Serine acetyltransferase (SATase) catalyzes the formation of O-acetyl-L-serine (OAS) which is the essential precursor of cysteine biosynthesis. There are five SATase isozyme genes on Arabidopsis genome. Among these, SAT-c, SAT-p, and SAT-m were characterized by differences of subcellular localization and the sensitivity to the feedback inhibition by cysteine. By the analyses of enzymatic properties using recombinant proteins of SAT106 and SAT#5, both of these enzymes synthesized OAS from acetyl-CoA and serine, although the affinities of these enzymes for their substrates were very low. Both of SAT106 and SAT#5 seem to be localized in cytosol. The activity of SAT#5 was feedback-inhibited by cysteine, whereas SAT106 was not subjected to this feedback inhibition. To reveal the role of each SATase isozymes, we are currently dealing with the expression analysis of each SATases under various stress conditions and the analysis of the knock-out mutant plant of each SATases.

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When challenged with the fungus Colletotrichum higginsianum, Arabidopsis accession Columbia plant (Col-0) developed brown necrotic lesions, including a yellow halo around the lesion. The Arabidopsis accessions showed variation in response to C. higginsianum. Arabidopsis accession Eil-0 was highly resistant to C. higginsianum. In the Eil-0 plant inoculated with the fungus, cell death did not appear as a rapid response, and the level of camalexin accumulated was low. Analyses using RNA gel blotting and cDNA microarray suggested that the defense responses against C. higginsianum occur mainly through the JA/ET-dependent pathway. Segregation analysis of progeny from crosses between Eil-0 and Col-0 plants was performed to determine the genetic basis of resistance. The resistance was dominant and seemed to be conferred by a single locus. Arabidopsis and C. higginsianum provide an excellent pathosystem for studying the molecular and cellular bases of plant-pathogen interactions

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Using the rice 9000 cDNA microarray, gene expression profile of rice leaves in early stage after rice blast fungus infection was analyzed. We used the 4th leaves of the susceptible rice cultivar (Nipponbare), resistant cultivar (IL7, which is the isogenic line containing the resistance gene Pi-i), and Nipponbare pretreated with ACC, which is a possible molecule for induced resistance. The result showed expression of about 500 clones was up- and down-regulated in infected Nipponbare. In these, many putative clones involved in metabolism, stress response and signal transduction were found. From the results, we were interested in sugar metabolism after the infection, and determined the levels of sugars and starch, indicating the amount of sucrose was considerably decreased in the rice leaves during resistance response.

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The elicitor activity against tobacco BY-2 cells was found in an autoclaved and filtrated Alternaria alternata culture medium. The object of this study is isolation and analysis of the elicitor active compounds from the A. alternata culture medium. The culture medium was autoclaved and filtrated to remove the fungus body. A 99% methanol-insoluble fraction of the filtrated solution showed an elicitor activity. The active fraction was separated by an extraction with 75% methanol, removing protein and dialysis. Finally, the elicitor was isolated by the reverse phase HPLC and gel filtration HPLC. The elicitor activity was measured by BY-2 chitinases induction. The isolated elicitor was composed mostly of carbohydrates and showed elicitor activity with the concentration of 10-30 ng/ml. Hydrolysates of the isolated elicitor were dirived the alditol acetates and analyzed by a gas chromatography. As the results, the elicitor active fraction was composed mainly of glucose (about 80%).

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The tobacco BY-2 chitinases were induced in intra and extracellular fractions by the addition of the pathogenic fungus Alternaria alternata cultured medium. We isolated three of these intracellular chitinases (TBC-1,2,3) by heat treatment, acid treatment, colloidal chitin affinity, ion-exchange chromatography, and hydrophobic chromatography. These molecular masses were estimated to be 28.8, 33.3, and 28.5 kDa, respectively. N-terminal amino acid sequences were analyzed. Based on this sequence, proper primers were designed used for successive TBC-1 cDNA analysis. TBC-1 cDNA was cloned encording 1013 bp full-length cDNA including 831 bp putative ORF resion. Compared with those of other proteins, TBC-1 showed homology to class IV chitinases of other plants but not already reported tobacco chitinases. Therefore, TBC-1 was thought to be a novel chitinase of tobacco. By northern blot analysis, TBC-1 gene expression was induced by a fungal elicitor from A. alternata.

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We have generated approximately 10,000 activation tagging rice lines. Among them, a lesion mimic mutant, which we designated Lesion mimic 1 (Lmm1), was further characterized. Lmm1 was dominant and the mutant phenotype was co-segregated to the T-DNA in the T1 generation. An ORF was located about 500 bp downstream of the T-DNA and the lesion mimic phenotype correlated with the enhanced expression of the mRNA. The encoded protein shows sequence similarity to a protein whose expression is induced by hypersensitive reaction in tobacco. In addition, transcriptional levels of PR proteins were increased and phytoalexins, both momilactone A and sakuranetin, accumulated in Lmm1. Further, T1 plants which exhibit lesion mimic were resistant to rice blast, which strongly suggests the correlation between the encoded protein and defense mechanism.
This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project MP-1202).

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Lesion mimic mutants develop spontaneous cell death without pathogen attack. Some of the genes defined by these mutations may function as regulators of cell death. To understand the molecular mechanism of cell death in lesion mimic mutants, we isolated a lesion initiation 3 (len3) mutant. The lesions in len3 plants develop under short day and long day conditions. The len3 plants were smaller than wild-type plants and expressed cytological and biochemical markers that associated with disease resistance response. The len3 plants constitutively expressed the PR genes and showed enhanced resistance to virulent pathogens.

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To clarify the plant defense mechanisms, we isolated and characterized a single recessive T-DNA tagged Arabidopsis mutant, cad1 (constitutively activated cell death), which shows spontaneously activated PR (pathogenesis-related) genes expression and cell death in vascular bundle. CAD1 gene encodes a protein with a significant homology to the Complement and Perforin in MACPF (Membrane attack complex and Perforin) domain, is a member of uncharacterized gene family in higher plant. MACPF domains have been identified in many animal species, and to be involved in innate immunity. Expression of the CAD1 gene was observed at vascular bundle of developing leaves and roots. Furthermore, genetic analysis using npr1-1 (nonexpresser of PR gene) and 35S-NahG plants revealed that the CAD1 gene regulate a novel pathway of cell death and defense activation in Arabidopsis.

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The hypersensitive response (HR) involves well-organized programmed cell death (PCD). PCD in animals and plants is thought to share components that have caspase activities. However, no plant proteinases exhibiting caspase activities had yet been identified. To identify a functional homolog of caspase in plants, we investigated hypersensitive cell death in TMV-infected tobacco plants. A caspase-1 inhibitor abolished the hypersensitive cell death. A biotinylated caspase-1 inhibitor that was infiltrated into the tobacco leaves bound to the 38- and 40-kDa components of the leaves. We found these components corresponded to two forms of vacuolar processing enzyme (VPE). VPE is a cysteine proteinase responsible for maturation and/or activation of vacuolar proteins. VPE inhibitor also abolished the hypersensitive cell death. The VPE activity appeared rapidly at the early stage of the HR and declined before the appearance of lesions. These results suggest that VPE exhibiting caspase-1-like activity is essential for TMV-induced hypersensitive cell death.

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We previously demonstrated that polyamines degraded by polyamine oxidase contribute to hypersensitive cell death by producing H2O2 during HR against tobacco mosaic virus infection. To further investigate relationships among polyamines, H2O2 and hypersensitive cell death, we employed tobacco cultured cells (BY2 cells). When BY2 cells were treated with cryptogein, an elicitor of HR, hypersensitive cell death was obvious. However, this was inhibited by α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. Polyamines were found to accumulate in apoplast of cryptogein-treated BY2 cells. In the presence of DFMO, however, such accumulation was almost suppressed. Polyamine oxidase activity was also detectable in apoplast. Generation of H2O2 was detected as early as 15 min after elicitation and this was also suppressed by the inhibitor, although effects were only apparent after 3 h or later. These results suggest that polyamines are a direct source of H2O2 in the late phase of HR.

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Systemic acquired resistance (SAR) is one of plant defense system, following salicylic acid (SA) accumulation and SAR marker genes expression. To investigate the influences of environmental stress on SAR induction, we analyzed effects of abscisic acid (ABA) on SAR. In this study, we used 1,2-benzisothiazole-3(2H)-one 1,1-dioxide (BIT), for SAR induction. BIT activate upstream of SA in SAR. ABA treatment suppressed BIT-induced PR genes expression and SA accumulation, indicating that ABA suppressed the induction of SAR by BIT. BTH ,benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, a SAR inducer, activates downstream of SA in SAR signaling pathway. ABA treatment also suppressed BTH-induced SAR in Arabidopsis. These results suggested that ABA downregulates SAR signaling pathway. Furthermore, environmental stress such as osmotic stress induced by mannitol suppressed the induction of SAR.

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To clarify the molecular mechanism of the gravitropism in higher plants, we have isolated many mutants from Arabidopsis with abnomal shoot gravitropism. The shoot gravitropism 2 (sgr2) recessive mutant exhibits abnormal gravitropic response in hypocotyls and inflorescence stems. We have demonstrated that the endodermal cells containing sedimentable amyloplasts are the site of gravity perception in Arabidopsis shoots. However, amyloplasts did not sediment in sgr2. The SGR2 encodes a protein homologous to bovine phosphatidic acid-preferring phospholipase A1, which localizes mainly to vacuolar membrane. To investigate the molecular function of SGR2 in the gravitropism, we performed a genetic screen for the second mutation suppressing the sgr2-1 phenotype. The kiritsu (krt) mutations suppressed abnormal gravitropic response semidominantly and aberrant amyloplasts movement in the endodermal cells of sgr2-1. This suggests that KRT genes are involved in the function of vacuole and in the movement of amyloplasts.

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Root gravitropism is an inevitable feature for proper plant growth. However, the mechanism to perceive the gravity and to transmit this signal in roots is not clear. According to the hypothesis that root collumella cells percept the gravity, we searched genes that are ectopically induced in root collumella cells from Arabidopsis T-DNA enhancer trap lines. As a result, we isolated the specific expression genes from 7 lines that induced GFP in root collumella cells. Using three sets of nest primers designed from the T-DNA (Gal4-GFP) sequence, we obtained 45 highly specific amplifications after Tail-PCR. Sequence determination showed that 2 lines had the T-DNA region inserted near the genes for a Myb family transcription factor and an auxin responsive protein, respectively.
We are now searching the knockout lines and identifying the promoter regions for these genes.

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We have demonstrated that microgravity conditions in space resulted in automorphosis and reduction of auxin polar transport activities of epicotyls in etiolated pea (Pisum sativum L. cv. Alaska) seedlings. In addition, automorphosis was phenocopied by the application of auxin polar transport inhibitors. These results strongly suggest that automorophosis is closely related to auxin polar transport. Strenuous efforts to know the regulatory mechanisms of auxin polar transport in pea seedlings at molecular bases resulted in successful identification of PsPIN2 and PsAUX1 encoding putative carrier proteins for auxin polar transport. Significantly high homology was found on deduced amino acid sequences between AtAUX1 and PsAUX1. On the other hand, PsPIN2 showed higher homology with AtPIN3 and AtPIN4 than with AtPIN1. Based on the results of gene expression of PsPIN2 and PsAUX1 under various gravity conditions, a possible role of gravity in regulating auxin polar transport will be discussed.

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In higher plants, it has been known that roots and shoots show positive and negative gravitropism, respectively. However, whether leaves show gravitropism or not is unclear.
To examine the gravitropism of leaves, we analyzed the motion of rosette leaves of Arabidopsis thaliana after changing the direction of gravity. Under continuous light, direction of the expansion of rosette leaves was dependent on the shoot axis, whereas leaves moved in short time in gravity-dependent manner under dark, suggesting the interaction of light signal and gravity on the direction of leaf expansion. In petiole, amyloplasts, which are believed to act statolithe for gravity, were found only in the basal end of a particular cell layer, and sedimented according to the direction of gravity. Based on these data, nature of gravitropism in leaves will be discussed.

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We identified four genes encoding proteins partially-similar to LHCPs in the green alga Chlamydomonas reinhardtii, and examined changes in the mRNA levels after a transfer from low to high light conditions. The level of the three Lhc-like mRNAs increased transiently with maximum at 1 hour after the transfer. Amino-acid sequences of two of them are similar to those of ELIP and HLIP, which have been proposed to be involved in protection against light stress in higher plants. The other gene does not correspond to any known genes. Expression of this novel gene was much more sensitive to high light than that of the other genes tested. The expression was not induced by reactive oxygen species (ROS), and the high-light response was not affected by photosynthetic electron transport inhibitors, suggesting that ROS and redox of the electron transport may not the high-light signal for this gene.

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Activation tagging is another method for insertional mutagenesis. Because enhancers in T-DNA inserted into genome can activate transcription of nearby genes, it is expected that plant have dominant gain-of-function mutations and acquire resistant phenotype to environmental stress. We have created about 50,000 independent Arabidopsis lines transformed with a tetrameric enhancer T-DNA and performed functional analysis of genes of these lines in collaboration. We newly tried to isolate different sensitive mutants to UV-B radiation from this Arabidopsis activation tagging lines.
After 14 days plants of each line were irradiated with white light (6 W/m2) including UV-B (0.25 W/m2) for 35 hours, we searched plants that showed increased or diminished UV damage of appearance, especially bleaching of leafs, for 3 days under white fluorescent light in 16-h-light/8-h-dark cycles. As a result of selection of approximately 5,000 lines, we isolated some hypersensitive and resistant mutants to UV radiation.

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The maintenance of genomic integrity requires coordination between DNA repair and replication. We investigated expression patterns of plant (rice) DNA repair genes such as CPD photolyase, UV-DDB1, CSB, PCNA, RPA32, FEN-1 and ORC1. We found that all the genes tested were expressed in proliferating tissues, but only CPD photolyase was expressed in non-proliferating tissue. The removal of DNA damages in both non-proliferating tissue and the proliferating tissues was observed under light conditions. In the dark, DNA damage in non-proliferating tissue was not repaired efficiently, but that in the proliferating tissues was removed rapidly. These results suggested that photoreactivation is the major DNA repair pathway in non-proliferating cells while dark repair is active in proliferating cells. Using a rice 22 K custom oligo DNA microarray, we found that most DNA repair genes were more strongly expressed in proliferationg tissues. These results indicate that plant dark repair is coordinated with DNA replication.

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We previously showed that cyclobutane pyrimidine dimer (CPD) photolyase is deficient in UVB-sensitive rice, and that the CPD photolyase deficiency might result from a mutation of CPD photolyase. The CPD photolyase activity in UVB sensitive Surjamkhi (Indica) was significantly lower than that in UVB resistant Sasanishiki (Japonica). There were two variations at codon 126 and 296 in the deduced amino acid sequence of CPD photolyase between both cultivars. We confirmed that these mutations affected the CPD photolyase activity. To test whether the difference in the CPD photolyase activity causes the difference in UVB sensitivity in rice, we carried out the genetic analysis of correlation between UVB-sensitivity and genotype of CPD photolyase using F2 and F3 plants generated by reciprocally crossing Sasanishiki and Surjamkhi. As a results, we found that UVB sensitivity and the activity of photolyase were tightly linked in rice plant.

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The error-prone DNA translesion synthesis (error-prone TLS) has been well characterized in yeast and mammalians, but not in higher plants. Recent finding of an AtREV3-disrupted mutant (rev3-1) in Arabidopsis suggested that the error-prone TLS is significant for tolerance to DNA damages in higher plant. To clarify the details of the error-prone TLS in higher plants, we analyzed the T-DNA inserted Arabidopsis mutants defective in AtREV1 or AtREV7 genes, which are thought to be involved in the error-prone TLS system. The AtREV1 mutant (rev1-1) was sensitive to UV-B, gamma-ray and MMC. These physiological responses of rev1-1 were quite similar to those of rev3-1. The results indicated that AtREV1 functions in the error-prone TLS in Arabidopsis. We will also present a couple of results which characterize the T-DNA inserted AtREV7 mutant.

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LexA is a repressor involved in SOS response, which has been extensively studied in Escherichia coli. Although many cyanobacteria have a lexA-like gene in genome, little is known about the SOS response in cyanobacteria. We already reported by DNA microarray analysis that LexA-like Sll1626 in Synechocystis sp. PCC 6803 was involved in transcriptional regulation of pil genes but not the SOS-related genes. In this work, we expressed and purified LexA homologs of Synechocystis (Sll1626) and Anabaena sp. PCC 7120 (Alr4908). When Anabaena Alr4908 was incubated, autocleavage products increased with time, especially at high pH, in consistent with the findings on the E. coli LexA. Meanwhile, Synechocystis Sll1626 did not produce any cleavage products. These results suggest that the LexA-like Sll1626 of Synechocystis, which is functional as a repressor, is regulated by yet unknown mechanism rather than the SOS-responsive manner.

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Global expression profiles of the flood stresses, standing (FS) and lay down (FD) were analyzed using 22K rice oligo microarray. Combined results from two flood stresses revealed 1,166 genes with greater than 2-fold change over (Up regulation) or less than 0.5-fold change over (Down regulation) control. Numbers of genes after each stress treatment (24hr) is as follows, 962 genes (5%) were changed in FS and 469 genes (2.3%) were in FD. Commonly up-regulated genes in both two flood stresses were 130 genes, including peroxidase and glucose biosynthesis etc. Commonly down-regulated genes were 132 genes, including nitrate reductase and transcription factor-like gene etc. Results of cis-element analyze and the related transcription factors will be presented.

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To determining possible minute amounts of DNA lesion caused by the natural sunlight, sensitive ELISA is the key. ELISA responses of cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4PP) have been discovered to be strikingly amplified when sodium chloride is applied together with DNA to ELISA plates. The salt effect occurred most effectively on Immulon 4HBX plates, slightly less effectively on 2HB and scarcely on 1HB. With Nunc plates, even a slight suppression was observed. Examination of various kinds of inorganic salts on 4HBX plates led us to conclude that both Na+ and Cl- are likely functional in amplification. Exceptionally potassium bromide and potassium iodide were promotive, while lithium chloride was ineffective. The rate of amplification was identical regardless of the extent of lesion at a fixed salt concentration, and hence this novel effect of salts serves for sensitizing ELISA of CPD and 6-4PP.

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In order to understand the specific functional roles of CDPK, we have initiated a high-throughput yeast two-hybrid screening program to identify CDPK substrates and interacting protein. Wildtype, catalytically impaired, and constitutively active kinase mutants were used as baits in a robotic-based, interaction-screening program in which 18 million prey clones from two different libraries are tested for each bait. Using six different versions of AtCPK4 we have identified a total of 370 interactions and 30 different in-frame prey fusion proteins. The use of constitutively active AtCPK4 baits was found to improve the efficiency of recovering positive interactors relative to the wildtype kinase. To date, several candidate AtCPK4 substrates have been confirmed in vitro including Di19, an abscisic acid-independent, drought-inducible protein (Gosti et al., 1995), a GTPase type chloroplast outer envelope receptor, AtToc33, which is known to be phosphorylated at Ser181 (Jelic et al., 2003), and a serine-rich protein of unknown function.

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The development of a system capable of synthesizing and characterizing any desired protein on a preparative scale is one of the most important endeavors in biotechnology today. Recently we prepared a cell-free system from extensively washed embryos, and indeed found that the system became far more active. More recently, we are successful to develop a novel cell-free system for the high-throughput (HT) synthesis and screening of gene products by optimizing the mRNA UTRs and a new strategy to construct PCR-generated DNAs. Examples for the assessment of protein folding by NMR and measurement of set of kinase activities provided to show that the system is capable of producing functional proteins. Furthermore, the system made us possible to invent a robotic system, GenDecoder, for HT protein production automatically. The cell-free technology, reported here, has a potential basis for functional proteomics.

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In two-dimensional polyacrylamide gel electrophoresis (2-DE), peptides with the same primary structure are often separated as several spots because of the post-translational modifications. Immunoblotting and MALDI-TOF mass spectrometry analysis revealed that the RuBisCO large subunit gave two spots in 2-DE gel of proteins in the crude extracts of plant leaves; one was at pI 6.0, similar to that predicted from its sequence, and the other was unfocused around pI 4.3-4.5. The post-translational modifications reported for RuBisCO could not explain the appearance of the low-pI spot. Thus further analysis of the low-pI spot was carried out here. The low-pI spot was not detected in 2-DE with purified RuBisCO. When the low-pI spot extracted from the isoelectric focusing gel was subjected to 2-DE, the peptide in the low-pI spot moved to pI 6.0. We are investigating the cause of the appearance of the low-pI spot with crude extracts.

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Plant growth is limited in acid soil because of phosphorus (P) deficiency and aluminum (Al) toxicity. Though specific gene expression of rice (Oryza sativa L.) under aluminum and phosphate stress was well investigated, response via protein expression needs further analysis.
To evaluate the differential expression of protein, 1) rice in the seventh leaf stage were subjected to +P treatment, -P treatment, or +Al treatment. Samples were taken at 0,12,24,72,120 hours after each treatment started and at 10 days after treatment in the case of ±P treatment.2) Root proteins were extracted using Tris-HCl/NaCl buffer, then concentrated. The extract was resolved in 7M Urea/2M Thiourea buffer. 3) These proteins were separated by using two-dimensional electrophoresis, then quantified by digital analysis and analyzed by MALDI-TOF-MS or Edman sequencing. As a result, 15 proteins were identified (November, 2003). Based on these data, we examine metabolic changes in low phosphorus or high aluminum treatment.

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It has been well established that the scutellar tissues play the important roles for the breakdown and uptake/utilization of storage starch in germinating rice seeds. Previously, we described about the initial proteomic analysis of abundant proteins expressed in the scutellar tissues. Here, we report the proteomic identification of time-dependent increased and abscisic acid (ABA)-induced proteins in the scutellar tissues. Glycolytic enzymes, TCA cycle-ATP producing enzymes, sucrose synthesis-related enzymes and ABA-inducible proteins were identified as the scutellar proteins increased during 6 days germination. ABA treatment caused a significant increase of dry weight and protein contents of the scutellar tissues. Most of the ABA-induced proteins detected were identical to the time-dependent increased enzyme proteins. Based on these results, we considered that ABA stimulates a function of the scutellum of germinating rice seeds. We also mention about the results obtained by the analysis of rice viviparous mutants.

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Double-stranded RNAs (dsRNA) that contain promoter sequences can trigger transcriptional inactivation in trans of gene driven by a homologous promoter. Small RNAs derived from dsRNA are implicated in this dsRNA-mediated transcriptional gene silencing phenomenon and in de novo methylation of the target promoter, but the molecular mechanism(s) is still unclear. To investigate the silencing machinery, a tissue specific promoter system was developed. From a mutant screen, 3 mutants, drd1, drd2, drd3 (defective in RNA-directed DNA methylation), which show a reactivation of the silenced promoter activity were obtained. In all of these mutants, non-CpG methylation of the target promoter is almost eliminated while CpG methylation is unaffected. DRD1 was identified as a novel putative chromatin remodeling factor that belongs to a plant specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. The possible role(s) of DRD1 in the RNA-directed de novo DNA methylation pathway will be discussed.

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Detergent-insoluble glycolipid-enriched complex (DIG) is the term to explain a putative membrane subdomain formed through lateral association of sphingolipids and cholesterol. In mammals and yeast, particular proteins such as GPI-anchored proteins, ion channels, and membrane receptors are localized to DIGs via specific sorting pathways. Thus, it is believed that DIGs serve as the functional sites for membrane trafficking, cell polarity, signaling, and cell-to-cell communication. Here, we examined whether or not such membrane subdomains are present in plant cells, and ask physiological roles, if any, of such DIGs in plants with the aid of proteomic approaches. We isolated and characterized DIG-components from Arabidopsis. GC-MS and LC-MS analyses demonstrated distinct compositions of sterols and fatty acids in the DIGs. Also, DIG-specific proteins were identified by 2D-PAGE, and the primary structures were determined using MALDI-FT-ICRMS.

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Stable isotope labeling methods possess methodological advantages because of its secure and diverse properties, whereas radio isotope labeling methods widely used in biological studies have disadvantages in terms of these aspects. Nuclear Magnetic Resonance (NMR) is a powerful method for identification of labeled metabolic flux and their structures. We focused major metabolic nucleus in plants, carbon and nitrogen, and performed their multi-dimensional NMR analysis. Briefly, we harvested ¹⁵N labeled seeds of Arabidopsis thaliana, and observed specific nitrogen movements upon water absorption or light irradiation in vivo. Furthermore, ¹³C labeled samples exhibit distinct metabolic difference between wild type and a mutant strain of Arabidopsis thaliana. A validity of NMR in plant metabolomics will be discussed.

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In the RIKEN Genomic Sciences Center, more than 50,000 activation tagged mutant lines of Arabidopsis have been produced for the saturated mutagenesis of the genome. To find the function of a gene, systematic analysis of phenotypes is vital. However, conventional phenotypic analysis of mutant screens depends mainly on qualitative descriptions after visual observation of morphological traits. We have proposed a new methodology of phenotypic analysis based on precise three-dimensional (3D) measurement by a laser range finder and automatic data processing. In this report, we exhibit systematic quantification of leaf morphological traits based on 3D morphological parameters, and analysis of the traits between several types of Arabidopsis. The proposed method enables us to obtain quantitative and precise descriptions of plant morphologies compared to conventional two-dimensional measurement. The method will open a way to find new traits from mutant pools or natural ecotypes based on 3D data.

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RPA (Replication Protein A) has a multi-function protein involved in DNA replication, transcription, DNA repair, DNA recombination and so on. RPA is three subunits protein consists in 70kDa, 32kDa and 14kDa in human. We report cloning of two types of RPA70kDa subunit (OsRPA70a, b), RPA30kDa subunit (OsRPA32) and RPA14kDa subunit (OsRPA14) in rice (Oryza sativa). In animal and yeast, 70kDa subunit has just one type, but in plant, 70kDa subunit has two types. Two types of RPA70kDa subunit differ the expression tissues. OsRPA32 binds to OsRPA70b stronger than OsRPA70a. And T-DNA insertion line in Arabidopsis thalina RPA70a homologues is lethal, but theirs RPA70b homologues is not lethal. And AtRPA70b mutant is sensitive for UV and MMS. So, two types of RPA70kDa subunits may have different function in DNA repair mechanism.

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Homologous recombination is an essential process for the maintenance and variability of the genome. Rad51 play a central role in homologous recombination by carrying out the pairing of homologous DNA molecules and initiating the strand exchange reaction. In human, five members of Rad51 family (Rad51 paralogs; Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3) have been isolated. These proteins form a functional complex and interact with Rad51.
To better understanding homologous recombination in higher plants, we have characterized Rad51 paralogs using Yeast two-hybrid system and RT-PCR analysis in Arabidopsis. In this study, we have characterized T-DNA or Ds transposon knockout mutants of Rad51 paralogous genes (AtRAD51C and AtXRCC2) in Arabidopsis.

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Chromatin assembly factor 1 (CAF-1) is a three-subunit complex, consisting of FAS1, FAS2 and MSI1, in Arabidopsis thaliana, that interacts with histones H3 and H4 and targets them to DNA after replication and DNA repair synthesis. In contrast to human and budding yeast, little is known about relationship between CAF-1 and DNA repair systems in higher plants. In this study, we analyzed the γ-ray sensitivity and frequency of homologous recombination (HR) that is one of the DNA repair systems in Arabidopsis CAF-1 mutants (fas1 and fas2). In contrast to budding yeast, fas1 and fas2 mutants showed increased sensitivity to γ-ray irradiation compared to wild-type plants. Expression of the AtRAD51 gene, which plays a central role in HR, was increased in fas1 and fas2 mutants in the absence of DNA damage treatment. Recombination assay revealed that intrachromosomal HR in planta is 40-fold stimulated in fas1 and fas2 mutants.

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Rad51, eukaryotic homologues of RecA, plays key roles in DNA double-strand break (DSB) repair via homologous recombination (HR) as recombinase. We previously reported two rice Rad51 genes (OsRAD51A1 and OsRAD51A2) which were induced by DNA damaging agents. We analyzed the effect of OsRad51 gene expression using transgenic rice plants. Expression of OsRad51A2 promoter::gusA chimeric gene in rice calli and plants was quickly and remarkably up-regulated by DSB inducers such as gamma-ray and bleomycin. Antisense expression of OsRad51A1 cDNA conferred growth retardation at vegetative and reproductive phases in T2 transgenic rice plants. Those T2 transgenic lines also displayed hypersensitivity against gamma-ray irradiation. These results indicated that OsRad51A2 expression is controlled at transcription level by its 5'-upstream sequence containing the possible DNA-damage responsive element, and that lowering OsRad51 transcript levels could raise sensitivity toward DSB inducers by inhibiting formation of HR-machinery sufficient for DSB repair.

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Homologous recombination (HR) machinery is composed of so-called Rad52 epistasis group proteins in eukaryotes. In plant kingdom, none of the genes located downstream of HR proteins have been isolated. Here, we characterized Snf2p/Swi2p family genes, OsRad54 and OsRad54B, and XPF endonuclease superfamily gene, OsMus81. Transient expressin of GFP::OsRad54 and GFP::OsRad54B cDNAs localized GFP-fluorescence exclusively in onion nuclei, suggesting their role(s) in nuclei. RT-PCR analysis revealed that OsRad54 and OsMus81 were induced by gamma-ray causing double strand breaks. Yeast two-hybrid analysis detected strong protein-protein interactions in OsRad51A1-OsRad54 and OsRad54-OsMus81 pairs, moderate interaction in OsRad51A1-OsRad54B, but not in OsRad54B-OsMus81. These results implied important roles of OsRad54 DNA-helicase protein as a bridge between DNA-exchanging reaction with OsRad51A1 and crossover formation with OsMus81 in the HR process. Biochemical analyses are in progress to elucidate precise functions of those proteins.

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Plant cell proliferation is regulated by nutrient availability, and expression of cyclin D is regulated by sugars by hexokinase-dependent pathway. Nucleolin is a global regulator of ribosome synthesis and its expression in yeast and animals is linked with cell proliferation. A large fraction of Arabidopsis mRNAs that increased significantly within 6 hr-treatment with sucrose was occupied by genes in "protein synthesis" including a gene for nucleolin (AtNucl.-1) and about 200 genes for ribosomal proteins (RPs). Glucose, but not 2-deoxyglucose, also caused increase in mRNAs of these genes, suggesting the hexokinase sensor-independent manner of regulation. A disruptant of AtNucl.-1 contained significantly higher levels of pre-rRNA sequences that are removed during processing, and showed phenotypes such as decreased growth, narrow leaves and increased number of stems. In addition, sugar-induced changes in mRNAs for RPs were lost in the disruptant, suggesting the involvement of AtNucl.-1 in the regulation of expression of RPs.

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The roots of Arabidopsis seedling exhibit wavy growth patterns on tilted agar surfaces by continual touching to the surface. The wav2 mutant shows a shorter-pitch wavy growth pattern in comparison with that of wild type. We reveal that, in this research, helical growths in respond to touch stimulation, especially right-handed growth, are enhanced in the mutant. Curvatures of in respond to light and gravity are also enhanced in the root of wav2. Those results suggest that WAV2 is involved in the mechanism to suppress the helical growths, especially right-handed growth, in the roots, and that it regulates the changes of the growth pattern of roots in respond to various environmental stimuli through that mechanism. Map-based cloning revealed that WAV2 encodes a novel protein with a transmembrane domain. In this meeting, we discuss the function of WAV2 by addition of results on analyses of gene expression and subcellular localization of WAV2.

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Turions (overwintering buds) of aquatic plant pondweeds (Potamogeton distinctus A. Benn.) can elongate under anoxia, showing high tolerance for low oxygen stress. During the anaerobic growth rapid starch degradation and active sucrose metabolism occur. Among enzymes involved in carbohydrate metabolism sucrose synthase (SuSy) is the most significant in the increase of enzymatic activity.
SuSy is one of the anaerobic proteins. SuSy plays a role to provide sugars to glycolysis by sucrose cleavage. Moreover, SuSy associated with the plasma membrane has a possible role to supply sugars for cellulose synthesis. We isolated two distinct SuSy genes from pondweed tissues and found that only one gene enhances the expression under anoxia. These results suggest that SuSy enhanced during the anaerobic growth is involved in both energy production and cell growth.
Furthermore, isolation of genes which are specifically expressed in the anaerobic growth is now in progress by a PCR-based subtraction method.

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In this study, ascorbate peroxidase (APX), L-galactono-1,4-lactone dehydrogenase (GLDH), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GSH) in broccoli florets were investigated in the gene expression after harvest, treatments with 10% sucrose and irradiation of various light intensity. The decreases in ascorbate and sucrose contents in florets were caused at 48 h after removing leaves from intact plants with simultaneous suppression of the gene expressions of chloroplastic APX, MDAR, DHAR and mitochondrial GLDH, but not when the plants were kept intact or leaves attached. In addition, ascorbate contents were kept high and the gene expressions related to chloroplastic and mitchondrial ascorbate metabolism were enhanced by feeding 10% sucrose or high light in harvested broccoli florets.

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We studied the regulation system of biosynthetic pathway of ascorbate (AsA) in Arabidopsis plants which were cultured for 2 weeks on the MS medium containing 3% Suc under normal conditions. When the plants were transferred to the dark for 48 h, the level of AsA decreased. However, the transfer of dark-adapted plants from the dark to the light for 48 h did not allow AsA to restore to the initial level. The DCMU treatment also decreased the AsA level under light conditions.
To investigate the effect of exogenous sugar on the AsA level, two-week-old plants were transferred to the MS medium in the absence of Suc in the dark for 48 h. As a result, the AsA level decreased. Interestingly, the AsA level was increased to the initial level by reillumination for 48 h. There was no difference in transcript levels of enzymes in the AsA biosynthesis between both conditions.

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We studied the signal transduction system of the expression of cytosolic ascorbate peroxidase (cAPX) in tobacco plants. The levels of cellular H₂O₂ and the cAPX transcript were increased by the treatment with 3-aminotriazol or paraquat in the tobacco plants. The treatment with DBMIB also increased the transcript level of cAPX under low-light intensity. These results indicate that the cAPX expression is regulated by the redox status of plastquinone pool and/or cellular H₂O₂ level. Next, we used transgenic tobacco plants expressing the E.coli catalase or thylakoid-membrane bound APX in chloroplasts, which showed the increased tolerance to photooxidative stress. There was no difference in increase in the transcript level of cAPX at the first 1 h under the high-light irradiation between the wild-type and both transgenic plants. We report the effects of the enhancement of H₂O₂-scavenging capacity in chloroplasts on the induction of cAPX expression under high-light intensity in the transgenic plants.

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We obtained the transgenic rice plants overexpressing the OsHCTR (NADPH-dependent HC-toxin reductase) gene. Previously, we reported that these plants showed tolerance to the biotic and the abiotic stresses. Last year, we reported that the overexpressing this gene resulted in the increase of NAD(P)(H) level and the activation of NAD synthetase. Here, we analyzed the maize plants possessing mutated Hm1 gene (homologue of OsHCTR gene). The result that the Hm1 mutation (hm1 / hm1) was also associated with loss of stress tolerance due to lowered level of NAD. Metabolites analysis also showed that aspartic acid level was increased, suggesting that amino acid may be a precursor for NAD. Based on this result, Both Hm1 and OsHCTR gene are functionally important to sustain stress tolerance in higher plants.

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Rice plants overexpressing OsHCTR increased the amount of NAD(P)(H) by the activation of NAD synthetase and NAD kinase. Moreover, transgenic rice had high tolerance against several stresses such as H₂O₂, UV, submergence, NaCl, and rice blast. Since reduced form of NAD(P) is a great source of redox in vivo, we were prompted to investigate NAD(P)H-producing pathway. Generally, NADH is produced in TCA cycle and glycolysis, whereas NADPH is produced pentose-phosphate pathway and malic enzyme. The activity of NADH-producing enzymes in TCA cycle decreased and malic enzyme was strongly activated in transgenic line, indicating that NADPH-producing route was accelerated. Analysis of numerous metabolites revealed upregulating glutathione (GSH) level in transgenic line. We also confirmed the activation of GSH reductase and GSH peroxidase. Thus, acceleration of GSH cycle using increased NADPH level may contribute to the ROS tolerance.

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In chloroplast stroma, thioredoxin (Trx) regulates various enzyme acitivities including chloroplast ATP synthase and the four Calvin cycle enzymes, FBPase, GAPDH, PRK and SBPase, via thiol-disulfide exchange reaction. This regulation system is known as thiol modulation. Two Trxs in stroma, named Trx-f and Trx-m, function for this redox regulation. On the process of the reduction of the target protein, a reduced form Trx exchanges the disulfide bond and di-thiols with the target proteins and gives the reduced form target proteins. We had developed a new method to capture the possible target proteins using mutant Trx-immobilized resin, and succeeded to identify a number of new target proteins for chloroplast Trx.
We next intended to study the redox regulation system in thylakoid lumen. HCF164 is a thioredoxin-like protein localized in thylakoid lumen. We analyzed biochemical property and redox regulation of HCF164, and surveyed the target proteins for HCF164 in thylakoids.

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Thioredoxin is a ubiquitous, disulfide oxidoreductase with two redox active cysteines at the active center (-WCGPC-). In cyanobacteria and in chloroplasts of higher plants, thioredoxin is involved in light-dependent regulation of various enzymes. In addition, redox transfer via thioredoxin is important for an anti-oxidative stress system in the aerobic organisms. We had investigated the possible target proteins of thioredoxin of cyanobacteria in the whole cell lysate of Synechocystis sp. PCC6803 using immobilized mutant of thioredoxin and identified two peroxiredoxin like proteins as the major targets. In the present study, physiological significance of these proteins as anti-oxidative stress system in cyanobacteria was confirmed in vitro and in vivo.

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Effects of methylviologen on global gene expression of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 were studied by DNA microarray. In our previous report, we found that the Fur-homolog, Slr1738, plays a regulatory role in induction of sll1621 in response to oxidative stress. Here, detailed analysis of the DNA microarray allowed us to identify sll1620 and slr0589 as possible target in addition to sll1621. By gel mobility shift assay, we demonstrated that the His-tagged Slr1738 specifically bound to the upstream regions of sll1620 and slr0589. Furthermore, we found another set of oxidative stress-responsive regulon: transcriptional regulator Sll0088 and slr0074-slr0075. This was confirmed by specific binding of the His-tagged Sll0088 to the upstream region of slr0074 in gel mobility shift assay.

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We have found that vernalization stimulates the synthesis of reduced glutathione (GSH) and synthesized GSH induces the bolting of the rosette plant Eustoma grandiflorum. In this study, we investingated whether GSH specifically induces bolting. The application of GSH increased the bolting frequency of non-vernalized plants, but that of oxidized glutathione had no such effect. Like GSH, the application of cysteine, a precursor of GSH, induced bolting, while neither dithiothreitol (DTT) nor 2-mercaptoethanol induced bolting. The inductive effect of vernalization on bolting was nullified by the addition of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. When GSH was applied at the same time as BSO, the bolting frequency was restored. However, DTT or cysteine had no restoring effect on bolting in BSO-treated plants. Thus, the effect of BSO could be reversed only by GSH, but not by other thiols. These suggest that vernalization-induced bolting is specifically regulated by GSH.

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We showed that changes in the glutathione redox state are required for the transdifferentiation of mesophyll cells into tracheary elements (TEs) in the Zinnia experimental system. To verify significance of the cellular redox regulation by glutathione in planta, we generated transgenic plants with altered glutathione redox ratio by introducing a chimeric gene construct where a gene for dehydroascorbate reductase (DHAR), or chloroplastic or cytosolic glutathione reductase (GR) was fused to the CaMV35S promoter. DHAR oxidizes GSH and GR regenerates GSH from GSSG. All transgenic plants generated showed retarded root formation, compared to non-transgenic plants. Conforcal laser scanning microscopy showed that those transgenics had the reduced number of cells in the root meristematic region. Taken together, changes in the glutathione redox state regulate the rate of root elongation by modulating the rate of cell division. We are also investigating on TE differentiation occurring in the vicinity of the root meristematic region.

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