Cyclins are known as one of the most important regulators of the eukaryotic cell cycle. In our initial studies using synchronized cultures of tobacco BY2 cells, we identified cis element called MSA (M phase-specific activator) that is necessary and sufficient for activation of promoters from cyclin B genes. Beside the cyclin B genes, many other G2/M phase-specific genes are under the transcriptional control mediated by the MSA elements. A group of c-Myb-like transcription factors bind to the MSA motifs and regulate promoter activities of G2/M phase-specific genes in tobacco. We are currently analyzing Arabidopsis knockout mutants in which c-Myb-like genes are disrupted in various combinations. A double knockout mutation for two related c-Myb-like genes caused severe defects in nuclear division, division plane determination, and cytokinesis. The results of our reverse genetic studies of the c-Myb-like genes are consistent with their roles in transcriptional regulation at the G2/M in BY2 cells.

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Sulfur is an essential macro-nutrient required for the synthesis of amino acids, proteins and lipids in primary metabolisms. It is usually taken up from soil in the form of sulfate. Uptake and internal transport of major nutrients, such as nitrogen, sulfur and phosphorus, are organized by multiple forms of transporter proteins bearing diversified functions in specific cell-type and organelles. Recent molecular physiological studies have identified the functional properties of plant sulfate transporter family proteins though the aid of functional genomics tools in Arabidopsis. SULTR1;1 and SULTR1;2 were the two essential components of the high-affinity sulfate uptake system at the root surface. The low-affinity subtype, SULTR2;1, was co-localized with SULTR3;5 in the root vasculature, and was functional for root-to-shoot transport. SULTR4;1 and SULTR4;2 facilitate release of sulfate from the vacuoles, controlling sulfate reserve in the root tissues. The function of SULTR1;3 was relevant to the phloem transport of sulfate.

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Arabidopsis terminal flower2 (tfl2) mutants show photoperiod insensitive early-flowering phenotype, as well as several other pleiotropic phenotypes. We found that the early flowering of tfl2 is caused mainly by ectopic expression of the FT gene, a floral pathway integrator and that TFL2 competitively counteracts the activity of CO, an FT activator to repress ectopic FT expression. Molecular cloning of TFL2 showed that it encodes a Heterochromatin Protein 1 (HP1) homolog. Gene expression analyses using DNA microarrays, however, did not show an increase in the expression of heterochromatin genes in tfl2 mutants but instead showed the upregulation of the several floral homeotic genes. This multiple genes repression by TFL2 causes the pleiotropic phenotype of the tfl2 mutant. Our results demonstrate that despite its homology to HP1, TFL2 is involved in the repression of specific euchromatin genes and not heterochromatin genes in Arabidopsis.

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Brassinolide, the most biologically active brassinosteroid, is formed by a Baeyer-Villiger oxidation of castasterone. However, such enzyme has not been identified. The formation of castasterone from 6-deoxocastasterone via two-step oxidations is catalyzed by cytochrome P450 monooxygenases (CYP85As). A null mutation in the tomato CYP85A1 gene, extreme dwarf (d^x), causes severe dwarfism due to brassinosteroid deficiency, but this mutant still produces normal fruits. GC-MS analysis showed that d^x fruits, as well as the wild type, produce brassinolide at high levels, indicating that brassinolide is synthesized in the fruits independently of CYP85A1. We have identified a new CYP85A gene that is specifically expressed in tomato fruits. This newly identified CYP85A, transformed into yeast, catalyzed the Baeyer-Villiger oxidation of castasterone to brassinolide, as well as the conversion of 6-deoxocastasterone to castasterone. Thus, this enzyme has been identified with the brassinolide synthase of tomato which is a multifunctional P450 enzyme catalyzing three consecutive oxidations.

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Both animals and plants use steroids as signaling molecules for their growth and development. Animal steroids are mainly perceived by members of the nuclear receptor superfamily of transcription factors. In plants, BRI1, a plasma membrane-localized leucine-rich repeat (LRR) receptor kinase, is a critical component of a receptor complex for brassinosteroids (BRs). To date, no evidence has shown direct binding of BRs to BRI1. Here, we present evidence for direct binding of active BRs to BRI1 using a biotin-tagged photoaffinity castasterone, (BPCS), a biosynthetic precursor of brassinolide. Binding studies using BPCS and recombinant BRI1 fragments reveal that the minimal binding domain for BRs is a 70 amino acid island domain (ID) localized between LRR21 and LRR22 in the extracellular domain, together with its C-terminal flanking LRR, (ID-LRR22). Our results demonstrate that BRs bind directly to the 94 amino acids comprising ID-LRR22 in the extracellular domain of BRI1.

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We have isolated, so far, 10 rice bri1 mutants, including d61-1 and d61-2 which were already reported. Accompanying with the phenotype severeness, the length of leaf sheath, the number of the crown roots and lateral roots of seminal root were decreased. We also analyzed the bri1 null mutant, d61-4. The amount of castasterone (CS), an active brassinosteroid (BR), in shoot of d61-4 was approximately 30-fold of the wild-type plant, while CS in root was about the same level. The expression of OsDWARF gene, which encodes C6 oxidase was increased in shoot than in root. As rice BRI1 homologue genes, BRI2 and BRI3 were expressed in root, defect in BRI1 defect may be complemented partly by these genes in this mutant. In d61-4, abnormal embryo development after 5DAP was also observed.
This work was in part supported by PROBRAIN.

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CYP90B1/DWARF4 catalyzes C-22 hydroxylation, the rate-limiting step of brassinosteroid (BR) biosynthesis in Arabidopsis. We have previously reported a rice CYP90B1/DWARF4 homolog, OsDWARF4, and its loss-of-function mutant, osdwarf4-1. The phenotype of osdwarf4-1 suggests that at least one functionally redundant gene is existed in the rice genome. A OsDWARF4-like gene identified by database screening is identical to CYP724B1/D11, which was recently identified as the gene corresponds to rice semi-dwarf mutant, d11. Because d11 was also showed mild phenotype similar to osdwarf4-1, we suspected that OsDWARF4 and CYP724B1/D11 function redundantly in rice. In the present study, we generated and characterized the osdwarf4-1 d11 double mutant. Based on these experiments, we will discuss the biological function of OsDWARF4 and CYP724B1/D11 in BR biosynthesis.

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The major catabolic pathway of abscisic acid (ABA) is initiated by hydroxylation at C-8' by ABA 8'-hydroxylase. Recently, Arabidopsis CYP707A genes have been shown to encode ABA 8'-hydroxylases. We examined the structural requirements for substrate recognition of ABA 8'-hydroxylase using 45 ABA analogs. The analogs were incubated with a microsomal fraction of CYP707A3 expressing insect cells, and all analogs were tested for their ability to act as an inhibitor. ABA 8'-hydroxylase recognized strictly the C-8' and C-9' in ABA. The carboxyl group at C-1 was necessary for binding to the enzyme, whereas C-6 and 1'-hydroxyl group were not essential for effective binding. The lack or enlargement of C-7' and the substitution at C-3' affected the binding to the enzyme, although it is not necessarily fatal. The present findings will be useful for developing the inhibitors of ABA 8'-hydroxylase.

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The key enzyme for ABA catabolism is ABA 8'-hydroxylase. In Arabidopsis , four ABA 8'-hydroxylases (CYP707A1-4) have been identified. To reveal the role of each CYP707A, we analyzed ABA contents and phenotypes in mutants and examined their expression site by using CYP707A promoter::GUS. In vegetative tissue, ABA level and drought tolerance of cyp707a1 and cyp707a3 were increased compared to those of wild type. Moreover, its double mutant exhibited even stronger tolerance to drought stress. These results indicate that CYP707A1 and CYP707A3 play an important role during drought stress. Although only CYP707A2 has thought to play an important role in seed, ABA level in cyp707a1 was higher than that in cyp707a2. Moreover, cyp707a1cyp707a2 exhibited increased hyperdomancy and accumulated 50-fold more ABA content than wild type. These results suggest that both CYP707A1 and CYP707A2 are indispensable for regulating ABA levels before germination.

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Stomata open and close under several environmental conditions. Among them, ABA is known to close stomatal pores in response to drought stress. Since ABA signaling is inhibited by ethylene during seed germination, we investigate the cross-talk between ABA and ethylene in stomatal closure of Arabidopsis thaliana epidermal peels. Preopened stomata closed almost completely within 5 minutes in response to ABA but ethylene gas treatment suppressed the ABA-induced stomatal closure. Time course observation showed that under ethylene treatment, the rate of stomatal closure was not so affected but the stomatal closure stopped halfway. In the other hand, when we examined a change of fresh weight under drought stress in planta, plants exposed to ethylene gas in advance showed elevated amount of water loss compared to those of control plants. Therefore, ethylene may act as a negative regulator in the ABA signaling pathways to suppress the stomatal closure.

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The phytohormone abscisic acid (ABA) regulates physiologically important stress and developmental responses. To address the mechanism of ABA response, we isolated 4 novel ABA hypersensitive mutants named ahg (ABA hypersensitive germination). ahg2 showed clear ABA hypersensitivity not only in germination, but also at later developmental stages, and displayed pleiotropic phenotypes, small leaves, and short stems and roots. In ahg2, ABA was more accumulated and ABA-inducible genes were upregulated. Map-based cloning of AHG2 revealed that this gene encodes a poly(A)-specific ribonuclease is involved in mRNA destabilization and degradation. Detailed analysis of the ahg2 mutation suggests that this mutation causes reduced AHG2 expression. Interestingly, the expression of AHG2 was induced by treatments with ABA, high salinity, and osmotic stress. These results suggest that AHG2 has a pivotal role in the downregulation of some specific genes by destabilizing mRNAs to maintain proper ABA and stress responses.

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We isolated Arabidopsis ABA hypersensitive mutants using ABA analog. One of these mutants, ahg3 is analyzed in this study. This mutant exhibits less germination or growth activities at the presence of ABA. Characterization of ahg3 revealed that ahg3 have enhanced sensitivity to salinity and osmotic stress. We conducted mapping of ahg3 and identified a point mutation in the AtPP2CA gene that encodes a PP2C. In vitro PP2C activity of the recombinant ahg3-1 protein was hundred times lower than that of wild type protein. ahg3-2, a T-DNA insertion mutant, showed ABA hypersensitive phenotype similar to ahg3-1. We compared the ABA sensitivity of ahg3-2 with that of other T-DNA insertion mutants of ABA-related PP2Cs. ahg3-2 showed the strongest phenotype at post-germination growth phase. This result indicates that among several PP2Cs that are implicated in ABA response, AHG3/AtPP2CA may play a major role in the ABA signaling pathway at germination and post-germination growth.

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To get more insight into the ABA signaling mechanism, we carried out screens for Arabidopsis mutants with altered ABA response. Using an ABA analog, PBI-51, we isolated several ABA hypersensitive mutants named ahg (ABA hypersensitive germination). ahg11, one of these mutants, showed ABA and salt hypersensitivity at seed germination. The corresponding gene for ahg11 was identified by map-based cloning. The predicted gene product is a PPR (pentatricopeptide repeat) protein belonging to a large protein family. The majority of PPRs in Arabidopsis are predicted to function at either chloroplasts or mitochondria. Recently, some PPRs with RNA-binding ability have been reported. Transient expression of an AHG11-GFP fusion protein showed that AHG11 is targeted to mitochondria. This result suggested that AHG11 might be involved in mitochondrial RNA processing. It is unknown and interesting how mitochondrial RNA processing is implicated in ABA response. A detailed analysis of this mutant is now ongoing.

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There are 10 starch synthase (SS) isozymes in rice. With the exceptions of GBSSI, SSI and SSIIa, their functions in starch biosynthesis are poorly understood. SSI and SSIII account for the major SS activity in developing maize endosperm. The SSIII-deficient mutant dull-1 in maize is well known, but no SSIII mutants in rice have been identified so far. In this study, we isolated an SSIIIa mutant from about 40,000 knockout rice population. In the mutant, Tos17 was inserted in exon 1 of the SSIIIa gene and the SSIIIa band on Native-PAGE/SS activity staining gel was completely absent. The proportion of α-polyglucan chain with DP of DP6-8, 17-18, 31-70 in amylopectin of endosperm starch was reduced and DP10-15, 20-29 was increased compared to wild type ones. The functions of SSIIIa of starch biosynthesis in rice endosperm are discussed.

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Recent investigations have shown that most of ADPG linked to starch biosynthesis accumulates in the cytosol in source leaves, and is produced by sucrose synthase (SuSy). This finding is consistent with the occurrence of a gluconeogenic pathway wherein SuSy is involved in the de novo production of ADPG, whereas both plastidial phosphoglucomutase (pPGM) and ADPG pyrophosphorylase (AGP) play a prime role in the scavenging of starch breakdown products. To further test this hypothesis, we have compared the ADPG content in both Arabidopsis and potato WT leaves with those of the starch-deficient mutants with reduced pPGM and AGP. Importantly, ADPG levels in all the starch deficient lines were normal as compared with WT plants. Collectively, the accumulated data show that (a) SuSy exerts a strong control on the starch biosynthetic process and (b) SuSy, but not AGP, catalyzes the production of cytosolic ADPG linked to transitory starch biosynthesis in source leaves.

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Endocytosis as a mechanism for nutrient uptake in heterotrophic cells was investigated using suspension culture cells of Acer pseudoplatanus L, the endocytic inhibitors wortmannin and LY294002 and the fluorescent endocytosis marker Lucifer Yellow-CH (LY). Time-course analysis of sucrose uptake in intact walled cells revealed a two-phase process involving an initial 90 min wortmannin- and LY294002-insensitive sucrose uptake period, followed by a prolonged phase of rapid sucrose accumulation which was greatly inhibited by the two endocytic inhibitors. Rates of sucrose and LY accumulation were virtually identical. In addition, LY incorporation increased as a function of external sucrose concentration. When sucrose was substituted by other sugars or amino acids, uptake of LY greatly diminished, indicating that sucrose itself is the primary signal of endocytosis. These results demonstrate the existence of a sucrose inducible endocytic process as a viable mechanism for solute transport into the vacuole of storage cells.

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Shikonin, a red naphthoquinone pigment, is a secondary metabolite produced in a perennial herbal plant L. erythrorhizon. In the biosynthesis of shikonin, the geranylation step catalyzed by PGT (PHB: geranyltransferase) is a regulatory key step. This enzyme activity is regulated by several chemical and physical elements, in particular it is strongly down-regulated by light irradiation. We have recently shown the expression pattern of LePGT, the gene encoding PGT in L. erythrorhizon, by in situ hybridization; i.e. LePGT is specifically expressed in the epidermis of root tissues where shikonin accumulates. Northern analysis also showed that LePGT expression was strongly suppressed by light irradiation. To further clarify of the light-regulation mechanism and tissue-specificity of LePGT expression, we isolated the promoter region of the gene by TAIL-PCR, and established reporter gene constructs, using GUS and GFP genes. We will report the promoter activities in transgenic Arabidopsis and tobacco plants.

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The biosynthesis of legume-specific 5-deoxyflavonoids requires the co-action of chalcone polyketide reductase (PKR) and chalcone synthase (CHS) to produce 6'-deoxychalcone. In contrast to extensive studies on CHSs, no detailed information of PKR genes is available. Thus, we studied PKR genes of a model legume Lotus japonicus.
Homology-based PCR yielded two cDNAs, which are ca. 80% identical to known PKRs. Genome analysis showed that four PKR genes (PKR1-4) form a cluster within a 58 kb region. The comparison of exon-intron structures suggests that PKR3 should be a pseudogene. Expression analysis revealed constitutive expression of PKR4 and transient increase of PKR1 and PKR2 mRNAs after elicitation. This result is consistent with the existence of W-box motif involved in plant defense responses in the promoters of PKR1 and PKR2. PKR-transgenic petunia displayed decreased amounts of anthocyanins and the emergence of 6'-deoxychalcone.

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The expression of chalcone synthase (CHS) is stimuated by developemental signals and a variety of environmental stimuli, and the resultant flavonoids have wide range of physiological functions. In particular, leguminous plants are known to synthesize antimicrobial isoflavone, phytoalexin in response to pathogen attack. CHS1, a member of soybean CHS gene family is found to be transcribed after both UV light irradiation and elicitor treatment. Functional analysis of 5'deletions have delimited the sequences nessesary for elicitor inducibility.
We have isolated SBZ1 which binds to promoter region of french bean CHS15 and soybean CHS7 containing both the G-box and the H-box, and becomes a substrate for phosphorylation in vitro. Here we show that recombinant SBZ1 could bind to CHS1 promoter region. Furthermore, this binding to CHS1 promoter was stronger than to CHS7, which indicates that SBZ1 might be involved in elicitor inducibility of CHS1.

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Phenylalanine ammonia-lyase (PAL) acts as the key enzyme in regulating metabolic flow from the primary metabolism to phenylpropanoid metabolism, and the genes encoding PAL are regulated by both environmental and developmental cues. It has been revealed that the expression of gDcPAL1 gene in suspension-cultured carrot cells is induced by dilution effect, elicitation and UV-B irradiation, and box-L₁ and box-L₅ in the gDcPAL1 promoter play an important role to regulation of gDcPAL1 expression. We isolated DcMYB1 transcription regulatory factor, which binds to both box-L₁ and box-L₅ and have studied on the function of DcMYB1. In this study, it was indicated that gDcPAL1 promoter was activated by DcMYB1 binding to the plural box-Ls in gDcPAL1 promoter and that DcMYB1 acted as the transcriptional activator concerning to the induction of expression of gDcPAL1 by several stresses, they were dilution effect, elicitation and UV-B irradiation.

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Aurones are a class of flavonoid derivatives, and confers bright yellow coloration to flower in some ornamental plants such as Antirrhinum majus. An Antirrhinum polyphenol oxidase homolog, aureusidin synthase (AmAS1), catalyzes synthesis of aurones from chalcones. However, transgenic plants overexpressing AmAS1 gene alone failed to produce aurones and yellow color flowers. Here we have revealed that a novel Antirrhinum chalcone 4'-glucosyltransferase (Am4'CGT) provide chalcone 4'-glucoside to AmAS1 and that Am4'CGT plays a critical role for aurone biosynthesis. Overxpression of Am4'CGT and AmAS1 genes is sufficient to produce aureusidin 6-glucoside in transgenic torenia. Furthermore, overexpression of Am4'CGT and, AmAS1 and simultaneous RNAi of anthocyanin biosynthetic genes result in bright yellow flower accumulating aureusidin 6-glucoside. This is the first report of successful generation of clear yellow flowers with genetic engineering. Subcellular localization study of Am4'CGT and AmAS1 strongly suggests that chalcone 4'-glucoside is synthesized in cytosol and is transported to vacuole.

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The sepal color of Hydrangea macrophylla changes easily depending on environmental conditions. The most interesting phenomenon of the color development of hydrangea is that the same components (delphinidin 3-glucoside, 3-acylquinic acid, 5-acylquinic acid, Al³⁺) develop a variety of sepal colors from red through purple to blue. Recently we found that in purple colored sepals of hydrangea red, purple, and blue colored cells coexist. To clarify the mechanism of cell color variation in purple colored sepals we prepared protoplast mixture of various colored cells. We recorded an absorption spectrum of a cell, vacuolar pH, composition of the anthocyanin and the co-pigments, Al³⁺ in the each colored cell. Between the color and vacuolar pH no correlation was observed. The bluer the cell, the more the amount of 5-acylqunic acid increased. The sepal color might be affected by many factors, such as pH, somposition of co-pigment and Al³⁺.

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We established a system in which anthocyanin synthesis of regenerated torenia shoots, which derived from the leaf discs in vitro, was induced on the medium containing high concentration of sucrose. In this system, ABA could also induce anthocyanin synthesis without adding the high concentration of sucrose. Determination of endogenous ABA in the regenerated shoots producing anthocyanin in the presence of high concentration of sucrose revealed that the amount of endogenous ABA transiently increased just before the induction of anthocyanin synthesis. These results suggested that change in the amounts of endogenous ABA might play an important role in the induction of anthocyanin synthesis in the regenerated torenia shoots. In the previous study, we performed cDNA subtraction to isolate the genes up-regulated in torenia regenerated shoots producing anthocyanin. In this study, gene expression profiles of 1000 clones obtained by cDNA subtraction were analyzed by cDNA microarray.

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DOPA 4, 5-dioxygenase (DOD) is the most important enzyme involved in betalain biosynthesis. The cDNA coding DOD was isolated from common portulaca (Portulaca grandiflora) by cDNA subtraction method (Christinet, et al., Plant Physiol 134: 265-274). It was confirmed by complementation experiments using the particle bombardment that the gene was concerned with betalamic acid synthesis in vivo, but there has been no reports to detecte DOD activity in vitro. In this study, we isolated MjDOD cDNA from four o'clock (Mirabilis jalapa) by PCR method using the degenerate primer based on the conserved amino acid sequence among plant DOD genes. A MjDOD cDNA was cloned into yeast expression vector and introduced into Saccharomyces cerevisiae. A crude extract from the recombinant yeast strains was added to a reaction mixture containing L-DOPA. The color of the reaction mixture turned to yellow and a reaction product in the mixture was betalamic acid confirmed by HPLC.

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Two types of red pigment, anthocyanins and betacyanins, never occur together in the same plants. Although anthocyanins are widely distributed in higher plants, betacyanins are characteristic constituents of most species of Caryophyllales.
We have cloned cDNAs of DODA (Dopa-dioxygenase) and PAP (Phytolacca americana polyphenol oxidase) which might be involved in betacyanin biosynthesis from Phytolacca americana. Phytolacca DODA cDNA showed high degree of homology (approximately 70%) with DODAs from other Caryophyllales, and had a conserved motif found in DODAs of betacyanin synthesizing plants. PAP showed very high degree of homology (95%) with known PAP1. Spatial and temporal expression of DODA and PAP in various organs of Phytolacca plants has been investigated by semi-quantitative RT-PCR. Accumulation of DODA transcripts has been found not only in betalain-containing cells but in green or white cells. Close correlation between betacyanin accumulation and expression of PAP was observed.

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Nicotine, a major alkaloid in tobacco plants, acts as a defensive compound against insects. Nicotine is produced in tobacco roots and transported to leaves for storage. We found that expression of two genes (NtMATE1, NtMATE2) was specifically suppressed in the nic1nic2 mutant root. These genes encode similar proteins, which are homologous to multidrug and toxic compound extrusion (MATE) family transporters. Expression of these genes was tightly controlled by NIC regulatory genes and observed specifically in the wild type root. Promoter::GUS expression analysis demonstrated that NtMATE1 was expressed all tissues except for epidermis in the differentiated region. In NtMATE1 over-expressing transgenic tobacco BY-2 cells, NtMATE1 was localized to the tonoplast. Analysis of nicotine secretion in NtMATE1 over-expressing BY-2 cells and transgenic tobacco roots in which NtMATE1 expression was reduced by RNAi suggest NtMATE1 promotes excretion of nicotine from the vacuole, and consequently facilitates translocation of nicotine from roots to aerial parts.

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Quinolizidine (Lupin) alkaloids (QAs) are a group of secondary compounds which are mainly found in the family Leguminosae. In Lupinus plant, tigloyl esters of alkaloids are found as major forms among ester-type alkaloids, which are produced by action of tigloyl-CoA:13α-hydroxymultiflorine/13α-hydroxylupanine O-tigloyltransferase (HMT/HLT). We report here, for the first time, molecular cloning and characterization of HMT/HLT. HMT/HLT has been purified from L. albus. The internal amino acid sequences were determined, allowing design of primers for RT-PCR. Using an amplified fragment as the probe, a full-length cDNA clone was isolated from cDNA library. The functional analysis was carried out with the recombinant gene product heterogeneously expressed in E. coli. We confirmed that the recombinant enzyme catalyzed the acyltransfer reaction from tigloyl-CoA to 13α-hydroxymultiflorine and 13α-hydroxylupanine. The substrate specificity, the assays with inhibitors and the nucleic acid hybridization analyses of HMT/HLT will be also presented.

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Cardiolipin (CL) is a phospholipid localized in the mitchondrial inner membrane of eukaryotes. CL is suggested to be involved in many mitochondrial functions. However, the gene for CL synthase (CLS) has not been cloned from multicellular eukaryotes, and the CLS mutant has also not been reported. To know physiological roles of CL in higher plants, we have identified the Arabidopsis gene encoding the CLS and isolated mutants in which the CLS gene is disrupted.
As a result of searching candidate genes in Arabidopsis genome, AtCLS was predicted as the CLS gene. We have shown that the AtCLS gene encodes a eukaryotic CL synthase, that is targeted into mitochondria. Moreover, we have analyzed knockout mutants of AtCLS and found that they show an embryo-lethal phenotype. These findings demonstrate that CL plays a critical role in mitochondria during plant develpment .

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Ubiquinone (UQ) is a component of the mitochondrial respiratory chain, and is composed of a quinone moiety and an isoprenoid side chain. The length of side chain varies depending on the organism it derives, and is defined by the nature of the polyprenyl diphosphate synthase. In this study, decaprenyl diphosphate synthase gene (ddsA) from Gluconobacter suboxydans was introduced into rice in order to change its UQ side chain length from nine to ten. In plants, UQ biosynthesis thought to be occurred in mitochondria or ER-golgi system. Therefore, we produced three kinds of transgenic rice plants expressing DdsA targeted into mitochondria (S14-DdsA), golgi apparatus (CTS-DdsA) or cytoplasm (35S-DdsA). By western analysis, we obtained S14-DdsA and CTS-DdsA plants accumulating DdsA, but could not obtain such a 35S-DdsA plant. HPLC analysis of revealed that UQ content of CTS-DdsA plants were indistinguishable from wild type, while S14-DdsA plants produced UQ-10 (UQ content: 10 μg/gFW).

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In higher plants, Z,E-mixed polyisoprenoids, including dolichol and polyprenol, are biosynthesized, showing two different distribution patterns in the chain length, C_50-60 and C_70-120. However, physiological role of Z,E-mixed polyisoprenoids in higher plants is hardly elucidated. To understand the physiological functions of Z,E-mixed polyisoprenoids, we have cloned and characterized 6 cDNAs encoding cis-prenyltransferase (CPT), which catalyzes formation of the structural backbone of Z,E-mixed polyisoprenoids, in Arabidopsis thaliana.
Crude proteins prepared from yeast strains harboring each AtCPT except AtrCPT4 showed CPT activity in vitro, catalyzing the formation of C_80-100 polyprenyl products. In contrast, AtCPT4 showed CPT activity, catalyzing the formation of C_50-60 polyprenyl products. Transposon Ds-inserted mutant of AtCPT4 showed increased elongation of petioles and hypocotyls under long-day condition, while no significant difference between mutant and wild-type grown in the dark. Moreover, expression of AtCPT4 was repressed in the seedling grown in the dark, but rapidly induced by irradiation of white light.

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Molecualar mapping of a gene (Als1) responsible for Al resistance in rice was conducted by using a rice mutant(als1) sensitive to Al. F2 seeds were derived from a cross between the mutant and Kasalath. Bulk segregant analysis was performed by pooling equal amounts of DNA from 10 Al-sensitive or 10 Al-resistant F2 plants. A total of 59 InDel markers were selected to examine polymorphism among the Al-sensitive and Al-resistant bulk, the mutant, and the Kasalath. As a result, the marker R6M44 on the long arm of chromosome 6 was linked to this gene. Co-segregating analysis using the 27 Al-sensitive F2 plants showed that R6M44 had a distance of 24.7 cM from (Als1). To further map this gene, two polymorphic InDel markers MaOs0615 and MaOs0619 were developed. Linkage analysis showed that (Als1) was located between MaOs0615 and MaOs0619, with a distance of 3.8 cM and 1.9 cM, respectively.

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Al sensitivity was compared between rice (cv. Nipponbare) and wheat (cv. ET8), and rice exhibited much higher tolerance to Al during primary root development from seed, but not during root elongation of seedlings than wheat.
To identify rice gene(s) regulating Al tolerance during primary root development, we obtained 8 Al-sensitive candidates from the 1020 Nipponbare TOS17 line of T3 generation. The segregation pattern of Al sensitivity of these candidates was investigated in T3 population seeds derived from one spikelet, and 2 lines exhibited 1(Al sensitive): 3(Al tolerant) segregation, suggesting that Al sensitive phenotype of these mutants is due to single recessive mutation.
In mature plants grown in normal soil, no significant difference in plant length and tiller number was observed between these mutants and wild type Nipponbare, suggesting that these Al-sensitive mutations does not affect normal growth.

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Deduced functions of AtBCB and NtGDI1 gene for Al stress were investigated. The cDNAs fused to a GFP gene in plant cells showed that the AtBCB and NtGDI1 protein exist in cell membrane and cytoplasmic region, respectively.
Non-transformant (Ler) accumulated lignin by Al stress, while the AtBCB transformant showed a constitutive produce of lignin in root. Microscopic observation of Al treated roots indicated that a deposition of lipid-peroxides was clearly low in the area where lignin was accumulated. We supposed that lipid peroxidation was probably eliminated by lignin.
Temperature sensitive yeast sec19- mutation was complemented by the NtGDI1 gene at 37 degerees. Sec19- mutant showed Al sensitive at 32 degrees and this phenotype was also complemented by NtGDI1 gene. Since Sec19 protein functions for a vesicle transport, we suppose that NtGDI1 protein can function for Al efflux in both yeast and Arabidopsis.

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Excess salt is harmful for plants. However, halophytes have developed the mechanism of salt tolerance to adapt themselves to high salinity. Sheep grass belongs to Poaceae and it can grow under 500 mM NaCl, although the remarkable structural feature, such as salt grands or bladders, was not observed. Previously, we cloned the salt-inducible plasma membrane protein 3 genes (AcPMP3) from sheep grass. Expression of AcPMP3 was up-regulated under salt, drought, cold, ABA and H₂O₂ treatments. To investigate the function of AcPMP3 under salt stress, salt sensitive yeast (YR93-31; Δpmp3, Δnha1, Δpmr2) was transformed with AcPMP3. Consequently, overexpression of AcPMP3 could recover salt tolerance in YR93-31 under high NaCl and KCl conditions, and decrease cellular Na⁺ and K⁺ accumulation. AcPMP3 mRNA was abundant in mesophyll and root epidermis cells, and it might regulate Na⁺ and K⁺ accumulation in their cells under salt stress.

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OsPI1 (Oryza sativa Phosphate-limitation Inducible gene 1) cDNA, 375bp full-length, has been isolated from P-deficient rice roots using a cDNA microarray. The objective of this study was to analyze the function of OsPI1. P-deficiency resulted in OsPI1 mRNA being rapidly and systemically accumulated. The expression pattern corresponded with that of the novel P-deficient inducible genes designated as TPSI1/Mt4 family. The results of in situ hybridization of P-deficient rice indicated a higher accumulation of OsPI1 mRNA in young tissues, which require a larger amount of P. Sequence features and in vitro translation results indicated that OsPI1 is not the usual coding RNA, but has a specific function as the RNA molecule. We conclude that the OsPI1 gene has an important function in adaptation for P-deficiency that involves the regulation of other functional genes responding to P-deficient conditions. Preliminary results about the effect of OsPI1 knockout by RNAi will be presented.

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Iron-uptake mechanism in higher plants is mainly divided into two: one consists of H+-excretion, reduction of ferric ion and the ferrous ion uptake (Strategy 1). The other one consists of biosynthesis/excretion of Mugineic acids and uptake in the conjugation with ferric ion (Strategy 2). Here, we used two genes representing those strategies, NtIRT1 and HvIDS2, to examine the expression specificity responding to three types of iron-deficiency (1. transplant to Fe-deficiency condition after grown in a normal condition; 2. transplant to Fe-deficiency condition with washing out the apoplasmic Fe; 3. transplant to cadmium added Fe-deficiency condition) in the respective native mother plant. Results demonstrated that NtIRT1 did not express without apoplasmic Fe even the plant was in a Fe-deficiency condition, while HvIDS2 expressed whenever the plant was in a Fe-deficiency condition. Result from the further experiment using a transformed tobacco, which expressing HvIDS2p/GUS fusion gene, will be also discussed together.

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We have previously identified the novel cis-acting elements IDE1 (iron-deficiency-responsive element 1) and IDE2 through promoter analysis of barley iron-deficiency-inducible IDS2 gene using transgenic tobacco system. To analyze the function of IDE1 and IDE2 in rice plants, IDS2 promoter fragments containing IDE1 and IDE2 were introduced into rice. A fragment containing one copy each of IDE1 and IDE2 was shown to be sufficient to confer strong iron-deficiency-inducible expression in rice roots and leaves. The expression was dominant in vascular bundles and root exodermis, resembling the expression pattern of other rice genes related to iron acquisition via mugineic acid family phytosiderophores (MAs). Rice iron-deficiency-inducible genes detected by microarray analysis included genes involved in every biosynthetic step of MAs. Promoters of those genes possessed sequences homologous to IDE1 and/or IDE2 at high frequency, suggesting coordinately regulated expression of Graminaceous genes related to iron acquisition.

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Sulfoquinovosyl diacylglycerol (SQDG) is the sole lipid with a sulfur atom in chloroplasts and cyanobacteria. Roles of SQDG have been defined in the photosynthetic apparatus (Sato 2004). We here examined the physiological significance of SQDG in a green alga Chlamydomonas reinhardtii after the shift of the cells to sulfur-free condition. The sulfur-starvation brought about conversion of SQDG that had occupied 12% of the sulfur-atom in the cells into the other cellular compounds. However, the synthetic activity of SQDG remained unaltered with an increase in the transcript level of the gene for UDP-sulfoquinovose synthase, one of the genes for SQDG synthesis. These results implied that, under sulfur-starved condition, preexisting SQDG serves as the sulfur-source, and that de novo synthesized SQDG plays some crucial role.
N. Sato (2004) Roles of the acidic lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol in photosynthesis: their specificity and evolution. J. Plant Res. (vol.6)

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SULTR1;1 high affinity sulfate transporter is highly regulated in the epidermis and cortex of Arabidopsis roots responding to sulfur deficiency (-S). To identify the cis-acting element involved in the -S inducible expression, SULTR1;1 promoter was analyzed using transgenic Arabidopsis. Deletion and gain-of-function analysis using luciferase reporter gene revealed that the 16bp sulfur responsive element (SURE) from -2777 to -2761 of SULTR1;1 was sufficient and necessary for the -S response. The SURE contained an ARF binding sequence, but was not responsive to auxin, indicating its specific function in the -S response. The base substitution analysis indicated the significance of a 5bp sequence (GAGAC) within the ARF binding site as a core element. Microarray analysis of early -S response in roots indicated the presence of SURE core in the promoter regions of -S inducible genes, suggesting the existence of a common regulatory mechanism inducing -S response.

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The Cys-rich peptide, Phytochelatin (PC) has been shown to play an important role in heavy metal detoxification. However, Arabidopsis transgenic plants overexpressing the PC synthase gene failed to show an increase in Cd tolerance. This suggests that novel factors in addition to PC remain to be identified to improve the heavy metal detoxification in plants.
We have successfully isolated a novel Arabidopsis Cd-sensitive mutant (cds1) by carbon ion beam mutagenesis, which causes large base pair deletions to occur at random loci in the genome. cds1 mutant plants show the same phenotype as PC synthesis mutant plants; they are more sensitive to Cd and Hg than wild type, and also show a slight increase in Zn sensitivity. Genetic analyses and GSH synthesis inhibitor studies revealed that the cds1 phenotype was caused by a recessive single mutation, which was different from those of PC-GSH synthesis mutants.

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SmtA, a metallothionein homologue of Synechococcus PCC 7942, binds heavy metal ions and is involved in response to the heavy metal stress. SmtA has two metal-binding regions, namely, the N-terminal (Met1-Cys47) region and C-terminal region (Gly43-Gly56), which binds two and one molecules of Zn²⁺, respectively. We have constructed the polypeptides which include tandemly-connedted N-terminal regions of SmtA and analyzed their metal binding ability.
The number of Zn²⁺ bound to isolated recombinant proteins increased depending on the number of included N-terminal regions. For example, a modified SmtA with additional 10 N-terminal regions bound 26 Zn²⁺ molecules. The binding mode of Zn²⁺ was estimated to be similar to that in the native SmtA. In addition, E. coli expressing either the native or modified SmtA contained increased amount of Zn²⁺, indicating that these proteins are functioning in accumulating heavy metals in the cells.

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Punch holes on peach leaves caused by Cu treatment is similar to the lesion spots on a leaf exposed to Xanthomonas campestris pv. pruni . We hypothesized that Cu treatment caused programmed cell death (PCD). First, we investigated the relation between Cu concentration and an aria of lesion on a leaf after Cu treatment. The lesion was not observed with Cu below 200 μ M but the aria of lesion spot was constant when leaf was treated with over 300 μ M Cu. Second, we tested whether Cu affected PCD or not. It is known that VPE (vacuolar processing enzyme) has caspes1 activity and plays an important roll as a determinant of PCD. We treated leaf with 500 μ M Cu with/without caspase-1 inhibitor. The lesion was almost never seen on the leaf with caspase-1 inhibitor. These results suggest Cu treatment causes hyper sensitive reaction on the peach leaves.

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Glutamate dehydrogenase (GDH) catalyzes the amination of
2-oxoglutarate and the deamination of L-glutamate. After the discovery of glutamate synthesis cycle, the function of GDH has been debated. GDH of Brassica napus consists with a and b subunits that are encoded by two genes (gdh1, 2) and forms seven isoforms (GDH1~7). In this report, we examined to identify signals to change
the isoform patters and determined the expression of a subunit polypeptide and the corresponding gdh2 gene.
SA and JA treatments induced GDH7 significantly. Once, the leaf tissue was wounded, any SA, JA and ethylene inhibitors could not suppress the induction of GDH7. Treatment with SA and JA in acid solution induced GDH7 significantly and the isoenzyme patterns were almost identical to those of protoplasts. Changes in GDH isoenzyme patterns of protoplasts reflected the amount of a subunit and gdh2
mRNA.

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Generally, the peroxidase(PO) activity in higher plants is known to be upregulated by environmental stresses. PO proteins are encoded by a multigene family, and most of physiological functions of PO isozymes have yet been unclear. In this study, we analyzed the expression of PO genes of poplar as a model tree. ESTs generated from the stress-treated leaves of Populus nigra were subjected to the screening of poplar PO clones, and 198 PO candidates were isolated. Of the candidates clones many were found to be 3'-UTR splicing variants. These PO genes were also confirmed stress-inducible by northern analysis. We are also isolating PO cDNA from the leaf, shoot and xylem of P. alba grown under natural conditions. The stress-induced expression and organ-specific expression of these PO genes will also be discussed.

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To date, many genes that are transcriptionally activated upon wounding have been identified, but their biological functions are not necessarily clear yet. In order to understand the molecular basis of wound response, we have identified genes whose transcripts accumulated at very early stages of wounding in tobacco plants. In the present study, we focused on one of such particular genes, WI4.
WI4 consists of 221 amino acids, and has a bHLH (basic-helix-loop-helix). Using dual-luciferase assay, WI4 was demonstrated to repress luciferase transcription in BY2. WI4 was shown to localize in the plastid. In leaves, WI4 transcripts and proteins transiently and locally accumulated 1 h after wounding. In contrast, in non-photosynthetic tissues, its transcripts were constitutively accumulated. Transgenic tobacco, that over-expressed WI4, showed abnormal phenotype with pale-green leaves, no stems and roots. These results suggested that WI4 is a transcriptional repressor that functions in plastid, possibly regulating transcription of photosynthetic genes.

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We previously identified a specific gene encoding a WRKY-type protein, that was immediate-early and transiently activated upon mechanical wounding in tobacco, and designated as Wizz (wound-induced leucine-zipper zinc-finger). In order to examine expression pattern during wound-response, we generated transgenic tobacco plants with Wizz promoter::LUC and 35S::LUC. Results indicated dissected promoter as short as -270::LUC to possess transcription activity. Consequently, 4 types of transfomants with further deleted constructs, -217::LUC,-155::LUC,-97::LUC, and -TATA::LUC were generated and their activities are under examination. The second project is to identify genes that are regulated by WIZZ. To this end, We constructed overexpression transgenic lines and, using its cDNA together with that from wild-type control, We screened a microarray, containing approximately 4,000 genes originated from N. sylvestris and 48 wound-response genes from N. tabacum. A preliminary hybridization showed a considerable number of genes to be differentially expressed between the two, and their identity is now under progress.

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Exposed to unfavorable environments such as pathogen attack, plants quickly establish the defense system. The MAP Kinase (MAPK) cascade plays a critical role in the signal transduction for cellular responses. In tobacco plants, WIPK is one of the representative MAPKs, and well characterized. However, the downstream factor(s) of WIPK has not so far been identified, leaving the whole signaling system rather obscure.
Using the yeast two hybrid screening system, we obtained one particular protein, which specifically interacts with WIPK, and designated as NtWIF. In order to examine the biological function, we generated NtWIF over-expressed transgenic tobacco plants, which were found to produce a high level of salicylic acid. Consequently, transcripts of PR-related genes were up-regulated. Results suggested that NtWIF is involved in regulation of salicylic acid-responsive genes. Further screening of affected genes by micro-array is currently under the progress.

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Gene targeting by homologous recombination (HR) is a powerful tool for reverse genetics. We have developed a gene-targeting system in rice based on large-scale transformation with strong positive-negative selection and succeeded in modifying the Waxy gene. To access the applicability of our targeting system, we are trying to knockout the Alcohol dehydrogenase genes, Adh1 and Adh2, which are arranged as direct repeats separated by 30 kb on chromosome 11. Previously, we reported our attempt of targeted modification of both Adh1 and Adh2 separately and results including Southern analysis of the targeted T0 plants (2004). Here we report the analysis of T1 plants having Adh2 targeted as well as phenotypic characterization of Adh1 modified plants. Based on these results, we will discuss the implications of HR in somatic rice cell, which is involved in T-DNA mediated foreign genes integration.

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We are currently analyzing Arabidopsis metabolism-related genes with a combination of transgenic suspension-cultured T87 cells and transcriptome/metabolome analyses. Here we established a high-troughput protocol of Agrobacterium-mediated transformation of the T87 cells. In our routine procedure, a person works five days for 24 Agrobacterium strains carrying individual manipulated genes for transformation of plant cells and picks more than ten transgenic calli two or three weeks later. Moreover, we developed a new protocol for cryopreservation of transgenic T87 cell lines, by which a person can handle 200 vials with transgenic suspension-cultured cells and stock them in liquid nitrogen within a single day. The protocols we developed would be crucial to high-throughput analyses, especially of the metabolisms observed in the T87 cells, and could be applicable for other plant suspension-cultured cell lines.

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In the previous annual meeting, we reported a newly developed protocol for cryopreservation of Arabidopsis suspension-cultured cells. In this study, we evaluated how the preservation affected gene expression and metabolite production by transcriptome and metabolome analyses. Independent three experiments were carried out for the evaluation. After recovering the cells from liquid nitrogen, the transcripts were analyzed using the Agilent 22K microarrays. The transcripts of 46 genes that exhibited more than 2 fold changes were reproducibly detected among the triplicates, while duplicated self hybridization detected only 4 genes, indicating that the cryopreservation protocol affected gene expression pattern of the genes. Gas chromatography mass spectrometry analyses of metabolites of the cell lines also indicated some changes in metabolites. The results provided us fundamental information of the effects of the cryopreservation protocol.

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