R. sphaeroides can grow both under respiration and photosynthesis conditions. Elimination of reactive oxygen species (ROS), therefore must be critical for their growth. In this study, we focused on R. sphaeroides peroxiredoxin (Prx) to get insight into the antioxidative stress system of this bacterium, since Prx is reported as an important antioxidant protein for various organisms.
On the genome of R. sphaeroides, two genes encoding TypeII Prx and PrxQ have been assigned. Both the recombinant Prxs could catalyze the reduction of various ROS using thioredoxin. Growth rate of PrxQ disruptant was slower than that of wild type. In addition, the accumulation of the photopigment synthesis in the disruptant was significantly lower than that of wild type. These results suggest that PrxQ functions as a main quencher for the ROS in this bacterium, and the redox balance in the cell must affect the regulation of photopigment synthesis in this bacterium.

View full abstract

Mg-chelatase catalyzes the insertion of Mg²⁺ into protoporphyrin IX and is composed of three subunits, CHLI, CHLD and CHLH. Mg-insertion by Mg-chelatase requires ATP hydrolysis attributed by CHLI, which primary determines the rate of Mg-insertion. Determination of three-dimensional structure of Rhodobacter BCHI revealed that the protein is a member of AAA⁺-protein family. Our previous study showed that CHLI1 of Arabidopsis is a target protein of chloroplast thioredoxin. In this study, we identified one critical disulfide bond involved in the redox regulation of ATPase activity based on the analysis of point mutation of 4 cysteine residues of CHLI1. These cysteines are expected to be located in close proximity within the C-terminal regulatory domain based on the BCHLI structure. Thus, the identified one crucial disulfide bond located in the C-terminal domain of CHLI1 may be involved in thioredoxin-dependent redox regulation, which may regulate the ATPase activity of CHLI1.

View full abstract

We have reported previously that transgenic Arabisopsisplants expressing ADP-ribose/NADH pyrophosphatase (AtNUDX2) had increased tolerance of oxidative stress. Here we studied in detail the relationship between activities of ADP-ribose/NADH pyrophosphatase (AtNUDX2, 6, 7) and tolerance of oxidative stress. The AtNUDX2-overexpressing plants suppressed the accumulation of free ADP-ribose under oxidative stress caused by 3 μM paraquat treatment. The levels of NADH in the AtNUDX7-overexpressing and knockout plants were considerably low and high, respectively, under normal conditions. High levels of NADH were also observed in the AtNUDX6-knockout plants. Interestingly, the activities of poly (ADP-ribosyl)ation (PAR) in the AtNUDX7-overexpressing and the knockout plants were clearly enhanced and suppressed, respectively, under normal conditions. These results indicate that the AtNUDX2 protein plays an important role in the removal of free ADP-ribose, and AtNUDX6 and 7 proteins function in the activation of PAR via hydrolyzation of NADH, resulting in increased tolerance of oxidative stress.

View full abstract

NAD(P)(H) (nicotinamide adenine dinucleotides) is well-known cofactor involved in electron transport and redox reaction. It was recently reported NAD(P)(H) plays major roles in post-translational modifications and signal transduction. Nonetheless, little is known about NAD biosynthesis and catabolism. NAD synthetase (NADS) is an enzyme that converts a NAD precursor (NaAD) to NAD in a final step of the NAD biosynthesis. At1g55090 in Arabidopsis thaliana encodes a putative NADS homologous to QNS1 of Saccharomyces cerevisiae with 54 % identity. Complementation tests and analyses of the enzyme activity using Δqns1 yeast strain revealed that At1g55090-encoding protein was the bona fide NADS of A. thaliana. We also found that AtNADS promoter activity was spatially regulated. The expression pattern was well consistent with AtNMNAT, which converts NaMN to NaAD. Taking all information together, we will discuss about the role of NAD biosynthesis on plant development.

View full abstract

NAD kinase (NADK) is an only enzyme, which synthesizes NADP from NAD. Measurement of photosynthetic parameters of the nadk2 mutant, which is defective in chloroplast NADK, revealed that the effective efficiency of photosynthetic electron transport was decreased and non-photochemical quenching was increased. We therefore quantified xanthophylls and the xanthophyll composition was aberrant in nadk2 mutant. Furthermore, we investigated carbon and nitrate metabolism in nadk2 mutant and NADK2-overexpressing plant (OX). Carbohydrate levels were decreased in nadk2, while that were increased in OX. Likewise, the amounts of glutamate and related amino acids were also paralleled the expression level of NADK2. These results suggest that overexpression of NADK2 activates both carbon fixation and nitrate assimilation, and NADK2 is essential for regulation of chloroplast-specific function.

View full abstract

Seed germination has been known to require H₂O₂, but it remains unclear how the plant senses H₂O₂ to regulate germination. Since protein tyrosine phosphatase (PTPase) that is easily inactivated in oxidative state, we considered it as a candidate factor sensing H₂O₂ for the regulation of seed germination. To investigate the function of AtPTP1, a typical and single PTPase in Arabidopsis, in seed germination, we isolated T-DNA inserted mutants of AtPTP1 and generated the transgenic plants overexpressing the gene. The mutant seeds showed a higher germination frequency compared to wild-type seeds in a medium containing ABA, whereas the transgenic seeds showed delayed germination. Taking into account that recombinant AtPTP1 was inactivated by H₂O₂ and that ABA induces H₂O₂ generation, these results are considered to suggest that the AtPTP1 is involved in the ABA responses in seed germination.

View full abstract

2'-deoxymugineric acid (DMA) is a chelater of Fe³⁺ and plays an important role in uptake of Fe in gramineous plants. Nicotianamine (NA) chelates many kinds of metals and has an significant role in metal homeostasis in higher plants. DMA and NA are usually quantified by HPLC and UV absorption, and its quantification limit is 100 pmol. Consequently we need large amount of samples to measure DMA and NA by HPLC. A highly sensitive and simple quantification method for DMA and NA was developed using liquid chromatography/erectrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS). Fluorenylmethoxycarbonylation of DMA and NA enabled their retention on a reversed phase column. A internal standard, N-nicotyllysine (NL) was used to ensure reliable quantification by giving a linear calibration curve drawn between the DMA/NL and NA/NL molar ratios injected and the DMA/NL and NA/NL area ratio in mass chromatograms. DMA and NA are quantified at the same time.

View full abstract

Glutathione (GSH) is involved in many aspects of plant growth and development. Transport and compartmentalization of GSH is essential to perform all these functions. We have cloned a GSH transporter from rice (OsGTL1). OsGTL1 is a putative member of oligopeptide transporter family and encode a polypeptide of 757 amino acids divided in 12 transmembrane domains. Electrophysiological measurements using Xenopus leavis oocytes showed that OsGTL1 is a functional GSH transporter. Microarray and Northern blot analysis revealed that the expression of OsGTL1 is upregulated in response to Fe-deficiency. GUS analysis driven by OsGTL1 promoter showed that expression of OsGTL1 is upregulated in response to Fe-deficiency especially in root tips of Fe-deficient rice. These results raised the possibility that GSH may have functions apart from that of an antioxidant and could play a role in Fe-deficiency tolerance in rice.

View full abstract

Fe deficiency is a major abiotic stress in crop production. Although responses to Fe deficiency in graminaceous plants, such as increased production and secretion of mugineic acid family phytosiderophores (MAs), have been described, the gene regulation mechanisms related to these responses are largely unknown. We characterized the Fe-deficiency-inducible bHLH transcription factor OsIRO2. In rice, the overexpression of OsIRO2 resulted in higher MAs secretion and higher tolerance to Fe deficiency, while repression of OsIRO2 resulted in lower MAs secretion and hypersensitivity to Fe deficiency. Microarray analysis demonstrated that OsIRO2 regulates 59 Fe-deficiency-induced genes, including the genes involved in Fe(III)MAs transport system. Some of those genes, including two transcription factors, possessed the OsIRO2-binding sequence in their upstream regions. OsIRO2 possesses a homologous sequence of the Fe-deficiency-responsive cis-acting elements (IDEs) in its upstream region. We propose a novel gene regulation network for Fe-deficiency responses, including OsIRO2, IDEs, and the two transcription factors.

View full abstract

It is thought iron uptake is mainly regulated by long-distance signals sent from shoots. Expression of iron uptake genes coding iron regulated transporter (NtIRT1) and ferric chelate reductase (NtFRO1) are induced in roots of iron deficient tobacco. To elucidate the role of the signals for iron uptake in roots, we performed the expression analysis of the genes in intact tobacco plants with leaf excision and hairy roots under various conditions. In consequence, it was suggested that the iron uptake is regulated by promotive signals which are synthesized in iron deficient leaves regardless of leaf position and sent to roots. Meanwhile iron deficient hairy roots induce the gene expression, suggesting that roots have their own iron sensor and regulate iron uptake. We propose the most appropriate model that iron uptake signals are synthesized in whole plants and the amount of the signals correlates with the size of plants.

View full abstract

Iron-deficiency tolerance of gramineous plants is well correlated with the amount of mugineic acids secreted. However, iron-deficiency symptom of barley and rice was different though the iron content of rice increased by cultivation with barley in the same container. We predict that iron availability in leaves also involved in the mechanism of iron-deficiency tolerance in barley. In the previous report, we suggested that iron availability is different among gramineous plant species. In this work, we compared the expression patterns of iron storage protein ferritin between rice and barley under various iron nutritional condition. In rice, ferritin expression significantly increased by iron excess, whereas ferritin expression in iron deficient barley was the same level as in other iron nutritional condition. The relation between ferritin and iron availability will be discussed.

View full abstract

Resistance to aluminum (Al) in barley is achieved by the secretion of citrate from the roots, however, genes responsible for this process have not been identified. We conducted a microarray analysis to compare gene expression between Al-resistant (Murasakimochi) and -sensitive (Morex) cultivars. We found that several transporter genes were highly and constitutively expressed in the Al-resistant cultivar. Among them, one of the transporter genes was expressed about 20-fold higher in the Al-resistant cultivar. A positive correlation was observed between the Al-activated citrate secretion and the expression of this candidate gene in eight cultivars differing in Al resistance. When the cRNA encoding this gene was injected to Xenopus oocytes with citrate, current change was observed upon Al exposure. Immunostaining showed that the protein encoded by this gene was localized in the epidermis of the roots. These results suggest this gene is involved in the Al-activated citrate secretion in barley.

View full abstract

Rice is the most Al tolerant species among small-grain cereal crops, but neither the mechanisms nor the genes responsible for the high Al tolerance are understood. We previously cloned an Al-tolerant gene (Als1) using an Al-hypersensitive mutant. When this gene was introduced into the mutant, the Al tolerance was recovered. This gene was mainly expressed in the roots and the expression was up-regulated by a short exposure (2 h) to Al. In a transgenic rice carrying Als1 fused with GFP under the control of Als1 promoter, the signal was observed in the plasma membrane of all cells at the root tips. Immunostaining also showed that Als1 is located in all cells of the root tips. Furthermore, Al was detected in the cytoplasm of all root cells of the mutant but not in the wild-type rice. These results suggest that Als1 is involved in the exclusion of Al from the cells.

View full abstract

So many Al stress tolerance mechanisms for plants have been proposed, but we believe that characterization of the response mechanism is also very important to solve this problem. In this study, we tried to isolate the genes encoding transcription factors related to the gene-expression of the Al inducible Arabidopsis gene, AtGST11, using our unique screening system, bio-panning.
A cDNA library was constructed using T7 phage and mRNA derived from Al treated Arabidopsis and repeatedly screened three times by a DNA (promoter region of AtGST11; p-AtGST11)-protein binding reaction (bio-panning). DNA sequencing indicated that they were an unknown protein, a RING finger protein and HD-Leucine zipper protein. A gel-shift assay using p-AtGST11 and these purified proteins indicated that they can bind to the region of -200 to 0 from ATG site in p-AtGST11 and suggested that they may function for the expression of AtGST11 under Al stress.

View full abstract

In wheat root apices, aluminum (Al) activates ALMT1 to export malate. To elucidate the activation mechanism by Al, we compared the function and the amino-acid sequences of the ALMT1 of wheat, rye and Arabidopsis to identify the amino acid residues involved in the activation of ALMT1. Cultured tobacco cells were transformed with rye orthologs which exhibited 90% identity with wheat ALMT. The transformants which highly expressed rye ALMT1 gene released malate in response to Al as much as the transformants with wheat ALMT1. Furthermore, it has been reported that the ALMT1 ortholog of Arabidopsis (50% identity with wheat ALMT1) functions as Al-activated malate transporter. We hypothesized that the negatively-charged amino acids (Asp, Glu) interact with Al ion, and found several negatively-charged amino-acid residues conserved among these ALMT1 proteins. A malate efflux capability will be investigated in transgenic tobacco cells expressing the mutagenized ALMT1 proteins which are substituted these amino-acid residues.

View full abstract

As factors inducing growth injury on plants under the high pH above 9, deficiencies of P, Fe, etc. and ammonia toxicity have been well documented. At the same time, occurrence of hydroxyl ion toxicity has been assumed frequently although evidence was lacking. Therefore, we investigated whether the hydroxyl ion toxicity occurs in rice and tomato in the range of pH 9-11. Result showed that the growth injury was induced by hydroxyl ion toxicity at the pH above 9. Then, mechanism of growth injury induced by hydroxyl ion toxicity were investigated. It was found that water absorption was inhibited within 12 hours above pH 9 (tomato) and pH 10 (rice). In addition, root elongation was inhibited during 8 days growth. These results demonstrate that hydroxyl ion toxicity exists in rice and tomato. However, MDH activity in the crude homogenates of leaves, stem and roots were not affected significantly.

View full abstract

Boron (B) is both essential and toxic to plant growth. Through transcriptome and semi-quantitative PCR analysis with low- and high-B treated Arabidopsis thaliana, we identified nine genes whose transcript accumulations were induced in roots or rosette leaves under boron excess. These genes included a heat-shock protein and MATE efflux family transporters. We also obtained T-DNA insertion lines in more than ten genes whose expressions were induced by low- or high-B treatetments. Among the lines, root growth of wrky6-3 carrying T-DNA insertion in the third exon of WRKY6 gene was poorer than that of wild-type under boron deficiency but not under a control condition. We also confirmed that boron deficiency-specific retardation of root elongation of wrky6-1 and wrky6-2 (kind gift from I. Somssich), establishing role of WRKY6 in low-B response. Promoter activity and transcript accumulation of WRKY6 was induced near the root tip under the boron deficiency.

View full abstract

Boron (B) cross-links pectic polysaccharides at the rhamnogalacturonan II regions and thereby contributes to build the cell wall. However, it remains unknown how B deficiency triggers various metabolic disorders and brings about cell death. To understand this mechanism, we have analyzed the responses of cultured tobacco BY-2 cells under B deficiency.
When 3-day-old cells were transferred to a B-free medium, dead cells started to increase from 24 h after the treatment. We previously showed that the genes for antioxidative enzymes are upregulated in low B-acclimated cells, which suggests that oxidative damages are involved in low B stress. In the present study we found that lipid peroxides accumulated in -B cells by 18 h, prior to the increase in the dead cell population. In addition, an antioxidant butylated hydroxyanisole reduced cell death. These results indicate that oxidative damage does occur under B deficiency as the direct cause of cell death.

View full abstract

Transcriptome analyses of the model legume Lotus japonicus by cDNA array (Kouchi et al. 2004) and SAGE (Asamizu et al. 2005) have led us to identify twenty transcription factors (TFs) whose expression is up regulated in the nodulation process. Expression profile of the TFs was analyzed quantitatively by realtime RT-PCR. We found expression of seven TFs was induced as initially as 3 hrs after the Mesorhizobium loti inoculation. We observed expression of three of the early responding TFs was affected in symRK but not in nin plant, indicating a possibility that induction of these TFs takes place prior to the NIN activation. We obtained nodulation-defective mutants for one ERF gene by screening the Lotus TILLING line. Overexprssion of this gene in transformed hairyroots resulted in significant increase of nodule number compared to non-overexpressed vector control. We are focusing on this gene to elucidate its function using the erf mutated plant.

View full abstract

Legume plants have an ability to fix atmospheric nitrogen via symbiosis with soil microbes. The secretion of flavonoids from legume roots is the initiation of nodule formation process, but almost nothing is known about the membrane transport mechanism of flavonoid molecules from root cells. We performed the first biochemical characterization of the transport mechanism of flavonoid from legume roots using soybean and the signal flavonoid genistein as a model system.
Plasma membrane vesicles were purified by fractionation of microsomes prepared from soybean roots on a discontinuous sucrose density gradient. The genistein transport by plasma membrane vesicles, which was analyzed by HPLC, was critically dependent on the hydrolysis of MgATP. The activity was inhibited by a typical inhibitor of ABC transporters vanadate, while it was insensitive to ionophore, suggesting the involvement of an ABC transporter in the secretion of flavonoids from soybean roots.

View full abstract

To prevent excessive nodulation, leguminous plants have a systemic regulatory system called "autoregulation" of nodulation (AON). In Lotus japonicus, Har1 has been shown to be involved in AON and the har1 mutants display hyper-nodulation phenotype by the lack of AON. The har1 mutants are also hyper-infected by symbiotic AM fungi and parasitic nematodes in roots, suggesting that AON signal(s) also play the inhibitory role against other kinds of infections. Here, we report that Har1 also participates in the defense mechanism against Pseudomonas syringae.
Leaves of wild type L. japonicus were susceptible to Pseudomonas syringae pv. pisi but not to pv. glycinea. In contrast to the wild type plants, leaves of har1 mutants were severely infected by P. syringae pv. glycinea. In addition, har1 mutants were more susceptible to P. syringae pv. pisi than wild type. These findings suggest that Har1 participates in shoot defense mechanisms.

View full abstract

Recent studies of symbiotic mutants of Lotus japonicus have led to isolation of a number of host genes, which are required for endosymbioses with both rhizobia and arbuscular mycorrhiza. These genes are termed "common sym genes" and thought to be essential for early signaling processes required for successful infection of plant cells by symbiotic fungi and bacteria. The seven common sym genes identified so far are all conserved in rice that is known as a mycorrhizal plant. On the other hand, non-mycorrhizal plant Arabidopsis thaliana loses half of them. This suggests that common sym genes are universally involved in plant-arbuscular mycorrhiza symbiosis.
To confirm the involvement of common sym genes in rice-arbuscular mycorrhiza symbiosis, we isolated and characterized their rice orthologues, OsCASTOR, OsPOLLUX and OsCCaMK. Furthermore, OsPOLLUX and OsCCaMK deficient rice mutants, isolated from the Tos17 mutant library, show symbiotic defective phenotypes in arbuscular mycorrhizal infection.

View full abstract

Among the several bacterial secretion systems known to date, Type III secretion system (T3SS) are found in a number of pathogens of animals and plants and constitute needle-like to inject pathogenicity proteins for establishing bacterial niche. Genes for T3SS have been identified in some rhizobial strains and shown to participate in secretion of proteins called Nops however, biochemical functions of the Nops are mostly unknown. Determination of Mesorhizobium loti MAFF303099 genome revealed presence of T3SS gene cluster and posed us questions if and how these T3SS genes have roles in symbiosis. As a first step to elucidate symbiotic roles of T3SS in M. loti, we have conducted, 1) construction of T3SS mutants of MAFF303099, 2) infection tests with a number of host legumes, and 3) construction of over-expression system of T3SS in vitro to obtain Nops.

View full abstract

About 13,000 full-length cDNAs of rice were mixed and ligated to an expression vector cassette. They were introduced into Arabidopsis by Agrobacterium-mediated floral dip transformation to generate Rice-Arabidopsis FOX (full-length over-expressor) lines. Three-week-old T2 seedlings of approximately 12,000 FOX lines were screened for resistance to a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 by dip inoculation. More than 20 resistant lines were selected after 2-3 rounds of screening and the rice cDNAs inserted in their genomes were identified. The rice cDNAs identified by this procedure are being re-transformed into Arabidopsis and rice for overexpression to confirm the disease resistance phenotypes. At present, some rice lines separately overexpressing two of the four cDNAs identified in the screening of the Rice-Arabidopsis FOX lines have shown resistance to the pathogen, Xanthomonas oryzae pv. oryzae, causing rice blight.

View full abstract

The hypersensitive response (HR), a form of programmed cell death that occurs during immune responses in plants, is characterized by a distinct cellular morphology, including plasma membrane shrinkage, nuclear condensation, and DNA fragmentation. Initiation of HR cell death requires de novo protein synthesis, suggesting that the induction of HR cell death involves a transcriptional network regulated by a key factor. PCR-subtraction and cDNA microarray analyses revealed that plant specific transcription factor OsNAC4 is specifically induced during HR cell death. Overexpression of OsNAC4 caused HR-cell death accompanied with DNA fragmentation. In cell lines in which OsNAC4 levels have been suppressed by RNAi, HR cell death was markedly decreased in response to incompatible bacterial strains. In addition, recognition of plant immune initiation signal causes accumulation of OsNAC4 in the cytosol and its translocation from the cytosol to the nucleus. These data indicate that OsNAC4 is a positive regulator of HR cell death.

View full abstract

Plant hypersensitive cell death (HR cell death) is a form of plant programmed cell death and plays a major role in plant immunity. We previously demonstrated that HR cell death is initiated by a plant specific transcription factor, OsNAC4. To clarify mechanism of HR cell death induction by OsNAC4, we compared global gene expressions in OsNAC4 RNAi line and in a control line using a 22,000 elements oligonucleotide microarray. Variations of 143 genes were abolished in the RNAi line during the incompatible interaction. All genes were upregulated in control line by inoculation of the incompatible N1141 strain, indicating that OsNAC4 positively regulated expression of these genes. We analyzed in depth the sequence motif of all 143 genes and only one cDNA encoding putative endonuclease (OsHEN1) was found. When OsHEN1 was expressed in rice cells, clear nuclear DNA cleavage was observed. These data indicate that OsNAC4 induced DNA degradation through OsHEN1.

View full abstract

Rice recognizes flagellin, a main component of a bacterial flagellum, and subsequently induces immune responses, such as generation of reactive oxygen species and expression of defense-related genes. We have previously reported that rice receptor kinase, OsFLS2, is functional orthologue of Arabidopsis FLS2 which percepts flg22 peptide derived from the N-terminal region of flagellin. However, in spite of expression of OsFLS2 mRNA in rice, flg22 induced little immune response in rice. To explain this discrepancy, we made OsFLS2 overexpressed rice and analyzed the immune response to flg22. As a result, overexpression of OsFLS2 in rice caused stronger immune responses than the wild type. This result indicates that low sensitivity of rice to flg22 is attributed to low amount of OsFLS2.

View full abstract

We have previously shown that OsRac1, a small GTPase Rac of rice, is involved in disease resistance. In this study, we find that RAR1, an important component of R gene mediated disease resistance, HSP90 and 70, the molecular chaperones, MAPK6, a rice MAPK homologous to tobacco NtSIPK and Arabidopsis MAPK6 and Sti1 (Hop in mammalian cells) are all found in OsRac1 complex. Functional studies of RAR1, Sti1 and HSP90 using RNAi method and geldanamycin (GDA), the HSP90 specific inhibitor, indicate that they have critical role in rice innate immunity and involve in OsRac1 related defense signaling pathway. On the other hand, treatment of GDA but not suppression of RAR1 disassociates OsRac1 protein complex, indicating that HSP90 but not RAR1 function is required for the complex formation. Together, our results suggest a key role of OsRac1 protein by forming the defensome with important components in the early steps of defense signaling.

View full abstract

Elucidation of the plant defense signaling network controlling responses to pathogen infection, has been challenging for several biological reasons. First, plant defense signaling pathways are highly interconnected. Second, plants receive multiple stimuli associated with pathogen infection including pathogen-associated molecular patterns and effectors delivered directly into plant cells. Third, part of the signaling network is robust to perturbations.
To reveal the structure and function of this signaling network, we have employed a strategy using parallel and quantitative data collection. Our objective is to collect hundreds of observations about the behavior of the system after introducing specific perturbations. To this end, we have developed a high-performance, inexpensive custom microarray and have initiated gene expression profiling combined with a reverse genetics strategy. Analysis of known mutants as reference points within the signaling network revealed a model for the topology of the network and key players controlling signaling pathways utilized to process different stimuli.

View full abstract

Plants change expression of numerous genes upon pathogens attack, and many of the changes compose plant defense mechanisms. The hrcC mutant of the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 is defect in injecting type III effectors (T3Es) into the plant cell, and plant responses induced by the mutant strain is considered mostly based on recognition of pathogen-associated molecular patterns (PAMPs). It is not clear whether PAMPs have any effects on salicylic acid (SA) -mediated responses, which are induced by recognition of some T3Es. Several Arabidopsis defense mutants were analyzed by mRNA profiling using a small-scale custom microarray after inoculation of the hrcC strain. We found that induction of some genes by the hrcC strain depends on the ICS1 gene, which is essential for the pathogen-induced SA synthesis. This strongly suggests that PAMPs can induce SA-mediated defense responses. We will also discuss expression profiles of other Arabidopsis mutants.

View full abstract

KNOX genes play an important role in formation and maintenance of the shoot apical meristem (SAM), and their ectopic expression in leaf results in its abnormal morphology. Thus, the SAM-specific expression of KNOX genes is essential for normal development of plants. To understand the mechanism we identified and analysed a rice mutant resembling KNOX-overexpressors. The mutant knox leaf expression1 (kle1) showed ectopic expression of KNOX genes and produced very small shoot. kle1 was seedling-lethal and showed defects in SAM maintenance and leaf morphology. KLE1 was shown to encode a fatty acid elongase similar to Arabidopsis FIDDLEHEAD. KLE1 was specifically expressed in an L1 layer of shoot during embryo, vegetative and reproductive development. These results suggest that KLE1 regulates shoot development by controlling fatty acid composition of an L1 layer and that a signal, which controls shoot development, might be transmitted from an L1 layer to inner cells.

View full abstract

Trichomes (epidermal hairs) function as a physical barrier against herbivores in Arabidopsis thaliana. Furthermore, it has been reported that wounded plants develop new leaves with increased trichome density. Such upregulation of trichome density is dependent on both wound-inducible biosynthesis and SCF^COI1 complex-mediated signaling of jasmonic acid.
We developed a sensitized genetic screening to obtain mutants specifically defective in the plastic regulation of trichome density. Here we report the characterization of a mutant, unarmed9 (urm9). urm9 could not normally increase their trichome density in response to wounding or jasmonic acid treatment. urm9 exhibited otherwise normal response to jasmonic acid, such as accumulation of anthocyanin pigment, supporting that URM9 gene is specifically involved in the regulation of trichome density. Double mutant analyses indicated that URM9 acts as a positive regulator of trichome differentiation in concert with GL1 and GL3. Map based approach to clone URM9 is underway.

View full abstract

Transdifferentiation of matured cells to a totipotent stem cell is a well-known feat of plants, but the molecular mechanisms are mostly unexplored. We established a moss Physcomitrella patens as a model for the transdifferentiation, in which differentiated leaf cells of a dissected leaf turn into an apical stem cell within a couple of days. We developed a multiple-point timelapse imaging system to capture the entire process. Leaf cells that faced to the dissected dead cells were easy to change to a stem cell. The transdifferentiation did not require any exogenous phytohormones under the appropriate light conditions. The effective wavelengths of the light were in the blue and red regions, suggesting the involvement of cryptochrome and phytochrome mediated signaling. We also found a nuclear swelling during the transdifferentiation process and the changes of DNA contents during the process will be presented.

View full abstract

Plant stem cells continuously produce numerous cells, which subsequently differentiate into matured cells. Differentiated plant cells still have a great potency of transdifferentiation to an apical stem cell under the proper conditions. Differentiated cells of flowering plants change to calli, in which a small number of cells transdifferentiate to meristem cells. The small amount caused the problem to identify the factors for the transdifferentiation.
To identify critical factors affecting the transdifferentiation process, we established an experimental system with the moss Physcomitrella patens, of which a leaf cell changes to an apical meristem cell without a cell division. We constructed the custom microarray containing 10,000 genes for photo-signaling, cell cycle, DNA metylation, chromatin remodeling, histone modification, phytohormone reaction, and transcriptional regulation. Clustering analysis revealed several patterns of gene expression during the process. We also investigated the epigenetic regulation by histone modification using ChIP-PCR and the result will be presented.

View full abstract

Land plants landed around 480 million years ago. The evolution of developmental genes in land plants is still a challenging issue, because genome information was only available in angiosperms, which originated around 200 million years ago. Recently, whole genomes of the green algae Chlamydomonas reinhardtii, the moss Physcomitrella patens, and the lycopod Selaginella moellendorffii were mostly sequenced and genome-wide comparisons of developmental genes became possible. We used 700 Arabidopsis thaliana developmental genes as queries and searched homologous genes in other organisms. Phylogenetic trees of the homologs were constructed using the neighbor-joining and maximum likelihood methods, and the existence of potential orthologous genes was examined. Conservation and divergence of developmental genes in land plants will be discussed.

View full abstract

Asymmetric cell division in a stem cell generates two different daughter cells; one is a self-renewed stem cell daughter and the other is a differentiated, non-stem cell daughter. Although unequal distribution of mRNA or proteins has been known to play a pivotal role to specialize each daughter cell, such molecules in plants remain largely unknown. The isolated protoplasts of P. patens divide asymmetrically to generate stem cells and differentiated non-stem cells, assuring a good system for the study of molecular mechanisms for asymmetric cell division. We transiently overexpressed P. patens cDNAs in the protoplasts and identified 58 genes as candidates involved in asymmetric cell division. For those candidates, we made cDNA-YFP knock-in transgenic plants by using gene targeting technique to investigate protein localization under a control of their native promoters. We found nine proteins accumulated preferentially in the stem cells but not in differentiated protonemal cells.

View full abstract

Iron, zinc, manganese, cupper are required for development of reproductive organs and seeds. Metal chelators are also required for metal transport to reproductive organs and seeds. Mugineic acid family phytosiderophores are metal chelators biosynthesized in graminaceous plants, and NA, a chelator of metals, is ubiquitously present in higher plants. Deoxymugineic acid (DMA) is found in rice plant and is produced from NA in two steps catalyzed by nicotianamine aminotransferase (NAAT) and DMA synthase (DMAS).
In this study, we investigated the gene expressions of OsYSL2, a transporter of Fe(II)-NA and Mn(II)-NA, OsYSL15, Fe(III)-DMA transporter, OsIRT1, Fe(II) transporter, OsZIP4, Zn(II) transporter, OsNASs, nicotianamine synthase, OsNAAT1 and OsDMAS1 in inflorescences and seeds of rice plant to illuminate the role of NA and MAs at reproductive stage in monocot. Distribution and transport of metals were analyzed by tracer experiments and a synchrotron X-ray fluorescence microprobe.

View full abstract

CURLY LEAF (CLF) and SWINGER (SWN) are factors of the Polycomb complex that plays key roles in chromatin remodeling by modulating histone methylation. The clf swn plants form somatic embryos after germination at 23^oC. The LEAFY COTYLEDON1 (LEC1) expressed in the embryos is a key gene not only for the normal embryogenesis but also for the somatic embryogenesis.
We found that the formation of somatic embryos in the aerial part of the clf swn was suppressed by low temperature (10 ). The LEC1 was mis-expressed in a whole part of the clf swn plants at 23^oC. By contrast, the LEC1 expression in the aerial part of the clf swn was reduced at 10^oC, consistent with the suppression of the formation of somatic embryos. These results suggest that the de-methylation of histones may cause the ectopic expression of the LEC1 and play a key role in the somatic embryogenesis.

View full abstract

During the embryogenesis, the basic body plan of an organism develops from a unicellular zygote. In higher plants, the zygote divides asymmetrically, generating two daughter cells of different fates: the small apical daughter will form the aerial organs of plants, whereas the large basal cell will produce all the below-ground roots. We identified Arabidopsis homeobox genes, WOX2 (WUSCHEL-related homeobox2) and WOX8, whose dynamic expression pattern during embryogenesis reflects the apical-basal axis development. We have used these genes as tools to investigate how asymmetry of cell fates originates during early embryogenesis.

View full abstract

The assembly of newly replicated and repaired DNA into chromatin is essential for development, differentiation and growth in eukaryote. Although the mechanisms of deposition of histones by the histone chaperones have recentry defined based on genetical and biochemical studies in Saccharomyces cerevisiae, Drosophila and mammals, their physiological function in plants is not well understood. To investigate the roles of anti-silencing function 1 (ASF1a, ASF1b), the histone H3/H4 chaperones and FASCIATA 2 (FAS2), the second subunit of chromatin assembly factor 1 (CAF-1) in Arabidopsis, we characterized asf1a and asf1b mutants, double or triple mutants with fas2. We observed that double mutant of either one of asf1 with fas2 is defective in pollen development and triple mutant of both asf1 with fas2 displays embryonic lethal phenotype. Our results suggest that ASF1 and CAF-1 play critical roles in the gametogenesis and embryogenesis.

View full abstract

Two cDNA clones PF10056 and PF11634 having some homology to BURP-domain protein transcript were found in peach fruit. The deduced amino acid sequences (444 and 427 amino acids, respectively) showed 50% identity to RD22 from Arabidopsis in the C-terminal half. In addition, "CHX₁₀CHX₂₅CHX₂₅CH" (X: arbitrary amino acid) commonly found among BURP-domain proteins was also exist near C-terminal region. In the N-terminal half, sequences of 28 amino acids were repeated 5 times or five and half times. The clones expressed strongly in immature fruit or young leaf although expression was dropped latter half of fruit development. Expression in flower bud was low comparing to young fruit.

View full abstract

It is well known that the size of meristem is regulated by CLAVATA (CLV) signaling pathway in Arabidopsis. On the other hand, the genetic mechanisms of such regulation are poorly understood in other plants. We have been studying the function of FLORAL ORGAN NUMBER1 (FON1) and FON2 genes in rice. Our previous findings indicated that the genetic regulation of the CLV signaling pathway is principally conserved between rice and Arabidopsis. Furthermore, our data also suggest that some modifications are required for constructing a model in rice. To elucidate the mechanism of meristem maintenance further, it is essential to identify other genes that regulate meristem size. Here, we report the isolation of a gene that suppresses the fon2 mutation and its function. Our results strongly suggest an existence of an extra signaling pathway independent that of FON2-FON1 to regulate maintenance of the aerial meristems in rice.

View full abstract

Inflorescence architecture is an important agronomic trait because it is reflected in grain yield in rice. Little is known how inflorescence morphology is determined.
Characterization of the rice aberrant panicle organization 1 (apo1) mutant revealed that the decreased number of spikelet results from precocious transition of meristem identity from inflorescence to spikelet in apo1. Smaller main axis inflorescence meristem causes abnormal phyllotaxy of lateral inflorescence meristem.
In apo1 , lodicules replace stamens and carpels are indeterminately differentiated. APO1 also regulates floral organ identity and determinacy. Double mutants analyses and spatial expression patterns analyses of floral organ identity genes demonstrate that APO1 positively regulates Class C floral organ identity gene. APO1, FLORAL ORGAN NUMBER 1 and FON1 and FON2 cooperatively regulate cell proliferation and determinacy in rice floral meristem. We identified APO1 gene by map-based approach and we discuss its functions.

View full abstract

We identified a cleistogamy mutant of rice harboring a missense mutation in the class B MADS-box gene SUPERWOMAN1 (SPW1), which specifies lodicule (an equivalent of petal) and stamen identities. In this mutant, spw1-cls, stamens are normal but lodicules are transformed homeotically to lodicule-glume mosaic organs, thereby engendering cleistogamy. Introduction of the wild-type SPW1 gene into spw1-cls complemented the lodicule phenotype. Furthermore, introduction of the spw1-cls version of SPW1 (SPW1^cls) into spw1-1, a null allele of spw1, recapitulated the cleistogamy phenotype. We analyzed relationship of SPW1^cls with counterpart class B genes, OsMADS2 and OsMADS4, using yeast two-hybrid assay and in situ hybridization.

View full abstract

Specification of floral organs in rice is controlled by mechanisms similar to the so-called "ABCDE morel" of Arabidopsis thaliana. Although many MADS-box genes are known to be involved in the regulation, its detail is still unclear. OsMADS6 is one of rice class G MADS-box genes whose function is unknown. We have reported that the OsMADS6 RNAi lines showed alteration in the identities and numbers of several floral organs.
In this research, by using double mutants of OsMADS6 and other MADS-box genes, we investigated the relationship between OsMADS6 and other MADS-box genes. Also, we analyzed interaction of rice MADS-box proteins by yeast two-hybrid experiment. We discuss the function of class-G MADS-box genes and their interaction with other MADS-box genes, in rice floral organ determination.

View full abstract

The leaf in rice plant develops almost in one dimension, because cell division is largely restricted in its base. Therefore, it is easy to follow the process of vascular development in the leaf. We screened rice mutants defective in vascular formation of leaves. As the result, we succeeded in identifying 12 lines of vascular defective mutants.
In this paper we report a mutant showing a defect in leaf venation pattern. In this mutant, the intervals of commissural veins in the leaf are narrow and almost 10% of them are aggregated. Chromosomal walking revealed the causal gene of the mutant. This gene was unique in rice and predicted to encode a LRR-RK. In Arabidopsis, there are 5 homologous genes. Based on results obtained so far, we discuss the function of the LRR-RK in commissural vein formation.

View full abstract

Although polar auxin transport was known to play a pivotal role in vascular pattern formation, its molecular mechanism is not sufficiently elucidated. To understand the molecular mechanism of vascular pattern formation, we have analyzed van3 mutants characterized by fragmented veins. We have shown that VAN3 encodes an ARF-GAP protein that function on the TGN. As a first step toward characterizing molecular function of VAN3, we performed phenotypic analysis of various alleles of van3 mutants. This analysis suggested an indispensable role of PH domain in establishing vascular continuity. We have previously shown that PH domain in VAN3 strongly binds to PI4-P. Interestingly, mutation in the CVP2 that encodes an enzyme producing PI4-P phenocopies the van3. These results indicate that PI4-P plays essential function to establish the vascular continuity. We also isolated new protein that functions in the PI4-P-mediated process. Taken together, we will discuss the role of phosphoinositide-signaling in vascular development.

View full abstract

In Zinnia elegans cell cultures, single mesophyll cells transdifferentiate into tracheary elements in response to auxin and cytokinin. From the culture medium, we identified a dodeca-peptide designated TDIF, which inhibits tracheary elements differentiation.
To detect the localization of mRNAs and peptides in tissues, we first prepared a specific antibody against TDIF. Using the antibody and a cDNA-probe, we performed immunostaining and in situ hybridization to the zinnia tissues. Furthermore, we tried to reveal the function of TDIF, using molecular genetic approaches for Arabidopsis CLE41 and CLE44 peptides, which are the same sequence as TDIF, and CLE42 and CLE46 peptides, which are highly homologous to TDIF. Now, We are making transgenic plants suppressing the expression of their causal genes and those having their prmoter::GUS fusion. Based on these results, we will discuss the in vivo function of TDIF.

View full abstract

Postembryonic development in plants is dependent on the activity of shoot and root meristems. The Arabidopsis CLAVATA3 (CLV3), a CLV3/ESR-related (CLE) ligand, is responsible for the regulation of shoot meristem organization. We generated variable lengths of chemically synthesized CLV3 peptide and revealed that 12aa sequence of CLE domain is sufficient for CLV3 function. We also examined other 25 chemically synthetic CLE peptides, which correspond to the predicted products of 30 Arabidopsis CLE genes, and found functional redundancy between these peptides. Here, we show the effects of chemically synthetic CLE peptides on shoot and root meristems and discuss about CLE function in plants.

View full abstract

Leaf organizations in monocots are greatly diversified from those in dicots, and their structural relationships have been a matter of debate. In order to genetically dissect the leaf organization in monocots, we focused on "unifacial leaf" development, which was repeatedly evolved in a number of divergent species in monocots. Unifacial leaf develops bilaterally or radially symmetric leaf blade, in which leaf surface consists only of the abaxial side. From our observation, it was revealed that all of the leaf axes (i.e. adaxial-abaxial, distal-proximal, and central-lateral) are disorganized in unifacial leaves. Thus, comparative analyses between unifacial and normal bifacial leaf development should provide a number of information about leaf organization in monocots. In this report, we will discuss the mechanism of unifacial leaf development mainly based on the histological analyses, and will introduce our molecular genetic approaches to reveal the leaf organization in monocots.

View full abstract