Jasmonic acid (JA) is a plant hormone responsible for the defense from pathogen and insect attacks. It is synthesized at high level immediately after the mechanical wounding of plant tissue. The initial step of JA biosynthesis is a lipolytic reaction by lipase(s) that releases linolenic acid from chloroplast membrane lipids. One of these lipases is the DAD1 involved in the JA production at flower opening, but the dad1 mutant fully generates JA after wounding, suggesting that the lipase other than DAD1 is responsible for the wound induction. In the Arabidopsis genome, there are 11 lipase genes highly homologous to the DAD1, and six of them (DAL1-DAL6) are predicted to be chloroplast proteins. We examined the expression of these genes and showed that the DAL2, 3, 4 are constitutively expressed in leaves, whereas the DAL6 and DAD1 are induced after wounding. Making multiple knockouts of these genes is in progress.

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Jasmonates are lipid-derived compounds regarded as "non traditional" plant hormones which exert numerous effects on plant growth and development. In recent years, it is well known that jasmonates are important as a mediator of wound- and stress-induced signal transduction and induce expression of a variety of genes involved in defensive response. And it also has been revealed that jasmonates plays an important role in fruits ripening. In some fruits, It is reported that the endogenous concentration of jasmonates increases during the onset of fruits ripening, and that the application of exogenous concentration of jasmonates stimulates ethylene production and color change.
To determine if jasmonates affect the expression of the genes involved in ripening, such as ethylene biosynthesis, we examined the change of the mRNA levels encoding these genes. Treatment of breaker stage tomato disks with methyl jasmonate promoted the accumulation of mRNA for ACC oxidase, but ACC synthase 4.

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Jasmonic acid (JA) and methyl jasmonate (MeJA) are singaling compounds under stress conditions such as wounding and pathogen responses. Our previous report has shown that MeJA treatment induced genes involved in jasmonates biosynthesis, tryptophan biosynthesis, and phytohormone metabolism such as auxin and salicylic acid.
For larger scale analysis, we screened jasmonates-responsive genes (JRGs) using cDNA macroarray covering 8,384 loci, and identified 300 genes as JRGs. In order to analyze JRGs in more detail, we focused on genes, which related to amino acid biosynthesis or secondary metabolism.Consequently, we found that jasmonates induced many genes involved in paticular metabolic pathways such as auxin biosynthesis, indole-glucosinolate biosynthesis, sulfur metabolism, and ascorbate metabolism in mRNA level. We measured contents of thiol compounds and activities of ascorbate recycling enzymes in jasmonate-treated plants, and found that JA treatment increased their contents and activities. The results indicate that jasmonates activate these metabolic pathways in Arabidopsis thaliana.

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Several types of jasmonic acid (JA) derivatives, including JA-amino acid conjugates, a JA-biotin conjugate, a JA-dexamethasone heterodimer, and a JA-fluoresceine conjugate, were prepared as candidates for molecular probes to identify and analyze JA-binding proteins. These JA derivatives except JA-fluoresceine conjugate exhibited significant biological activities in rice seedling assay, soybean phenylalanine ammonia-lyase-inducing assay, and/or rice phytoalexin-inducing assay. These results indicate that these JA derivatives might be useful probes to identify and analyze JA-binding proteins. It was also indicated that the activity spectra of prepared compounds were different from each other, suggesting that different types of JA-receptors function in the respective bioassays.

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Expressed sequence tag (EST) library was prepared from a model tobacco BY-2 cell culture. This EST library includes approximately 10,000 partial cDNA clones that are now opened to public users at (http://mrg.psc.riken.go.jp/strc/). We also carried out multiple microarray analyses with the selected EST clones, and with particular interest in the plant hormone-regulated gene expression. As one example, BY-2 cells cultivated under auxin-deficient conditions were treated with stress-related hormones salicylic acid, abscisic acid and methyl jasmonate. The expression patterns induced by these hormones with a special focus in commonly and/or differentially regulated genes, and the correlation of gene expression with the hormone-induced phenotypes, will be discussed.

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To understand the function of Chl a in Acaryochloris marina in which Chl d is a major pigment, we measured delayed fluorescence (DF) and estimated a pigment content. A. marina was grown in BG 11 medium under incandescent light with intensities of 5, 15, and 35 μE/(m² sec). DF in the nanosecond time range was measured with a time-correlated single-photon counting method. Pigment content was estimated by HPLC. The DF was observed only in the wavelength region of Chl a both in A. marina and Synechocystis sp. PCC 6803, and the DF completely disappeared by heat-treatment in both species. Analyses on pigment content revealed that the Chl a/Pheo a ratio decreased as decrease in light intensities, but never lowered than one. These clearly indicated that the primary electron donor of the PS II in A. marina is Chl a as in another cyanobacteria.

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An extrinsic 33-kDa component protein (PsbO) in photosystem II is essential for photosynthetic oxygen evolution. The extrinsic 33-kDa protein is present in all oxygen-evolving organisms, and plays a central role in the stabilization of the manganese cluster. In Arabidopsis, the extrinsic 33-kDa proteins are encoded by two genes, psbO and psbO2. Immunoblot analysis showed that PsbO was more abundant than PsbO2. Furthermore, an Arabidopsis mutant with a defect in psbO showed the lower photosynthetic activity, suggesting functional differences of two proteins as well as those in expression. Thus, we produced the recombinant PsbO and PsbO2 without transit peptide in E. coli and purified them to characterize their functions. The reconstitution experiment using spinach PSII particles without their extrinsic 33-kDa proteins indicated that the restoration of oxygen evolution activity with PsbO2 was lower than PsbO. Further analysis is on going.

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The psbP gene family encoding the extrinsic 23-kDa proteins in the oxygen-evolving complex of photosystem II consists of four isogenes in Nicotiana tabacum. Tobacco psbP genes are divided into two groups (minor group-I; 1A and 5B: major group-II; 2AF and 3F) by the sequence similarity. We have analyzed the physiological function of these psbP genes using group-specific PsbP-silenced transgenic tobacco plants obtained by RNA interference (see Yamamoto et al., JSPP Annual Meeting 2004).
Preliminary analyses in T0 and T1 transgenic plants using a PAM fluorometer showed significantly higher F₀ and the decreased photosynthetic electron transport rate in group-II-specific PsbP-silenced tobacco than that in control tobacco, whereas group-I-specific PsbP-silenced tobacco was indistinguishable from control tobacco. These results suggest the difference in the function or in the level of expression between the two groups of psbP. Further characterization with T2 plants is currently under way.

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Photosystem II, a large membrane protein complex, catalyzes the important reaction of water oxidation to molecular oxygen. Many PSII subunits are conserved between plants, green algae, and cyanobacteria. The extrinsic proteins that comprise the oxygen evolving complex differ between cyanobacteria and chloroplasts. In the current accepted model, higher plants and green algae have PsbO, PsbP and PsbQ, while cyanobacteria contain PsbO, PsbV and PsbU. Interestingly, homologs of both PsbP and PsbQ have been found according to previous genomic and proteomic analysis in cyanobacterium, Synechocystis 6803. To clarify the function of these two proteins in cyanobacterial PSII, we have analyzed isolated PSII complexes from knockout mutants of both proteins. In this presentation, we demonstrate that the functional roles of PsbP and PsbQ proteins are conserved between cyanobacteria and plants and that five lumenal extrinsic proteins are present in cyanobacterial PSII. [Supported by National Science Foundation]

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The green algal extrinsic 23 and 17 kDa proteins directly bound to photosystem II (PSII) independent of the other extrinsic proteins, which is largely different from those of spinach PSII (Suzuki et al., Plant Cell Physiol. 44, 76-84, 2003). In order to elucidate the possible causes responsible for the differences, cross-reconstitution experiments were performed with different combinations of the extrinsic proteins and the green algal (Chlamydomonas reinhardtii) or spinach PSII. These results showed that the green algal extrinsic proteins could not bind to spinach PSII directly, indicating that their direct binding depends principally on PSII intrinsic proteins. In addition, we performed crosslinking experiments with a water-soluble carbodiimide. These results showed that the extrinsic 23 kDa protein is in close proximity with the large subunit of cytchrome b559, indicating that the latter provided a possible binding site for the 23 kDa protein in the green algal PSII.

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The extrinsic 33 kDa protein which plays important roles in maintaining the stability and activity of Mn cluster electrostatically binds to PSII intrinsic protein. We have reported that the positive charges on the protein are important for electrostatic interaction with PSII intrinsic proteins by chemical modification of charged amino acids, and estimated that Lys residues in six domains of the 33 kDa protein are commonly involved in the binding in both spinach and red algal PSIIs. In this study, we examined which Lys residues in the six domains directly participate in the interaction by site-directed mutagenesis. The 33 kDa protein substituted 15Lys and 164Lys to Gly could not functionally bind to PSII, while the protein substituted 198Lys to Gly functionally bound. On the basis of these results, we will discuss Lys residues on the 33 kDa protein responsible for the electrostatic interaction with PSII intrinsic proteins.

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PSII-H is present in photosystem II (PSII) core complex in cyanobacteria and plants. The role of PSII-H was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The photoautotrophic growth of the psbH-disruptant was much slower than that of wild type. Cells and thylakoids from the mutant showed about half the oxygen-evolving activity of wild type. While the PSII complex of wild type was mainly collected as dimer by anion exchange chromatography, the PSII complex of the psbH-disruptant was not recovered as dimer but as two monomeric forms. One was an inactive PSII complex lacking 33 kDa, cyt c550, and 12 kDa extrinsic proteins. The other was a PSII complex retains the oxygen-evolving activity and the extrinsic proteins. Moreover, PSII-X was absent in the PSII complexes of the psbH-disruptant. These results imply that PSII-H stabilizes the dimeric PSII complex associated with PSII-X and extrinsic proteins.

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We constructed and characterized Synechocystis mutants, in which Ala344 of D1 protein was replaced by glycine, valine, and aspartate (A344G, A344V, A344D) and His-tag was attached to CP47, to elucidate the role of C-terminus Ala344 of D1 protein as a putative ligand to Mn-cluster in PSII. Under moderate light conditions (50 μE m^-2s^-1), all the mutants grew photoautotrophically, however, A344G and A344D mutants could not grow photoautotrophically at increased light intensity of 200 μE m^-2s^-1. A344G and A344D mutants exhibited thermoluminescence Q-band (S₂Q_A^-) and B-band (S₂Q_B^-) at temperatures higher by 5^oC than those of the control strain. No change in protein compositions of the purified PSII particles were detected in the mutants. These results suggest that the Gly or Asp substitution affects the properties of Mn-cluster. To examine the effects of mutations on the structure of Mn-cluster, we are conducting EPR spectroscopic characterization of Mn-cluster.

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We examined the effects of the site-directed mutation at the D1-Ala344 (A344G, A344V, A344D) of the Synechocystis sp. PCC6803 on the structure of the Mn-cluster using the flash-induced FTIR difference spectroscopy. Mid-frequency (1800 - 1000 cm^-1) S₂/S₁ spectra of these mutants were distinctively different from wild-type spectrum in the carboxylate stretching region (1600 - 1300 cm^-1). In addition, a negative band at 1112 cm^-1 was strongly induced in A344G mutant, and amide I and II bands were significantly modified in A344V mutant. These results suggest that subtle differences of side chains of the C-terminus residue characteristically modified the coordination structure of the Mn-cluster. Furthermore, the low-frequency (650 - 350 cm^-1) S₂/S₁ spectrum demonstrated that bands at 606/625 cm^-1, assigned to a Mn-O-Mn cluster mode, were largely affected in A344G mutant, suggesting that the structure of the Mn-cluster itself is changed considerably.

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EPR of the Mn multiline in the S₂-state of single crystals Synechococcus vulcanus has been observed at 6 K by W-band EPR spectrometer. The multiline composed of 21 lines, showing that four manganese contribute to the multiline. The hyperfine constant does not depend on crystal orientation but the positions of hyperfine lines are changing with orientation. No multiline EPR was observed in a frozen solution of the same sample. Similar spectra were observed in the S₀-state reduced by NH₂OH. These results are now under simulation assuming g-anisotropy.

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In PSII, the secondary quinone electron acceptor Q_B is reduced by the primary quinone Q_A, and after two-electron reduction, Q_B is protonated and released from the protein. The structural relevance of the Q_B binding site to this Q_B function still remains unclear. Here, we have studied the structure and reaction of Q_B using FTIR spectroscopy. FTIR difference spectra of S₂Q_B^-/S₁Q_B and of S₂/S₁ were measured using PSII preparations from spinach or the cyanobacterium T. elongatus, and a Q_B^-/Q_B spectrum was calculated as a double difference. In this spectrum, the CO stretching band of Q_B^- was observed at 1480 cm^-1, the frequency 2 cm^-1 higher than that of Q_A, indicating the difference in H-bonding interactions between Q_A^- and Q_B^-. In addition, a positive C=O band of a COOH group was observed at 1747 cm^-1, suggesting that a nearby Asp or Glu residue is partially protonated upon Q_B^- formation.

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In the oxygen-evolving complex of PSII, two water molecules are cleaved into four protons and one molecular oxygen through a light-driven cycle of S states (S₀-S₄). To study the proton release steps in this cycle, we have examined the pH dependence of the S-state transitions using FTIR spectroscopy. FTIR difference spectra upon 1st- to 4th-flash illumination were measured using PSII core complexes from Thermosynechococcus elongatus at pH4-10. The efficiencies of S-state transitions at each pH were estimated by fitting the spectra with the standard spectra measured at pH6. The results showed that the efficiencies of S₂->S₃, S₃->S₀ and S₀->S₁ transitions decreased by lowering pH, but were less sensitive at higher pHs. This is consistent with the view that protons are released in the S₂->S₃, S₃->S₀ and S₀->S₁ transitions.

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It has been reported two fluorescence emission bands at 685 nm(F685) and 695 nm(F695) in photosystem II(PSII) at 77 K, emitted from chlorophyll a in the core antenna CP43 and CP47, respectively. This study, we measured the fluorescence dynamics of a Arabidopsis thaliana leaf in the picosecond time region at 5 K, 40 K and 77 K. We found a new band at 687 nm(F687). With increasing temperature, the decay rate of F687 became faster so that in a later time region it could not be distinguished from F695 because of the overlap with F695. We also observed F687 in the spinach PSII and Synechocystis sp. PCC 6803 at 5-77 K. Each bands in cyanobacteria decay faster than those of higher plants. We will propose a model for the excitation-energy transfer dynamics in PSII, accounts for the rise and decay kinetics of the fluorescence bands.

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Photosynthetic oxygen evolution is carried out by machinery that is composed of four manganese ions. A molecular oxygen is produced by a series of reactions with five distinct intermediate state labeled S₁ (i=0-4).
We have found a g=2 ESR signal with 200 G width in Ca²⁺-depleted photosystem (PS) II. The ESR signal was produced by illuminating the Ca²⁺-depleted PS II in S₁ state at 245 K for a short period. By annealing the sample, the signal disappeared with trapped Y_Z radical signal, and S₂ multiline signal appeared. We will discuss the origin of the g=2 broad signal in S₁ state.

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Photosynthetic water oxidation is catalyzed by the tetranuclear Mn-cluster of photosystem (PS) II. Two water molecules are oxidized to an oxygen molecule through five intermediates states (S₀ - S₄). In the present study, flash-induced FTIR difference spectra for the S-state cycle and effects of H₂¹⁸O-substitution on the spectra were examined using PS II core particles from Thermosynechococcus elongatus. Mid-frequency (1800 - 1000 cm^-1) difference spectra for S₁-to-S₂, S₂-to-S₃, S₃-to-S₀, and S₀-to-S₁ transitions showed characteristic vibrational modes from backbone polypeptides and amino acid residues associating with the Mn-cluster, but were scarcely affected by H₂¹⁸O-substitution. In low-frequency (650 - 350 cm^-1) difference spectra, 650 - 600 cm^-1 bands changed in their sign and intensity during the S-state transitions and showed considerable isotope shifts upon H₂¹⁸O-substitution, indicating that these bands reflect the changes of the Mn-cluster core modes and/or Mn-water modes that are involved in the water oxidation chemistry.

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In Neurospora crassa light signal can affect several morphogenesis including stimulation of carotenoid synthesis in mycelia, and 2)perithecial polarity. We have reported that mutation in nucleoside diphosphate kinase, ndk-1P72H showed lack of perithecial polarity. We present that superoxide and hydrogen peroxide may be included in the process of light signal transduction. Using a mutant in superoxide dismutase (Cu/Zn SOD), sod-1 we detected that the production of carotenoid in sod-1 under light was accelerated compared with that of wild type. The photoreceptor mutant wc-1 and wc-2 showed no production of carotenoid in the mycelia. However, double mutant, wc-1, sod-1 and wc-2 sod-1 did not show stimulation of carotenoid synthesis. The vvd mutation in VVD gene, which suppress the function of wc-1 and wc-2, showed enhanced production of carotenoid in the mycelia. The double mutant, vvd, sod-1 showed additive stimulation of carotenoid synthesis.

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As for aquatic photosynthetic organisms, different sensors for photo-movement and photomorphogenesis were acquired according to each phylogenetically diversified organism. Recently, archaeal-type rhodopsin from Chlamydomonas reinhardtii and BLUF-domain-containing adenylyl cyclase from Euglena gracilis were identified as sensors of photomovement. In phototactic zoospores from brown algae, posterior flagella are green-fluorescent, which indicates the accumulation of flavin molecules. Action spectra suggested that flavoprotein in posterior flagella was a promising candidate for sensor of phototaxis. In this study, we tried to isolate and characterize the flavoprotein(s) from green-fluorescent flagella of the brown alga Scytosiphon lomentaria. Zoospores were released from the matured algal thalli. Detached flagella by Vortex treatment were frozen and suspended in Bis-Tris-propane buffer. Soluble fraction including flavoprotein was chromatographed. Fluorescence spectra and SDS-PAGE of eluates from ion-exchange and gel-filtration columns suggested that 41kDa protein was flavoprotein. Fluorescence and TLC analyses indicated that FMN was non-covalently bound to the protein.

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In order to elucidate the function of cryptochromes (cry) in rice, we had isolated all rice CRY genes (OsCRY1a, OsCRY1b and OsCRY2 ) and have analyzed the their expression patterns at transcription level.
Here, we examined tissue specific localization of each CRY protein by the western analysis. We transformed rice with expression vectors which overexpressed OsCRY1a or OsCRY1b genes and analyze the effect of cryptochromes on the early photogenesis in rice. We have noticed that coleoptile elongation is inhibited by blue-light irradiation. We compared blue-light sensibility on the inhibition of coleoptile elongation between OsCRY1a or OsCRY1b overexpressed transgenic rice and WT. Very low level of blue light irradiation didn't inhibit the coleoptiles elongation in the WT plants, while OsCRY1a or OsCRY1b overexpressed transgenic plants showed the inhibition. These results indicate that the possibility OsCRY1a and OsCRY1b are involved in the inhibition of coleoptile elongation by blue-light irradiation.

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The cpt1 mutant of rice was isolated in our laboratory as a mutant showing no coleoptile phototropism and severely impaired root phototropism. Map-based cloning of the CPT1 gene indicated that cpt1 mutation is linked to a deletion of 208 kb at position 85 centimorgan on chromosome 2. This region included a gene homologous to Arabidopsis NPH3 (AtNPH3), known to be essential for hypocotyl phototropism. The cpt1 mutant transformed with genomic DNA of rice NPH3 (OsNPH3) showed normal phototropisms of coleoptiles and roots, demonstrating that OsNPH3 is CPT1. The deduced amino acid sequences of OsNPH3/CPT1 and AtNPH3 showed 58% identity. Database and sequence analysis indicated that rice has at least 36 NPH3 homologs and that OsNPH3 is an ortholog of AtNPH3. The fact that mutation in NPH3 genes results in severely impaired phototropism in both Arabidopsis and rice indicates that the product of NPH3 is essential for phototropism of higher plants.

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Phototropins act as photoreceptor in blue light (BL) dependent responses in plants. However, components involved in phototropin signaling are largely unknown. Using a yeast two-hybrid screening, dynein light chain like protein (vfDLC) of Vicia faba was isolated as an interactor of vfphot1a. DLC is a subunit of the dynein complex which translocate along microtubules. Far western and two-hybrid analysis revealed that vfDLC binds to the N-terminal region of vfphot1a but not to vfphot1b. Interaction between GST-vfDLC and vfphot was confirmed by pull-down assay. Northern analysis revealed that the transcription level of vfDLC was higher in guard cells than in other cell types. vfDLC-GFP showed fluorescence spots on the plasma membrane in guard cells. Treatment of plant materials with microtubule-depolymerizing herbicides partially inhibited BL dependent H⁺-pumping and stomatal opening. These results suggest that vfDLC may be a component of BL dependent signal transduction in stomatal opening.

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Blue light (BL), absorbed by phototropins, activates the plasma membrane H⁺-ATPase in guard cells, and drives stomatal opening. However, there is no biochemical evidence for early events of phototropins in stomatal guard cells. In vivo phosphorylation analysis using ³²P-labeled guard-cell protoplasts from Vicia faba revealed that Vfphots were reversibly phosphorylated in response to BL, and that phosphorylation of vfphots was faster than that of the H⁺-ATPase. We found that the 14-3-3 protein was bound to Vfphots upon phosphorylation. Amino acid substitution analysis using recombinant Vfphots (Vfphot1a and Vfphot1b) expressed Escherichia coli showed that binding of 14-3-3 protein requires phosphorylations of Ser358 residue for Vfphot1a (RRKS₃₅₈) and Ser344 residue for Vfphot1b (RRKS₃₄₄), which are localized between LOV1 and LOV2, respectively. We conclude that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 binding are likely to be key step of BL responses in stomata.

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Phototropins (phot1 and phot2), as blue light (BL) receptors, mediate phototropism, chloroplast relocation and stomatal opening in Arabidopsis. We provide biochemical evidence for the binding of 14-3-3 protein to Arabidopsis phot1. Overlay assay using a recombinant 14-3-3 protein showed that the 14-3-3 protein bound to phot1 in etiolated seedlings, leaves, and guard cells within 1 min after the start of BL pulse. The half-saturating intensity of BL was 7.2 μmol m^-2 s^-1 (30 s). Interestingly, the 14-3-3 proein did not bind to phot2 in any fluence rate of BL. Furthermore, phot1 was co-precipitated with the 14-3-3 protein in the etiolated seedlings in response to BL, suggesting that the 14-3-3 protein binds to phot1 in vivo. These results indicate that the binding of 14-3-3 protein to phot1 is common to phot1-mediated responses. Overlay assay using recombinant phot1 revealed that binding site(s) localizes in the region between LOV1 and LOV2.

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Phototropins (phot1 and phot2), autophosphorylating protein kinases, mediate stomatal opening through activation of the plasma membrane H⁺-ATPase via phosphorylation in response to blue light (BL). However, the mechanism by which the signal from phototropins is transduced into H⁺-ATPase activation is unknown. We investigated the effect of tautomycin (TAU), a specific inhibitor of type1 and type2A protein phosphatases, on BL-dependent responses to elucidate the role and location of protein phosphatase in BL signaling. Stomatal opening in epidermal strips and H⁺ pumping in Vicia guard cell protoplasts in response to BL were strongly inhibited by TAU. Furthermore, TAU inhibited both BL-induced phosphorylation of the H⁺-ATPase and subsequent binding of 14-3-3 proteins. However, BL-induced autophosphorylation of phototropins was not affected by TAU. These results suggest that a TAU-sensitive protein phosphatase functions as a positive regulator in guard cell BL signaling downstream phototropins and upstream the H⁺-ATPase.

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Blue light (BL)-dependent H⁺ pumping by guard cells, which drives stomatal opening, is inhibited by phytohormone abscisic acid (ABA). We investigated this response in respect with activity of the plasma membrane H⁺-ATPase, and hydrogen peroxide (H₂O₂) generated by ABA in Vicia guard-cell protoplasts (GCPs). BL-dependent phosphorylation of the H⁺-ATPase and subsequent binding of a 14-3-3 proteins were inhibited by ABA at 10 μM or H₂O₂ at 1mM in similar degrees. Inhibitions of BL-dependent H⁺-pumping and phosphorylation of the H⁺-ATPase by ABA in GCPs were partially reversed by ascorbic acid. A single-cell analysis revealed that H₂O₂ is generated by ABA in GCPs mainly from guard-cell chloroplasts. Fusicoccin-dependent H⁺ pumping and binding of a 14-3-3 protein to the H⁺-ATPase were inhibited slightly by both ABA and H₂O₂. These findings suggest that H₂O₂ may be involved in ABA-inhibition of BL-dependent H⁺-pumping through decrease in phosphorylation levels of the H⁺-ATPase.

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phototropin 1 (phot1) and phot2, which are blue-light receptor kinases, function in blue-light-induced hypocotyl phototropism, chloroplast relocation, and stomatal opening in Arabidopsis thaliana. Previous studies have shown that the proteins RPT2 and NPH3 transduce signals downstream of phototropins to induce the phototropic response. However, the involvement of RPT2 and NPH3 in stomatal opening and in chloroplast relocation mediated by phot1 and phot2 was unknown. Genetic analysis indicates that RPT2 is involved in the phot1-induced phototropic response and stomatal opening, but not in chloroplast relocation or phot2-induced movements. Biochemical analyses indicate that RPT2 is purified in the crude microsomal fraction as well as phot1 and NPH3, and that RPT2 makes a complex with phot1 in vivo. On the other hand, NPH3 is not necessary for stomatal opening or chloroplast relocation. Thus, these results suggest that phot1 and phot2 choose different signal transducers to induce three responses.

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It is well known that blue and red light can open stomatas through the osmotic regulation. In the previous study, we have found that in guard cells the cortical microtubules were organized as radial arrays during daytime, suggesting their role in the stomatal opening. However, it was not clear which light induced the cortical microtubular organization. Then, we examined the relationship between the cortical microtubular organization and the stomatal aperture under the blue or red-light condition in Vicia Fava L.. As a result, both the blue and red lights could organize the cortical microtubules, the stomatal aperture was higher under the blue light. Furthermore, using Arabidopsis plants stably expressing GFP-tubulin fusion protein, we found that the blue light induce the radial array more clearly.
These results suggest the blue light regulation of stomatal opening through the organization of cortical microtubules.

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Blue light receptors in Arabidopsis include two types of proteins, cryptochromes and phototropins. Previous studies have suggested that the cryptochromes cry1 and cry2 function in photomorphogenesis and that the phototropins phot1 and phot2 regulate photo-induced movements. Receptors in the same family have redundant functions although their responses to the fluence rate of blue light differ. To uncover novel functions of blue light receptors that may be concealed by their functional redundancy, we conducted a gain-of-function analysis using multiple mutants of blue light receptors. Comparison of the responses of the quadruple mutant cry1 cry2 phot1 phot2 to blue light with those of related triple mutants revealed that cryptochromes function in novel blue-light-dependent random-hypocotyl-bending and that phototropins function in one photomorphogenesis: cotyledon expansion. Microarray analysis suggested that cry1 and cry2 independently function as key regulators of early blue-light-induced genes, whereas phot1 and phot2 play subsidiary roles in transcriptional regulation by blue light.

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psbD contains a blue-light-responsive promoter (psbDLRP), differentially recognized by SIG5, a nuclear-encoded sigma factor of PEP. SIG5 transcription is induced by weak blue-light (5 μmolm^-2s^-1) via cryptochromes in Arabidopsis. Besides the weak blue-light, psbDLRP activation, however, requires blue light non-specific illumination at high intensity. Here we examine how these two distinct lights are involved in psbDLRP activation. SIG5 transcript abundance increased to a constant level at 120 min after blue-light illumination (5 μmolm^-2s^-1) with 30-min induction time, while no transcript from psbDLRP was detected. Further illumination of the resulting plants with red-light (50 μmolm^-2s^-1) immediately induced psbD transcription, which was stimulated by DBMIB. These results indicate that PEP assembled with SIG5 transcribes from psbDLRP, only when plastoquinone pool is reduced by photosynthetic electron transport. It is likely psbD transcription is regulated via a blue-light signal transduction pathway in nucleus and the redox state in chloroplasts.

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Phototropin (phot) is a plant blue-light receptor. Higher plants has two phot isoforms, phot1 and phot2, each of which has two chromophoric domains named LOV1 and 2 in the N-terminal half, and the C-terminal half works as a serine/threonine kinase. Photoreaction of LOV domains may alter their stracture to activate kinase. To make this clear, small-angle X-ray scattering was measured with phot1 and phot2 of Arabidopsis. Results indicates that LOV1 fragments of both phot1 and phot2 exist in a dimeric form and do not exhibit any detectable light-induced structural change. On the other hand, LOV2 is in a dimmer in phot1 and a monomer in phot2. Futhermore, LOV2 fragment of phot1 shows marked changes in scattering patterns under light illumination, indicating global structure changes during the photoreaction. These results will be discussed in connection with the different roles of LOV domains in regulating the kinase activity of phot.

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Phototropin (phot) is a blue-light photoreceptor for tropic responses, relocation of chloroplasts and stomata opening in plants. Phot has two chromophoric domains named LOV 1 and 2 in the N-terminus half, and a serine/threonine protein kinase domain in the C-terminus half. Phot has been shown to be autophosphorylated in response to blue light, however, no other substrate has been detected. Bacterially expressed Arabidopsis phot2 kinase domain (KD) with GST-tag (GST-KD) phoshorylated casein, a common in vitro substrate of ser/thr protein kinase. Using this in vitro analysis system, the roles of the each LOV domain were studied. GST-LOV2-KD phosphorylated casein in a light-dependent manner that was abolished by introduction of mutation blocking photochemical reaction of LOV2. GST-LOV1-LOV2-KD also showed light-activated protein kinase activity. Blocking the photochemical reaction of LOV1 had little effect on the kinase activity. In contrast, that of LOV2 brought significant decrease of the activity

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Phototropin (phot) is a blue-light receptor in plants. Phot has two FMN binding domains named LOV domain. In response to light absorption, a covalent adduct forms between a cysteine and FMN C(4a), which is called S390. According to the crystal structures of the LOV2 domain of Adiantum phytochrome3 (phy3), a chimeric phytochrome/phototropin, in the dark and S390 states, the side chain of Gln1029 switches hydrogen bond with FMN.
The structural changes were measured in the Q1029L mutant by low-temperature FTIR spectroscopy. It was implied that hydrogen-bond of FMN C(4)=O group is not formed in Q1029L. Upon formation of S390, hydrogen bond of C(4)=O becomes weaker in both WT and Q1029L. On the other hand, the protein structural changes of Q1029L were smaller than those of WT at room temperature.
These results suggest that the hydrogen-bonding interaction of Gln1029 with FMN plays an important role in the protein structural changes of phy3-LOV2.

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Arabinan and galactan are side chaines of pectic polysaccharide, rhamnogalacturonan I (RG-I). They may involve in cell adhesion. But little is known about biosynthesis of galactan and arabinan. Bioassay of galactan is performed by determing incorporated ¹⁴ C-Gal from ¹⁴ C-labeled UDP-Gal. We prepared 2-aminobenzamide (2AB) -labeled galactooligosaccharides as acceptor substrates and determind galactosyltransferase (GalT) activity by HPLC, LC-ESI-MS, ¹ H NMR spectroscopy and enzyme hydrolysis. GalT transferred up to 14 Gals to the non reducing end of 2AB-labeled galactooligosacharides. The newly incorporated Gal residues were β-1,4 linked. 2AB-labeled RG-I having Gal residues was an acceptor substrate, suggesting that the GalT was capable to adding Gal residues to RG-I galactan side chains.

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Rhamnogalacturonan I, a component of pectic polysaccharides, consists of a backbone of repeating disaccharide unit, →αGalA1→2αL-Rha→, attached with sugar-chains of (1→4)-linked β-Galp and/or (1→5)-linked α-L-Araf residues at C-4 of L-Rha residues. The side-chains regulate intercellular attachment, thereby affect the architecture of cell walls. We have analyzed the enzymatic properties of a membrane-bound galactosyltransferase (GalT) prepared from etiolated soybean hypocotyls (Planta, in press). We have attempted to solubilize and purify the GalT protein to identify the corresponding gene.
Activity of GalT was assayed by measurement of the amount Gal groups transferred from UDP-[¹⁴C]Gal onto β-(1→4)-galactan as an acceptor. By the treatment of the microsomal fraction with 0.75% Triton X-100, more than 70% of GalT activity could be solubilized. Subsequent chromatography of the solubilized fraction on a CM-Sepharose column followed by affinity chromatography separated a large amount of impurity from the enzyme protein.

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We have investigated β-galactosyltransferase (GalTase) involved in the synthesis of arabinogalactan-proteins whose basic carbohydrate structure comprises a β-(1→3)-galactan, branched with consecutive (1→6)-linked β-Galp residues, to which α-L-Araf residues are attached through (1→3)-linkages.
The enzymatic reaction was carried out in a mixture composed of a microsome fraction prepared from etiolated soybean hypocotyls, UDP-[¹⁴C]Gal, and β-(1→3)-galactan as an acceptor at 25°. The Gal transfer occurred maximally at pH 5.7 in the presence of 15 mM Mn²⁺ and 0.75% Triton X-100 (specific activity about 500 pmol/min/mg protein). Digestion of the radiolabeled product with exo-β-(1→3)-galactanase released radiolabeled β-1,6-galactobiose as the main product, indicating preferential transfer of Gal groups onto the β-(1→3)-galactan chains through β-(1→6)-linkages, creating branching points. β-1,3-Galactooligomers served as acceptor while β-1,6-oligomers were inactive.

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Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans found in higher plants. AGPs consist of large amounts of carbohydrate components (>90%) having β-3,6-galactan as the backbone frame, whose fine structures and molecular functions are remained to be determined.
We have cloned an endo-β-1,6-galactanase (Tv6GAL) from Trichoderma viride and an α-L-arabinofuranosidase (RsAraf) from radish as useful tools for the structural and functional analyses of AGPs. RsAraf releases L-arabinofuranose residues from native AGPs purified from radish [1]. Tv6GAL hydrolyzes β-1,6-galactosyl side chains of α-L-arabinofuranosidase-treated AGPs. Tv6GAL appeared to be a novel member of family 5 glycosyl hydrolase based on its amino acid sequence [2]. We are constructing transgenic Arabidopsis by introducing RsAraf and/or Tv6GAL to analyze the molecular functions of the carbohydrate moieties of AGPs.
[1] Hata K. et al. (1992) Plant Physiol. 100: 388-396
[2] Kotake et al. (2004) Biochem. J. (in press)

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Plant polygalacturonase (PG) is known to function in leaf abscisson and defense gene activation. Polygalacturonase β-subunit (PGβS) tightly associates with PG and has been proposed to interact with the catalytic subunit of PG to inhibit its activity or to prevent access of the enzyme to cell wall pectins.
We extracted proteins from tobacco leaf cell walls by treatment with LiCl. In the fraction, the amount of 42K protein slightly increased during leaf expansion. cDNA encoding the 42K protein had high similarity with tomato PGβS. The tobacco PGβS homologue gene was expressed during leaf expansion. We tried to induce silencing of the PGβS homologue gene using tomato mosaic virus-based vector, which harbored mRNA of PGβS homologue. Nicotiana benthamiana showed cell death in the inoculated leaf, and showed retarded development in upper leaf, which was speculated as silencing effect. We expect that the tobacco PGβS homologue play a role in leaf development.

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XTH (Xyloglucan endotransglucosylase/hydrolysase) is a class of enzymes that can mediate endotransglucosylation and/or hydrolysis of xyloglucan molecules. In Arabidopsis thaliana, 33 genes encoding XTH proteins have been identified. Individual members of this gene family were shown to exhibit distinct expression profiles, temporally and spatially. Each XTH member is considered to play the role individually in plant morphogenesis. To gain insight into roles of individual members of the gene family, we have isolated and analyzed phenotypes of homogeneous T-DNA insertion lines, which cover 24 XTH genes. We found that the xth12 mutant has defect in root growth and the xth27 mutant has lesions in leaves. On the other hand, no macroscopic phenotype was observed in other xth mutants. The result indicates that most of XTH genes are redundant functionally, but some XTH genes play important roles individually in specific aspects during the plant growth and development.

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The plant cell wall is a dynamic architecture constructed by many kinds of proteins, most of which are encoded by multi-gene families. Although the complete sequencing of Arabidopsis genome has revealed thousands of putative cell-wall related genes, functions of most of these genes are not known. In order to investigate their roles in the cell wall formation, we extracted cell wall proteins from Arabidopsis suspension -cultured cells with 1M KCl solution. We also extracted proteins from the protoplast which had been allowed to regenerate cell wall for 1h and 3h, respectively. Using the 2D-PAGE and MALDI-TOF MS technologies, we separated, mapped and identified these proteins, and found 72 protein spots in the native cell wall, 176 and 213 protein spots in the 1h and 3h regenerated protoplast, respectively. Based on these results, we discuss roles of these proteins in individual step during the cell wall formation.

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Polysaccharide-linked hydroxycinnamoyl esters (PHEs) can form covalent ester-ether bridges between lignin units and polysaccharides to provide additional cross-linking within cell walls of gramineous plants. These covalent cross-links are important for the mechanical support of aerial parts and also in the resistance to compressive gravitational forces; they contribute to the termination of wall extensibility, to the strengthening of cell walls, and to prevention of the entry of pathogens into cells. Details of the biosynthesis of PHEs and the molecular mechanisms responsible for the temporal and spatial control of the biosynthesis of PHEs remain unclear. In this study, as a part of program characterizing the biosynthetic regulation of plant cell wall component, we examined the polysaccharide-linked hydroxycinnamoyl esters biosynthetic pathway and its molecular mechanism of genetic regulation in rice plant using rice one gene recessive mutant, Fukei 71.

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Gibberellin (GA) regulates root growth. GA promotes the growth of Pisum sativum roots in the presence of ancymidol, an inhibitor of GA-biosynthesis. To investigate the role of cell wall proteins in root growth, we analyzed, by SDS-PAGE, the major bands of apoplastic proteins of pea roots treated with GA and/or ancymidol. GA-affected major proteins were sequenced and identified by homology search. Eight of eleven proteins affected by GA were sequenced. Three proteins homologous to pectin methylesterase (garden pea, mung bean) and to peroxidase (spinach, soy bean) were enhanced by GA. Five proteins were suppressed by GA. These were homologous to peroxidase (Medicago), chitinase class III (tobacco, rice, azuki) and cysteine proteinase (pea, Arabidopsis). Since nine of 13 proteins found in database contained signal sequences, most major proteins analyzed were conceived as apoplastic proteins with less contamination with cytoplasmic proteins. Role of these proteins in cell wall extension will be discussed.

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Dehydrodiconiferyl glucoside (DCG) is a neolignan isolated from Vinca rosea tumor cells that induces cell division. We isolated cDNA of a peroxidase, AoPOX1, secreted to cell walls during asparagus somatic embryogenesis. AoPOX1 exhibited the activity to convert coniferyl alcohol (CA) to dehydrodiconiferyl alcohol (DDCA). DDCA is aglycone of DCG. We hypothesized the autocrine mechanism in somatic embryogenesis via neolignan molecule (Takeda et al. 2003). There are two possible pathways for DCG biosynthesis; 1) CA is converted to DDCA then glucosylated in apoplast; 2) Coniferyl alcohol glucoside (CAG) is synthesized in symplast and CA and CAG are coupled by apoplastic peroxidase. The glucosyltransferase activity was not detected in the cell-wall protein fraction. The glucosyltransfelase activity in the symplast and concentration of DCG or CAG in the medium are under investigation.

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Suspension-cultured cells of azuki bean utilize sucrose and glucose but neither galactose nor mannose. Galactose inhibits sucrose synthesis in cells but mannose appears lacking this effect (Inouhe and Kato 2003). In this study, we compared the possible effects of mannose on growth and sucrose concentration of cells in the liquid and solid cultures. We also determined the phosphomannose isomerase (PMI) activity as a candidate enzyme converting mannose to sucrose via sugar phosphates. Azuki bean cells in liquid medium containing 1% mannose had very slow growth rate and decreased sucrose level. By contrast, the cells inoculated on agar medium containing mannose proliferated actively after a month of nascent period and increased the intracellular sucrose level. We found that the PMI activity in the mannose-adapted cells increased as a function of culture period. However, similar activities were detected in cells grown in sucrose medium. Mannose-adaptation appeared to involve the other enzymatic pathway(s).

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Redox status in apoplast appears to affect cell wall metabolism and hence cell growth in plants. To examine this possibility, we examined effects of two antioxidants, ascorbic acid (AsA) and glutathione (GSH) on cell elongation in epicotyl segments of azuki bean under light and/or dark conditions. We found that neither AsA nor GSH stimulated cell elongation in darkness. However GSH strongly stimulated it under the light unlike AsA. GSH mimicked auxin effects because it alone stimulated cell elongation with no IAA application. Externally applied GSH increased the intercellular level of GSH in the segments. However, BSO, the specific inhibitor of GSH synthesis, did not inhibit cell elongation of epicotyl segments. These results suggested that GSH might stimulate cell elongation of stem segments by changing redox status in apoplast or by modifying some process linking to the transport of GSH to symplast.

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Previously, we reported that a peroxidase isoenzyme (cationic cell-wall-bound peroxidase, CWPO-C) from poplar callus preferentially oxidizes sinapyl alcohol and sinapyl alcohol polymer as substrates. In this study, CWPO-C cDNA sequence was cloned by the PCR technique, RT-PCR and RACE. Competitive PCR analysis showed that CWPO-C gene was expressed in lignified organs. However, wound stress did not induce CWPO-C gene transcription. These results showed that CWPO-C gene constantly expresses in lignified organs. Western analysis using polyclonal antiserum against CWPO-C showed that this antiserum recognized CWPO-C protein and that this peroxidase ionically bound to the cell walls of developing xylem. Immunohistochemical analysis showed that this peroxidase mainly located fiber cells in lignifying tissue. We will discuss this peroxidase isoenzyme as a lignification-specific peroxidase isoenzyme in woody angiosperms.

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Adjacent cellulose microfibrils are thought to be cross-linked by xyloglucans and the separation of microfibrils during elongation has been thought to require enzymes that cleave xyloglucan or loosen its binding to microfibrils. By overexpressing xyloglucanase and producing a knockout mutant of xyloglucan, it would be possible to study the function of xyloglucan in the primary and secondary walls of higher plants. The expression of xyloglucanase constitutively in Populus alba increased the lengths of stem and petiole and enlarged palisade parenchymal cells compacting in the limited space of the leaf. Degradation of xyloglucan network not only decreased the wall elasticity in the elongating stem but also increased the elasticity of secondary xylem. The increased elasticity corresponded to an increase in specific gravity, showing that the degradation of xyloglucan might cause an increase in cellulose density in the secondary xylem.

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