Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes. In plants, MAPK cascades are known to function in various biotic and abiotic stress signaling. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPK, 10 MAPKK and 60 MAPKKK genes in its genome. However, detailed functions of each MAPK cascades are still unknown. We have been focused on MKK3, one of the Arabidopsis MAPKK, and found the novel MKK3-MPK6 cascade by using in vitro activation assay. We also demonstrated that MKK3-MPK6 cascade functions in vivo by using DEX mediated conditional expression transgenic system. DEX-treated MKK3 DD plants did not show HR-like cell death phenotype as those found in DEX-treated MKK4 DD plants. Microarray analysis revealed that MKK3 and MKK4 cascade might regulate different sets of downstream genes. Possible function of MKK3-MPK6 cascade will be discussed.

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Volatiles released from a plant induce gene expressions and root-growth inhibitions in other plants. However, molecular mechanism of the volatile reception has not been elucidated. We assume that G-protein-coupled receptors (GPCRs), odorant receptors in animals, might detect volatiles also in plants. We found six GPCR-like genes, AtGPCR1-6, on Arabidopsis thaliana genome. Transcripts of these genes were detected in tissues of A. thaliana. Expressions of AtGPCR1 and AtGPCR6, however, were less in flowers and buds than other tissues. This is also the case of the expression of GPA1 (α subunit gene of heterotrimeric G protein) , suggesting that AtGPCR1 and AtGPCR6 have functional relations with GPA1. On the other, AtGPCR2 and AtGPCR5 were more expressed in flowers and buds. Bornyl acetate, a plant terpenoid, induced the expression of LOX (lipoxygenase gene) in wild type, but not in a mutant of AtGPCR5 which is knocked out by inserting T-DNA.

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Two open reading frames, alr0295 and alr2325, were found to encode the cAMP receptor proteins (CRPs) in the genome of the filamentous cyanobacterium Anabaena sp. PCC 7120. These open reading frames were named ancrpA and ancrpB, respectively, and those translated products were showed cAMP- and DNA-binding affinity. Since cAMP-binding affinity of AnCrpA is higher than that of AnCrpB, cAMP signaling in Anabaena sp. PCC 7120 is thought to be mediated mainly by AnCrpA. Therefore, the target genes for AnCrpA were identified using a DNA microarray method. The results showed that the transcription of some nitrogen fixation-related genes were repressed in the ancrpA disruptant. Electrophoretic mobility shift assay revealed that AnCrpA bound to the 5' upstream regions of target genes. It is concluded that AnCrpA regulates the expression of the nitrogen fixation-related genes.

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Oxidative stress due to reactive oxygen species and, in particular, to H₂O₂ has a major impact on all biological systems. DNA microarray analysis revealed that incubation of cyanobacterial cells of Synechocystis sp. PCC 6803 with 0.25 mM H₂O₂ dramatically changed the pattern of genome-wide expression of genes. It induced the expression of 77 genes with induction factor higher than 4.0. Screening of the mutant libraries of histidine kinases (Hik) and response regulators (Rre) by means of the H₂O₂-inducible expression of genes revealed that five Hiks and three Rres are involved in the perception of H₂O₂ signals. Hiks and Rres regulate the expression of approximately 70% of the H₂O₂-inducible genes. Further analysis will be discussed.

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We previously identified that a histidine kinase, Hik33, regulates the gene expression at low-temperature in the cyanobacterium Synechocystis sp. PCC6803. In this study we rigidified the plasma membrane by disruption of the desA and desD genes for Δ12 and Δ6 acyl-lipid desaturases, respectively, in Synechocystis. DNA microarray analysis revealed that cold-inducible genes could be divided into three groups according to the effects of the rigidification of membrane lipids. The first group included genes whose expression was not induced by cold in wild-type cells but became strongly cold-inducible in the mutant cells. The second group consisted of genes whose cold inducibility was moderately enhanced and the third group consisted of genes whose cold inducibility was unaffected by the mutation. Genes regulated by Hik33 were categorized into the second group, suggesting that the activity of Hik33 might be regulated by the membrane rigidity induced by cold.

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Jasmonic acid (JA) is a plant hormone which regulates various developmental processes in plants, and it acts as a signal molecule in plant defense against herbivores and pathogens. We isolated a cDNA for the JA-responsive gene RERJ1 from suspension-cultured rice cells. RERJ1 encodes a transcriptional regulator with a bHLH motif. Analyses of the phenotypes of rice plants expressing sense- and antisense-RERJ1 mRNA strongly suggested that RERJ1 is involved in the inhibition of shoot growth caused by exogenously applied JA. However, the other biological functions of RERJ1 remain to be clarified. In this study, it was indicated that in rice plants RERJ1 was upregulated by exposure to stresses such as wounding, salt, and drought as well as JA, but that RERJ1 was not responsive to abscisic acid. We also carried out microarray analysis using RNA from sense-RERJ1-overexpressing rice calli to survey the target genes of RERJ1.

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The initial event of cytokinin response in Arabidopsis cells is composed of the His-to-Asp phosphorelay from receptor histidine kinases to transcription-factor-type (type-B) response regulators including ARR1. We reported in the last annual meeting that all the type-A response regulator genes in Arabidopsis are primarily responsive to cytokinins and can be directly activated by ARR1. Although artificial activation of ARR1 induces various cytokinin-related phenomena including ectopic shoot formation, pathways from the regulation by ARR1 to the phenomena are still difficult to be supposed mainly because an overall view of the genes regulated by ARR1 remains unclear. To identified ARR1 target genes other than those for type-A proteins, we compared two batches of transcripts prepared from plants in which ARR1 is artificially activated or not by the High Coverage Expression Profiling (HiCEP) analysis. We will report on the novel ARR1 target genes and discuss how they are related with cytokinin-induced phenomena.

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We have reported that transgenic Arabidopsis overexpressing salinity-responsive DDF1 gene (35S::DDF1) show dwarfism due to gibberellin deficiency caused through induction of AtGA2ox7 gene. DDF1 encodes AP2 DNA-binding domain that is highly similar to that of DREB/CBF transcriptional activators. We have also reported that RD29A gene, which has DRE motifs in their promoter, is induced in the 35S::DDF1 plants, indicating DDF1 is capable of binding DRE and/or DRE-Like motif. Transactivation assay showed that transient overexpression of DDF1 induced the expression LUC reporter gene under the promoter of RD29A or AtGA2ox7 in Arabidopsis leaves. Gel shift assay showed that DDF1 bound the DRE motif of the RD29A promoter or DRE-like motifs found in the AtGA2ox7 promoter. These results indicate that DDF1 is transcriptional activator, directly capable of activating these target genes.

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Arabidopsis ht1 was isolated as a hot phenotype under low CO₂ concentration. Its stomatal response to CO₂ was significantly inhibited. The mutated HT1 gene was speculated to encode a protein kinase. To determine whether the HT1 proteins have a phosphorylation activity, we performed in vitro kinase assay. The data indicated that the HT1 has both autophosphorylation and phoshprylation activity. ht1-1 has a mutation site which converts R to K residue within the catalytic subdomain VIb. This conversion greatly impaired the phosphorylation activity in vitro. We constructed a kinase negative mutant of HT1 gene, in which the lysine codon at the predicted ATP-binding site was replaced by a tryptophan codon (HT1:KW). Over expression of the HT1:KW in normal plants results in suppression of the sensitivity to CO₂. These results indicate that the HT1 kinase plays an important role in the CO₂ signaling.

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The 26S proteasome is an active proteolytic complex consisting of a 20S core protease capped at ends by a 19S regulatory particle. The 19S is made up of six ATPases (RPT) and, at least, 11 non-ATPase subunits. In Arabidopsis genome, the RPT2 subunits are encoded by two genes, AtRPT2a and AtRPT2b.
We found that rice RPT2a is expressed in the sugar treated embryos by microarray analysis. In Arabidopsis, T-DNA inserted knockout mutant of AtRPT2a (rpt2a) showed hyper-response to sugars and the AtRPT2a expression was largely enhanced by sucrose application, while the AtRPT2b did not. The rpt2a displays abnormally root apical meristem and leaf shapes, since halted root mutant is the same allele of the rpt2a (Ueda et al. 2004 Development 131: 2101-2111), indicating that sugar signaling which is partially associated with plant development is mediated through proteasome-dependent programmed proteolysis.

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We have measured sub-microsecond time-resolved absorption spectra of the mixtures of various carotenoids and bacteriochlorophyll a (BChl a), by pumping at Q_y. These carotenoids have different number of conjugated double bonds (n = 9-13). We have also measured the LH2 antenna complexes which contains different carotenoids (n = 9-11), isolated from 4 kinds of the purple photosynthetic bacteria. As the result, in the solutions, not only the triplet-energy transfer from BChl a to carotenoid, but also the "back" triplet-energy transfer from carotenoid to BChl a were observed. On the other hand, in the LH2 complexes, only the signal of the triplet carotenoid is observed, but no signal of triplet BChl appeared. This shows extremely efficient triplet-energy transfer in LH2. In each case, carotenoids having a longer conjugated chain dissipated the triplet energy more efficiently.

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We have isolated the Core (RC-LH1) complexes from purple photosynthetic bacteria, which contain carotenoids having different number of conjugated double bonds (n), Rhodobacter sphaeroides 2.4.1 (n = 10), Rhodopseudomonas acidophila (n = 11) and Rhodospirillum rubrum (n = 13), and measured their submicrosecond time-resolved absorption spectra exciting at the Q_y absorption. The time-resolved spectra exhibited three components. A long-lived component was ascribed to the radical cation of bacteriochlorophyll a. The other two can be ascribed to triplet state of carotenoids, based on the typical spectral pattern of T_n ← T₁ absorption. The transient absorption on the shorter-wavelength side shifted systematically depending on the conjugation length; their wavelengths corresponded to those of the carotenoid T_n ← T₁ absorptions in the LH2 complexes. The transient absorption on longer wavelength may be due to T₁ carotenoid in the reaction center.

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Time-resolved EPR spectra of 15-cis-spheroidene bound to the reaction center (RC) from Rhodobacter sphaeroides 2.4.1 were recorded. A 4-component analysis of the spectral data by singular-value decomposition followed by global fitting identified the triplet transformations of Car, i.e., ³Car(I) → ³Car(R) → ³Car(II) →, in which ³Car(R) functions as a leaking channel of triplet population. The sequential transformations were ascribed, by simulation of the zero-field splitting parameters using a polyene model, to the conformational changes of (0°, 0°, 0°) → (+20°, –20°, +20°) → (+45°, –40°, +40°), concerning the rotational angles around the cis C15=C15', trans C13=C14 and trans C11=C12 bonds. Inhomogeneous orientation of the Car–RC assembly was identified by spectral simulation, a fact which suggests the generation of a magnetic dipole moment during the conformational changes. Thus, the rotational motions around the central double bonds play a key role in the triplet-energy dissipation.

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The electronic-absorption spectra of all-trans carotenoids with different numbers of conjugated double bonds (n), including a spheroidene derivative (n=8), nurosporene (n=9), spheroidene (n=10), lycopene (n=11), anhydrorhodovibrin (n=12) and spirilloxanthin (n=13) were recorded at a high concentration (ODmax ** 20cm-1). After data-fitting we observed absorptions due to the **hidden** singlet states on the red side of the 1Bu+ absorption. The energies of those states exhibited a linear relation as function of 1/(2n+1). We assigned those states to the 2Ag-, 1Bu- and 3Ag- states based on comparison with our previously resonance-Raman and fluorescence results (Tokutake Sashima, et. al., J. Phys. Chem. B 2000, 104, 5011; Ritsuko Fujii, et. al., J. Phys. Chem. A 2001, 105, 5348). In the 2Ag- state, we observed a series of vibrational progression from **=0 to **=3.

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Phycobilisome has been suggested to transfer energy not only to PSII but also to PSI, although little is known about the molecular mechanism. Phycobilisome rods and the core are associated with each other via rod-core linker polypeptides (CpcG). Last year, we showed that CpcG1 and CpcG2 have distinct roles in phycobilisome assembly in Synechocystis sp. PCC 6803. Here we present their physiological roles focused on energy distribution between PSII and PSI. While phycobilisomes with CpcG1 contained all the set of components, those with CpcG2 lacked major core components. Spectral analysis suggested that phycobilisome with CpcG2 retained a minor allophycocyanin component, which will be discussed in the presentation. Fluorescence spectra demonstrated a significant defect in energy transfer to PSI in ΔcpcG2 in contrast to a defect in transfer to PSII in ΔcpcG1. This indicates that CpcG2 together with the minor allophycocyanin component plays an important role in energy transfer to PSI.

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Heat-stable photosynthetic light-harvesting membrane protein (LH2) that has a cylindrical structure of 7 nm diameter was introduced into the inorganic nanoporous material that has honeycomb-like hexagonal cylindrical interior channels with a diameter of 7.9 nm. LH2 was purified by the detergent treatment of the membranes of a thermophilic purple photosynthetic bacterium Thermochromatium tepidum by HPLC column chromatography. The measurement of N₂ adsorption indicated that most of LH2 molecules were adsorbed inside the nano-pores. Absorption spectrum of LH2 inside nono-pores was similar to free LH2 except for a slight red shift (3 nm) of B850 band. The energy transfer from B800 to B850 was very rapid. The ring structure as well as the antenna function of LH2, therefore, seems to be well maintained inside nano-pores. Fluorescence decay of the adsorbed LH2 was remarkably faster (27 ps). Fluctuation of LH2 structure inside nano-pores will be discussed.

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The enzymatic activity of a methyltransferase, BchU, which catalyzes methylation at the C-20 position of a chlorin moiety, was investigated in vitro. The bchU gene derived from a photosynthetic green sulfur bacterium, Chlorobium tepidum, was over-expressed in Escherichia coli as a His-tagged protein (His₆-BchU), and the His₆-BchU was purified. The His₆-BchU catalyzed the methylation of zinc bacteriopheophorbide d at the 20 position in the presence of S-adenosylmethionine to give zinc bacteriopheophorbide c. However, metal-free bacteriopheophorbide d could not be methylated by the BchU, indicating that the central metal in the chlorin ring should be required for the recognition in the BchU catalyzed methylation. The zinc chlorin possessing a 1-hydroxyethyl group at the 3-position was methylated more efficiently than the other substrates used in this study. We would like to discuss the role of BchU in the bacteriochlorophyll c biosynthetic pathway.

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An S-adenosylmethionine (SAM)-dependent methyltrasferase, BchU, catalyzes methylation at C-20 position of chlorin moiety in bacteriochlorophyll c biosynthetic pathway, however, the methylation mechanism underlying the bacteriochlorophyll c biosynthesis is poorly understood. We have determined the crystal structure of BchU in order to elucidate its reaction mechanism at the molecular level.
Recombinant BchU from Chlorobium tepidum was overexpressed in E. coli, purified, and crystallized. We collected diffraction data using synchrotron radiation at SPring-8 and determined the crystal structure at 2.3 A resolution. The structure of BchU consists of two domains; N-terminal domain and C-terminal domain. The N-terminal domain is involved in dimerizaion and the C-terminal domain contains a typical Class I motif. In addition, we determined the BchU structure in complex with SAM. These structural features and analysis of putative substrate-binding pocket provide invaluable information for the methylation mechanism of BchU.

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Protochlorophyllide (Pchlide) oxidoreductase catalyzes the penultimate step of chlorophyll a biosynthesis. Most oxygenic phototrophs including cyanobacteria have two structurally unrelated Pchlide reductases, a light-dependent Pchlide reductase (LPOR) and a nitrogenase-like light-independent (dark-operative) Pchlide reductase (DPOR). Here we show in-vitro activity of cyanobacterial DPOR and its oxygen sensitivity for the first time. An LPOR-lacking mutant of the cyanobacterium Plectonema boryanum was cultivated under high light bubbling with nitrogen (containing 2% CO₂), and the soluble fraction was prepared in an anaerobic chamber. The Pchlide reduction activity was successfully detected in the soluble fraction in an ATP-dependent manner. Upon exposure to the air, the DPOR activity in the soluble fraction was drastically decreased with a half-life of approximately 15 min. Oxygen sensitivity of DPORs from cyanobacteria and photosynthetic bacteria will be discussed in view of the evolution of oxygenic photosynthesis.

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In Synechocystis sp. PCC 6803, promoter activities of photosystem (PS) I genes decrease under high-light and increase under low-light conditions. To know the mechanism of the coordinated regulation of PSI genes, we investigated promoter architectures of psaAB genes encoding reaction center subunits and psaD gene encoding a small subunit of PS I. It was revealed that both psaAB and psaD genes have two promoters (P1 and P2). Although both promoters in psaAB showed light response, the regulatory mechanism was different between them: P1 was activated specifically under low-light conditions, whereas P2 activity was repressed under high light. As for psaD gene, activity of P1 was very weak and did not decrease under high light. P2 showed light response and its activity was up-regulated under low-light conditions. Although the regulatory mechanism seems similar between P1 of psaAB and P2 of psaD, we could not find common cis-elements for these two promoters.

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The RC-bound tetraheme cytochrome subunit of a purple bacterium, Blastochloris viridis, was successfully synthesized in Rubrivivax gelatinosus cells. The redox midpoint potentials (E_m's) of 4 hemes in the chimeric RC were almost identical with those in the B. viridis RC, in which the hemes are linearly arranged from the core proteins as c559 (+380 mV), c552 (+30 mV), c556 (+320 mV), and c553 (-50 mV). To clarify the physiological significance of such high-low-high-low arrangement of E_m's of hemes, amino acids located near the hemes were mutationary replaced to change their E_m's. More than 20 mutants were obtained. In a mutant, R264L, in which the 264th Arg was replaced by Leu, the E_m of c559 was lowered to 130 mV and P⁺ was rereduced with a rate 20 times slower than in the native chimera when c556 works as the donor. This mutant, however, grow photosynthetically.

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One of important functions of carotenoids is the protection against photooxidative damage in photosynthetic organisms. Nevertheless, the correlation between the chemical structure of carotenoids and their protective function against photooxidative damage, have been investigated insufficientry. To assess functional features of cyclic carotenoids, we attempted to construct a mutant of the purple photosynthetic bactererium, Rubrivivax gelatinosus (IL144), that accumulated lycopene, and then lycopene cyclase gene (crtY) from Erwinia uredovora was introduced into the mutant. This mutant accumulated β-carotene in cell, membrane and pigment proteins. This fact shows that the cyclic carotenoid can be used for growth of the purple bacterium that originally has only acyclic carotenoids. Resistance against singlet oxygen of the mutant strain seemed to be higher than that of the lycopene accumulating strain, although that against photodamage of the both strains were similar to each other.

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In cyanobacteria, small transcriptional regulators that consist solely of a helix-turn-helix motif of LuxR-family type are highly conserved. In Synechocystis sp. PCC 6803, a small LuxR-type regulator, Ssl0564, regulates the expression of several genes such as ndhD2 encoding a subunit of NAD(P)H dehydrogenase, rpe encoding pentose-5-phosphate-3-epimerase and katG encoding catalase-peroxidase when the reducing side of photosystem I is in oxidized state. It was revealed that purified Ssl0564 becomes dimeric form by the treatment with oxidizing agents and three cysteine residues at C-terminal region seem to be important for dimerization. The quantification of sulfhydryl groups of Ssl0564 using DTNB showed that oxidized and reduced form of Ssl0564 contain 0.15±0.11 and 3.14±0.32 mol of thiol/mol of the protein, respectively. We suppose that the activation of Ssl0564 is achieved by the formation of intra- and/or inter-molecular disulfide bonds at C-terminal three cysteine residues.

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N₂-fixing cyanobacteria can produce hydrogen as an inevitable by-product of nitrogenase reaction. In order to improve photobiological H₂ productibity by decreasing reabsorption of hydrogen, we have created three hydrogenase mutants from Anabaena PCC 7120 (ΔhupL, ΔhoxH, ΔhupL/ΔhoxH) and shown that the ΔhupL and ΔhupL/ΔhoxH produced H₂ at a rate 4-7 times that of wild-type. However, such high production activity stage lasted for only about 10 hours. Homocitrate is liganded to the catalytic MoFe₇S₉ cluster in nitrogenase, and inactivation of homocitrate synthase decreases efficiency of nitrogen fixation leading to increased hydrogen production in Klebsiella pneumoniae. We have created three mutants (ΔnifV1, ΔnifV2, ΔnifV1/ΔnifV2) on the ΔhupL background. One of the mutants showed higher and more lasting hydrogen productivity over the parental strain under Ar or N₂.

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The PAS domain is one of the important signaling modules that monitor changes in light, redox potential, oxygen or small ligands. The PAS domain superfamily is highly diverse in sequence and in length but is conserved in the basic three-dimensional structure. Recently, structure and function of some PAS domains are revealed. We formerly detected a large number of PAS domains from the filamentous cyanobacterium Anabaena sp. PCC 7120, although most of them have no obvious similarity with the PAS domains of known function. To predict novel sensory PAS domains, we performed comparative genomics analysis with closely-related species. As a result, we extracted 25 putative sensory PAS domains. In the present study, we expressed and purified these 25 PAS domains as a His-tagged protein. Analyses of absorbance spectra and metal ions revealed novel ligand-binding PAS domains. Sensing mechanism of these ligand-binding PAS domains will be discussed.

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We previously revealed that the YABBY gene DROOPING LEAF (DL) plays an essential role in rice carpel specification. To further understand the genetic mechanism of floral organ specification in rice, we studied the functions of the two paralogous C-class genes, OsMADS3 and OsMADS58, because C-class genes play predominant roles in carpel specification in eudicots.
Major phenotype of loss-of-function mutants of OsMADS3 is homeotic transformation of stamens into lodicules in whorl3, while that of suppression lines of OsMADS58 gene is indeterminate organ development and abnormal carpel morphology in whorl4. These results indicate that functions of the two C-class genes are diversified to work differently in each whorl. In addition, expression analyses suggest that DL and C-class genes function independently. We also found the involvement of the C-class genes in lodicule differentiation in rice flower.
The molecular mechanism of the functional diversification of C-class gene activities will be discussed.

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<Arabidopsis> flowers are composed of four types of floral organs, sepals, petals, stamens, and carpels, which are placed in concentric whorls. Each type of organ has characteristic shape and forms, and the position is defined in each whorl. Little is known about developmental mechanisms controlling the position and shape of floral organs. To identify genes involved in the mechanisms, we are analyzing <fl51> mutant showing defects in sepal and petal shape. In <fl51>, four sepals and petals are narrower and longer than those of wild type, though their identities are normal. Sepal primordia are smaller, and their position shifted toward either the abaxial or adaxial sepal primordium, suggesting that <FL51> is required for the formation of sepal primordia. <FL51> encodes one of protein components of a spliceosome. Expression pattern of <FL51> will be presented and we will discuss about function of <FL51> in sepal and petal development.

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TAF10 is one of the TATA box-binding protein (TBP) associated factors (TAFs), which constitute TFIID with a TBP. Previously we isolated Arabidopsis thaliana TAF10 (atTAF10), which was single-copy gene closely related to other organisms' TAF10, and revealed that atTAF10 is expressed transiently in the developmental processes of lateral roots, rosette leaves and most floral organs. Here we will report the several abnormal morphologies observed in the transformants in which the proper expression of atTAF10 was disturbed. For example, terminal flower, over-production of immature leaves and emergence of carpel-like structures were observed in both atTAF10-over-expressed- and atTAF10-antisense-suppressed transformants. In these transformants, the expression of WUSCHEL (WUS) and AGAMOUS (AG) was up-regulated. Collectively, the results suggest that atTAF10 is a "selective" TAF in plants, which is involved in normal development of several tissues through regulating WUS and AG expression.

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In higher plants, leaves are formed from the shoot apical meristem (SAM). However, some species in Streptocarpus (Gesneriaceae) do not retain SAM, while leaves are formed via rosette leaves from a kind of intercalary meristem named groove meristem. Thus, the process of leaf formation is intriguing from the viewpoints of developmental biology. In this study, we used Streptocarpus parviflorus, a rosulate species and followed the process of the first leaf formation. It was formed in the axil of the macrocotyledon. When the leaf formation was classified into two stages, leaf primordium was first bulged, while lamina and petiolode were differentiated in the second stage. Notably, tunica-corpus-like structure was observed at the bulged structure. We have particularly interested in examining the origins of respective organs with the detection of BrdU incorporation. The significance of such temporal and spatial changes of cell division activity was discussed.

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Leaves of terrestrial plants have diverse and complex patterns of leaf venation. Although many descriptive studies of venation exist, little is known about the molecular mechanisms underlying the formation of venation pattern. The Arabidopsis vascular-enhancer-trap line, in which GFP is expressed specifically in the procambium and young vasculature, was mutagenized with EMS. By observing GFP-lit-up veins in leaves, we are screening mutants defective in vascular development. We obtained mutants and classified them into at least three groups according to their phenotypes. In group 1 mutants, there are fewer veins in leaves (nov, 1B-22, 2B-17, 3B-55, 621C-27, 623A-11). In group 2 mutants, veins in leaves are fragmented (621A-4, 621B-25, 621C-6, 621C-20, 622B-2). In group 3 mutants, veins in leaves are thicker (1B-11, 1D-1, 622A-3). Characterization of phenotypes of these mutants and mapping the mutant loci are in progress. We will discuss possible functions of the affected genes in vascular development.

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To understand vascular pattern formation, we have analyzed van3 mutants of Arabidopsis characterized by fragmented veins. We have shown that VAN3 encodes an AZAP type ARF (ADP ribosylation factor)-GAP (GTPase activating protein) that function in the trans Golgi network. As the first step toward elucidating the function of Arabidopsis AZAP type ARF-GAPs (AZAPs) systematically, here we observed the expression patterns of all the four Arabidopsis AZAPs using the GUS reporter genes.
In leaf primordia, VAN3 was expressed uniformly. As the primordia developed, the expression became restricted to vasculatures. Similar expression patterns were observed in the other AZAPs. In roots, however, VAN3 is expressed in the elongation zone, while the other AZAPs are expressed in QC and columella cells. These results indicate that AZAPs function in different root cells. Now efforts are underway to analyze knockout mutants of AZAPs. Taken together, we will discuss the role of AZAPs in plant development.

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There is less molecular information about vascular formation in monocotyledonous plants, compared with that in dicotyledonous plants. The development of the leaf in the monocotyledon progresses almost in one dimension, because cell division occurs at the base of leaves. Therefore, it is easy to follow the process of vascular development in the leaf. In order to elucidate the vascular formation system in the monocotyledon, the research was carried out by using the rice plant as a model.
We investigated the pattern formation and internal structure of the vascular tissue with the cleared leaf blades and the continuous sections in the basement of the rice plant. Based on this information, we screened mutants defective in vascular formation. As the result, we succeeded in identifying 11 lines of vein pattern mutants and a line of vascular structural mutant. From these results, we will discuss the mechanism of vascular formation in rice leaves.

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Vascular cells differentiate from the procambium. In order to clarify the mechanism of vascular patterning, it is necessary to understand the early stages of procambial development and provascular development just prior to procambium. To identify genes expressed at the early stages of provascular/procambial development, we have created Arabidopsis enhancer-trap lines and screened for the lines expressing a reporter GFP in the provascular/procambium cells. So far, we obtained 367 lines in which GFP fluorescence was detected in the provascular/procambium cells and/or vasculature. We identified the T-DNA insertion sites in 36 lines. Genes near the insertion sites encode, for example, transcription factors, proteases and AUX/IAA proteins. Concerning the lines in which GFP was expressed in vascular initial and provascular/procambium cells, we are currently performing linkage analysis between GFP expression and their T-DNAs. We report GFP expression patterns of the aforementioned lines, their T-DNA insertion sites, and results of the linkage analysis.

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In order to identify genes involved in early step of vascular formation, we screened 51000 gene trap lines with GUS as a reporter gene.We isolated two groups of candidate lines, group 1 (show GUS staining pattern preferentially along procambial cells in developmental stage 19 embryo; 3 lines), and group 2 (show abnormal vascular development; 3 lines). We reported previously identification of the tag insertion loci for group 1 lines (GGH, RING finger, unknown gene). Here, we report identification of causal genes of group 2 lines as CYP86, AtKTN, and an unknown gene. We also analyzed detailed expression patterns of these lines. Group 1 genes as well as AtKTN show GUS staining pattern preferentially along procambial cells or meristematic tissues of mature plants. However, each line shows differential expression pattern in root apex region, which may represent different stages in procambial development.

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The final shapes of plant organs are results of elaboration of the regional regulation of cell division and differentiation. The cincinnata (cin) mutant of snap dragon has altered leaves with uneven curvature of the surface. The cin leaves have excess cell division in the leaf tip and margin. Because CIN codes TCP transcription factor, it is clear that TCP is involved in the determination of shape of organs. However, no systematic functional analyses of this family have been conducted. We applied CRES-T system to Arabidopsis TCPs for analysis of functional role of TCP family on plant development. Expression of the chimeric TCP repressor induced defects in the shape of multiple organs including cotyledon and leaf. These organs did not have clear specification of cell shape according to the position. These results indicate that the TCPs are required for the regional regulation of cell division and differentiation during organ development.

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Initiation of lateral meristems is indispensable for plant development. To elucidate underlying molecular genetic mechanisms, we analyzed a relationship between two transcription factors, MOC1 and LAX.
In the moc1 mutant, initiation of panicle branches and tillers are partially suppressed, and defects are observed only in the panicle branching in the lax mutant. On the contrary, initiation of all lateral meristems is completely suppressed in the moc1 lax double mutant. The microarray analysis enabled us to find out common downstream factors for MOC1 and LAX. These two genes are expressed in and around cells where lateral meristems arise during reproductive and vegetative phase. By in situ hybridization, we found that MOC1 expression in lax was normal whereas LAX expression in moc1 was suppressed in the vegetative phase. These results suggest that MOC1 and LAX act independently in the reproductive phase, and MOC1 regulates LAX in the vegetative phase.

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During reproductive phase in rice, the shoot apical meristem generates primary branches, which generate secondary branches and spikelets. A spikelet has a pair of glumes, 1 lemma, 1 palea, 2 lodicules, 6 stamens and 1 pistil from outside to inside. Meristem activity should be maintained properly to generate normal branches and spikelets. lonely guy (log) mutants show abnormal branching pattern and the reduction of floral organ number. Floral meristems in log show flat structure compared with wild type rice and cease the differentiation of floral organs prematurely. This indicates that LOG functions as a regulator of meristem activity during reproductive phase. We found that LOG encodes a homologue of lysine-decarboxylase (LDC), which catalyzes the synthesis of cadaverine, a kind of polyamine. There are only a few reports describing the involvement of polyamine in plant morphogenesis. Our finding indicates that cadaverine may play an important role in plant morphogenesis.

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The architecture of rice is affected by organs differentiated from axillary meristems. LAX is essential for initiation of axillary meristems and lax has reduced number of lateral organs in the panicle. LAX, expressed in an adaxial boundary of the axillary meristem, encodes a bHLH transcription factor. However, on the basis of amino acid sequence, it is predicted that LAX does not bind to DNA and functions as an inhibiter of another bHLH protein through formation of a heterodimer. To determine its function at molecular level, we are studying genes working downstream of LAX and directly controlled by LAX. We are also identifying heterodimer partners of LAX. A microarray analysis through an induction system showed that LAX induces various transcription factors and auxin signaling genes. This result suggests a possibility that LAX mediates auxin signaling pathway at a boundary of the axillary meristem.

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Lateral shoot from axillary meristem (AM) on axil of leaves is an important organ to construct diverse bodies adapting to various environmental conditions. To make clear the molecular mechanism of AM-formation, we analyzed novel Arabidopsis semi-dominant mutant uni-1d. The uni-1d heterozygote looked like a sea urchin, named U-NI in Japanese, due to many short lateral shoot forming in axil of vegetative leaves and cotyledon. UNI encoded a novel NBS-LRR family protein and uni-1d has missense mutation causing a gain-of-function. Protein of this family has been understood as involving only disease resistance. Indeed, pathogen related genes are expressed strongly in uni-1d, suggesting uni-1d showed constitutive defense response. These results suggest that UNI may involve in both AM-formation and defense response.

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The typical body plan of higher plants is composed of the main axis attached by numerous lateral organs. Formation of lateral organs creates boundaries that separate the primodia from the tissue of the main axis. The role of the boundaries in lateral organ formation is not well understood. In the puchi mutant of Arabidopsis, ectopic bracts and spitules are formed at the base of flower primordia. In addition, the base of lateral roots is expanded in puchi, mainly due to an increased number of cells. We cloned the PUCHI gene and it encodes a putative transcription factor. PUCHI expression is detected in the boundary between the inflorescence meristem and flower primordia, and between the primary root and lateral root primordia. These results suggest that PUCHI functions in the boundaries to repress bract and spitule formation in flowers and restricts cell proliferation at the base of lateral root primordia.

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Extensive studies have provided evidence that caspase activity is involved in programmed cell death of plants as animals. However, no plant proteinases themselves that exhibit caspase activity have been identified. To identify a functional homolog of caspase in plants, we investigated hypersensitive cell death in tobacco mosaic virus (TMV) -infected tobacco plants. We present here that a vacuolar processing enzyme (VPE) which is a cysteine proteinase responsible for maturation and/or activation of vacuolar proteins, is a proteinase exhibiting caspase-1 activity, and that VPE is essential for virus-induced hypersensitive cell death. VPE deficiency decreased caspase-1 activity and prevented virus-induced hypersensitive cell death. The disintegration of the vacuolar membranes occurred in the leaves before visible lesions were formed. VPE deficiency suppressed the disintegration of the vacuolar membranes in the TMV -infected leaves. These results suggest that VPE is involved in vacuolar collapse, which triggers hypersensitive cell death.

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In spite of importance of programmed cell death, few orthologs of animal apoptosis regulators have been found in plants. We have identified two novel genes encoding proteins homologous to mammalian inhibitor of apoptosis protein (IAP), Arabidopsis thaliana IAP-like protein (AtILP) 1 and 2 in the Arabidopsis genome. ILP genes also existed in other organisms including budding yeast, nematode and vertebrates and constitute a novel gene family. Like baculovirus IAP repeats (BIR) in IAPs, ILP proteins contain two (usually one complete and one separated) highly conserved BIR-like domains (BLDs). BLDs, like BIRs, have a putative C2HC-type zinc finger, a derivative of C2H2-type zinc finger. Phylogenetic analyses based on C2H2-type zinc finger motifs indicated that ILPs are putative paralogs of IAPs. These findings suggest the conservation of ILPs in wide-range of eukaryotes, including plants, and their closely related functions to IAPs.

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Apical dominance is a phenomenon in which a terminal bud inhibits the outgrowth of axillary buds. Auxin and cytokinin are thought to have a major role in this process. Based on our previous data we concluded that a role of auxin delivered from terminal bud is to control cytokinin biosynthesis in the stem by repression of isopentenyltransferase (IPT) expression. To elucidate how auxin represses PsIPT expression, we generated transgenic Arabidopsis containing the GUS reporter gene under the control of promoter region of PsIPT2. Because GUS protein seems to be stable in plant cells, we examined GUS transcript levels. GUS was expressed in 10-day-old transgenic Arabidopsis seedlings and was repressed by IAA treatment. The GUS expression pattern was the same as that of PsIPT2 expression in the pea stem. These results indicated that Arabidopsis have molecular mechanisms of repression similar to those of auxin on PsIPT2 expression in pea.

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Although auxin has been considered to regulate many processes in plant development and has been studied for over a centry, the biosynthesis of auxin has remained elusive. Recently it has been reported that YUCCA catalyzes a key step of auxin biosynthesis through the analysis of Arabidopsis mutant, yucca, which overproduces the auxin.
To the auxin biosynthesis and biological function in rice, we isolated OsYUCCA-like1 gene and analyzed its expression pattern in rice, we also produced the overproducer of the OsYUCCA-like1 gene. The plants showed the formation of excess root hairs and abnomal gravitropism, which are similar phenotype to the Arabidopsis yucca mutant.
Increased expression of DR5-GUS in the overproducers also indicated the elevated level of auxin.
Based on these results, we concluded that the OsYUCCA-like1 gene is involved in the step of auxin
biosynthesis pathway in rice.

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Cytokinins induce cell division and differentiation of the plant in the presence of auxin. By using a selective ¹³C-labeling system for isoprenoids from the methylerythritol phosphate (MEP) pathway in plastids or the mevalonate (MVA) pathway in the cytosol, we have previously demonstrated that the prenyl side chain of trans-zeatin (tZ) is mainly derived from dimethylallyl diphosphate in the MEP pathway, but also partially produced through the cis-trans isomerization of cis-zeatin from the MVA pathway in Arabidopsis seedlings. In the present study, we found that Agrobacterium sends its CK biosynthesis enzyme into plastids to create a new tZ biosynthesis pathway using an intermediate of the MEP pathway, hydroxymethylbutenyl diphosphate, in the host plant. We will present new findings from labeling experiments in Arabidopsis seedlings, Catharanthus cell cultures and tomato crown gall using ¹⁸O-labeled precursor.

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To explore the genetic factors affecting postembryonic root formation, we screened Arabidopsis mutants with irregular adventitious and lateral root formation. The ar-C22 mutation showed retarded growth of primary roots, no production of lateral root, and adventitious root formation. Although the primary roots and hypocotyls in ar-C22 mutant showed a vascular system with fewer cells, adventitious roots showed normal vascular system and branched secondary roots. The hypocotyls of ar-C22 showed auxin accumulation in vascular system. Application of auxin induced the lateral root formation in the primary root of ar-C22 mutant. Gene analysis revealed that ar-C22 is a new ahk4/cre1/wol allele known as cytokinin receptor. All AHK genes, AHK2∼4, are ubiquitously expressed in vascular system of hypocotyl and the ahk2ahk3ahk4 triple mutant showed similar root morphology to ar-C22 mutant. These results suggest that retarded vascular system of ar-C22 hypocotyls leads the accumulation of auxin resulting in the adventitious root formation.

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The Ti-plasmid of Agrobacterium tumefaciens has IPT gene, Tmr, on the T-DNA region, which is integrated into the genome of host plants after infection. To understand the cytokinin biosynthesis pathway in Agrobacterium-infected cells, we studied biochemical properties and subcellular localization of Tmr in comparison to Arabidopsis IPTs (AtIPTs). Overexpression of Tmr in Arabidopsis caused predominant accumulation of trans-zeatin (tZ)-type cytokinins, whereas those of AtIPT1, 3, 4, 5 and 7 resulted in accumulation of isopentenyladenine-type ones. Recombinant Tmr was able to utilize both DMAPP and HMBDP as a substrate with similar Km values in vitro, whereas DMAPP was a much better substrate for AtIPTs. Unexpectedly, we have found that Tmr is localized in plastids in the host plant cells in spite of the absence of an apparent transit peptide. These results demonstrate that, after Agrobacterium infection, Tmr is targeted to plastids to produce tZ directly using HMBDP in the MEP pathway.

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It was recently demonstrated that certain histidine kinases (designated as AHK2-AHK4) serve as primary receptors for one of the classical plant hormones, cytokinin. In addition to these cytokinin receptors, Aravidopsis has a number of phosphorelay components (e.g., AHP and ARR), which together constitute a cytokinin-responsive His-to-Asp phosphorelay signaling circuitry. The hallmarked cytokinin-induced genes are ARRs encoding type-A response regulators. Otherwise, nothing is known about downstream components of the cytokinin-mediated phosphorelay circuitry. To identify such cytokinin-associated genes, we carried out extensive microarray analyses by employing not only wild-type plants but also transgenic plants, in which the cytokinin-mediated phosphorelay circuitry was genetically manipulated (e.g., ARR21-C-ox, ARR22-ox). The results of such combinatorial microarray analysis revealed a number of interesting genes, each of which was rapidly upregulated in response to cytokinin through His-to-Asp phosphorelay. By logically explaining the strategy of such combinatorial microarray analysis, the genome-wide list of cytokinin-induced genes will be presented.

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Isopentenyladenine (iP) nucleotides, which is synthesized by isopentenyltransferase (IPT), are hydroxylated to trans-zeatin (tZ) nucleotides by a cytochrome P450 monooxygenase. The cytokinin (CK) hydroxylase had been supposed to be constitutively expressed because of the predominant accumulation of tZ-type CKs in transgenic plants overexpressing bacterial IPT. Isolation and expression analysis of CYP735A1 and CYP735A2 genes encoding CK hydroxylase in Arabidopsis revealed that the expression was organ specific and regulated by phytohormones, such as auxin, ABA and CK. These results suggested that CK metabolism might be regulated at the hydroxylation step. To identify the tissues or cells expressing CK hydroxylase genes, we generated transgenic Arabidopsis lines expressing GUS or GFP gene controlled by the promoter of CYP735As. We will also discuss the possibility of a long-range transport of CKs via xylem vessels comparing expression site of AtIPTs and CYP735As.

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Mitochondria and plastids (organelles) were derived from free-living bacteria. They play the roles as energy converters after division. The mechanism of organelle division is unknown for long time. I discovered that mitochondrial and plastid divisions can be clearly separated into organelle nuclear (nucleoidal) division and organellokinesis (mitochondriokinesis and plastidokinesis). The mechanical apparatus that regulates organellokinesis has remained undetermined. In 1986, the plastid-dividing apparatus (PD ring) for plastidokinesis and in 1993, the mitochondrial-dividing apparatus (MD ring) for mitochondriokinesis were first identified by me in the red algae. Moreover, recent studies (2001, 2003)have revealed that bacterial FtsZ ring forms in stroma whereas dynamin ring functions at the cytosolic or stromal side of the division site. These results showed that the dynamic trio:FtsZ, PD/MD and dynamin rings control the organelle division. The information of 3 genomes in Cyanidioschyzon merolae to be sequenced will help elucidate the fundamental mechanisms of eukaryotic cells.

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