To understand the mechanism of cadmium tolerance and accumulation in higher plants, a large-scale screen was conducted with a T-DNA insertional collection of Arabidopsis ecotype Col-0 mutants. One line, hsc1 displayed increased sensitivity to cadmium. The average delta root elongation of the mutant was almost half of that of wild type with 50 μM Cd. Thermal asymmetric interlaced PCR was used to detect a T-DNA insertion locating in the 7th intron of an open reading frame of a gene annotated to encode triosephoshate isomerase (TPI) on Chromosome 3. TPI catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. RT-PCR indicated that the TPI transcript accumulates predominantly in the shoots of wildtype treated with 100μM Cd for one week but cannot be detectable in the shoots or roots of hsc1.

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O. brevis bxa1 gene encoding a CPx-ATPase was isolated and characterized in our group. To clarify the gene function for various heavy-metal stresses more detail, several experiments shown below were performed using constructed yeast transformants.
The bxa1 transformant was more sensitive only to Cd stress than a control strain carrying a vector. Fluorescence microscopic observation indicates that Bxa1::GFP fusion proteins localize mainly in endoplasmic reticulum (ER) membrane. Moreover, SEM (scanning electron microscopy) observation suggested that expression of Bxa1 in yeast causes a change of cell size, cell shape and cell viability and that Cd stress causes a promotion of these changes. Protein synthesis related to cell wall and cell membrane formation may be affected by an accumulation of Bxa1 in ER, because ER is an important organ for many protein syntheses in various metabolisms.

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In order to phytoremediate Cd-contaminated soils, we need a wide range of information about plant responses to Cd. Aiming to isolate Arabidopsis mutants that show altered responses to the heavy metal, we prepared Cd-gradient agar plates (CGAPs), in which a gradation of Cd concentration was formed at the media surface. CGAPs enabled us to isolate some interesting Arabidopsis mutants. The T-32 line was one of Cd tolerant mutant lines that were isolated in our previous study. Subsequent study showed that T-32 seedlings were tolerant not only to Cd, but also to Ni. When the T-32 plants were fed with 30 μM of Cd for 24 h, they accumulated Cd at a concentration about 2.5 fold higher than WT. Chromosomal location of the mutation responsible for the Cd-tolerant and accumulating phenotype is now under investigation.

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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. To explore a novel heavy metal detoxification mechanism, we screened for an Arabidopsis Cd-sensitive mutant (cds1) by carbon ion beam mutagenesis, which causes large base pair deletions at random loci in the genome. Genetic analyses and GSH synthesis inhibitor studies revealed that the cds1 phenotype was different from those of PC synthesis mutants. Moreover, Cd content in shoots of cds1 cultured on Cd-containing media was similar to wild type, suggesting that Cd-sensitive phenotype of cds1 was not attributed to the Cd transport system from roots to shoots. In the rough mapping, cds1 was localized to approximately 300kbp region of chromosome 4. One of the genes was found to have a 2.5kbp deletion in the promoter region.

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Reed ( Phagmites communis ) is highly tolerant to cadmium. The cadmium concentration of leaf was one-third of that of stem after 7 d exposure to 100μM CdCl₂. Half of the total cadmium in the stem localized in cell wall and another half in subcellular. These results suggest that the stem of reed has detoxication mechanism to intracellular cadmium. Cadmium in the stem was found in the fraction of MW < 3000 and 10000 to 50000, and the cadmium complexes in MW 10000 to 50000 fraction were not thiol compounds. On the other hand, the sugar content of MW 10000 to 50000 was increased in response to cadmium stress. These results suggest that the stem of reed produce non-thiol sugar compound binding to cadmium in response to cadmium stress, thus reed could accumulate cadmium in stem and keep low concentration in leaf.

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Copper is an essential trance metal for all living organisms. Cu/Zn superoxide dismutases (CSD1 and CSD2) are involved in the scavenging of reactive oxygen species in cytosol and chloroplast respectively and their expression is down-regulated under copper deficient conditions. In this work, we demonstrated that one of microRNA, miR398, was expressed under low-copper conditions, and degraded CSD1 and CSD2 mRNA directly. In addition, iron superoxide dismutase (FeSOD) is expressed instead of Cu/ZnSODs and completes the function under copper deficient conditions. On the other hand, plastocyanin (PC) involved in photosynthetic electron flow is transcribed constitutively in any copper conditions. From our results, we propose that copper is preferentially transferred to PC under copper deficient conditions, and miR398 is involved in this regulation.

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Copper is an essential element that plays critical role in various biological processes, but its accumulation in soil causes growth inhibition of the roots. To identify major genes regulating variation of Cu-tolerance among natural variation, we performed QTL analyses using two sets of RILs of A.thaliana (Ler/Col and Ler/Cvi). One of the QTL shared by both populations on the lower arm of chromosome 1 (QTL1) can explain about 40% of the Cu-tolerance phenotypic variation of Ler/Cvi RILs. The QTL1 collocate a Cu-exporting ATPase, HMA5, which had been characterized its critical function of Cu-tolerance by reverse-genetics approach by Salk-Institute group. Complementation test by crossing between T-DNA insertion mutant and parental lines can fit the additive effect of the QTL1. These results indicate HMA5 is QTL gene for Cu-tolerance. The QTL1 interact lower arm of the chromosome 5 that includes several candidate genes that can functionally interact with HMA5.

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Arabidopsis thaliana metal tolerance protein 1 (AtMTP1, 43 kDa) is a member of the cation diffusion facilitator (CDF) family. We previously reported that AtMTP1 was localized in vacuolar membranes and played a key role for Zn tolerance. Heterologous expression of AtMTP1 in a Zn-sensitive zrc1cot1 double mutant strain of Saccharomyces cerevisiae complemented the zinc tolerance in the strain. Vacuolar membrane vesicles prepared from yeast cells of AtMTP1/zrc1cot1 actively incorporated ⁶⁵Zn²⁺ in a pH gradient dependent manner, indicating that AtMTP1 functions as a Zn²⁺/H⁺ exchanger. However, it is unclear that how AtMTP1 senses zinc concentration and transports zinc ions from the cytosol, where the Zn²⁺ concentration is is maintained at a very low level. AtMTP1 contains a long His-rich loop between transmembrane domain IV and V. Analysis by site-directed mutagenesis revealed that the His-rich loop is necessary for zinc transport and that several resides are essential.

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The OsNAC6 gene is a member of the NAC transcription factor gene family in rice. Expression of OsNAC6 is induced by abiotic stress including cold, drought, and high salinity. OsNAC6 gene expression is also induced by wounding and blast disease. Both transgenic Arabidopsis and rice plants overexpressing OsNAC6 exhibited growth retardation. The results of microarray analysis revealed that many abiotic- and biotic-stress-inducible genes were upregulated in Arabidopsis and rice plants overexpressing OsNAC6. A transient transactivation assay showed that OsNAC6 activates the expression of at least three genes including a gene encoding peroxidase. The transgenic rice plants overexpressing OsNAC6 not only showed an improved tolerance to dehydration and high-salt stresses, they also exhibited increased tolerance to blast disease. By utilizing stress-inducible promoters, such as the OsNAC6 promoter, it is hoped that inducible overexpression of OsNAC6 in rice can improve stress tolerance without growth retardation.

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RSOsPR10 (Root Specific Oryza sativa PR 10) was originally found as the root specific protein induced by drought and salt treatment in rice. The mRNA was induced specifically in roots by drought, salt, probenazole, rice blast fungus and jasmonic acid (JA), but not by low temperature, abscisic acid and salicylic acid (SA) treatment (Hashimoto et al., PCP, 2004). In this study, we confirmed that the expression of RSOsPR10 by salt was induced vie JA and that the induction was suppressed by SA using hebiba, JA-deficient mutant. Moreover, we analyzed the localization of RSOsPR10 in rice seedlings by immunohistochemical approach. The protein was detected in the cortex cells that surrounded vascular bundle in the root after salt and JA treatment, but was not detected in the shoot. We also confirmed the suppressive effect of SA on the RSOsPR10 induction by salt or JA treatment in tissue level.

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In higher plants, various Z,E-mixed polyisoprenoids, including dolichol and polyprenol, are biosynthesized. However, physiological function of Z,E-mixed polyisoprenoids in higher plants is hardly elucidated. To understand the physiological roles of Z,E-mixed polyisoprenoids, we characterized Arabidopsis thaliana cis-prenyltransferases (AtCPTs) which catalyze the formation of the basic backbone of Z,E-mixed polyisoprenoids. Among AtCPTs analyzed so far, AtCPT5 showed a novel medium-chain CPT activity, catalyzing the formation of C_30-35. In addition, AtCPT5 were induced in response to low temperature and hyper osmotic stress.
To reveal the roles of AtCPT5 in the stress responses, transgenic Arabidopsis lines with high expression of AtCPT5 driven by CaMV 35S promoter were prepared. Their root growths were repressed with increasing of the levels of AtCPT5 activity. Investigating the response to various abiotic stresses, such as low temperature, high salinity, high osmolality and abscisic acid treatment revealed the 35S:AtCPT5 transgenic lines acquired a tolerance to high osmolality.

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Regulation of ABA biosynthesis is crucial for plant's adaptive responses to osmotic stresses. Arabidopsis 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3) is known to be the rate-limiting enzyme in ABA biosynthesis under drought stress conditions. However, regulatory mechanisms underlying drought induction of NCED3 is still unknown. In the present study, we investigated the expression and localization of AtNCED3, AtABA2 and AAO3 in turgid and dehydrated plants using specific antibodies against each enzyme. Western blotting showed that AtABA2 and AAO3 were expressed in turgid plants, whereas AtNCED3 was undetectable. Induction of AtNCED3 was observed in rosette leaves after 30 min of dehydration. The expression patterns of ABA biosynthetic enzymes were investigated by an immunohistochemical technique. In dehydrated leaves, the immunofluorescent signal for AtNCED3 was observed in vascular bundles. In addition, AtABA2 and AAO3 were also localized in the identical tissues. These results indicate that drought induced ABA production occurs in vascular systems.
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SAPKs, rice SnRK2 protein kinases, are rapidly activated by hyperosmotic stress and ABA. A rice cDNA library was screened by the yeast two-hybrid method for SAPK-interacting proteins. One of the positive clones encoded a phosphatidylinositol 4-kinase-ß (PI4K-ß). Because phosphoinositides are implicated in the signal transduction of hyperosmotic stress and ABA, we chose this clone for further analysis. A GST-fusion protein with a fragment of PI4K-ß was found to be phosphorylated by SAPK2 in vitro. We constructed an Arabidospsis double knockout line with T-DNA insertions into the two genes highly homologous to the rice PI4K-ß clone to investigate the in vivo functions of PI4K-ßs. The double knockout line exhibited a dwarf phenotype and osmotic strength-dependent abnormalities in cell elongation/expansion of the root cortex and epidermal cells including root hairs. These results suggested possible involvement of PI4K-ß in the signal transduction of hyperosmotic stress. Further characterization of this mutant is being performed.

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Plants perceive gradients in air moisture by their roots and elongate them toward water sources to effectively obtain a limited amount of water in soil (hydrotropism). A simple bioassay with an air-moisture gradient was developed to examine hydrotropic responses of roots in Arabidopsis thaliana seedlings. In this assay, hydrotropic responses of the mutants defective in AKT2 gene, which encodes a voltage-gated K⁺ channel, were slower than those of wild type. QRT-PCR showed that the expression of AKT2 in roots was increased by dehydration stress. Application of abscisic acid (ABA) to seedlings increased the AKT2 expression, whereas ABA inhibitors reduced it. Studies with ABA mutants also suggest that ABA participates in signaling response to hydrotropic stimulus. In addition, the expression of AKT2 gene in the elongation zone of roots was examined to clarify the role for AKT2 and ABA in hydrotropism.

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It was shown that desiccation tolerance of M. polymorpha cells is increased by preculture in medium with 0.5M sucrose, and this effect of sucrose is partly replaced by the same concentration of mannitol. M. polymorpha cells were desiccated on silica gel and water content of the cells decreased to 0.1g/gDW after 24hrs of desiccation. In non-precultured cells, no survival of cells was observed after desiccation. However, approximately 100% and 20% of survival were observed in the cells precultured with 0.5M sucrose and 0.5M mannitol, respectively. Sucrose content of precultured cells was about four times higher than that of non-precultured cells. When the cells precultured with 0.5M mannitol were washed with medium containing 0.5M sucrose and then desiccated for 24hrs, the survival of cells was almost 100%. These results suggest that intra- and extracellular sucrose act an important role on the development of desiccation tolerance in M. polymorpha cells.

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Since betaine is synthesized in chloroplast, its transport among plants would be important. However, the molecular mechanisms of betaine transport in plants are largely unknown. Here, we isolated betaine transporter from a halotolerant cyanobacterium Aphanothece halophytica (ApBetT) and its properties were compared with those of mangrove Avicenia marina (AmBetT). It was found that ApBetT specifically catalyzed the transport of betaine. Sodium markedly enhanced betaine uptake rates whereas potassium and other cations showed no effect, suggesting that ApBetT is a Na⁺-betaine symporter whereas AmBetT is a H⁺-betaine symporter. pH dependence of betaine uptake activities of ApBetT were high at alkaline pH whereas those of AmBetT were high at low pH. The fresh water cyanobacterium, Synechococcus cells overexpressing ApBetT showed NaCl-activated betaine uptake activities with enhanced salt tolerance allowing growth in sea water supplemented with betaine. Based on these data, physiological role of ApBetT and AmBetT will be discussed.

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Betaine is an important osmoprotectant, and synthesized by many plants in response to abiotic stresses. Almost all known biosynthetic pathways of betaine are two-step oxidations of choline, recently a novel biosynthetic pathway of betaine from glycine, catalyzed by two N-methyltransferase enzymes, was found in a halotolerant cyanobacterium Aphanothece halophytica. Recent studies suggest that for the accumulation of large amount of betaine, supply of precursors such as choline, ethanolamine, and serine as well as choline transport across chloroplast envelop are important. Here, the potential role of serine supply for betaine synthesis was examined in Arabidopsis plants. Arabidopsis plants expressing two N-methyltransferase genes accumulated betaine to a high level in roots, stems, leaves, and flowers, and improved the seed yield under stress conditions. Effects of precursor supply on the accumulation of betaine were examined and their results will be presented.

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The cell wall is important for maintaining cell shape and rigidity. Although the structural framework of the cell wall consists largely polysaccharides, proteins may also participated in networks of the wall. In order to understand the function of the cell wall proteins, we identified dark-grown hypocotyl of black gram (Vigna mungo L.) cell wall proteins. The cell wall proteins were extracted with 3 M LiCl (pH 6.8) and applied to two-dimensional gel electrophoresis. Five protein spots were analyzed for NH2-terminal amino acid residues and 10 amino acid residues were determined. Based on the residues information, we have cloned and sequenced the genes of two of them (P1 and P5). P1 showed homology with unknown protein of Medicago sativa and P5 with curculin-like (mannose-binding) lectin of Medicago truncatula. The functional roles of P1 and P2 in wall rigidity or cell elongation are made investigation.

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Stress-extension rate (SER) characteristics of frozen-thawed segments from the elongation zone of inflorescence stems of Arabidopsis thaliana measured with the programmable creep meter (PCM) by the ramped sweep method. The SER curves were essentially the same as those of the hypocoytls of cowpea, soybean and cucumber although the maximal rupture tension and extension rate were low (around 16 gf at pH 6.8) and high (up to 25 % h^-1 at pH 4.8), respectively. The curve depended strongly on pH indicating that the extension of Arabi cell walls were also facilitated by acids. However, we found that the Lockhart approximation to the Arabi SER-curve was not effective. We defined the differential extensibility and the apparent yield tension and developed an algorism for calculating them. The wall yielding properties of Arabi cell walls were found to be adequately characterized with these new parameters that closely depended on tension.

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α-L-Arabinofuranosidase is an enzyme that is able to hydrolyze arabinosyl residues from pectin or hemicellulose. The loss of arabinosyl residues from cell wall polysaccharides was observed in many kinds of fruit and it seems to be related for fruit softening or textual changes. It is also reported that the existence of arabinosyl residues in cell wall polysaccharides affected the adhesion of each cell.
In tomato fruit, α-L-arabinofuranosidase activity was detected both ripened and developing fruit. α-L-Arabinofuranosidases purified from higher plants were classified in glycoside hydrolase family 3 or 51. Family 3 α-L-arabinofuranosidases seem to be bi-functional enzyme with β-xylosidase activity. Therefore, this enzyme may act against the release of xylosyl residues in vivo in addition to arabinofuranosidase activity. However, the role of the enzyme is unclear. We cloned α-L-arabinofuranosidase cDNAs exhibiting different expression pattern. Substrate specificities of each clone will be discussed.

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Recent studies have established that boron in plant cells occurs as the borate-rhamnogalacturonan II (RG-II) complex, but the physiological function of the complex has yet to be determined. 3-Deoxy-D-manno-oct-2-ulosonic acid (KDO) is a specific component monosaccharide of RG-II. Mutant plants lacking KDO should have altered RG-II structure and will be useful to study the function of B-RG-II complex. To obtain such a mutant we have been characterizing the KDO biosynthesis in Arabidopsis.
KDO cytidylyltransferase (CMP-KDO synthetase; CKS, EC 2.7.7.38) activates KDO prior to its incorporation into RG-II. Arabidopsis has one copy of CKS (At1g53000). With the three independent T-DNA insertion lines examined, no homozygous individuals were found and progeny from heterozygotes showed a 1:1 segregation ratio. When heterozygous males were crossed with wild-type females, cks mutation was not transmitted. These results indicate that cks mutation impairs pollen function and suggest that B-RG-II is essential for reproduction.

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Intercellular attachment is an essential process in the morphogenesis of multicellular organisms. Little is known about the biosynthesis and assembly of pectins in cell walls. nolac-H18 mutant, which is defective in intercellular attachment and in glucuronic acid of pectin, showed drastic reduction of the formation of borate cross-linking of rhamnogalacturonan II (RG-II). The gene tagged with T-DNA, named NpGUT1 (pectin-glucuronyltransferase1), encodes a glucuronyltransferase and was predominantly expressed in apical meristems. Transcript levels of NpGUT1 during the cell cycle were examined with synchronized tobacco BY-2 suspension cultured cells. The accumulation of NpGUT1 mRNA showed a single peak in phase during the cell cycle. The dexamethasone-induced expression of the NpGUT1 antisense gene resulted in the aberration of cell shape and cell division. These results indicate that NpGUT1 is important for the cytokinesis and cell cycle.

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The enzymatic activity and the gene expression of pectin methylesterase (PME) in Populus 0-B cells which can grow in the medium without boron were examined. The activity of PME was higher than that of 1/1-B cells which required enough boron (100 μM), as well as 1/20-B cells tolerant to low boron condition (5 μM). However changes in the activity were different from 1/20-B. The activity decreased until 6 days after subculture and then increased. The gene expression of PME was similar to 1/20-B. Namely a large amount of PME mRNA was accumulated in 8-10 days and gene expression was observed until the later stage of culture period in which no gene expression was observed in 1/1-B. From these results, it was indicated that the induction mechanism of PME as the adaptation to the boron deficiency was present in 0-B as well as 1/20-B.

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Plant cell walls are dynamic structures that involved in the morphology, growth, and development of cells and tissues. We produced the mutants that were abnormal in intercellular attachment by activation tagging. We introduced pPCVICEn4HPT vector into Nicotiana tabacum and obtained seven mutant lines that had defective and fragile organs. We named them shoolac (shoot with loosely attached cells). For investigation of variation of shoolac1 we isolated cell walls, fractionated them to pectin and hemicellulose fractions and subjected each fraction to sugar composition analysis. The arabinose contents of 4M KOH-soluble hemicellulosic fractions of shoolac1 were higher than that of wild one, 4M KOH-soluble hemicellulosic fractions were digested with xyloglucan-specific endoglucanase and the released xyloglucan oligosaccharides were analyzed by LC-MS and ¹H NMR spectroscopy. Shoolac1 xyloglucan had almost two times higher arabinose content than the wild type. This result indicates that increase in arabinose content in xyloglucan affects cell adhesion.

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Using T-DNA tagging method with leaf disk culture of haploid Nicotiana plumbaginifolia, we have produced some nolac (non-organogenic callus with loosely attached cells) mutants that showed the weak intercellular attachment and lost the ability to form organs. The gene tagged with the T-DNA in nolac-K4 mutant, named NpLRX1 (LRR-EXTENSIN 1), is a novel tobacco gene that encodes a cell-wall protein containing chimeric leucine-rich repeat (LRR) and extensin domains. Tobacco leaf disks transformed with NpLRX1-RNAi construct displayed aberrant adventitious buds and disorganized cell morphogenesis with large intercellular spaces. The shapes of NpLRX1-RNAi-transformed BY-2 cells were irregular, and the arrangement of cortical microtubules was clearly aberrant in these cells. When the LRR domain of NpLRX1 was expressed in BY-2 cells or leaf disks, the similar phenotypes were observed. NpLRX1 may affect the dynamics of the cytoskeleton and have a role in the communication between cytosol and cell wall mediated by LRR domain.

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In Spirogyra, terminal cells differentiate to be rhizoids. Rhizoids are formed by repeated branching. In the presence of oryzalin, a microtubules depolymerizing drug, spherical rhizoids are formed. DCB, a cellulose synthesis inhibitor, caused burst at the tip of rhizoids. Synthesis of callose was not inhibited by DCB. This result indicates that cellulose synthesis is necessary for rhizoid elongation. Next, we observed the cellulose microfibrils. In the inner surface of the rhizoid, bundled microfibrils were observed, and those orientations were independent of microtubules. In the swelled rhizoid caused by oryzalin, bundled microfibrils were also observed. On the other hand, in the tip of the rhizoid, the microfibrils oriented to the same direction of as that of microtubules. We suggest that orientation of cellulose microfibrils at the tip region has a vital role in rhizoid morphogenesis.

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Previously we reported that CWPO-C (cationic cell-wall-peroxidase) from poplar oxidizes sinapyl alcohol and polymeric substrates unlike other plant peroxidases. The characteristics of CWPO-C indicate that the catalytic mechanism is quite different from other peroxidases.
In this study, we investigated the catalytic mechanism of CWPO-C by the homology modeling and chemical modification technique. The chemical modification toward Tyr decreased guaiacol oxidation ability of CWPO-C, whereas it did not affect that of HRP. Interestingly, the Tyr modification for CWPO-C strongly decreased the oxidation of syringaldazine and 2,6-dimethoxyphenol rather than the guaiacol oxidation. Furthermore, TNM-modified CWPO-C did not oxidize ferrocytochrome c. These results indicate that Tyr residue mediates the oxidation of macromolecule substrates. The homology modeling shows that Tyr177 and Tyr74 locate near the heme and are exposed on the protein surface among four Tyr residues in CWPO-C. These results suggest that Tyr177 and/or Tyr74 residue is the active site of CWPO-C.

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Secondary cell walls in fibers and tracheary elements constitute the most abundant biomass on the land. While a number of genes involved in the biosynthesis of secondary cell wall components have been identified so far, little is known about the molecular mechanisms underlying the coordinated expression of these genes. Here, we identified the Arabidopsis NAC domain protein, SND1 (for secondary wall–associated NAC domain protein), which is a key transcriptional activator regulating secondary cell wall synthesis in fibers. SND1 is expressed specifically in interfascicular fibers and xylary fibers in stems and dominant repression of SND1 causes a drastic reduction in the secondary wall thickening of fibers. Ectopic overexpression of SND1 results in activation of the expression of secondary cell wall biosynthetic genes, leading to massive deposition of secondary cell walls in non-fiber cells.

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Polysaccharide-linked hydroxycinnamoyl esters (PHEs) are accumulated in cell walls of cortical fibers and vascular cells in rice. PHEs are thought to be very important cell wall components, which strengthen the cell wall and prevent pathogen attack. We found that a rice dwarf mutant Fukei71 (d50) abnormally accumulated PHEs in cell walls of irregular shaped-internode parenchyma cells. The genetic approach revealed that D50 gene encodes an inositol polyphosphate 5-phosphatase (IP5P). Expression pattern of D50 gene was examined by RT-PCR and promoter analysis. RT-PCR showed that D50 is expressed in roots, leaf blades, leaf sheaths, stems, and flowers at almost the same levels. Promoter-GUS analysis confirmed the expression of D50 mainly in meristematic tissues of young internodes. Currently we are determining the intracellular localization of the D50 protein by particle bombardment with CaMV35S::D50::YFP construct.

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Recently, the molecular mechanism of photoperiodic flowering pathway of rice is just now uncovering. Under short day condition, induction of flowering requires the gene expression of OsGI, Hd1 , Ehd1 and Hd3a.
But this knowledge is study result of some representative cultivars. Cultivated rice, which is widely distributed in the world, shows a lot of diversity among the same species. Therefore this study focused on the natural variation of photoperiodic flowering pathway in rice.
To investigate natural variation in rice, we investigated the expression level of flowering time genes and flowering time of the core collection. The results showed that strong correlation was observed between the levels of Hd3a expression and the flowering time. The same analysis using representative cultivars also suggests that variation of flowering time genes causes diversity of flowering time. The relationship between the diversity in flowering time and the function of these genes will be discussed.

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Rice (SDP) has the three key regulators of flowering time, OsGI, Hd1 and Hd3a have been shown to play important roles in the photoperiodic control of flowering in rice. Overexpression of Hd3a in rice was shown early flowering phenotype, therefore Hd3a is considered as switch gene of induction of the flowering. Although Hd3a encodes a protein with sequence similarity to the PEBP (phosphatidylethanolamine-binding protein) protein, Hd3a biochemical function is unknown. We have obtained transgenic rice expressing Hd3a :: gus and found that GUS activity was detected in an area surrounding the vascular tissue in the leaf. Hence, we introduced Hd3a cDNA fragment and Hd3a:GFP fusion cDNA into rice under the control of rolC, Cab and RPP16 promoter. The transgenic plants show early flowering phenotype. These results suggest that the expression of Hd3a in the leaf and cells surrounding the vascular tissue might be sufficient to promote the induction of flowering.

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Rice Flowering Locus T 1 (RFT1) is the closest homologue of Heading date 3a (Hd3a) gene, which promotes floral transition in short-day (SD) of rice. Double RFT1-Hd3a RNAi plants did not flower even at 300 days after sowing, suggesting that RFT1 is a novel regulator of photoperiodic flowering in rice. RFT1 expression was very low in wild-type plants, however, in Hd3a RNAi plants, a marked increase of RFT1 expression was found at the late stage and complemented the lack of Hd3a expression, promoting flowering. Furthermore, we found that changes in chromatin modification at the 5'UTR of RFT1 locus were observed when RFT1 expression was increased in Hd3a RNAi plants. These results indicate that photoperiodic flowering of rice in SD is regulated by the intergenic communication between Hd3a and RFT1 through chromatin modifications.

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We performed the night-break (NB) experiments and carried out molecular analysis of flowering time genes in rice. NB caused delay of flowering by the down-regulation of Hd3a without affecting OsGI or Hd1 expression pattern, and phyB transduced NB signal to down-regulate Hd3a and inhibit flowering in rice (Ishikawa et al 2005). To understand roles of phytochrome in rice flowering in detail, we examined phytochrome mutant of rice using monochromatic light. NB effect induced by red light on Hd3a is dependent on the photon fluence and reversed by subsequent far-red light irradiation. We also analyzed Hd1 over-expressing rice (Hd1-ox) and found thatHd1-ox caused delay of flowering in SD with down-regulation of Hd3a. In contrast, when Hd1 was overexpressed in phyB mutant, flowering was promoted. These results suggest that phyB dependent signal modulates the activity of Hd1 in rice flowering. Roles of phytochrome on rice flowering will be discussed.

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In Arabidopsis, phyB, cry2 and phyA are important photoreceptors for flowering regulation. Recent reports have demonstrated that these photoreceptors regulate stability of CO protein and FT gene expression. However, how these photoreceptors regulate FT expression is still unclear.
We found that a T-DNA insertion line (SALK) exhibited dramatic late flowering phenotype under continuous white light and named it as the tdu mutant. The tdu mutant showed low FT expression and late flowering phenotype, although CO expression was normal. Analyses of several alleles, overexpressors and silencing lines demonstrated that the gene into which T-DNA was inserted was indeed the causal gene. The TDU gene encodes a novel nuclear protein containing glutamine-rich region, which is often seen in transcription activators. Homologues of TDU are found in other plant species. Moreover, analyses of hypocotyl elongation, flowering time under monochromatic light and genetic analyses suggested that TDU functions downstream of cry2.

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In Arabidopsis, information from various pathways is integrated at the transcriptional regulation of floral pathway integrator genes including FT. FT encodes a protein of the PEBP/RKIP family. FT functions interdependently with another flowering regulator gene FD, which encodes a bZIP transcription factor and is expressed in shoot apex. FT is expressed in vascular tissues of cotyledons and leaves, however, when expressed in the shoot apex, FT can complement ft loss-of-function mutant phenotype. We demonstrated that FT and FD proteins interact in nucleus in transgenic Arabidopsis plants by BiFC. Furthermore, flowering of 35S::FT:GR transgenic plant was dependently promoted in dexamethasone. These results suggest that FT acts in the nucleus with FD at the shoot apex to promote flowering. We are currently screening genes of which expression is regulated by FT by microarray analysis.

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Dominant late-flowering mutant fwa is an epigenetic mutant that ectopically expresses FWA due to promoter hypomethylation. To gain clues to the molecular basis of late-flowering phenotype in fwa, we performed microarray analysis using fwa epiallele and 35S::FWA. The transcription profile suggests that late-flowering phenotype of fwa is not due to the mis-regulation of transcription. Yeast two-hybrid analysis and in vitro pull-down assay showed that FWA protein can specifically interact with FT protein. These results suggest that ectopically-expressed FWA inhibits floral transition by interfering with the FT function through protein interaction. fwa-101D had no effect on the precocious-flowering phenotype of 35S::TSF, 35S::CiFT, and 35S::Hd3a. Therefore, effect of FWA protein seems very specific to FT. No interaction was observed between FWA protein and TSF protein. Specific interaction of FWA with FT enabled us to analyze FT protein function through specific inhibition.

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Photoperiodic induction of flowering requires light perception in leaves, followed by transmission of mobile signal from leaves to the apex, where floral meristems are initiated.
In Arabidopsis, floral pathway integrator FT acts mainly in the photoperiod pathway through transcriptional regulation by CONSTANS. FT encodes a 20kD protein of the PEBP/RKIP family and is expressed in vasculature of cotyledon and leaf where light is perceived. Recently, it was reported that FT mRNA is transported from leaf to the apex.
To examine whether the effect of FT to promote flowering is transmissible across long distance, we performed micrografting experiments. Using 4-day-old seedlings, two-shoot, 'Y-shaped' grafts were assembled on hypocotyls of ft-1 recipient. We observed that flowering of ft-1 recipients was accelerated by grafting scion with functional FT (either endogenous gene or transgene). Whether FT promotes its own long-distance action is currently being investigated.

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AtC401 was isolated as an Arabidopsis homologue of the Pharbitis nil C401 (PnC401) that is a gene of which encodes a leaf protein closely related to photoperiodic induction of flowering, and shows a circadian rhythm at the transcriptional level. AtC401 consists of a kinase domain in the N-terminal half and 12 times of pentatricopeptide repeat (PPR) in the C-terminal half. AtC401 protein physically interacts with CONSTANS, through PPR motif, in Yeast. To investigate the function of AtC401, atc401 mutant, AtC401-overexpressor(OX), double mutant co-1/atc401, CO-OX/atc401, CO-OX/AtC401-OX, and co-1/AtC401-OX were made, and analyzed the flowering time. Although AtC401-OX showed almost no difference compared to wild type Columbia, and CO-OX showed early flowering, CO-OX/atc401, and CO-OX/AtC401-OX showed late flowering phenotype. These results suggest that AtC401 functions as a floral regulator, by associating with CONSTANS.

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In Arabidopsis thaliana, CONSTANS (CO) plays a central role in photoperiodic flowering through positively regulating the floral switch gene, FT. We first identified two cis-elements required for CO activation in the FT promoter by transient assays of Arabidopsis seedlings. We further identified a 10bp essential core consensus sequence as the true target of CO from the 21bp element II, one of the identified cis-elements, using its derivatives with scanning mutations. We next found several genes which have more than two consensus sequences in their promoters and exhibited distinct expression patterns between co mutant and the wild type in the public Arabidopsis microarray data. Finally, we next verified them as downstream genes of CO by transient analysis. The Hd1 gene, a rice ortholog of CO, can activate FT promoter in this transient assay. Therefore, we are now investigating on the cis-element recognition between CO and Hd1 in the transient analysis.

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In Arabidopsis, LKP protein family consists of three members, LKP1/ZTL, LKP2 and FKF1, and is known to be involved in the regulation of circadian clock, flowering time and hypocotyl elongation. These proteins contain three functional domains, LOV domain, F-box motif and kelch repeats. The mutation of FKF1 gene results in late flowering and decreased level of CO mRNA. Recent studies have shown that FKF1 is involved in the regulation of flowering time by degradation of CDF1 protein, a negative regulator of CO gene expression. Overexpression of LKP1/ZTL and LKP2 resulted in delayed flowering under LD condition, although both ztl and lkp2 plants flowered normally. LKP2 and CO proteins were shown to be co-localized in the nuclei, and an yeast two-hybrid analysis demonstrated that the F-box motif and the kelch repeats in LKP2 interacted with CO. Here, we report the flowering time of transgenic plants that overexpress LKP2 fused with GFP.

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Recent studies revealed that endocytosis is involved in the many plant functions of higher order, although detailed molecular mechanisms of endocytosis remains almost unknown. In Arabidopsis, many Rab GTPases including Rab5 members, Ara7, Rha1 and Ara6, regulate endocytosis. The SNARE is another key player in the endocytic traffic. AtVAM3/SYP22 encodes a SNARE protein localized both on the vacuolar membrane and pre-vacuolar compartments. atvam3/syp22 shows wide spectrum of phenotypes including wavy leaves, semi-dwarfism, excessive differentiation of myrosin cells and late flowering. We have reported that ara7 and rha1 enhance the phenotypes of atvam3/syp22, whereas ara6 suppresses them. This suggests that the wide range of phenotypes observed in atvam3/syp22 is caused by the defect in the endocytic pathway. In this meeting, we will present our recent results on the relationship between the endocytic pathway and regulation of flowering, which are suggested by the detailed analysis of the late flowering phenotype of atvam3/syp22.

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An Arabidopsis mutants yellow variegated 1 (var1) and var2 exhibit a leaf-variegated phenotype. The responsible genes encode different FtsH metalloproteases (FtsH5 and FtsH2, respectively) involved in the quality control of photosystems. To understand the mechanisms leading to leaf variegation, we isolated the suppressor of var2. Interestingly, it also suppresses the variegation of var1 and var1/var2. The mutation was found in a gene encoding a chloroplastic translation initiation factor 2 (cpIF2). We showed that this protein indeed acts as a cpIF2 in vivo. The suppression of leaf variegation caused by the loss of FtsHs was most likely attributed to reduced protein synthesis in chloroplasts. This hypothesis was further supported by the observation that chloroplast translation elongation factor G mutant also suppressed the leaf variegation of var2. Based on these results, we propose that the balance between protein synthesis and degradation in chloroplasts is crucial for variegation formation.

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An Arabidopsis leaf-variegated mutant var2 results from the loss of FtsH2, an ATP-dependent metalloprotease in thylakoid membranes. Here, we report physiological and biochemical characterization of the white sectors of var2. We successfully visualized plastids in the white sectors by expressing chloroplast-targeted GFP, suggesting that those plastids retain protein transport machinery. Observation of plastid DNAs by a DNA-specific fluorescent dye DAPI showed that plastid DNAs in the white tissues were organized as nucleoids typically detected in undifferentiated plastids. RT-PCR and immuno-blot analyses also showed that chloroplast genes as well as nuclear genes were expressed in the white tissues, although the levels of photosynthetic proteins appeared to decrease. These results collectively suggested that the white sector is an active tissue formed by viable cells. We currently examine if there are any changes in protein composition between the white and green sectors by proteomic analysis.

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Chloroplast is an organelle that is important for growth of plants. In this study, to understand the molecular mechanism of the chloroplast development, we identified and characterized a variegated mutant of Arabidopsis thaliana, 5-31. From this mutant, two types of cotyledon, albino like and wild like ones are observed. The former died before bolting because of its inability of photosynthesis. The latter developed variegated true leaves and grew slower than wild. In the variegated leaf, the number of chloroplasts was greatly reduced. But when the mutant grows in the media supplemented with 0.3% sucrose, it's observed to increase the number of chloroplast in the white sector, resulting in a mild variegated phenotype. The recovery of the number of chloroplasts was more obvious when it grows on a 1% sucrose media. This result shows that chloroplast development in the white sector is influenced with the concentration of sugar in the cell.

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The flexibility of the thylakoid membrane of Arabidopsis thaliana during growth was reported at this meeting last year. Surprisingly, the reorganized "curly structure" found was subsequently observed in a large variety of land plants. In order to understand the functional differences between the original "linear" and the reorganized "curly" thylakoids, chlorophyll content, photosynthetic efficiency and starch content of leaves were examined. The chlorophyll content in the latter was rather higher than in the former and the a/b ratio was less in the latter. No definite differences in the CO₂ assimilation rate or the parameter values of Fv/Fm, qp, ΦII and NPQ were found. However, the chloroplast with "linear thylakoid" accumulates much more starch than that with "curly thylakoid". Concluding we propose that mature chloroplasts in leaves of land plants occur in two forms and may change from one form to the other.

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It is not yet entirely clear how grana stacks are formed, and several models had been proposed, such as the disk piled model, the helical model, and so on. The Arabidopsis mutant pcb2, which is deficient in divinyl protochlorophyllide 8-vinyl reductase, not only accumulates divinyl chlorophylls instead of chlorophylls but also lacks distinctive grana stacks. In general, LHC II complexes are believed to be involved in the development of the grana stacks, and the LHC II complexes in the pcb2 have an abnormality. This abnormality may be explained by the altered chlorophyll-protein interaction caused by the difference of molecular structures between divinyl chlorophyll and normal chlorophyll. In addition, the decreased number of granal layers and their abnormal radius in pcb2 provided new insight to the mechanism that maintains the cylindrical shape of grana stacks. We discuss a new model of grana stack formation.

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To study the functions of novel nuclear-genes involved in chloroplast development and photosynthesis, we systematically analyzed a series of albino or pale green(apg )mutants screened from Ds-transposon tagged lines in Arabidopsis thaliana. In this study, we focused on the apg11 and apg12. The amino acid sequence of APG11 showed 51% identity and 65% similarity with the APG12 in the C-terminal 100 amino acid region. The chloroplast of apg11 contained few internal thylakoid membranes, and the chloroplast proteins related to photosynthesis were not detected in by immunoblot analysis. The N-terminal amino acid region of APG11 and APG12 were predicted to be a transit peptide targeting chloroplast . When the p35S::APG12Tp-GFP chimeric gene introduced into the epidermal cells of A. thaliana leaves by particle bombardment, GFP fluorescence signals were observed in the chloroplast. Those results suggest that the APG11 and APG12 are essential for chloroplast development and thylakoid biogenesis.

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Nuclear-encoded genes related to the plastid-structure/function (e.g. Lhcb, RbcS) were coordinately regulated with the functional status of plastids. An unknown signal, called "plastid signal", has been hypothesized for this regulatory mechanism. Analysis of gun mutants that have defects in this signaling has revealed the involvement of the tetrapyrrole intermediates (MgProtoIX) as a signaling factor. However, the accumulation of MgProtoXI and MgProtoIXMe in chlM and crd1 mutants respectively does not repress Lhcb expression. This suggests that MgProtoIX(Me) level does not simply account for the regulation of Lhcb.
MgProtoIX synthesis is catalyzed by Mg-chelatase which is a multi-protein complex consists of ChlH(Gun5)-ChlD-ChlI and Gun4. Recently Gun5 is reported to function as the abscisic acid (ABA) receptor. Detailed analysis of plastid-signal and ABA-related phenotype of Mg-chelatase mutants and other tetrapyrrole mutants will be reported. We will also present the characterization of newly isolated gun mutants.

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It is generally believed that plastid evolved from the endosymbiosis of a unicellular photosynthetic bacterium into an ancient eukaryotic cell. In the present-day plastid genome encodes fewer than 100 ORFs, and most genes that were encoded by the bacterial ancestor have been moved to host nuclear genome. Therefore, the nucleus controls most aspects of plastid gene expression. On the other hand, it has been known that plastid send signals to the nucleus to control nuclear gene expression. However, little is known about the signaling components that regulate the expression of nuclear genes. To elucidate the signaling pathway from plastid to the nucleus, we took advantage of a mutant defective in the major protein import receptor. In this mutant, ppi2, the expression of nuclear genes encoding plastid proteins is compromised. We performed detailed expression analysis using ppi2 mutant. We show that a specific pathway was affected in the ppi2 mutant.

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The rice virescent-1 (v₁) mutant is temperature-conditional and develops chloroplast-deficient leaves under the restrictive temperature. Positional cloning and structural analyses indicate that V₁ encodes a 32 kDa protein that accumulates in chloroplast. C-terminal region of V₁ revealed structural similarity to bacterial RNA-binding proteins. V₁ protein accumulated abundantly at early P4, and rapidly decreased inversely as contents of leaf chlorophyll and RubisCO increased drastically, but was barely detectable in mature leaves at P5 and P6 stages. V₁ accumulation is enhanced largely by low-temperature (20°C), and is suppressed by high-temperature (30°C) treatment. In v₁ mutant grown at 20°C, maturation of 16S and 23S rRNA was impaired and precursor rRNAs predominantly accumulated at the P4 leaves. In such v₁ mutant, chloroplast translation/transcription capacity were severely suppressed. These observations suggest that V₁ is involved in the maturation of chloroplast rRNAs activated in the early P4 stage of leaf development.

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