ICE1 is a transcription factor regulating expression of DREB1A and cold-inducible genes and cold tolerance. To elucidate a detail mechanism of ICE1-dependent cold signaling, we carried out yeast two hybrid screening to isolate ICE1-interactors. Three ICE1 interactors have a typical bHLH domain, suggesting that they are transcription factors. T-DNA insertional mutants of these bHLH genes were more tolerant than wild-type plants under freezing temperature. In the mutant plants, DREB1A transcripts were up-regulated. A recent article demonstrates that ICE1 is involved in stomatal development. However, these mutants did not show any defects in stomatal development, but one of them has more stomata than wild type plants. These results demonstrate that two of the bHLH act as cold response-specific negative regulators and another bHLH is involved in negative regulation of both cold responses and stomatal number.

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We established the in vitro system for vessel element transdifferentiation in Arabidopsis Col-0 suspension cells. Through a microarray analysis of the system we identified a number of genes with drastic changes in expression during the transdifferentiation. Moreover, we revealed that VASCULAR-RELATED NAC-DOMIN7 (VND7) encoding a NAC-domain transcription factor functions as a master regulator for vessel formation. However, the regulatory mechanism of VND7 expression is still largely unknown. Therefore, in this study we aimed to identify transcription factors that regulate the VND7 expression. We selected 58 transcription factor genes with up-regulated expression during vessel formation in addition to VND family genes, VND1 to VND7, for the particle bombardment-based transient assays with the VND7pro:Luciferase construct. As a result, we found that several genes regulate the VND7 expression positively, which include ASL18/LBD30 and ASL20/LBD18 previously shown to induce the VND7 expression and all VND family genes. We are now further analyzing the transcriptional relationship among these transcription factors.

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The ERF-associated amphiphilic repression (EAR) motif is a plant specific repression domain (RD). The minimum core sequence is 6 amino acids and it acts as a repressor when fused with heterologous DNA binding domain. However, the mechanism of repression via EAR-motif (RD) is not known and it should be clarified whether a plant specific mechanism of transcriptional repression may exist. We attempted iTRAQ proteome analyses to identify factor(s) that interact with RD, and found that plant specific histone deacetylases, a histone demethylase and several nuclear proteins of unknown function were isolated. We are analyzing protein-protein interactions between these factors and RD, respectively, both in yeast and in vitro system. A sub-nuclear localization of representative proteins is examined by confocal microscopy. We present genetic evidences, that the nuclear protein involves in the RD related repression, using transgenic Arabidopsis and mutant lines. We will discuss a possible mechanism of transcriptional repression caused by the RD, including changes of histone codes, such as acethylation and methylation.

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In the winter-annual accessions of Arabidopsis, an active allele of FRIGIDA (FRI) elevates expression of FLOWERING LOCUS C (FLC), a repressor of flowering, and thus confers a vernalization requirement. FLC activation by FRI involves methylation of Lys 4 of histone H3 (H3K4) at FLC chromatin. Many multicellular organisms contain two classes of H3K4 methylases, a yeast Set1 class and a class related to Drosophila Trithorax. Here, we demonstrate that ARABIDOPSIS TRITHORAX-RELATED7 (ATXR7), a putative Set1 class H3K4 methylase, is required for proper FLC expression. The atxr7 mutation partially suppresses the delayed flowering of a FRI-containing line. The rapid flowering of atxr7 is associated with reduced FLC expression and is accompanied by decreased H3K4 methylation at FLC. Previously, it has been reported that lesions in ATX1, which encodes a Trithorax class H3K4 methylase, partially suppress the delayed flowering of winter-annual Arabidopsis. We show that the flowering phenotype of atx1 atxr7 double mutants is additive relative to those of single mutants. Therefore, both classes of H3K4 methylases appear to be required for proper regulation of FLC expression.

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The Ubiquitin (Ub)/26S proteasome pathway plays an essential housekeeping role to eliminate the proteins which are damaged or misfolded. It is also essential for most, if not all, aspects of cellular regulation by removing rate-limiting enzymes and dismantling existing regulatory networks as a way to fine-tune homeostasis, adapt to new environments, and redirect growth and development. This pathway has been implicated in a range of cellular networks, including cell cycle regulation, hormone response, photomorphogenesis, and pathogen resistance. Each proteasome subunit is presumed to contribute a specific function to overall proteasome activity, although these specific functions still remain largely unknown. We found that one of the proteasome subunits likely plays a role in regulating expression of transgenes inserted in the genome. The relationship between the proteasome and this gene regulation will be discussed.

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It is known DNA methylation controls expression of some genes during endosperm development. However, it remains to be clarified whether DNA methylation controls expression of embryogenesis-related genes in higher plants. In this study, we investigated DNA methylation status of FUS3 locus during seed maturation in Arabidopsis.
From Methylome database, it was found the upstream region of FUS3 is methylated. The FUS3 5'-upstream region showed a hypermethylation in mature seed. To test whether the hypermethylation of the FUS3 5'-upstream region down-regulates expression of FUS3 we made transgenic lines, which were induced the hypermethylation on this region. By the analyses of the transgenic lines, FUS3 expression level showed a negative correlation to DNA methylation level. Furthermore, we examined the contribution of DNA methyltransferase for de novo DNA methylation at the FUS3 5'-upstream region by using some mutants. As a result, it was found that DRM2 is involved in de novo DNA methylation at the region during seed maturation. From these results, we suggested that DNA methylation via DRM2 is involved in the control of FUS3 expression during seed maturation in Arabidopsis.

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CTP:phosphorylcholine cytidylyltransferase (CCT, EC 2.7.7.15) is a rate-limiting enzyme in the CDP-choline pathway to phosphatidylcholine biosynthesis. In Arabidopsis thaliana, CCT is encoded by CCT1 and CCT2. We previously reported that the double knock-out mutant cct1-1 cct2 shows epigenetic homeosis that converted stamens and petals into carperoids and sepaloids, respectively (Sekiguchi et al., JSPP meeting, 2008). The mutant phenotypes resemble that of the B class gene mutants apetala3 and pistillata in the ABC flower morphogenesis model. Thus, B class genes might be down-regulated in cct1-1 cct2 mutants. We analyzed the transcript levels of APETALA3 (AP3) and PISTILLATA (PI) by RT-PCR and found that the level of AP3 transcripts is down-regulated in cct1-1 cct2 plants in parallel with the severity of homeosis. Methylation analysis showed that another AP3 promoter region that is located upstream of our previous region is highly methylated in cct1-1 cct2 plants. These results suggest that DNA methylation in the AP3 promoter causes epigenetic homeosis in cct mutants.
⁴Present address, Graduate School of Agricultural and Life Sciences, The University of Tokyo.

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Post-translational modifications of histone N-tail affect chromatin and gene activities. Histone deacetylase affects gene silencing and heterochromatin formation in eukaryotes. In Arabidopsis, histone deacetylase 6 (HDA6) is involved in epigenetic gene silencing. We studied the enrichments of histone H4 acetylation in hda6 mutant on the genome-wide by ChIP-chip system. These genome-wide analyses show that about 160 independent peaks of histone H4 hyperacetylation, located on the TSS were detected. These were correlated with gene expression in hda6 mutant. Notably, HDA6 was required for the repression of transposable elements such as Sahdu and Copia near the centromere. In order to reveal the genome-wide histone modification sites, specifically targeted by HDA6, ChIP-chip analyses are in progress. We would like to discuss the role of histone modification status in the gene regulation mechanism in Arabidopsis.

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The ddm1 mutation in Arabidopsis thaliana causes genome-wide DNA hypomethylation, and by succeeding self-pollination, various types of developmental abnormalities are observed. One of such developmental abnormalities, bonsai (bns) was caused by loss-of-function of BNS gene. Recent study revealed that BNS gene was ectopically hypermetylated in ddm1 background. Thus, ddm1 mutation has two opposite effect; genome-wide DNA hypomethylation and local DNA hypermethylation.
To dissect the mechanism of ddm1-induced DNA hypermethylation, we combined the ddm1 mutation with mutations in various components of DNA methylation machineries, such as DNA methyltransferases, RNAi machineries, and histone modifiers. Interestingly, mutations in known components of RdDM (RNA-directed DNA methylation) did not suppress the induction of hypermethylation into BNS locus. On the other hand, hypermethylation in BNS was not induced in ddm1 cmt3 and ddm1 kyp mutants. Our results indicate that BNS locus was hypermethylated by CMT3-KYP mediated pathway.

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In RNA silencing pathway which is widely conserved among eukaryotes, the specific interaction between a Dicer-family protein and a dsRNA binding protein plays an important role. In this study, we biochemically analyzed the function of Arabidopsis thaliana dsRNA binding protein 4 (DRB4) in the dsRNA cleavage activity of Dicer-like 4 (DCL4). We prepared crude extracts from Arabidopsis seedlings, and incubated them with 500bp dsRNA derived from actin gene. As a result, the extracts from wild-type plants produced 21nt small RNA while the extracts from dcl4-2 and drb4-1 mutants did not. Then, we purified the DRB4 complexes from crude extracts by co-immunoprecipitation using anti-DRB4 antibody. The DRB4 complexes produced 21nt small RNA from 500bp dsRNA in vitro. DCL4 complexes purified by anti-DCL4 antibody showed cleavage activity similar to the DRB4 complexes. The DCL4 complexes from the drb4-1 extracts could not produce any small RNAs, but the addition of recombinant DRB4 to these complexes recovered the activity of 21nt small RNA production. These results suggest that DRB4 plays an essential role in the DCL4-dependent dsRNA cleavage and production of 21nt small RNA.

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Although cell reprogramming has long been observed in plants, the molecular machinery is mostly unexplored. To investigate the precise mechanisms of plant cell reprogramming, we focused on cold shock domain protein 1 of the moss Physcomitrella patens (PpCSP1), which is highly expressed in the apical stem cells. PpCSP1 transcripts were found to be up-regulated during the reprogramming process, where differentiated leaf cells were reprogrammed to apical stem cells. YFP signals of transgenic lines in which YFP gene was inserted to fuse to PpCSP1 were predominantly accumulated in the cells. We also revealed that PpCSP1 expression is post-transcriptionally repressed by the 3' UTR. The transgenic line in which the 3' UTR of PpCSP1 was removed from the genome, accumulated the transcripts. Cell reprogramming of this line was observed not only in the excised surface of the leaves as seen in wild type, but also from the entire leaves. Such an ectopic reprogramming supports the idea that PpCSP1 is a reprogramming promoting factor. Since PpCSP1 contains highly conserved RNA binding domains, its target RNA could be the key factor for the reprogramming process.

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Nonsense-mediated mRNA decay (NMD) is a well-known eukaryotic surveillance mechanism that eliminates aberrant mRNAs that contain a premature termination codon (PTC). UP-Frameshift (UPF) proteins, UPF1 and UPF3, are essential for normal NMD function. Here, we noticed that, in Arabidopsis, most of the mRNA-like noncoding RNAs (mlncRNAs) have the features of NMD substrate. We examined transcriptomes of two Arabidopsis mutants, upf1-1 and upf3-1, using whole genome tiling arrays. The results expectedly showed that many mlncRNAs were upregulated in both mutants. Thus, it is indicated that mlncRNAs are targeted by NMD system.
The 5'-to-3' RNA degradation by NMD is mediated by an exonuclease, XRN1, in animals. Arabidopsis has three genes encoding XRN enzymes. We examined expression profiles of three mutants, xrn2, xrn3 and xrn4, using tiling arrays. However, significant upregulation of mlncRNAs was not detected in all mutants. A previous report suggested that FRY1 protein promotes XRN activity. Increased accumulation of some mlncRNAs was detected in fry1 mutants. Thus, these results imply that three XRNs redundantly act on degradation of NMD-targeted RNAs.

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Control of gene expression is exerted by multiple steps such as transcription, processing and post-transcriptional events including mRNA export, mRNA degradation, translation, and post-translational events. Recent discovery of small RNAs has enhanced the impact of post-transcriptional regulation, in particular, control of mRNA stability. We first investigated control of mRNA decay in cold stress response in Arabidopsis. mRNA half-life can be determined by hybridizing microarrays to probes corresponding to total RNAs over a time course following inhibition of transcription. We use this strategy, called mRNA decay array, with Arabidopsis suspension cell culture (T87) to isolate genes regulated at the level of mRNA stability after cold stress treatment. The suspension cell culture showed the similar response to cold stress as the intact plants at early stages of their growth. The result implies that the system is suitable for understanding the cold response at the single cell level. This microarray analysis revealed that several transcripts are stabilized or destabilized efficiently enough to change the steady state level of mRNAs.

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NIP5;1, an aquaporin-like gene, is a boron (B) channel required for normal plant growth under low B conditions and its transcript accumulation is .upregulated under low B conditions (Takano et al., 2006). We have already reported that at least three distinct elements in NIP5;1 promoter regions were involved in the expression of three different portions of roots under low B condition and 5'untranslated region (5'UTR) (312 bp) is involved in NIP5;1 transcript accumulation in response to low B in last meeting.
To elucidate mechanisms of NIP5;1 regulation in response to B more detail, a series of truncated 5'UTR were generated, were fused to the GUS reporter gene under the control of cauliflower mosaic virus 35S RNA promoter and were introduced into Arabidopsis. The GUS expression of transgenic plants inserted 312 bp 5'UTR is higher in low B condition than in high B condition. On the other hand, the GUS expression of transgenic plants inserted 5'UTR but not including +184-+197(13bp) is high in both low and high B conditions. These results suggested that at least +184-+197 regions of 5'UTR are important for NIP5;1 transcript accumulation in response to low B.

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BBR/BPC proteins comprise a class of transcription factors that are confined to the plant kingdom. They have been identified due to their specific binding to a conserved simple di-nucleotide sequence repeat DNA-element (GA/TC)n.
Three distinct domain structures could be identified common to most BBR/BPC proteins: A N-terminal putative activation/interaction domain, a nuclear localization sequence (NLS) and a highly conserved basic DNA-binding domain, which is structured as a typical zinc-finger-like motif at its C-terminus.
Based on their differing N-terminal domains, the BBR/BPC family can be subdivided into distinct groups. Phylogenetic analysis on the DNA-binding domain sequence strongly supports this division.
BBR/BPC proteins exhibit group-wise expression patterns that are indicative of a high degree of functional redundancy between the group members.
Group II proteins form homo-/hetero-oligomeric structures in the nucleus and the nucleolus, which are mediated by a novel type of coiled coil-interaction domain. Target site analyses of putative binding motifs suggest a role for BBR/BPC proteins in regulating other transcription factor or hormone signalling related genes.

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We have been studied the mechanism of petal color change of Ipomoea tricolor cv. Heavenly Blue during flower-opening period. We have already reported the involvement of ItNHX1 in the pHv increase and found synchrony of petal-color change from red to blue, pHv increase, K<+>+</+> accumulation, and cell expansion growth during flower-opening period. We concluded that ItNHX1 exchanges K<+>+</+>, but not Na<+>+</+>, with H+ to accumulate an ionic osmoticum in the vacuole, which is then followed by cell expansion growth. To clarify whether this system involving NHX was comporehensive in other morning glories during flower opening period, we studied with morning glory petals of Ipomoea tricolor cv. Pearly Gate and red flowers of Ipomoea nil cv. Scarlet O'Hara. We analyzed their vacuolar pH change and ion contents of epidermal cells from -24 h to 0 h and found that in both case vacuolar pH increased and K<+>+</+> content became higher at 0 h compared with those at -24 h. We will discuss the role of NHXs in cell growth during flower opening of morning glory petals with special reference with NHXs.
K. Yoshida et al. Proc. Jpn. Acad. Ser. B, 85, 187 (2009).

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The Arabidopsis mutant stop1 (for sensitive to proton rhizotoxicity1) carries a missense mutation at an essential domain of the histidine-2-cysteine-2 zinc finger protein STOP1. Transcriptome analyses revealed that various genes were down-regulated in the mutant, indicating that STOP1 is involved in signal transduction pathways regulating aluminum (Al)- and proton-responsive gene expression. Critical genes for Al tolerance in Arabidopsis, AtALMT1, ALS3 and AtMATE co-down-regulated in the mutant. On the other hand, various gene belong to ion homeostasis, pH-regulating pathways and cell wall stabilization process were down regulated in the mutant. CIPK23, regulating a major K transporter, and a sulfate transporter, SULT3;5, were down-regulated in the mutant. In addition, integral profiling of the metabolites and transcripts revealed that pH-regulating metabolic pathways, such as the GABA shunt and biochemical pH stat pathways, are down-regulated in the mutant. These changes could explain the proton hypersensitivity of the mutant and would make the mutant more susceptible in acid soil stress than other Al-hypersensitive knock-out lines.

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In order to develop the methods for bioremediation of acidic soil using leguminous plants, we isolated a total of 23 proton tolerant (PTO) genes by functional screening of a soybean cDNA library with Escherichia coli expression system. We identified their orthologous genes in Arabidopsis thaliana by the blastp program. The orthologs of PTO49 includes three paralogs designated AtPTO49a, AtPTO49b, and AtPTO49c. The identity of the predicted amino acid sequences between AtPTO49a and AtPTO49b is 83 %, and the two paralogs are clustered into the same branch as PTO49 in the phylogenetic tree. The three paralogs showed the same organ specificity of expression. However, a knockout mutant of atpto49b showed obvious hypersensitivity to acid stress and morphological abnormality of leaf whereas atpto49a exhibited only mild acid sensitivity. These results suggests that each paralogous PTO49-like gene has distinct function.

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Graminaceous plants including rice utilize a chelation strategy to acquire Fe from the soil. The roots of these species release low-molecular weight compounds, called mugineic acid family phytosiderophores (MAs), which bind to and solubilize Fe(III) in the rhizosphere. Previously, we reported that rER-vesicles which were proposed to be involved in MAs biosynthesis were observed in iron-deficient rice roots, and OsNAS2:sGFP fusion protein, the enzyme participating in the MAs synthesis pathway, was localizes as small spots in the cytoplasm of the transgenic rice roots (Nozoye et al., JSPP 2008). Two motifs involved in cellular vesicle transport (tyrosine motif and di-leucine motif) were conserved in all NAS protein identified so far. In this report, we introduced the mutations into these motifs and observed the localization of mutated proteins in the transgenic rice root cells. The mutated OsNAS2 in tyrosine motif localized in the vesicles, however these vesicles cohered and could not move. On the other hand, the OsNAS2 mutated in di-leucine motif did not localize to these vesicles. It was suggested that MAs-vesicles were systematically transported in the rice root cells.

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Phytoremediation is known as a tool to clean-up excess pollutants from the environment. To use plants more efficiency for phytoremediation, it is helpful to know how plants obtained the resistance against pollutants. In this study, we focus on one of water and/or soil pollutant, selenium (Se), and try to identify the gene participate in plant Se-resistance by QTL analysis. Recombinant inbred lines (RIL) were made from progenies from F1 hybrid between two Arabidopsis thaliana ecotypes (selenium resistant, Col-0 and selenium sensitive, Ws-2). Among 205 F7 RILs, we measured root length of selected 99 RILs grown with or without selenite. Simultaneously, we performed genotype analysis of each RIL using 33 SSLP or CAPS markers. QTL analysis of RILs showed a loci located on chromosome 4 (close to ciw7 marker) significantly involve in the resistance to Se. Moreover, semi-fine-mapping analysis of RILs indicate that genes involve in Se resistance is located between two markers, FCA8s and ciw7, on chromosome 4. Since there are about 450 candidate genes in the identified region, we hereafter carry out fine-mapping analysis at the region.

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Growth and development in plants are supported by continuous supply of dividing cells in meristems. The cell division activity is maintained under environmental stress conditions. However, underlying mechanisms for the maintenance of meristematic activity in such circumstances are not well understood. In this study, we show that RSS1, which is required for environmental stress tolerance in rice, is involved in the machinery to ensure cell division activity under salinity. RSS1 encodes a novel protein with D-box, which is important for cell cycle-dependent protein degradation, and is preferentially expressed in dividing cells at G1 to S phase. In the shoot basal region of rss1, progression of cell cycle is inhibited at G1 under salinity. In the root tip of rss1, both meristematic and elongation zones were diminished depending on salinity. This is mimicked in wild-type root treated with DNA synthesis inhibitor, indicating that decrease of cell division activity accounts for the rss1 phenotype under salinity. The role of RSS1 in the mechanisms to ensure cell cycle and for stress tolerance will be discussed with the action of cytokinins, as well as evolutionary history of angiosperms.

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The accumulation of compatible solutes such as glycine betaine (betaine) and proline is one of the adaptive strategies for salt and drought stresses. It was shown that betaine is translocated in barley plants. However, little is known about the mechanisms of betaine transport at molecular level in barley plants. We have previously isolated the gene encoding a betaine/proline transporter, HvGB/ProT1 from barley plants. The purpose of this study is to elucidate the physiological role of HvGB/ProT1.
The transport assays using yeast strain 22574d showed that HvGB/ProT1 transports betaine and proline coupled with H⁺. Onion epidermal cells expressing HvGB/ProT1:GFP fusion protein indicated that HvGB/ProT1 is localized at the plasma membrane. The expression level of HvGB/ProT1 was higher in old leaves than in young leaves. On the other hand, the expression level was low in roots. Moreover, we determined the tissue-specific expression of HvGB/ProT1 by in situ hybridization. HvGB/ProT1 was expressed in xylem parenchyma cells and mestom sheath in leaves. On the other hand, in root tip, cross sections showed that expression of HvGB/ProT1 is restricted mainly to lateral root cap.

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The metal-accumulating trees are important plants for restoring contaminated soil because of their high biomass; however, they have not been well characterized. We examine here the metal tolerance and the growth rate of willow plants, including Salix eriocarpa, S. gilgiana and S. integra, in the presence of Cd. The synchrotron radiation-based μ-X-ray fluorescence (XRF) analysis revealed the accumulation pattern of Cd at the tip of the serration in leaves, and the phellogen and/or the phelloderm under the stem surface in S. gilgiana. μ-X-ray absorption near edge structure (XANES) spectra of Cd in all the accumulation sites are close to that of Cd ion coordinated by O ligands. Although sulfur is generally considered to detoxify Cd in the herbaceous plants, the present results suggest that the chemical form of Cd was not Cd-S in all the accumulation sites. S. gilgiana is one of the candidates for phytoremediation to restore metal contaminated soils in Japan. The present work adds a novel facet to phytoremediation technology by revealing a Cd accumulation mechanism in the woody metal accumulator.

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Athyrium yokoscense (At) is a metal hyper-tolerator/accumulator fern indigenously grown in Japan and eastern Asia. Here, cadmium accumulation in the fern was kinetically analyzed using the positron-emitting tracer imaging system (PETIS). Results showed a clear difference in Cd accumulation kinesis between At and some major crops (e.g. rice and tobacco). That is, major crops slowly absorbed Cd in roots and then comparatively quickly dispersed it among whole plant body, whereas At absorbed Cd in roots within surprisingly short time, and kept the most parts in rootstock at least during the observation period. At that time, the Cd absorption rate into roots (mol h^-1 plant^-1) was linearly dependent on the Cd concentration in the medium (0.1-100 μM) for At. Interestingly, concentrations of some potentially competitive metals in the media (e.g. Fe, Cu, and Zn) did not influence the Cd absorption rate in At. These results would relate the mechanism of Cd hyper-tolerance and/or Cd hyper-accumulation of At.

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An Arabidopsis mutant, itn1, is less sensitive to exogenous abscisic acid (ABA) than wild type plants. ITN1 encodes a plasma-membrane protein with an ankyrin-repeat motif which is implicated in protein-protein interactions. In order to clarify the molecular function of ITN1, we searched interacting partners of ITN1 by yeast two-hybrid screening. As a result, a putative transcription factor, RTV1, was identified as one of candidate proteins. Bimolecular fluorescence complementation analysis shows that RTV1 interacts with ITN1 at the plasma membrane in onion epidermal cells. In an Arabidopsis mutant deficient in RTV1, the ABA induction of PR1, known as a stress-responsive gene, occurred earlier than that in wild-type plants. By contrast, in the itn1 mutant, the ABA-induced expression of PR1 was suppressed. These suggests that RTV1 represses the PR1 expression at an early stage of ABA response, and that ITN1 induces the PR1 expression by interacting with RTV1 and inhibiting its activity at the later stage. Possible functions of the interaction between ITN1 and RTV1 in the ABA response will be discussed.

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Among cytsolic ADP-ribose/NADH pyrophosphatases (AtNUDX2, 6, and 7) in Arabidopsis, AtNUDX6 prefers NADH to ADP-ribose as a physiological substrate and its expression is induced by pathogenic attacks. Here, we explored physiological role of AtNUDX6 in the immuno response. The expression levels of AtNUDX6 were increased by treatment with 0.5 mM salicylic acid (SA), but not by various stressful conditions, suggesting that AtNUDX6 is involved in the SA signaling. The expression of NPR1-dependent SA-induced genes (SAIGs) paralleled that of AtNUDX6 under normal conditions and SA treatment, while those of NPR1-independent SAIGs did not change together with expression of AtNUDX6. Furthermore, the expression of TRX-h5 (thioredoxin 5) which catalyzed SA-induced NPR1 oligomer-to-monomer reaction paralleled that of AtNUDX6 under normal conditions and SA treatment. An inverse correlation was observed between the levels of ICS1 and AtNUDX6 under both normal conditions and SA treatment. These findings indicated that AtNUDX6 positively regulates the NPR1-dependent SA signaling via modulation of the NADH metabolism in plant immune response.

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Rice WRKY45 is a key transcription factor that mediates salicylic acid (SA) defense signaling pathway, and protein phosphorylation is possibly involved in its regulation. Our previous results showed that OsMPK6 was specifically activated by SA in rice cultured cells. However, since OsMPK6 has been implicated in various signaling pathways, its upstream kinases (MAPKKs) presumably play a key role in determining the pathway specificity of MAP kinase cascades. To investigate an SA-pathway-specific MAP kinase cascade leading to WRKY45 activation, we have functionally characterized rice MAPKKs.
In vitro phosphorylation assays using 4 recombinant MAPKK proteins expressed in E. coli showed that OsMPK6 was most strongly phosphorylated by OsMKK10-2. A GST-pulldown assay revealed that OsMKK10-2 strongly binds to OsMPK6. We also showed that dexamethazone-induced expression of OsMKK10-2 proteins resulted in the phosphorylation/activation of OsMPK6 and the upregulation of WRKY45 transcript levels in transgenic rice cells. We will also discuss the signaling specificity of OsMKK10-2-OsMPK6 cascade and its role in disease resistance.

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To clarify the processes involved in plant immunity, we have isolated and characterized Arabidopsis mutant, nsl2 (necrotic spotted lsion 2; we previously reported as the cad1), which shows a phenotype that mimics the lesions seen in the hypersensitive response. Inoculation of nsl2 mutant plants with virulence pathogen shows that the NSL2 mutation results in the restriction of bacterial growth (Plant Cell Physiol. 2005, 46: 902-912).
We started identification of the NSL2 interactors. Yeast two hybrid screening revealed that NSL2 interact with NAP1. Characterization of NSL2 interactors will be reported.

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Phytopathogenic fungus capable of producing trichothecenes can be found throughout the world, and include certain species of Fusarium. Trichothecenes has a sesquiterpenoid ring structure, and can be classified according to the presence or absence of characteristic functional groups.
Type A trichothecenes, such as T-2 toxin, caused rapid and prolonged activation of MAPKs, and triggered the cell death by activation of an elicitor-like signaling pathway in Arabidopsis. However, a MAP kinase cascade in the response to trichothecenes is unknown. Novel MAPKKK (MKD1) was identified as a subunit of AtNFXL1 protein complex. We had revealed that AtNFXL1 functions as a negative regulator of the trichothecene-induced defense response. The mkd1 mutant enhanced susceptibility to Fusarium sporotrichioides compared with wild type. Furthermore, accumulation of T-2 toxin was increased in mkd1 mutant. The MKD1 gene is ubiquitously expressed in all organs examined, and highly expressed in stomata and vascular bundles. In gel kinase assays showed that the activation of MPK3 and MPK6 in mkd1 mutant by T-2 toxin was decreased compared with wild type.

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To characterize the molecular function of OsPti1a in disease resistance, we investigated the localization of OsPti1a in rice cultured cells. OsPti1a was mainly localized in plasma membrane (PM), especially to detergent resistant membranes (DRMs) that were involved in regulation of signal transduction. OsPti1a in microsomal fraction including DRMs was phosphorylated, suggesting that OsPti1a functions on the PM through protein kinase cascade. HAstrepII-tagged OsPti1a at the N-terminal did not complement ospti1a mutant phenotype. Because N-terminus of OsPti1a has a putative lipid modification site that is important for binding with PM, we investigated whether N-terminal of OsPti1a is important for its cellular localization. In contrast to wild type protein, N-terminal deleted OsPti1a was localized in the cytosol fraction. These results suggest that the localization of OsPti1a in PM is important for negative regulation of defense signaling in rice. Additionally, OsPti1a was detected in the large complex about 250-450 kDa in microsomal fraction, suggesting that OsPti1a functions in the PM-associated complex in disease resistance.

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Pathogens deliver a number of effector proteins into plant cells to suppress immune responses. Resistant plants are able to recognize the effectors and induce strong immune responses including the oxidative burst and programmed cell death. Although many genes involved in plant-pathogen interactions have been identified, little is known about the mechanisms of effector recognition and subsequent signal transduction. Because the function of effectors is often not predictable from the amino acid sequences, it is difficult to study the mechanism of interaction between effectors and plant proteins and how effectors suppress immune responses. To elucidate the molecular basis of the interactions, we screened 261 proteins for their solubility and performed protein structural analyses. We will report the structural features of Phytophthora-derived effector proteins and their putative function.

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RAR1-SGT1-HSP90 complex is essential for disease resistance triggered by resistance (R) proteins in plants. Recent reports showed that SGT1-HSP90 complex is also necessary for innate immunity in animals induced by R protein homologues, Nod family proteins. Here, we present the structural and functional characterization of the plant RAR1-SGT1-HSP90 complex. NMR and X-ray crystal structural analysis clarified (1) the structure of CS domain of SGT1 which binds to both RAR1 and HSP90, (2) the structure of SGT1-HSP90 core complex and (3) the structure of RAR1-SGT1-HSP90 core complex. Mutational and biochemical analyses showed that the interaction between RAR1 and SGT1 enhances the formation of RAR1-SGT1-HSP90 complex and also enhances the interaction of SGT1 to R protein. Moreover, RAR1 mutations that abrogate the interaction with SGT1 compromise resistance against virus. These results suggest that RAR1 interaction with SGT1 is a critical step for assembly and function of the RAR1-SGT1-HSP90 complex and is required for innate immunity in plants.

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In order to clarify the role of symbiotic association in desiccation tolerance of photosynthetic partners in lichens, responses to dehydration in a chlorolichen (Ramalina yasudae) and its photobiont (Trebouxia sp.) was studied. Responses to dehydration in the isolated Trebouxia sp. were different from R. yasudae. That is, (1) the photosystem II (PSII) reaction was totally inhibited in R. yasudae when photosynthesis was completely inhibited by desiccation, but it remained active in Trebouxia sp. (2) Dehydration-induced quenching of PSII fluorescence was smaller in the Trebouxia sp. compared to that in R. yasudae. (3) The isolated Trebouxia sp. showed a higher sensitivity to photoinhibition than R. yasudae in the dehydrated condition. We analyzed about the water extract of R. yasudae and found out that arabitol which contained large amount in water extract accelerated quenching of PSII fluorescence of Trebouxia in the dehydrated state. The phenomenon was confirmed by the analysis of picosecond time-resolved fluorescence spectra. In other words, it indicates that accumulated arabitol in lichen body play a important role against dehydrated tolerance of Trebouxia.

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Root exudates influence the surrounding soil microbial community. Recent evidence shows the involvement of ATP binding cassette (ABC) transporters in root secretion of phytochemicals. In this study, we analyzed the effects of ABC transporter mutants of Arabidopsis on the microbial community in their native soils. After two generations, we observed by using automated ribosomal intergenic spacer analysis that the abcg30 (pdr2) mutant significantly altered the soil microbial communities compared with the wild type. The profile of root exudates of abcg30 significantly differed from those of the wild type. Microbial taxa in soils were determined by pyrosequencing, and revealed that abcg30 mutant cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria and enriched in bacteria involved in heavy metal remediation. Whole genome expression analyses of abcg30 roots revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated while some sugar transporters were down-regulated in roots. Some transporter and transcription factor genes were further analyzed in RT-PCR. (Badri et al. 2009 Plant Physiol)

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We tried to perform the chemical genetics screening by Brz (brassinazole) that was synthesized as the first specific inhibitor of brassinosteroid biosynthesis. Target of Brz220 is the cytochrome P450 enzyme encoded by DWF4. In order to analyze in detail the mechanisms of brassinosteroid signal transduction, we screened for mutants that showed longer hypocotyls than wild type when grown with Brz220 in the dark, and designated bil mutants (Brz-insensitive-long hypocotyl). We identified a semidominant mutant, bil5, from fast neutron-treated lines. Hypocotyl elongation of these plants on Brz medium was at least twice that of the wild type. Adult bil5 plants showed pale green and thin leaves, and thin and shortened inflorescences. Furthermore, we screened for mutants that showed pale green cotyledon than wild type when grown with Brz220 in the light, and designated bpg2 mutants (Brz-insensitive-pale green2). By analyses of the two mutants, we would like to discuss the molecular mechanism of chloroplast regution in brassinosteroid signalling.

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Brassinosteroids (BRs) are steroid hormones of plants that regulate plant growth and development. BRs bind to leucine-rich repeat kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1) localized to the plasma membrane, activate transcription factors in collaboration with cytosolic kinases and phosphatases, and regulate BR-responsive gene expression, but the details regarding the BR signaling pathway from perception to nuclear events remain unknown. To identify additional components involved in BR signal, we had analyzed various dwarf and semidwarf mutants from Arabidopsis mutant, which is insensitive to the BR synthetic inhibitor Brz. Brz causes deetiolation and dwarf phenotype similar to those of BR-deficient mutants. We had tried to screen mutants that showed to etiolate on medium containing Brz. Here we identify a bil4 (Brz-insensitive-long hypocotyl 4) mutant from activation tagging line. bil4 mutant exhibited outwardly curving, narrow leaves and shorter, thinner inflorescences. From genetic and functional analyses to bil4 mutant, we would like to clarify in detail the mechanism of brassinosteroid signaling transduction.

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Brz was synthesized as the first specific inhibitor for brassinosteroid biosynthesis. We had identified Brz insensitive mutant bil1 from EMS-mutation lines, and bil5 from fast neutron-mutation lines. To identify additional components of the brassinosteroid signaling, trial to screen new mutants as Brz-insesitive-long hypocotyls in the dark was applied to Arabidopsis activation-tag lines.
The semidominant mutant bss1(Brz-sensitive-short1) showed normally etiolated hypocotyl same as wild type without Brz in the dark. However, with Brz, bss1 hypocotyl became about 50% length of wild type in the dark. In the weak light, hyocotyl of bss1 showed about 50% length of wild type without Brz. These results suggested that bss1 hypocotyl is hypersensitive to Brz and the light. In the later growth stage, bss1 showed extremely dwarf phenotype by inhibition of axis elongation. Further analysis is in progress.
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Brassinosteroids (BRs) are the only steroid hormones in plant which widely distribute across the plant kingdom. From a number of structure-activity relationship analyzes, the activity as BR was largely affected by the structure of A-ring, B-ring and the side chain. In this work we found that two B-ring modified BRs, Iso-carbabrassinolide (Iso-carbaBL) and 6-deoxobrassinolide (6-deoxoBL) show different BR activity between Arabidopsis and rice. The activity as BR was measured by the det2 hypocotyl elongation assay for Arabidopsis and the lamina joint inclination assay for rice. In Arabidopsis, the BR activity of Iso-carbaBL was strong as that of castasterone (CS), while in rice, Iso-carbaBL showed no BR activity. The BR activity of 6-deoxoBL is stronger than that of brassinolide (BL), the most active BR, in Arabidopsis, while the BR activity of 6-deoxoBL in rice was weaker than that of CS. We investigated the binding activity of Iso-carbaBL and 6-deoxoBL to Arabidopsis and rice BRI1 and feedback regulation of BR biosynthetic genes by these chemicals to clarify the function of these synthetic BRs as BR in Arabidopsis and rice.

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We have characterized BR-upregulated rice basic helix-loop-helix genes BU1 (previously called OsBU3) and BU17. Analyses of rice plants overexpressing (BU1:OX) and suppressing BU1 indicated that BU1 controls bending of the lamina joint and acts as a positive regulator of BR response. BU17:OX showed erect leaves, suggesting that BU17 may act as a negative regulator of BR response. Here, we describe the role of BU1 and BU17 in BR signaling.
The induction of BU1 by exogenous BL did not require de novo protein synthesis and was weaker in a BR receptor (OsBRI1) mutant and a G protein alpha (RGA1) mutant as compared to WT. These results indicate that BU1 is a novel primary response gene to two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 was expressed in the lamina joints, phloem in floral organs and leaf blades, and epithelial cells in embryos.
Compared to WT, the expression level of BU17 was higher in BU1:OX, but lower in RNAi plants suppressing BU1 gene family; this suggests that BU17 may be located downstream of BU1 gene family. However, BU17 may play a role opposite to BU1, since BU17:OX displayed erect leaves.

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The rice lamina joint inclination assay is well known as an extremely sensitive system for brassinosteroid (BR) bioassay, although it was originally developed as an assay for auxin. The synergy between BR and auxin in the rice lamina joint inclination assay has also been reported, however, the molecular mechanism of this synergism has remained elusive. Here we demonstrate that auxin treatment transiently increases the expression of the rice BR receptor gene, OsBRI1, and its translational product, OsBRI1. The other phytohormones, such as gibberellin, cytokinin, and jasmonic acid did not affect the expression of OsBRI1, although the BR treatment decreases the expression of OsBRI1. On the other hand, auxin treatment did not affect the expression of BR biosynthetic and catabolic enzyme genes. These results suggest that auxin stimulates the BR sensitivity by increasing the expression of BR receptor, OsBRI1.

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Auxin is known to play a critical role in various development aspects in plants. To better understand the mechanisms of auxin responses in plants, we used the liverwort Marchantia polymorpha L., which is one of the basal land plants and has less complex developmental processes than angiosperms. Application of artificial auxin, naphthaleneacetic acid (NAA), at 1 μM to gemma resulted in rhizoid formation not only on the bottom side of thallus, but also on the top side. In contrast, auxin tranport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), at 10 μM reversed the dorsiventral axis of thallus. These results suggest that auxin is involved in determination of the dorsiventral axis during thallus development. To visualize auxin response, the β-glucuronidase (GUS) was expressed under the control of the soybean auxin-inducible promoter, GH3, in M. polymorpha. Transgenic plants carrying GH3::GUS showed GUS activity in an NAA-dependent manner. Histochemical staining of GUS revealed that auxin responses are different among tissues and developmental stages in M. polymorpha.

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The plant hormone auxin regulates many aspects of plant growth and development. Recent progress has provided a scheme that F-box proteins, TIR1/AFBs, function as auxin receptors and degrade transcriptional repressors, AUX/IAAs, allowing ARFs to regulate transcription of auxin-responsive genes. However, auxin signaling in seed plants turned out to be highly complex, and their genetic redundancy prevents our understanding of the whole story. The liverwort, Marchantia polymorpha, belongs to a group of basal land plants and has a simple body plan and low genetic redundancy. We identified key genes for auxin-signaling in M. polymorpha, MpTIR1, MpIAA, MpARF1, and MpARF2, indicating the auxin-signaling machinery in M. polymorha is basically the same as in seed plants. Sequence comparison suggests a possibility that MpARF1 is regulated by miR167 as ARF6 and ARF8 in Arabidopsis. MpIAA has a glutamine-rich region in addition to the four functional domains conserved among AUX/IAAs. Expression of MpIAA that has a mutation in a putative degron reduced sensitivity to auxin. Using these plants with altered auxin-response, the effect of auxin on morphology of M. polymorpha is being investigated.

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The Small Acidic Protein 1 (SMAP1) gene is responsible for the anti-auxin resistant mutation, aar1, and encodes a factor that mediates the synthetic auxin 2,4-D response. The previous results suggested that SMAP1 functions upstream of the AUX/IAA degradation step in auxin signaling pathway. To gain an insight into the function of SMAP1, the aar1 mutant was crossed with known auxin-related mutants. The double mutants, aar1-1 tir1-1, aar1-1 ecr1-1, and aar1-1 aar3-2 showed more resistant to 2,4-D than the corresponding single mutants in root growth assay. On the other hand, the axr1 aar1 double mutants showed severe morphological defects with a lack of root meristem formation. Similar phenotype was observed in the offspring generated by crossing axr1-12 and the SMAP1 RNAi transgenic lines. The overexpression of SMAP1-GFP fusion gene under 35S promoter relieved pleiotropic morphological phenotypes of axr1-12 such as dwarf, multiple shoots, altered flower structure, low fertility and reduced auxin response. The results suggested that the function of SMAP1 is tightly linked to that of AXR1 in genetic level.

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Auxins, a group of phytohormones, regulate plant growth and development. For biosynthesis of a naturally occurring auxin, indole-3-acetic acid (IAA), Arabidopsis has four possible pathways: the YUCCA (YUC) pathway, the CYP79B pathway, the indole-3-pyruvic acid pathway and the indole-3-acetamide pathway. IAA biosynthesis-suppressed mutants such yuc quadruple knockout lines show IAA-deficient phenotype, but IAA levels are not greatly affected in these mutants at the normal growth condition. To address this question, we have analyzed the levels of IAA metabolites in Arabidopsis IAA biosynthesis mutants. We found that IAA-amino acid conjugates are significantly decreased in the CYP79B-deficient mutants. The levels of IAA-amino acid conjugates were dramatically increased in the YUC2-overexpression mutants. These results indicate that the IAA metabolic pathways may have important roles to maintain IAA levels in IAA biosynthesis-deficient mutants.

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Auxins are a group of phytohormones that act in many aspects of plant growth and development. Indole-3-acetic acid (IAA) is a naturally occurring auxin, whose biosynthetic pathway is not fully understood. To elucidate the IAA biosynthetic pathway, we have been developing methods for the analysis of labile IAA intermediates by LC-ESI-MS/MS. Indole-3-acetaldehyde (IAAld) has been proposed as a common IAA biosynthetic intermediate, but not much is known about how IAAld is produced in plants. Here we present a direct analysis method for IAAld in plants using LC-ESI-MS/MS. Using our IAAld analysis method, we detected IAAld in Arabidopsis without any derivatization. When stable isotope-labeled indole or tryptophan was fed to Arabidopsis, IAAld was efficiently labeled with stable isotopes. We are currently analyzing the levels of IAAld in various IAA biosynthesis-related mutants to identify the IAA biosynthetic pathway involving IAAld.

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We screened four semi-dominant semi-dwarf mutants of rice through MNU mutagenesis. Map-based cloning revealed that the dwarf phenotype in these mutants is caused by gain-of-function mutations in the N-terminal DELLA/TVHYNP region or the C-terminal GRAS domain of SLR1, and thus named them as Slr1-d1~4. A Gibberellin (GA) response test showed that Slr1-d are still responsive to GA, although at a reduced rate compared to the wild type. Additionally, degradation rate of SLR1-d protein were slower than SLR1. Since interaction between SLR1 and GA receptor GID1 is known to be essential for SLR1 degradation, interaction studies were conducted using the yeast two hybrid system, where reduced interaction abilities of the SLR1-d protein with GID1 were observed compared to SLR1. These results suggest that the semi-dwarf phenotype of Slr1-d mutants are caused by reduced interaction between Slr1-d protein and GID1, which lead to delayed degradation of SLR1-d protein.This work was supported by the Targeted Proteins Research Program and KAKENHI(18107001).

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It has been proposed that the binding of GA to GID1 induces interaction between GID1 and DELLA protein, a repressor of GA signaling, and resulting in the degradation of DELLA protein. We isolated and focused one suppressor mutant of gid1, Sgd-1. Sgd-1 carried one amino acid substitution (P99S) in the OsGID1 locus, which causes an interaction between GID1 and DELLA in the absence of GA. We found another mutant GID1 with P99A that has the highest GA-independent DELLA interacting activity. Through the analyses of OsGID1P99A or P99S by Y2H assay and Surface Plasmon Resonance, we revealed that these mutated GID1s have a close state of the lid without GA resulting in DELLA interaction.
It has been reported that AtGID1b also interacts with DELLA in GA independent manner. Mutation analysis revealed that such GA-independency depends on a similar mechanism of the OsGID1P99A-DELLA interaction. We also found that some dicot GID1s have similar GA independent GID1-DELLA interacting activity.
A part of this research is supported by target protein, KAKENHI (18107001), and new agricultural project (IPG0003).

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Strigolactones are endogenous growth regulators that inhibit shoot branching, as well as being communication signals for symbiosis and parasitism in the rhizosphere. Studies using chemical inhibitors and mutants have suggested that strigolactones are biosynthesized from carotenoids. However, the isoprenoid origin of the strigolactone carbon skeleton has not been proved directly in a metabolism study. To address this question, a ¹³C-labeled precursor specific to the mevalonate or methylerythritol phosphate (MEP) pathway was fed to Lotus japonicus cultured roots in the presence of a chemical inhibitor that blocks each endogenous pathway. We found that ¹³C-deoxyxylulose, but not ¹³C-mevalonolactone, was efficiently incorporated into 5-deoxystrigol, indicating that the MEP pathway principally provides isoprene units to strigolactones.

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We have previously shown that strigolactones, a group of carotenoid-derived terpenoids, act as hormones or their biosynthetic precursors that regulate shoot branching by inhibiting axillary bud outgrowth. Studies using rice tillering dwarf mutants sueggested that DWARF14, encoding a member of the α/β-hydrolase superfamily, regulates shoot branching in a step downstream of strigolactone biosynthesis; d14 mutant plants accumulate strigolactone at higher levels than does wild-type plants and their tillering phenotype is not rescued by strigolactone treatment. To explore whether D14-related gene(s) regulates shoot branching in the strigolactone-dependent pathway in other plant species, we are currently studying physiological roles of D14 homologs in Arabidopsis through reverse genetics.

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Hormonal control of axillary bud growth has been extensively studied for many years. In particular, an involvement of two hormones, auxin and cytokinin, has been well known. On the other hand, recent genetic and physiological analysis using grafting techniques revealed that a graft transmissible branch inhibiting hormone plays a major role in the control of shoot branching. The chemical nature of the predicted branch inhibiting hormone was revealed recently.
Five tillering dwarf (d) mutants of rice, dwarf3 (d3), d10, d14, d17 and d27 , show an accelerated shoot branching and a reduced plant height in common due to the failure in biosynthesis, perception or signal transduction of strigolactone. fine culm1 (fc1) mutant also shows an increased shoot branching, resembling the five d mutants. FC1 is an ortholog of maize teosinte branched1 (tb1) , and encodes a transcription factor containing a TCP domain.
As a first step to reveal how strigolacton controls shoot branching, we analyzed phenotypes of the five d mutants and fc1 in detail. Moreover, a possible interaction between FC1 and strigolactone will be discussed.

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