Under abiotic stress conditions such as drought and high salinity, ABA levels increase in plants, and ABA regulates the expression of many genes that function in the stress tolerance. In the promoters of such ABA-regulated genes, a conserved cis-element designated ABRE (ABA-responsive element), which controls ABA-responsive gene expression, has been identified. Arabidopsis cDNAs encoding bZIP-type transcription factors referred as ABRE-binding (AREB) proteins were isolated using the yeast one-hybrid screening method. Among these transcription factors, expression of AREB1, AREB2, and ABF3 was upregulated by ABA, dehydration, and high-salinity stresses in Arabidopsis plants. Based on results of analyses of both gain- and loss-of function mutants of AREB1, AREB2, and ABF3, here we report that these AREB family transcription factors are involved in the ABA/stress signaling. Moreover, taken together with the information on downstream target genes obtained by microarray analyses, we will discuss the role of each AREB transcription factor.

View full abstract

A glasswort (Salicornia europaea), one of the halophytes belonging to the Chenopodiaceae, accumulates a large amount of alkaline and alkaline-earth metals in vacuoles, and tolerates high salt environments. We examined in this study the accumulation of heavy metals in the shoot of S. europaea. S. europaea were aseptically grown on Murashige-Skoog solid media reduced in nitrogen content to one-tenth and supplemented with 0.3 M NaCl. Six-day old seedlings were supplied with one of heavy metals. Fresh weight, dry weight, and metal content of the shoot of S. europaea were estimated at 28 days after sowing. Application of Mn²⁺, Cd²⁺, Zn²⁺, Pb²⁺, Cr³⁺, Co²⁺, Ni²⁺, Cu²⁺, and Hg²⁺ at the concentrations of 10, 2, 1.5, 1, 0.5, 0.5, 0.5, 0.2, and 0.02 mM, respectively, inhibited 50% of the plant weight. S. europaea accumulated Mn²⁺, Cd²⁺, and Zn²⁺ of 5.5, 1.1, and 0.54 mg g^-1 dry matter in shoots, respectively.

View full abstract

The available iron (Fe) in the calcareous soils is very low because of high pH. Fe deficiency is the major agricultural problem all over the world. Graminaceous plants secrete mugineic acid family phytosiderophores (MAs) for Fe acquisition from the soil by chelating. Rice, which is the main crop in the world, secretes very few amount of MAs and consequently not tolerant to Fe deficiency. Barley secretes higher amount of MAs and is tolerant to Fe deficiency. We have produced several transgenic rice lines tolerant to Fe deficiency by introducing barley genes involved in MAs synthesis into rice. To further test the tolerance of these lines on farm, we performed a field experiment to evaluate these transgenic rice plants for two years. We have succeeded in the selection of the transgenic rice tolerant to Fe deficiency in the calcareous soils.

View full abstract

When incinerated ashes contact with water, leaching of chemical toxic factors such as high alkaline pH, salts and heavy metals may occur. We have assessed the safety of incinerated ash-leachates by giving them to rice plants. Consequently, high concentration of Cu was one of the toxic factors in ash-leachates. Thus we examined the effects of excess Cu to rice plant. Rice roots were treated with the hydroponic solutions containing 10 μM, 45 μM or 130 μM of CuCl₂ individually. After 1 day of the treatments, both photosynthesis and transpiration of leaves declined with increasing Cu concentration in the hydroponic solution. From the results of DNA microarray analysis, genes related to defense, lipid metabolism or stress response were up-regulated, and those related to photosynthesis or transport were down-regulated by Cu-doses. For the further discussion, accumulation of chemical elements in rice plants will examine. (Supported by the leading project of the MEXT.)

View full abstract

Environmental pH is a key factor influencing plant growth and development. In our previous study, we found that Arabidopsis germinated seeds or seedlings grow well over the pH range 4.2 to 10 and are capable of neutralizing acidified or alkalized culture media.
In this study, we examined whether proton pumps such as plasma membrane H⁺-ATPase, vacuolar H⁺-ATPase, and vacuolar H⁺-PPase, were involved in the neutralization of culture media. Treatment of seedlings with vanadate promoted the neutralization of acidic medium (pH 4.2), whereas fusicoccin slowed down it. In contrast, the application of vanadate to seedlings hindered the neutralization of alkaline medium (pH 10.0), and fusicoccin promoted it. These results suggest that plasma membrane H⁺-ATPase primarily functions in the neutralization of alkaline environments.
Using Arabidopsis mutants, we are now investigating the involvement of various transporters including plasma membrane Na⁺/H⁺ antiporter (SOS1) in medium neutralization.

View full abstract

Graminacious plants secrete mugineic acid family phytosiderophores (MAs) from their roots to solubilize the external insoluble iron and absorb the chelated iron. The secretion of MAs from root increases under Fe-deficient condition and followed the circadian rhythm. The particular vesicles derived from rER were observed to accumulate at the cell periphery near the cell membrane in Fe-deficient barley roots before sunrise. Immunocytochemical analysis on nicotianamine synthase (NAS), nicotianamine aminotransferase (NAAT), deoxymugineic acid synthase (DMAS) and IDS3, enzymes in MAs biosynthesis pathway, revealed that these enzymes were induced in Fe-deficient root epidermal cells. The subcellular localization of NAS and NAAT were determined to be on the membrane and inside of vesicle, respectively. DMAS and IDS3 were localized to the vicinity of the vesicle. We concluded that this particular vesicle could be related to the MAs synthesis. The polar vesicle transport may regulate the diurnal rhythm of MAs secretion from barley roots.

View full abstract

Reeds are perennial grasses which can grow in saline and keep shoot Na⁺ content low. To analyze Na⁺ distribution in reed, radioisotope tracer experiments were carried out. More ²²Na was distributed into stem and leaf sheath during short or long uptake periods. For pulse chase experiments, plants were loaded with ²²Na for 1 week under saline condition and transferred to chase solution with salt. The radio activities in root were quickly decreased during chase period, but these in shoot were not significantly decreased. Considering that shoot Na⁺ content of reed reach the maximum level at three days after salt treatment, our results suggested that reeds could keep Na⁺ content in shoot low due to restriction of Na⁺ transport from root to shoot rather than to retranslocation of Na⁺ from shoot. We will also discuss the real-time Na⁺ behavior using PETIS (Positron Emitting Tracer Imaging System) under continuous saline stress.

View full abstract

We have been established the Ni tolerant BY-2 cells, termed NIT cells. Recently, it was demonstrated that the increased nicotianamine (NA), which is a plant derived chelator of metals, can confer Ni tolerance to plants. To evaluate whether NA also contributes to the Ni tolerance of the NIT cells, the expression level of nicotianamine synthase gene, NtNAS, was determined by the northern analysis in the wild-type (WT) cells and the NIT cells. The accumulation of NtNAS mRNA in the NIT cells was considerably higher than that in the WT cells. Using the HPLC, we further determined the concentrations of NA in the both types of cells. Although the content of NA in the WT cells was below the detection limit, the NIT cells had extremely small amount of NA. We will further discuss the correlation between the Ni tolerance and NA among a dozen of the cloned NIT cells.

View full abstract

The Fox Hunting System (Full-length cDNA over-expresser gene-hunting system), a system for random over-expression of a normalized Arabidopsis full-length cDNA library, was applied for the functional analysis of stress-inducible transcription factors. An agro-library containing forty three stress-inducible transcription factors was constructed and used for transformation of Arabidopsis plants. Several salt-tolerant lines expressing F39 gene were screened from the T1 transgenic plants. T2 lines expressing F39 gene also showed obvious salt tolerance. Moreover, based on the microarray analysis and the analysis of loss-of-function mutants, an important role of F39 in salt stress responce will be discussed.

View full abstract

Reactive oxygen species (ROS) are easily generated by reduction of molecular oxygen in living cells. Because of ROS damaging cellular compounds, organisms have been developed multiple mechanisms of detoxification. Because bryophytes are considered as the first land plants, and adaptive responses to oxidative stress are crucial in their evolution. We analyze superoxide dismutase (SOD) which catalyzes the disproportionation of superoxide radicals. We found that copper repressed FeSOD activity in chloroplasts of a moss, Barbula unguiculata. This copper mediated repression involves regulation at the level of transcription. We cloned promoter region of FeSOD gene. Deleted analysis of FeSOD promoter using transgenic plants showed that the copper-mediated transcriptional repression element(s) were located between -318 to -199. Furthermore, two tandem copies of -318 to -259 or -258 to -199 conferred copper responsiveness on the heterologous actin promoter. We identified GTACT sequences within these regions are responsible for copper-mediated transcriptional repression.

View full abstract

High level Cd-accumulation in plant without toxicity is a specific and rare phenomenon known in only a few wild species. Further investigations on Cd-accumulation can contribute to the application of phytoremediation. In this point of view, we analyzed the characteristics of Cd accumulation and tolerance in Vicia villosa, a green manure crop. Two cultivars of V. villosa, two Vicia species and five legumes were treated with 50μM Cd hydroponically for 4 days. V. villosa represented higher Cd-accumulation than other legumes did. When V. villosa was grown under different Ca conditions (0.5-3mM) with 100μM Cd, 3mM Ca treatment enhanced the Cd accumulation and alleviated Cd toxicity in leaves. In 3mM Ca treatment, V. villosa showed drastic accumulation of both Cd and Ca after the 4th day of Cd treatment. Involvement of Ca accumulation pathways in Cd uptake by V. villosa was also examined

View full abstract

Cd-hyperaccumlulation is a rare phenomenon and only several wild plants are known as a Cd-hyperaccmumulator. The mechanisms involved in Cd-hyperaccumulationi are little understood. In our previous work, Avena strigosa was indicated as a novel Cd-accumulating crop with superior tolerance against Cd. We herein characterized the abilities of Cd accumulation and tolerance in different cultivars of A. strigosa and A. sativa. Plants were grown hydroponically in a growth chamber for 3 weeks and then, treated with ranged concentrations of Cd (0-100μM). Total Cd concentrations and Cd-distribution to the cell wall fraction were analyzed by ICP-MS. One cultivar of A. strigosa exhibited greater Cd accumulation in the leaves than other oats, sorghum and rice without showing toxicity. In leaves of this cultivar, superior capacity of apoplastic Cd accumulation as well as unknown vacuolar transport systems, were suggested to play a significant role in Cd tolerance.

View full abstract

Excess boron is toxic to living organisms, but its mechanisms of actions remains largely uncovered. We reported inhibition of splicing as one of the molecular mechanism of boron toxicity. As a new approach, we screened Arabidopsis mutants sensitive to high boron. Growth of Arabidopsis Col-0 was examined on media containing a range of concentrations of boron. Root growth was partly inhibited with 3 mM boric acid. 20,000 ethyl methanesulfonate mutagenized F2 seeds were grown on media containing 3 mM boric acid and the lines with reduced root growth were transferred to media containing normal (0.03 mM) concentration of boric acid. Those lines with recovered root growth were selected and progenies were tested for segregation, extents of phenotypes. Among the lines obtained, we selected seven lines for further analysis. At least four loci were represented among these lines.

View full abstract

Arsenic is a toxic element that is widely distributed in the soil and fresh water environments. Although arsenate has been used for screening of photosynthetic mutants in Chlamydomonas reinhardtiiby Prof. Togasaki, it is still unclear why photosynthetic mutants exhibit arsenate-resistance. In this study, we observed that cells incubated in the dark are more resistant to arsenate than in light. In the dark, the inhibition of photosynthetic activity by arsenate and the arsenate uptake were both about a half of those in light. Furthermore, in the presence of both phosphate and arsenate (1mM each), inhibition of phosphate uptake by arsenate and that of arsenate uptake by phosphate in the dark were larger than in light. This is considered to be the cause of the lower inhibition rate of photosynthetic activity in the dark than in light, suggesting photosynthesis has a positive effect on arsenate uptake.

View full abstract

In the early growth stage of etiolated Alaska pea seedlings germinated and grown in a horizontal or an inclined position under 1 g conditions, an increased auxin level estimated by auxin-inducible gene (PsIAA4/5) expression was found in the elongating side of epicotyls, resulting in bending to the direction of negative gravity vector. In epicotyls showing automorphosis under simulated microgravity conditions on a 3D clinostat, expression of PsIAA4/5 was observed all around epicotyls. Polar auxin transport (PAT) in the proximal side of epicotyls was responsible for the determination of the growth direction of epicotyls both under 1 g and simulated microgravity conditions. In agravitropic mutant, ageotropum, which shows automorphosis-like epicotyl bending, PAT in the proximal side of epicotyls was not affected by simulated microgravity conditions. These results suggest that PAT in the proximal side of epicotyls regulates auxin distribution, resulting in graviresponse in the early growth stage of etiolated pea seedlings.

View full abstract

The NPR1 plays a central role in salicylic acid (SA)-mediated systemic acquired resistance (SAR) in Arabidopsis. NPR1 is localized in the cytoplasm as an oligomer in uninduced state. Upon SAR induction or SA treatment, NPR1 translocates to the nuclei as a monomer leading to upregulation of its target genes. Overexpression of NH1, a rice ortholog of NPR1, increases rice resistance against a bacterial pathogen, Xanthomonas. However, the role of NH1 in defense responses of rice remains obscure.
We showed that RNAi-mediated knockdown of NH1 compromised BTH-inducible resistance against rice blast. Immunodetection using anti-NH1 antibody revealed that NH1 proteins were accumulated in the cytoplasm as a monomer. Moreover, NH1 localization was not affected by BTH treatments. These data strongly suggest that the mechanism controlling NH1 localization is different from that of NPR1 in Arabidopsis. Experiments are in progress to identify NH1-dependent BTH-inducible genes using gene expression profiling.

View full abstract

Arabidopsis disease resistance protein RPM1 recognizes AvrRpm1, a type III effector released from Pseudomonas syringae and activates defense responses such as rapid hypersensitive cell death at infection site and accumulation of antimicrobial compounds in systemic tissues. In spite of comprehensive genetic studies, its signaling cascade is still unclear. To overcome redundancy and lethality problems in genetics, we introduced chemical genomics, an alternative approach for gene identification using organic compounds that disrupt particular biological process in combination with functional genomics and biochemistry. The use of unbiased diverse small molecule libraries allowed us to target broad range of proteins. We established a cell death assay system suitable for high throughput screenings using Arabidopsis suspension cells. Twelve thousand chemicals provided from Chembridge and Microsource were screened and various cell death inhibitors and potentiators were isolated. These potentiators can be applied for agricultural agents that protect crops against various pathogens.

View full abstract

WRKY proteins are a large family of transcription factors that have been suggested to have a regulatory function in the response to pathogen infection and other stresses. OsWRKY53 is a chitin elicitor-induced rice WRKY gene that we isolated by microarray analysis. To examine whether OsWRKY53 activated transcription of genes, we performed a transient assay by particle bombardment method. As the results of the transient assay, it was suggested that OsWRKY53 functioned as an activator of target genes in vivo, and that the transactivation of the reporter gene was enhanced in response to elicitor treatment. On the other hand, we demonstrated that the elicitor-induced expression of OsWRKY53 was reduced in cpm2 that is thought to be a jasmonic acid (JA)-deficient mutant. This suggests that JA plays an important role in the elicitor-induced expression of OsWRKY53. Now, we are trying to identify an elicitor / JA-responsive region of OsWRKY53 promoter.

View full abstract

WRKY proteins are a large family of transcription factor that have been suggested to be involved in the responses to pathogen infection and other stresses. So far, we isolated two elicitor-responsive WRKY genes (OsWRKY53, OsWRKY71) from suspension-cultured rice cells. Using microarray analysis and quantitative RT-PCR, we revealed that several defense-related genes were upregulated in both 35S-OsWRKY53 and 35S-OsWRKY71 transgenic rice cells. We also showed by transient gene expression assays that OsWRKY71 possibly functions as a transcriptional repressor, while OsWRKY53 was suggested to function as a transcriptional activator. These results suggest that OsWRKY53 and OsWRKY71 cooperatively function in the regulation of expression of these defense-related genes in a unique manner. Identification of a repressor domain of OsWRKY71 is also now underway.

View full abstract

Induced disease resistance is activated by some stimuli and protect the whole plant from the attacks by various types of pathogens such as bacteria, fungi and viruses. In this study, we analyzed the effect of endophytes on disease resistance of host plants. Rice plant treated with endophytes exhibited resistance against the rice blast disease caused by Magnaporthe grisea . The detailed mechanisms of resistance induction by endophytes and suppression of disease development have been under investigation.

View full abstract

Production of C6-aldehydes in plants is induced after infection of pathogen. When Arabidopsis plants were treated with C6-aldehyde, expressions of several defense genes and accumulation of a phytoalexin were induced, and resistance against Botrytis cinerea was also enhanced. By monitoring the defensive response with (E)-2-hexenal-treatment in many Arabidopsis mutants, we found that some mutants were insensitive against the treatment. Mutation of glutathione synthetase I or glutathione reductase 2 (GR2) caused deterioration of response against C6-aldehydes in Arabidopsis. Total glutathione level is reported to contribute to some of defense responses in Arabidopsis. Furthermore, reduction of glutathione activates NPR1-dependent defensive response in Arabidopsis. However, (E)-2-hexenal-treatment resulted in no increase in total glutathione level and in transcriptional level of GR2 in Arabidopsis. (E)-2-Hexenal-induced defensive response might be thus independent of total glutathione amount or NPR1. Probably, the defensive responses depend on other pathway relating to glutathione synthesis.

View full abstract

Systemic acquired resistance (SAR) is one of plant defense systems and induced from the pathogen infection part through salicylic acid (SA) accumulation. In Arabidopsis, it is demonstrated that SAR induction is suppressed by environment stress response mediated by abscisic acid (ABA). In this study, the effects of ABA on SAR in tobacco plants were investigated. The resistance against tobacco mosaic virus (TMV) and the expression of SAR marker gene (PR-1) were induced by BTH and BIT, SAR inducers, however, these were suppressed by ABA treatment. In addition, the accumulation of SA by the BIT treatment was also suppressed by ABA.

View full abstract

The plant activator is very useful for plant disease control and management. However, development of the plant activator is very difficult and challenging because it has no toxic effect against fungi and bacteria and works only through the plant. We tried development of screening systems for plant activator using plant immune system. We developed a reporter gene-assay system using promoters of the defense-related genes. To validate the strategy, we performed subsequent analysis using an Arabidopsis microarray consisting of 1,200 full-length cDNA clones representing putative defense-related and regulatory genes. Furthermore, we prepared a B. rapa cDNA microarray using ca. 2 K cDNA clones selected from 2,166 non-redundant sequences of cDNA library of Chinese cabbage. We applied B. rapa microarray to evaluate the performance of Arabidopsis microarray for the screening of plant activators. In this report, we will suggest that this novel system can be used to screen for candidate plant activators.

View full abstract

Cylindrocladium black rot (CBR) of soybean is seen all over the world. Symptoms of CBR include chlorosis of the root, followed by complete wilting of the plants, and death. It is difficult to remove the pathogenic fungi, Cylindrocladium parasiticum, for its facultative parasitism. The control by cultivation and breeding of resistant varieties has been tried, but it is not yet enough. C. parasiticum exist in every soybean field of Japan, but actual disease incidences are usually not so high. C. parasiticum infect most of soybean plants up to 4 week after sowing, but the symptoms appear from 8 week after sowing. These facts suggest that soybean intrinsically has the resistant ability against to CBR. This histological study revealed that additional periderm developed and that it functioned enough to prevent the infection of C. parasiticum. We will discuss the conditions and the mechanisms for CBR outbreak.

View full abstract

We previously showed that the promoter of the Ri-plasmid-borne agropine synthase gene (Ri-ags) of Agrobacterium rhizogenes had tissue-specificity to root and wound-responsiveness in leaf and stem in tobacco. Because of very rapid emergence of mRNA after wounding and unnecessariness of de novo protein synthesis the wound response was thought to be a primary one. Because a signal transduction pathway for this wound response in tabacco was not elucidated, we planned to analyze it with Arabidopsis genetically. In this study, we introduced a fusion gene of the Ri-ags promoter and the GUS reporter into Arabidopsis, then investigated the expression of the fusion gene in T2 plants. As in a tabacco plant, a high GUS activity was observed in roots. But, no wound response was observed in leaves. Thus, in Arabidopsis the Ri-ags promoter is thought to be controlled differently than in tabacco.

View full abstract

Bacterial wilt is caused by a soil borne pathogen, Ralsotonia solanacearum, damaging the production of major crops such as potato, tomato, and eggplant. The infection occurs mainly at roots via wounds or secondary roots. In early stages of infection, the pathogens multiply in the vascular element and produce extracellular polysaccharides clogging their vascular system. The pathogenicity and molecular mechanisms of the infection of R. solanacearum have been well studied through molecular genetic approaches and the genome sequencing project, while information about plant responses during the infection or development of disease resistance is very limited. Here, we report genome-wide gene expression analyses performed with tobacco plants during the infection of either an incompatible- or a compatible-strain of R. solanacearum, using DNA array filter sets with 12,158 non-redundant tomato EST sequences. Changes in the metabolic profiles through transcriptome regulations are discussed as an integrated disease resistance response in plants.

View full abstract

The interactions between plants and incompatible pathogens often lead to hypersensitive response, in association with rapid and localized cell death, hypersensitive cell death, at the infected sites of host tissues. However, little is known about molecular mechanism that operates hypersensitive cell death in plants. We previously reported that a vacuolar processing enzyme is essential for the execution process of hypersensitive cell death through disrupting the vacuole (1-3). To investigate the involvement of the vacuolar system in hypersensitive cell death, we inoculated avirulent bacteria into Arabidopsis mutant plants that exhibited abnormal vacuolar system. In several mutants, the hypersensitive cell death was delayed than in wild-type plants. We discuss the involvement of vacuolar system in plant defense systems.
(1) Hatsugai et al. (2004) Science, 305, 855.
(2) Hara-Nishimura et al. (2005) Current Opinion Plant Biol., 8, 404.
(3) Hatsugai et al. (2006) Apoptosis 11, 905.

View full abstract

We have isolated nodule specific genes from Astragalus sinicus which forms indeterminate-type nodules. One of the clones, AsNODf32, encodes a cysteine protease that strongly expresses in a senescent zone of the nodules. It is important to compare AsNODf32 with homologues from other legumes that form determinate-type nodule for understanding the functions. Therefore, we identified GNf037h07 from EST database of model leguminous plant Lotus japonicus. In this study, we constructed a plasmid which expresses GNf037h07:GFP fusion protein constitutively to examine the subcellular localization of GNf037h07. The fusion protein expressed in cultured cells of tobacco BY-2 localized in cytoplasm of rapidly growing cells and in the vacuole of senescent cells. These findings suggest that GNf037h07 works in the vacuoles of mature nodules. We are now studying leader sequence of the protease that determines growth-phase-dependent subcellular localization of the protease using GFP fusion proteins.

View full abstract

Lipid rafts are relatively resistant to solubilization at low temperature in nonionic detergent. These domain are known to be enriched in sterols and sphingolipids and postulated to be involved in transmembrane signaling. The aim of this study is to analyze the components of lipid raft domains associated with defense responses of rice.
We fractionated lipid raft proteins from rice cultured cells by using Triton-X 100 and sucrose density-gradient ultracentrifugation and identified the proteins contained raft fraction by mass spectrometry. Also, as our previous works indicated that OsRac1, a small GTP-binding protein, functions as a key regulator of defense signaling in rice, we analyzed the raft fractions isolated from constitutive active OsRac1 (CA-Rac) and dominant negative-OsRac1 (DN-Rac) expressing rice cultured cells. We investigated whether the raft components among Wild type, CA-Rac and DN-Rac cells were different.

View full abstract

Hypersensitive cell death is known as a kind of defense responses and a typical programmed cell death in plants. It has been indicated that cell death signaling is transduced via Hsp90, MAPK (NtMEK2-WIPK/SIPK) cascade and then mitochondria, although almost other factors which function between these steps, are not identified yet. Many pathogenic bacteria utilize the type III secretion system to inject effector proteins into the host cells, and their main role is considered to disturb host defense systems including hypersensitive cell death. We have isolated plural cell death suppressors from type III effectors of Ralstonia solanacearum which is a bacterial pathogen causing severe damage in solanaceous species using tobacco hypersensitive cell death induction systems, and then analyzed the mechanism of the suppression. It is expected that the identification of the targets for cell death suppressors would provide unknown factors involved in hypersensitive cell death signaling.

View full abstract

Receptor-like kinases (RLKs) belong to the large gene family and play important roles in several biological processes such as growth, development and defense responses. Recently, several plant RLKs have been proven to be required for plant-pathogen or plant-symbiont interactions. To understand the diversity and function of RLKs of legume, we tried to identify RLK gene family in the genome of model legume Lotus japonicus and classified them by their protein domains. By comparing Lotus RLK genes to that of Arabidopsis, we found that several RLK subfamilies which proposed to be involved in the response to rhizobium or plant pathogen have expanded in Lotus genome. Among these subfamilies, we focused on LysM and LRRXII RLK family to understand phylogenetic and microsyntenic relations with Medicago and Arabidopsis. In this study, we will present the diversity of Legume RLKs and suggest their importance in plant-microbe interactions.

View full abstract

Legumes engage in root endosymbioses with both nitrogen-fixing rhizobial bacteria and phosphorus-acquiring arbuscular mycorrhizal fungi. Genetic programs in host plants for establishment of both symbioses share common symbiosis genes, mutations of which cause impaired symbiosis phenotype. Symbiosis-receptor kinase (SYMRK) was identified as a common symbiosis gene from a legume Lotus japonicus. The symrk mutant phenotype indicates that SYMRK functions in an early signaling network responding to the microbal symbiotic signals, but the direct SYMRK downstream components have not been identified prior to this study.
To identify SYMRK interacting partners, we performed yeast two hybrid screening using the SYMRK intracellular domain as a bait. We identified 4 homologues of the Drosophila Seven in Absentia (SINA) as SYMRK interactors. We designated these genes as Seven In Absentita homologue of Lotus japonicus (SIAL) 1 to 4. SIAL1-4 showed different interaction specificities to SYMRK, suggesting multiple roles of SIAL proteins in root symbioses.

View full abstract

Salicylic acid (SA) plays important roles both in the hypersensitive response and in the systemic acquired response (SAR). Two pathways are proposed for the biosynthesis of SA in Arabidopsis: the benzoic acid (BA) pathway and the isochorismate pathway. In the latter, a mutant defective in isochorismate synthase has been isolated and this mutant is known to be involved in SAR. However, mutants related to the former pathway have not been identified. To study the BA pathway in plant immune responses, we isolated the BA-sensitive mutant bah1 (benzoic acid hypersensitive 1). The bah1 mutant accumulated high levels of SA and expressed the PR1 gene after the application of BA, suggesting that the BA pathway for SA biosynthesis is activated in the bah1 mutant. Moreover, the bah1 mutant was resistant to Pseudomonas syringae.

View full abstract

N-acetylchitooligosaccharides elicit several defense responses in suspension-cultured rice cells (Oryza sativa cv Nipponbare). We have reported the isolation of two elicitor responsive genes, CIGR1 and CIGR2, of GRAS family in rice and analyzed the detailed mode of expression. To study the role of CIGR2 in defense, we generated CIGR2-RNAi transgenic rice plants where CIGR2 expression was significantly silenced and analyzed the phenotype when inoculated rice blast fungus, Magnaporthe grisea. The number of granulated cells, a cytological marker of hypersensitive cell death was significantly increased in the incompatible interaction, indicating that CIGR2 negatively regulates cell death. We will show some results on a gene that is likely to be regulated by CIGR2.

View full abstract

The nodule number of legume roots is tightly regulated through root-to-shoot long-distance signaling. Hypernodulation mutants deficient in the negative regulation of nodulation, also termed autoregulation of nodulation, have been isolated and characterized. However, genes responsible for autoregulation have not been identified besides HAR1 of the model legume Lotus japonicus and its orthologous genes from other legumes. HAR1 has been shown to regulate the nodule number by the shoot genotype. Thus, the isolation of root-regulated hypernodulation mutants would extend our understanding of nodule autoregulation at molecular level.
In the present study, novel L. japonicus hypernodulation mutants were isolated by C⁶⁺ ion beam irradiation. Grafting experiments allowed us to find two root-regulated hypernodulation mutant lines 716-1 and 1836-1. Both mutants showed no dwarf phenotypes, but a lower lateral root number than wild-type MG-20. Mapping of the causative genes indicated that 716-1 and 1836-1 have mutations in different loci.

View full abstract

The infection process of the rice blast fungus into rice plant passes the stage of attachment of the spore to the leaf surface, germination, the appresorium formation, and the infectious hyphae formation. We analyzed the response of rice against blast fungus by using mutants that are arrested at each stage of infection. The accumulation of H₂O₂ and the phytoalexin in the rice was not seen in mgb1 that germinates but does not form the appresorium, whereas the accumulation of H₂O₂ beneath the appresorium was seen in mst12 that form no penetration peg. Differential expression of genes as well as the generation of H₂O₂ and phytoalexins was observed at 2 hrs after inoculation of wild type strain, Guy11, in an R-gene dependent manner. These results indicated that rice recognizes signals from pathogen before appresorium formation and the initiation of invasion is critical for the phytoalexin generation.

View full abstract

Reef-building corals are an obligatory symbiotic system in which symbiont's photosynthesis is essential. It has been reported sometime that many reef-building coral species include endolithic algae within their skeleton. In spite of its potential significance, inter-relationship between endolithic algae and host corals is still obscure. We report here that endolithic algae bring about a positive effect on the host coral Acropora digitifera. In order to investigate effects of endolithic algae to host corals, we compared endolithic algae infected and non-infected groups of A. digitifera. Infected group maintained a higher maximal quantum yield (Fv/ Fm) than that of non-infected at optimal temperature (26ºC and 28ºC). In high temperature conditions (30ºC and 32ºC), however, there were no significant differences between both groups. Recovery rate of Fv/ Fm from damaged conditions was similar in both groups at water temperatures tested.

View full abstract

Nodule development in leguminous plants is induced by Nod factors secreted by rhizobia. Plant mutants unable to form nodule primordia involving cortical cell division are called as non-nodulation (Nod^-) mutants. Nod^- mutants have defects in some components of Nod factor signaling pathway ranging from Nod factor perception to gene expression. In Lotus japonicus, ten genes responsible for this pathway have been identified. In order to isolate a novel mutant with some genomic deletion, we attempted ion beam mutagenesis using an early flowering ecotype Miyakojima MG-20. Irradiation of C⁶⁺ at 80 Gy resulted in successful isolation of 5 Nod^- mutants. Among them a mutant 3122 has 1,366 bp deletion in MtNsp1-like sequence of L. japonicus. The LjNsp1 gene encodes a putative transcription factor of the GRAS family. The expression analysis of LjNsp1 and the interaction with LjNSP2 in yeast two hybrid system will be reported.

View full abstract

Arbuscular mycorrhizal (AM) fungi establish ecologically important symbiotic associations with the majority of land plant species, allowing improved uptake of phosphate (Pi) from the soil in exchange for plant-assimilated carbohydrates. Additionally, AM fungi endow plants with tolerance to pathogens and stress. Soybean is the major leguminous crop in the world, the yield of which being 1.8 x 10⁸ t. Investigation of molecular mechanisms of AM symbiosis is the basis to save fertilizers and chemicals.
AM-inducible Pi transporter genes in soybean have not been reported. We constructed a cDNA library of soybean AM roots. Using degenerate oligonucleotide primers, we amplified cDNA fragments for soybean Pi transporters. Then, we screened the cDNA library using not only the cDNA fragments but also Pi transporter genes of M. truncatula and L. japonicus as probes. As a result, we isolated a number of Pi transporter genes. Their phylogenetic relationship and expression patterns will be presented.

View full abstract

Plants recognize pathogen infection and induce rapid and strong defense responses including a localized cell death called hyper sensitive response. This defense system is controlled by resistance (R) proteins that recognize, directly or indirectly, pathogen effector molecules. Genetic approaches have led to the identification of several components of the R-protein triggered pathway. Among those identified, RAR1, HSP90 and SGT1 are required for resistance mediated by multiple R proteins recognizing viral, bacterial, oomycete or fungal pathogens. RAR1 and SGT1 interact with each other and with HSP90. The CS domain of SGT1, which binds both RAR1 and HSP90, has structural similarity to P23, a co-chaperon of HSP90. Silencing of RAR1, SGT1 or HSP90 in Nicotiana benthaminana plants resulted in reduction of R-protein levels. These results may suggest that the RAR1-SGT1-HSP90 complex works for stabilizing R proteins as a chaperon complex. The function of RAR1-SGT1-HSP90 complex will be discussed.

View full abstract

To perform comparative sequence and transcriptome analyses between Brassica rapa and Arabidopsis thaliana, we prepared a B. rapa cDNA microarray using 1,820 (ca. 2 K) cDNA clones selected from 2,166 non-redundant sequences of cDNA library of Chinese cabbage. The gene expression during infection with fungal pathogen Colletotrichum higginsianum and treatments with signaling molecules was analyzed using 2 K B. rapa and 1.2 K Arabidopsis cDNA microarrays. In B. rapa, the results suggested a large correlation coefficient between compatible pathogen C. higginsianum-infection and the treatment with salicylic acid, methyl jasmonate, or ethephon. The expression profiles of 145 counterpart gene sets between the B. rapa and Arabidopsis were distributed in the self-organizing map analysis. The 28 % of them indicated similarities in the two species transcriptome. These expressed sequence tag (EST) and microarray data should provide a valuable resource for functional genomics on the crops.

View full abstract

Legume-nodulating rhzobia must cope with ROS not only during free-living aerobic conditions but also during establishment and maintenance of nitrogen fixing symbiosis, since host legume plant might employ ROS as defense measure against invading microorganisms. Number and enzymatic properties of catalases vary among rhizobial species. The sequence of Mesorhizobium loti MAFF303099 revealed that this organism contain two predicted catalase genes, mlr2101 and mlr6940. The latter shows homology to B. japonicum katG and locates adjacent to an oxyR-homologue mll6938. We intend to reveal how M. loti MAFF303099 use these probably distinct catalases to cope with ROS and if there is any universal strategy for utilization of anti-oxidant enzymes among rhizobia. Here, we describe effects of insertion or deletion-insertion mutagenesis and expression analysis for two M. loti .catalases
Our results indicated that mlr6940 has important role in free-living condition, whereas mlr2101 might have role(s) under no-proliferating conditions including nitrogen-fixing conditions.

View full abstract

Many plant secondary metabolites show strong antimicrobial activities and cytotoxicity. They are potentially also toxic to plants but plants producing those active compounds seem to be insensitive to their own metabolites, suggesting that they have a species-specific detoxification mechanism. Cultured cells of Coptis japonica, which produce a yellow isoquinoline alkaloid berberine and accumulate it in the vacuole, have strong berberine tolerance, where as it is highly toxic to plants that do not produce berberine. In order to clarify the detoxification mechanism for own secondary metabolite, we tried to isolate berberine tolerant genes using C. japonica cell culture system as a model. To isolate those genes, a yeast functional screening using a cDNA library constructed with a shuttle vector was performed. One cDNA clone, which conferred clear berberine tolerance, encoded a galactinol synthase (CjGolS). The accumulation level of berberine in CjGolS-expressing yeast was less than 40% compared to the control.

View full abstract

Low oxygen conditions caused by submergence or poor soil drainage can reduce crop productivity. The response of metabolic processes and the morphological adaptations to anaerobic conditions have been investigated extensively. However, the regulation of signal transduction components and transcriptional factors, especially in early stage, is poorly understood. In this study, we have screened early responsive genes to low oxygen stress in rice. Total RNA from rice root treated under anaerobic conditions for 30 min was analyzed using GeneChip Rice Genome Array (Affymetrix). Approximately 800 genes were identified as anaerobic inducible. Genes associated with metabolism were about 20% of the induced genes and included genes known to encode anaerobic proteins (e.g., ADH1, PDC1, LDH, and AlaAT). Putative genes for transcriptional factors and genes possibly related to signal transduction were estimated about 10%, respectively. These results indicate the complexity of regulation mechanism for anaerobic response in plant.

View full abstract

Pondweed (Potamogeton distinctus A.Benn.) displays tolerance to anoxia. Elongation of overwintered turions is promoted by oxygen-free conditions, and it is influenced by chemicals which have effects on a plasma membrane (PM) H⁺-ATPase, suggesting that PM H⁺-ATPase plays an important role in survival under anoxic conditions. We examined the activity of PM H⁺-ATPase, its protein amounts, and phosphorylation levels of the domain binding 14-3-3 proteins in pondweed turions and etiolated hypocotyls of pea (Pisum sativum) which cannot survive over one day in anoxia. In pondweed turions, the PM H⁺-ATPase activity and its phosphorylation levels were maintained under anoxia. On the other hands, both fusicoccin and okadaic acid, which promoted PM H⁺-ATPase activity, had slightly promotive effects on survival of pea hypocotyls under anoxia, suggesting that a role of PM H⁺-ATPase is important for anoxic tolerance of plant cells.

View full abstract

Alkylphenol ethoxylates are biodegraded to alkylphenols in the environment. The 4-t-octylphenol (4-t-OP) is found to show endocrine disrupting action with estrogen-like properties and to be severely toxic to aquatic organisms. We have studied whether 4-t-OP affects gametogenesis of Chlamydomonas reinhardtii cells seen after nitrogen-starvation.
The mating ratios of gametes cells at the early stages of gametogenesis were increased by addition of 4-t-OP at the concentrations of 10^-11 to 10^-9 M, but were significantly decreased by that above the concentration of 10^-6 M. Thus, we analyzed the expression of gametogenesis-related genes (NIT1, NAR3, FUS1, MID, GAS28) during gametogenesis, in the range of 4-t-OP concentrations at which the mating was stimulated. Effects of 4-t-OP on the expression of these genes were dependent on the mating types, mt⁺ and mt^-: some genes of mt^- were expressed more intensely than those of mt⁺. These results suggest that 4-t-OP enhances the expression of gametogenesis-related genes sex-specifically.

View full abstract

Tomato and eucalyptus represent key species with great potential for conducting genomic research. As a member of the International Tomato Sequencing Project, we launched on sequencing of the chromosome 8. Our mission is to sequence the euchromatin, the estimated size of which is 17Mb. As of Nov. 2006, we finished 40 BACs (4.4Mb), and 20 BACs are in sequencing pipeline. The genome size of eucalyptus is estimated to be 650Mb. The Whole Genome Shotgun sequencing strategy was applied to produce the draft sequence. To date, three million reads were produced. As the third project, we launched on the genome sequencing of twenty-one endophytic bacteria. Some of them have been reported to contribute to the nutrition and increase the disease resistance of host plants. Sequencing of threefold coverage of each genome was achieved by producing approximately 108Mb draft sequences in total. We will present the latest status of our projects.

View full abstract

Soybean (Glycine max) is one of the most important crops for its high content of protein and oil. However, genetic and genomic analyses of soybean are delayed due to its complex and large genome. The objective of our project is to integrate EST-derived SSR markers to a genetic linkage map of soybean to analyze complex soybean genome and to investigate the synteny between soybean and other legumes. Up to now, 5044 primer pairs, generating 100-300bp amplified fragments including SSR, have been screened. As a result of screening, 445 functional markers were obtained and 444 markers of them were mapped to a genetic linkage map which was constructed based on 94 soybean recombinant inbred line (RIL) plants. Using EST sequence, several linkage groups could be compared with the Lotus japonicus genome in silico. This result indicated that EST-derived SSR markers was powerful tool for comparative genomics between soybean and other legumes.

View full abstract

Sugi, Cryptomeria japonica D. Don, is one of the most commercially important conifers, and it covers 44 % of all cultivated forests in Japan. Sugi pollinosis is the most serious allergic disease in Japan. Recently, male-sterile mutants of C. japonica whose pollen is hardly released have been selected. To clarify the molecular mechanisms of male-sterility in C. japonica, we have constructed a full-length cDNA library using normalized RNAs derived from normal male flowers at various developmental stages. We obtained expressed sequence tags (ESTs) of 19,484 cDNA clones. Assembling with PHRAP and BLASTN program resulted that these ESTs represented 10,466 transcripts. Approximately 75% of these transcripts showed similarity (BLASTX E-value ≤ 1e-5) to reported sequences in the public databases such as UniProt and TAIR. We will prepare a DNA microarray using this EST data to analyze the profiles of gene expression in normal and sterile male flowers of C. japonica.

View full abstract

Tomato and Arabidopsis differ morphologically and genetically in the course of evolution. We previously demonstrated that tomato has numbers of genes that have low similarity to Arabidopsis genes by analyzing full-length cDNAs of miniature tomato Micro-Tom. In this study, we focused on functional analysis of these tomato genes using virus-induced gene silencing (VIGS) to obtain insights into molecular bases of plant diversity. We obtained Micro-Tom full length cDNA sequences from tomato databases MiBASE and KaFTom. And then, 85 full-length cDNA clones that have low similarity (>1e-10) to Arabidopsis genes at amino acid sequence level were selected for VIGS assay. Partial sequences of these cDNAs were inserted into tobacco rattle virus-based VIGS vector, and then VIGS in Micro-Tom plant was induced by agro-infiltration. By agro-infiltration to cotyledon, gene silencing was induced not only in leaves, but also in fruit. We report examples of morphological changes seen in VIGS-induced tomato plants.

View full abstract