumulate storage products such as starch in immature seeds, resulting in no embryo and emdosperm. Using a microarry system, we comprehensively analyzed gene expression in immature seeds of the antisense SPK transformants. We found that expression of 125 genes were remarkably changed in the transformants as compared with the wild-type. These genes were classified into the six groups by their predicted functions. These expressions were also determined at booting stage, early stage of development, and middle stage of seed development.

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Storage proteins in cotyledon of legume plant are degraded by proteases expressed during seed germination and are mobilized to the embryonic axis for its growth. In aleuron layers of rice or barley, expression of proteases is induced by gibberellin (GA) and its induction is antagonized by abscisic acid (ABA). We examined whether GA and ABA regulate expression of the proteases in cotyledons of germinated bean seeds with RNA blot analysis of the genes for 7 cysteine proteases, EP-C1, CP1, CP2, CP3, CP4, LLP1 and LLP2. Our result indicated that expression of the 6 genes except of CP4 was repressed by GA biosynthesis inhibitors or ABA, suggesting that GA and ABA regulates mainly the expression of the proteases in germinated cotyledons. We will report effects of inhibitors of ethylene action on the expression of the genes, because ethylene is known to be involved in regulation ofgermination.

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Roles of seed coat on interspecific differences in germination behaviors were surveyed in seeds of a series of species of genus Dioscorea. Each species has specific distribution area from southern warm places to northern cold places in East Asia. Even in species of the southern warm places, their deeply dormant seeds were able to fully germinate if their seed coats were removed. Lanolin coating instead of removed seed coat had no effect on germination. No differences in density of intercellular spaces of seed coat among species were observed by SEM. The function of seed coat is likely to not be physical barrier for water and gases. Observations by a light microscope suggest that some cells in the four cell layers of seed coat undergo certain physiological process. Seed coat may cause interspecific differences of germination behaviors in relation to north-south distribution of species.

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We obtained a monoclonal antibody, mAb3-1-1, against caleosin which is an oil body protein in sesame seeds and has a molecular weight of 27 kd in a cultivated strain. Protease treatment of caleosin proteins by V₈ or trypsin showed that the epitope recognized by mA3-1-1 is a terminal region of caleosin proteins. Moreover, during germination process a peculiar disappearance of caleosin proteins was observed in a wild strain, Sesamum radiatum as well as in a cultivated strain, S. indicum. The mAb3-1-1 reacted with the caleosin of 27 kd in the cultivated strains, but reacted with 25 kd, 17 kd and 15 kd bands in the wild strains instead of 27 kd. It is conceivable that 17 kd and 15 kd bands are oleosin proteins in oil bodies. The relationship between caleosin and oleosin in sesame oil bodies will be discussed.

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Isopentenyl pyrophosphate, a precursor of isoprenoids can be synthesized via mevalonate (MVA) pathway in the cytosol and non-mevalonate (MEP) pathway in plastids. MVA pathway depends on 3-hydroxy-3-methylglutaryl-Coal reductase (HMGR) for the production of mevalonate as the first committed step. HMGR is encoded by small divergent gene families in higher plants. Arabidopsis encodes two HMGR genes, namely HMG1 and HMG2. To understand the role of the individual members of HMGR genes, we screened Arabidopsis T-DNA tagged lines and isolated hmg1 and hmg2 mutants. hmg1 mutant plant showed, 1) inhibition of root elongation and expansion of cotyledon in seedling, 2) dwarf and sterile phenotype, 3) rapid senescence in mature leaves and internodes. In addition, rapid induction of a senescence associated gene was observed in hmg1 seedling after dark treatment. No distinct phenotype was shown in hmg2 mutant. Detailed studies on expression of the gene and isoprenoids profiling in hmg1 plant are underway.

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In higher plants, isoprenoids are synthesized via mevalonate pathway and non-mevalonate pathway. Sterols are formed in cytoplasm via the former pathway. The key enzyme on mevalonate pathway is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). HMGR is regulated on the transcriptional and post-translational level. HMGR is inactivated by AMP-activated protein kinase (AMPK) in mammals. In plants, however, it is not known whether HMGR is regulated by AMPK-homologous kinases (SnRK) in vivo. Therefore, we analyzed Arabidopsis SnRKs, Akin10, Akin11 and Akin12. Kinase assay using HMGR C-terminal peptide as a substrate revealed that Akin10 and Akin11 could phosphorylate HMGR but Akin12 could not. To determine organs where Akin10, Akin11 and Akin12 are expressed, we examined the RT-PCR analysis. Akin10 and Akin11 are expressed ubiquitously. On the other hand, Akin12 is expressed in immature siliques. These results suggested that Akin10 and/or Akin11 are functional HMGR kinases. Now, we are screening and analyzing insertion mutants of these genes.

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We isolated a rice mutant, gid2, which showed a severe dwarf phenotype. The gid2 mutant showed very little response to exogenously treated GA. In gid2 mutants, level of bioactive GA was also accumulated.
Map-based cloning revealed that the GID2 gene encoded a putative F-box protein. 4.2Kb of DNA fragment containing the entire region of this gene complemented the gid2 phenotype. The deletion of nucleotides in two gid2 alleles introduced a novel stop codon in its coding region.
As previously reported, negative regulator of GA signaling, SLR1, is degraded by GA-dependent manner. In gid2, SLR1 was highly accumulated and GA-dependent degradation of SLR1 was not observed, whereas the comparatively small amount of SLR1 detected in wild-type plants degraded following GA treatment. These results suggest that GA-dependent degradation of SLR1 mediated by SCF complex involving GID2.
This work was in part supported by PROBRAIN.

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SLENDER RICE1 (SLR1) encodes an orthologous protein to GAI/RGA previously isolated as a repressor for GA response in Arabidopsis. Recent our studies have demonstrated that SLR1 represses the GA response in nuclei, but degrades rapidly when exposed to GA, suggesting that the GA response is de-repressed by the GA-dependent degradation of SLR1.
In order to investigate how GA signal is transmitted to degrade SLR1, we isolated two kinds of mutant named as GA-insensitive dwarf 1and 2 (gid1 and 2). Recently, we cloned the GID2 gene which encoded an F-box protein, a component of SCF ubiquitin ligase. In gid2 mutant, SLR1 protein was highly accumulated. There were two forms of the accumulated SLR1 in gid2, nascent and phosphorylated. These results indicate that SLR1 may be a target for the SCF^GID2 and phosphorylation may trigger degradation of SLR1 in a similar manner as reported in yeast.
This work was in part supported by PROBRAIN.

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Rice gid1 mutant shows extremely dwarf phenotype, which is insensitive to GA. GID1 was isolated by a map-based cloning and predicted to encode a serine hydrolase. From yeast two hybrid assay, GID1 was considered to interact directly with SLR1, which is a negative regulator in GA signaling. Here, we will report the functional relationship between GID1 and GID2, which encodes a F box like protein involved in the degradation process of SLR1.
The amount of SLR1 protein in gid1 was much less than that in gid2, while gid1 showed much severer dwarf phenotype than gid2. We also performed the phenotypic analyses of GID1 and GID2 over-expresser in gid2 and gid1 mutant background, respectively. Based on these results, we predicted that GID1 attacks SLR1 to inactivate its repressor activity in a GA dependent manner.
This work was in part supported by PROBRAIN.

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Seed dormancy and germination are controlled by multiple environmental and endogenous factors. In Arabidopsis, both gibberellin (GA) and cold treatment (4˚C, dark) promote seed germination. We have previously shown that the AtGA3ox1 gene, encoding a GA 3-oxidase catalyzing the final step to produce bioactive GAs, is upregulated in response to cold treatment. Consistent with the increase in AtGA3ox1 expression, endogenous GA4 levels were elevated following cold treatment. To predict how cold treatment stimulates germination through GA action, GA-dependent gene expression was monitored using DNA microarray analysis. The GA-responsive genes during cold treatment included those previously determined to be GA-responsive at 22˚C in the light, but also included a new set of genes that appear to be unique to the low temperature environment. These results suggest that GA biosynthesis and response are modulated under different environmental conditions to control seed germination in Arabidopsis.

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Gibberellin affects a lot of phases of plant development such as germination, stem elongation and flowering. Thus its biosynthesis and signal transduction must be controlled strictly. We are interested in GA3oxidases, which directly catalyze production of active gibberellins, and the expression of some of them is under GA negative feedback regulation. Analysis of this feedback regulation of GA3oxidase may uncover the mechanism regulating the amount of endogenous gibberellin. We already have shown by promoter deletion assay that the region between 1000 bp and 800 bp of AtGA3ox1 promoter is necessary for GA negative feedback regulation. By further deletion of this region, the 50 bp region within this 200 bp was shown to be necessary for GA negative feedback regulation. Yeast one-hybrid screening using this region identified a DNA binding protein, which contained an AT-hook motif and novel domain. More detailed analysis is under way.

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We have previously shown that gibberellin (GA) is required for tissue-reunion in the cortex of cut hypocotyls in cucumber and tomato seedlings, and that cotyledons are necessary for the tissue-reunion in hypocotyls. In this study, we analyzed expression of GA biosynthesis genes and endogenous GA contents to examine the contribution of cotyledons to providing GAs for the tissue-reunion in hypocotyls. Quantitative RT-PCR analysis indicated that transcripts of LeGA20ox1 and LeGA3ox2 genes (encoding GA 20-oxidase and GA 3-oxidase, respectively) accumulate at relatively high levels in cotyledons in tomato seedlings. To study the role of cotyledons in GA content in the hypocotyl directly, cotyledons were removed from 7-day-old cucumber seedlings, and endogenous GA levels in the hypocotyls were determined. Relative to intact seedlings, those without cotyledons contained reduced amounts of bioactive GA₄ and its precursors. These results suggest that cotyledons play a role in determining GA content in cucumber and tomato hypocotyls.

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The primary biosynthetic reaction of cytokinin is catalyzed by isopentenyltransferase (IPT). In Arabidopsis thaliana, seven genes (AtIPT1, AtIPT3 to AtIPT9) were identified as the biosynthesis genes.In the last year's meeting, we reported differential localization of AtIPTs in plastids and mitochondria. To understand physiological function of each AtIPT, we tried to determine the expression site of the AtIPTs with transgenic Arabidopsis. We constructed AtIPT promoter::GFP chimeric genes and introduced into Arabidopsis. In the transgenic plants, AtIPT1 expressed in elongation area and vascular stele of roots. AtIPT3 expressed in the companion cell of phloem over a whole seedling. AtIPT5 expressed in lateral root primodium of perycycle. AtIPT7 expressed in vascular stele and companion cell of phloem in root. These results suggest that the AtIPTs are differentially regulated in terms of the spatial distribution and that the variety implies physiological differentiation of each AtIPT.

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Availability of inorganic nitrogen has crucial effects on growth and development of plants. Phytohormones act as mediators of changes in the internal and external environments of plant body. Cytokinin has been considered to have a function to communicate the nitrogen availability because correlation between the level of cytokinins and the nitrogen status has been reported in various plant species.
Adenylate isopentenyltransferase (IPT) is thought to catalyze the limiting step of cytokinin biosynthesis. To gain some insights into the nitrogen-responsive change of cytokinin level, we analyzed the expression pattern of these genes (AtIPT1, AtIPT3-8) in Arabidopsis by real-time PCR. In the seven genes, the accumulation of AtIPT3 mRNA was coordinated with that of cytokinin during recovery from nitrogen starvation. Since the expression of GFP gene fused to the promoter of AtIPT3 gene showed similar pattern, the nitrogen-responsive accumulation is regulated at least in parts by transcriptional level.

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It has been uncovered that the His-Asp phosphorelay plays an important role in the signal transduction of plants in response to the stimulus such as phytohormones and osmotic stress. Eukaryotic type of His-Asp phosphorelay is composed of three types of factors; sensor His-kinase, His-containing-phosphotransmitter and response regulator (RR). The type-B family of RRs has been shown to function as transcriptional factor. To date, the details of the signaling pathways mediated by type A-family of RRs remain to be elucidated. In maize, the cDNAs encoding for the type-A family of RRs (ZmRR1,2,4,5,6,7) and type-B family of RRs (ZmRR8,9,10) were isolated and the expression patterns of these genes are analyzed. We performed yeast two-hybrid screen to isolate the interacting proteins with ZmRR1. One of the interacting proteins, ZmHD1, contained HD domain that might function as metal-dependent phosphohydrolase. The biochemical characterization on ZmHD1 will be presented.

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An arabidopsis mutant, rrd4, which was originally isolated as being temperature sensitive for root redifferentiation, is characterized by cytokinin-dependent defects of dedifferentiation at the restrictive temperature. This suggests that the RRD4 gene functions in sequestrating negative effects of cytokinin on dedifferentiation. Chromosome mapping located the RRD4 in the 64-kbp region at the 23-cM position of chromosome I. Sequence analysis of CDSs in this region identified an amber mutation in F20D23.23 of the mutant genome. Among T2 plants derived from the transgenic rrd4 mutant, into which a wild-type genome fragment containing F20D23.23 had been introduced, the presence of the introduced DNA and the ability of callus formation at the restrictive temperature were co-segregated. These results have led to the conclusion that the RRD4 gene corresponds to F20D23.23. The primary structure indicates that this gene encodes a protein similar to a murine tuftelin-interacting protein, TIP39.

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The water-oxidizing reaction in the oxidizing side of Photosystem 2 (PS2) is catalyzed by the manganese cluster. A minimum size of the water-oxidizing complex still remains to be unsolved, although its functional unit is considered to consist of PS2 protein components such as two reaction center proteins, the chlorophyll-binding proteins, Cyt b559 and PS2 extrinsic proteins.
We treated spinach PS2 membranes with sucrose monolaurate (SML), to isolate the PS2 core complexes depleted of CP43 and the functional manganese cluster. The low-molecular-mass intrinsic proteins containing L protein (designated as SML extracts) were also isolated during a preparation process of PS2 core complexes. The L protein seems to function in Q_A activity. Thus, we reconstituted PS2 core complexes with SML extracts, resulting in repair of both the reducing and oxidizing side of PS2. Furthermore, followed by photoactivation of these reconstituted preparations, restoration of the water-oxidizing activity was obtained.

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The molecular mechanism of photosynthetic water oxidation still remains unclear. For its clarification, it is important to reveal the reactions of water at molecular level. Here, we attempted to detect the reactions of water in the water oxidizing complex (WOC) using Fourier transform infrared spectroscopy (FTIR).
FTIR difference spectra of flash-induced S-state transitions were measured using hydrated PSII core complexes from Synechococcus elongatus. The spectra showed a differential signal at 3617/3588 cm^-1 upon 1st flash and negative peaks at 3634, 3621, 3612 cm^-1 upon 2nd, 3rd and 4th flash, respectively. These bands downshifted by ~940 and ~10 cm^-1 upon D₂O and H₂¹⁸O exchange, respectively, indicating that the bands arise from water coupled to the Mn-cluster. The different wavenumbers and the mostly negative intensities were interpreted as due to substrate water with specific structures. Thus, FTIR is a promising method to investigate the mechanism of photosynthetic water oxidation.

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Pulsed Electron Electron Double Resonance was applied to three spins system to determine the relative positions of electron transfer components in Photosystem II. Y_Z radical and Q_A^- were trapped after illumination of Tris-treated Zn-substituted PS II for 20 s at 253 K. The time profile observed in spin echo height for Y_D-Y_Z-Q_A system as function of time of application of the second π pusle was analyzed based on the dipolar interaction between three sspins. Using the known distances 38 A for Y_D-Q_A and 30 A for Y_D-Y_Z, Best fitting was obtained for distance 34 A of Y_Z-Q_A. The same method was applied to the Y_D-Q_A-Chl_Z system. The distance 34 A for Chl_Z-Q_A was obtained.
The results for three spin sytems and the distances and their orientations for two spin sytems were combined. The derived configuration of electron transfer components will be compaired with X-ray data.

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In higher plant PSII, the 33 kDa protein directly binds to PSII, but the 23 and 17 kDa proteins associate with PSII through the 33 kDa protein and through both 33 and 23 kDa proteins, respectively. In contrast, in green algal PSII, each of the 33, 23 and 17 kDa proteins directly binds to PSII independent upon the other extrinsic proteins (Suzuki et al. P.C.P. in press). In this study, we performed cross-reconstitution experiments of these extrinsic proteins with green algal and higher plant PSIIs to clarify the difference of their binding properties. The results showed that higher plant 23 and 17 kDa proteins directly bind to green algal PSII by itself, while green algal 23 and 17 kDa proteins cannot directly bind to higher plant PSII. This indicates that the difference of binding properties of their extrinsic proteins to PSII is responsible for intrinsic proteins.

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By means of in vitro reconstitution with spinach PS II particle, we previously found that a small subunit protein, PsbL in PS II complex is involved in the primary electron transfer at the donor side of P680 at pH lower than 5.5. In this study, using a reverse genetic approach, we have inactivated the psbL gene in the tobacco chloroplast genome to investigate the function of PsbL in vivo.
The homoplasmic psbL knockout plants (ΔpsbL) could grow on sucrose containing medium, but it displayed a clear mutant phenotype. While young leaves were normally green, older were almost completely white. The PSII activity of ΔpsbL determined by chlorophyll fluorescence at room temperature with PAM was about 10% of the wild type plant, irrespective of young or old leaves. The function of PsbL in the PSII will be discussed in the combination of the in vitro and in vivo data.

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An extrinsic 33-kDa component protein in photosystem II is essential for photosynthetic oxygen evolution. It is present in all oxygen-evolving organisms, and plays a central role in the stabilization of the manganese cluster. In Arabidopsis thaliana, 33-kDa proteins are encoded by two genes, psbO and psbO2. We characterized the Arabidopsis mutant with a defect in one of them, psbO, and an intact psbO2. The mutant showed considerably retarded growth, suggesting that there is a functional difference between them. Immuno-blot analysis also detected two signals with slightly different mobility in wild type, but only fast moving signal in the mutant. Both PsbO and PsbO2 were expressed as a mature protein fused with GST in E. coli, to characterize their properties. Fused proteins were isolated with affinity chromatography, cleaved with enterokinase and purified ion exchange chromatography. Further characterization is on going.

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In green algae and higher plants, three extrinsic proteins of OEC33, 24, and 18 associate with photosystem II and play important roles. In order to understand their functions, it is essential to solve their 3D structures. For this purpose, it is needed to grow good-quality crystals suitable for X-ray structure analysis. Here we tried to purify and crystallize OEC18, which has not been crystallized so far. At first, we purified OEC18 from spinach and obtained microcrystals with a length of 100μm. Since the thin crystals were not large enough for X-ray experiments, we expressed and purified OEC18 of maize by E. coli as a GST fusion protein. After cleavage of the protein with thrombin, we obtained some microcrystals under several conditions. We will optimize the crystallization conditions to obtain X-ray quality crystals. In addition, we will report the purification and crystallization of OEC33 from spinach.

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    The extrinsic 23-kDa protein in an oxygen-evolving complex (OEC23) of photosystem II (PS II) has a function of retaining calcium and chloride ions, which are essential cofactors for photosynthetic oxygen evolution. OEC23 is found in PS II complex from higher plants and green algae but not in that of cyanobacterium and other ancestral photosynthetic organisms.
    In order to elucidate its structure and the molecular evolution, OEC23 was crystallized by the hanging-drop vapour-diffusion technique. Diffraction data to 2.0Å were collected from a native crystal belonging to space group P2₁22₁, with unit-cell parameters a=74.15, b=91.36, c=52.16Å. A heavy-atom derivative was prepared, and the phase problem was solved by multi-wavelength anomalous dispersion (MAD) experiments using synchrotron X-ray radiation at SPring-8 (Hyogo, Japan). The crystal structure of OEC23 was determined at 2.0Å resolution.

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Cytochrome b-559 is an integral protein of photosystem II complexes (PSII). However, the number of cytochrome b-559 (cyt b-559) associated with the PSII reaction center has been subject of controversy. Some studies have concluded that there is one heme equivalent of cyt b-559 per reaction center, some have found two, and others have reported intermediate value.
In this study, PSII complex highly active in oxygen evolution was purified from a thermophilic cyanobacterium, Synechococcus vulcanus (S.v.) and used to determine the cyt b-559 contents. Our work shows that, depending on the method used to estimate pheophytin a and reduction minus oxidation difference absorptoin spectra of cyt b-559 and cyt c by deconvolution of the original spectra, the S.v. preparation contains 1.0 cyt b-559 and 1.0 cyt c per reaction center.
Moreover, we compare the cyt b-559 content in a cyanobacterial and a spinach PSII preparation.

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During the donor-side photoinhibition of spinach photosystem(PS) II, the reaction center D1 protein cross-linked with the antenna chlorophyll-binding protein CP43 of PSII lacking the oxygen-evolving complex (OEC) proteins. The cross-linking did not occur upon illumination of PSII that retained the OEC33, or when OEC33-depleted PSII was reconstituted with the OEC33 prior to illumination. These results suggest that D1, CP43 and the OEC33 are located in close proximity at the lumenal side of PSII. The present data show that the spatial arrangement of D1 and CP43 at the lumenal side of PSII in spinach chloroplasts is consistent with the assignment of these proteins recently proposed on the crystal structures of PSII from cyanobacteria. Moreover, the data suggest that the binding condition and positioning of the OEC33 in the PSII from higher plants, are different from those in cyanobacteria.

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It has been considered that, during the photoinhibition and repair cycle of PS II, the extrinsic proteins are released from PS II upon photoinhibition and rebind to a newly reconstituted PS II. Under illumination with strong light (4 mE m^-2 s^-1) of spinach PS II membranes, OEC33 was gradually released from PS II and suffered from some damage, resulting in a smeared band in a silver-stained gel of SDS-PAGE. Among the released OEC33, only a fraction that exhibited the smeared band lost the ability to bind to PS II. The presence of scavengers of superoxides or chelating agents during illumination did not affect the protein release but specifically suppressed the damage. Furthermore, exposure to hydroxyl radicals damaged OEC33 in the same way as observed under strong illumination. These results suggest that hydroxyl radicals generated from superoxides damage OEC33 that has been released from PS II under strong illumination.

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At high temperatures, the photosystem II reaction center D1 protein is cleaved into a 23kDa and a 10kDa fragments. Since the cleavage seems to occur at the DE-loop of the D1 protein, it is likely that the mechanism of the cleavage is the same as that in the 'primary cleavage' of the D1 protein during acceptor-side photoinhibition. In this work, we compared the heat-stimulated cleavage and degradation of the D1 protein with those of the acceptor-side photoinhibition in detail.
The results with protease inhibitors show that an extrinsic serine-type endopeptidase is associated with the heat-induced primary cleavage of the D1 protein, and an intrinsic protease is responsible for the secondary degradation of the 23kDa fragment. These results suggest that heat-induced proteolysis of the D1 is actually very similar to the proteolysis in the acceptor-side photoinhibition of photosystem II.

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As reported in last year, O₂-evolving system of spinach chloroplasts could reversibly be heat-inactivated at 50^oC and restored the activity by photo-reactivation. Next, further lower temp at 45^oC was expected for the reversible heat-inactivation as D.Nash, M.Miyao and N.Murata (Biochim. Biophys. Acta vol-807 (1985) 127-133) reported that Cl^- ion requiring PSII-particles was heat-inactivated at 45^oC. So, in this study, Cl^- requiring chloroplasts were prepared by washing them with hypotonic Cl^- ion free solution and successfully heat-inactivated the chloroplasts at 45^oC which were reversibly photo-reactivated to restore the O₂ evolving activity. The site of reversible heat-inactivation was determined to be at PSII by measuring the light-induced chlorophyll fluorescence rise (Fv-Fo) and O₂-evolving Hiil reaction. The chloroplasts could be protected from the heat-inactivation by adding Cl^- and Ca²⁺ ions.

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Phosphatidylglycerol (PG) is the only phospholipid in thylakoid membranes and its function for photosynthesis is great of interest. We previously isolated a pgsA mutant of Synechocystis sp. PCC 6803, defective in biosynthesis of PG. We have already demonstrated that PG plays an important role for the function of plastquinone Q_B in the photosystem II (PS II).
In this study, we have studied the function of PG in the formation of PS II complex. In the mutant, photoinhibition was easily induced after deprivation of PG and it was mainly caused by impairment of repair step of photosynthetic machinery. We further analyzed molecular background of this photoinhibition and demonstrated that dimerization of PS II complex was limited in the mutant under high light condition. These results suggest that PG plays an important role for maintenance of photosynthetic machinery through dimerization of PS II complex.

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A cDNA for putative cytochrome P450, down-regulated during Agrobacterium tumefaciens-mediated tumorigenesis, was isolated from a potato tuber. The gene product was assigned the name CYP72A29 based on the deduced amino acid sequence. Phylogenetic analysis revealed that CYP72A29 is unique to solanaceous plants. Northern blot analysis indicated that the expression of the CYP72A29 was transiently up-regulated by wounding in tuber discs, and then gradually increased by further incubation. Inoculation of tuber discs with the wild type or IAA-overproducing mutant strain of A. tumefaciens caused a marked suppression of the accumulation of CYP72A29 mRNA. The down-regulation occurred when the discs were treated with 2,4-D, suggesting that auxin may regulate the expression of the CYP72A29. The accumulation of CYP72A29 mRNA was also suppressed by treatment with arachidonic acid, a potent elicitor. RT-PCR analysis revealed that the transcripts were most abundant in sprouts and eyes, and hardly detectable in leaves, flower buds and stems.

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The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a method based upon the tracer experiment using stable isotope-labelled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenylalanine-d₅, and the change in the ratio of stable isotope-labelled compound to non-labelled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The biosynthetic flux of p-CO and CGA were determined 1.15 and 4.63 nmol / g fw / h, respectively.

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It is known that plants detoxify several compounds with several modifications such as hydroxylation or glutathionation, and accumulate them in the cells. We report here the metabolism of exogenously added harmful compound naphthols in tobacco cells. Most of added naphthols were accumulated in the cell as glucosides with further modification. The glucosylation activities against naphthols increased in proportion to the concentration of naphthols in the culture medium.
To investigate further, the metabolite of naphthol was purified from the naphthol-treated tobacco cells, and determined the structure as malonylglucoside. Strong malonylation activity was detected in the cell-free extract of tobacco cells using naphtholglucoside as a substrate. Furthermore, when naphthol was added to cell cultures of Sophora and Lithospermum, the added naphthol was accumulated as malonylglucoside in the cells. These results suggested the existence of the metabolizing system that accumulates such xenobiotic compounds as malonylglucoside in plant.

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Here we report comparison of phenolic metabolite composition between shikonin-producing (Mp) strain and non-producing (MpW) strain, and also functional analysis of LEPS-2, a novel cDNA preferentially expressed in the Mp strain, in order to clarify variation of shikonin biosynthesis ability present in Lithospermum erythrorhizon callus cultures.
Major peaks of soluble methanol extracts of Mp callus were identified, including hydroxycinnamoyl esters and naphthoquinone pigments. In contrast, in MpW callus the contents of these compounds were remarkably low except for lithospermic acid. However, expression levels of the genes encoding enzymes involved in phenylpropanoid pathway showed no significant difference between these two callus strains.
Expression of LEPS-2 was highly correlated with shikonin production in cultured cells. Furthermore, LEPS-2 protein was localized in the apoplast fraction of the cell walls, where shikonin were also stored. Therefore, LEPS-2 protein may be involved in the intra-cell wall trapping, transports, and/or accumulation of shikonin pigments.

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Prenylation plays an important role in the biosynthesis of plant secondary metabolites and quinones, e.g. ubiquinone. Recently, we have isolated two cDNAs of prenyltransferase (LePGT-1 and -2) which prenylates p-hydroxybenzoate (PHB) in the biosynthesis of naphthoquinone, shikonin, from Lithospermum erythrorhizon. Both LePGT showed similar expression pattern in plant and responses to several factors which regulate the shikonin biosynthesis. Both LePGT showed strict substrate specificity for geranyl diphosphate as prenyl donor. A detailed enzymatic characterization of recombinant LePGTs expressed in yeast, however, revealed that these two LePGTs showed apparent different Vmax and the Km values for both prenyl acceptor and donor. To understand the functional differences of two LePGTs, we constructed 5 mutant LePGTs which had a deletion in the conserved region for eukaryotic prenyltransferase. The microsomal fractions of yeast transformants harboring these constructs did not show the enzyme activity. Further characterization of recombinant proteins are in progress.

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The nicotinic acid synthesis is used in both secondary and primary metabolisms. A part of nicotine is derived from nicotinic acid in tobacco, whereas the primary metabolite NAD is also synthesized from nicotinic acid. In spite of the importance of this metabolism, the biosynthetic pathway has not been established in higher plants. We found expression level of quinolinate synthetase (QS)-like gene was lower in low-nicotine tabacco mutant nic1nic2 than that in wild type. cDNA homologues to QS and L-aspartate oxidase (AO), which are both involved in the nicotinic acid synthesis in bacteria, were obtained from RIKEN Arabidopsis cDNA library and were found to complement growth of E. coli mutants deficient in QS or AO genes. Our results suggest that in higher plants nicotinic acid is produced from L-aspartate, as in bacteria. This pathway is different from the one starting from L-tryptophan in mammals and fungi.

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Nicotine, a major alkaloid in tobacco plants, acts as a defensive compound against insects. Nicotine is produced in the tobacco roots and transported to the leaves for storage. Nicotine level is controlled by two unlink loci, Nic1 and Nic2. We analyzed the difference in the expression pattern between wild-type root and low alkaloid mutant (nic1nic2) root by using fluorescence differential display technique. We found several genes were reduced in expression in the mutant root. Among them, two genes (NtMATE1, NtMATE2) encode similar proteins, which contain 12 putative transmembrane segments, with homology to multidrug and toxic compound extrusion (MATE) family transporters. These proteins are very similar to TRANSPARENTS TESTA 12, which may control the vacuolar sequestration of proanthocyanidins in Arabidopsis thaliana. Expression of these genes was tightly controlled by NIC regulatory genes and observed specifically in the wild type root. These results suggest that these transporter-like proteins participate in nicotine transport.

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Tobacco plants produce enhanced levels of nicotine when they are wounded. Tobacco nic mutants produce extremely low level of nicotine. Putrescine N-methyl transferase (PMT), which catalyzes the methyl transfer from S-adenosyl methionine to putrescine, is one of the genes upregulated by NIC genes that control nicotine biosynthesis. The PMT gene expression increases following jasmonate treatment likely reflecting jasmonate's role in the wounding signal transduction. We used a transient assay strategy in tobacco BY-2 protoplasts to determine the PMT promoter DNA sequence involved in jasmonate response. Four copies of a 24bp PMT promoter sequence (from -172bp to -149bp upstream of the start methionine) fused to CaMV35S minimal promoter was sufficient to mediate methyl jasmonic acid response. The A622 gene has similar expression pattern to PMT but its function has not yet been clarified. We will also report analysis of the A622 promoter.

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Nicotine-biosynthesis genes are mainly expressed in tobacco roots and their expression is enhanced by methyl jasmonic acid (MeJA). Study using tobacco low-nicotine nic mutants have revealed that expression of nicotine-biosynthesis genes is positively regulated by NIC loci and that the JA cascade modulating nicotine-biosynthesis works NIC loci-dependently. Because NIC loci partially function in MeJA-treated leaves, we compared the mRNA population in the MeJA-treated leaves of wild-type tobacco and those of the corresponding low-nicotine nic mutant by differential display to survey genes involved in NIC-dependent JA-response. A cDNA showing NIC-dependent and JA-induced expression was isolated, and its corresponding gene was named as JEI1. Wounding enhances the JEI1 expression in the leaves rapidly and transiently as well as MeJA. Treatment with elicitors and protein synthesis inhibitor also increases JEI1 transcript level. The protein encoded by JEI1 is a novel protein that has two protein-protein interacting domains.

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Recently, it has been proposed that trigonelline (Tg) has various physiological functions, such as a signal in the response to oxidative stress, compatible solutes in drought and salt stress and as a leaf-closing factor. However, biosynthesis of Tg and its regulation have not yet been clearly demonstrated. In the present study, we examined the distribution and biosynthesis of Tg in coffee plants. The concentration of Tg was highest in young leaves and decreased with leaf development while its level in coffee fruits was increased with maturation. The content in roots was low. In coffee leaves, 60% of exogenously supplied [¹⁴C]-labelled nicotinic acid and nicotinamide was converted to Tg during an 18 h incubation. Tg synthesis from [¹⁴C]-nicotinamide was also demonstrated in both isolated seeds and pericarps. Tg synthesis was highest in green coffee beans and its activity was significantly reduced in red ripen fruits.

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The caffeine(1,3,7-trimethylxanthine) biosynthetic pathway contains three S-adenosylmethionine(SAM)-dependent methylation steps. Caffeine synthase(CS), which is investigated by our group, is the SAM-dependent N-methyltransferase. CS is involved in the last two steps(N-1 and N-3 methylation) of caffeine biosynthesis in tea leaves.
In this study, we isolated tea caffeine synthase(TCS) homologous gene BTS1 from cacao(Theobroma cacao) leaves that accumulate theobromine(3,7-dimethylxanthine) as the predominant purine alkaloid by RACE. The estimated molecular mass of BTS1 was 41 kDa and it was similar to that of TCS and CTS which was theobromine synthase from coffee. The identity of amino acid sequence between BTS1 and TCS1, BTS1 and CTS1 were 55 and 35%, respectively. Recombinant BTS1 catalyzed the conversion of 7-methylxanthine to theobromine, suggesting that BTS1 was theobromine synthase.

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Of thirteen steps in the biosynthesis of berberine, the antimicrobial tetrahydrobenzylisoquinoline alkaloid, two are catalyzed by cytochromes P450 (P450). Interestingly, one of them catalyzes a cyclization of an ortho-methoxyphenol, which is unique and a difficult reaction to mimic in organic chemistry. We report here the isolation of this novel methylenedioxy ring-forming P450 cDNA from cultured Coptis japonica cells.
Two P450 cDNAs were isolated from a cDNA library of high berberine-producing Coptis japonica, using PCR fragments amplified with primers designed for the conserved region of P450. Their gene products were designated CYP80B2 and CYP719 by P450 nomenclature committee. Recombinant CYP80B2 produced in yeast showed (S)-N-methylcoclaurine-3^'-hydroxylase activity as CYP80B1 isolated from California poppy had. Recombinant CYP719 showed the methylenedioxy ring-forming activity, which converts (S)-tetrahydrocolumbamine to (S)-tetrahydroberberine. Further enzymological analysis showed that CYP719 had high substrate affinity and specificity, indicating that it is specific to berberine biosynthesis.

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Phototropin has two chromophoric domains named LOV1 and 2, each of which bears a FMN, in its N-terminal half. C-terminal half is a serine/threonine kinase. Absorption of photon initiates photochemical reactions of FMN that may induce conformational changes in the protein moieties responsible for the regulation of kinase activity. To investigate the details of the early photoreception process, we have started biophysical analyses using UV-visible absorption, FT-IR and CD spectra of the LOV domains. We have already reported that the thiol-flavin adduct but not thiolate-flavin (Schwartz et al., 2001) adduct is formed between C4a of the isoalloxazine ring of FMN and thiol of the only one cystein in the LOV. In the present paper, we will show the adduct formation induces significant conformational changes in the protein moieties of phototropin in contrast to the figure obtained by X-ray crystallography with little conformational change in them.

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Blue light induces leaf (or leaflet) movement in leguminous plants. Physiological and biochemical studies have demonstrated that the movement is carried out through the volume changes of pulvinar motor cells. However, the molecular mechanism of blue-light induced leaf movement has remained unknown. To know the above-mentioned mechanism, we have screened mutants from model legume plant Lotus japonicus and obtained a slow leaf-movement to blue light irradiation mutant laziness. Nictinasty of the laziness mutant was similar to that of the wild type, and the other blue light responses, hypocotyl phototropism, chloroplast relocation and inhibition of hypocotyl elongation, were also normal. To identify the LAZINESS locus, we are performing rough mapping. In addition, we isolated Lotus CRYs and PHOTs, and generated transgenic plants expressing anti-sense gene to analysis the function of blue light receptors in Lotus. Blue light responses of the transformants will be reported.

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phot1 and phot2 are blue-light (BL) receptors that mediate phototropism, chloroplast movements, and stomatal opening in Arabidopsis thaliana. Physiological studies have suggested that cytosolic Ca²⁺ plays a pivotal role in these BL-induced responses. We examined a BL-induced increase in cytosolic Ca²⁺ in leaves of transgenic A. thaliana of wild types, phot1 and phot2 mutants, and phot1 phot2 double mutants expressing the Ca²⁺-sensitive luminescent protein aequorin. The results indicate that phot1 increased the cytosolic Ca²⁺ at lower fluence rates and phot2 at higher fluence rates. The contribution of phot1 in the absence of phot2, calculated from the percent differences in the elevation of cytosolic Ca²⁺ between the phot2 mutant and phot1 phot2 double mutant, is similar to its contribution in the presence of phot2, the percent differences between wild type and phot1 mutant, suggesting that phot1 and phot2 act independently in mediating the BL-induced increase in cytosolic Ca²⁺.

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Phototropins (phot1 and phot2) are suggested to be multifunctional blue light (BL) receptors that mediate phototropism, chloroplast movement, and stomatal opening. Any of these BL responses was not observed in phot1phot2 double mutant lacking both phototropins. To investigate the role of phot1 in BL response of stomata in vivo we transformed Arabidopsis phot1phot2 double mutant with wild-type phot1, and determined BL-dependent stomatal responses. We found that stomatal response was restored by the transformation and that the phot1 protein was expressed in the double mutant. These results suggest that phot1 mediates BL responses of stomata in vivo. To investigate further whether the kinase domain of phot1 is essential in the BL responses of stomata, we have transformed double mutant with phot1 mutant gene, which lacks protein kinase activity. Functional analyses of BL response of stomata are under investigations in transgenic plants carrying kinase mutation.

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Blue light (BL) activates the plasma membrane H⁺-ATPase via phosphorylation, and induces stomatal opening. Recently, we showed that phototropins (phots) act as BL-receptors in stomatal opening using phot1phot2 double mutant of Arabidopsis thaliana. However, BL-responses using guard-cell protoplasts from Arabidopsis (AtGCPs) has not been investigated. In this study, we isolated AtGCPs and investigated H⁺-pumping in response to BL. The H⁺-ATPase, phot1 and phot2 were identified in AtGCPs using specific antibodies. H⁺-pumping in AtGCPs reached maximum 2 min after the start of BL (100 μmol/m²/sec for 30 sec), and sustained for 10 min. An inhibitor of protein kinase, staurosporin, at 5μ M inhibited H⁺-pumping. These results confirmed the identical properties of H⁺-pumping shown in GCPs from Vicia faba. Furthermore, we investigated BL-responses in phot1phot2. BL did not induce H⁺-pumping, although the H⁺-ATPase was observed in GCPs. These results indicated that phot1 and phot2 act as BL-receptors in stomatal opening.

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Blue light (BL), absorbed by phototropin (phot), activates the plasma membrane H⁺-ATPase via phosphorylation of the C-terminus in guard cells, and drives stomatal opening. However, there is no biochemical evidence for early events of phot in stomatal guard cells. We isolated cDNAs encoding phot, named vfphot1 and vfphot2, from guard cells of Vicia faba. In vivo phosphorylation analysis using 32P-labeled guard-cell protoplasts revealed that vfphots were reversibly phosphorylated in response to BL, and that phosphorylation of vfphots was faster than that of the H⁺-ATPase. BL-induced phosphorylations of vfphots and H⁺-ATPase showed similar fluence dependency. Staurosporin, an inhibitor of protein kinase, and diphenyleneiodonium chloride, an inhibitor of flavoprotein, inhibited BL-induced phosphorylations of vfphot and H⁺-ATPase with similar concentration-dependencies. From these results, we concluded that vfphots act as BL-receptors mediating stomatal opening and that BL-induced phosphorylation of vfphots might be required for the activation of H⁺-ATPase.

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Phototropin (phot) is known to mediate important blue-light responses such as phototropism, chloroplast positioning and stomata opening in higher plants. We have recently identified a phot homologue (Cr-phot) in the monocellular green algae, Chlamydomonas reinhardtii. To investigate the biological activity of Cr-phot in distantly related plant species, we expressed the CrPHOT gene in the Arabidopsis phot1 phot2 mutant and examined whether it complemented the mutation. We first chose several lines in which the curled-leaf phenotype observed in the phot1 phot2 mutant was complemented. These lines were subjected to RNA gel blot and immunoblot analyses and were classified into high, medium and low expressers. The phototorpic and the chloroplast positioning responses were restored in the high but not in the medium and low expressers. Hence, Cr-phot is capable of mediating some blue-light responses in Arabidopsis, indicating that certain aspects of signal transduction are conserved between these two distantly related species.

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