Seed storage proteins are synthesized on the endoplasmic reticulum (ER) as precursors and then transported to protein storage vacuoles, where they are processed into mature forms. Here, we isolated and characterized an Arabidopsis mutant, maigo5 (mag5), that accumulated the precursors of storage proteins in dry seeds. mag5 seed cells developed many novel structures with high electron-dense cores. Immunoelectron microscopy revealed that these structures are MAG bodies previously found in the seeds of mag2 (1) and mag4 (2). Because MAG bodies are derived from the ER, we considered that mag5 mutant has a defect in storage proteins exit from the ER. We used map-based cloning and identified the MAG5 gene, which encodes an unknown protein. Both MAG2 and MAG4 are involved in the protein transport between ER and Golgi body: MAG2 is necessary for retrograde transport, while MAG4 is necessary for anterograde transport. Our findings suggest that MAG5 is required for intracellular transport between ER-Golgi body.
(1) Li L. et.al, Plant Cell 2006;18(12):3535-3547.
(2) Takahashi H. et.al, Plant Cell Physiol 2010;51(10):1777-1787.

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GEF (Guanine nucleotide Exchange Factor) participates in vesicular transport. Rice glutelin is synthesized on endoplasmic reticulum as a precursor, which is transported via Golgi to protein storage vacuole. In glup6 mutation for the gene encoding GEF, the proglutelin is accumulated in the endosperm. But the function of GEF to the intracellular transport of proglutelin is not clear. Immunofluorescence microscopy of the glup6 endosperm demonstrated that the novel structure including proglutelin was observed in glup6 endosperm. This structure included also β-glucan, which is synthesized at the Golgi apparatus. Confocal microscopy of glup6 endosperm indicated that the structure included the membrane protein of the prevacuolar compartment and the Golgi. These results suggest that the proglutelin trafficking from the Golgi is significantly distorted in glup6. The granules including proglutelin were observed within the cell wall in early developing stage, while the granules were disappeared in late developing stage.
On a conclusion, GEF functions in the formation of endomembrane system in rice endosperm and participates in intracellular transport of proglutelin to PSV.

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Rab GTPases regulate the intracellular membrane trafficking events and are activated by Guanine nucleotide exchange factor (GEFs). The trafficking of rice glutelin involves membrane traffic pathway, which has been mainly described as that glutelins are initially synthesized on the endoplasmic reticulum as 57-KD precursor, then transported via the Golgi apparatus to protein storage vacuole (PSV), proteolytically cleaved and accumulate in PSV. In our previous study, both of glup4 and glup6 mutants for Rab5a and GEF, respectively, showed the novel structure containing the proglutelin in the endosperm. The immune precipitation analysis of the Glup6-GEF and Glup4-Rab5 proteins revealed that both proteins were interacted each other, suggesting that the Glup6-GEF activates Glup4-Rab5. The microscopy analysis of the endosperm in double recessive type of glup4 and glup6 mutants showed more severe novel structure containing the proglutelins than each single mutant. These results suggest the possibility that the Glup6-GEF activates also the other Rab proteins and the Glup4-Rab5a is activated also by the other GEF proteins.

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Recent studies reveal that endocytosis is important for a variety of plant functions. However the underlying molecular mechanisms and the significance in plant development still remain largely unknown. Rab5, a subfamily of Rab GTPases, regulates a variety of endosomal functions as a molecular switch and key regulator of endocytosis. Mammalian Rab5 GTPase is known to regulate not only endosomal fusion but also signaling through endosomes. Rab GTPases are activated by specific guanine nucleotide exchange factors (GEFs), which accelerate the exchange of GDP for GTP. Vps9 domain, a catalytic core for activation of Rab5, is conserved in all Rab5 GEFs identified thus far. We have already demonstrated that the Arabidopsis VPS9a can activate all of three Arabidopsis Rab5 members (ARA6, ARA7, and RHA1). VPS9a comprises the conserved VPS9 domain at the N-terminus and a C-terminal region with no similarity to known domains. We found that truncation of the C-terminal region of VPS9a confers an increased GEF activity toward ARA6. Detailed functional analysis of the C-terminal region of VPS9a is now underway.

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Post-Golgi organelles play fundamental roles in various plant functions of higher order. However, our knowledge on molecular mechanisms of their biogenesis and trafficking system interconnecting them is still limited. We are studying molecular mechanisms of endocytic and vacuolar transport pathways with a special focus on plant-unique RAB and SNARE molecules. A. thaliana has two types of RAB5 members, conventional RAB5 and plant unique ARA6. Distinct subcellular localization of ARA6 from conventional RAB5 suggested functional differentiation between these two RAB5 groups (Ueda et al., 2004). On the other hand, VAMP727 is a plant-unique R-SNARE, which is characterized by an insertion of 20 amino acids in its N-terminal longin domain. We have already reported that VAMP727 forms a SNARE complex with VAM3, VTI11, and SYP51 on a subpopulation of PVCs closely associated with the vacuolar membrane (Ebine et al., 2008). In this meeting, we will report our recent results on a regulatory role of ARA6 in VAMP727 complex formation, and a role of ARA6 in environmental stress responses.

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RAB GTPases, including RAB5, are known to act as molecular switches through cycling between GTP-bound and GDP-bound states. The GTP-bound state is considered as an active form, with which effector molecules specifically interact to induce downstream reaction. In mammalian cells, many RAB5 effectors are known to trigger various downstream phenomena. RAB5 is conserved in a broad range of eukaryotic organisms including plants. In addition to the conventional-type RAB5 homologs, land plants possess plant-unique RAB5 homologs (ARA6 in Arabidopsis thaliana) (Ueda et al., 2001). Our previous studies indicated that ARA6 and conventional-type RAB5 regulate different membrane trafficking pathways, which could be integrated by RAB5 effector molecules. However, plant RAB5 effectors have not identified thus far. To elucidate the molecular basis of RAB5-regulated trafficking pathways, we attempted to isolate effector molecules of ARA6. By yeast two-hybrid screening, we have identified seven effector candidates. In this meeting, cell biological and physiological significance of these effectors will be presented.

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SNARE molecules are key regulators of vesicular traffic, which execute membrane fusion between transport vesicles/donor organelles and target organelles. These molecules are classified into four subtypes (Qa-, Qb-, Qc- and R-SNARE) according to their structure. One of major plant R-SNARE families, VAMP7, consists of two subgroups, VAMP71 and VAMP72. In seed plants, structurally distinct VAMP72 molecules harboring an insertion comprising ~20 amino acids in its N-terminal longin domain are also conserved. We previously reported that this type of VAMP72 molecule in A. thaliana, VAMP727, is involved in membrane trafficking around endosomes. We further indicated that the inserted sequence in the longin domain is essential for VAMP727 function. To investigate how plants acquired VAMP72 molecules with the insertion during evolution, we are studying VAMP72 members in basal plant lineages including the liverwort, Marchantia polymorpha. In this meeting, we are reporting results of functional analyses of VAMP72 members in M. polymorpha.

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SNARE proteins, evolutionarily conserved membrane fusion proteins, are classified into four subgroups (Qa-, Qb-, Qc-, and R-SNAREs). R-SNAREs are further divided into two classes: longins with the N-terminal longin domain and brevins lacking the longin domain. Longins are also classified into three subgroups, SEC22, YKT6, and VAMP7. Plants possess only longins in their genomes; A. thaliana harbors 18 R-SNAREs, 12 of which belong to the VAMP7 group consisting of VAMP71 and VAMP72 members. While functions of several VAMP72 members have been already reported, precise functions of the VAMP71 group remain totally unknown. We have already demonstrated that only VAMP714 of VAMP71 members is localized in the Golgi when expressed in protoplasts, while other VAMP71 members are localized on the vacuolar membrane. In this study, we isolated VAMP714 homologs from several land plant lineages for information on diversity of plant VAMP71 members, and compared subcellular localization of their products. We will discuss the possible mechanism of functional diversification of the VAMP71 group during evolution.

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SYP2 proteins are a subfamily of Qa-SNAREs that are possibly responsible for protein trafficking between prevacuolar compartments (PVC) and vacuoles. Arabidopsis thaliana SYP22/VAM3/SGR3 and SYP21/PEP12 proteins function independently, and are yet both reported to be essential for male gametophytic viability. Here, we systematically examined the redundancy of three SYP2 paralogues (i.e., SYP21, 22, and 23). Surprisingly, no visible phenotypes were observed, even in the double knockout syp21/pep12 syp23/plp. A deficiency of either SYP21/PEP12 or SYP23/PLP in a syp22 background resulted in a defect in vacuolar protein sorting. A GFP-SYP23/PLP fusion localized to the cytosol. The immunoprecipitation analysis showed that SYP23/PLP interacted with vacuolar Qb- and Qc-SNAREs, VTI11 and SYP5, respectively, suggesting that SYP23/PLP is able to form a SNARE complex anchoring the membrane. Thus, SYP2 proteins, including cytosolic SYP23/PLP, appear to function redundantly in vacuolar trafficking and plant development.
(1) Shirakawa, M. et al, Plant Cell Physiol., 50, 1319-1328 (2009).
(2) Shirakawa, M. et al, The Plant Journal., in press.

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Clathrin is a coat protein of a clathrin-coated vesicle (CCV), which sorts cargo proteins into various endosomes from trans-Golgi network and plasma membrane in mammalian, yeast and plant cells. The clathrin coat is composed of a microscopically visible structure with tree-legged shape, called triskelion. This triskelion comprises three clathrin heavy chains (CHCs), each harboring a single clathrin light chain (CLC). CHCs are well conserved among eukaryotic cells while CLCs vary in similarity, suggesting the latter may have an organism-specific function, but an interaction between CLC and CHC in Arabidopsis thaliana has not been analyzed in detail. In this study, the interaction of both types of Arabidopsis clathrin molecules was analyzed. Yeast two-hybrid and bimolecular fluorescence complementation analyses revealed Arabidopsis CLC actually interacted with CHC. Deletion analysis of CLC (1-258) showed an internal region of CLC (82-144) interacted with hub of CHC (1086-1705), suggesting the similar tendency of interaction of plant clathrin chains to that in mammalian cells. The analysis of the interaction of Arabidopsis CLC and CHC molecules will be discussed in this presentation,

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The endoplasmic reticulum (ER) is the site of protein synthesis in the eukaryotic secretory pathway. The ER possesses an elaborate quality control system that recognizes and eliminates aberrant proteins generated in the ER. An ER-resident Hsp40, ERdj3, plays important roles in the ER quality control as a partner for BiP (Hsp70 in the ER) in mammalian cells. We identified AtERdj3B as an ortholog of mammalian ERdj3 and found that the knock out mutants of AtERdj3B became sterile when they were grown at elevated temperature. Analyses of the aterdj3b mutants showed that defects in pollen release from the mutant anthers caused the sterility. Transmission electron micrographs showed deposition of aberrant materials related to the pollen coat on the surface of pollen grains, which resulted in tight adhesion of pollen grains to the mutant anthers. Analyses using an AtERDJ3B_pro:GUS construct revealed that AtERdj3B is highly expressed in tapetum cells, which play key functions in pollen coat production. We found that tapetum specific expression of AtERdj3B suppressed the sterility of the aterdj3b mutants. These results suggest importance of the ER quality control system in tapetum functions.

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PtdIns 3, 5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P2 from PtdIns 3-P, and functions in vacuole/lysosome homeostasis. We show that expression of Arabidopsis FAB1A/B in S. pombe fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-GFPs localize to the endosomes in root epidermal cells of Arabidopsis. Reduction in the expression levels of FAB1A/B impairs vacuolar acidification and endocytosis. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B. The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis.

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Seed vernalization promotes seed germination and flowering in various plants, and glutathione is required for vernalization-promoting flowering. Here we show that major changes in gene expression following seed vernalization involve glutathione in Arabidopsis thaliana. Seed vernalization treatment of wild-type seeds changed certain genes during seed germination, which are defined as "seed-vernalization-responsive (SVR) genes". Regardless of seed vernalization treatment, a similar gene expression pattern was observed in seed germination of transgenic plants overexpressing the gamma-glutamylcysteine synthetase gene GSH1, coding an enzyme limiting glutathione biosynthesis, under the control of the cauliflower mosaic virus 35S promoter. Taken together, SVR gene expression is modulated by glutathione. Based on changes in the amount and redox state of glutathione in wild-type and transgenic seeds treated and untreated with seed vernalization, we discuss the importance of oxidized redox states of glutathione in seed vernalization.

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We have previously reported that glutathione is associated with regulation of photosynthesis in Arabidopsis thaliana. Here we show that exogenous GSH and GSSG can increase leaf CO₂ fixation in A. thaliana in a manner different from each other. Wild-type plants (Columbia) were supplemented with GSH or GSSG by weekly subirrigation until five weeks passed after sowing. Compared to no supplementation, GSSG and GSH increased seed and biomass yield. GSSG and GSH also promoted leaf-area-based CO₂ fixation with increasing nitrogen and chlorophyll contents in five-day-old plants. GSH raised Chl a/b ratio, whereas GSSG did not cause any significant change in Chl a/b ratio. GSH-supplemented plants had about 2-fold more Rubisco content than that of non-supplemented plants, while GSSG-supplemented plants had 1.3-fold more Rubisco than the control plants. Taken together, these indicate that both GSH and GSSG have an increasing effect on photosynthesis, but that the mechanisms are different from each other.

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Excess nitrogen fertilization promotes vegetative growth, resulting in reduction of seed yield due to the decrease in the light utility efficiency. However, application of GSH or GSSG whose amount was equivalent to the excess nitrogen increased seed yield with little significant plant size in Arabidopsis thaliana. To investigate the effect that had not been seen when excess nitrogen supplied, we performed growth analysis of Arabidopsis wild-type plants with and without subirrigation of GSH or GSSG. In 4-week-old plants with subirrigation of GSH or GSSG, the leaf blades grew more round. Flowering was not markedly influenced by GSH or GSSG. At fifth week after imbibition, GSH or GSSG-subirrigated plants bolted and had increased nitrogen content, followed by remarkable increase in aerial biomass. Taken together, GSH and GSSG can increase biomass during the reproductive period without changing the length of vegetative period. We will also report the growth analysis results in details.

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Elongation factor G (EF-G) is a key protein in translational elongation. We have recently found that EF-G is inactivated via oxidation of two specific Cys residues and subsequent formation of an intramolecular disulfide bond in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we examined the role of EF-G (Slr1463) in the response of protein synthesis and PSII to strong light that gives rise to oxidative stress. When EF-G was overexpressed, the synthesis of the D1 protein de novo was enhanced under strong light, whereas the synthesis of almost all other proteins was suppressed. In this mutant, the level of another EF-G homolog Sll1098 decreased. When a modified EF-G (Slr1463) in which Cys105, a target of ROS, had been substituted by Ser was expressed together with wild-type EF-G, the synthesis of the D1 protein de novo was enhanced under strong light with no significant effects on the global synthesis of proteins. Moreover, the extent of photoinhibition of PSII in mutant cells was alleviated without any changes in the rate of photodamage to PSII. These observations suggest that the modified EF-G might enhance the repair of PSII during photoinhibition.

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Elongation factor G (EF-G), a key protein in translational elongation, is inactivated by reactive oxygen species via formation of an intramolecular disulfide bond between two specific cysteine residues in Synechocystis sp. PCC 6803. The oxidized EF-G can be reduced and reactivated by thioredoxin. However, it remains to be clarified whether EF-G is reduced by reducing equivalents derived from the photosynthetic transport of electrons in vivo. We attempted to establish method by which the redox state of EF-G can be monitored in vivo. After Synechocystis cells were exposed to light at various intensities, cells were supplemented with methanol to fix the redox state of proteins. Cells were broken and SH-groups of proteins were modified with PEG-maleimide, and then the reduced and oxidized forms of EF-G were detected immunologically. When the intensity of light was raised, the ratio of reduced form of EF-G increased. However, under strong light, the ratio of oxidized form increased. These observations suggest that EF-G might be regulated by reducing equivalents that are generated by the photosynthetic transport of electrons and mediated by thioredoxin.

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Thioredoxin (Trx) localized in stroma, is reduced by ferredoxin and ferredoxin-thioredoxin reductase. The required reducing equivalents are provided by photosynthetic electron transfer system. Reduced form Trx activates various thiol-enzymes in stroma by way of reduction and directly supplies reducing equivalents required for the reaction cycle of peroxiredoxin as well, which functions as detoxification system of reactive oxygen species. In addition, we showed that stroma Trx functions as a part of a trans-membrane reducing equivalent transfer system in higher plant chloroplasts. HCF164 that is a membrane anchored thioredoxin-like protein faced to thylakoid lumen side, receives reducing equivalents from stroma Trx. In this study, we analyzed the membrane protein CcdA, a candidate component of this reducing equivalents transfer system itself. We discuss the electron transfer mechanism across the thylakoid membranes.

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The levels and/or redox state of NADP(H) in chloroplasts must be properly regulated, since it is essential not only for primary metabolism including photosynthesis, but also for antioxidative systems. Recently we found that an Arabidopsis chloroplastic Nudix hydrolase, AtNUDX19, has the pyrophosphohydrolase activity toward NADPH and its expression is increased with increasing light intensity. Here, to clarify the involvement of AtNUDX19 in photoacclimation, we isolated and characterized a knockout mutant lacking AtNUDX19 (KO-nudx19).
Under normal and high-light (100 and 800 μmol/m²/s, respectively) conditions, levels of NADPH were higher in the KO-nudx19 than in the wild-type plants, while levels of NADP⁺ were lower in the mutants. Interestingly, the initial activities of enzymes in the Calvin cycle, and the activities of CO₂ fixation and photosynthetic electron transport were increased in the KO-nudx19 under high-light, resulting in enhanced growth of the mutants. Moreover, the activities of antioxidative enzymes were increased in the KO-nudx19. These findings suggest that AtNUDX19 acts as a crucial regulator for photoacclimation.

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Recently we demonstrated that chloroplastic NADPH pyrophosphohydrolase, AtNUDX19, is involved in the regulation of photosynthesis and antioxidative system during photoacclimation through hydrolysis of NADPH, suggesting that the levels and/or redox states of NADP(H) is crucial for regulating stress responses. Here we revealed the regulatory mechanisms of such stress responses by a microarray analysis in a knockout mutants lacking AtNUDX19 (KO-nudx19) grown under normal and high-light conditions.
The expression levels of >600 genes were altered in the KO-nudx19 plants compared with the wild-type plants under normal and high-light. A large number of genes encoded factors related to hormonal responses (jasmonic acid: JA, abscisic acid: ABA, and salicylic acid: SA). The KO-nudx19 plants were insensitive to treatment with JA and ABA, and hyper-sensitive to wounding and osmotic stress. Furthermore, the KO-nudx19 plants showed hyper-sensitive to treatment with SA. These findings indicated that the regulation of levels and/or redox states of NADP(H) by AtNUDX19 is essential for modulating plant stress responses through hormonal regulation.

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We have demonstrated that Arabidopsis SR proteins, atSR45a (plant-specific type SR protein) participates in a spliceosomal assembly through other splicing factors and its expression is markedly induced by high-light stress (Tanabe et al., 2009). These facts suggest that atSR45a is involved in the regulation of high-light stress responsive-alternative splicing events (AS). Here, to identify AS events of genes regulated by atSR45a, we compared gene expression profiles in the wild-type as well as the knockout atSR45a (KO-sr45a) plants under high-light stress using the tiling array.
The expression levels of 235 genome regions in the KO-sr45a plants were altered significantly compared with those in the wild-type plants. By semi-quantitative RT-PCR analysis, we confirmed changes in the transcript levels or AS patterns of the genes in response to high-light stress. In addition, temporal correlation between the expression levels of atSR45a and the AS efficiencies in these genes were observed in the progression of high-light stress. The sequencing analysis of the AS variants showed that atSR45a plays a principal role in switching of splicing in the intron retention events.

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Ozone (O₃) is the main photochemical oxidant that causes leaf damage in many plant species. It has been thought that O₃-induced visible leaf injury causes decrease of photosynthesis, resulting reduction of grain yield. However, recent study showed that degree of visible leaf injury is not necessarily corresponding to reduction rate of grain yield in O₃-exposed rice, suggesting that these may be regulated in different mechanisms. To identify gene loci that affect rice grain yield under O₃ exposure condition, QTL analysis was performed using Sasanishiki/Habataki chromosome segment substitution lines (CSSLs). As the result, we identified the loci, which affects O₃-induced yield loss, near RM3430 marker in chromosome 6. Previous study showed that APO1 gene, locating near this marker, influences rice grain yield through affecting primary rachis number. Thus, expression analysis of APO1 was carried out. In young panicles, transcript level of APO1 was the highest and its expression was significantly suppressed in O₃-exposed Habataki but not in O₃-exposed Sasanishiki. This indicates that reduction of grain yield in O₃-exposed Habataki may be caused by suppression of APO1 expression by O₃.

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Plants undergo continuous exposure to various biotic and abiotic stresses in their natural environment. These stresses often cause accumulation of reactive oxygen species (ROS), leading to the development of oxidative stresses. To understand the crosstalk in biotic and abiotic stress signaling, we focused on analysis of oxidative stress response. Methyl viologen (MV) is a herbicide that catalyze the formation of superoxide free radical, a major ROS.
We found an ecotype Nos-d to show resistance to methyl viologen. The Nos-d also showed tolerance to osmotic stress, but not to salinity or stress related hormones. Nos-d also showed tolerance to another oxidative stress-related chemicals, but the difference between Nos-d and Col was very slight. To isolate another MV-tolerant accessions, MV tolerance of Arabidopsis accessions distributed from RIKEN BRC was evaluated. Several MV tolerant accessions were identified. Analysis using Nos-d x Col F2 population suggested that the resistant phenotype is linked to a single locus. Mapping of the locus of MV tolerance is now in progress to understand the tolerance mechanism.

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Symbiosis between legume plants and rhizobia causes the development of new organs, nodules, which function as an apparatus for symbiotic nitrogen fixation. In this study, the roles of auxin in nodule development in Lotus japonicus have been demonstrated using molecular genetic tools and auxin inhibitors. The expression of an auxin-reporter fused to GUS was analyzed in L. japonicus roots, which showed a strong signal in the central cylinder of the root, whereas upon rhizobium infection, the generation of GUS signal was observed at the dividing outer cortical cells during the first stage of nodule cell divisions. When nodules were developed to maturity, strong GUS staining was detected in vascular tissues of nodules, suggesting a distinct auxin involvement in the determinate nodule development. Numbers and the morphology of nodules were affected by several auxin inhibitors. The common phenotypical alteration by these auxin inhibitors was the strong inhibition in forming lenticels, which are normally developed on the nodule surface from the root outer cortex. These results indicate that auxin is required for the normal development of determinate nodules in a multidirectional manner.

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A plant hormone abscisic acid (ABA) inhibits nodule formation of Lotus japonicus. The five genes of L. japonicus were screened as ABA responsive gene by using cDNA macroarray of L. japonicus. Four of them, Class 1 chitinase (PR3), PR10.2 C Protein (PR10.2c), Chalcone synthase (CHS), and β-1,3-glucanase (Glu1), were involved in plant defense system and ORF2 bases 1807-2850 was unknown function.
Transformed hairy roots of L. japonicus were generated with anti-sense genes. The anti-sense hairy roots of LjGlu1) and CHS showed increased nodule number and enhanced nitrogen fixation activity. The anti-sense of ORF2 bases 1807-2850 also gave enhanced nitrogen fixation activity to the hairy roots. The expression level of these three genes in anti-sense hairy roots was 53% lower for LjGlu1, and around 30% lower for other two genes compared with those of wild type. In this study, RNAi transformed hairy roots harbouring RNAi constructs and nodule formation and nitrogen fixation were determined.

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Symbiotic nitrogen fixation in nodules that contain symbiotic rhizobia enables legumes to thrive in nitrogen-poor soils. However, it is so energy consuming that plants control nodulation at both local and systemic levels. Mutants deficient in such controls exhibit a range of hypernodulation phenotypes.
We isolated a novel hypernodulation mutant "plenty" from the M2 progeny of Lotus japonicus MG-20 mutagenized by ion beam irradiation. Grafting experiments demonstrated that the root of plenty was responsible for its hypernodulation phenotype. The plenty mutant suggested an unknown nodulation regulating pathway because of additive numbers of nodules in plenty;har1 double mutants, its partial tolerance to external nitrates and its normal triple response to ethylene.
The plenty mutant developed nodules of the same size as MG-20, though the number of nodules increased. This pattern is unlike other hypernodulation mutants such as har1, klv or tml. A biomass assay suggested that many normal-sized nodules in plenty were hindrances to plant growth, since they resulted in hyperactivity of nitrogen fixation per plant.
Map-based cloning of the PLENTY gene is being conducted.

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Leguminous plants develop root nodules to establish the endosymbiosis with nitrogen-fixing bacteria. We have reported the identification of a novel leucine-rich repeat receptor-like kinase (LRR-RLK), KLAVIER (KLV), which negatively and systemically controls the number of nodules in Lotus japonicus. In leaf, KLV was predominantly expressed in the vascular tissues, as with another LRR-RLK gene, HAR1, which also regulates nodule number. Genetic analyses indicated that KLV and HAR1 function in the same genetic pathway to govern the negative regulation of nodulation, and are epistatic to the effect of overexpression of LjCLE-RS1 or LjCLE-RS2 that suppresses nodulation.
In this study, transient expression analysis in Nicotiana benthamiana indicated the physical interaction of KLV and HAR1. These results suggest that the potential KLV-HAR1 receptor complex regulates symbiotic nodulation transmitting the root-derived signals.

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Legumes establish symbiotic association with N₂ fixing soil bacteria, termed rhizobia, resulting in the root nodule formation. In the nodule cells, rhizobia differentiate into endosymbiotic form, bacteroids and then start to fix N₂. During the symbiosis, the bacteroids need to be supplied mineral nutrients from the host plant.
Nitrogenase, a key bacterial enzyme for N₂ fixation, requires molybdenum (Mo) for its activity. Mo must be provided from the host plant to bacteroids. However, Mo transport mechanism(s) of the host plant to the bacteoids is still unclear.
Recently, we identified a Mo transporter from Arabidopsis thaliana (AtMOT1) (Tomatsu et al., 2007). To clarify the molecular base of Mo transport mechanism(s) required for the establishment of N₂ fixing symbiosis, we identified Mo transporter in L. japonicus. We found four AtMOT1 homologue genes in L. japonicus genome (ST51-54). Expression of the ST54 was detected only in nodules and was increased during nodule development. In contrast, expression of remaining three genes was detected in all tested organs. It is expected that the ST54 is involved in Mo transport to bacteroids.

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Nitrogen fixation activity of rhizobia inside the root nodules is strictly controlled by interactions with the host plants. The early steps of the interactions between rhizobia and host legumes have been revealed by genetic method using symbiotic mutants of the model legume plants such as Lotus joponicus. Meanwhile, the information about the host plant genes that are crucial for the interactions in the later stages of symbiosis, such as nodule organogenesis and the control of nitrogen fixation is still limited. The Fix^- mutants, which form apparently normal nodules that contain endosymbiotic rhizobia but exhibit low or no nitrogen fixation activity, are the useful materials to identify the plant genes involved in development of rhizobial nitrogen fixation under symbiotic conditions.
In this study, we characterized a novel Fix^- mutant of Lotus joponicus, Ljsym104. This mutant forms small white nodules with low nitrogen fixation activity, and shows nitrogen deficiency symptoms under symbiotic conditions. Further characterization of Ljsym104 mutants and the causal gene, LjSYM104, will be presented.

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Lotus japonicus NIN is one of key regulators for nodulation. nin mutants display aberrant root hair curing and defects in infection thread (IT) formation and in cortical cell division (CCD). We have demonstrated that NIN has abilities to induce IT formation and CCD. However, NIN protein function and its molecular mechanisms, by which NIN regulates nodulation processes, have not yet been elucidated, although the secondary structure prediction of NIN protein has suggested that it may act as a transcription factor. When we expressed NIN fused with GAL4-DNA binding domain in tobacco cells, expression of a reporter gene, of which promoter possesses GAL4-binding DNA sequences, was induced, indicating that NIN is a transcriptional activator. We searched genes whose expression is upregulated by NIN using publicly available L. japonicus transcriptome data base and found 19 genes as candidates for NIN targets. ChIP assay and EMSA showed that two genes, at least, were directly targeted by NIN. DEX-inducible form of NIN protein induced expression of these genes depending on DEX. Furthermore, we are analyzing function of these factors in nodulation processes.

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Nitrogen-fixing symbiosis between legumes and rhizobia is initiated by the recognition of rhizobial Nod factors (NFs) by host plants. NFs are diversely modified derivatives of chitin oligosaccharide, a fungal elicitor that induces defense responses in plants. Recent evidence has shown that both NFs and chitin elicitors are recognized by structurally-related LysM-receptor kinases. Transcriptome analyses of Lotus japonicus roots indicated that NFs not only activate symbiosis genes but also transiently activate defense-related genes through NF receptors. Conversely, chitin oligosaccharides were able to activate the symbiosis genes independent of NF receptors. Analyses using chimeric genes consisting of the LysM receptor domain of a Lotus NF receptor, NFR1, and the kinase domain of an Arabidopsis chitin receptor, CERK1, demonstrated that the substitution of a portion of the alphaEF helix in CERK1 with the amino acid sequence, YAQ, from the corresponding region of NFR1 enables L. japonicus nfr1 mutants to establish symbiosis with Mesorhizobium loti. Together with these results, we will discuss the molecular evolution of LysM receptor kinase.

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Flavonols, a subclass of flavonoids, have crucial roles in UV protection and modulation of flower color. They are known to have roles in leguminous plants-rhizobia symbiosis. Recently, it was also suggested that they act as auxin transport regulators during nodulation. We identified the genes of Lotus japonicus coding flavanone 3-hydroxylase (FHT1 and FHT2) or flavonol synthase (FLS1, FLS2 and FLS3) to uncover the role of flavonols during nodule development. Their expression levels, except for FLS3, were transiently up-regulated at 2 days after inoculation with wild-type Mesorhizobium loti, whereas they kept increasing after 4 days with nodA or nodD disruption mutants. These results suggest that the expression of the genes coding biosynthetic enzyme of flavonol was suppressed by Nod factor and induced by unidentified factor derived from M.loti. The expression of GUS gene driven by FHT1 or FLS1 promoter clarified that FHT1 is expressed in various organs, and that FLS1 is specifically expressed in shoot and root apices. Furthermore, we are now preparing FLS1 transgenic hairy roots to examine the impact of the modification of flavonol biosynthesis on root nodulation.

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There believed to be around 10^8-9 of bacteria present per gram of soil. Therefore, it is inevitable for plants to encounter with various soil bacteria during root development. Previous reports demonstrate that plants have evolved systems that sense and respond to environmental stimuli including soil bacteria. Studies on interactions between plants and soil bacteria, however, have been rather limited to elucidation of molecular mechanisms involved in mutualism between plants and either root nodule bacteria or arbuscular mycorrhizal fungi, leaving the possible effect of vast majority of soil bacteria onto plant growth and development mostly uncharacterized. We report here that Bacillus subtilis can inhibit the growth of Arabidopsis and rice without direct contact, possibly by volatile compounds. This inhibitory effect is dependent on the culture condition of the bacteria. Moreover, we found that these two plants also exhibit similar growth retardation when co-cultured with Agrobacterium tumefaciens, suggesting that the plant's response to volatiles of soil bacteria might be regulated by a mechanism that is conserved among plant kingdom.

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Microbial volatiles promote accumulation of exceptionally high levels of starch in leaves. This phenomenon (designated as MIVOISAP) is inhibited by cycloheximide and cordycepin. Transcriptomic analyses revealed that Arabidopsis MIVOISAP is accompanied by drastic transcriptome reprogramming. Using different Arabidopsis mutants we investigated the impact in MIVOISAP of down-regulation of starch genes and of genes involved in the main signaling pathways. These analyses revealed that starch content increase in microbial volatiles-treated hy1 and hy2 photoreceptor mutants, and in mutants impaired in starch synthases class III (SS3) and IV (SS4), jasmonic acid (JA) sensing, and plastidial NADP-thioredoxin reductase C (NTRC) was low, whereas starch content increase was highest in bam1, isa3 and phs1 mutants impaired in starch breakdown enzymes. The overall data showed that MIVOISAP involves a photocontrolled, transcriptionally and post-transcriptionally regulated network wherein JA, SS3, SS4 and NTRC-mediated changes in redox status of plastidial enzyme(s) play important roles. We also provide evidence that microbial volatiles activate starch degradation during starch over-accumulation

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A phytohormone, abscisic acid (ABA) induces stomatal closure to reduce transpirational water loss via a fine-tuned signal transduction in guard cells. In this study, we report that gene disruption of an Arabidopsis histidine kinase, AHK5 causes ABA hypersensitivity of guard cells. In the ahk5-1 mutant, ABA-induced stomatal closure was enhanced. We investigated plasma membrane ion channel activities of the ahk5-1 mutant guard cells using a patch clamp technique. Changes of cytosolic free calcium concentration in guard cells were also monitored using a fluorescent calcium indicator, yellow cameleon 3.6. The results suggest that AHK5 functions as a negative regulator of ABA signaling in Arabidopsis guard cells.

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The cuticle is a hydrophobic layer on the aerial surface of plants, and contains cuticular wax, which is a mixture of aliphatic very long-chain fatty acids and their derivatives. It plays a critical role in preventing non-stomatal water loss. However, molecular mechanisms underlying the cuticle development under various environmental conditions have remained largely unknown. The amount of total cuticular waxes on the leaves of drought-tolerant wild watermelon grown in high light condition was 2-fold larger than those grown in the low light condition. Moreover, drought treatment resulted in the 22-fold increase in the amount of total waxes compared to those in the low light condition. The real-time PCR analyses revealed that two key genes for the wax synthesis, i.e., β-ketoacyl-CoA synthase which catalyzes a committing step for the fatty acid elongation, and aldehyde decarbonylase for the conversion of long-chain aldehyde to alkane, were significantly up-regulated under drought. These observations suggested that the wax-related genes were coordinately up-regulated under drought, for the fortification of cuticle layer in this plant.

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In Arabidopsis, flavonoid biosynthesis is regulated by MYB transcription factors such as MYB12 for flavonol and MYB75/PAP1 for anthocyanin. We have generated the transgenic lines over-expressing both MYB12 and PAP1 (WOX1-1, WOX1-2) to characterize them besides the lines of each single MYB gene over-expression (MYB12OX and pap1-D by integrated analysis of metabolomics and transcriptomics. The integrated analysis showed flavonoid-specific overaccumulation and induction of expression of stress-related genes in the overexpression plants. Abscisic acid (ABA) was not changed among Col-0 and the TF-overexpressing plants. These results implied flavonoid biosynthetic pathway has interactions to stress response mechanism, and induction of flavonoid biosynthetic pathway improves the ability of abiotic stress mitigation. As the result of abiotic stresses (drought, oxidation and salt stress) experiments, the overexpressing plants showed the stress-mitigating ability to the abiotic stresses.
Flavonoid biosynthetic pathway has the feed-forward interaction to stress response mechanism.

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In order to analyze root development under water deficit condition, we developed a novel system to treat soil-grown plants readily with draught. When rice plants were grown in the containers, called "Root box", seminal roots of them were more elongated in water- stressed condition than in control condition. Number of crown roots was decreased in the water-stressed condition. Microarray analysis has revealed that number of auxin-related and auxin-inducible genes were up-regulated in root tips of water-stressed rice plants. These genes include ILR1 and IAR3 encoding enzymes which catalyze removal of amino acid moieties from IAA-amino acid conjugates. Consistent with this observation, level of IAA was higher in the root tips of the water-stressed rice plants, suggesting that the elevated level of IAA is one of key factors for the enhanced root elongation under water-stressed condition. Since there were no difference in length of cortical cells in root tips between control and the water-stressed conditions, the enhanced root elongation under water-stressed condition may be caused by increased cell division.

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Drought is the major environmental threat to agricultural production and distribution worldwide. Plant dehydration-stress adaptation is a complex biological process that involves global changes in gene expression and metabolite composition.
To discover novel master genes that regulate dehydration responsive pathway, we analyzed co-expressions of Arabidopsis genes using our dehydration-transcriptome data. We found 120 correlated-genes modules by co-expression analysis. In these modules, we analyzed gene-to-gene correlations of dehydration-repressed genes. Promoter analysis of these dehydration-repressed genes showed that Site II (GGNCCC) motifs were frequency found in their promoter regions. Site II is the predicted target sequence of TCP transcription factor. Now, we are analyzing an Arabidopsis stress-inducible TCP and its roles in abiotic stress response.

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DREB2A is an Arabidopsis transcription factor that expresses in response to water- and heat-stresses and contributes to acquisition of tolerance against these stresses via transcriptional activation of stress-specific target genes. DREB2A is considered to be inactivated under non-stressful conditions by its negative regulatory domain and activated in response to stress signals. However, signals that determin inactivation and activation of DREB2A are not clear.
DREB2B is a homolog of DREB2A that occurred through gene duplication in Brassicaceae. We have reported DRE-dependent transactivation activity and negative regulation of DREB2B is weaker than those of DREB2A. Several differences in amino acid sequences of the DNA-binding domain and the negative regulatory domain between DREB2A and DREB2B could result in the differences in the properties. To determine key amino acid residues responsible for the differences, we substituted these residues of DREB2A into corresponding residues of DREB2B, and compared the properties between mutant and wild-type proteins. We also report about analysis of transgenic Arabidopsis that express constitutive active forms of DREB2A and DREB2B.

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DREBs are transcription factors that interact with a cis-acting DRE sequence and activate the expression of downstream genes involved in water or temperature stress responses and tolerance in Arabidopsis thaliana. Previously, we performed a comprehensive analysis of DREB2-type genes in rice. We reported that transgenic A. thaliana plants overexpressing OsDREB2B showed enhanced expression of stress-responsive genes and improved drought- and heat shock-stress tolerance. These results suggest that OsDREB2B is a key gene encoding a transcription factor that functions in stress tolerance in rice. However, transgenic rice expressing OsDREB2B at high levels did not show significantly improved stress tolerance as compared to control plants, though OsDREB2B showed high transactivation activity in a transient transcriptional activation analysis using rice protoplasts. It is possible that OsDREB2B requires a modification for full activation in rice plants. Now we analyze the accumulation level of the OsDREB2B proteins in transgenic rice.

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We have shown that an agravitropic pea mutant, ageotropum, seedlings showed automorphosis-like growth and auxin polar transport of the first internode of etiolated epicotyls was substantially suppressed to lower than 50 % of that in etiolated Alaska pea epicotyls. We have also isolated novel cDNAs containing the complete open reading frames of a putative auxin influx facilitator, PsAUX1, and putative auxin efflux facilitators, PsPIN1, PsPIN2 and PsPIN3, from etiolated epicotyls of ageotropum. No deletion and insertion of nucleotides in these genes isolated from ageotropum pea was found compared to those in Alaska pea. Expression of PsAUX1, PsPIN1, PsPIN2 and PsPIN3 genes in the first internode of etiolated ageotropum and Alaska pea epicotyls were almost same during early growth stage of the seedlings. These results strongly suggest that automorphosis-like growth and reduced auxin polar transport in etiolated ageotropum pea epicotyls do not depend on structure and function, and gene expression of PsAUX1 and PsPINs but on different mode of actions of other molecules regulating auxin polar transport and/or dynamics of PsAUX1 and PsPINs between ageotropum and Alaska pea seedlings.

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In the root, the transport of auxin from the tip to the elongation zone (shootward) governs gravitropic bending. Shootward polar auxin transport, and hence gravitropism, depends on the polar deployment of the auxin efflux carrier, PIN2. In Arabidopsis thaliana, PIN2 has the expected shootward localization in epidermis and lateral root cap; however, this carrier is localized towards the root tip (rootward) in cortical cells of the meristem, a deployment whose function is enigmatic. We use pharmacological and genetic tools to cause a shootward relocation of PIN2 in meristematic cortical cells without detectably altering PIN2 polarization in other cell types or PIN1 polarization. This relocation of cortical PIN2 was negatively regulated by the membrane trafficking factor, GNOM, and by the regulatory A1 subunit of type two-A protein phosphatase but did not require the PINOID protein kinase. When GNOM was inhibited, PINOID abundance increased and PP2AA1 was partially immobilized, indicating both proteins are subject to GNOM-dependent regulation. Shootward PIN2 specifically in the cortex was accompanied by enhanced shootward polar auxin transport and by diminished gravitropism.

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We have tried to isolate genes involved in shoot gravitropism by means of microarray. Gene expression profiles of Arabidopsis mutants, sgr1/scr, sgr7/shr and eal1, have been compared to those of wild type, and we focused several genes named DGE (DOWN-REGULATED GENE IN EAL1). These three mutants lose shoot gravitropism because of defects in formation of endodermis that is the gravity sensing tissue of shoot. We have demonstrated that DGE1 homologous to Oryza sativa LAZY1 is involved in gravitropism of Arabidopsis stem.
Here, we analyzed DGE2 encoding a protein that has a short homologous region to DGE1. It is conserved in angiosperm although DGE2 has no known functional domains or motifs. DETL (DGE TWO-LIKE GENE) that has 54% identity to DGE2 is found in Arabidopsis genome. We analyzed T-DNA insertion mutants for DGE2 and DETL. Since lateral roots of dge2 detl double mutant tend to elongate horizontal direction, these genes are expected to be involved in root gravitropism. In addition, gravitropic response of shoot, hypocotyl and root in dge1 dge2 detl triple mutant dramatically reduced. Now we are carrying out further physiological analysis of the triple mutant.

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To reveal the defense mechanisms of Pinus thunbergii with the pine wood nematode (PWN), Bursaphelenchus xylophilus infection, we have conducted the transcriptome analysis to the PWN-infected resistant and susceptible clone. In previous studies, there has been little approach from the view of transcriptional level, though the difference of defense response between resistant and susceptible clones has been indicated by anatomical and metabolically approach. Kusumoto et al (2010) reported that the phenol accumulation in xylem parenchyma cell of the infected resistant clone was more apparent than that of susceptible clone, and the collapsed ray parenchyma cell in resistant clone was littler than that of susceptible. In this study, we conducted the suppression subtractive hybridization (SSH) analysis using ESTs of the PWN inoculated resistant and susceptible clones. For the time course sampling scheme, it was designed at 1, 3, 7, 14 day post-inoculation based on the previously reported phenotypic data. In this report, we present a characterization of the difference of defense response between resistance and susceptibility in P. thunbergii with PWN infection.

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Root-knot nematodes (RKNs) are a major parasite of agricultural crops, causing galls or "knots" formation throughout the root system. Finding a novel biological system for controlling the infection process is important to develop new method for limiting the RKN damage worldwide. The motile J2 stage juveniles invade into the host plant root near the root cap, penetrate intercellulary to the area of cell elongation where they initiate permanent feeding sites by injecting stylets. The molecular mechanism underlying this infection process, particularly the host plant and RKN interaction is virtually unknown.
Tomato is a major host for the infection by RKNs. Recently a dwarf cultivar Micro-Tom is regarded as the model tomato and bio-resources such as mutant populations, a TILLING platform, full-length cDNA and genome sequences are developed mainly by the National BioResource Project. By accessing to these resources and information, we surveyed for host genes that potentially have a role in regulating the RKN infection process. We are assessing the genes' effect on the infection rate by RNAi or by utilizing the mutant. The current status of the study is presented.

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Root-knot nematodes (RKNs) are parasitic animals that invade root systems of many agricultural plants. RKNs are effective parasites because they induce permanent feeding sites inside plant roots as juveniles, and then spend their whole life-cycle at this single site. During initial infection, RKNs interact with root tips and move between cells to reach a final target destination near developing vascular cells. It is thought that successful infection by RKNs involves a suppression of defense responses. We have investigated this hypothesis by quantitative measurement of host defense response during invasion of tomato roots by the northern root-knot nematode, Meloidogyne hapla. Control experiments revealed that the defense response differs in roots from that typically observed in aerial tissues. During RKN invasion, a significant induction of the "root defense marker genes" was observed, demonstrating that a susceptible host does indeed recognise early RKN parasitism. These results indicate that suppression of host defense is not required for RKNs to establish an infection site, and that these signalling networks may even by used to guide the infection process.

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Plants infested by herbivores emit the special blend of volatiles, including green leaf volatiles different from volatiles emitted from intact plants. The blend of volatiles functions, for instance, as alarm signal for intact plants surrounding infested plants to induce the defensive response against upcoming herbivore attack. To clarify the molecular basis of induced defense, we used tomato plants, Solanum lycopersicum cv. Micro-Tom, and common cutworm, Spodoptera litura, a herbivorous insect of tomato plants, as a model system for volatile-induced defense. Last year, we present the induced defense in the plants exposed to herbivore-infested volatiles against subsequent infestation by common cutworms and the accumulation of glycoside in exposed plants probably related to the defense. In this report, we show the induced defense and glycoside accumulation in intact plants by exposure of only one compound, Z3-hexenol, which is a constituent of the blend of herbivore-infested tomato volatiles. These findings suggest that the Z3-hexenol presumably works as a signal compound emitted from damaged plants to their surrounding intact plants.

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In response to herbivory, plants emit a specific blend of volatiles that attracts natural enemies of herbivores. The emission of the volatiles, so called herbivore-induced plant volatiles (HIPV), can be considered as one of the induced indirect defenses of plants against herbivores. In plant-pathogen interactions, brown spots are seen at infected sites on leaves by hypersensitive reaction (HR), one of the defense responses. Similar brown color can be seen in plant-spider mite interactions. For instance, either brown or white scars are observed on the lima bean leaves infested by Kanzawa spider mites. We selected two strains of the spider mites by the colors of the scar: red (to induce brown scars) and white strains. Each strain induced different blends of HIPV in lima bean plants. Based on analysis of expression of defensive genes and quantification of salicylic acid, combination of jasmonate- and salicylate-related signaling pathway partly explains the difference of HIPV from plants infested by each strain. We will discuss the differential early events in the response to damage by either strain based on the observation of Ca2+ and H2O2 by a confocal laser scanning microscopy.

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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 (JA), ethylene (ET), and salicylic acid (SA) on plant tolerance to thrips feeding. We also introduce the development of the Brassica full-length cDNA resources and the database for systematic consolidation of Chinese cabbage and Arabidopsis (ABRANA : Arabidopsis and BRAssica Network Access).

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