In order to find the genes involved in salt tolerance of rice, we carried out the screening of a rice full-length cDNA expression library through functional complementation in yeast. The yeast expression library was constructed using Gateway System (Invitrogen) from the rice full-length cDNA library of NIAS that contains about 28,000 clones. The library was transformed in salt-sensitive yeast strain G19 (Δena1-4) and the transformants were selected on SD plate containing 0.5 M of NaCl. In the screening, a cDNA clone encoding a homologue of the Arabidopsis potassium channel protein KAT1 (OsKAT1) suppressed the salt-sensitive phenotype of G19. The transformants accumulated lesser amounts of Na⁺ than mutant cells under 0.5 M NaCl condition, whereas the K⁺ amount of these cells were almost the same. These results suggest that OsKAT1 may be participated in maintenance of cytosolic ion homeostasis during salt stress.

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Wheat aluminum(Al)-activated malate transporter (ALMT1) is localized on plasma membrane. Hydrophobicity profile of ALMT1 protein shows predicted 5 to 8 transmembrane regions. To elucidate the transmembrane topology of this protein, we examined the orientation of C-terminal domain of ALMT1 protein in cultured tobacco cells which expressed the ALMT1 protein tagged with the epitope at the C terminus. The accessibilities of the C terminus region of the ALMT1 protein to anti-epitope antibody and the anti-ALMT1 antiserum raised against the ALMT1 C-terminal 218 amino acid region were examined in the absence or presence of a detergent to infiltrate these antibodies into cells. The immunofluorescence signals against these primary antibodies were detected at the plasma membrane similarly in the presence or absence of the detergent. These results suggest that C terminus of ALMT1 protein faces toward the outside of the membrane.

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We have recently isolated the wheat ALMT1 gene which confers Al tolerance and encodes a novel plasma-membrane-bound protein facilitating malate efflux from root apex under Al exposure. To examine whether the ALMT1 ortholog confers Al-activated efflux of organic anions in other Al-tolerant plants, we focused on rye which is more tolerant to Al than wheat and secretes both malate and citrate under Al exposure. The time-dependent enhancement of expression of the rye-ALMT1 ortholog was significantly correlated with the enhanced effluxes of citrate and malate during Al treatment. These results suggest that the rye-ALMT1 ortholog is involved in the Al-dependent secretion of organic anions. By use of the methods of RT-PCR and cDNA library screening, we cloned three different cDNAs of the ALMT1 ortholog which showed 90% nucleotide identity to the wheat ALMT1-1. The analyses of expression patterns and functions of these ALMT1 orthologs in rye are now in progress.

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Aluminum (Al) causes root growth inhibition, but the toxicity mechanism has not been elucidated. In this study, a mechanism of the Al-induced inhibition of sucrose uptake was investigated in tobacco cells. Firstly, the characteristics of sucrose uptake was investigated in cells suspended in the calcium solution which contained various concentrations of ¹⁴C-Sucrose, but was adjusted osmolality with mannitol to the same level. The kinetics of sucrose uptake suggests two systems of high (at 1~3mM sucrose) and low (at 25~75mM sucrose) affinities. The high affinity uptake was inhibited almost completely with CCCP, but not with Wortmaninn, suggesting that high affinity uptake is via sucrose-proton cotransporter, but not endocytosis. Al inhibited both high and low affinity uptakes by more than 60% after 30 min exposure. These results suggest that tobacco cells have high and low affinity systems of sucrose uptake, and that Al immediately inhibits both systems.

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Arabidopsis genome contains ca. 130 ATP-binding cassette (ABC) protein genes. AtABCA1 of Arabidopsis, the counterpart of human ABCA1 functioning as a major regulator of cellular cholesterol homeostasis, is a single copy gene, whereas its orthologue is not found in yeast and even in rice genome. This occurrence suggests that AtABCA1 has physiological functions specific to dicots, or other members of ABC protein may complement the roles of ABCA1 in monocots. In this study we analyzed the gene expression of AtABCA1. The AtABCA1 expression was specifically up-regulated by abscisic acid. AtABCA1 promoter::GUS trasngenic plants showed that AtABCA1 preferentially expressed in the vascular tissues of root, stem, and leaf through out the growth stage from seedling to adult plant. In addition, strong expression was also observed in mature pollen. We are determining the subcellular localization of AtABCA1 by use of GFP-fusion protein expressed in Arabidopsis stable transformant.

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The first step of the sulfate assimilation pathway is catalyzed by ATP sulfurylase (ATPS). Four ATPS genes exist in Arabidopsis thaliana genome. ATPS activity has been detected in chloroplasts and in cytosol in Arabidopsis; however, all of the isoforms appear to encode a transit peptide for chloroplast targeting. Thus, the origin of the cytosolic form of ATPS is still uncertain. In this study, to clarify the physiological role of each ATPS isoform in Arabidopsis, we carried out gene expression analysis of the isoforms and functional analysis of atps mutants. The gene expression was detected by real-time quantitative PCR analysis. ATPS4 showed a specific pattern, in which the expression is low at two-week-old leaf, but is high at six-week-old leaf and silique. We isolated T-DNA inserted mutants of ATPS2 and ATPS3. The accumulation of cysteine and glutathione decreased about 80% in the obtained mutants compared to wild-type plant.

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Serine acetyltransferase (Serat) catalyzes the formation of O-acetylserine (OAS) from L-serine and acetyl-CoA, leading to synthesis of cysteine. In the genome of Arabidopsis, there are five Serat genes (Serat1;1, 2;1, 2;2, 3;1, 3;2). Until now, we have isolated T-DNA knockout mutants in each Serat gene. The results from gene expression analysis and metabolite profiles on these mutants suggested that each Serat gene has distinct role for cysteine biosynthesis. To investigate their roles further, we crossed those mutants to obtain double knockout mutants (serat1;1serat2;1, serat1;1serat2;2, serat2;1serat2;2). Serat1;1, 2;1, 2;2, which are thought to be major isoforms, are localized in cytosol, plastids, mitochondria respectively. In serat2;1serat2;2 mutants, expression levels of Serat3;2 was increased and OAS, Cys and GSH contents were remarkably decreased. Such result was not observed in serat2;1 or serat2;2 single mutants, implying that Serat2;1 and Serat2;2 complemented their function each other. We are currently analyzing the other two mutants.

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Acetylcholinesterase (AChE) was recently purified and cloned from maize, which is the first direct evidence of the plant AChE. Purified AChE was 88-kDa homodimers consisting of 42-44kDa polypeptides. The full-length cDNA of maize AChE gene is 1471 nucleotides, encoding a protein having 394 residues, including a signal peptide. In this study, enzymatic characterization of maize AChE was compared with AChE from Macroptilium atropurpureum and electric eel as representative of dicotyledon and animal AChE. The plant AChE hydrolyzed acetylcholine, propionylcholine, acetylthiocholine, propionylthiocholine, but not S-butyrylthiocholine and the AChE-specific inhibitor neostigmine bromide competitively inhibited its activity, implying that maize AChE functions in a similar manner as an eel AChE. However, both plant AChE showed a lower affinity to substrates and inhibitor than an eel AChE, and not exhibited the inhibition by excess substrate condition. Thus structural difference around active site between plant and animal AChE might be involved in differential inhibitory effect.

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PhospholipaseA₂(PLA₂) hydrolyzes glycerophospholipids specifically at the sn-2 position to yield free fatty acids and lysophospholipids. In animals, PLA₂s are classified according to the primary structure, Ca²⁺ dependence and cellular localization. Secretory PLA₂ (sPLA₂) is Ca²⁺ dependent and low molecular mass enzyme. Animal sPLA₂ commonly contains Ca²⁺ binding domain and catalytic domain. We have recently cloned two cDNAs encoding sPLA₂ from the tobacco. They also have the Ca²⁺ binding domain and the catalytic domain in their primary structures. In this study, we constructed bacterial expression system for these sPLA₂ and investigated their enzymatic properties. Interestingly, the one hydrolyzed the fatty acyl ester bond at the sn-1 and -2 positions of glycerophospholipids. They essentially required Ca²⁺ for their enzymatic activities. They showed different pH optimum and substrate specificity. We introduced site-directed mutations into some conserved amino acid residues to generate recombinant sPLA₂ mutants and studied their enzymatic properties.

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Phospholipase A₂ (PLA₂) hydrolyzes the fatty acyl ester bond at the sn-2 position of glycerophospholipids. In many plant species, the existence of PLA₂ has been ascertained by advance in genome project and molecular biology. However, there is little information on the physiological role of plant PLA₂, in contrast to many reports on the functions of animal PLA₂. We have recently cloned two putative secretory PLA₂ (sPLA₂) cDNAs from tobacco. However, it was difficult to analyze mRNA expression because of small amount of their transcripts. In this study, we investigated the tissue specificity of mRNA expression by using a quantitative RT-PCR. Both sPLA₂ mRNA expressions were high in flower, while they were low in root, leaf and stem. Furthermore, PLA₂ activity was also much higher in flower than in other tissues. We now investigate the response of both sPLA₂ to stress on tobacco plant and cultured cells.

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Delta 22-unsaturated sterols, containing a double bond at the C-22 position in the side chain, occur specifically in fungi and plants. In yeast, cytochrome P450 (CYP61) has been known as the sterol Δ²²-desaturase in the ergosterol biosynthetic pathway. In this study, we performed a comprehensive sterol profiling to identify functional homologues of CYP61 in plants. Thus, we prepared a series of P450 transgenic lines of Arabidopsis and studied differential effects on the sterol profiles in planta. In the overexpression lines of either CYP710A1 from Arabidopsis or CYP710A11 from tomato, stigmasterol accumulated to 6- to 32-fold higher levels compared with wild type. The promoter::GUS reporter assays suggested strict tissue specific expression patterns of the Δ²²-desaturase genes in Arabidopsis, implicating developmental stage specific involvement of the Δ²²-sterols.

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Higher plants possess a large set of the class III plant peroxidases. These peroxidases are thought to participate in a wide rage of physiological functions, however, the most of their functions have been unclear. Using proteomic analysis, we surveyed the localization of plant peroxidase in various organs in Populus alba. Peroxidase isoenzymes were extracted from the various organs and fractionated by a Concanavallin A Sepharose column. Each peroxidase protein was identified by the Peptide Mass Fingerprint analysis. All organs expressed anionic peroxidase isoenzymes dominantly, and they are composed in a small cluster of phylogenic tree of poplar peroxidases. Interestingly, each organ has different peroxidase constitution. These results suggest that individual peroxidase isoenzyme in this cluster is regulated under different transcriptional or translational control.

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RTX toxins produced by Gram-negative bacteria contain GGXGXDXUX motif sequences for Ca²⁺-binding and undergo reversible conformational changes by release and rebinding of Ca²⁺ ions. On the other hand, RTX proteins of cyanobacteria, whose structure and intracellular localization are similar to those of RTX toxins, are non-toxic and reported to be involved in cell motility. In the case of Synechocystis sp. PCC 6803, however, expression of RTX protein Sll1951 is suppressed in the motile wild type strain (WT), while the protein is abundantly expressed in the non-motile glucose-tolerant strain (GT) (above 10% of total proteins). From these facts, the involvement of cyanobacterial RTX proteins in cell motility is unlikely to be a general feature in Cyanobacteria. In this study, sll1951 was disrupted by insertion of a transposon. Resulting changes in the phenotype are reported in the presentation.

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Dicer proteins are involved in RNA silencing. They produce siRNAs and miRNAs, and, in animals, they interact with a dsRNA-binding protein (RDE-4 and R2D2). In Arabidopsis thaliana there are 4 members of the Dicer family and 5 members of the RDE-4/R2D2-like dsRNA-binding protein family. We have studied the biochemical properties of these eight gene products: four Arabidopsis Dicer-like proteins (DCL1-4) and four RDE-4/R2D2-like dsRNA-binding proteins (HYL1/DRB1, DRB2, DRB4 and DRB5). We have reported that DCL1, DCL3 and HYL1/DRB family proteins exhibit dsRNA-binding activity, DCL1, DCL4, HYL1/DRB1 and DRB4 localize in the nucleus, and HYL1/DRB family proteins bind to DCL family proteins. HYL1/DRB1 binds specifically to DCL1, and DRB4 most strongly binds to DCL4 in vitro. Here, we show that DRB4 interacts specifically with DCL4 in vivo by immunoprecipitation. The result suggest that a Dicer functions with a dsRNA-binding protein (HYL1/DRB family) in plant.

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We are studying RNA-mediated transcriptional gene silencing to understand effects of siRNA on various chromatin in rice. RNA-mediated TGS is a chromatin-based gene silencing, triggered by siRNA containing sequences that are identical to the promoter. We chose a total of 8 target genes and produced transgenic rice that accumulated siRNA containing sequences of target promoter. Our results indicated that 35S::GFP and Se5 were silenced, but the other 6 target genes were not silenced. Furthermore, we investigated the correlation between the silencing and epigenetic modifications. DNA methylation analysis showed that all 8 target promoters were methylated. ChIP analysis indicated that histone H3 deacetylation was observed in only the 35S target promoter, which was silenced. Now, we are analyzing the Se5 target promoter, which was silenced, for histone modifications. We also investigate the effects of DNA methylation or histone deacetylation inhibitors on RNA-mediated TGS. The epigenetic function of siRNA will be discussed.

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Cytosine methylation plays critical role in epigenetic regulation. Generally methylated cytosine represses the extension step in mRNA synthesis to suppress the gene expression.
We succeeded to induce RNAi that is targeted against spectinomycin resistance related aadA gene by introducing inverted repeat (IR) trnasgene in Chlamydomonas reinhardtii. However, without rearrangement of the target gene and the silencer, silencing intensity drifted severely through somatic cell divisions. After period, sub-clones originated in a particular transformant revealed various extent of silencing intensity. Comparative analysis of sub-clones showed that CG-methylation is heavily accumulated at IR region and the amount of the accumulated hairpin RNA is limited when the clone showed restrained RNAi. Moreover, accumulation of 3'-truncated immature IR transcripts was prominent in such transformants. While, target aadA gene is not methylated at all. Treatment by TSA enhanced accumulation of hairpin RNA. This indicates that CG-methylation collaborated with deacetylated histone repressed the transcription of the silencer.

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A rice epigenetic mutant, Epi-d1, shows a chimeric phenotype, which is combined with the normal and dwarf tillers in one plant body. Positional cloning and allelic test revealed that the Dwarf1 (D1) gene is causal for this phenotype. Switching -on and -off of the D1 expression frequently occurs in this mutant, that is, D1 is expressed in the normal tillers, and not in the dwarf tillers. We detected defference in the methylation status in the 5'-flanking region of the D1 gene between dwarf and normal tillers. We here show a rice epigenetic mutant, Epi-d1 and discuss the cause.
The result suggest that the methylation status at this region of the D1 gene is often changed reversibly and the non-methylated D1 gene can express its product but the methylated gene does not.
We will discuss relationship between unique sequence structure around the D1 gene and its ad hoc methylation.

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Transgenes stably integrated into chromosome are often silenced, a phenomena is called gene silencing. Gene silencing has been attributed to several factors, including differences in chromosome position, repeat sequences, and copy number. We transformed Arabidopsis plants with pBI121 (CaMV35S-GUS and NOS-NPTII) using Agrobacterium-mediated transformation, and carefully selected ten lines containing single 'complete' (intact, non-truncated, non-rearranged) copies of the transgene located at different chromosome positions. These lines showed very similar levels of transgene expression, and gene silencing was not observed. In a subsequent study, intercrosses of isolated lines were performed to generate plants harboring one to eight copies of the transgene. One to five copies of the GUS gene were introduced in the genome, positive correlation between gene copy number and GUS activity was observed. However, six or more copies of the GUS gene resulted in silencing.

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In eukaryotic nucleus, genomic DNA is assembled into chromatin, nucleoprotein complex including histone. Chromatin assembly factor-1 (CAF-1) supports the assembly of nucleosome on to the DNA in vitro. Anti-silencing function 1 (Asf1) was identified as a component of RCAF, the chromatin assembly factor dependent on CAF-1. We have previously shown that FASCIATA1 (FAS1) and FAS2 genes encode two subunits of the Arabidopsis counterpart of human CAF-1. ASF1a and ASF1b encode the homologues of human Asf1a and Asf1b respectively, and we report the characterization of asf1a and asf1b mutants, double or triple mutants with fas2. asf1a and asf1b display no significant phenotype, but in double mutant of them, pointed, narrow, and serrated leaves similar to which in fas2 mutant were observed. The double and triple mutant of asf1 with fas2 display male sterility and early-embryonic lethal phenotype, respectively. The role of Asf1 and CAF-1 in Arabidopsis will be discussed.

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Heterosis (hybrid vigor) is often observed in F ₁ individuals obtained from distantly related parents and it exhibits superior characteristics in various aspects of growth and environmental responses. The molecular basis of heterosis has been explained with either additive or synergistic expression of genes peculiar to each parent. However, many different organisms show the hybrid vigor in the basic activity of cells and individuals including growth rate and body size, therefore, there can be a biologically common mechanism of heterosis. To elucidate this hypothesis, we characterized the growth of F ₁ rice seedlings obtained by crossing between a japonica (Nipponbare) and an indica (Kasalath) rice under a model environment using an automated growth monitoring system. Heterosis was observed in the growth rate of the third leaf and it was specifically reduced on application of an inhibitor for histone deacetylases that affect global chromatin structures.

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NtWIF was isolated as a down-stream transcription factor of WIPK from tobacco plants. To identify phosphorylation site(s) of NtWIF, we introduced mutations into the potential MAPK target site, Thr-Pro located in the N-terminus. When Thr-Pro site was replaced by Ala-Pro, Thr-Ala or Ala-Ala, transcriptional activities were completely lost even in the presence of WIPK.
Subsequently, we looked for target genes of NtWIF. Structually, the N-terminus of NtWIF shows a high similarity to the plant specific DNA binding domain of Auxin response factor (ARF). Gel shift assay using AuxRE motif containing TGTCTC sequence, that other ARFs recognize and bind to, showed that the full-length NtWIF was able to bind to AuxRE motif. Moreover, co-transfection of AuxRE-Luc reporter plasmid and NtWIF resulted in activation of reporter gene expression. These results suggested that target genes of NtWIF possess the AuxRE motif in their promoter regions.

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Proteins synthesized in the endoplasmic reticulum (ER) are correctly folded before translocation. If protein folding is incorrect, genes for the ER-resident chaperones such as BiP are induced. This phenomenon is known as the ER stress response. Studying molecular mechanism of the ER stress response in Arabidopsis, we identified a bZIP transcription factor AtbZIP60 induced by tunicamycin. AtbZIP60 has a putative transmembrane domain in its C-terminal region, suggesting proteolysis triggers its activation. Indeed, a truncated form without C-terminal region (AtbZIP60ΔC) fused with GFP localized to the nucleus. In addition, overexpression of AtbZIP60ΔC activated BiP promoters in a transient assay although full length did not. Immunoblot analysis indicated that AtbZIP60 protein was cleaved by tunicamycin and DTT treatment, supporting above hypothesis. Determination of subcellular localization is under progress.

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We have revealed that distinct DNase I hypersensitive site (DNase I HS) was found in competent (expressed or inducible) gene promoter and was not found in incompetent (unexpressed nor inducible) gene promoters in Arabidopsis. Next, to understand the regulatory roles of histone modifications in transcriptional activation and competence, we performed chromatin immunoprecipitation analysis. Chromatin structural analysis of the heat-inducible HSP18.2 promoter revealed that the DNase I HS was pre-formed upstream of the cis-acting element in its inactive state, upon activation, histone H3 and H4 were acetylated, nucleosome(s) downstream of this DNase I HS was disrupted resulting in the exposure of the cis-elements and transcription start site. These results suggest that the DNase I HSs regulate transcription by providing accessible sites for the binding of chromatin remodeling factors. Regulatory roles of the presence of the DNase I HS, transcription factor binding and histone modification states will be discussed.

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Matrix attachment region (MAR) is DNA sequence bound to the nuclear matrix. It is considered that the sequence of MAR is about 1 kb-long and is existed at intervals of 50-100 kb on a chromosome. In this study, to investigate the function of MAR, we identified many candidates of MAR using the genome tiling array which covers the long arm of the Arabidopsis chromosome V. About the probe DNA used for the array, the protoplast prepared from Arabidopsis were dissolved by Triton X-100 and digested genome DNA by nuclease, and then co-precipitated DNA with nuclear matrix by centrifugation were purified. We confirmed the binding ability of candidates of MAR to the nuclear matrix in vitro. We analyzed the 20 kb genomic region ( 4 genes are included ) containing a candidate MAR in detail, three MARs existed. Here, we will report the position relation between these MARs and four genes.

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In plants, the primary enzyme of carbon fixation, RuBisCO (riblose-1,5-bisphosphate carboxylase/oxygenase), is encoded as two subunits, rbcL and rbcS. It is reported that the ML tree based on the amino-acid sequence of RbcL deduced from the nucleotide sequence alignment makes two clusters; R-type (red linage) cluster and G-type (green linage) cluster. G-type RuBisCO coding genes constitute rbcL-rbcS operon, while R-type RuBisCO coding genes constitute rbcL-rbcS-cfxQ operon. The cfxQ is predicted to be necessary for the expression of RuBisCO, but the function of CfxQ has not been known.
cDNA analysis and complete genome sequence of Cyanidioschyzon merolae 10D indicate that cfxQ is coded both in the plastid genome and in the cell nucleus. In this study, we purpose the function of two cfxQ.

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Photosynthetic genes are considered to be transcribed actively in the light condition whereas not in the dark. To search the sequential accumulation patterns of the transcripts of photosynthetic genes, we isolated the transcripts of Cyanidioschyzon merolae 10D in 12h-light and 12h-dark condition for every two or four hours, and measured the amount of transcripts by northern hybridization analysis. The transcripts of psbA and psbB-T decreased gradually in the dark period, and suddenly increased in the light period. The transcripts of psbO, psbU and psbC decreased suddenly in the dark period but gradually recovered. That of psbA was constant in all the period. We are now analyzing other kinds of transcripts of photosynthetic genes. The grouping of genes by the transcriptional patterns under light and dark conditions are discussed.

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In plants, Sec-pathway is known for one of the preprotein translocation pathway into the thylakoid lumen. This pathway contains SecA, SecY and SecE proteins. In Cyanidioschyzon merolae, whose genome sequence has been completed, contains secA genes both in the cell nucleus and the plastid genome. Phylogenetic analysis of the three genes revealed that plastid-encoded SecA, SecY and SecE were closely related to those in plants while nuclear-encoded secA was related to the mycobacterial secA2 which concerns auxiliary secretion factor in bacterial Sec-pathways. Northern hybridization analysis showed that the two secAs, secY and secE were actively transcribed. The secA mutant of E. coli was not suppressed by the two secAs in C. merolae. The overexpression of plastid secA in E. coli inhibited the growth while nuclear secA did not. The results suggest that the two secAs are different in their functions.

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A glucose-tolerant strain of Synechocystis sp. PCC 6803 grows both photoautotrophically and heterotrophically consuming glucose as the source of reduced organic carbon. Additionally, light is necessary for the maintenance of heterotrophic growth. The objective of this study is to elucidate the effect of the light and glucose on the expression of fructose-1,6-bisphosphate aldolase (fbaA), which is one of the glycolytic enzymes.
The fbaA mRNA was induced by the addition of glucose with the light. On the other hand, glucose addition in the dark induced the transcription so slow that the transcript slightly appeared after the addition of glucose. The fbaA mRNA was appeared by light-pulse for 5 min without glucose addition, however, decreased gradually in the dark without glucose. Therefore, both light and glucose were required for the full induction of fbaA transcription, and glucose may also work for the stability of fbaA mRNA.

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ent-Kaurene is a hydrocarbon precursor for gibberellins. We have previously reported that some plants emit ent-kaurene into the headspace and that the airborne ent-kaurene is able to fully complement the dwarf phenotype of the Arabidopsis ga1-3 and ga2-1 mutants.
To address whether structural differences in the diterpene hydrocarbon affect the emission into the headspace, we generated transgenic plants overproducing a heterologous diterpene cyclase in the plastid. GC-MS analysis indicated that the transgenic plants are able to release heterologous diterpene hydrocarbon to the atmosphere. In addition, we demonstrated in the transgenic plants that the released hydrocarbon is predominantly produced through the MEP pathway, using a feeding experiment that enabled us to label downstream isoprenoids with ¹³C isotopes through the MVA and MEP pathways, respectively. We are currently screening Arabidopsis mutants with altered ability to emit ent-kaurene into the headspace.

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The recessive 'tall rice' mutant, elongated uppermost internode (eui), is morphologically normal until its final internode elongates drastically at the heading stage. To determine the molecular mechanisms underlying the tall eui phenotype, we measured endogenous gibberellin (GA) levels in the uppermost internodes of wild-type and eui plants. GC-MS analysis showed that eui internodes contained a much higher level of GA₁ and GA₄ than did wild-type internodes, indicating that the eui phenotype was caused by elevated levels of bioactive GAs. Map-based cloning revealed that the Eui gene encodes a previously uncharacterized cytochrome P450 monooxygenase. Using heterologous expression in yeast, we found that EUI catalyzed 16α,17-epoxidation of non-13-hydroxylated GAs. Consistent with the tall phenotypes of the eui mutants, 16α,17-epoxidation reduced the biological activity of GA₄ in rice, demonstrating that EUI functions as a GA deactivating enzyme.

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Deepwater rice is cultivated in Southeast Asia where serious flood occurs periodically. To avoid flood condition, deepwater rice elongates its internode along with the rising water. QTL analysis, using a deepwater rice cultivar in Bangladesh and a wild rice species that carry deepwater characteristics, revealed one major QTL for internodal elongation on the long arm of chromosome 12 in both rice strains. This indicates that the QTL on chromosome 12 is the key locus for internode elongation under water submergence condition in deepwater rice. Nearly isogenic line (NIL) carrying this region confirmed the existence of this QTL and indicated that it is inherited as a dominant manner. As all deepwater rice characteristics were observed in this NIL plant, the QTL locus may function as a master gene for deepwater characteristics. Using this NIL, the relationship between the major QTL gene and plant hormones, ethylene and gibberellin, will be discussed.

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ACC synthase (ACS) is a rate-limiting enzyme of ethylene biosynthesis pathway. We found that LeACS2, a wound-inducible ACS in tomato, was immediately phosphorylated after translation at Ser-460, and that half-life of phosphorylated LeACS2 was longer than non-phosphorylated LeACS2, suggesting that the turnover of LeACS2 protein in the cell is regulated by phosphorylation/dephosphorylation. Thus, we attempted to identify the protein phosphatase involved in LeACS2 turnover in this study. Biotin-tagged phosphorylated peptide (Biotinyl-454KNNLRL(pS)FSKRMYD467-CHO) was synthesized based on LeACS2 sequence. The 32 kDa protein which bound to the peptide was detected from the extract of wounded tomato fruit tissue and the amount of the protein was dependent on the peptide concentration, suggesting that the 32 kDa protein may a catalytic subunit of Ser/Thr protein phosphatase based on the molecular mass. We speculate the 32 kDa protein is involved in dephosphorylation of LeACS2 and try to identify the 32 kDa protein.

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trans-Zeatin (tZ) is one of cytokinins (CKs) that induce cell division and differentiation of the plant in the presence of auxin. We have recently demonstrated that tZ-type CKs are mainly produced using dimethylallyl diphosphate from the plastidic methylerythritol phosphate (MEP) pathway in Arabidopsis seedlings. Interestingly, tZ-type CKs were also partially produced through the cis-trans isomerization of cis-zeatin (cZ)-type CKs, of which prenyl side chains are derived from the cytosolic MVA pathway. To elucidate CK biosynthesis in cultured cell lines of plant, we carried out feeding experiments using ¹³C-labeled precursors of the MEP/MVA pathways. We will present the results from feeding experiments in Arabidopsis T87 cells and tobacco BY2 cells which do not require exogenous CK in the medium.

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Cytochrome P450 monooxygenases (P450s) play crucial roles in Brassinosteroid (BR) biosynthesis from campesterol to brassinolide. Recent molecular genetic studies for BR-deficient mutants of Arabidopsis, rice, tomato and garden pea have identified several P450 genes (CYP85A, 90A, 90B, 90C, 90D, and 724B) so far. Among the BR-biosynthetic P450 genes, CYP85As and CYP90B1 have been characterized by functional expression in yeast cells and E. coli, respectively. Here, we report biochemical characterization of a P450, C-23 hydroxylase. The P450 was expressed in insect cells. The P450 activity was measured in an in vitro assay reconstituted with NADPH:cytochrome P450 reductase. The reaction products were analyzed by GC-MS. The P450 catalyzed the C-23 hydroxylation of 6-deoxocathasterone and cathasterone to 6-deoxoteasterone and teasterone, respectively. We have also determined the substrate specificity of the C-23 hydroxylase. To our knowledge, this is the first report of the biochemical characterization of the C-23 hydroxylation by the P450.

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The evidence showing that phototropism is caused by blue light-induced local accumulation of growth inhibitor(s), interfering with the action of evenly distributed auxin, has been presented and the search for growth inhibitor(s) has been evolved in several plant species. Caprolactam and 8-epixanthatin have been isolated and identified as candidates for inhibitors in sunflower seedlings. Recently, we have confirmed the presence of growth inhibitor(s) showing stronger activity than these compounds. Here, we isolated a new potent, blue light-induced growth inhibitor from sunflower hypocotyls and identified as 8-O-β-D-glucopyranosyl-1,9,14-pentadecatriene-4,6-diyne-3,8-diol (designated "helian") from ¹H and ¹³C NMR and HRFABMS spectra. It inhibited the growth of cress root at concentrations higher than 2.6 Χ 10^-6 M. The distribution of helian was much higher in the illuminated halves than in the shaded ones in phototropically stimulated sunflower hypocotyls. These results suggest that helian may play an important regulatory role in phototropism of sunflower hypocotyls.

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Although Arabidopsis thaliana is well known as a model plant, few bioactive substances of this plant have been reported except for some phytohormones. In our search for bioactive substances from A. thaliana, four new glycolipids, arabidopsides A~D (1~4) have been isolated from this plant. Compounds 1~4 were monogalactosyl and digalactosyl diacylglycerides containing 12-oxophytodienoic acid (OPDA) and/or dinor-oxophytodienoic acid (dn-OPDA). Further search for bioactive substances of A. thaliana resulted in isolation of two glycolipids 5 and 6, and the structures of 5 and 6 were elucidated by spectroscopic data and chemical means.
Arabidopsides A (1), B (2), and D (4) exhibited inhibitory effect on the growth of the root of cress, while arabidopside A (1) promoted chlorophyll degradation of oat leaf, and the senescence promoting activity was more effective than OPDA, and as strong as methyl jasmonate. it was suggested that arabidopside A (1) play important roles in leaf senescence A. thaliana.

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Progesterone is an animal steroid hormone that functions as a corpus luteum hormone. Recently, we identified progesterone from Arabidopsis, rice, tomato and pea, suggesting that progesterone is a ubiquitous constituent of plants. To clarify the biological function of progesterone in plants, Arabidopsis was germinated and grown in the presence or absence of progesterone under the various light conditions. Arabidopsis hypocotyl and petiole showed elongation in some conditions. These functions might be similar to brassinolide functions. Analysis of possible function of progesterone for another plant organ is in progress. In addition, to clarify the molecular mechanism of progesterone bioactivity for plant, we analyzed candidate genes related to progesterone biosynthesis by microarray analysis

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Allelopthic potential of C. junos fruit waste was investigated for searching its recycling method. The peel powder of C. junos fruit inhibited the root and shoot growth of six test plant species. The putative compound causing the inhibitory effect of C. junos fruit peel was isolated and identified as abscisic acid-β-D-glucosyl ester (ABA-GE). ABA-GE inhibited the growth of six test plants and its inhibitory activity was about one third of that of (+)-ABA. The growth inhibitory activity of peel extract separated from C. junos fruit waste was greatest and followed by that of segment cover and seeds from the waste. ABA-GE concentration was greatest in the peel extract, followed by the segment cover extract and seed extract, suggesting that there was a good correspondence between concentrations and inhibitory activities of the extracts. Therefore, the C. junos waste may have allelopathic potential, and may be potentially useful for weed suppressive agent.

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GAs are phytohormons that play an essential role in promoting seed germination. The seed germination of various plants has been shown to require reactive oxygen species (ROS) such as O₂^- and H₂O₂. Based on DNA micoarray analysis and germination test, we have reported that H₂O₂ promoted the seed germination of Arabidopsis and induced genes involved in GA biosynthesis. O₂^- was generated following seed imbibition.
To dissect further the role of O₂^- generated during seed germination in Arabidopsis, we examined the effects of DPI, an inhibitor of NADPH oxidase, on O₂^- generation and the transcript level of genes involved in GA biosynthesis. O₂^- generation was slightly suppressed by DPI and correlated with gene expression involved in GA biosynthesis. These are likely to suggest that O₂^- generated during seed germination contributes to the regulation of gene expression of GA biosynthesis and O₂^- is produced NADPH oxidase.

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The CYP707As encode ABA 8'-hydroxylases, the key regulatory enzymes in ABA catabolism. To reveal the roles of four Arabidopsis CYP707As (CYP707A1-A4) in seeds, we analyzed the expression and mutant phenotypes of these genes. Previous study has demonstrated that CYP707A2 is the major isoform for the ABA catabolism after seed imbibition. Interestingly, the cyp707a1 dry seed accumulated higher ABA levels compared to the cyp707a2. Expression analysis during seed development revealed that CYP707A1 and CYP707A2 were differentially expressed at mid-maturation and late-maturation stage, respectively. The mutant analysis demonstrated that degree of seed dormancy was correlated with the ABA levels in imbibed seed rather than those in dry seed. Among cyp707a mutants examined, ABA-hypersensitivity was most prominent in the cyp707a2 during germination, whereas the cyp707a1 was most strongly inhibited by exogenous ABA during early seedling growth, indicating CYP707A1 acts as the major player for ABA catabolism in postgermination growth.

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In germinated cereal aleurone, gibberellins induce gene-expression for hydrolytic enzymes to mobilize stored molecules. Functions of GA-primary genes, such as GAMyb, act key roles leading to this regulation. To dissect true mechanism of GA-mediated gene-expression, regulation of primary gene-expression themselves have been studied. Previous analyses indicated that enhancer activities present in a large intron should confer GA-response of primary genes, showing a similarity with known-genes regulated by chromatin organization. Following a hypothesis that chromatin-based control would be important for initial GA-actions, DNA methylation of GAMyb has been evaluated. Bisulfite sequencing revealed limited DNA methylation in the promoter region. Methylation-level of seedling was higher than that in aleurone. Asymmetric-type of methylation appeared to partially reduce with treatment of GA. Total extent of DNA methylation showed a correlation with differences between embryo and endosperm nuclear-types, implying that polyploidy of endosperm aleurone tissues should contribute high-response to GA.

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Previous studies showed meiotic recombination frequencies were affected by sex, age and temperature in many different organisms. We established the method to detect genome-wide meiotic recombination in Arabidopsis. Using this method, we have already shown meiotic recombination frequencies were changed depending on sex and age; lower in female meiosis than male meiosis and in young age female meiosis than old age female meiosis.
To make clear the aging effect, we examined the meiotic recombination frequencies at main and lateral shoots generated from rosette leaves in long-day condition and at main shoot in short-day condition.
From these data, we would like to discuss the aging of shoot apical meristem involved in homologous recombination during meiosis.

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Mitochondria and plastids in plant cells have their own DNAs (organellar DNAs). Male gametophyte (pollen) consists of vegetative cell and generative cell. The content of these organellar DNAs in both cells is known to decrease during pollen maturation, however, the molecular mechanisms controlling such an organellar DNA metabolism remain to be understood. We screened Arabidopsis mutants in which the organellar DNA, detected by DNA-specific dye (DAPI) staining, are retained in mature pollen grains. Several mutants were isolated in which DAPI-stained signals were observed in the cytoplasm of vegetative cells. Such signals were not observed in wild-type pollen grains. Double staining of DAPI and mitochondrion-preferable dye (DiOC₆) revealed that DAPI-stained signals are derived from plastid DNAs but not from mitochondrial DNAs. One of the mutants exhibited the leaf-variegated phenotypes. We will report on the electron microscopy study and the progress of map-based cloning of the responsible genes.

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The T-DNA insertion line in NMNAT (nicotinate/nicotinamide mononucleotide adenyltransferase) gene was found to produce less seeds. This allele only inherits maternally leading to male gametophytic malfunction. Hence, this allele was named MALE GAMETOPHYTIC MALFUNCTION (MGM). The rate of living pollen grains derived from mgm anthers was similar to wild type anthers. However, mgm affects pollen lifespan after release from anthers. Namely, a half-life of pollen from wild type and mgm were 85 hours and 40 hours, respectively. In vitro test revealed that only 40% of mgm pollen showed full tube growth after 16 hours. Furthermore, less pollen tube reached the micropyles when mgm or wild type stigma was pollinated with mgm pollen, resulted in decreased relative fertility than wild type pollen. Our results indicate that NAD is essential for the pollen tube growth, which eventually essential prerequisite for seed production.

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K034 is an asexual mutant of dioecious Silene latifolia. It has asexual and female-like flowers in the ratio of roughly nine to one. The asexual flower has no matured stamens and carpels whereas the female-like flower has two carpels. Both asexual and female-like flowers are similar to the male flower in the early stage. They have smaller fourth whorls than the female has. In the late stage, both carpel and stamen primordia are suppressed in the asexual flower but the carpel primordium in the female-like flower continues to grow. As stamens of the wild-type male are elongated, SLM1 and SLM2 were expressed in the stamens but neither of them was expressed in stamens of the asexual flower. We examined 11 STSs scattered on the Y chromosome in K034. Six STSs are present but five STSs are absent. These five locate on the p-arm of the Y chromosome.

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Silene latifolia is a dioecious plant that has X and Y sex chromosomes. When the female plant is infected with smut fungus, its female flower elongates the stamens to be a suspected hermaphrodite flower. We isolated S. latifolia homologs (SlLFY, SlWUS, SlUFO etc.) of floral homeotic genes, and examined their temporal and spatial expression-patterns in the male, the female, and the infected female by using in situ hybridization. One of them, SlUFO was expressed in whorls 2 and 3 regardless of sex at the stage 2 when the stamen and carpel were not differentiated. The number of surface cells in the whorl 4 where SlUFO expression was not observed is 23 in the male flower, but 42 in both female and infected female flowers. The whorl 4 in the infected female flower is the same size of that of the female flower and bigger than that of the male flower.

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MADS-box genes encode transcription factors and are involved in development of flowers in flowering plants. It has been reported that MADS-box genes are expressed in gymnosperms, a fern, a moss and green algae. It is important to clear the role of MADS-box genes in non-flowering plants for understanding the evolution of MADS-box genes and the evolution of flower. However, there is no report of the functional analyses of MADS-box genes of non-flowering plants. In this study, we analyzed six MIKCc-type MADS-box genes (PPM1, PPM2, PpMADS1, PpMADS5, PpMADS6, and PpMADS-S) of a moss, Physcomitrella patens. Expression analysis of knock-in GUS-fusion proteins revealed that they were expressed in protonema, gametophore, rhizoid, gametangia and sporophyte. There is no notable morphological difference in each knock-out line. However, we found that the number of gametophores which produced sporophyte in ppm1ppm2ppmads1 triple knoce-out line was decreased. Phenotypic analyses of multiple knock-out mutants will be presented.

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Pistillody, homeotic transformation of stamens into pistil-like structures, has been reported in the cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) with the cytoplasm of wild relative species Aegilops crassa. The induction of pistillody is suppressed by the Rfd1 gene located on the long arm of chromosome 7B in wheat cultivar Chinese Spring (CS). Because of the absence of Rfd1, the alloplasmic line of CS ditelosomic 7BS ((cr)-CSdt7BS) lacking the long arm of chromosome 7B exhibits pistillody in all florets, whereas the euplasmic CS ditelosomic 7BS (CSdt7BS) with wheat cytoplasm forms normal stamens. Pistillody is caused by the alteration of the class B MADS box gene expression pattern, which should be due to the function of mitochondrial gene(s) in the Ae. crassa cytoplasm. Here, we identified mitochondrial chimeric genes, orf256-coxI and nad1-ND6, specifically expressed in the young spike of the pistillody line by using the cDNA subtraction technique.

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We have identified two novel genes which highly express in the rice anther by microarray analysis using cDNA prepared from reproductive organs. These genes were named as OsCER1 and OsCER1B since they have sequence similarity to the Arabidopsis CER1 gene. CER1 is known to be involved in the wax biosynthesis in the Arabidopsis pollen, stem, and leaf, whose mutation has been demonstrated to result in a conditional male sterile. We found two mutant alleles of the rice OsCER1B in which Tos17 was inserted in the exon region. Fertility of these mutants was drastically decreased probably because of defects in a pollen formation. We are now analyzing the pollen structures of these mutants to elucidate the function of OsCER1B in the rice pollen formation. In addition, we identified two more genes related with CER1 in the rice genome. Gene expression profiles of four CER1-related genes are under investigation.

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Vacuolar processing enzyme (VPE) is responsible for the maturation of vacuolar proteins. Arabidopsis δVPE was specifically and transiently expressed in two cell layers of the seed coat (ii2 and ii3) at an early stage of seed development. At this stage, cell death occurs in ii2 and ii3 cell layers. In a δVPE-deficient mutant, cell death of the two layers of the seed coat was delayed. Immunocytochemical analysis localized δVPE to electron-dense structures inside and outside the walls of seed coat cells that undergo cell death. We investigated the protein compornents of the structures. Interestingly, δVPE exhibits caspase-1-like activity, which has been detected in various types of plant cell death. Our results suggest that, at the early stage of seed development, δVPE is involved in cell death of limited cell layers, the purpose of which is to form a seed coat.
S. Nakaune et al., The Plant Cell, 2005, vol.17, 876-887

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