Precise pigment analyses of a cyanobacterium Gloeobacter violaceus PCC 7421 were performed. Phe a and Chl a' were found to be present as minor pigments like other cyanobacteria, suggesting the photosystems of this cyanobacterium are similar to other cyanobacteria. Menaquinone-4 (MQ-4) was detected in the PS I particles of G. violaceus, but phylloquinone was absent. MQ-4 should be the secondary electron acceptor of PS I in G. violaceus. The stoichiometry of MQ-4/Chl a' was estimated to be two, indicating that two molecules of MQ-4 were present in PS I. The genome sequences of G. violaceus showed the absence of genes homologous to the phylloquinone biosynthesis enzyme, thus an unknown pathway of the MQ-4 biosynthesis would be present in this cyanobacterium.

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ESR and ENDOR spectra of P740⁺ in photosystem I (PS I) of Acaryouchloris marina were studied. The ENDOR spectrum of P740⁺ that seems to be a special pair of chlorophyll (Chl)d was compared with those of P700⁺ that is a dimer of Chl a and Chla' in spinach PS I. EPR and proton ENDOR spectra of purified Chla⁺ and Chld⁺ were also investigated. The numbers of ENDOR separations in P700⁺ were absent in the P740⁺ signal. The difference can be ascribed to the different chemical structures of Chla and Chld; a formyl group in Chla is replaced by a vinyl group in Chld. Electronic structure of P740⁺ was discussed based on the results.

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Pigment components of electron donors P740 and P720 in PS I and PS II reaction centers, respectively, were studied by laser photolysis of thylakoid membranes of Acaryochloris marina that has Chl d as the major pigment. We measured laser-induced difference spectrum of P720 in the presence of ferricyanide. It showed negative peaks at 684 and 715 nm, but no change at 740 nm. Further addition of ascorbate induced the laser-induced absorption change of P740 together with P720. The difference spectrum of P740 was calculated. It seems to be composed of bleach of Chl d special pair at 740 nm, shifts of Chl d accessory and Chl a acceptor pigments. The P720 spectrum seems to be interpreted as the bleach of Chl d special pair at 725 nm, Chl d accessory shift and the shift of the acceptor Pheophytin a (or Chl a).

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We studied photosystem (PS) I /II in Acaryochloris cells with different phycobilion contents. This oxygenic prokaryote has Chl d as a major pigments. Although the special pair in PS I have been identified as Chl d dimmer P740 in this organism, little is known about the components in PS II. The relationship between phycobilin and PS II is unknown too. We measured the activity and amount of the pigments in two types of cells of Acaryochloris with different ratios of phycobilin to Chl d. The ratio of PS I/PS II was determined based on the measurements of Y_D^* and P740⁺ ESR signals. Low temperature absorption and the fluorescence spectra, delayed fluorescence from PS II were also measured. Features of PS I and PS II reaction centers were discussed based on these results.

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The triplet state in PS II forms on one of the two accessary chloropylls, that was confirmed by EPR study of oriented membranes. Each accesssory chlorophyll belongs to D1 and D2 proteins, respectively. However which chlorophyll is active has not yet been determined. Pulsed EPR detects the dipolar interaction between a radical pair from which the distance ican be derived.
This work reports the result of ESEEM study of D1D2Cytb559 reaction center complexes with an acceptor quinone DMBIM reconstituted. The reduced quinone forms spin polarized signal induced by the tirplet state synchronized with laser flash. The obtained distance of 26 A is ascribed to the triplet state on the D1 protein and the reduced quinone.

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The primary donor Chl of PSII, P680, has an extremely high oxidation potential (E_ox) of ~1.2 V, which enables water oxidation. How such a high potential was achieved in the evolution of PSII remains unanswered. We have investigated the cause of the high P680 potential using a quantum chemical method. The dielectric-constant (ε) dependence of Chl E_ox was estimated by DFT calculations. A sharp increase in E_ox with decreasing ε was observed at ε < 5, whereas E_ox was rather constant at higher ε. From this relationship, the E_ox of Chl in ideal hydrophobic proteins (ε = 2) was estimated to be ~1.5 V. This indicates that the E_ox of Chl is originally very high in nonpolar environments. Indeed, the protein environment around P680 is significantly hydrophobic in comparison with that around bacterial P, suggesting that reducing the polarity around P680 was a major factor to achieve its high potential.

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In PSII, the secondary quinone electron acceptor Q_B is singly reduced upon flash illumination, and a subsequent flash induces its double reduction followed by protonation and release from the protein. The detailed mechanism of the Q_B reactions in PSII remains unclarified. In this study, we have measured almost pure Q_B^-/Q_B FTIR spectra without donor-side signals and studied the Q_B reactions. Flash-induced Q_B^-/Q_B difference spectrum was measured using Mn-depleted PSII core complexes from Thermosynechococcus elongatus. The obtained spectrum showed a characteristic positive peak at 1745cm^-1 in the typical COOH region. However, the peak was unaffected by deuteration, indicating that it does not arise from a COOH group but probably arise from the C₁₀=O stretch of a nearby pheophytin. The observation that the COOH region was little affected by deuteration indicates that unlike bacterial reaction centers, proton uptake by carboxylate groups upon Q_B^- formation does not take place in PSII.

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Site-directed mutagenesis in the photosystem II (PSII) was performed using the thermophilic cyanobacterium Thermosynechococcus elongatus. PSII lacking the redox active tyrosine Tyr_D was engineered by substituting phenylalanine for tyrosine at D2-160. The effects of the Y160F mutation were characterized. The t_1/2 of oxygen release on the S₃→S₀ transition was estimated to be 1-2 ms in both WT’ and Y160F, although the doubling time of Y160F cells was approximately twice as slow as that of WT’ cells. Flash-induced absorption changes around 430 nm revealed the displacement of the P_D1⁺P_D2 ↔P_D1P_D2⁺ equilibrium in favor of the left side. The FTIR difference spectrum of P₆₈₀⁺/P₆₈₀ showed that the carbomethoxy C=O stretch of chlorophyll downshifted about 10 cm^-1 upon mutation. The rate of P₆₈₀⁺ reduction by Tyr_Z in the mutant slightly increased. These results suggest that minor structural perturbations perhaps in the H-bonding network between Tyr_D and P₆₈₀ take place upon Y160F mutation.

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Structural changes of α-carboxylate of the D1 C-terminal alanine-344 (Ala344) during S-state cycling of photosynthetic oxygen evolving complex were selectively measured using light-induced Fourier transform infrared (FTIR) difference spectroscopy with specific ¹³C-labeling of α-carboxylate of Ala in photosystem II core particles from Synechocystis sp. PCC 6803. Several bands effected by ¹³C-labeling in S₂/S₁ FTIR difference spectrum at 1360-1300 cm^-1 were assigned as those originated from the C-terminal α-carboxylate. The bands showed characteristic changes during S-state cycling. They appeared prominently upon the S₁-to-S₂ transition and to a lesser extent upon the S₂-to-S₃ transition, but reappeared with the opposite sign upon the S₃-to-S₀ transition. No obvious isotopic band appeared upon the S₀-to-S₁ transition. These results indicate that the α-carboxylate of C-terminal Ala344 ligates, in a unidentate manner, a Mn ion that is oxidized upon the S₁-to-S₂ transition and reduced reversely upon the S₃-to-S₀ transition.

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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 low-frequency (1000-370 cm^-1) FTIR difference spectra for the S-state cycle and effects of H₂¹⁸O-, D₂¹⁶O-, D₂¹⁸O-substitution on the spectra were examined using PS II core particles from Thermosynechococcus elongatus. Low-frequency difference spectra for S₁-to-S₂, S₂-to-S₃, S₃-to-S₀, and S₀-to-S₁ transitions showed characteristic vibrational features depending on each water-substitution. Close inspection of the isotopic bands revealed that bands only sensitive to ¹⁶O/¹⁸O exchange or H/D exchange may be ascribed to mainly Mn cluster core vibrations or modes from amino acid side chains and polypeptides. Furthermore, bands sensitive to both ¹⁶O/¹⁸O and H/D exchanges may be attributed to the interaction between Mn and substrate water and/or water-derived chemical species involved in the water oxidation chemistry.

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The extrinsic proteins present in the luminal space of thylakoids are shown to play crucial roles in optimizing the water-oxidizing activity in photosystem II (PSII). In higher plants, PsbO, PsbP and PsbQ proteins are the major components of luminal extrinsic proteins, and they have been extensively analyzed from biochemical and structural standpoints in the past decades. However, their physiological functions in vivo have been poorly understood. In this study, we have applied the RNA interference (RNAi) technology to generate transgenic tobacco plants, psbOir, psbPir and psbQir, in which the expression of psbO, psbP and psbQ genes was selectively and significantly silenced. Both psbOir and psbPir plant displayed distinct phenotypes such as drastic reduction in the growth rate and pale green-colored leaves. On the other hands, there was no obvious visible difference between the psbQir and the wild-type plants. Further analyses about these RNAi tobacco plants are on going.

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PsbP is an extrinsic 23-kDa protein in the oxygen-evolving complex of PSII and reported to retain ions essential for the water-splitting reaction. In amphidiploids Nicotiana tabacum, PsbP is nuclear-encoded by a multigene family of four members, which could be divided into two groups by the sequence similarity (group-I: 1A and 5B; group-II: 2AF and 3F). All PsbP members are accumulated and group II comprises major component of PsbP in N. tabacum. To clarify the reason why N. tabacum has four PsbP members, group-specific PsbP-silenced plants were generated by RNAi. When compared to control plants, both group-specific PsbP-silenced plants showed marked decrease of PSII activity, while group-II-silenced plants showed more significant decrease of PSII activity. This result indicates that expressions of all PsbP members are required for the maximum PSII activity in N. tabacum. Further functional characterization of two PsbP groups is on-going.

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In plants, pollen has two sperm cells to perform double fertilization. Together with the two sperm nuclei and a vegetative nucleus, pollen consists of three nuclei at the time of fertilization. In contrast, two types of mature pollen grains exist, either containing one or two generative nuclei. Such difference in the nucleoploidity of mature pollen grains may affect pollen viability. We isolated an Arabidopsis mutant that has altered nucleoploidity. This mutant, nikaku, segregated normal tri-nuclear pollen grains and abnormal di-nuclear pollen grains to 1:1. Our genetic analysis showed that the di-nuclear phenotype was transmitted only maternally, and the transmission rate was always 1:1. These results suggest that pollen mitosis II is impaired in the mutation. Analysis of DNA contents in di-nuclear pollens revealed that the mutation blocked DNA replication, suggesting that nikaku may affect pollen mitosis II at an early step.

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A male-sterile mutant of Arabidopsis thaliana was isolated by means of T-DNA tagging. Filament elongation at anthesis was defective in the mutant, although pollen fertility was not defective. This result indicates that male sterility was not caused by pollen itself but by filament elongation. A transmission electron microscope analysis revealed that primexine formation was significantly reduced, and that sporopollenin was randomly deposited onto the microspore plasma membrane. The sporopollenin that failed to deposit aggregated and accumulated within the locule and on the locule wall. However, completely reticulate pattern was observed at anthesis. MOJYAO gene is considered to be DET2 gene which is involved in brassinosteroid biosynthesis.

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We have cloned a new member of the RAD2/XPG nuclease family, OsRAD, from rice. OsRAD possesses two nuclease domains that are conserved in the RAD2/XPG nuclease family. OsRAD belongs to class 4 of the RAD2/XPG nuclease family, and OsRAD homologues were found in animals and higher plants. To elucidate the function of OsRAD, we generated rice OsRAD-RNAi transgenic plants in which OsRAD expression was silenced. Most of the OsRAD-RNAi plants displayed low fertility, and some of them were male-sterile. OsRAD-RNAi plants lacked mature pollen, resulting from a defect in early microspore development. A OsRAD-GFP fusion protein was localized in the nucleus, and the OsRAD promoter was specifically active in the anthers. Furthermore, a recombinant OsRAD protein possessed both single-stranded and double-stranded DNA-binding activities. Our results suggest that OsRAD plays an essential role in DNA metabolism required for early microspore development in rice.

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Yeast ATG6, isolated as an autophagy-related gene, is allelic to VPS30, a component of phosphatidylinositol 3-kinase (PI3K) complex. Screening of T-DNA insertion mutants of Arabidopsis ATG6 has identified no homozygous mutant lines. Approximately 50% of the seeds in the progeny of selfed-heterozygous plant were heterozygous, characteristic of a male gametophytic mutant phenotype. This idea was confirmed by crossing experiments between AtATG6 heterozygous and wild type plants. DAPI staining of mature pollen grains from AtATG6 heterozygous plants detected no phenotypically aberrant pollen. However, efficiency of pollen germination in AtATG6 heterozygous plants seemed to be reduced compared to wild type. When AtATG6 heterozygous plants were crossed to quartet1 mutant, normal and aborted pollen grains from ATG6/atg6/qrt/qrt plants seemed to segregate 2:2 in the tetrads: less than 2 pollen grains in each quartet germinated. These results suggested that ATG6/VPS30 plays critical roles in pollen development.

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Apical growth of tobacco pollen tubes is not uniform but exhibits regular growth rate fluctuations with a typical period of 45 seconds. In tobacco, as in lily, calcium signals at the tip show similar periodicity to growth rate, show by the Cameleon calcium reporter (Watahiki et al., 2004). To investigate how this calcium signal might be mediated, a calmodulin domain protein kinase (CDPK or CPK) was isolated from Nicotiana plumbaginifolia. NpCPK17 cDNA was fused with CFP and expressed in growing tubes. Based upon fluorescence Imaging and cell fractionation analysis, pollen NpCPK17 was found to be associated with the plasma membrane and tip-localised vesicle clouds. NpCPK17 possesses myristolylation sites essential for membrane localisation. Distribution of NpCPK17-CFP at the pollen tube tip changed dynamically with a similar frequency to growth fluctuation. These results suggest a mechanism for a possible feedback loop between phosphorylation and tip growth.

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Seedlessness is an important agricultural trait on fruits to determine their market price. A citrus cultivar 'Hira Kishu' produces seedy fruits. However, a relating cultivar 'Mukaku Kishu' (Citrus kinokuni hort. ex Tanaka cv.
Mukaku Kishu) produces seedless fruit by embryo abortion during development after fertilization. We have developed several seedless citrus cultivars with this characteristic. Linkage analysis reveals that single dominant gene determines the seedlessness. A DNA marker linked to the trait successively mapped on our citrus linkage map. We evaluated expression of cultivar-specific genes in both cultivars to
reveal the molecular detail. Extracted RNAs from ovules at 8 weeks after fertilization on the both cultivars were provided for cDNA subtraction with suppression subtractive hybridization (SSH) protocol. After elimination of non-specific cDNA by MOS protocol, about 200 cDNA clones for both forward and reverse subtraction were sequenced. Clustering analysis identified putative cultivar specific cDNA clones.

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Silene latifolia is a dioecious plant that has X and Y chromosomes. Female plants (XX) initiate development of stamens by infection of the dimorphic smut fungus Microbotryum violaceum. Until stage 7, no floral modification is observed between healthy and infected females. At stage 8, the healthy female does not develop stamens, whereas the infected female initiates to develop stamens. Expression of the floral-B function gene, SLM2, an ortholog of PISTILLATA in Arabidopsis, was examined by in situ hybridization. Until stage 7, SLM2 was expressed on whorl 2 and 3 of the healthy and infected females. At stage 8, SLM2 transcript was supressed in the stamen primordia of the healthy females. However, developing stamen of the infected female expressed SLM2 as intense as developing petals. The suppression of SLM2 expression would be removed by the smut-infection independent of the presence of Y chromosome.

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Two homeodomain leucine zipper (HD-Zip) genes were isolated and characterized from a cDNA library of the male flower buds of S. latifolia. Two genes, SlHDL1 and SlHDL2, encode proteins characterized as HD-Zip transcription factors. Seven cDNAs were isolated by cDNA subtraction of healthy male buds with the female buds infected with the dimorphic smut fungus Microbotryum violaceum. Five cDNAs are predicted to encode a chalcone-synthase-like protein, a glycosyl hydrolase family 17 protein, a proline-rich protein APG precursor, a strictosidine synthase family protein, and a mandelonitrile lyase protein. In situ hybridization showed that they are expressed in tapetum, tetrad and pollen grains between meiosis and microspore mitosis, timing that corresponds to a burst in tapetal activity in the developing anthers. The genes isolated will be useful for the isolation of tapetal-specific promoters, and revealing the interactions between the male-sterility restoration factor and the fertility related genes.

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MADS-box genes encode transcription factors and are involved in development of flowers in flowering plants. It has been reported that MADS-box genes are expressed in gymnosperms, a fern, a moss and green algae. It is important to clear the role of MADS-box genes in non-flowering plants for understanding the evolution of MADS-box genes and the evolution of flower. However, there is no report of the functional analyses of MADS-box genes of non-flowering plants. In this study, we analyzed three MIKCc-type MADS-box genes (PPM1, PPM2, and PpMADS1) of a moss, Physcomitrella patens. Expression analysis of knocked-in GUS-fusion proteins revealed that PPM1 and PPM2 were expressed in shoot apex of gametophore, antheridia, and young sporophytes and that PpMADS1 was expressed in young sporophytes. There is no notable morphological difference in each knock-out line. Phenotypic analyses of double and triple knock-out mutants will be presented.

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The INDETERMINATE protein, ID1, plays a key role in regulating the floral transition in maize. ID1 is the founding member of a plant-specific zinc finger protein family that is defined by a highly conserved amino acid sequence called ID domain. The ID domain includes a cluster of three different type of zinc fingers separated from a canonical C2H2 finger by a long spacer; ID is distinct from other ID family proteins by having a much longer spacer.
We determined the binding sequence of ID1 by in vitroDNA selection and amplification binding assays and analyzed the DNA binding properties of ID1 and ID family proteins. Our results demonstrate that maize ID1 and ID family proteins have novel zinc finger configurations with unique DNA binding properties.

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The regulation of floral organ number is closely associated with floral meristem size. Although molecular genetic mechanisms that regulate maintenance of the meristems have been well studied in Arabidopsis, the regulation of such events in monocots is poorly understood. To elucidate the mechanism of meristem maintenance in monocots, we have been studying the function of FLORAL ORGAN NUMBER1 (FON1) and FON2 genes in rice. A mutation in either fon1 or fon2 cause an enlargement of the floral meristem size, resulting in an increase in the number of floral organs. We have already reported that FON1 encodes LRR type receptor-like kinase that orthologous to Arabidopsis CLAVATA1 (Suzaki et al. Development 131:5649-5657, 2004). Here, we report the isolation of FON2 and its function. Taken together the previous finding about FON1, the results obtained here strongly supported the idea CLAVATA signaling system regulating floral meristem maintenance is conserved in the grass family

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FLORICAULA (FLO) of Antirrhinum and LEAFY (LFY) of Arabidopsis play major roles in the reproductive transition and floral meristem identity. In maize, two duplicated FLO/LFY homologs play conserved roles with those in the dicot species. On the other hand, RFL, a FLO/LFY homolog in rice, might be involved in panicle branching. To understand the function of FLO/LFY homolog in wheat, we isolated WFL (Wheat FLO/LFY) and analyzed its expression pattern. In situ hybridization analysis revealed that WFL RNA expression was observed in all layers of young spikes excepting spikelet formation sites as axillary meristem, and not detected in spikelet primordia in the later stages. As development of floret proceeds, the WFL RNA was detectable in the developing palea. The WFL expression pattern indicates that WFL is associated with spikelet formation rather than floral meristem identity, and may play a novel role in developing palea.

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The Arabidopsis ACL5 gene, whose inactivation causes a severe defect in the elongation of stem internodes, encodes a polyamine biosynthetic enzyme, spermine synthase. Although polyamines are recognized as being necessary for cell growth and proliferation, how they control the plant development remains to be understood. To elucidate the mechanism, we have isolated and characterized suppressor of acl (sac) 51d-54d mutants. In this study, we show that these sac mutations have no effect on the stem growth defects in a series of phytohormone-related mutants including axr2, gai, and dim. This result suggests that the function of these SAC genes is independent of phytohormone signaling pathways. Because the acl5 mutant contains spermine produced by another spermine synthase SPMS, the effects of sac mutations on the acl5 spms double mutant that has no spermine were examined. The result will be presented.

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Upon submergence, a heterophyllous aquatic plant of Ludwigia arcuata produce narrow submerged-type leaves, with a reduction of the number of epidermal cells in transverse direction. Such reduction has been ascribed to the effect of ethylene.
In this study, we examined at what extent ethylene affects the orientation of the division planes in developing leaves, whereby developmental plasticity was considered in the third young leaves. Two days after addition of ethylene, divided cells, in which division planes were parallel to the leaf long axis decreased in a whole leaf level. Furthermore, the ratio of division planes transverse to the leaf long axis increased in the basal portion of the third young leaves, while division plane parallel to the leaf long axis decreased near the central region. These outcomes indicate that ethylene actually affected the direction of division planes, resulting in changing leaf shape.

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The cpm1 mutant of rice is impaired in phytochrome-mediated inhibition of coleoptile growth and in the process of anthesis causing partial sterility (Biswas et al., 2003). The CPM1 locus was mapped to a 143cM region of chromosome 3, in which the ALLENE OXIDE SYNTHASE1 (AOS1) gene is located. The cpm1 mutant was found to have a point mutation in this gene, which is expected to alter one of the amino acid residues conserved in all the reported AOSs. We complemented the cpm1 mutant with the OsAOS1 genomic DNA that included the promoter region. The complemented mutant recovered the phytochrome response and complete fertility, demonstrating that OsAOS1 corresponds to CPM1. The cpm1 mutant had a normal level of OsAOS1 transcripts. It is expected that the mutated protein has a reduced enzyme activity. This possibility is now under investigation. We will also discuss the role played by OsAOS1 in photomorphogenesis.

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Gibberellin(GA) regulates shoot and root growth and flowering of many plants. Since the physiological function of GA in the growth and development of tea trees, particularly in roots, is not well known, we studied the morphological and growth-enhancing effects of GA in combination with the inhibitor of GA-biosynthesis to decrease the endogenous GA level. Root-applied GA3 strongly promoted shoot growth but little affected root elongation itself. However, the inhibitor of GA-biosynthesis promoted flower bud formation and inhibited shoot and root elongation with remarkable thickening of root apices. These inhibitor effects were completely reversed by GA3. The possibility was presented that the shoot and root growth, and flower initiation are controlled by restraining the GA level of tea plants and the method may contribute the efficient production and propagation of tea trees.

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We have previously shown that the cortex of cut hypocotyls completely unite in 7 days, and that leaf gibberellin (GA) is required for this process in cucumber and tomato seedlings. To better understand how the tissue-reunion takes place, histochemical and gene expression analyses were performed using Arabidopsis cut flowering stem. The lowest internode of the main flowering stem was cut through half of its diameter with a micro-surgical knife, and gene expression during the reunion process was analyzed with full-genome microarray. Our microarray analyses of the tissue-reunion showed differential gene expression between cut and non-cut flowering stems, and changes in gene expression during reunion process. The analysis also showed that a number of genes involved in cell division and cell wall modification were up-regulated during the reunion process, and a subset of these up-regulated genes were previously determined to be GA- responsive.

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We have cloned gibberellin (GA) 3β-hydroxylase cDNA, (HvGA3ox), a homolog of the rice gene OsGA3ox from germinating barley seeds. HvGA3ox cDNA was expressed in Escherichia coli capable of oxidizing GA9 to GA4 and GA20 to GA1. Deduced amino acid sequence analysis showed that HvGA3ox was identical to HvGA3ox2 reported by Spielmeyer et al. (2004). Northern analysis showed that HvGA3ox2 was expressed only in germinating seeds, not in other organs. Typical feedback regulation was not observed in seedling treated with either GA3 or uniconazole. HvGA3ox2 mRNA was detected from 12 hours after imbibition just before the expression of a-amylase gene, Amy1. In situ hybridization of germinating seeds revealed that HvGA3ox2 mRNA was localized in the epithelium. Our results suggest that GA biosynthesis in epithelium is important for the expression of α-amylase in germinating barley seeds, and HvGA3ox2 encodes the key enzyme of this event.

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GAMYB was first isolated as a positive transcriptional regulator of gibberellin (GA)-dependent α-amylase expression in barley endosperm, and its molecular properties has been extensively characterized. Here, we report the role and the regulation of GAMYB and structurally related GAMYB-like proteins in rice growth. We found that GAMYB and GAMYB-like genes contain a target site for a microRNA, miR159, and overexpression of miR159 in rice plant revealed not only the regulatory mode of miR159 on these genes but also the role of these genes in rice growth. On the other hand, microarray experiment revealed that almost all of GA-regulated genes did not respond to GA in the endosperm of gamyb. This suggests that GAMYB is involved in the regulation of almost all GA-mediated gene expression in rice endosperm.
This work was in part supported by PROBRAIN.

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RSG is a bZIP transcriptional activator, which regulates shoot growth by controlling GA level. Suppression of RSG resulted in severe dwarfism. We have demonstrated 1) RSG interacts with 14-3-3 proteins, which are broadly conserved regulatory factors, through phosphorylated serine-114(PS114) in RSG; 2) Binding with 14-3-3 inhibits the transport of RSG to nucleus; 3) RSG is not statically sequestered in the cytoplasm by 14-3-3, but dynamically shuttling between nucleus and cytoplasm; 4) endogenous level of GA regulates function of RSG by control of intracellular localization.
To clarify mechanism which regulate RSG nuclear export through endogenous GA level, commitment of 14-3-3 protein to the mechanism are analyzed. Because RSG interacts with 14-3-3 protein via PS114 in RSG, antibodies which specifically recognize PS114 in RSG (aPS114) are prepared. Immunoblotting using aPS114 has revealed that phosphorylation of RSG are elevated in the process of nuclear export via GA.

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Seed germination is controlled by multiple environmental and endogenous factors. We have previously shown that red-light upregulates the gibberellin (GA) biosynthesis gene AtGA3ox1 and AtGA3ox2, and downregulates GA deactivation gene AtGA2ox2. In situ RNA analysis showed that mRNA of these genes are mainly detected in the cortex of embryonic axis. This suggests that the cortex may function as the major cell type that regulates endogenous GA levels. Recently, we have identified new 2-oxidases, AtGA2ox9-10, both of which catalyze the conversion of GA₁₂, an intermediate of bioactive GAs, into GA₁₁₀, an inactive GA. We found that AtGA2ox9 and AtGA2ox2 were both downregulated, while AtGA3ox1 was upregulated, during cold treatment after imbibition. These results indicate that multiple GA-metabolic steps regulate endogenous levels of bioactive GAs, which in turn control seed germination, in response to environmental signals.

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We have been studying on GA signaling in rice. We found that SLR1, a repressor for GA signaling, is degraded in a GA-dependent manner through SCF^GID2 complex. Recently, we showed that the phosphorylation of SLR1 is promoted by GA in gid2 and the phosphorylated SLR1 is specifically interacted with recombinant GST-GID2. Here, we will report the phosphorylation sites of SLR1 and the role of its phosphorylation.
By using ³²P-labeled SLR1 in gid2 and transgenic plant, we found that the phosporylated amino acid is serine and the phosphorylation sites locate on both GA signal perception and regulatory domain. The phosphorylation was observed even under GA deficient condition and kinase inhibitors did not inhibit the degradation of SLR1. Furthermore, dephosphorylated SLR1 protein also bound to GID2 in vitro. These results indicate the phosphorylation of SLR1 protein is not essential for the interaction with GID2. (supported by PROBRAIN)

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Rice SLR1 (SLENDER RICE 1) gene encodes a DELLA protein, which belongs to subfamily of GRAS protein superfamily and functions as a repressor of gibberellin (GA) signaling. SLR1 has been considered to be a sole DELLA-type GA-signal suppressor protein in rice. Using the rice DNA database, however, we identified a homologous sequence to SLR1, SLR1-like (SLRL), in the rice genome. SLRL does not have the DELLA motif but its primary structure is highly similar to the C-terminal portions of DELLA proteins. Overexpression of SLRL in transgenic rice plants induced a dwarf phenotype with increased level of OsGA20ox2 gene expression, indicating that SLRL also acts as a repressor of GA signaling. The expression of SLRL was positively regulated by GA level and preferentially occurred in reproductive organs. Based on these results, we discuss the functional role of SLRL in GA signaling.
This work was in part supported by PROBRAIN.

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Because gibberellin(GA) regulates various aspect of plant growth, endogenous amount of GA and its signal transduction must be regulated appropriately. Gibberellin 3-oxidase, GA3ox catalyzes a final step of biosynthesis of active GA and might be a crtical point to control endogenous amount of active GA. Arabidopsis containes four deduced GA3oxes. One of them, AtGA3ox1 is under negative feedback regulation at transcription. To elucidate molecular mechanism of this regulation, we carried promoter deletion assay using AtGA3ox1 promoter and found 50 bp cis region necessary for feedback regulation. Minimal promoter fused with this region gained feedback responsibility. Novel AT-hook trans factor was obtained by one hybrid screening using this region. This AT-hook protein behaved as a transcriptional activator and dimereized in yeast. We demonstrated that AT-hook protein binds to the cis region specifically by gel shift assay and determined the sequence necessary for AT-hook protein binding to AtGA3ox1 promoter.

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To understand the relationships between chromatin condensation and gene expression states, we evaluated the degree of chromatin condensation as DNase I sensitivity at a 500 bp resolution across a 80 kb Arabidopsis genomic region containing variously-expressed 30 protein-coding genes. The general DNase I sensitivity was at a similar level irrespective of gene expression states, suggesting that the chromatin condensation is relatively uniform. However, surprisingly, distinct DNase I hypersensitive sites (DNase I HSs) were found at both sides of most genes. Further analysis of several well-characterized genes showed that the DNase I HSs were present in transcriptionally-competent (expressed or inducible) gene promoters and absent from incompetent (not inducible) gene promoters. These results suggest that the DNase I HSs regulate gene expression potential by modulating the accessibility of the DNA elements to transcription regulators.

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We succeeded to induce RNAiin Chlamydomonas reinhardtii by introducing inverted repeat DNA construct as a silencer for aadA gene that confers spectinomycin resistance. The highest concentration of spc. was reduced from 3oo micro g/mL to 60 micro g/mL by the induced RNAi. However after 30-50 timed of somatic cell division, cells that survive on the plate containing even 200 micro g/mL of spc reached to about 5 %. Relationship between the threshold and the amount of dsRNA/siRNA was observed apparently, and also extent of cytosine methylation in the inverted repeat construct was clearly affiliated. CG but not CNG methylation was concentrated in the hairpin structure region. Therefore, DNA methylation was probably induced by DNA-DNA interaction and extent of methylation seems change and does not converge through cell divisions.Incomplete RNAi suggests that C. reinhardtii has no RNA dependent RNA polymerase and RNA dependent DNA modification system.

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p-SINE1, a rice retroposon, is transcribed by RNA polymerase III (pol III). We have previously reported that the amount of p-SINE1 transcripts dramatically increased by treatment of cells with DNA-methylation inhibitors. We examined DNA methylation of a p-SINE1 member whose sequence is identical to the transcripts, and found that cytosine residues, not only in CpG sequences but also in CpHpG or CpHpH sequences, are methylated specifically in p-SINE1. The features of the p-SINE1 methylation were similar to those of RdDM (RNA-directed DNA methylation), induced by short interfering RNA (siRNA). To examine the involvement of siRNA in regulation of p-SINE1 expression, we performed northern blot analysis with RNA isolated from rice cells, and found siRNA which hybridized to the p-SINE1 sequence. The amount of the siRNA apparently decreased by the treatment with DNA-methylation inhibitors. These results suggest the possibility that siRNA is involved in regulation of the p-SINE1 expression.

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Proteins that specifically bind dsRNA are involved in RNAi, and are characterized by a conserved dsRNA-binding motif (dsRBM). We have studied the biochemical properties of such gene products, each containing dsRBMs: four Arabidopsis Dicer-like proteins (DCL1-4), HYL1/DRB1 and three of its Arabidopsis homologs (DRB2, DRB4 and DRB5). Previously, we reported that DCL1, DCL3, HYL1/DRB1 and the four HYL1 homologs exhibit dsRNA-binding activity. The dsRBMs of human dsRNA-binding proteins is reported to function as a protein-protein interaction domain. We show that DRB4 interacts specifically with DCL4, and HYL1/DRB1 most strongly interacts with DCL1. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1/DRB1 and DRB4 localize in the nucleus. The presented data suggest that each member of the DRB protein family may individually modulate Dicer function through heterodimerization in vivo. We are now undertaking immunoprecipitation assay to prove such specifically interactions in vivo.

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We study RNA-mediated TGS to understand effects of siRNA on various genes in rice. RNA-mediated TGS is the chromatin-based gene silencing, triggered by siRNA which contains sequences identical to promoter regions. We produced transgenic rice plants which accumulated promoter-derived siRNAs. We chose a 35S::GFP as a target transgene and OsRac genes as endogenous target genes. Our results indicated that 35S::GFP was silenced, but endogenous OsRac genes were not silenced. However, OsRac target promoter DNA also were methylated de novo, like a 35S target promoter. In addition, results of ChIP analysis of both transgene and endogenous target promoter indicates that degree of histone H3 acetylation and H3 methyl-lysine 4 was lower in 35S promoter. These results suggest that histone modification, but not DNA methylation, is associated with RNA-mediated TGS of the transgene.

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Eukaryotic precursor tRNAs containing introns are processed in the nucleus and the mature forms of the tRNAs are exported to the cytoplasm. It has been shown in budding yeast that tRNA splicing machinery is located in the inner membrane of the nuclear envelope. Surprisingly, the latest study has revealed that the yeast tRNA splicing endonuclease is predominantly located on the outer membrane of mitochondria.
In order to explore an intracellular localization of the plant tRNA splicing machinery, two different kinds of GFP fusion genes (i.e., AtSen1::GFP and AtSen2::GFP) were constructed based on the Arabidopsis genes coding for the splicing endonuclease subunits. These plasmids were introduced into the onion epidermal cells with a particle gun. Fluoresent observation disclosed that these fusion proteins are both located in mitochondria, like in the yeast. Furthermore, transient assay with plant tRNA ligase::GFP-fusion genes is under way.

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In plants, primary enzyme of carbon fixation, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), is encoded as two subunits, rbcL and rbcS . It is reported that the ML tree based on amino-acid sequence of RbcL deduced from the nucleotid sequence alignment makes two clusters; R-type (red linage) cluster and G-type (green linage) cluster. G-type RuBisCO operon genes constitute rbcL-rbcS operon, while R-type RuBisCO genes rbcL-rbcS-cfxQ operon. The cfxQ is predicted to be necessary for the expression of RuBisCO.
cDNA analysis and complete genome sequence of Cyanidioschyzon merolae 10D indicated that cfxQ is coded both in the plastid genome and an the cell nucleus. In this study, we purpose to clear function of two cfxQ , respectively.
Northern hybridyzation analyses of the two genes show the timing of expression of plastid cfxQ is different from that in the cell nucleus.

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Arabidopsis genome contains many ABC protein genes. P-glycoprotein-like (PGP) subfamily has been drawing attentions of plant hormone researchers concerning the auxin transport activity. In this study, we analyzed the gene expression and function of AtPGP4, a PGP type ABC protein. The AtPGP4 expression was up-regulated by both auxin and cytokinin. AtPGP4 promoter::GUS transgenic plants showed that AtPGP4 was strongly expressed in the root, and limited mainly to epidermal cells. AtPGP4 was localized at the plasma membrane, which was proved by sucrose density gradient fractionation and immunoblotting. The localization of AtPGP4 was also supported by immunolocalization analysis. The root development of atpgp4 mutants was clearly reduced compared to the wild type. Atpgp4 mutants displayed reduced basipetal auxin transport, whereas AtPGP4 overexpressing seedlings showed a substantial increase in basipetal auxin transport. We are now analyzing this transport activity more in detail.

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ER body is an ER derived organelle, which might be responsible for defense systems, because they are induced by wounding. A proteomic analysis showed that both PYK10 and PBP1 (At1g16420) were decreased in their levels in Arabidopsis nai1 mutant, which has no ER bodies. PYK10, which is localized in ER bodies, is a beta-glucosidase with three oligosaccharide chains. On the other hand, PBP1 is a cytosolic lectin.
During incubation of the extract from Arabidopsis roots, PYK10 molecules were separated into two forms; active form and inactive form. The amount of active form of PYK10 was less than that of inactive form of PYK10. Most of the active PYK10 molecules formed huge aggregates during the incubation. An immunoprecipitation with anti-PYK10 antibodies revealed that PBP1 was interacted with PYK10. These results suggest that PBP1 is responsible for aggregation of inactive PYK10, but not for aggregation of active PYK10.

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We previously reported that Rubisco-LSU was specifically cleaved at the peptide bond between Phe-40 and Arg-41 in lysates of wheat chloroplasts incubated in darkness. In this study, we attempted to purify and identify the protease localizing in chloroplasts which involves in the degradation of Rubisco-LSU at the same site. We first prepared the fluorescence quenching substrate having the proximal sequence of the cleavage site of the LSU to detect the protease activity. The protease was purified from 500 g of spinach leaves through ammonium sulphate precipitation, hydrophobic interaction chromatography and gel filtration. Protease activity was inhibited by EDTA and o-phenanthroline. SDS-PAGE analysis showed two polypeptides (MW 80 kDa, 103 kDa) were involved in the final fraction. Then we isolated each polypeptide, and determined their partial amino acid sequences. The amino acid sequence of 103 kDa polypeptide was identical one of the metalloproteases found in Arabidopsis thaliana.

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A novel cDNA clone (OsNInv) encoding a neutral invertase was isolated from rice. The deduced amino acid sequence of the OsNInv cDNA clone showed high similarity to other plant alkaline/neutral invertases. Semi-quantitative RT-PCR revealed that the level of OsNInv expression was quite low and the pattern of its expression was not organ specific. Analyses using three types of web software for the prediction of protein localization in the cell, Predotar, TargetP, and PSORT, strongly supported the possibility that OsNInv is transported into the mitochondria. Transient expression of a fusion protein combining the amino terminal region of OsNInv with sGFP demonstrated that the N-OsNInv::GFP fusion protein was transported into the mitochondria efficiently. The results of this study revealed that the amino terminal region of OsNInv possesses the mitochondrial targeting signal and suggested that OsNInv protein is accumulated in the mitochondria.

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In bacteria and plants, Sec-, Tat- and SRP-pathway are known for protein translocation. SecA is essential component of in Sec-pathway. SecA operates to translocate preprotein into thylakoid lumen in plants. Primitive red alga Cyanidioschyzon merolae is unique in that secAgene exsists both in the nucleus and the plastid genome. The purpose of this study is to clarify the difference of two SecA. SecAs are investigated by the method of phylogenetic analysis, reverse transcript PCR, real time PCR and complementation test of Escherichia coli secA Ts mutant. The result showed that both secAs were transcripted. Phylogenetic analysis showed that secAin the nucleus was related to the mycobacterial secA2 which concerns auxiliary secretion factor in Sec pathway. Both of the two secA did not complement E.coli secA mutant. E.coli was rethal when plastid secA overexpressed while not nuclear secA. The results suggested that the two secAs have different function.

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Sulfur responses in higher plants have been well studied. Most of the proteins and compounds which function in the sulfate assimilation pathway have been characterized, However, the mechanisms to sense or regulate sulfur status are not understood although some are known in other organisms. In this study, we focused on Sac3 identified as a sulfur regulator in Chlamydomonas reinhardtii. We studied one of ten Sac3 homologs in Arabidopsis thaliana to determine if the gene is involved in sulfur response in higher plants as well. Two independent T-DNA inserted mutants in the relevant gene did not induce the increased accumulation of sulfate transporter Sultr2;2 mRNA under sulfur deficiency. Furthermore, under the deficiency, the O-acetyl-L-serine level was elevated in the mutants compared to the wild type. These results suggest that this gene is involved in sulfur response and that the mechanism of sulfur regulation may be conserved between these two different species.

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Protein phosphorylation and dephosphorylation are most widely used regulatory mechanism in signal transduction. It has been implicated that MAP kinase cascades are involved in the regulation of plant disease resistance. MAP kinases are activated by upstream MAPK kinases. However, few negative regulators of plant MAP kinase have been reported. In the present study, we characterized protein phosphatases that inactivated StMPK1, a potato MAP kinase activated by various elicitors. StMPK1 was inactivated by crude extract from potato suspension cultured cells. The inactivation of StMPK1 was inhibited serine/threonine phosphatase inhibitors. Recently, alfalfa MP2C has been shown to be responsible for the dephosphrylation of activated MAPK. We isolated potato MP2C from potato cDNA library using homology-based PCR cloning. The potato MP2C dephosphorylated and inactivated StMPK1, suggesting the involvement of potato MP2C in resistant reactions.

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