Sulfur is an essential nutrient required for plant growth. Plants have developed a regulatory mechanism that activates sulfur assimilation for survival under sulfur deficiency (-S). However, the molecular mechanisms controlling the -S-inducible gene expression is almost unknown. To identify the key regulator, we isolated Arabidopsis mutants showing aberrant responses to -S. The expression of SULTR1;2 sulfate transporter was visualized as GFP fluorescence in a transgenic plant expressing SULTR1;2 promoter-GFP fusion gene. This plant was used as a parental line for the EMS mutagenesis. The isolated mutant, slim1, lacked the -S response of SULTR1;2 expression. SLIM1 encoded a transcription factor localized in the nucleus. Microarray analysis of the slim1 mutant indicated that majority of the -S-responsive genes were regulated by SLIM1. The plant growth on -S was affected by the slim1 mutation, but was stimulated by over-expression of SLIM1. These results indicate that SLIM1 is a positive regulator of -S response.

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We performed microarray analysis with roots and rosette leaves of Arabidopsis thaliana which were exposed for one day to excess boron or deficient boron. When we compared gene induction ratios under each boron stress on each gene, there was positive relationship between these two inductions. On the other hand, there existed genes which are specifically upregulated by boron excess or boron deficiency. In roots, genes including At5g64170 were upregulated specifically by boron excess and genes including NIP5;1 were upregulated specifically by boron deficiency. In rosette leaves, genes including AtPT2 were upregulated specifically by boron excess and genes including At3g11340 were upregulated specifically by boron deficiency. Fifteen genes involved in senescence or defense response, which include WRKY6, were upregulated in rosette leaves by boron stresses. There were also boron-induced transcription factors. We are working with these genes to elucidate the mechanism of Arabidopsis to acclimatize to conditions with boron stresses.

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Phosphate (Pi) is one of the most important growth-limiting nutrients. When plants suffer from phosphate limitation, overall phospholipid contents that correspond to 30% of total Pi storage is decreased and, conversely, contents of some non-phosphorus galactolipids such as digalactosyldiacylglycerol (DGDG) increase significantly. Although several studies reported that Pi starvation induces enzymes for galactolipid biosynthesis, the mechanisms for phospholipid degradation is yet unknown.
We identified a novel non-specific phospholipase C (NPC) family in Arabidopsis. Among them, NPC4 and NPC5 were both induced by Pi starvation but differentially localized. While NPC4 was responsible for the bulk NPC activity induced by Pi starvation, only npc5 showed decreased DGDG accumulation upon Pi deprivation. Thus, these isozymes are differentially play roles in the Pi starvation-inducible phospholipid degradation in vivo.

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When white lupin plants face P-deficiency, they develop 'cluster roots7, which has an exudative ability of organic acids and acid phosphatase. However, responses of cluster roots to lack of other nutrients remain still unclear. This study aims to estimate function of cluster roots of white lupin grown under P- or N-deficient conditions. White lupin plants were grown in soils fertilized with (Control; C) or without P (-P) or N (-N).
Phosphatase activity was highest in -P senescent cluster roots, and was similar level between -N and Control, suggesting that increase of phosphatase activity in cluster roots is specific in P-deficiency. It was shown that peptidase activity in the rhizosphere soil was increased during low available-N with a few nodules, indicating that organic-N were utilized prior to the activation of N-supply from nodules. The peptidase activity in the rhizosphere soil was similar level between cluster and non-cluster roots.

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It is proposed that P-deficiency inducible gene is regulated by the long-distance signaling from shoots, because these are repressed by high status of shoot P though low P concentration around roots. However regulation mechanisms of P-deficiency inducible gene expression is not detailed. We have isolated a P-limitation inducible novel rice gene OsPI1. The aim of this study is to investigate expression pattern of OsPI1. P re-supply and split-root experiments were conducted using P-deficient rice plants. Roots and shoots were collected continuously, and then P-status and accumulation of OsPI1 transcripts were analyzed. In re-supply treatment, P was absorbed and OsPI1 was repressed immediately. P was increased in +P split-roots and shoot, and OsPI1 was repressed. In -P split-roots, significant changes of P-status and OsPI1 accumulation were not found during 12h. It is indicated that OsPI1 is regulated not only by the long-distance signaling, but also by the local P-status in roots.

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It has previously been shown that desiccation tolerance of cultured cells of M. polymorpha is increased by preculture in a medium with 0.5M sucrose. It is also suggested that stress-induced proteins are involved in the development of desiccation tolerance in plant cells. Water content of M. polymorpha cells decreased to 0.1 g/gDW after desiccation on silica gel for 24 hrs. Survival of cells after desiccation was 0% in the cells without preculture, however, approximately 80% in the preculture with 0.5M sucrose. Expression patterns of heat-stable proteins were changed when cells were precultured in this medium. When cells were precultured in this medium plus 10 μM cycloheximide, survival was approximately 80% after desiccation for 4 hrs, but almost 0% after desiccation for 24 hrs.
These results suggested that de novo synthesized proteins during the preculture participate in fully development of desiccation tolerance in cultured cells of M. polymorpha.

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Some bryophytes are known to have high desiccation tolerance. In order to find out the mechanism of the tolerance, we have investigated changes in various photosynthetic activities caused by dehydration using bryophytes grown under different water environments. We found that PSII fluorescence is quenched and the PSII reaction center (PSIIRC) activity is lost by dehydration, which seems to be important to protect PSII under dry conditions. However, even in desiccation-tolerant species, partial dehydration by sorbitol treatments give the state where the photosynthetic activity was lost but the PSIIRC activity was remaining. We investigated the effect of high light at this state, and found that PSIIRC was practically deactivated by nonphotochemical quenching (NPQ), which was not recovered by dark incubation. This NPQ was found not to be caused by the xanthophyll cycle and to be very important for desiccation tolerance. Here we will report on details of this NPQ.

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Roots of higher plants seem to be advanced mechanisms to drought stress, since the root system is in direct contact for resistance to the soil. In this study, we analyzed drought-responsible proteins in the root tissues of wild watermelon (Citrullus lanatus sp. No101117-1) by the proteomic approach. Wild watermelon were exposed to the drought stress and the root soluble proteins were subjected to 2-D gel electrophoresis. The intensities of 35 spots were increased 1 day after the stress (Type I) and those of 42 spots were increased after 3 days (Type II). By LC-MS/MS analysis, cell division- and metabolism-related proteins were identified in Type I spots. On the other hand, various HSPs, antioxidative enzymes, and proteases were found in Type II spots. These results suggest that wild watermelon roots change the strategy in drought resistance from the induction of the adaptive to resistance mechanisms, during the progress of the stress.

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Wild watermelon (Citrullus lanatus sp. No101117-1) is highly resistant to drought stress, but contribution of the root system to the resistance remains to be characterized. We have demonstrated by proteomic analysis that expression of aRan GTPase (CLRAN) is increased by drought stress. Here, we analyzed developmental mechanisms of wild watermelon roots under drought stress and physiological function of CLRAN. The root elongation of wild watermelon was promoted under the drought stress. In comparison to domesticated watermelon, wild watermelon showed lower sensitivity to exogeneous ABA in the elongation of primary root and the lateral root initiation. CLRAN was expressed mainly in root meristematic and elongation zones, and the expression was increased to 1.5-fold in root tips by drought stress. The Arabidopsis overexpressing CLRAN showed suppressed ABA sensitivity. These results indicate that wild watermelon plants have the root development system for drought avoidance response in which CLRAN functions as a key-regulator.

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Citrulline is an efficient hydroxyl radical-scavenging compatible solute which accumulates in the leaves of wild watermelon during drought/strong light stresses. In plants, citrulline metabolic pathway is composed of eleven enzymes. However, how these enzymes are regulated for massive accumulation of citrulline in wild watermelon remains unclear. In this study, we determined the changes in activities of all the eleven enzymes in wild watermelon leaves during drought. The activities of the first and second enzymes in the citrulline pathway, N-acetylglutamate synthase and N-acetylglutamate kinase, and activity of the enzyme forming carbamoyl carrier, carbamoyl phosphate synthetase, increased approximately 7-, 7-, and 3-fold, respectively, during drought. In contrast, the activity of citrulline-catabolism enzyme, argininosuccinate synthase, decreased one-fifth under drought. These results suggest that citrulline accumulation is triggered by the increase in the influx of substrates into the synthetic pathway, and decrease in the efflux of citrulline into the catabolic pathway.

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To improve soil environment, utilization of terrestrial cyanobacteria is considered as a practical means. Nevertheless, the mechanism of desiccation tolerance of this organism has not yet been elucidated. We have analyzed the global expression profiles during dehydration in the filamentous cyanobacterium Anabaena sp. PCC 7120. Rehydration process, during which cells recover from damages by the stress, is considered to be important for dehydration tolerance besides enduring during dehydration. We performed DNA microarray analysis during rehydration. Many genes were upregulated during rehydration. Among them, we focused on the ancrpB, whose gene product is a transcriptional factor activated by cAMP. Intracellular cAMP concentration increased transiently when rewetting cells. The recovery of oxygen evolution activity during rehydrartion in ancrpB disruptant was lower than that in wild type, suggesting the importance of the gene regulation by AnCrpB. We will discuss the cAMP signaling during rehydration as well as expression profiles during dehydration and rehydration.

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F-box proteins regulate various cellular processes by degradation of target proteins by ubiquitination in Skp1-Cullin-F-box protein (SCF) complexes of ubiquitin/26S proteasome pathway. Recent studies showed that ~700 F-box protein genes are present in Arabidopsis thaliana genome, and that the F-box proteins play critical roles in various aspects of plant growth and development. However, no reports have been published on the F-box proteins involved in drought stress response.
Recently, we identified the genes encoding 4 Stress-Inducible F-box Proteins (SIF1~4) and 5 Recovery-Inducible F-box Proteins (RIF1~5) by microarray analyses (Oono et al. 2003, 2006). Expression of the SIF1 gene was up-regulated by drought, high-salinity and ABA treatments. Knock-out mutants of the SIF1 gene showed hypersensitivity to osmotic stress (200mM sorbitol). The roles of SIF1 in the stress responses will be presented.

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We found more than 50 predicted NAC proteins in rice. These proteins were classified into 11 subgroups Groups A to K by sequence similarity. Microarray analysis revealed that five genes including OsNAC6 among six genes in the Group A were induced by dehydration, high-salinity, and low temperature. Northern and promoter:GUS analysis showed that the expression of the OsNAC6 gene was also induced by abscisic acid, jasmonic acid, and wounding. The transactivation assay using a yeast system revealed that the OsNAC6 protein function as a transcriptional activator. The transient assay showed that the OsNAC6-sGFP fusion protein is localized in the nucleus. Transgenic Arabidopsis and rice plants overexpressing OsNAC6 showed dwarf phenotype. The results of microarray analysis revealed that many abiotic- and biotic-stress-inducible genes were up-regulated in the OsNAC6-overexpressed Arabidopsis and rice plants. These results indicate that OsNAC6 functions as a transcriptional activator under biotic and abiotic stresses in plants.

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Phytohormone abscisic acid (ABA) promotes stomatal closure to adapt plants to drought. Recent studies revealed that protein phosphorylations have an important role in ABA signaling, but a few phosphoproteins have been characterized in detail. In this study, we investigated ABA-induced protein phosphorylations in Vicia guard cell protoplasts using 14-3-3 protein, as a probe. We found that a 61 kDa protein bound to 14-3-3 protein tightly in response to physiological concentration of ABA within a few minutes. This reaction was found exclusively in guard cells, and inhibited by the protein kinase inhibiter, K-252a. Furthermore, reactive oxygen species (ROS), such as H₂O₂, as secondary messengers of ABA signaling in guard cells, and inhibitors of ROS production had no effect on this reaction. Thus ABA-induced phosphorylation of 61 kDa protein may participate in the upstream of ROS production or in the parallel pathway of ABA signaling in guard cells.

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Abscisic acid (ABA) plays an important role in seed maturation, seed dormancy, stomatal closure, and stress response. RPK1, a leucine-rich repeat (LRR) receptor kinase in the plasma membrane, is upregulated by ABA in Arabidopsis. We analyzed the phenotypes of T-DNA insertion mutants and RPK1-antisense plants. Repression of RPK1 expression in Arabidopsis decreased sensitivity to ABA during germination, growth, and stomatal closure; microarray and RNA gel analysis showed that many ABA-inducible genes are downregulated in these plants. Furthermore, overexpression of the RPK1 LRR domain alone or fused with the BRI1 kinase domain in plants resulted in ABA-sensitive phenotypes. We then produced the C-terminal hemagglutinin (HA)-tagged RPK1 construct driven by the CaMV35S promoter and introduced into Arabidopsis plants. These plants overexpressing RPK1 showed weak growth retardation and increased ABA sensitivity in root growth and stomatal closure. We will present further details of the plants overexpressing RPK1-HA.

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The receptor protein kinase 2 (RPK2), an Arabidopsis LRR receptor-like kinase (LRR-RLK), is the only homolog of the receptor protein kinase 1 (RPK1) which is a key regulator of ABA signaling in Arabidopsis. Two RPK2 knockout mutants (rpk2-1 and rpk2-2) showed male-sterility and bushy shoot growth. Abnormal lignification was observed on the surface of rpk2 anther and it might cause the defect of anther dehiscence. Tapetum abnormally fatted in rpk2 anther after meiosis and normal pollen maturation was inhibited. We identified 255 downregulated genes (fold decrease > 3) in rpk2 flowers by a microarray experiment and revealed that many genes for lignin biosynthesis and cell wall degradation enzymes were downregulated. Furthermore, several genes for ABA- or stress-related proteins, such as RD20 and LEA proteins, are also downregulated. These results indicate that RPK2 is essential for floral development and that RPK2 may be involved in ABA signaling pathway.

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The phytohormone abscisic acid (ABA) regulates physiologically important stress and developmental responses. To address the mechanism of ABA response, we isolated 4 novel ABA hypersensitive mutants named ahg (ABA hypersensitive germination). ahg1 showed hypersensitivity to ABA, NaCl, KCl, mannitol, glucose and sucrose in germination and post-germination growth but not in adult plants, indicating its function in seed. The ahg1 seed accumulated slightly more ABA before the stratification and showed increased seed dormancy. Map-based cloning of AHG1 revealed that this gene encodes a novel protein phosphatase 2C (PP2C). We already have shown that ahg3 has a point mutation in the AtPP2CA gene encoding another PP2C that have pivotal role in ABA response in seed. We will discuss the relationship between AHG1 and AHG3/AtPP2CA in ABA response.

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The phytohormone abscisic acid (ABA) regulates physiologically important stress and developmental responses. To address the mechanism of ABA response, we isolated 4 novel ABA hypersensitive mutants named ahg (ABA hypersensitive germination). ahg2 showed clear ABA hypersensitivity not only in germination but also at later developmental stages. Map-based cloning of AHG2 revealed that this gene encodes a poly(A)-specific ribonuclease (AtPARN). Interestingly, the expression mRNA levels of stress- and salicylic acid-inducible genes in ahg2 were upregulated compared to WT. ahg2 also showed SA hypersensitivity in germination and resistance against biotrophic pathogen P. syringae pv. tomato but not against necrotrophic pathogen B. cinerea in plant. In addition, ahg2 plant accumulated more SA as well as ABA. We will discuss the role of AHG2/AtPARN in abiotic stress and biotic stress.

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Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes. In plants, MAPK cascades are known to function in various biotic and abiotic stress signaling. Arabidopsis genome-sequencing project has revealed the existence of a large number of MAPK family genes in its genome. However, detailed functions of each MAPK cascades are still unknown. We have been focused on MKK3, one of the Arabidopsis MAPKK, and found the novel MKK3-MPK6 cascade. Analyzing the phenotype of MKK3DD plants and the regulated gene expressions of MKK3, we also demonstrated that MKK3-MPK6 cascade mediates JA signaling. Exogenous application of JA activated MPK6 and this activation was severely impaired in mkk3 mutant and JA-insensitive coi1 mutant. Plants overexpressing either MKK3 or MPK6 were insensitive, by contrast, either mkk3 or mpk6 showed the increased sensitivity to JA. These results indicated that the MKK3-MPK6 cascade plays a crucial role in JA signaling in Arabidopsis.

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C6-aldehydes can induce defense genes and induce accumulation of antifungal substances in Arabidopsis. In order to elucidate the signaling pathway induced by C6-aldehydes, we compared the responses of mutants deficient in the phytohormone signaling pathways; i.e. jar1-1, etr1-1, npr1-1, or pad2-1. When compared with the wild type (WT) plant, inductions of the defense genes in response to C6-aldehydes were significantly repressed in jar1-1 and pad2-1, but not in etr1-1 nor in npr1-1. After C6-aldehyde treatment, significant accumulations of camalexin could be observed with WT and npr1-1, but only partial accumulation could be observed with etr1-1, and jar1-1. pad2-1 showed little accumulation of camalexin after the treatment. The treatment could also enhance resistance of etr1-1, jar1-1 and npr1-1 against Botrytis cinerea, but failed to enhance the resistance with pad2-1. Thus, it was suggested that ETR1-, JAR1- and PAD2-dependent signalings were activated by C6-aldehyde treatment, but NPR1-dependent signaling was not.

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The ontogenic change in photosynthetic activity of temperate seagrasses was assessed using pulse-amplitude modulated (PAM) fluorometer: Diving-PAM. Seeds of Zostera marina, Zostera asiatica and Zostera japonica were cultured in outdoor tanks. The effect of age on photosynthetic activity was determined by minimum fluorescence (F), maximum fluorescence (Fm'), variable fluorescence (&DeltaF), effective quantum yield (&Phi_PSII), electron-transport rate (ETR) and leaf absorptivity (AF). Leaf chlorophyll pigments (a, b, a+b & a:b) were also measured. Fluorescence yields, leaf absorbance and chlorophyll pigments increased with age.In contrast, the effective quantum yield (&Phi_PSII) and electron-transport rates (ETR) decreased with age. Highest effective quantum yield and electron-transport rates were observed in younger tissue than in older tissue. Z. japonica had the highest yield and electron-transport rates, hence, found to be productive. The results demonstrate that PAM fluorometry is indeed an effective technique in determining temporal changes in the photosynthetic activity of seagrasses over time.

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State transition in oxygenic photosynthesis is a short-term mechanism of excitation energy redistribution between photosystem I (PSI) and II (PSII). It is hypothesized that preferential excitation of PSII translocates mobile light-harvesting complex from PSII to PSI. Although there are several LHCII polypeptides in higher plants and algae, biochemical evidence for which types of LHCII polypeptides are mobile, is weak. Here, using the green alga Chlamydomonas reinhardtii cells locked into state 1 and state 2, we established the procedure to separate chlorophyll-protein complexes under a mild condition and found a new PSI-LHCI supercomplex specifically from state 2 cells which is larger than the normal supercomplex and contains the mobile LHCII polypeptides. These mobile polypeptides were identified as CP26, CP29 and LHCII TypeII by mass spectroscopic and immunochemical analyses. A molecular mechanism on the translocation of LHCII complexes between PSI and II during state transition will be discussed.

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State transition, the redistribution of light-harvesting complex II (LHCII) between photosystem I (PSI) and photosystem II (PSII), is one of the photoacclimation mechanisms in plants and algae. To explore the dynamic changes in the supermolecular organization during state transitions, biochemical isolation of both PSI and PSII supercomplexes including the light-harvesting complexes is demanded. However, there has been as yet no report obtaining the highly pure PSII-LHCII supercomplexes during state transtions. In this study, we establish a new, simple, rapid procedure to isolate highly pure PSII-LHCII supercomplexes from a Chlamydomonas mutant carrying a hexahistidine-tag at C-terminus of CP47. The differences in the size and protein compositions of the supercomplexes between state 1 and state 2 will be reported.

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Plant photosynthesis is reliant on the dynamic assembly and stability of the four multisubunit complexes located in the thylakoid membranes, which work in unison to generate ATP and reducing equivalents required for the Calvin cycle reaction occurring in the stroma. The role of a number of stromal factors in the transcriptional, post-transcriptional and post-translational regulation of membrane complex assembly is well documented, but the contribution of lumenal factors to this process is relatively unknown. The thylakoid lumen has recently been found to containnearly 20 distinct immunophilins, the majority of which are FKBP-type immunophilins. We isolated Arabidopsis mutants lacking the AtFKBP20-2 isoform and show that they display a severe and specific defect in the content of the PSI complex, characterized by an alteration in the stoichiometry of core and antenna subunits. Recent evidence for the role of additional lumenal immunophilins in photosynthetic regulation will also be presented.

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Chloroplast F₁ (CF₁) equips several regulation mechanisms; redox regulation, the specific inhibition and acceleration by phytotoxic tentoxin, and inhibition by the internal inhibitory subunit epsilon. In order to investigate these regulation mechanisms thoroughly, we constructed the expression system for the subcomplex of F₁-ATPase from cyanobacteria Thermosynechococcus elongatus BP-1 (TSF₁). In addition, we successfully introduced the regulatory region of the gamma subunit of the chloroplast CF₁ into the gamma subunit of this complex (TSF₁-redox). ATP hydrolysis activity of the obtained TSF₁-redox was nicely redox regulated. The effect of tentoxin to TSF₁ was similar to that for CF₁. Hence TSF₁ and TSF₁-_redox mimic many features of CF₁, and they will be a useful tool for the study on the regulation mechanisms of CF₁ at the single molecule level. The inhibitory effect of the epsilon subunit on rotation of the gamma subunit will be also discussed.

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Whereas light energy absorbed in photosystem II (PSII) is used for photosynthetic electron transfer and carbon dioxide fixation, the excess proportion of absorbed energy is dissipated as heat. This thermal dissipation, which can be detected as non-photochemical quenching (NPQ) in chlorophyll fluorescence, is controlled by PsbS. To reveal the quantitative relation between the electron transport activity and NPQ formation, we produced the psbS-silenced rice using RNA interference.
When we divided the fate of absorbed light energy in PSII to electron transport (P), dissipation in antenna (D) and that at PSII reaction center (E) according to Damming Adams et al (1996), RNAi transformations showed the decrease in (D) with the increase in (E), and the similar level in (P) in comparison with the wild type. These results suggest that NPQ do not down regulate the electron transport activities at PSII of rice.

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In oxygenic photosynthesis, NAD(P)H dehydrogenases are involved in cyclic electron transport around photosystem I, which may play an important role in acclimation to oxidative stress. None of them, however, have been purified and characterized. Native PAGE analysis followed by activity staining for NAD(P)H dehydrogenases revealed that a thermophilic cyanobacterium Thermosynechococcus elongatus contains several NAD(P)H dehydrogenases. Among them, activities of a 140 kDa band and a 70 kDa band were increased significantly in the cells grown under high light intensity in the presence of methyl viologen. This result suggests that these enzymes play roles in protection against oxidative stress, thus they may be involved in cyclic electron transport around photosystem I. Both enzyme complexes were purified by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE. Subunit composition and enzymatic properties of these enzymes will be discussed.

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NAD(P)H dehydrogenase (NDH) of chloroplasts is involved in PSI cyclic electron transport. Eleven and three subunits are encoded by the chloroplast genome and nuclear genome, respectively. Comparison with the bacterial NDH complex indicates the existence of some unidentified subunits functioning in electron donor binding in plants. An Arabidopsis gene, CRR7 was identified in the mutant which specially lacks NDH activity. In crr7, the level of the NDH complex was reduced. CRR7 (At5g39210) encodes a protein containing 156 amino acids with a plastid targeting signal and localized to the thylakoid membranes. CRR7 is hydrophilic protein without any transmembrane domain and does not contain any known motifs. CRR7 is conserved in cyanobacterial genomes. CRR7 was unstable within the crr2-2 , in which the expression of ndhB was impaired. These results strongly suggest that CRR7 is a novel subunit of the chloroplast NDH complex.

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Chloroplast NDH complex is a homologue of mitochondrial NADH dehydrogenase and involved in photosystem I cyclic electron transport. In higher plants, 14 subunits of the NDH complex have been identified. However, these subunits contain no electron donor-binding site and electron donor for the NDH complex also has not been decided. Arabidopsis crr1 was isolated as a mutant lacking NDH activity, using chlorophyll fluorescence imaging. Because CRR1 contains chloroplast a targeting signal and a NAD(P)H-binding motif, the CRR1 is a possible candidate for electron donor-binding subunit of the NDH complex. Interestingly, CRR1 is homologous to DHPR (dihydrodipicolinate reductase) involved in lysine biosynthesis, although it does not have DHPR activity. In addition, whereas genes for DHPR are expressed in all tissues, CRR1 is expressed only in photosynthetic tissue as well as other genes for the NDH subunits. Now we are studying the possibility that CRR1 is a subunit of the NDH complex.

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Previously, we identified 3, 8-divinyl protochlorophyllide a 8-vinyl reductase (DVR) gene from Arabidopsis.
DVR reduces an 8-vinyl group on chlorophyll to an ethyl group. The substrate of DVR is assumed to be DVPchlide a, and chlorophyll biosynthesis pathway was constructed based on this assumption. However, real substrate of DVR is not determined because the enzymatic experiment has not been done with purified enzymes or recombinant proteins.
In order to determine the chlorophyll biosynthesis pathway, we carried out enzymatic experiments using the recombinant DVR protein and chlorophyll intermediate molecules. These experiments showed DVR had high activity with DVChlide a, but low or no activity with other pigments. We also investigated the flow of chlorophyll intermediates in Arabidopsis. Conversion of DVPchlide a to MVPchlide a required several hours but DVChlide a was reduced within a few seconds.
On the basis of these experiments, we will propose revised chlorophyll biosynthesis pathway.

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Chlorophyllide a oxygenase is a rieske-type monooxygenase that catalyzes conversion of chlorophyll a to chlorophyll b. It has been demonstrated that the levels of CAO protein control accumulation of chlorophyll b. CAO protein is consisted of three domains, namely, A, B and C domains. We hypothesized that A and B domains are involved in the regulation of the CAO levels, while C domain has a catalytic function. In order to identify the functions of A and B domains, we constructed a series of modified CAO sequences that were fused with GFP and overexpressed them in Arabidopsis thaliana. We analyzed the CAO protein levels in these transgenic plants by confocal microscopy and immunoblotting, and found that the B domain is not necessary for the regulation of the CAO protein levels, although this domain contains a phydrophylic region that resembles the PEST sequences known as the targets of the ubiquitin degradation system.

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In higher plants, chlorophyll a is converted to chlorophyl b through 7-hydroxymethyl chlorophyll a, and chlorophyll b returns to chlorophyll a again through 7-hydroxymethyl chlorophyll a. This conversion system is called chlorophyll cycle.
With the aim of identifying the enzymes and the regulatory factors that are involved in chlorophyll cycle, we have carried out screening EMS-mutagenized Arabidopsis thaliana. We isolated a mutant that accumulated 7-hydroxymethyl chlorophyll a.7-hydroxymethyl chlorophyll a accumulated more than 10 times in this mutant compared with wild type during 3-day-dark-induced senescence. 7-hydroxymethyl chlorophyll a accumulated more than several times without dark-induced senescence. The size of this mutant was smaller than that of wild type, and its leaves were softer and paler than those of wild type. We found that this mutated phenotype was recessive.
Currently, fine mapping of this gene is being undertaken.

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nyc1 was isolated in rice as the recessive mutation that inhibited chlorophyll breakdown during senescence. Such a mutant is called 'stay green' mutant. In nyc1, the inhibition of Chlb breakdown was prominent but Chla breakdown was also inhibited to some extent. Consequently, the Chla/b ratio became nearly 1 at the very late stage of senescence. In nyc1, Rubisco large subunit and Chla binding proteins were degraded but LHCII remained stable even at the very late stage of senescence. HPLC analysis revealed that the degradation of not only Chla and Chlb but also carotenoids that bind LHCII tightly was inhibited in nyc1. These data collectively suggest that nyc1 is the mutant involved in LHCII stability during senescence.

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Biosynthetic pathway of Chl d in Acaryochloris marina has not be chaified yet, although Chl d is thought to be systhesized from Chl a.
Recently we came across the formation of Chl d from Chl a in aqueous acetone with papain². Oxidative cleavage of a C=C double bond of a vinyl group in Chl a is necessary to yield Chl d. Chl a has many C=C bonds, so that it is too difficult to oxidize the sole C=C bond. In this study, we examined the reaction of Chl b and Phe a.
Similar reaction was observed in Chl b and Phe a with papain, although the reaction were very slow². Our finding will provide insight into the unsolved key question as to the biosynthetic pathway of Chl d via Chl a in A.marina.

1.Kobayashi et al., Photosynth. Res., 84, 201-207 (2005)
2.Koizumi et al., Sci. Tech. Advanced Materials, in press.

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Chlorin ring structure of chlorophyll a is formed by the reduction of porphyrin D-ring by light-independent protochlorophyllide reductase, a nitrogenase-like enzyme, in most photosynthetic organisms. Bacteriochlorophyll a has bacteriochlorin ring structure that is formed by the reduction of chlorin B-ring. Phenotypic analysis of mutants indicated that the three genes, bchX, bchY and bchZ, which show similarity to those of structural genes of nitrogenase, are involved in the chlorin B-ring reduction. However, there has been no biochemical evidence. We tried to reconstitute the chlorin B-ring reduction with purified proteins. BchX was purified as a single polypeptide, and BchZ was co-purified with BchY. When both components, BchX and BchY-BchZ, were incubated with chlorophyllide a, ATP, ATP-regeneration system and dithionite under anaerobic conditions, chlorophyllide a was converted to a new pigment with longer wavelength. Involvement of second nitrogenase-like enzyme in bacteriochlorophyll biosynthesis will be discussed.

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The cyanobacterium Gloeobacter violaceus is different from other cyanobacteria in that it doesn't have thylakoid membranes, and the photosynthesis reaction is carried out on cytoplasmic membranes. The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:Pchlide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). In this study, we purified POR from G. violaceus using Escherichia coli overexpression system, and determined its kinetic and substrate binding properties. The purified protein is fully active and the activity is light dependent. The evolutionary implication of POR will be discussed based on the comparative analysis of enzymatic properties between G. violaceus POR and those of other cyanobacteria and plant PORs.

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In higher plants, red algae, and cyanobacteria, phytobilins are utilized for photoreceptor and/or light harvesting pigments. Phytobilins are synthesized by ferredoxin-dependent bilin reductases (FDBRs) from biliverdin ΙΧα (BV). Phycocyanobilin:ferredoxin oxidoreductase (PcyA), one such FDBR, successively reduces two sites of BV, the vinyl group of D-ring and the A-ring, to produce phycocyanobilin. This indicates that PcyA recognizes the slight difference between the vinyl and methyl groups, and controls the sequence of the reductions.
We have determined the crystal structure of PcyA from Synechocystis sp. PCC 6803 in complex with BV at 1.51 Å resolution, revealing the first tertiary structure of an FDBR family member. The binding site of ferredoxin, and the mechanisms of recognition and reduction of BV will be discussed. Additionally, the features of molecular surfaces and active sites of other FDBRs, which were derived by homology modeling, were compared with those of PcyA.

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After the completion of the whole genome sequence of model plants, it is now possible functional genomics approach through the comprehensive analyses of metabolites (metabolomics) and gene expression (transcriptomics). We have already integrated metabolomics and transcriptomics of PAP1-overexpressed transgenic plants, two putative glycosyltransferase genes induced by PAP1 expression were confirmed the gene functions, experimentally⁽¹⁾. On the other hand, transcriptome coexpression database (ATTED) was released in last year. In this research, we inferred a coexpression framework model including the putative phenylpropanoid metabolic genes and up-regulated genes by PAP1, and subsequently speculated each gene function. Metabolic profiling of the targeted gene T-DNA insertion mutants were performed using high-throughput analysis based on UPLC-Q-TOF MS. Subsequently, detailed analysis of metabolite changing in the mutants revealed the changes in metabolo-types, indicating the 2 glycosyltransferases and 1 dehydratase gene functions.
1. Tohge et al., Plant J. (2005) 42: 218-235

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It is well known that genes involved in a certain biosynthetic pathway are expressed coordinately. By coexpression analysis, we attempted to identify the function of unknown glycosyltransferase genes related in flavonoid metabolism.
In Arabidopsis, flavonoids are highly modified by glycosylation. The structures imply that at least eight glycosyltransferases (GT) are involved in this pathway and only four genes were identified. GTs are encoded by 119 genes in Arabidopsis. Their primary structures are not sufficient to estimate their functions.
By coexpression analysis of public database, ATTED, we found a GT gene that is highly correlated with flavonoid biosynthetic genes. The amino acid sequence is similar to that of flavonol-7-glucosyltransferase. The T-DNA insertion mutants didn't contain flavonoid 7-rhamnosides. These results show that the gene encodes flavonoid-7-rhamnosyltransferase (At7RhaT). Real time PCR analysis showed that transcripts of At7RhaT are accumulated abundantly in buds. It's consistent with the flavonoid accumulation pattern in Arabidopsis organs.

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Delphinidin 3-(6''-malonyl)glucoside-3',5'-diglucoside-6'''-p-coumaroyltransferase (3'AT) has been assigned to a role in conversion of ternatin C5 into ternatin C3. This enzyme activity was detected for the first time from the petals of butterfly pea using 1-O-acylglucose as acyl-donor. The purified 3'AT had heteromeric structure comprised of 31- and 24-kDa subunits on SDS-PAGE. Biochemical and enzymatic characters suggested that 3'AT was a kind of 1-O-acylglucose-dependent acyltransferases which constitute a class of serine carboxypeptidase-like acyltransferase (SCPL-AT). We employed a PCR-based strategy using primers designed on conserved regions of SCPL proteins to clone the gene. The isolated cDNA clone, CtAT1, encoded 31- and 24-kD subunits containing the sequences for three tryptic fragments derived from purified 3'AT protein. The cDNA encoded a SCPL protein with a N-terminal signal sequence, three possible N-glycosylation sites, and Ser-His-Asp putative catalytic triad. Expression of the cDNA in a baculovirus expression vector produced an active protein displaying 1-O-acylglucose:anthocyanin 3'-O-glucoside-O-acyltransferase activity.

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Carnation (Dianthus caryophyllus) petals come in several color and especially pink and red colors consist of anthocyanin. The anthocyanin pathway gene from chalcone synthase through UDPglucose:flavonol 3-O-glucosyltransferase or other sugar addition steps, have been well characterized. However, S gene that was thought to determine dark or pale color in petal has not been clarified. Previously, we isolated GST (Glutathione S-Transferase) gene (DcGST4-1) from dark red carnation. In this study, we isolated two types of mutated DcGST4 (DcGST4-2, DcGST4-3) from pale color carnation. These three DcGST4s were introduced into petal of pale color carnation by particle bombardment and transiently expressed. Only DcGST4-1 formed dark red spots in the petal of pale color carnation. This result indicated that two types of DcGST4 mutation might be cause of pale colors formation in petal of carnation, and that GST4 might be S gene.

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Two types of red pigments, anthocyanins and betacyanins, never occur together in the same plant. The accumulation of flavonols in the Caryophyllales suggests that the step(s) of anthocyanin biosynthesis from dihydroflavonols to anthocyanins could be blocked in the Caryophyllales. We have shown that the Caryophyllales had genes encoding functional DFR and ANS and the genes are not expressed in most tissues and organs in Spinacia oleracea.
To clarify the regulatory mechanism of DFR and ANS expression, we have analyzed promoter region of DFR and ANS in the Caryophyllales. The promoter region contains several motifs, which a transcriptional factor could bind. Yeast one-hybrid assay showed that PhAN2 and PhJAF13, which were anthocyanin regulators, could bind to the DFR and ANS promoters of spinach. The transient assay in Phytolacca americana cells showed that the promoters were not activated by PhAN2 and PhJAF13.

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The coordinate induction of flavonoid biosynthesis with shikimate pathway, serving phenylalanine as precursors for flavonoid biosynthesis, in response to environmental stimuli suggests that the expression of genes involved in both pathways coordinate under the common transcriptional control. 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, the first enzyme in the shikimate pathway, is encoded by multiple copies of the gene with different patterns of expression of tissue or cells and induction following environmental stimuli. However, the organization and differential regulation of individual genes in the DAHP synthase multigene family during development and in response to different environmental stimuli has remained obscure. We have isolated DAHP synthase homologues from Lotus japonicus Gifu-129. Sequence analysis of PCR products suggests that at least five distinct DAHP synthase genes exist in L. japonicus. The analysis of expression patterns of each homologue in response to different environmental stimuli is in progress.

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An isoflavan vestitol is a phytoalexin of the Lotus spp. including a model legume Lotus japonicus. Despite of extensive studies on isoflavonoid metabolism, detailed mechanism of the isoflavan biosynthesis has been unclear. In this study, homologous sequences (PTR1 and PTR2) of phenylcoumaran benzylic ether reductase (PCBER), an enzyme of lignan pathway catalyzing an analogous reaction to pterocarpan reduction, were selected from a L. japonicus EST library, and substrate specificity of the proteins was examined. At the amino acid levels, PTRs are 60% identical with both PCBER and isoflavone reductase. Crude extracts of E. coli expressing PTRs catalyzed the formation of vestitol from a pterocarpan (-)-medicarpin and NADPH. Lignans and 2'-hydroxyformononetin were not used as substrates. Thus, PTRs showed pterocarpan reductase activity. RT-PCR analysis using L. japonicus seedlings displayed the constitutive accumulations of PTR mRNAs with or without reduced glutathione-treatment. Further characterization of the enzymes is in progress.

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The flower color could be modified to be deep yellow/orange by introducing carotenoid/xanthophyll biosynthetic genes into the pale-yellow colored petunia, whose petal contains carotenoid. Especially, ketolase gene (crtW) from Agrobacterium aurantiacum caused remarkable change of the color of the petal and is useful to detect the expression of the gene product. Histochemical analysis demonstrated that white-colored petunia contains few leucoplasts in petal cells and that in pale-yellow colored petunia, there are chloroplasts in cells near vein and other cells contain chromoplasts. No change was observed in the plastid of crtW-transformed petunia showing orange color flower. Moreover, in order to generate deep-orange colored petunia, various transit peptides were added to crtW and the transformants were obtained. Various flower color value of the tarnsformants revealed that there is compatibility of the transit peptide sequences exists in the expression of the crtW product.

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Plant cell develops a large vacuole as the cell enlarges, and cytoplasm is localized around the cell cortex as a thin layer. Recent progress in visualization of the tonoplast has revealed complex vacuolar structures and suggested that cytoskeletons participate in the regulation of the tonoplast structures. However the relationship between the vacuolar structure and cell growth and differentiation is still unclear. In this study, we successfully visualized the tonoplast by fluorescent dyes and AtVam3p-GFP fusion gene in Physcomitrella patens, and revealed that complex structures of the tonoplast were conspicuous at growing tips, around developing septum, and in dense regions of chloroplasts. The cytoskeletal inhibitors affected the tonoplast structures and protoplasm distribution. We currently analyze changes of the tonoplast structures and distribution, when protonemata regenerate from protoplasts, and will discuss how the localization of cytoplasm and vacuole is regulated during cell differentiation and cell growth.

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Stomatal movement originates from changes in the shape of the guard cells and is discussed in combination with vacuole. We observed vacuolar structures in guard cell during stomatal opening and closing using Vicia and transgenic Arabidopsis line expressing GFP-AtVam3p. Detailed observation of confocal images demonstrated numerous intrar-vacuolar membrane structures in the closed stomata while they mostly disappeared in the open-stomata. Introduction of a morphological parameter, vacuolar complexity, could evaluate the stomatal morphological changes numerically. A 3-D reconstruction using our software REANT revealed a unified vacuolar structure even when they seemed to be compartmented at the single confocal image in the closed stomata. Furthermore, calculation of the vacuolar volume and its surface area by REANT showed a parallel increase and decrease of the vacuolar volume during stomatal opening and closing while the surface area was maintained. These results suggest that stomatal vacuoles maintain their membrane as intra-vacuolar structures when closing stomata.

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In higher plants, vacuoles increase their volume according to cell enlargement and become large vacuoles that occupy most of the cell volume. To observe the vacuolar dynamics in living cells, we have already established a transgenic BY-2 cell-line expressing a GFP-AtVam3p fusion protein (BY-GV), and have also developed 3-D reconstruction software, named REANT. These methods allowed us to reveal unique vacuolar structures including TVM (tubular structure of vacuolar membrane) in mitotic cell and vacuolar networks developed in elongating miniprotoplasts. In this study, vacuolar movements were quantified to explore the physical mechanism of the formation and development of these vacuolar structures. We developed analyzing software tools for time-sequential series of fluorescent images of vacuolar membrane and bright-field images. Frequency analysis using discrete wavelet transform enabled us to evaluate vacuolar movements without image segmentation. Vacuolar movements were regulated by spatio-temporal factors: cell cycle progression, vacuolar structures and actin microfilaments.

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Blue light-induced membrane hyperpolarization in guard cells through the activation of plasma membrane H⁺-ATPase drives K⁺ uptake and elicits stomatal opening. Hyperpolarization-activated Ca²⁺channel in guard cells has been reported, however, it is not investigated whether the membrane hyperpolarization induces the Ca²⁺ uptake through this type of Ca²⁺ channel. In this study, we showed the blue-light-induced changes in cytosolic Ca²⁺ concentration in guard cell protoplasts isolated from Arabidopsis thaliana, which has been transformed with Ca²⁺-sensitive luminescent protein, aequorin. At lower level than physiological concentration of extracellular K⁺, blue light-dependent Ca²⁺ uptake was observed, however, it was absent at the physiological level of K⁺. When we added the K⁺ channel blockers, the Ca²⁺ uptake was observed even in the presence of higher concentration of K⁺. These results indicate that blue light-dependent hyperpolarization can induce Ca²⁺ uptake but K⁺ uptake is predominant under physiological condition.

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Blue light induces stomatal opening through the activation of the plasma membrane H⁺-ATPase. Recent investigations have demonstrated that blue light activates the H⁺-ATPase via phosphorylation on threonine residue in the C-terminus with subsequent binding of 14-3-3 protein to the phosphorylated C-terminus in guard cells. However, protein kinase and protein phosphatase, which catalyze phosphorylation and dephosphorylation of the H⁺-ATPase, are still unknown. In this study, we investigated in vitro phosphorylation and dephosphorylation of the H⁺-ATPase. The H⁺-ATPase was phosphorylated in the presence of ATP in the isolated microsome. An inhibitor of protein kinase, K-252a, had no effect on this phosphorylation. Dephosphrylation of the H⁺-ATPase was also found in the isolated microsome, and was inhibited by EDTA, but not by calyculin A, suggesting that type 2C protein phosphatase may catalyze dephosphorylation of the H⁺-ATPase. We will report analyses of the H⁺-ATPase complex by immunoprecipitation and gel filtration.

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