We have reported that Arabidopsis transcription factors DREBs control expression of many stress-responsive genes. Overexpression of DREB1A in transgenic Arabidopsis activated the expression of the target stress-responsive genes and resulted in improved stress tolerance. In rice, we isolated cDNAs for DREB homologues, OsDREBs.
We generated transgenic rice overexpressing the DREB or OsDREB genes. These transgenic rice revealed drought, high-salt and cold stress tolerance and contents of various osmoprotectants in the transgenics are higher than those of control plants. We carried out microarray analysis using the transgenics and identified several target genes of DREB in rice. These results indicate that the DREB gene family is quite useful for improvement of tolerance to environmental stresses in rice.
However, these plants overexpressing DREB genes show growth retardation and dwarf phenotype. To minimize the negative effect on plant growth, we generated transgenic rice overexpressing DREB genes driven by a stress-inducible promoter.

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Regulatory mechanisms of stress responses in rice largely remain unclear, compared to those in Arabidopsis. Using rice microarray, we identified many abiotic stress-responsive genes. Among them, a gene for a bHLH transcription factor repressed expression under drought stress condition. The bHLH protein showed a high sequence homology with Arabidopsis PIF3-like 5 (PIL5), and possessed a PIL motif at N-terminus, driving us to name the transcription factor OsPIL5.
To identify the function of OsPIL5, we generated transgenic Arabidopsis plants overexpressing OsPIL5 and observed their phenotypes. The transgenic plants showed growth promotion and decreased tolerance to drought stress. In contrast, a dominant loss-of-function Arabidopsis mutant of OsPIL5 with a repression domain exhibited growth retardation and increased tolerance to drought stress. It is possible that OsPIL5 functions as an important growth regulatory factor in response to drought stress in rice. Currently, using transgenic rice plants, we are analyzing function of OsPIL5.

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Root tissues of higher plants have advanced adaptive and defensive mechanisms to drought stress, since root development is usually less inhibited than shoot growth under such conditions. Wild watermelon plants (Citrullus lanatus sp. No101117-1) can survive severe drought. In this study, we analyzed drought-responsible proteins in the root tissues of wild watermelon by the proteomic approach. Two-week-old wild watermelon plants were exposed to the drought stress by stopping watering for 3 days and the root extracts were subjected to 2-D gel electrophoresis. In the soluble fraction, the intensities of 66 spots were increased by the stress treatment. Interestingly, most of the proteins that were induced after 1 day of the stress were those predicted to be involved in metabolic pathways and root development. On the other hand, various HSPs and ROS-scavenging enzymes were found in the spots that were treated much longer.

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Drought/strong-light stresses cause an accumulation of citrulline, an efficient hydroxyl radical scavenger, in wild watermelon leaves to 30 mM. To understand the mechanism for its accumulation, we characterized glutamate N-acetyltransferase (GAT) involved in the simultaneous reactions of the first and fifth steps in the citrulline biosynthesis. The 7000-fold-purified GAT enzyme from the watermelon leaves was composed of two different polypeptides. Analysis of the corresponding cDNA cloned revealed that the two polypeptides were derived from the single precursor polypeptide, suggesting that the GAT precursor is autocatalytically cleaved like other GATs from microorganisms. The green fluorescence protein assay revealed that the first 26 amino acid sequence at the N-terminus of the precursor functions as the chloroplast transit peptide. The GAT showed thermostability up to 70^οC and was not inhibited by citrulline and arginine at physiologically-relevant high concentrations unlike Arabidopsis GAT. We discuss contribution of GAT to citrulline accumulation during drought/strong-light stresses.

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Tulip plants require low temperature to initiate stalk elongation and flowering. If tulip bulbs are stored without being exposed to low temperature, their stalk elongation will be inhibited, and flowering will not occur or will be abnormal. When cell solutions extracted from bulbs stored at low and high temperature were analyzed with the psychrometer, it was found that cells of bulbs stored at low temperature contained much more osmotically active solutes than those stored at high temperature.
Water-soluble carbohydrate components were analyzed with UV-MALDI and ESI TOF-MS, while they were extracted with water from scales of tulip bulbs stored at low and high temperature and the bulbs after planting. MS analyses revealed distinctive signals of oligosaccharides derived from starch degradation. Series of oligosaccharides signals were modified by the ratio of adduct cations of Na+ and K,+ depending on temperature treatments and stage of bulb and stalk development.

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It has long been argued whether the permeability, Lp, of Chara, fresh water green algae, is anisotropic (Lp for influx is significantly larger than that for efflux).
In the present report I introduce a non-invasive with high temporal resolution (100msec) technique for measuring the change of turgor pressure in response to the step change of extracelluar osmotic pressure (Ogata 2004 submitted to PCP). Using this technique, it was found that there was no difference in Lps between influx and efflux across the plasma membrane. The apparent rectification in LpÅ@seems to be derived from; 1) the temporal change in concentration of the solute around the surface of membrane, the water motive force is changed; 2) the spatial deviation of the solute number along the length of the cell sap, a counter water motive force should be expected; and 3)the visco-elastic properties of the cell wall.

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Ribulose 1, 5-bisphosphate carboxylase/oxigenase (RuBisCO) is the key enzyme of photosynthetic carbon assimilation. RuBisCO is composed of eight small subunits encoded by nuclear gene rbcS and eight large subunits encoded by chloroplast gene rbcL. To understand the relationship between the expression of rbcS and rbcL genes at different developmental stage in leaves, we generated transgenic tobacco plants harboring antisense rbcS under the control of dexamethasone (DEX) -inducible promoter. In these plants, the amount of endogenous rbcS mRNA can be artificially reduced by induction of antisense rbcS mRNA using DEX treatment. These transgenic plants were cultured in a hydroponic growth medium with or without DEX. In this study, we report the effect of rbcS mRNA reduction on the expression of rbcL gene and the accumulation of RuBisCO in young and mature leaves.

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Phylogenetic analysis of translated rbcL sequences divides ribulose-1,5-bisphasphate carboxylase/oxygenase (RuBisCO) into four clades. Forms I through III include the amino acid residues that are required for catalytic activity of RuBisCO and are capable of catalyzing both the carboxylation and oxygenation of ribulose-1,5-bisphasphate. Form IV lacks the residues involved in binding of the phosphate group on C5 and the functional loop 6 conserved in photosynthetic RuBisCO, and has no carboxylation and oxygenation activity. Form IV is thereby referred to as the RuBisCO-like protein (RLP). We found that RLP from Bacillus subtilis catalyzed the 2,3-diketo-5-methylthiopentyl-1-phosphate enolase reaction in the methionine salvage pathway. We created several mutated RLPs where a few lysine residues essential for RuBisCO and some residues were changed to other amino acid residues. Some mutated RLPs lost the enolase activity. Thus well conserved, catalytic lysines of RuBisCO also play key roles in RLP.

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To characterize photosynthesis in various organs of rice plants, soluble proteins from six different organs were compared by two-dimensional gel electrophoresis. Organs examined were leaf blade (LB), leaf sheath (LS), stem, rachis-branch including rachis (RB), lemma + palea (LP), and root. Either immunoblotting or microsequencing identified 44 spots, which included spots of nine and 14 enzymes involved in the Calvin cycle and carbon/nitrogen metabolism, respectively. Comparison of relative abundances of these spots indicated some unique features in green organs other than LB. The relative abundances of Calvin cycle enzymes in LS differed from those in other green organs, and the level of Rubisco activase was very low in RB. LP and stem shared common features, namely, the abundance of the chloroplastic NADP-ME, the absence of the chloroplastic carbonic anhydrase, and very low levels of the chloroplastic GS2, which could be indications of operation of the C₄-like pathway in these organs.

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A survey of the complete nucleotide sequence of the rice genome revealed the presence of five genes that encode phosphoenolpyruvate carboxylase (PEPC), four (Osppc1-4) of which encode the plant-type PEPC while one (Osppc-b) the bacterial-type. The plant-type genes have homologous exon-intron structures, an indication of their evolution from a common ancestor gene. Expression of each Osppc gene was analyzed by RT-PCR. The results indicate that expression of Osppc1, Osppc3 and Osppc-b was very low in all organ tested, while Osppc2 and Osppc4 were highly expressed in green organs. It was also found that expression of Osppc2 in leaf blade showed diurnal variation while that of Osppc4 was constitutive. Expression analyses of Osppc2 and Osppc4 using the reporter gene are now in progress.

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Phosphoenolpyruvate carboxylase (PEPC) undergoes activity regulation through reversible phosphorylation. Although the phosphorylation was considered to be enhanced by light in the leaves of C₃ plants, we found that the maize PEPC expressed in transgenic rice leaves was phosphorylated at night. The phosphorylation was induced by nitrate addition irrespective of light conditions, and when grown with ammonium as a sole nitrogen source, the extent of phosphorylation did not show clear diurnal oscillation. These results suggest that the PEPC phosphorylation in rice leaves is not under the control of light but tightly correlates with the assimilation of nitrate. Expression analysis of three rice PEPC kinase genes (OsPPCK1-3) indicated that the expression of OsPPCK2 was induced by nitrate while that of OsPPCK3 was upregulated at night. We propose that OsPPCK2 and OsPPCK3 share roles in controlling the PEPC phosphorylation in rice leaves in response to physiological conditions.

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PEPC catalyzes the primary CO₂-fixation reaction in C₄ photosynthesis. Zea mays PEPC (ZmPEPC) is feedback-inhibited by L-malate (MA) in an allosteric manner. Plant enzyme is also regulated by phosphorylation of Ser near the N-terminus by a specific protein kinase (PEPCk), which reduces the sensitivity to MA.
We constructed two mutant PEPCs, ΔN_1-34ZmPEPC, plant specific N-terminus region truncated mutant of ZmPEPC, and +N_1-34EcPEPC, N-terminus region of ZmPEPC attached to E.coli PEPC (EcPEPC). ΔN_1-34ZmPEPC showed reduced sensitivity to MA. Although almost the same amount of ³²P as wild-type ZmPEPC was incorporated into +N_1-34EcPEPC, its sensitivity to MA was not affected by phosphorylation. These results suggest that not only N-terminus but also the other plant-specific sites may be involved in the desensitization of ZmPEPC, followed by phosphorylation. The phosphorylation of wild-type ZmPEPC was inhibited either by ΔN_1-34ZmPEPC or by wild-type EcPEPC. It suggests that PEPCk recognizes some sites of ZmPEPC, besides N-terminus.

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Photosynthetic organisms produce phosphoglycolate by photorespiration (by the oxygenase activity of rubisco) under air. The compound is a strong inhibitor of photosynthesis, and phosphoglycolate phosphatase (PGPase) deficient mutants cannot grow under air, even in Chlamydomonas that has CO₂-concentrating mechanism (CCM) to suppress photorespiration. The activity of Chlamydomonas PGPase (PGP1) increases shortly after transfer to low CO₂, following the increase in the mRNA level, similar to CCM enzymes, although the mechanism of Pgp1 expression is not clear. In order to help define the mechanism, as Pgp1 is likely to play an important role in the regulation of both photorespiration and CCM, we determined and compared the DNA sequences of Pgp1 gene and the 5'-upstream region in both wild-type 2137 and pgp1-1 mutant N142, and also compared the Pgp1 upstream sequence with those of photorespiratory and CCM enzymes. Possible isozymes of PGPase are also discussed based on their gene sequences.

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The cellular content of glycogen and sucrose in Synechococcus elongatus PCC 7942 was determined using an enzymatic method. The amount of glycogen in the cells was progressively increased in the late growth phase (OD₇₃₀ > 2). Sucrose serves as an osmoregulatory compound under high salinity environment in certain cyanobacteria and was not detected when Synechococcus cells were grown in a low-salt medium. The synthesis of sucrose was observed in 30 minutes after the addition of 0.2 M NaCl. The intracellular sucrose content attained to a constant level (35 nmol/ml/OD₇₃₀) in 8 hours. The addition of 0.2 M NaCl abolished the growth of the mutant defective in glycogen accumulation. The sucrose synthesis was observed in the high salt concentration but its rate was slower than in the wild type cells. The result suggested that the polysaccharide accumulated in the cells had the vital role during the growth under high salinity environment.

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The green alga Chlorella contains a storage polysaccharide, starch, which consists of amylose and amylopectin. Effects of various environmental factors during the growth on the intercellular content and structure of starch were investigated. When high CO₂ (3%)-grown cells were transferred to low CO₂ ( 0.04%, air level ) condition, pyrenoid and pirenoid starch developed, and starch content and the ratio of amylose to amylopectin increased. The chain length distribution of amylopectin was also affected by CO₂ concentration during the growth. After NH⁴⁺ addition or in the dark, starch content decreased and the rate of amylose to amylopectin increased. Furthermore, expression of granule-bound starch synthase (GBSS), which is considered to be responsible mainly for the amylose synthesis, was determined. The result suggests that GBSS expression is upregulated at the transcritional level in the low CO₂-condition.

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In search of new genes essential for C4 photosynthesis, we have isolated several C4 plant-abundantly expressed genes by transcriptome analysis between Flaveria trinervia (C4) and F. pringlei (C3). Among them we found out a novel gene, encoding a CP12 domain-containing protein, of which expression is specific in C4 species. Its predicted amino acid sequence was clearly distinct from other CP12 molecules. Therefore we named this gene as FtCP12L. Using specific antibody for FtCP12L, its C4-plant specificity was also confirmed. Comparative expression analyses betweenFtCP12 and FtCP12L revealed that FtCP12L was expressed in both mesophyll cells and bundle sheath cells and its pattern was different from that of FtCP12, suggesting its function being not involved in the Calvin cycle. Now we are trying to detect and to identify the complexes containing FtCP12L for elucidation of its physiological function in C4 plants.

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To understand how the root growth direction changes in response to environmental stimuli, we characterized an Arabidopsis mutant, wav2 and wav3, whose roots show abnormal wavy growth patterns on inclined agar medium.
The roots of the wav2 mutant show the shorter-pitch wavy growth pattern on inclined agar and bent with larger curvature in gravitropic and phototropic responses than those of the wild-type. The cell-file rotations of the wav2-1 root in the wavy growth pattern were enhanced in both right-handed and left-handed rotation. WAV2 encodes a protein with a transmembrane domain and an α/β-hydrolase domain. Expression analyses showed that WAV2 was expressed strongly in adult plant roots and seedlings, especially in the root tip, the cell elongation zone, and the stele. Our results suggest that WAV2 negatively regulates stimulus-induced root bending through inhibition of root tip rotation.
In addition, we introduce recent studies on the wav3 mutant in this meeting.

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An agravitropic mutant of morning glory (Pharbitis nil), weeping, has defect in shoot gravitropism. Our previous results showed that weeping were also defective in endodermis development and in shoot circumnutation. Such defects in circumnutation were also observed in endodermis-less Arabidopsis mutant, scarecrow (scr). In search of the mutated gene of weeping, cDNAs for SCR homolog from morning glory (PnSCR) were cloned from both wild type and weeping. We found an amino acid insertion in PnSCR obtained from weeping. To investigate whether this insertion inactivates the PnSCR functions, we performed complementation test by introducing either wild type PnSCR or weeping type PnSCR, respectively, to Arabidopsis scarecrow mutants. As a result, none of the defects were rescued by weeping type PnSCR, whereas wild type PnSCR rescued them. This result indicates that defects in gravitropism, endodermis development, and circumnutation observed in weeping are attributable to the loss of function of PnSCR.

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In plants, a large part of fatty acids in membrane is comprised from polyunsaturated fatty acids. In this study, protein modification caused by polyunsaturated fatty acid peroxidation products was examined by immunochemical analysis. Results obtained by a model experiment using reaction mixture contained model protein (BSA or Rubisco) and C18 unsaturated fatty acid methyl ester showed that protein modification was most effectively caused by peroxidation of linolenic acid (18:3) and were extremely promoted over 33°C in the presence of a metal-catalyzed oxidation system (Fe³⁺/ascorbate/O₂). Detectable protein adducts were derived from reactive aldehydes such as malondialdehyde, acrolein and crotonaldehyde. When isolated thylakoid membrane was peroxidized at 37°C in the presence of a metal-catalyzed oxidation system, protein modification by malondialdehyde was also detected in intact plants stressed at 40°C under illumination. Thus, protein modification might account for a part of damage of heat stressed plants.

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We have previously reporterted that one of the three dnaK homologues ( dnaK1, dnaK2 and dnaK3 ) in cyanobacterium Synechococcus sp. PCC7942, dnaK2, was found to be induced during heat shock, high light and salt stresses. Primer extension mapping revealed several transcription start site of dnaK2 while no sequence homology with the Escherichia coli sigma-70-like or heat shock promoters was observed. It was suggested that other factors might relate to heat response of the dnaK2 gene.Transcriptional reporter assay using thermostable beta-galactosidase gene, bgaB, revealed that, in addition to the upstream regulatory sequence, the region corresponding to the N-terminal coding sequence was required for substantial expression and heat induction of the dnaK2 gene. Moreover, the 20bp sequence element in the upstream region was highly conserved among cyanobacterial dnaK and this element was also necessary for heat induction of the dnaK2 gene.

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Hsp90 plays important roles in diverse pathways in eukaryote. However, functions of its prokaryotic counterpart HtpG are not understood. In cyanobacteria, we reported previously that HtpG protein increased in response to various stress conditions such as heat, high light, oxidation and iron-deficiency. In addition, htpG null mutant revealed reduced production of phycocyanin and chlorophyll.
Our systematic analysis of protein-protein interactions by using yeast two-hybrid analysis suggested a specific interaction between HtpG and HemE (uroporphyrinogen decarboxyrase). HemE is a key enzyme in that its activity facilitates the porphyrin and chlorophyll metabolism. To analyse the role of HtpG in the HemE activity, the cell lysate of ΔhtpG strain was assayed and compared to that of the wild type strain by measuring the level of uroporphyrin. The results indicated that the enzymatic activity of HemE was higher in the htpG mutant suggesting a negative effect of HtpG on HemE activity.

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Heat stress in plant results in repression of most mRNA translation and the preferential translation of heat shock protein mRNAs. The 5' untranslated region (5'UTR) of HSP mRNA is well known to confer the preferential translation during heat stress. In this study, we showed that the m7G-capped mRNA with 5'UTR of Arabidopsis heat shock protein (HSP81-3) gene was translated efficiently under heat stress condition by using Arabidopsis T87 protoplast transient expression assay. To investigate the function of HSP81-3 5'UTR in more detail, we performed the same transient expression assay using uncapped mRNA or dual luciferase assay vector for measuring IRES activity. As a result, HSP81-3 5'UTR conferred the temperature dependent increase of both translation efficiency of uncapped mRNAs and IRES activity. Thus, activation of the IRES activity depending on temperature may contribute the effective translation of HSP81-3 mRNA during heat stress which inhibits cap-dependent translation.

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We recently isolated an Arabidopsis frt1 mutant that showed enhanced freezing tolerance in mature leaves, but not in young leaves, due to elevated sugar levels relative to the wild type. However, previous result suggested a possible problem in sugar translocation pathways rather than sugar metabolism. Because of such unique phenotype, we herein identified the FRT1 gene from T-DNA-flanking sequences, which successfully rescued the frt1 phenotype. FRT1 encoded a putative enzyme involved in cell wall catabolism, whose homologs constitute a large gene family in Arabidopsis. However, mutation of the most homologous gene showed no frt1-like phenotype, reinforcing the unique function of FRT1. RNA gel blot analysis showed that the FRT1 transcripts were detectable at ambient temperature but diminished at low temperature, suggesting that the putative FRT1-dependent pathway is dispensable at low temperature or its down regulation might have unknown positive effects on sugar accumulation at low temperature.

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Rice lip19 encoding a 148 amino acids-bZIP protein has been isolated as one of low-temperature-induced genes. Its product, LIP19, seems to play a regulatory role in gene expression during cold stress, but none of its function has been uncovered. Currently we found that LIP19 lacks the common ability of bZIP transcription factors in tems of homodimerization and DNA binding. Thus, we searched for LIP19-interacting partner(s) and identified OsOBF1. So far, we have showed that (i) both LIP19 and OsOBF1 localize to nucleus, (ii) their spatial expression sites are same in rice leaf blades, and (iii) LIP19 directly interacts with OsOBF1 using in vitro pull down assay.
Here we show that DNA recognition sequence of LIP19-OsOBF1 heterodimer differs from that of OsOBF1 homodimer. We will also demonstrate LIP19-OsOBF1 interaction in vivo. Based on all the data obtained, we will discuss a role of LIP19 in a cold signaling pathway in rice.

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Freezing tolerance of cortical parenchyma cells of mulberry (Morus bombycis) drastically increases in winter. 27-kDa group 3 late embryogenesis protein (WAP27) accumulated in ER of the cells in winter.
In natural conditions, plants are subjected to prolonged freezing that at high subzero temperature results in prolonged exposure of cells to concentrated solutions. We therefore examined cryoprotection of recombinant WAP27 (rWAP27) on lactate dehydrogenase (LDH) activity against prolonged freezing. The rWAP27 preserved above 80% of LDH activity during prolonged freezing at -2, -4, -10, -20^oC. The cryoprotective activity of bovine serum albumin was nearly equal to that of rWAP27 during prolonged freezing at -20^oC, but decreased with freezing time at -2 and -4^oC. The cryoprotective activity of ovalbumin was also decreased with freezing time at -2 to -20^oC. The rWAP27 has a high cryoprotective activity to freeze-labile proteins against prolonged freezing.

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Abscisic acid (ABA) is known to play an important role in responses to environmental stresses. We are investigating mechanisms of ABA-induced freezing tolerance in the moss Physcomitrella patens. ABA induced physiological changes such as expression of various genes, accumulation of boiling soluble proteins and increase in soluble sugar contents.
We isolated mutants with reduced ABA sensitivity by UV mutagenesis. These mutants grew normally in a medium containing ABA, whereas the wild type showed inhibited growth. Freezing and osmotic stress tolerance in these mutants was lower than that of wild type, but the extents of tolerance varied among the mutants. In these mutants, levels of expression of stress-related genes, a few boiling-soluble proteins and soluble sugars after ABA treatment were reduced as compared with those of wild-type. The degrees of freezing tolerance were correlated with levels of proteins and sugars in ABA-insensitive mutants.

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Arabidopsis ABI3 and the homologous genes are known as transcriptional factor which expresses specifically in embryo from very early stage of embryogenesis. In our previous work, we had identified an embryonic promoter cis-element, Carrot Embryonic Element 1 (CEE1), in the C-ABI3 (carrot-ABI3) promoter. And we had shown that 150 bp fragment of ABI3 promoter region (Atd3-1L1) including two reverse-CEE1-like sequences (AEE1-1 and AEE1-2) is related to the ABI3 gene expression in embryo. In this study, we analyzed precisely the Atd3-1L1 region related to the embryonic expression. The result indicated that AEE1-1 sequence is important for gene expression in embryo. On the other hands, OsEE1 sequence which was found in OsVP1 promoter can bind with carrot CEE1-binding-factors. CEE1, AEE1-1 and OsEE1 are very similar sequence, which might be related to gene expression in embryos of various plants.

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In higher plants, transcriptional silencing by chromatin remodeling is suggested to be involved in the embryogenesis and in transition from vegetative to reproductive phase. PKL is a putative component of histone deacetylase (HDAC) complex which is related to transcriptional silencing. PKL represses the expression of LEC1 during seed germination and this repression of LEC1 leads to the postembryonic development of the embryos.
We analyzed the effects of HDAC inhibitors on seed germination in Arabidopsis. Treatment with HDAC inhibitors suppressed germination and did not the expression of LEC1. In LEC1 deficient mutant, the germination was not suppressed by the treatment with HDAC inhibitors. When HDAC inhibitor-treated seeds were transferred to inhibitor-free medium, the germinated seedlings showed abnormal development: formation of multiple-shoots and somatic embryo-like structures on the leaves. These results suggest that histone deacetylation is related to repression of LEC1 expression during germination and transition from embryonic to postembryonic development.

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Somatic embryogenesis is unique characteristics of plant cells. Embryogenic cells (EC) of carrot maintained in the medium containing 2,4-D regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. C-ESE1 (Carrot Early Somatic Embryogenesis 1)was isolated from early stage of somatic embryogenesis after 2,4-D depletion. Transgenic carrot EC with cosuppressed C-ESE1 expression showed that C-ESE1 might be involved in cell attachment in somatic embryogenesis and also affect the polysaccharide deposition on the cell wall.
Here we report the polysaccharides in the medium of C-ESE1-cosuppressed cell culture. The analysis of sugar composition of the polysaccharides isolated from the culture medium indicated that the polysaccharides isolated from C-ESE1-cosuppressed EC culture medium had considerably increased the fucose, arabinose and xylose, suggesting thatC-ESE1 cosuppression induced the loss of some polysaccharides and/or glycoproteins from the cell wall for cell-to-cell attachment.

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We have developed a carrot somatic embryo-forming system in which somatic embryos are directly formed when hypocotyl explants are treated with 2,4-D and then cultured in hormone-free medium. Searching for genes expressed specifically in 2,4-D-treated hypocotyls may reveal the mechanism of somatic embryogenesis. Differential display analysis revealed three genes, No.130, No.151 and No.209. No.130 and No.209 proteins had high similarities with alcohol dehydrogenases and BRI1-like lateral suppressor region Cs, respectively, but No.151 protein had hardly similarities with any proteins. No.130 and No.209 were expressed the highest before the globular stage of somatic embryogenesis and No.151, at the heart-shaped stage.
Because of similarity between No.209 and lateral suppressor region C, lateral suppressor also may acts during somatic embryogenesis. We identified the full-length carrot lateral suppressor-like protein cDNA. The expression pattern of this gene was similar to that of No.209 gene during somatic embryogenesis.

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It is well known that abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy in zygotic embryos. It has been reported that the VP1/ABI3 factor functions as a transcriptional factor on seed specific ABA signal transduction. We had suggested that a carrot homolog of the VP1/ABI3 factor, C-ABI3, is involved in the control of expression of some ABA-inducible genes in carrot zygotic and somatic embryos. Recently, we isolated a new kind of cDNA clone encoding the VP1/ABI3 factor from carrot somatic embryos and named it C-ABI3-like (C-ABI3-L). The expression of C-ABI3-L was observed specifically in embryogenic cells, somatic embryos and developing seeds. Furthermore, in the transgenic non-embryogenic cells showing ectopic expression of C-ABI3-L, the expression of some seed specific ABA-inducible genes was successfully induced by ABA-treatment. These results indicate that C-ABI3-L might function on ABA signal transduction in carrot embryos as well as C-ABI3.

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It is generally said that seeds with pale color coat have weaker dormancy than seeds with dark color coat. This dormancy enhancement was attributed to coat impermeability, physical strength or presence of germination inhibitor.Arabidopsis transparent testa (tt) mutants which have defect in flavonoid metabolism have been reported to have reduced dormancy. We have isolated tt7 allele (TRW71-1) as one of the high temperature resistant germination mutants, and found that tt4-1, tt5-1, tt6-1 and tt7-1 were high temperature resistant but tt3-1 and banyuls were not. Proanthocyanidin content in the seeds showed no correlation with the germination character. In addition, germination of tt4-1, tt5-1, tt6-1 and tt7-1 seeds were inhibited by auxin transport inhibitor, NPA, by a dose dependent manner at high temperature. These results suggest that distribution of auxin in Arabidopsis embryo have a contribution to regulation of germination at high temperature.

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GAs are phytohormons that play an essential role in promoting seed germination, during which the GAs biosynthesis is activated by such environmental factors as low temperature and light. On the other hand, the seed germination of various plants has been shown to require reactive oxygen species (ROS) such as O₂^- and H₂O₂. Previous studies have shown that ROS serve as an important molecule for cellular signal transduction, but the molecular mechanism for how ROS promote seed germination, especially in regard to GAs, remains to be elucidated.
Here we aim to elucidate the role of ROS generated during the germination of Arabidopsis seeds. According to DNA micoarray analysis and germination test, H₂O₂ promoted the seed germination of Arabidopsis and induced a set genes associated with GAs. O₂^- was actually generated following seed imbibition. These suggest that H₂O₂ generated via the disproportionation of O₂^- regulates the GAs-associated pathway promoting seed germination.

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Temperature is one of the critical environmental factors for seed germination. Like other winter annual species, germination of A. thaliana seeds is inhibited in high temperature conditions (thermoinhibition). We have found that thermoinhibition of Arabidopsis seeds required de novo synthesis of abscisic acid (ABA) during imbibition. Quantitative PCR analyses showed that three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5 and NCED9, were up-regulated by supraoptimal temperatures. To reveal contribution of NCED genes on thermoinhibition, we analyzed seed germination of each NCED gene knockout (T-DNA insertion) lines. The seeds of NCED9 knockout line showed high germination percentage (ca.70%) at 32oC, while almost no germination was observed with the seeds of wild type (Col) and NCED2 knockout line. In wild type seeds, NCED9 gene transcript was most abundant among the three NCED genes when imbibed at 34oC. These results suggest NCED9 gene plays a primary role on ABA accumulation and thermionhibition in Arabidopsis.

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Phytic acid (inositol hexakisphosphate; IP6), a storage compound of phosphate in seed, is accumulated in the vacuole during seed maturation. The synthesis and vacuolar transport mechanisms of IP6 are still unclear. It has been proposed that IP6 is synthesized as follows : first, glucose-6-phosphate is converted to inositol monophosphate (IP1) by MIPS, then, IP1 is sequentially phosphorylated by several inositol kinases to produce IP6. We prepared an anti-MIPS antibody and used it for immuno-electron microscopy and indirect immuno-fluorescent microscopy. MIPS localized in endosperm, but not in embryo where IP6 is accumulated. Immunoblot analysis for isolated embryo revealed that 2S albumin, 12S globulin and inositol (1,4,5) trisphosphate kinase mainly localized in embryo, while MIPS did not. Phytic acid was detected both in endosperm and embryo. These findings suggest that a part of products by MIPS synthesized in the endosperm was transported to embryo and phosphorylated there to produce IP6.

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Two dominant mutants that have short internodes of inflorescence and reduced apical dominant were isolated by screening of 50,000 of RIKEN activation tagging lines. The T-DNA insertion sites of these lines were near each other. Transcription of a gene that encodes a putative bHLH protein near the T-DNA insertion site was increased in these mutants. We made overexpressr of this gene under control of CaMV 35S promoter. The ectopic expression of this bHLH gene recapitulated the original mutant phenotype. We observed yellowish seed color in the overexpressor lines although we could not detect color change in activation tagged mutant. The yellowish seed color of the overexpressor lines were caused by reduction of tannin accumulation in seed coats. This observation suggested that overexpression of this bHLH gene might be involved in tannin biosynthesis or metabolism.

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Rice is a Si-accumulating plant. To isolate a gene which is responsible for Si uptake in rice, we have isolated a mutant (lsi1) which is defective in the xylem loading of Si (Ma et al., 2002). In the present study, the gene Lsi1 was isolated by map-based cloning technique. The cDNA was 1409 bp long and the deduced protein consisted of 298 residues. The program PSORT predicts that the Lsi1 encodes a membrane protein. This gene was not induced by Si and constitutively expressed mainly in the roots. To investigate cellular localization of LSI1, Lsi1 was fused with GFP and introduced the tobacco leaves and onion epidermal cells. Microscopic observation showed that LSI1 was localized in the memblane. Further functional analysis of this gene is currently being undertaken.

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Rice is a known Si-accumulator, however, the molecular mechanism responsible for high Si uptake by the roots is not understood. In the present study, we isolated a novel rice mutant (lsi2), which was defective in Si uptake. Physiological study showed that this mutant was defective in xylem loading of Si rather than the transport of Si from external solution to root cortical cells. Genetic analysis showed that the low Si uptake was controlled by a recessive gene. To map this gene, bulk segregant analysis was performed by pooling equal amounts of DNA from 10 low-Si uptake or 10 high-Si uptake F₂ plants. Both microsatellite and EST-PCR markers were used for mapping the gene. As a result, the gene was mapped to the chromosome 3, which location is different from that of Lsi1, another gene responsible for xylem loading of Si in rice. Fine mapping of this gene is being undertaken.

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Arabidopsis BOR1 is an efflux transporter of boron. Among six BOR1 paralogs present in Arabiodpsis genome, we studied role of BOR2 in B transport. Expression of BOR2 resulted in reduction in B concentration of yeast cells. It suggests that BOR2 encode an efflux-type B transporter, similar to the case of BOR1. Transgenic Arabidopsis lines carrying T-DNA in BOR2 showed growth retardation compared to wild type plants at low boron supply. Root cell elongation was severely inhibited under low boron supply in these lines. Transgenic Arabidopsis expressing GUS under control of the promoter has revealed that BOR2 are expressed in root tip, cortex cells in roots. We propose that this transporter exports B out of the cells into the apoplasm for effective formation of RGII-B dimer for cell elongation.

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Boron deficiency and toxicity hamper quality and productivity of crops world wide. Arabidopsis thaliana BOR1 is an efflux transporter responsible for effective transport of boron from roots to shoots. Expression of BOR1 in yeasts reduces concentration of cellular boron and improves growth under high boron. To examine if similar improvement can be observed in plants, BOR1 is expressed under the control of the cauliflower mosaic virus 35S RNA promoter in A. thaliana. Growth of the transgenic plants under boron excess condition was not significantly different from the non-transgenic plants. This is most likely to be due to degradation of BOR1 protein under high boron supply. Unexpectedly, the growth of transgenic plants under B-limitation was improved significantly. Fertility under limited boron supply was also improved. This method can be used for generation of crop plants for better performance under boron deficient conditions.

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Boron is among the essential elements for higher plants. Rice plants grow normally with relatively low supply of boron and boron concentrations in aerial portions are generally lower than those of dicots, though they are also susceptible to boron deficiency. To reveal the molecular mechanisms of boron transport in rice, we analyzed rice genes similar to BOR1. Four BOR1-like genes were found in the genome of rice and were named OsBOR1, 2, 3 and 4.
When expressed in yeast, OsBOR1-3 reduced concentration of boron in the cells suggesting that they are efflux transporter of boron. Tissue specificity of expression of the homologous genes differ among the OsBOR genes, suggesting differential roles of individual transporters. Analysis of several Tos17 tagged lines corresponding to the OsBOR genes suggest important role of OsBOR genes in boron transport and growth under boron-limiting conditions.

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Seed storage proteins are synthesized on rough endoplasmic reticulum as larger precursors. The precursors are transported to protein storage vacuoles (PSVs) by vesicle-mediated machinery, and then are processed into their mature forms. Recently, we identified AtVSR1 as a vacuolar sorting receptor for seed storage proteins in Arabidopsis (1). Storage protein precursors are accumulated in the extracellular space in the seeds of AtVSR1-deficient mutant. To identify the factors responsible for the receptor-dependent vacuolar sorting of storage proteins in maturing seeds, we applied reverse genetic approach with various T-DNA inserted lines of Arabidopsis. We characterized the storage proteins patterns accumulated in their mutant seeds by immunoblot analysis with specific antibodies against storage proteins. We screened some mutants that accumulated storage protein precursors in the seeds.
1) T. Shimada et al. Proc. Natl. Acad. Sci. USA, 100, 16095 (2003)

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The transport mechanism of plasma membrane proteins to vacuoles has not been well characterized in plants. To clarify how to degrade plasma membrane proteins (PIP2a and LTI6b), we monitored the endocytotic pathway in transgenic Arabidopisis plants that express a fusion protein (GFP-PIP2a or GFP-LTI6b) and wild-type tobacco BY-2 cells. Because of the rapid delivery of endosomes to the vacuoles, endosomes were scarcely detectable. We found that E-64d, an inhibitor of papain family proteases, caused the accumulation of endosomes in sucrose-starved BY-2 cells. This result indicates that E-64d attenuate the fusion of endosomes to vacuoles. In transgenic Arabidopsis plants, treatment with E-64d induced the accumulation of GFP-fluorescent endosomes and inhibited the degradation of the fusion proteins. We found that two papain homologues (ENPs) are localized in accumulated endosomes. These results suggest that ENPs facilitate the fusion of endosomes with vacuoles at the final step of the endicytotic pathway.

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Plant vacuoles are complicated and multi-functional organelle. The Arabidopsis shoot gravitropism 2 (sgr2) mutant isolated as a gravitropic mutant in the inflorecence stem and the hypocotyl, has defect both in amyloplasts movement and in vacuolar function in endodermal cells. Our previous study has suggested that amyloplasts movement important for gravity sensing is closely related to the dynamics of vacuoles. The SGR2 encodes a protein homologous to bovine phospholipase A1, which is localized mainly to vacuolar membrane and small organelles. To investigate the molecular function of SGR2, we performed a genetic screen for the second mutation suppressing the defect of sgr2-1 gravitropic phenotype. The kiritsu171 (krt171) mutation suppressed abnormal amyloplasts movement of sgr2-1. Based on the map position, we found a mutation in a gene encoding a putative acyltransferase containing an EF-hand motif and a transmembrane domain. Taken together, KRT171 and SGR2 may be involved in the modulation of vacuolar dynamics.

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Multiprotein bridging factor 1 (MBF1) is known to be a transcriptional co-activator that mediates transcriptional activation by bridging between a transcriptional activator and a TATA-box binding protein (TBP). Expressions of target genes regulated by changing of subcellular localization of MBF1s in Drosophila and human are previously reported. Arabidopsis genome also contains three subtypes of genes encoding MBF1s (AtMBF1s). We have already characterized their expression profiles in Arabidopsis. However, it is unknown if AtMBF1s are associated with regulation of gene expression by changing its subcellular localization.
To determine subcellular localization of AtMBF1s, we fused each three AtMBF1s with GFP and introduced plasmids into Arabidopsis and onion epidermal cells. The signal associated with GFP was observed in nuclei in both experiments. These observations suggest that AtMBF1s originally localize in nuclei and may play a role as transcriptional co-activator.

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The translocon at the inner envelope membrane of chloroplasts (Tic) plays a central role in plastid biogenesis by coordinating the sorting of nucleus-encoded preproteins across the inner membrane and coordinating the interactions of preproteins with the processing and folding machineries of the stroma. Beyond this general outline, the precise roles of known Tic proteins in translocation, sorting and preprotein maturation have not been defined. We have examined the function of a major Tic component, Tic110. We have demonstrated that the hydrophilic domain of Tic110 is soluble, located at the stromal side of the chloroplast inner envelope. It has also been demonstrated that Tic110 directly binds to preproteins. In this presentation, we demonstrate that Arabidopsis Tic110, atTic110, is essential for plastid biogenesis and plant viability. In addition, we will present data showing the mechanism of Tic110 during protein translocation across the inner envelope.

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The petiole curvature plays a major role in light interception, but the mechanisms are not clear. We examined the petiole curvature in Chenopodium album L. grown under two different light conditions (360, 50 μmol m^-2 s^-1).
To specify the light perception site for induction of the curvature, the lamina or petiole were separately illuminated with white light continuously. The curvature was induced most effectively when the abaxial side of petiole was illuminated. The amount of curvature depended on the growth light condition. Under continuous illumination of the abaxial side, the petiole kept on curving. In natural condition, however, the curvature may cease when the lamina receives optimal light intensity. We investigated the light perception site for cessation of the curvature. The adaxial side of petiole was the most sensitive site. Furthermore, blue light was effective to induce the curvature, and blue and red light were effective to stop the curvature.

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Phytochrome B is a major plant photoreceptor and its polypeptide is folded into two domains: the N-terminal photosensory domain and the C-terminal domain which has been widely believed to transduce the signal to downstream components. However, we have recently found that the N-terminal domain of Arabidopsis phyB, when dimerized and localized in the nucleus, triggered full phyB responses with much higher photosensitivity than the intact full-length phyB. In order to elucidate the molecular mechanism of the N-terminal domain signaling of phyB, we performed a genetic screening for long hypocotyl mutants under very weak red light by mutagenizing the transgenic Arabidopsis expressing the artificial N-terminal domain protein described above. We have so far screened at least 1200,000 M2 seedlings which is derived from approximately 200,000 M1 plants. Here we will report the details of screening method and some recent results on the isolated extragenic mutations.

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Phytochrome B (phyB) molecule consists of two domains: the N-terminal photosensory domain and the C-terminal domain, and the latter has been believed to transduce the signal to downstream components. However, our recent study has demonstrated that the N-terminal domain alone of phyB was fully functional when dimerized and localized in the nucleus. Moreover, a 450-amino acid N-terminal fragment of phyB effectively transduced the signal in the nucleus.
Here, we analyzed missense mutations within the N-terminal domain of phyB to identify the site critical for signaling. We mutagenized transgenic Arabidopsis expressing the N-terminal domain of phyB and screened for long hypocotyl mutants in red light. As a result, we identified several missense mutations within the N-terminal domain of phyB, some of which exhibited normal photochemical properties. Interestingly, they fell within a relatively small region, suggesting that this site is directly involved in phyB signal transduction.

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Phytochrome A (phyA), which mediates the far-red high-irradiance and very low fluence responses, accumulates in the nucleus upon light activation by far-red light. Phytochrome A is rapidly degraded mainly in the cytoplasm after red light illumination. We investigated the subcellular sites of phyA signal transduction and degradation. PHYA-GFP (PAG) was fused to either nuclear localization signal (PAGL) or nuclear export signal (PAGE) and expressed under the control of the PHYA authentic promoter in the phyA deficient mutant. Microscopic observation confirmed that PAGL and PAGE were localized to the intended sites. Hypocotyl analysis revealed that PAGL was more active than PAG whereas PAGE failed to elicit the response. Hence, phyA appears to transduce the signal in the nucleus for the far-red high-irradiance responses. Interestingly, both PAGL and PAGE were degraded rapidly with similar kinetics after irradiation with red light. Hence, light-dependent degradation can take place in both the sites.

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