Large variations in k_cat and the CO₂/O₂-specificity ( τ ) of Rubisco exist among enzymes from different photosynthetic species. Among them, Rubisco from higher plants have the lowest k_cat and highest τ and their variations have been small. However, we previously found a large variation in k_cat, and Galmés et al. (2005) also observed a significant variation in τ among higher plants including hemi-deciduous and evergreen trees. In this study, we examined simultaneously k_cat and v_c/v_o at 0.038%CO₂ and 21%O₂, and determined net Rubisco activity per enzyme protein at ambient air [k_cat(1-0.5_o/v_c)] from various plants. Nicotiana tabacum, Populus alba, Fagus crenata, and a few Eucalyptus species showed higher k_cat (50-70% higher than that of rice Rubisco). For v_c/v_o, P. alba, Abies Mariesii and Pinus pumila had 20% higher than rice Rubisco. The k_cat(1-0.5v_o/v_c) was the highest in P. alba, and 1.7-fold greater than that of rice Rubisco.

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The CO₂-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the complex multi-step reaction starting with enolization of ribulose 1,5-bisphosphate (RuBP). The RuBisCO-like protein from Bacillus subtilis (BsRLP) catalyzes the 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase reaction, which resembles the enolization of RuBP. Here we determine the general enzymatic properties of the BsRLP. BsRLP activity required Mg²⁺ for catalysis and was activated by CO₂ as well as RuBisCO. The four residues Lys175, Lys201, Asp203 and Glu204, essential for enolization reaction of RuBisCO were conserved in BsRLP and essential for BsRLP catalysis. The Lys123, the residue conserved in DK-MTP-1-P enolases, was also essential. Interestingly, the substrate, the product and the transition-state analog of RuBisCO (RuBP, phosphoglycerate and 2-carboxy-D-arabinitol-1,5-bisphosphate, respectively) exhibited competitive inhibition with respect to DK-MTP-1-P for BsRLP activity. These results suggest that BsRLP utilizes the same amino acid residues for its enolase reaction in similar ways as does RuBisCO for RuBP enolization.

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Marine diatoms possess a CO₂-sensing mechanism and transcriptional control in response to changes in the ambient [CO₂]. Structure and function of promoter region of a carbonic anhydrase gene in the marine diatom, Phaeodactylum tricornutum (Pptca1) was partially clarified and it has been demonstrated that the critical CO₂-response sequence was located downstream 70bp relative to the transcription-start site. This core-regulatory region comprises two putative cAMP-response elements, CRE1, 2, and a putative p300-binding site and was demonstrated that Pptca1 was under control of cytosolic cAMP in a repressive manner. A series of manipulated constructs of Pptca1 was ligated to the uidA gene and introduced into P. tricornutum. GUS activities of each transformants, grown in 5% CO₂ or air were measured. As a result, a new candidate of cis-element was found. In this talk, data on determination of these critical cis-elements for CO₂ response and their functions will be presented and discussed.

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Marine diatom is the most successful algae, which is responsible for 25% of global primary production. However, CO₂-response in marine diatoms is yet not studied extensively at molecular levels. In this study, in order to understand CO₂-response in marine diatoms, CO₂-responsive genes were screened by cDNA-AFLP (cDNA-amplified fragment length polymorphism) analysis, which compared cDNA populations between those from cells of the marine diatom phaeodactylum tricornutum grown in 0.5 % CO₂ and air. CO₂-responses of 49 cDNA fragments were so far confirmed. Of these, 28 fragments, including some genes which encode Calvin-cycle related protein (phosphoglycerate kinase and transketrase), were repressed in air. On the other hand, the remaining 21 genes, including some genes which encode nitrogen-fixation system related and amino-acid synthesis systems related protein (ferredoxin nitrite reductase and ammonia dependent asparagine synthetase), were induced in air. We also will discuss some homologues of known CCM-related genes in cyanobacteria and green algae.

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In conditions of CO₂-limiting stress, aquatic photosynthetic organisms induce active uptake systems for inorganic carbon (Ci) that enable the accumulation of Ci within the cell (Carbon-Concentrating Mechanism: CCM). In Chlamydomonas reinhardtii, the regulatory factor CCM1 (CIA5) is indispensable for induction of the CCM. Since the CCM1 was detected in fractions with high molecular weigh in vivo, it was possible that the CCM1 may form a complex with other unknown components. To know the molecular components of the putative CCM1 complex, the CCM1 was isolated by Tag-affinity purification method. It has been assumed that the CCM1-A and the CCM1-B were translated in vivo, because two types of the Ccm1 cDNA were isolated. Since the mass spectra derived from the CCM1-A were detected, the CCM1-A was translated in vivo. We discuss about the function of the CCM1 complex based on the findings by mass spectral analysis.

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When CO₂ supply is limited, aquatic photosynthetic organisms induce the carbon-concentrating mechanism (CCM) that facilitates CO₂ uptake to maintain photosynthesis. By using an open gas analysis system, we showed that a multicellular green alga, Volvox carteri, induces the CCM under CO₂-limiting conditions. Although it has been reported that a regulatory factor CCM1 (CrCCM1) is indispensable for induction of the CCM in a unicellular green alga, Chlamydomonas reinhardtii , the structure-function relationship of the CCM1 is poorly understood, because of lack of information on orthologues in other organisms. We have isolated a gene encoding for the CCM1 orthologue from V. carteri (VcCCM1), which shows 51.1% sequence identity with CrCCM1. There were two shared conserved regions: an N- and a C-terminal. Detail analyses focusing on these regions and complementation test using VcCCM1 in the C. reinhardtii ccm1 mutant are to be presented.

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Acclimation to low-CO₂ stress is achieved by monitoring of the environmental changes through putative CO₂-signal transduction pathways. Our focus is to understand how the changes in CO₂ levels are sensed by cells, and what are the roles of low-CO₂ responsive genes. Using cDNA array, we identified a gene, named as LciB, encoding a 48-kDa soluble protein, showed rapid induction during the acclimation process to the low-CO₂ stress conditions. LciB complemented the mutant, pmp1, which shows reduced CO₂-transport activity and high-CO₂ requiring phenotype under low-CO₂ conditions. LciB RNAi strains also showed lower growth rate under low-CO₂ conditions. We examined the subcellular localization of LCIB protein by immuno-histochemistry and also found that LCIB form a complex in vivo using a gel filtration assay. Based on the above observations, it is possible that LCIB protein is associated with unidentified cellular components necessary to efficient carbon transport under low-CO₂ conditions.

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In most aquatic habitats, plants use bicarbonate as a carbon source of photosynthesis, and hydoxyl ions are extruded to the cell exterior, resulting in alkalization of the external medium. In characean cells, hydroxyl ions are extruded at fixed positions, forming so-called alkaline bands. Lucas (1976) studied the effect of external potassium on the bicarbonate incorporation. At 10 m M KCl, bicarbonate incorporation was severely inhibited in most cells. However, it was not inhibited in some cells. In the present study, we analyzed the effect of KCl on alkaline band formation. In most cells, the plasma membrane was suddenly depolarized in the presence of 10 mM KCl. In such cells, formation of alkaline band was severely inhibited. In some cells, however, the membrane remained polarized during the measurement. In such cells, alkaline bands were normally formed. It was concluded that the membrane potential is necessary for alkaline band formation

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The moss Physcomitrella patens is one of useful model organism because genomic information and reverse genetic tools such as gene targeting are available. By using these, we study stem cell formation that traces the process of transdifferentiation from a leaf cell to a pluripotent stem cell as a model of plant totipotency. Although forward genetics using mutants is a powerful approach to identify novel factors, it is difficult to carry out it in P. patens because its genomic physical map and effective method of T-DNA tagging using Agrobacterium have not been established. To identify the factors concerned with stem cell formation in P. patens, we performed screening of the chemicals inhibiting stem cell formation. Using the chemical library including 10000 synthetic compounds with diversity, we selected about 500 compounds as candidates for inhibitors of stem cell formation. To characterize the chemical function, further analyses are undergoing.

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A stem cell is undifferentiated, self-renews, and also gives rise to a cell to be differentiated. Formation of several types of stem cells to produce different types of differentiated cells is properly regulated during development of multicellular organisms. However, molecular mechanisms for the stem cell characterization have been largely unknown.
Here we show that AINTEGUMENTA/PLETHORA/BABY BOOM (APB) orthologs PpAPBs (PpAPB1,2,3 and 4 are involved in the stem cell characterization in the moss Physcomitrella patens. Gametophore stem cells were induced by exogenous cytokinin in the wild type, while the quadruple disruptants did not form any gametophore stem cells with exogenous cytokinin application. These results suggest that the PpAPBs play a critical role in characterization of a gametophore stem cell. Meanwhile, the expression of PpAPBs is regulated by not cytokinin but auxin. We will discuss the roles of PpAPBs, auxin, and cytokinin in stem cell characterization.

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Myo-inositol is required for maintenance of the fundamental composition of cells in all organisms, such as cell wall polysaccharides and membrane phospholipids. Myo-inositol synthesis from glucose 6-phosphate is a two-step process involving conversion of glucose 6-phosphate to inositol 1-phosphate, and dephosphorylation of inositol 1- phosphate to myo-inositol. This last step is catalyzed by an enzyme myo-inositol monophosphatase (IMP). Arabidopsis IMP-like gene family has been reported in the GenBank, but physiological analysis of these enzymes has not been carried out. In this work, we identified three members of IMP-like genes in Arabidopsis genome database with significant amino acid sequence similarity to the known human IMP genes. We report the expression pattern of these AtIMP-like genes in growing plant organs by the analysis with real-time PCR and promoter::GUS transformants. Our results showed possible activities of AtIMP-like proteins in plants.

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Arabidopsis Cdf1 (cell growth defect factor-1) was isolated as a cell death inducer when expressed in yeast (Kawai-Yamada et al, JBC, 2005). In Arabidopsis genome, there are three Cdf-related genes (Cdf1-3). To investigate the cellular localization of Cdf family proteins in plants, GFP-fused Cdf1-3 were expressed in Arabidopsis. Microscopic analysis demonstrated that Cdf1-3-GFP proteins localized on plastid envelope. Furthermore, the analysis of T-DNA inserted plants demonstrated that the heterozygous Cdf1 mutant showed normal vegetative phenotypes under optimal growth conditions, but embryos homozygous for Cdf1 arrested at the globular stage. Such phenotype was complemented by the overexpression of Cdf1. These data indicate that plastid envelope protein Cdf1 is essential during embryogenesis. Furthermore, T-DNA inserted plants in Cdf1-3 demonstrated delayed phenotype in dark-induced leaf senescence. The possible function of Cdf family proteins in embryogenesis and senescence will be presented.

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In Arabidopsis thaliana, CURLY LEAF (CLF) and SWINGER (SWN) are known as Polycomb complex Group (PcG) factor that play key roles by modulating histone methylation. It is suggested that PcG regulate repression of embryonic traits by the fact that clf swn forms embryo-like structure (ELS) after germination, but the molecular mechanisms are still unclear.
We had already reported that the formation of ELS on shoot apical meristem of the clf swn was suppressed by cultivation at low temperature (10ºC). To identify developmental stage for repressing embryonic traits, the clf swn was transferred from 21ºC to 10ºC at various time after sowing. On the other hands, ELS were formed when the clf swn was transferred after the stage of cotyledon expansion, CLS formation was induced as the higher concentration of 2,4-D. These results suggest that auxin should be involved in ELS formation in clf swn at the stage of cotyledon expansion.

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We have identified "master" transcription factors that can induce xylem vessel cell differentiation. In this study, we established a cell differentiation system in which meta-xylem cells were induced synchronously and at high frequency in Arabidopsis suspension cells transformed with VND6, a gene encoding a muster transcription factor. This experimental system is greatly useful for analyzing cellular events such as secondary cell wall development and programmed cell death. Using this system, as an example, we will report on how secondary cell wall patterning is regulated by cytoskeleton in meta-xylem cells.

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The formation and maintenance of tissue structure requires cell-cellcommunication. TDIF, which is a dodeca-peptide belonging to the CLE family, is one of the factors involved in such communication. TDIF inhibits tracheary elements differentiation in Zinnia mesophyll cell culture. As the first step to understand the role of TDIF in vascular development in vivo, we used Arabidopsis thaliana for molecular genetic approach. Because Arabidopsis CLE41 and CLE44 contained TDIF amino acids sequence at the C-terminus, we have examined the expression pattern of CLE41 and CLE44 promoter::GUS fusion genes. In addition, we raised a TDIF-specific antibody and examined localization of TDIF peptide. The results obtained, indicated that specific cells related to the vascular system produced and secreted TDIF.

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The plant vasculature is produced continuously and has well-organized radial pattern, which are governed by cell-cell interactions. We have identified secretary molecules mediating cell-cell interactions using Zinnia xylogenic culture. Of them, TDIF, which was isolated as an inhibitory factor of tracheary element differentiation, is a dodeca-CLE (CLV3/ESR) peptide with two hydroxyproline residues.
To elucidate the in vivo function of TDIF, we performed a detailed analysis using chemically synthesized TDIF. The Arabidopsis seedling grown with TDIF showed abnormal xylem vessel formation, although the entire morphology, the venation pattern, and the phloem formation were not affected. These results suggest that TDIF may be a signaling molecule regulating the plant vascular formation by its specific inhibitory effect on the xylem vessel formation in situ.

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Eucalyptus is a major raw material for paper manufacturing in the world. To improve the fiber properties, we are studying the Eucalyptus transcription factors (TFs). Previously, we reported several TF genes which might be involved in fiber development. EcHB1, is one of the TFs, encodes a HD-ZIP II factor. In this report, we show the results of the characterization of the EcHB1.
To characterize the function of EcHB1, we analyzed the interactions between EcHB1 and promoters of fiber-related genes using FCS method. From this assay, we detected the interactions of EcHB1 and XTH, CCOMT promoters. The expression of XTH and CCOMT genes were up or down-regulated in 35S::EcHB1 transformed Eucalyptus cali. Fiber length of transgenic tobacco harboring 35S::EcHB1 was longer than that of wild type. These results suggest that the EcHB1 is a novel transcription factor related to fiber development.

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The plasmodesmata (PD) regulates the passage of biomolecules between neighboring cells. The primary PD refers to single-stranded, linear entities that are created during cytokinesis, whereas the secondary PD (2˚-PD) have branches or higher-order architectures that are transformed from the primary PD after cytokinesis. The 2˚-PD occurs in the cell wall of the companion cell (CC)-sieve element (SE) in source leaves, regulating sucrose passage into SE. To date, the molecular mechanism of 2˚-PD formation is little understood. We recently isolated restricted sucrose export1 mutants the showed enhanced sugar accumulation and freezing tolerance in the source leaves of Arabidopsis thaliana. rsx1 mutants showed nascent 2˚-PD that is arrested in the middle lamellar of the cell wall between CC and SE. RSX1 encodes a pectate lyase, which hydrolyzes low-esterified pectin. Because pectin forms middle lamellar in the cell wall, we propose that RSX1 assists 2˚-PD formation by punctuating the pectin middle lamellar.

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RSX1 is a pectate lyase (EC 4.2.2.2) that is required for the secondary plasmodesmata formation in the cell wall between companion cells (CC) and sieve elements (SE) in the source leaves of Arabidopsis thaliana. We herein described the enzymatic properties of RSX1, with reference to a crystallographic structure of the putative pectate lyase Jun a 1, a causative cedar pollen allergen. We found that a recombinant RSX1 protein with an N-terminal deletion exhibits pectate lyase activity toward the substrate polygalacturonate. The recombinant protein also exhibited a comparable enzyme activity toward low-esterified pectin and far less activity toward high-esterified pectin. Because pectin is highly methyl-esterified in the middle lamellar of cell walls, our results suggest that a pectin methylesterase should collaborate with RSX1 to make pits through the middle lamellar pectin for the secondary plasmodesmata formation.

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Elicitor-triggered transient membrane potential changes and Ca²⁺ influx through the plasma membrane are thought to be important during defense signaling in plants. However, the molecular bases for the Ca²⁺ influx and its regulation remain largely unknown. We identified a putative voltage-gated Ca²⁺-permeable channel (OsTPC1) in rice, and developed its overexperssors as well as its retrotransposon-insertional functional knockout mutant (Ostpc1). OsTPC1 has been suggested to determine sensitivity to a proteinaceous xylanase elicitor, TvX, and plays a role as a key regulator of TvX-induced defense responses including activation of a MAP kinase and hypersensitive cell death in suspension-cultured cells (Plant J. 2005 42: 798-809). Oligomicroarray transcriptome analyses revealed that TvX-induced expression of genes including those related to defense responses and metabolism including phytoalexin biosynethesis were suppressed in Ostpc1, highlighting the role of OsTPC1 in defense signaling in cultured rice cells.

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Momilactones and phytocassanes are major diterpenoid phytoalexins in rice. In momilactones biosynthesis, two cytochrome P450 monooxigenases (P450s) and a dehydrogenase have been found to be involved and the genes encoding these enzymes are clustered on chromosome 4. Likewise, six P450 genes form a gene cluster on chromosome 2 with the diterpene cyclase genes for phytocassanes biosynthesis, although functions of these P450s have not yet been clarified. Here, we report functional analysis of two P450s, CYP71Z6 and CYP71Z7, of the six P450s encoded by the gene cluster on chromosome 2. As CYP71Z6 and CYP71Z7 are highly homologous to each other, we attempted to generate their double knockdown RNAi lines. The CYP71Z6/CYP71Z7 double knockdown suppressed elicitor-inducible production of phytocassanes A and B, strongly suggesting that either or both of CYP71Z6 and CYP71Z7 are involved in phytocassane A and B biosynthesis.

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Jasmonic acid (JA) is a plant hormone that acts as a secondary signaling molecule in plant defense responses. RERJ1 that encodes a bHLH transcription factor was isolated as a JA-responsive gene from rice suspension cells. Previously, we reported that RERJ1 functions as a transcriptional activator and the expression of OsChia4a, which encodes class 4 chitinase, was induced in a RERJ1-dependent manner.
Here, we identified a JA-responsive cis element in the promoter region of OsChia4a by reporter-gene assay. The deletion analysis showed that the region from -515 to -265 bp in the promoter contains JA-responsiveness, and we found that the region contains three E-boxes. Subsequent mutation analysis of three E-boxes indicated that an E-box at -448 to -443 bp is essential for responding to JA and also for RERJ1-dependent expression of OsChia4a. Now, we are analyzing the binding of RERJ1 to this E-box by chromatin immunoprecipitation.

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The plant hormone jasmonic acid (JA) is known to have an important role as a signaling transducer in elicitor-inducible defense responses in rice. To date, it has been shown that a F-box protein COI1 functions in a central part of a JA signaling in some plants; however, in rice a molecular basis mechanism of the JA signaling remains unknown. In this report, we demonstrate that rice OsCOI1 homologs function in the JA signaling pathway by analyzing OsCOI1-reduced RNAi lines. In these lines, RERJ1, which is a JA-responsive bHLH transcription factor gene, was not induced its expression in response to exogenously applied JA. This result suggests that OsCOI1 plays an important role in JA signaling of rice defense responses. Expression profiles of other JA-responsive genes, phytoalexins production, and phenotypic observations in these OsCOI1 RNAi plants are under examination.

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Most resistance (R) genes encode nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins, which act as intracellular receptors for pathogen signals. However, it is largely unknown how NBS-LRR resistance proteins transduce pathogen signals to their downstream effectors and thereby execute innate immunity in plants. We have recently found that the small GTPase OsRac1 is a molecular switch in innate immunity of rice. Therefore, we searched for OsRac1-interacting molecules by affinity column chromatography, identified five NBS-LRR-like proteins, and named them OsRac1-interacting NBS-LRR-like protein (Orin) 1-5. Orin1 preferentially bound to the GTP form of OsRac1 through its NBS domain. Agroinfiltration of the wild type Orin1 induced the hypersensitive response (HR) in Nicotiana benthamiana, whereas introduction of a dominant negative form of OsRac1 suppressed Orin1-induced HR. These results suggest that OsRac1 forms a complex with NBS-LRR-type R proteins and acts as an effector of NBS-LRR in innate immune responses.

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In plants, perception of pathogen-associated molecule patterns (PAMPs) triggers innate immune responses that contribute to disease resistance. Previously, we showed that the small GTPase OsRac1 play key roles in defense signaling of rice. Overexpression of the constitutively active form of OsRac1 enhances reactive oxygen species production in rice cells treated by N-acetylchitooligosaccharide elicitor, a PAMP derived from rice blast fungus. Here we report the development of an intracellular fluorescence resonance energy transfer (FRET) sensor that can detect the activation of OsRac1 by N-acetylchitooligosaccharide elicitor in vivo . The FRET biosensor is composed of OsRac1, the CRIB domain of PAK1, which binds specifically to the GTP-bound form of Rac, and variants of the green fluorescent protein, Venus and SECFP, as FRET donor and acceptor, respectively. This biosensor would facilitate identification of the upstream regulators of OsRac1, such as guanine exchange factors (GEFs) and real-time monitoring of PAMP-induced defense responses.

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Basal and R gene-mediated resistance are two systems for plant defense against pathogen attacks. Rac GTPase, RAR1, SGT1 and Hsp90 have been shown to be important components of defense signaling. To further understand the relationship and context of important components, we identified and characterized Sti1/Hop and RWD as novel players in defense signaling. We found that Sti1-knockdown plants were compromised to virulent fungus, while Sti1-overexpressing plants became more resistant. R gene-mediated pathway was also affected by the loss of Sti1. Our data showed Sti1 interacted with Hsp90, and RWD interacted with SGT1 and RAR1, while Rac1 interacted with Sti1 and RWD. These results indicate the existence of an OsRac1-containing protein complex. We hypothesize that defense components may form a dynamic complex termed as 'defensome'. Defensome may be required for both basal and R gene-mediated resistance and its composition may vary depend on different triggers and signaling stages.

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OsPti1a negatively regulates both R-gene mediated and basal defense responses in rice. Cultured cells of ospti1a mutant are highly sensitive to H₂O₂ treatment, suggesting that OsPti1a may negatively regulate ROS signal pathway. Pti1a-interacting protein 1 (Pip1) that encodes AGC kinase interacts with OsPti1a and Pdk1, a regulator of AGC kinase. In vitro kinase assay showed that Pip1 phosphorylated OsPti1a and was phosphorylated by Pdk1. Therefore, the phosphorylation relay Pdk1-Pip1-OsPti1a is assumed to be involved in defense signaling. To investigate the function of Pip1 and Pdk1 in defense response, we constructed overexpressed lines of Pip1 and Pdk1. Cultured cells of Pip1-OE are highly sensitive to H₂O₂ treatment as well as ospti1a mutant. Disease susceptibility to compatible race of Magnaporthe grisea was decreased in both Pip1-OE and Pdk1-OE as compared with WT. These results suggest that a negative regulator OsPti1a is negatively regulated by Pip1-Pdk1 signaling in disease resistance.

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ER body is one of ER-related organelles that highly accumulate a specific protein, PYK10, a β-glucosidase with an ER-retention signal. Functions of ER bodies are still unclear. ER bodies have a limited distribution in Arabidopsis seedlings. They are present in the epidermal cells of cotyledons and hypocotyls (constitutive ER bodies) of young seedlings, and disappear in those of mature tissues. When mature leaves are wounded, ER bodies are induced around the wound site of the leaves (inducible ER bodies). Therefore, the induction of ER bodies might be involved in self-defense of the plants. To clarify the function of ER bodies, we compared constitutive ER bodies with inducible ER bodies. Numbers of ER bodies increased not only in local wounded cotyledons but also in the systemic cotyledons after wounding. We report here QPCR analysis for components of constitutive and inducible ER bodies and then discussed their possible functions.

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We focus on the leaf-variegated mutant var2 of Arabidopsis, to study the molecular mechanism leading to leaf variegation. We previously reported that the green sectors of the var2 variegated leaves accumulated high ROS under a normal growth condition. It was hypothesized that these ROS directly and/or indirectly play roles in an antibacterial activity. To test this hypothesis, we inoculated Pst DC3000 expressing GFP onto var2 leaves. At an early stage of inoculation, GFP was predominantly detected in white sectors but not in green sectors, likely due to the ROS accumulation in the green sectors. Bacterial counts in leaf extracts, based on the measurement of colonization, showed that var2 leaves were more tolerant to Pst DC3000 than the wild-type leaves. Thus, we present a possibility that although the mutation in var2 is defective in photosynthesis, the resulting ROS accumulation may be advantageous for a biotic stress.

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Striga spp. (witchweeds) are major agricultural pests which infest important crops in sub-Saharan Africa and parts of Asia. Striga germinates in response to host-derived signal compounds, Strigolactons, and forms an attachment organ, haustorium. Striga haustorium penetrates into a host root and makes host-parasite vasculature connections, which allow the parasites to suck water and nutrients up from the host roots.
Striga spp. have broad host ranges. S. hermonthica infests to Gramineous plants including rice, maize and sorghum but not to dicotyledonous plants. To understand host recognition mechanisms of S. hermonthica, we investigated its interaction with host and non-host plants. Although Arabidopsis and cowpea are non-hosts, S. hermonthica haustorium is able to reach to their steles, while Lotus japonicus blocks Striga infection at the cortical cell layer. Our histological analysis suggests possible 4 types of non-host interaction of S. hermonthica.
We will also introduce Expressed sequence tag (EST) project of S. hermonthica.

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Plants are exposed to many types of abiotic or biotic stresses. Many researchers have analyzed the mechanism of these stress responses using Arabidopsis plants. At the present day, creation of plants having tolerance for several abiotic or biotic stresses has reported. On the other hand, insect damage is very serious problem that decrease the crop yields. However, the mechanism of plant response to feeding damage has not been well understood.
We analyzed the interaction between Arabidopsis and western flower thrips (Frankliniella occidentalis), which is one of the most serious insect pests. Thrips is cell content feeding insect that penetrate single cells with stylet to suck out the contents. In addition, thrips transmit the virus from plant to plant. We focused on the function of the immunity-related plant hormones jasmonate , ethylene, and salicylic acid. We report here the function of these hormones in plant response to thrips feeding.

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To identify useful rice gene, we are established rice FOX lines using 13,000 non-redundant rice full-length cDNAs to introduce them into Arabidopsis plants.
To isolate useful rice genes involved in tolerance to UV-B stress, which cause to several damages to plants, we tried to screen UV-B-tolerant mutants from rice FOX lines.
We already isolated 49 UV-B-tolerant mutant candidates in T2-generation from ca. 7,000 lines by root bending assay screening. The isolated 50 cDNAs were retransformed into Arabidopsis. And we confirmed the phenotypes of 28 T2-retransformed lines. So far 15 T2-retransformed lines showed long root phenotype regardless UV-B irradiation. These lines contained unknown function genes or stress-signal related genes (CDPK and CIPK). One T2-retransformed line, containing Auxin-regulated gene, showed an altered UV-B response at bending of root.
This research is supported by the Special coordination funds for Promoting Science and Technology entitled Rapid identification of useful traits using rice full-length cDNAs.

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Rice cultivars vary widely in their UVB sensitivity and this has depended on CPD photolyase mutations. It is well known that wild rice species have a high degree of genetic variation, making them important reservoirs of potentially useful genes. In order to search useful gene of CPD photolyase with higher activity, we investigated the UVB-sensitivity in two cultivated species (O. sativa and O. glaberrima) and three wild rice species with AA genome (O. barthii, O. meridionalis and O. rufipogon) focusing on the CPD photolyase activity and genotype of CPD photolyase. As a result, although the UVB sensitivity, the CPD photolyase activity and genotype of the CPD photolyase varied widely among these rice species, the sensitivity to UVB radiation depends on the activity of CPD photolyase. The CPD photolyase activity greatly depends on amino acid residue at position of 126 and 296.

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UVB can damage DNA by causing formation of cyclobutane pyrimidine dimer (CPD). CPD photolyase, which mediates CPD repair path, is one of crucial factor for determing UVB sensitivity in rice. Although plant cell has different genome in nuclei, chloroplast and mitochondria, it is unclear whether CPD photolyase functions in each organelle. We previously reported that UVB-induced CPD levels on each organelle genome decreased by exposure of the blue irradiation in rice leaves. These results suggest that CPD photolyase transits and functions in nuclei, chloroplast and mitochondria.
In order to clarify the localization of CPD photolyase in plant cell, in this study, we performed a western blot analysis using anti-rice CPD photolyase antiserum, and analysis of the cellular targeting of CPD photolyase-GFP fusion protein. As a result, CPD photolyase bands were detected in crude extract prepared from each purified organelle. Furthermore, GFP fluorescence was detected in nuclei and mitochondria.

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CPD photolyase is a crucial factor for determining the sensitivity of plant to UVB. CPD photolyases classified into two classes, class I and class II, based on their similarity of amino acid sequence. Class I CPD photolyase has been studied well. However, little is known about class II CPD photolyase. To characterize rice class II CPD photolyase, the enzyme from rice leaves was purified. SDS-PAGE of the purified enzyme showed existence of two proteins of about 54- and 56-kDa, whereas rice CPD photolyase expressed from E. coli was a single 55-kDa protein. Western blot analysis using anti-rice CPD photolyase antibody showed that both proteins were CPD photolyase. Treatment with protein phosphatase revealed that the 56-kDa native rice CPD photolyase was phosphorylated, whereas the E. coli-expressed rice CPD photolyase was not. Furthermore, the purified native rice CPD photolyase had significantly higher CPD photorepair activity than the E. coli-expressed rice CPD photolyase.

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UV damages the plants by targeting the DNA, photosynthetic apparatus, a wide variety of physiological processes and generation of reactive oxygen species (ROS). Our previous study using tobacco cell cultures (Nicotiana tabacum L.; cell line, Bel-B and Bel-W3), involvements of ROS and calcium signaling in UV-C-dependent cell death induction were suggested. Here, we examined the impact of UV-C irradiation on the onset of programmed cell death mechanism in cell suspension cultures of Arabidopsis thaliana (Ecotype, Columbia). Data indicated that UV-C induces the cell death through the action of induced ROS. Further analysis with several mutants and transgenic cell lines suggested that salicylic acid signal transduction pathway is also involved.

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Regulatory mechanisms of stress responses in rice largely remain unclear. Using rice microarray, we identified many abiotic stress-responsive genes. Among them, a gene for a bHLH transcription factor down-regulated by drought stress has been studied. The bHLH protein showed a high sequence homology with Arabidopsis PIF1, driving us to name the transcription factor OsPIF1. Overexpression of OsPIF1 in transgenic rice promoted internode elongation. In contrast, dominant loss-of-function rice mutants with a chimeric repressor resulted in short length of the internode sections. In a transient experiment, the transcription of a GUS reporter gene driven by the promoter containing G-box elements was activated by OsPIF1, suggesting that OsPIF1 acts as a transcriptional activator. These data suggest that OsPIF1 functions as an important regulatory factor of plant growth in response to drought stress. Currently, we are analyzing down-stream genes of OsPIF1 by using 44k rice oligoarray with OsPIF1 transgenic rice plants.

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In higher plants, calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs) represent important relays in calcium signaling. We have found that Arabidopsis cbl1/cipk1 double mutant plants exhibit hypersensitive hypocotyl responses specifically to red and blue light. Expression analyses revealed reduced expression of the light-inducible genes CHS and CAB3 in cbl1/cipk1 mutant plants. To further elucidate the pathway in which CBL1 and CIPK1 function, we focused on HYPERSENSITIVE TO RED AND BLUE 1 (HRB1) because it has been reported that hrb1 mutant plants show similar hypocotyl responses as the cbl1/cipk1 plants to both red and blue light. HRB1 has been also shown to function in cryptochrome and phytochrome signaling pathways. Interestingly, BiFC analyses showed molecular interaction between CIPK1 and HRB1 in planta. These results suggest that CBL1 and CIPK1 function in the light signaling pathway.

This work is supported by the Alexander von Humboldt Foundation and the DFG (AFGN).

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BLUF domain is a flavin-binding domain that functions as a blue light. The structure of BLUF-domain protein TePixD Thermosynechococcus elongatus BP-1 was reported by Kita et al. [1]. Upon illumination, the absorption band of flavin shows the 10-20 nm red shift. Further illumination induced FAD radical [2]. The illumination of the TePixD at 80-150 K was found to induce a new EPR signal at g=2 region, which was composed of the main two lines separated by 85 gauss, estimated as 6.9 A of the inter-distance. Pulsed ENDOR results suggested that the signal involves the falvosemiquinone and tyrosine neutral radicals. It can be interpreted by the interaction between flavosemiquinone and Y8 neutral radical.
[1] Kita et al., J. Mol. Biol., 2005, 349, 1-9.
[2]Fukushima et al. Biochemistry, 2007, in press

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A cyanobacteriochrome CcaS is known to regulate the expression of phycobilisome linker gene (cpcG2) in Synechocystis sp. PCC 6803. We previously reported that CcaS shows the reversible photoconversion between a green-absorbing form (Pg) and a red-absorbing form (Pr). In this study, mass spectrometry and denaturation analysis revealed that CcaS binds PCB in a configuration of ZZZ and ZZE for Pg and Pr, respectively. Autophosphorylation of histidine kinase of nearly full-length CcaS was up-regulated by preillumination with green light. Similarly, phosphotransfer to the cognate response regulator CcaR was higher in the Pr form than Pg. From these results, we concluded that green-light activated phosphorylation from CcaS to CcaR induces the expression of cpcG2, leading to accumulate CpcG2-phycobilisome. We will discuss about the universality of the CcaS system, which is also found in Nostoc punctiforme PCC 73102.

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Cyanobacteria harbor GAF-containing photoreceptors that may bind a linear tetrapyrrole as a chromophore (cyanobacteriochrome). Previously, we reported the properties of the GAF domain of TePixJ of Thermosynechococcus elongatus that was expressed in Synechocystis. Purified holoprotein showed reversible photoconversion between the 433nm and 531nm-absorbing forms. Denaturation analysis showed that the chromophore of TePixJ is phycoviolobilin. Here, we report reconstitution of TePixJ_GAF apoprotein with synthetic phycocyanobilin. In the beginning of reconstitution, free phycocyanobilin was covalently bound to the apoprotein with concomitant formation of photoactive holoprotein between 430 and 545 nm-absorbing forms. Extensive incubation resulted in conversion to phycoviolobilin which shows photoconversion between 434 and 532 nm in holoprotein. These suggest that phycocyanobilin initially incorporated into TePixJ_GAF is distorted, leading to auto-isomerization to phycoviolobilin. Thus, we can conclude that the GAF domain of TePixJ has a triple function: lyase, isomerase and photoconversion.

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Phytochromes are red and far-red light receptors in plants. Cyanobacteriochromes, which are distantly related to phytochromes and present only in cyanobacteria, show various spectral properties. Of these, the putative phototactic photoreceptor, AnPixJ, covalently binds phycocyanobilin and shows reversible photoconversion between green-absorbing (Pg) and red-absorbing (Pr) forms. It is suggested that unorthodox green-red reversible photoconversion of AnPixJ undergoes the same Z/E isomerization as phytochromes. In this study, we obtained blue crystals of Pr form of AnPixJ by hanging-drop vapor diffusion method. The crystal structure was solved at 1.8 Å resolution by SAD method using anomalous scattering of iodide. The configuration of chromophore of AnPixJ is quite similar to that of bacteriophytochrome, although the local environment surrounding chromophore of AnPixJ is clearly distinct from that of bacteriophytochrome. This is consistent with the previous spectral studies. Possible photoconversion mechanism from phytochrome-like Pr form to unusual Pg form will be discussed.

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To better understand the functions of phytochrome, we used the liverwort Marchantia polymorpha, which is haploid and has a simple body structure, as a unique model plant. We isolated a phytochrome gene in M. polymorpha, MpPHY. Southern-blot analysis suggested that MpPHY is a single-copy gene, whereas higher plants carry two or more copies of phytochrome gene. Mpphy(N612) showed Red/Far-Red photoreversibility typical of functional phytochrome in vitro. A single amino acid substitution mutant of Mpphy(N612), MpphyY241H(N612), exhibited no photoreversibility. In transgenic plants expressing MpphyY241H, formation of rhizoid was promoted regardless of light condition, and the blue light response was supressed. The MpphyY241H-GFP fusion protein localized in the nucleus forming speckles. These results indicate that MpphyY241H functions as a constitutively active form in M. polymorpha, and Mpphy controls photomorphogeneses.

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We previously demonstrated interactions among peroxisomal biogenesis factors, Pex5p, Pex7p and Pex14p, peroxisomal proteins with two targeting signals, PTS1 and PTS2, in Arabidopsis thaliana using yeast two-hybrid analysis. In the present study, we introduced bimolecular fluorescence complementation (BiFC) assay using split yellow fluorescent protein (YFP) to visualize the interactions between these proteins in living plant cells. When nYFP-PTS1/PTS2-cYFP or nYFP-Pex7p/PTS2-cYFP fusion proteins were expressed transiently, onion epidermal cells showed punctate green fluorescent signals that were co-localized with tdTomato-PTS1. The result indicates that PTS1-PTS2 and Pex7p-PTS2 interacted with each other in vivo in the peroxisomes On the other hand, when cYFP-Pex5p and nYFP-Pex7p were expressed together, onion epidermal cells showed cytosolic green fluorescent signal, suggesting that Pex5p and Pex7p interacted with each other in the cytosol. In this report, we propose a revised model for the import of PTS-containing matrix proteins.

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Oil body is an organelle abundantly found in cells of fatty seed. This organelle has simple structure of having large quantity of triacylglycerol as seed reserved lipid surrounded by a monolayer membrane of phospholipid. It has been suggested that oil body is produced from specific domain of endoplasmic reticulum. However, detailed mechanism of oil body formation is still obscure. In this study, we tried to identify and characterize mutants containing oil body with aberrant morphology to understand mechanism of oil body formation at molecular level. We will describe phenotype of one of the mutants and discuss function of the mutated gene in relation to mechanism of oil body formation during embryogenesis.

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Arabidopsis seeds contain a lot of oil bodies, which are surrounded by a lipid monolayer and store lipids, mainly triacylglycerols, as nutrient sources for seed germination. Oil bodies possess oleosins as major structural proteins. Oleosins are suggested to have an important role of determination of oil-body sizes. To investigate a physiological function of oleosins, we isolated three oleosin-deficient mutants of Arabidopsis thaliana (ole1, ole2 and ole3) and generated double mutants by crossing single mutants. The plants and seeds of these mutants exhibited no visible phenotypes under normal growth condition. Each of single mutant exhibited a similar germination rate to ewild tyoe, , while the double mutant ole1 ole2 exhibited clearly reduced germination rates Electron microscopy revealed that the ole1 ole2 seeds had irregularly-shaped oil-containing structures. We suppose that these structures might inhibit seed germination of ole1 ole2. We will discuss about the effect of oleosin-deficiency on the seed germination.

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Nucleus is a highly heterogeneous structure, which is composed of double layered nuclear membrane, nuclear pore and nuclear matrix. Since nuclear architecture provides a physical framework for the genetic activities, it is important to know the mechanisms in which structure of the nucleus is built. We are taking two approaches to investigate the function of plant nucleus. First, we are trying to do proteomics analysis of plant nucleus to isolate structural proteins, which contribute building nucleus up. Isolated nucleus from Arabidopsis cultured cells were sequentially washed with high-salt, alkaline, and detergent-containing buffer for obtaining insoluble fraction in which structural proteins are expected to localize. Isolated proteins are being subjected to mass spectrometrical analysis to identify their sequences. Second, to visualize the dynamics of plant nuclear pore complex, we expressed chimeric genes encoding nucleoporin fused with fluorescent proteins in plant cells.

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Organellar DNA degradation in plants is sensitive to the physiological state such as developmental stage and senescence. However, the underlying mechanism is unknown so far. Angiosperm pollen is a good experimental system because organellar DNA is degraded dramatically during pollen development. We performed screening to isolate mutants defective in organellar DNA degradation. Pollens from a mutagenized population were stained with DNA-specific fluorescent dye, DAPI, and observed using a fluorescent microscope. Wild-type pollen showed DAPI signals from the nuclei of vegetative and sperm cells, while mutant candidates exhibited additional signals in the cytoplasm. Among the mutant candidates was the 1411 mutant that also showed additional variegation phenotype. The responsible gene of the 1411 mutant encoded the large subunit of the ribonucleotide reductase (RNR) enzyme. The enzyme ensures adequate and balanced deoxyribonucleotide pool. Characterization of pollens and variegation phenotypes in several RNR mutants including 1411 will be presented.

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In bacterial cells, RelA/SpoT catalyzes synthesis or degradation of guanosine 3',5'-bispyrophosphate (ppGpp) in response to environmental stresses such as amino acid starvation. ppGpp controls transcription of a large number of genes through direct interaction with RNA polymerase. This process which regulates gene expression is called stringent response. Recently a growing number of RelA/SpoT homologues, designated RSH, has been identified in plants. To reveal the role of stringent response in plant cells, we are investigating four RSHs (RSH1,2,3 and CRSH) in Arabidopsis.
As a result of promoter analysis, the four RSH genes of Arabidopsis were shown to express in green tissues and flowers. In addition, Arabidopsis CRSH knockdown mutant had defects in reproduction stage, which implies the involvement of the CRSH in seed formation. Besides, we observed induction of RSH2 mRNA expression in response to abiotic stresses. These results show that stringent response has an important role in higher plants' physiology.

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Although chloroplasts contain high concentrations (4-23 mM) of total Ca²⁺, stroma free Ca²⁺ concentration is maintained at sub-micro M. Sai and Johnson (2002 Plant Cell) previously demonstrated that the stromal Ca²⁺ levels were transiently elevated in response to the light-to-dark transition. Interestingly, dark-induced stromal Ca²⁺ elevation precedes the generation of cytosolic Ca²⁺ transients, suggesting the presence of Ca²⁺ communication between chloroplasts and cytoplasm. However, the underlying molecular mechanisms are largely unknown. We recently demonstrated that thylakoid membrane-localized CAS is involved in external Ca²⁺-induced cytosolic Ca²⁺ elevation and stomatal closure (Nomura et al., 2007 Plant J). Here we provide evidence that CAS is essential for dark-induced stromal Ca²⁺ elevation in chloroplasts. These results suggest that thylakoid-localized CAS plays a crucial role in the regulation of cytoplasmic Ca²⁺ levels by chloroplasts.

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