A rice mutant, rod-like lemma (rol) has pleiotropic phenotypes in spikelet. In rol spikelet, the lemma is replaced by a rod-like structure and the stamen has an abnormal anther on its top. These phenotypes seem to be associated with a defect of organ development along the adaxial-abaxial axis. Thus, rol mutation is likely to affect regulation of the adaxial-abaxial polarity.
Establishment of the adaxial-abaxial polarity is required for expression of the genes that function specifically in the adaxial or abaxial side. In rice, the OsETTIN (OsETT) genes, orthologs of Arabidopsis ETTIN/ARF3, are specifically expressed in the abaxial side. Expression analysis showed the OsETT genes were ectopically expressed in rol mutant. This suggests a possibility that the ectopic expression of the OsETT genes would be associated with morphological defects in rol mutant. To assess this possibility, we tried to suppress the OsETT genes in rol mutant by RNA silencing. The result demonstrated the suppression of the OsETT genes rescued some aspects of rol phenotypes. Thus, this result indicated that ectopic expression of the OsETT genes was one of the causes of rol phenotype.

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Morphologies of flowers and inflorescences in monocots are distinct from those of flowers and inflorescences in eudicots. Especially in grasses, the inflorescences comprise of unique structural units, the spikelet and the floret. The rice spikelet has one floret, which is subtended by two tiny glume-like organs, called sterile lemma and rudimentary glumes. These organs are specific to the rice. To elucidate the developmental mechanism of these organ and their evolutionary origin, we have focused on a rice mutant, in which these organs are aberrant. Here, we report phenotypic analysis of this mutant, isolation of the gene responsible for the mutation, and its function.

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Maintenance of undifferentiated stem cells is one of the important functions of plant apical meristems. We isolated a novel mutant, named meristem disorganization1-1 (mdo1-1), exhibiting abnormal shoot morphogenesis. The mdo1-1 homozygous plants showed pleiotrophic shoot phenotypes, such as aberrant phyllotaxis and plastochrone, and frequent stem fasciation. In the mutant shoot apical meristem, the surface cells were occasionally enlarged and canonical layer structure was disrupted, associated with elevated cell division activity. The mdo1-1 allele also affected function of root apical meristem. In the mutant root tips, quiescent center and initial cells were not maintained properly, resulting in the retarded growth of roots. Molecular cloning as well as complementation test indicated that the MDO1 gene encodes a relatively small protein found specifically in wide variety of land plants and that, in mdo1-1, tightly conserved amino acid in the orthlogs is substituted. These results, thus, suggest that the MDO1 gene function is crucial for maintenance of undifferentiated stem cells in apical meristems.

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Phosphate is essential for plant growth, but its availability in soil is often limiting. The plants growing under limiting phosphate supply modify their root architecture and physiological processes in order to acquire sufficient phosphate. We have examined whether altered iron availability could influence the root architecture and nutrient uptake in Arabidopsis under low phosphate condition. Low phosphate availability induced a change in root architecture that was characterized by a shorter primary root and a greater number of lateral roots. These changes intensified when iron availability was reduced below normal level. Aithough root architecture was changed, phosphate uptake rates were not affected by the change in iron availability. The expression pattern of two phosphate transporters namely, Pht1;1 and Pht1;4, were differentially altered by iron availability. The results indicate the possibility of iron-phosphorus interaction in the regulation of root architecture and nutrient uptake.

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SULTR1;2 high-affinity sulfate transporter facilitates sulfate uptake from the environment. In Arabidopsis, expression of SULTR1;2 was induced upon sulfur deficiency (-S), which enables the plants to survive under -S condition. The -S response of SULTR1;2 is controlled by SLIM1 transcription factor. Previously, we have identified cis-acting element involved in the -S-inducible expression of SULTR1;2, which contains WRKY binding sequence. In this study, we have tried to identify the WRKY transcription factors that control -S-inducible expression of SULTR1;2. Among the 72 members of WRKY transcription factor in Arabidopsis, 3 members exhibited the up-regulated transcript levels by -S in the wild type plants but not in slim1 mutant. Then we isolated T-DNA insertion mutants of 3 WRKY members, and analyzed SULTR1;2 expression and sulfate uptake activity. Both were significantly down-regulated under -S condition in these mutants comparing with wild-type plants, suggesting the involvement of these WRKY transcription factors in -S-inducible expression of SULTR1;2 and sulfate uptake.

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Rice requires high amount of Si for healthy growth and high yield by alleviating various stresses. So far, we have isolated and characterized three Si transporters from rice; Lsi1, 2 and 6. Here we report a novel Si transporter, Lsi3. Lsi3 shares 81% identity with Lsi2 at amino acid level. Similar to Lsi2, Lsi3 showed efflux transport activity for Si in Xenopus oocytes. GFP-Lsi3 fusion protein is localized to the plasma membrane. The expression of Lsi3 was mainly detected in the roots and upper nodes. Immunohistological staining revealed that Lsi3 is localized at the pericycle cells of the roots. The Si uptake as well as Si concentration in the xylem sap was slightly increased in a Lsi3-enhanced line, suggesting that Lsi3 is involved in the xylem loading of Si. At the nodes, Lsi3 is localized at the parenchyma cells between enlarged- and diffuse-vascular bundles. Since Si in transpirational flow is unloaded by Lsi6 expressing at the xylem transfer cells surrounding the enlarged vascular bundles, the cell-type specificity of localization of Lsi3 suggests that it is responsible for re-loading of Si to the diffuse vascular bundles, which are connected to upper nodes and panicles.

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High accumulation of silicon (Si) in the shoots including leaves and grains is required for protecting the plants from biotic and abiotic stresses. Silicon uptake by the roots is mediated by an influx transporter HvLsi1 and an efflux transporter HvLsi2 in barley. However, the transporters involved in the distribution of Si in the shoots have not been identified in barley. Here, we report that HvLsi6 and HvLsi2 are involved in the Si distribution. HvLsi6 is a homolog of HvLsi1, but expressed in the leaf blades, leaf sheaths and nodes in addition to the roots. In the roots, HvLsi6 is expressed in all cells, but in the leaf blades and sheaths, HvLsi6 is only localized at parenchyma cells of xylem vessels. At the node, it is located at the transfer cells surrounding the enlarged vascular bundles. On the other hand, HvLsi2 is localized at the parenchyma cell layer adjacent to the transfer cells with opposite polarity of HvLsi6. These results suggest that HvLsi6 is involved in xylem unloading of Si in leaf blade and sheath. In addition, both HvLsi6 and HvLsi2 are involved in inter-vascular transfer of Si at the nodes from enlarged vascular bundles to diffuse vascular bundles.

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Lipid-modified proteins play critical roles in regulating many cellular functions. Myristoylation is well-recognized form of lipid modification in eukaryote. In this process, myristate is attached to the N-terminus of the proteins. In the last conference, we have reported the construction of myristoylation system in wheat-embryo cell-free translation system. To determine the sequence specificity in the myristoylation consensus motif for plant, we next analyzed the combination of amino acids at position 3 and 6. We used G protein γ-subunit from Arabidopsis thaliana (AGG1) fused the myristoylation consensus motifs, MGXAA(A/S)AAAA (X shows each 20 amino acid.), as substrates and the susceptibility of these proteins to myristoylation was estimated by using wheat-embryo cell-free translation system. When Ser was located at position 6, 12 amino acids were permitted at position 3 to direct myristoylation, while only 2 amino acids (Asn and Gln) were permitted at position 3 when Ala was located at position 6. To obtain further information, analyses of the amino acids at other positions and removal of the initiating Met are now in progress.

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Tuberonic acid (TA) and tuberonic acid glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and have been shown to induce tubers. These compounds have been found in various plant species and are biosynthesized from jasmonic acid. However, the enzymes that catalyze the metabolism between TA and TAG are poorly characterizing. We have identified the TAG hydrolyzing activity in rice and the enzyme catalyzing this activity, TAG glucoside (OsTAGG), was purified. In this study, novel TAG glucosidase isozyme (OsTAGG2) was purified and enzymatic characteristics were compared with OsTAGG1. OsTAGG2 showed high identity to OsTAGG1 and showed high substrate specificity toward TAG analogs, which is common to OsTAGG1. Together, these data suggested that OsTAGG2 is a novel TAG hydrolyzing glucosidase in rice. To elucidate the physiological functions of OsTAGG1 and OsTAGG2, expression analysis of these genes under stress conditions such as wounding is under way.

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Nicotinamide adenine dinucleotide (NAD) plays important roles in regulating cell homeostasis and responses to various stressful environments. In Arabidopsis, genes involved in the NAD biosynthetic pathway have been fully determined. Out of these, a gene encoding nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT), AtNMNAT, is critical for the NAD biosynthesis because the NMNAT acts in de novo and salvage pathway. Previously, we reported an ABA-induced posttranscriptional activation of NMNAT in stomatal guard cells. In this study, we found that GST-NMNAT forms homomeric complex with enzymatic activity in vitro. Interestingly, expression of truncated NMNAT with no enzymatic activity suppressed endogenous NMNAT activity in vivo, meaning that truncated form of NMNAT acts as a dominant negative. Interestingly, the formation of complex was reppressed in an ABA-insensitive mutant where NAD decrease did not occur. These results suggest that the level of cellular NAD is maintained by regulating the biosynthetic activity through the modulation of NMNAT complex.

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GDP-mannose-epimerase (GME) catalyzes the inversion of configuration at carbon-3 and carbon-5 of D-mannose moiety, first committed step for L-ascorbate (Asc) biosynthesis branched off other metabolic pathways. The product, GDP-L-galactose is converted to Asc with four steps via L-galactose. The pathway is specific for plants and some algae species. Therefore, GME reaction is thought to be an important step.
We have isolated a cDNA clone encoding full coding sequence of GME from peach fruit. Transgenic tobacco plants those overexpress GME under the control of 35S promoter were generated. Overexpression of GME transcript was confirmed in 10 transgenic lines. In order to evaluate GME activity, [¹⁴C]-GDP-mannose was reacted with ammonium sulfate fractionated proteins, acid hydrolyzed and separated by silica TLC. Separation of radioactive spots were not uniform among preparations, resulting uncertainty in GME activity evaluation. Recombinant GME with HAT tag expressed in E. coli were similarly treated, and generation of [¹⁴C]-galactose was confirmed but the ratio of substrate:product was about 6:1. Asc contents in leaf tissue were similar to that in original line SR1.

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The first step of plant-specific protein O-glycosylation is the hydroxylation of proline residues catalyzed by prolyl 4-hydroxylases (P4Hs). It is necessary to prevent plant-specific glycosylation to produce functional non-plant derived proteins in plants. In this study, we investigated the effects of the inhibition and suppression of P4Hs in tobacco on growth and O-glycosylation of ectopically expressed sporamin, which is a sweet potato protein containing single O-glycosylation site.
The treatment of α,α'-dipyridyl, an inhibitor of P4Hs, suppressed the growth of tobacco plants significantly. To repress the expression of each type of P4H in tobacco, we transformed tobacco with RNAi constructs against either type 1 or type 2 P4H. We obtained transgenic plants whose mRNA level of either of the P4Hs were partially decreased. In these plants, no obvious growth defects were observed. Then, we crossed them with each other or with a tobacco plant expressing sporamin, and obtained progenies. We will report the effects of P4H inhibitor and the reduction of the expression of P4Hs on plant growth and protein O-glycosylation.

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Sucrose transporters play a pivotal role for this process. To understand the uptake of sucrose in plant cells we investigated the intercellular localization of tobacco sucrose transporter (NtSUT) orthologs, nameley NtSUT2 and NtSUT4 found in tobacco EST database. Microsomes prepred from tobacco BY-2 cells were separated by a sucrose density gradient centrifugation and used to analyze the distribution of these proteins by immunoblotting. Distribution pattern of NtSUT2 protein suggested that this protein is localized in the Golgi apparatus. In contrast, NtSUT4 was cofractionated with a marker protein of vacuolar membrane (VM). We next expressed the GFP or RFP fusion of these proteins in tobacco cells. The florescence of tagged NtSUT2 co-localized with that of a marker for trans-Golgi network, SYP41-YFP. The tagged NtSUT4 showed the same distribution with SYP21-YFP, which is localized with vacuolar membrane. We next found that both endogenous and tagged sucrose transporters were degraded under several nutrient-staved conditions. The degradation of NtSUT2 in sucrose-starved condition was prevented in the presence of autophagy inhibitors, E64 and 3-methyladenine, respectively.

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Recently, several approaches have been adopted to improve Agrobacterium-mediated transformation of rice in order to generate a large number of transformants needed for gene targeting studies. We have succeeded in establishing a highly efficient transformation system in rice by co-cultivating suspension-cultured small calli with Agrobacterium on a filter paper moistened with N6 media containing the extract of the calli. The calli were subcultured by transferring 0.2 ml (packed cell volume) of the calli into 15 ml fresh media in 100 ml Erlenmeyer flasks every three days. After 3 weeks, the calli were used for transformation. The bacteria were collected and suspended in AA medium containing the extract of calli. For Agrobacterium infection, the density of the bacteria was adjusted and the calli were washed with N6 media containing the extract of calli, immersed in a bacterial suspension. The calli were transferred on a filter paper moistened with N6 media containing the extract of calli. Efficiency of transformation was about 60 times higher than that of calli co-cultured in N6 co-cultivation medium without the extract of calli.

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Mimosa pudica rapidly folds its leaves when exposed to various external stimuli, such as mechanical, thermal and electrical stimulations. Although the mechanism of this unique leaf movement has attracted wide attention of botanists for centuries, molecular and genetic studies have been hampered by the lack of gene manipulation system. Here we report methods for reproducible plant regeneration and Agrobacterium-mediated transformation to generate stable genetic transformants. First, we determined the conditions for shoot and root induction. The highest frequency of shoot regeneration was observed when the cotyledons were cultured on half-strength MS medium containing appropriate NAA and BAP ratios. The regenerated shoots successfully rooted on half-strength Hoagland's medium. Secondly, we optimized transformation conditions. The highest transformation efficiency was obtained under the conditions in which the cotyledons scratched with a knife and the hypocotyls were sonicated, and subsequently transfected by A. tumefaciens strain LBA4404 harboring a super binary vector pSB111. These results serve as a basis for establishing an effective transformation system in M. pudica.

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With the advances of whole genome shotgun sequencing technique, many organisms have been sequenced and many more will be. However, the genome annotation, what gene are where on the genome, is yet a though process.
The draft genome of the moss Physcomitrella patens have been published with nearly 36 000 gene models (Rensing et al 2009). However, only 1/3 of the gene models had experimental support. We collected various transcript sequence data including full-length cDNA sequences, 3' EST with 454 system, and 5' SAGE data with oligo-capping methods to have a better annotation. Furthermore, we collected over 360 million 25-nt reads of mRNA 5' enriched tags and over 140 million 50-nt reads of equalized cDNA using the SOLiD system (Lifetechnologies).
The 50-nt reads were aligned with the reference genome, and we found that the intron can be recognized. We constructed a prototype system to construct gene models using these data. We are developing this system to combine other data and process whole genome.

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To identify transcription factors (TFs) that would interact with 5' upstream region of a gene, yeast one-hybrid screening (Y1H) is often used. For Y1H, we need to identify cis-regulatory region and multimerize it for placing them in upstream of reporter gene. In addition, large-scale screening is required because non-TF genes mostly occupy cDNA library. To overcome such problems, we constructed novel cDNA library that only contains TF genes. Results of our test experiments showed that several transcription factors were identified with efficiency more than 100-fold even though whole 500-1000bp promoter region was directly used as a probe. However, false-positive or false-negative result was sometimes observed in the yeast experimental system. Therefore we are now trying to develop new method using plant instead of yeast. We will report the results of comparison of these methods to identify upstream transcription factors.

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We have developed a methodology for extraction of promoter constituents based on distribution profile of short sequences (Yamamoto et al, BMC Genomics 8: 67 2007). One group of the extracted elements is named as Regulatory Element Group (REG) due to shared characteristics with known transcriptional regulatory elements. Because biological roles of considerable amount of REGs are not known, information of these functions is quite helpful for understanding plant promoters. In this article, we will report our recent activities for massive identification of transcriptional regulatory elements. Our analysis includes prediction of regulatory elements using private and public microarray data, construction of synthetic promoters driving a luciferase-based reporter gene, validation of in vivo function of the predicted elements. We hope our analysis will reveal function of REGs and identify novel regulatory elements that belong to non-REG type.

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We have developed a web-based analysis tool, KaPPA-View, which enables to view transcriptome and metabolome data on the same metabolic pathway maps (Tokimatsu et al 2005, Plant Physiol 138:1289-1300). Recently we released the newest version, KaPPA-View4, whose features were as follows.
1) Dramatically Improved Speeds: We totally reviewed the program architecture and successfully improved the processing speeds for all the analysis steps.
2) Support for the Macintosh OS: The SVG Viewer plug-in is no longer needed for map representation, enables to provide the same analysis environment for Windows, Macintosh, and Linux users.
3) Analysis with User Maps: Users can prepare their own pathway maps with the free drawing software "Inkscape", and utilize them for data analyses.
4) Utilization as an External Viewer from the Other Systems: KaPPA-View4 provides a function for direct data uploading and viewing from the other application programs without logging-in to KaPPA-View4 through the web page. This function enables developers of omics databases to utilize KaPPA-View4 as an external viewer to represent their data on the pathway maps.
KaPPA-View4: http://kpv.kazusa.or.jp/kpv4/

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We show two analytical conditions for CE/MS analysis with a fused silica capillary column to profile anionic metabolites at high speed and in high resolution. The first one aimed simultaneous analysis of a number of major anionic compounds including organic acids, sugar phosphates, nucleotides and coenzymes. Under this condition where ammonium formate (pH 8.0) was used as electrolyte, a standard mixture including 35 compounds could be analyzed less than 17 minutes, indicating that this is a rapid method comparable with the conventional method with polymer coated capillary column. However, the peaks of pentose phosphate isomers and hexose phosphate isomers were overlapped under this analytical condition. Therefore, we also employed another analytical condition where a mixture of ammonium acetate (pH 10.0) and methanol was used as electrolyte. Under this analytical condition, peaks for isomers could be separately determined; e.g. R5P, Ru5P, F6P, G1P, G6P. Gal1P and M6P. Taken together with the results of analyses of Arabidopsis leaf extracts and extracts from moss, we will discuss how these analytical conditions are useful in precise profiling of anionic metabolites.

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Using UPLC-TQMS, we have been established the widely targeted metabolomics based on the large scale MS/MS data (multiple reaction monitoring, MRM) of approx. 1,000 of standard compounds [1]. Moreover, we enhanced the method, and applied it to plant bioresoureces [2, 3]. But many plant metabolites were unknown, and the standard compounds were not available. Thus, the un-targeted MS/MS data (MS2T) for unknown plant metabolites were established by Dr Matsuda (http://prime.psc.riken.jp/) [4, 5]. This detected MS2T can convert to the predicted MRM conditions. In this study, the 2,788 MS2T data, which collected from Arabidopsis tissue samples, were converted as total 50,184 of predicted MRM, and we successfully confirmed high sensitivity- and selectivity- detection. More than 70% of MS2T was identified as the detectable MRM. These results suggested that the optimized MRM conditions from standard compounds and MS2T allowed us the ultra widely targeted metabolomics.
[1-3] Sawada et al., PCP2009a-c, [4] Matsuda et al., Plant J 2009, [5] Akiyama et al., In Silico Biol 2008

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Genetic modification (GM) is a recent development which allows specific genes to be identified, isolated, copied and inserted into other plants with a high level of specificity. Metabolomics approach, especially MS-based metabolomics approach, is useful to estimate unintended effects on the metabolome by the induced gene(s) in the modified plants, because of the wide-coverage of detection of metabolites. In this study, we conducted metabolite profiling using multiple MS platforms to assay the substantial equivalence of the control tomato (cv. Moneymaker) and the genetically modified tomato, which are over-expressing miraculin in a tomato fruit. We grew tomato plants under two different nutrition conditions, soil and hydroponic culture system. Tomato fruits were harvested on 2006, 2007 and 2008. Metabolite profiles obtained from the three different MS platforms were summarized automatically by MetMask (the metabolitemasking tool), which has been developed as a practical and flexible solution. We will present the results of comparison of GM tomato and other 6 cultivars including Moneymaker.

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The analysis of metabolites in single organelles has consequently presented a significant challenge. At the previous meeting, we reported the development of a single cell metabolomic analysis system by applying a single vacuole and cytoplasm isolated from a single giant internodal cell of the alga Chara corallina to the CE-MS (capillary electrophoresis / mass spectrometer) -based metabolomic technique. We determined that metabolite compartmentalization in vacuole and cytoplasm of C. corallina was different under changing light conditions. We are now investigating localization and dynamics of metabolites in a single cell under various environmental conditions such as pH and temperature. By using a microinjection technique, metabolite transportation across the vacuolar membrane can be directly detected, suggesting a previously unknown function of the vacuole. Our metabolomics approach provides novel insights on metabolic dynamics in a single organelle derived from a single cell. This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN).

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Vacuole plays an important role in maintaining the homeostasis of plant cell through the regulation of development of turgor pressure, the accumulation of various inorganic ions and metabolites and the degradation of discarded proteins. We have analyzed the contents of intact vacuoles directly and comprehensively by using CE-MS and FT-ICR-MS. The findings reveal the existence of organic phosphate compounds in the vacuole despite the presence of phosphatases. We have also prepared the transgenic Arabidopsis suspension-cultured cells over-expressing certain unknown tonoplast proteins and conducted the comparative analysis of the vacuolar metabolites of the wild-type and transgenic cells in order to elucidate the possible functions of the unknown membrane proteins. In the present study, possible mechanism of the entry of the phosphate compounds into the vacuole, using the genetically manipulated vacuoles, has been explored.

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Metabolome analysis using mass spectrometry such as GC-MS or LC-MS has been developed to identify metabolites in plants with an improved efficiency. Recently, CE-MS is reported to be a useful tool to identify plant metabolites, especially the charged species.
In this study, we performed a simultaneous quantitative analysis of metabolites such as various primary metabolites including amino acids, organic acids and phosphate compounds in A. thaliana shoot and root at different growth stages by using CE-ESI-QTOF. We also attempted the quantification of plant hormones together with primary metabolites. In case of non-target analysis in positive mode, 548 and 604 peaks were found in shoot and root, respectively. Of these, 25 and 23 compounds in shoot and root respectively could be identified e.g. as amino acids. In negative mode, 199 and 214 peaks in shoot and root respectively were identified. Of these, 7 compounds in shoot and 10 compounds in root were identified; these included organic acids, phosphate compounds and others. Indole-3-acetic acid (IAA) was also identified simultaneously with primary metabolites.

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Plants have developed their own defense strategies, because they have no immune cells. Here we provide a novel mechanism underlying cell-autonomous immunity, which involves the fusion of membranes of a large-central-vacuole with the plasma membrane, resulting in the discharge of vacuolar antibacterial proteins to the outside of the cells where bacteria proliferate. The extracellular fluid that was discharged from the vacuoles of infected leaves had both antibacterial activity and cell-death-inducing activity. Membrane fusion was triggered in a proteasome-dependent manner by the bacterial infection. This novel membrane fusion-based defense strategy provides plants with a mechanism for attacking extracellular bacterial pathogens.

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While bacteria secrete avirulence gene products called effectors, plants use resistance-gene products to recognize those effectors with strict specificity. After their recognition, plants induce a variety of defense pathways so-called "gene-for-gene" resistance. In this study, an EMS-mutagenized line of Arabidopsis thaliana expressing dexamethasone (DEX)-inducible avrRpt2, an effector of Pseudomonas syringae pv. tomato (Pst. ), was employed for searching a novel mutant with altered hypersensitive response (HR).
Under DEX treatment, one line out of approximately 1,000 mutated seeds showed no HR-induced cell death with moderate susceptibility to virulent bacteria Pst. DC3000. HR-induced cell death could not be detected in this mutant when it was inoculated with avirulent Pst. DC3000 expressing either avrRpm1 or avrRpt2; however, it was highly susceptible only to avrRpm1, but not to avrRpt2. This mutant was named asavrRpt2 specific resistance with no cell death 1 (anc1). To date, such phenotype, HR-alternation with extremely high susceptibility to a specific effector, has not been reported, and it may give new insight into the plant defense system.

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Ralstonia solanacearum causes bacterial wilt in several economically important solanaceous. To elucidate the molecular mechanisms of plant-R. solanacearum interactions, we isolate and analyze R. solanacearum-responsive genes from Nicotiana plants. In this report, we focused on NbSec14P with similarity to sec14p from yeast. Introduction of NbSec14P rescued temperature-sensitive growth mutant of yeast. Expression of NbSec14P was strongly induced Nicotiana benthamiana leaves inoculated with non-adapted R. solanacearum. In NbSec14P-silenced plants, growth of non-adapted R. solanacearum was accelerated. Bacterial growth and wilt symptoms by adapted R. solanacearum were also accelerated in the silenced plants. In the silenced plants, expression of EREBP and PR-4 genes was compromised. Activities of phospholipids metabolism-relate enzymes such as phospholipase C and D were also reduced in NbSec14P silenced plants. These results indicate that phospholipid metabolisms might have a role in regulation of the defenses in Nicotiana plants.

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Plasma membrane NADPH oxidase, RBOH (Respiratory Burst Oxidase Homolog), plays a pivotal role in reactive oxygen species (ROS) production in response to pathogen attacks. We indicated that a potato calcium-dependent protein kinase (StCDPK5) activates StRBOHB by direct phosphorylation of the N-terminal region (Kobayashi et al. 2007 Plant Cell). However, how StCDPK5 recognizes StRBOHB as a substrate remains to be defined. Tomato SlCDPK2 was identified as a CDPK that phosphorylates ACC synthase (SlACS2). Agrobacterium-mediated transient expression of SlCDPK2 did not induce StRBOHB-mediated oxidative burst in Nicotiana benthamiana, whereas bimolecular fluorescence complementation (BiFC) analyses showed that both StCDPK5 and SlCDPK2 interact with StRBOHB on plasma membrane. To determine the domains required for StRBOHB substrate specificity, a series of chimeric genes was constructed by reciprocal exchange of domains between StCDPK5 and SlCDPK2. The expression of chimeric genes including both variable and kinase domains of StCDPK5 induced StRBOHB-mediated oxidative burst. These results indicate that variable and kinase domains of StCDPK5 are responsible for activation of StRBOHB.

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PVS (potato vetispiradiene synthase) is a key enzyme for synthesis of isoprenoid phytoalexins lubimin and rishitin in potato. Complementary DNAs encoding PVS1 to 4 were isolated. The role of potato phytoalexins in defense response has not been fully understood. We developed transgenic potato plants knocked down PVSs by RNAi. Phytoalexin acumulation was deficient in the transgenic potato tubers after inoculation with Phytophthora infestans. Transgenic tubers showed trailing necrosis like cell death to infection of avirulent P. infestans and high susceptibility to virulent P. infestans. In wild-type leaves, avirulent P. infestans failed to penetrate the epidermal cells and HR cell death was observed in the attacked cells. In contrast, secondary hyphae and HR cell death were observed around mesophyll cells in RNAi leaf-avirulent P. infestans interactions, even though phytoalexin acumulation has not been reported in wild-type leaves. In addtion, RNAi leaves increase disease susceptibility to virulent P. infestans. These results suggest that phytoalexins contribute to disease resistance against P. infestans with other defense-related factors in not only potato tubers but also leaves.

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Plant bacterial pathogens equipped with the type III secretion system (TTSS) generally deliver different TTSS effector proteins into plant cells. These TTSS effector proteins modulate the function of crucial host regulatory molecules and allow bacteria to invade plant cells. So far, we have isolated 16 TTSS effectors from Xanthomonas oryzae pv. oryzae (Xoo ; Furutani et al.,). To identify Xoo TTSS effectors that inhibit host immune responses, we generated transgenic rice plants expressing each of 10 Xoo effectors. These transgenic plants were inoculated with the TTSS-deficient hrpX mutant of Xoo. Transgenic plants expressing Xoo effectors showed different levels of susceptibility to the hrpX mutant, indicating that these effectors have the abilities to inhibit PAMPs-triggered immunity (PTI) inside plant cells. Especially, transgenic plants expressing four of 10 effectors developed severe disease lesions of the hrpX mutant. Thus, it is likely that these four effectors may block the important steps of PTI in rice. Investigation of target protein in rice using yeast two hybrid system is in progress.

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In plants, perception of pathogen-associated molecular patterns (PAMPs) triggers innate immune responses that contribute to disease resistance. Previously, we showed that the small GTPase OsRac1, belonging to the Rac/Rop GTPase family, is a key regulator in rice defense signaling. However, the spatiotemporal dynamics of OsRac1 activation during the defense response is unknown. Here we report the development of an intracellular fluorescence resonance energy transfer (FRET) biosensor termed Raichu-OsRac1 that facilitates the in vivo monitoring of OsRac1 activation by elicitors. We showed that N-acetylchitooligosaccharide elicitor, a PAMP derived from the rice blast fungus, induced OsRac1 activation on the plasma membrane of rice protoplasts rapidly after treatment using Raichu-OsRac1. Moreover, we identified a guanine nucleotide exchange factor (GEF) that interact with OsRac1 by yeast two-hybrid assay. We confirmed that this GEF can activate OsRac1. Our results also indicated that the GEF interacts with a receptor like kinase. These data suggest that activation of OsRac1 by PAMP occurs at an early step in rice innate immunity.

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Plants have evolved innate immunity systems to fight pathogen infection. Pathogen-associated molecular patterns (PAMPs) receptors are receptor-like kinases (RLKs) in plants. However, the maturation and transport of PAMP receptors are not well understood. Here, we characterize the Hsp90 co-chaperone Hop/Sti1, a novel OsRac1 interactor. Hop/Sti1 and cytoplasmic Hsp90 bind the rice chitin receptor OsCERK1 in the endoplasmic reticulum (ER). The OsCERK1 complex is then transported to the plasma membrane (PM) via the Sar1-dependent trafficking system. Impairment of Hop/Sti1 and Hsp90 function reduces the efficiency of OsCERK1 transport to the PM. Hop/Sti1a acts in chitin-triggered defense and rice blast resistance. Our results suggest that Hop/Sti1 and Hsp90 may function to link PAMP receptors and Rac/Rop GTPases. The Hop/Sti1a-Hsp90 chaperone complex appears to have a highly conserved role in the maturation and transport of receptors and ion channels in plants and animals.

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We have previously shown that OsRac1, a small GTPase in rice, is involved in defense responses. We isolated OsRac1-interacting proteins and showed signaling network with OsRac1. Now, we analyzed whether the OsRac1 complex is composed and how it behaves. Cell cultures expressing constitutively active form of OsRac1 (CA-OsRac1) or dominant negative form of OsRac1 (DN-OsRac1) fractionated by gel filtration were analyzed. The CA-OsRac1 complex formed a higher molecular weight complex than DN-OsRac1 complex. Next, elicitor-treated cell cultures expressing wild type form of OsRac1 were analyzed. Results indicate that the OsRac1 complex forms a larger complex at 10 minutes after elicitor treatment, and the complex is dissociated at 20 minutes. Defensome components in fractions of high molecular weight complex were not altered by elicitor treatment. And we show OsRac1 is activated by elicitor treatment. These results suggest that components of the OsRac1 complex except for OsRac1 form a protein complex first, and when defense signaling is triggered by elicitors, OsRac1 forms a larger complex. After signaling is terminated, OsRac1 is likely to be dissociated from the preformed complex.

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Plant disease resistance (R) proteins act as intracellular receptors for sensing pathogen invasion, and trigger a variety of immune responses. However, the molecules that are activated by R proteins, and how these downstream molecules induce a variety of immune responses, are largely unknown. Here, we found that the small GTPase OsRac1 directly interacts with Pit, a nucleotide binding-leucine-rich repeat-type R protein that confers resistance to the rice blast fungus, at the plasma membrane. OsRac1 contributes to Pit-mediated reactive oxygen species production as well as the hypersensitive response, and is required for Pit-mediated disease resistance in rice. Furthermore, in vivo fluorescence resonance energy transfer experiments indicated that the active form of Pit induces the activation of OsRac1 at the plasma membrane in rice protoplasts. Thus, OsRac1 is activated by R proteins during pathogen attack, and this activation may play a critical role in R protein-mediated immunity in rice.

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One of the earliest plant immune responses is the oxidative burst, in which levels of reactive oxygen species (ROS) rapidly increase. ROS generation after recognition of avirulent strain was strongly suppressed by Ca²⁺ chelating agent or kinase inhibitor. In order to investigate the Ca²⁺ dynamics during plant immune responses, yellow cameleon 3.6 was transiently expressed in cultured rice cells. When the avirulent strain was infected to the rice cells, rapid increase of Ca²⁺concentration was observed, while a notable change was not observed by virulent strain. The data indicated the possibility that calcium-dependent protein kinase (CPK) possessing EF hand motifs and kinase domain was one of the candidate molecules involved in induction of immune responses. Among 29 CPK genes, 6CPKgenes were induced during plant immune responses. RNAi knock-down experiments showed that OsCPK12 regulated the ROS generation during the immune responses. Interaction between OsCPK12 constitute active form and Osrboh (rice respiratory burst oxidase homologs) which is involved in ROS generation was examined using BiFC technology. OsCPK12 interacted with N-terminal domain of OsrbohA.

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The hypersensitive response (HR) is a common feature of plant immune responses and a type of programmed cell death. We recently reported that plant-specific transcription factor OsNAC4 is a positive regulator of HR-cell death in rice. To clarify the induction mechanism of HR cell death mediated by OsNAC4, we first examined the subcellular localization of OsNAC4 during HR cell death. OsNAC4 was translocated into the nucleus during HR cell death in a phosphorylation-dependent manner. N-terminal and C-terminal region of NAC domain was important for the regulation of OsNAC4 translocation. Gene expression profiling comparing rice cells and OsNAC4 knock-down transformants using a rice cDNA microarray showed that OsNAC4 controls the transcription of at least 139 genes. To clarify the transcriptional regulatory mechanism, yeast two-hybrid analysis using OsNAC4 as bait was performed. OsNAC3, a member of the same subfamily with OsNAC4, was specifically interacted with OsNAC4. When OsNAC4 was overexpressed in rice, clear HR cell death induced. These data suggest the possibility that dimerization between OsNAC3 and OsNAC4 is necessary to the transcriptional regulation of HR-related genes.

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In higher plants, cis-zeatin (cZ) has been considered as low- or in-active cytokinin, based on traditional bioassays of cytokinin and studies using Arabidopsis. However, cZ and cZ derivates are accumulated in some plant species including maize and rice. In contract to known cytokinin receptors of Arabidopsis, maize cytokinin receptors are sensitive to cZ. Thus, further investigations of cZ is required to understand generality and diversity of the cytokinin metabolism and function in higher plants. In rice, cZ-O-glucoside (cZOG) and cZ riboside-O-glucoside (cZROG) are accumulated in high levels. In vitro assay of O-glucosyltransferase activity using recombinant protein showed that UDP-glucosyltransferase proteins encoded by at least three genes can catalyze O-glucosylation of cZ and cZ riboside (cZR). In transgenic rice plants ectopically overexpressing the each gene, accumulation of cZOG or cZROG was likely to be up-regulated. Together, we conclud that these UDP-glucosyltransferase proteins should function as cZ- or cZR-O-glucosyltransferases in rice.

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We conducted detailed quantification analysis of abscisic acid (ABA) by LC-MS/MS during Arabidopsis seed development. Determination of ABA levels in individual F₂ seeds generated by crossing wild type and an ABA deficient mutant aba2 showed that ABA accumulated in seeds during mid development is derived from maternal tissues. Despite active ABA biosynthesis in maternal tissues in this stage, ABA was highly accumulated in the embryo, suggesting that ABA is transported to the embryo from other tissues. On the other hand, during later stages of seed development, ABA is mainly synthesized in the embryo and/or endosperm although the amount is much lower than that synthesized during middle stage. We identified genes whose expression was regulated by endogenous ABA during seed development by microarray analysis and compared their expression levels in mutants that exhibit different degree of ABA deficiency depend on tissues. It was shown that the expression levels of ABA-regulated genes did not always correlate with endogenous ABA levels in whole seeds, suggesting that ABA synthesized in different tissues has distinct role on downstream gene expression.

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Phytohormones are key players in regulation of plant growth, development and defense response. In this study, we conducted comprehensive analysis of phytohormones regulating metabolic pathways using integrated omics data collected from Arabidopsis mutants of basic phytohormones, Auxin, Cytokinin, Gibberellin, Abscisic acid, Ethylene, Jasmonic acid and Brassinosteroid.
We used 28 hormone mutants and 4 wild types to collect gene expression profiles and metabolome profiles. The seven-day-old seedling grown in 1/2 MS medium were analyzed by DNA microarray (Affymetrix ATH1 GeneChip) and by LC-Q-TOF/MS, GC-TOF/MS, LCMS-IT-TOF/MS and CE-TOF/MS. Omics profile of each mutant was characterized by Ontology analysis. Furthermore, gene expression responses and metabolite accumulations related to specific hormone mutants were identified. We also analyzed co-expression pattern between genes and metabolites to extract metabolites accumulated in hormone-related pathways. We will present relationships between phytohormone functions and metabolic pathways.

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Raphanusanin (Ra) is a light-induced growth inhibitor involved in the inhibition of hypocotyl growth in response to unilateral blue-light illumination in radish seedlings.
Knowledge of the roles of Ra still remains elusive. To understand the roles of Ra and its functional coupling to light signaling, we constructed the Ra-induced gene library using the Suppression Subtractive Hybridisation (SSH) technique and present a comparative investigation of gene regulation in radish seedlings in response to short-term Ra and blue-light exposure. The comparative analysis showed that, among the transcriptional profiles of 33 highly Ra-inducible genes, 25 ESTs were commonly regulated by different intensities and duration of blue-light irradiation. The transcriptional profiles, coupled with the transcriptional regulation of early blue light, have provided the functional roles of many genes expected to be involved in the light-mediated defence mechanism. The biological role of Ra in plant resistance will be discussed.

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In recent research for bioactive substances of Arabidopsis thaliana , seven new oxylipins named arabidopsides A- G were isolated from the aerial parts of this plant. Arabidopsides A- G were monogalactosyl diacylglycerides or digalactosyl diacylglycerides containing 12-oxophytodienoic acid and/or dinor-oxophytodienoic acid which were known as precursors of jasmonic acid (JA) and have received much attention because they play important roles in regulation of developmental and defense gene expression of plants as JA and methyl jasmonate. On the other hand, it is known that plant volatile compounds like C6 aldehydes, alcohols, and acetates are released from plants in response to mechanical wounding and herbivore attack. In this research, we investigated the effect of volatile compounds as potent inducers for triggering accumulations of arabidopsides A , B, E, and G in mature leaves of A. thaliana . In addition, volatile compounds-induced arabidopsides were not detected in the some JA- signaling and JA- synthesis mutants.

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We performed proteome analyses on the pod husk of cacao (Theobroma cacao), a tropical tree which is cultivated primarily for its beans for making chocolate. Protein identification was done by de novo sequence similarity data searching approach due to incomplete genome database. Easy and yet unambiguous interpretation of MALDI-TOF PSD spectra was achieved with the aid of N-terminal sulfonation of tryptic peptides using 4-sulfophenyl isothiocyanate. Currently, we have identified 51 proteins from 89 major proteins analyzed. We also performed differential proteome analyses between two cacao clones, LAFI7 and ICS39. LAFI7 is a resistant clone against Conopomorpha cramerella, the most destructive insect pest of cacao in South-East Asian countries, while ICS39 is the most susceptible clone. Several spots in 2-DE have been found to be differentially expressed, specifically, L212 (24.7 kDa/6.3) in LAFI7 and i212 (21.4 kDa/6.4) in ICS39. We successfully de novo sequenced four tryptic peptides that are shared by both proteins. Database searches show that L212 and i212 have strong similarity to ASR proteins.

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It is often observed that a tree that had once penetrated a mesh of a chain-link fence has continued the thickening growth for long years to engulf part of the fence. We obtained a specimen of a tree trunk containing engulfed part of a fence. The trunk was from chinese elm (Ulmus parvifolia) with a diameter of about 25 cm, and the angle between the trunk axis and the fence plane was about 38 degree). An X-ray image of the specimen demonstrated for the first time that the metal wire of the fence engulfed by the trunk did not disappear nor was rotten, but stayed intact with some deformations. 2) X-ray CT scan images were obtained at 1 mm intervals along the axis perpendicular to the fence plane. The growth ring formation was highly disturbed by the wounding caused by the fence wires and then recovered a few years later. 3) Based on the results of the three-point bending test of the fence wire (JIS G3543 V-GS2) the force of the thickening trunk pressing the wire at the start of the engulfment was estimated to be as high as 190 N (19.4 kgf), and the mechanical work done until then was estimated to be about 2.3 J.

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Lateral roots of Arabidopsis temporary grow obliquely relative to the gravity vector after the initiation and then grow downward. We study mechanism controlling growth direction of lateral roots by analyzing those of mutant hy5. In hy5 lateral roots, transition from oblique to downward growth is retarded. We built a mathematical model by expressing bending rate as a linear combination of sine and cosine of deviation of growth direction from gravity vector. The model fitted well to the plagiogravitropic root reorientation, implying that a mechanism sensing the two components of gravity separately contributes to the plagiogravitropism. We further estimated gravitropic set-point angle (GSA) based on the model and found that GSA declined with root elongation, and that the decline was retarded in hy5. Lateral roots of hy5 and wild-type grew more downward when antiauxin BH-IAA was applied. Because BH-IAA inhibits degradation of Aux/IAA, Aux/IAA accumulation may function in earlier transition to downward growth. We also screened for revertants of hy5 with earlier transition from oblique to downward growth in lateral roots. We found FtsH11 as a candidate for such suppressor genes.

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Plants sense various environmental stimuli and integrate each response to determine growth direction of organs. We have previously shown interference between gravitropism and hydrotropism in seedling roots of several plant species including pea, cucumber and Arabidopsis. To reveal the mechanism of this interference, in this study, we exposed roots of several agravitropic Arabidopsis mutants to the gradient of water potential induced by placing agar plates with different water potentials side by side and analyzed their curvatures. Roots of axr2 mutant that had a gain-of-function mutation in AXR2/IAA7 gene encoding transcriptional repressor of auxin-inducible gene and tir1 afb1 afb2 afb3 quadruple mutant that lacked auxin receptors were defective in hydrotropism, suggesting both tropisms required auxin response. Root curvature of aux1 mutant was greater than that of the wild-type 6 to 24 h after exposure to water potential gradient. Because aux1 mutant was defective in auxin influx carrier, our results suggested that interference between gravitropism and hydrotropism depended, at least in part, on the amount of intracellular concentration of auxin.

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A number of genetic studies have identified many shoot gravitropism mutants in Arabidopsis, leading to findings of proteins which are involved in gravitropic response. But details of gravity perception and later signal transductions still remain unknown. A transcriptional repressor MSG2/IAA19 is involved in auxin responses in Arabidopsis. To identify regulators of IAA19 expression, we performed screening of EMS mutagenized lines of IAA19promoter::NLS-GFP and IAA19promoter::GUS transgenic plants. We isolated a novel mutant of shoot gravitropism, A78. It showed lower GFP signal intensity than WT, and real-time PCR analyses confirmed this result though its auxin responsibility was kept intact. Although A78 phenotype resembled to the agravitropic mutant eal1 (Fujihira et al. 2000) which lacks endodermal amyloplasts in hypocotyls, A78 contained as many endodermal amyloplasts as WT. Genetic analyses revealed that A78 was a recessive mutation. We mapped this mutation in a novel gene in chr5. A T-DNA insertion mutation in this gene did not complement the gravitropic defects of A78. Furthermore, a T-DNA mutant of A78's paralog showed abonormal phenotypes including spiral leaf petioles.

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When cucumber seeds germinate in a horizontal position, resulting seedlings develop a protuberance, termed the peg, on the lower side of the root-hypocotyl transition zone (TR zone), due to gravistimulation. We previously showed that auxin induced peg formation and proposed that decrement of auxin in the upper side caused by auxin efflux induces peg suppression. To reveal the mechanism how gravistimulation induces this asymmetric auxin redistribution in the TR zone, we analyzed the localization of CsPIN1, an auxin efflux facilitator expressed in endodermis of the TR zone, by immunohistochemical method. The results showed that CsPIN1 localization pattern was symmetrical in the endodermis of the TR zone in vertically grown seedlings. In contrast, in cucumber seedlings grown in a horizontal position, prominent CsPIN1 signals were detected in the endodermis in the upper side of the TR zone, all of which were localized at the lower membrane. These results suggest that the change in localization pattern of CsPIN1 due to gravistimulation promotes auxin efflux from endodermal cells in the upper side so that induces decrement of auxin in the upper side of the TR zone in cucumber seedlings.

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State transition is a photoacclimation mechanism for photosynthetic machinery. We previously isolated a protein supercomplex composed of photosystem (PS) I, its light-harvesting complex I (LHCI), and PSII light-harvesting complex II (LHCII) from a green alga Chlamydomonas reinhardtii in State 2. In this study, we tried to isolate a larger protein complex by using a different detergent, tridecyl maltoside. The results of sucrose density gradient and Western blot analysis indicated that the purified sample contained Cyt b₆f, FNR, and PGRL1 in addition to previously isolated PSI, LHCI, and LHCII. Gel filtration and disassembling experiments showed that all of these proteins are associated and forming a single protein supercomplex. The analyses of mutants (PSI-His, ΔCyt b₆f, ΔPSI) also supported such a protein supercomplex in State 2 wild-type cells. All of these protein components have been known to be essential for cyclic electron flow. Therefore, the isolated protein supercomplex in this study could drive cyclic electron flow. We will report the results of biochemical and kinetic analyses of this newly isolated supercomplex in this and next presentation, respectively.

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State transitions and cyclic electron flow (CEF) around photosystem (PS) I are flexible regulatory mechanisms in photosynthetic machinery. The coupling of State 2 transition and CEF by unknown mechanism has been estimated from their simultaneous activations in a green alga Chlamydomonas reinhardtii. From our recent results, it was revealed that Cyt b₆f and some other components in CEF form a supercomplex with PSI during State 2 transition. In this study, CEF activity in isolated PSI supercomplex was evaluated by spectroscopic analyses. By a light excitation of PSI, lumen side and stroma side electron transfer reactions were driven in the presence of mobile electron carriers (plastocyanin and ferredoxin) confirming that the isolated protein supercomplex was a functional CEF complex. We also observed that PSII antenna relocation from PSII to PSI was followed by CEF activation from in vivo analyses. We propose comprehensive regulation model of state transitions and CEF linked by a PSI supercomplex formation.

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