We recently found that moisture gradient or water stress immediately degrades amyloplasts in the columella cells of Arabidopsis and Raphanus roots, which caused the reduced gravitropism. Roots thus exhibit strong hydrotropism by overcoming gravitropism. It is known that not only gravitropism or hydrotropism but also phototropism play important roles in orienting Arabidopsis roots. In the present study, we examined whether degradation of amyloplasts involves in the interaction between phototropism and gravitropism. We found that phototropic response accompanied a significant reduction of starch in columella cells of Arabidopsis roots. Phototropic response and amyloplast degradation occurred due to the unilateral irradiation of white, blue or red light. Bilateral irradiation of those lights resulted in neither root bending nor reduction of starch in the columella cells. These results suggest that Arabidopsis roots display phototropism by overcoming gravitropism, due to degradation of gravisensors, amyloplasts, in the columella cells, as does hydrotropism.

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To elucidate the auxin asymmetry in the gravistimulated cucumber seedlings, we raised polyclonal antibodies of the auxin-carrier-homologs (CS-AUX1; influx carrier, CS-PIN1; efflux carrier) isolated from cucumber and examined the expression and localization of those proteins. The expression of CS-AUX1 protein was observed all around the transition zone, expect for vascular tissue, and more abundantly in the lower side of the transition zone between hypocotyl and root than the peg-suppressed upper side when seeds were germinated in a horizontal position. On the other hand, CS-PIN1 protein accumulated more abundantly in the epidermal and cortical cells of the transition zone of the peg-suppressed upper side than the lower side. CS-PIN1 protein was also detected in the vascular tissue. These results suggest that localized change in the balance of influx and efflux carriers of auxin causes auxin redistribution and subsequently regulates peg formation in cucumber.

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Cucumber seedlings develop a peg on the concave side of the bending transition zone (TR zone) between hypocotyl and root. We have previously shown that gravistimulation causes asymmetrical mRNA accumulation of auxin-inducible genes (CS-IAA1 and CS-IAA2) in TR zone when peg initiation occurs. To reveal the molecular mechanism of these genes expression, we examined the localization of mRNAs of auxin response factors (CS-ARFs) during peg formation by in-situ hybridization and compared them with that of CS-IAA mRNAs. In 18-h-old seedlings CS-ARF2 mRNA, as well as CS-IAA1 mRNA, accumulated in epidermal, cortical and vascular tissues of TR zone. In contrast, 24-h-old seedlings accumulated more CS-IAA1 mRNA on the concave side of TR zone than on the convex side, but no asymmetric mRNA localization of CS-ARFs was detected. These results suggest that CS-ARF2 activates CS-IAA1expression during peg formation in cucumber seedlings, and auxin directly or indirectly modulates its transcriptional activity.

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Under true microgravity conditions in space as well as simulated ones on a three-dimensional clinostat, etiolated pea seedlings showed automorphosis-like growth, which was phenocopied by auxin polar transport inhibitors on 1 g conditions. Since auxin polar transport in etiolated pea seedlings grown in space were substantially reduced, these suggest an important role of auxin polar transport in the regulation of automorposis. Auxin polar transport is regulated by putative PsPIN1 and PsAUX1 in etiolated pea seedlings. Significantly high homology on nucleotide sequences was found between PsPIN1 and AtPIN. Expression of PsPIN1 and PsAUX1 in etiolated pea seedlings was substantially observed. Studies of the effects of auxin polar transport inhibitors as well as simulated microgravity conditions on expression of PsPIN1 and PsAUX1 in etiolated pea seedlings are now on progress. A possible role of gravity for auxin polar transport in higher plants will also be discussed.

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We have shown that in Japanese flowering cherry grown under simulated microgravity condition, its mechanical rigidity of stem was remarkably decreased. In this study, factors related to photosynthesis in 4 weeks old seedlings of Japanese flowering cherry on earth and under simulated microgravity condition were measured. No significant difference between the mean photosynthesis rate in both was found. The factors related to photosynthesis ability of both plants such as photosynthesis rate, chlorophyll content, chlorophyll a/b ratio and the density of stomata, were almost similar. These showed that photosynthesis activity of the seedlings was not changed under simulated microgravity condition, although its mechanical rigidity was decreased under such condition.

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Interactions between organisms and species have been long known. Allelopathy is a phenomenon observed in many plants that emit specific chemicals acting on other organisms, including plants, animals and microorganisms, in either inhibitory or excitatory ways. We studied whether allelopathy is modified under altered gravity or not. If biosynthesis, emission, transport and sensing mechanism of allelopathic substances would be affected by gravity, many organisms and ecological system might show different behaviors based on the inter-organisms and -species interactions when they are exposed to microgravity. We evaluated the gravitational effects on allelopathy by applying pseudo-microgravity, generated by a 3D-clinostat, onto a plant-plant system. The allelopathic action of Mucunaon the growth of lettuce and Arabidopsis was compared between the 3D-clinostat experiment group and the ground control. Since the action of allelopathic substances, L-DOPA, was similar for the two group, gravitational effects on the transport and other process were further examined.

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The β subunit gene of soybean β-conglicinin of has been shown to be upregulated by sulfur deficiency at the level of transcription. Insertion of the 235 bp-sulfur responsive promoter region of this gene into cauliflower mosaic virus 35S promoter enabled us to observe response to sulfur in non-seed tissues (Awazuhara et al. 2002). In transgenic Arabidopsis NOB plants carrying this chimeric promoter fused with GFP gene, GFP fluorescence was increased by sulfur deficiency (Ohkama et al. 2002). We screened mutants with altered GFP fluorescence from M2 plants of EMS-mutagenized NOB plants.
nbm1-1 (NOB mutant) shows higher GFP fluorescence than parental NOB plants irrespective of sulfur condition. Expression of Sultr2;2, APR1. SAT1, endogenous sulfur-responsive genes, were also upregulated. genetic mapping located the nbm1-1 locus in upper arm of Chromosome 5. Awazuhara et al. (2002) Plant Sci. 163: 75-82, Ohkama et al. (2002) Plant Cell Physiol. 43: 1266-1275

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Expression of the gene encoding Glycine max seed storage protein beta-conglycinin beta subunit increases under sulfur deficiency(ΔS). The transgenic Arabidopsis line NOB carries GFP under control of the cauliflower mosaic virus 35S RNA promoter with insertion of the sulfur nutrition responsive region of the beta subunit gene promoter. GFP fluorescence in NOB lines increases under ΔS condition (Ohkama et al. 2002). nbm2-1, one of NOB mutants, showed higher GFP fluorescence when grown under both normal and ΔS conditions. Plant size was small. In this mutant, concentrations of O-acetyl-L-serine, a signal metabolite of ΔS, and GSH, another signal metabolite having the opposite effect of OAS, increased. Cysteine increased, sulfate and nitrate decreased in concentration. Accumulations of mRNAs for Sultr2;2, APR1 and SAT1 gene, the genes known to be upregulated by ΔS condition and related to sulfur metabolism, increased.
Ohkama, N. et al., Plant and Cell Physiology 43(11):1266-1275(2002)

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During sulfate starvation, enzymatic activities and mRNA levels of proteins in sulfur assimilatory pathway are enhanced. Sensing of sulfate levels and regulatory mechanisms of gene expression responding to sulfate availability have not been well characterized in higher plants. In Arabidopsis, the low-affinity sulfate transporter Sultr2;1 expressed in root pericycle and xylem parenchyma cells is highly up-regulated responding to sulfate starvation. The results of inhibitor studies suggested that the increase of Sultr2;1 mRNA by sulfur limitation is under the control of post-transcriptional regulation. Transgenic plants carrying the Sultr2;1 5'region::luciferase:: Sultr2;1 3'region were constructed. Luciferase activities increased upon sulfur starvation only when the 3'region was present. Plants carrying the construct of 5'region::GFP::3'region showed similar response. Sultr2;1 3'region contained DST-elements involved in the regulation of mRNA stability, suggesting involvement of post-transcriptional regulation in sulfur limitation response of Sultr2;1.

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When phosphate in soil is limiting, plants behave various responses to adapt this condition. However, the details of these responses are still unknown. In this study, to perform comprehensive and systematic analysis about these responses, cDNA microarray and quantitative Real-Time PCR analyses were conducted.
Rice plants grew by hydroponic culture with or without phosphate (1 ppm or 0 ppm, respectively), and then separated into shoots and roots. Total RNA was isolated and used for the analysis of gene expression. Expression of some genes related to phosphate absorption was induced by Pi-deficiency. It was confirmed that the expression of genes involving with carbon metabolism was changed in shoot under Pi starvation. On the other hand, no significant increase of expression of the genes related to organic acid exudation was observed under Pi-deficient condition.

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Plants developed strategy to recycle phosphorus in their organ to replenish the requirement of other organ (especially newly growing organ). We focused on the role of Ribonuclease (RNase) and Acid phosphatase (APase) for the decomposition of phosphorus compound to regulate plant phosphorus level. As many phosphorus compounds are closely related to carbon metabolism, the expression level of following enzymes are evaluated by RT-PCR, Triose phosphate translocator, Phosphoenolpyruvate translocator (PPT), Phosphoenolpyruvate carboxylase, Pyruvate kinase, NAD-Glycerol 3-phosphate dehydrogenase, NADP-Glycerol 3-phosphate dehydrogenase. Under phosphated deficient condition, the level of inorganic phosphate decreased dramatically, and the enzyme activity of RNase and APase increased. The content of hexose decreased, instead starch content increased under phosphate deficiency. As the expression level of PPT increased dramatically, the carbon flow from cytosol to chloroplast as phosphoenolpyruvate may play an important role to maintain carbon metabolism under phosphate deficient condition.

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When plants grow under low-P conditions, acid phosphatase (APase) expresses in root exudates and tissues, which contribute to hydrolyze organic phosphate compounds for P absorption and translocating into other tissues, respectively. Objective of this study is to investigate function of APase isoforms under low-P condition.
Lupin plants were grown by hydroponic culture with (+P treatment, 2 ppm) or without P (-P treatment, 0 ppm). Plants were separated to each organ. APase isoforms were separated by IEF. Various APase isoforms in roots and leaves increased by P treatment. Notable effects by -P treatment were not observed in cotyledons and stems. Only one isoform in pI 4.7 was detected in the exudate of -P roots. mRNA accumulation of three APases was analyzed. mRNAs of cell wall localized and phytate specific APase was observed in all organs. On the other hand, mRNA of secretory APase was detectable only in -P roots.

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In response to increased levels of Zn²⁺,SmtA, a metallothionein, is expressed in Synechococcus sp. PCC 7942, while ZiaA, a transporter of Zn²⁺,is expressed in Synechocystis sp. PCC 6803. Gene expressions for these proteins are regulated by repressor proteins, SmtB and ZiaR, respectively, both of which belong to ArsR family, but their levels of repression differ. To understand the different repression mechanism, we compared the cis-element in the promoter region of smtA and ziaA. The promoter region of smtA involved two inverted repeats and that of ziaA involved one similar repeat. Gel retardation assay revealed that SmtB formed four different complexes with the promoter region of smtA, whereas it formed two different complexes with that of ziaA. These results suggest that the formation of larger complexes of SmtB due to the two inverted repeats might repress the expression of smtA more tightly.

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SmtA, a metallothionein of the cyanobacterium Synechococcus sp. PCC 7942, binds heavy metal ions such as Zn²⁺ and is involved in regulation of the homeostasis of Zn²⁺. NMR analysis has indicated that SmtA has two metal-binding regions, namely, the N-terminal region (Met1-Cys47) and C-terminal region (Gly43-Gly56), which binds two and one molecules of Zn²⁺, respectively. We attempted to improve the binding capacity for Zn²⁺ by jointing several N-terminal or C-terminal regions tandemly by genetic engineering. The recombinant proteins fused with a GST were overexpressed in E.coli and purified. ICP-spectrometry analysis revealed that the binding number of Zn²⁺ increased by two in proportion to one N-terminal region and by one to one C-terminal region. Strikingly, a recombinant SmtA with additional 10 N-terminal regions bound 23 molecules of Zn²⁺. Tandem joint of the C-terminal region improved the binding efficiency of Zn²⁺ per the number of amino acid residue.

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CPx-ATPases related to heavy-metal transport are a new subgroup of P-type ATPase. Its existence in plant cell have hardly observed. In this study, we isolated CPx-ATPase gene relate to heavy metal transport from genome DNA of Oscillatoria brevis and characterized.
Sequence analysis of the CPx gene (Bxa1) showed that it consists of 1983bp ORF and eight transmembrane helix. It contains high conservative regions of phosphorylation site (DKTGTL), ATP binding site (GDGINDAP), phosphatase domain (TGES) and putative cation transduction channel (CPC). Instead of the CXXC motif that is present in the N terminal of most metal transport CPx-ATPase, Bxa1 contains histidine rich motifs. rQRT-PCR analysis shows Bxa1 mRNA was induced by both monovalent (Cu⁺, Ag⁺) and divalent (Zn²⁺, Cd²⁺) heavy metal ions. Experiments on heavy-metal tolerance in E. coli which recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals.

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We will present the transgenic plants expressing an yeast zinc transporter, ZRC1. The ZRC1 transports excessive zinc from cytoplasm into vacuole and functions to maintain intracellular zinc homeostasis. The zrc1-overexpressing yeast has been shown to tolerate not only zinc but also cadmium. Therefore, we tried to introduce the zrc1 gene into tobacco and analyzed their heavy metal accumulation. Although the level of expression of the ZRC1 in tobacco was low, some transformants showed higher amounts of cadmium accumulation compared with those of wild type tobacco. Moreover, the dry weight of some transformants increased significantly and consequently total accumulation of cadmium increased. These results indicate that ZRC1 reduces the cadmium concentration in the cytoplasm by pumping up the cadmium into the vacuole, resulting with normal plant growth. The zrc1 gene is one of the good candidates for producing hyper-accumulator used for phytoremediation.

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Lolium perenne and Poa pratensis are perennial grasses which are used to correct Fe-deficiency of fruit tree in calcareous soil by growing them along the tree. We investigated the response of these grasses to Fe-deficiency, focusing on the secretion of Fe-chelating substances from the roots. Both grasses secreted Fe-chelating substance from the roots in response to Fe-deficiency and the secretion was characterized by a temperature-controlled diurnal rhythm. Three kinds of phytosiderophores were secreted from Lolium perenne (cv. Tove); two of them were 2*-deoxymugineic acid (DMA) and 3-epi-hydroxy-2*-deoxymugineic acid (epiHDMA), while the third one was not identified previously. After purification, this unknown compound was identified as 3-hydroxy-2*-deoxymugineic acid (HDMA) by NMR. On the other hand, DMA, avenic acid A (AV) and an unknown compound were secreted from Poa pratensis (cv. Baron). The purification and identification of this unknown compound are currently being undertaken.

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An Arabidopsis mutant named short stem and midrib(ssm) shows a semi-dwarf phenotype. We identified the mutant gene by chromosome walking and found that it encodes a syntaxin SYP23/AtVAM3, which is localized on the vacuolar membrane. In the ssm mutant, the sixth intron of the SYP22/AtVAM3 gene is partially deleted. We found that the resulting intron sequence was not spliced and translated to 19 amino acids between the 6th exon (which encodes juxtamembrane domain) and the 7th exon (which encodes transmembrane domain).
Knockout mutants of SYP22/AtVAM3 are known to be gametophyte lethal. Further studies on the ssm mutant will provide a new clue for elucidating the function of plant syntaxin-related proteins.

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To elucidate the molecular mechanism of the shoot gravitropism, we have isolated many shoot-gravitropism(sgr) mutants in Arabidopsis. The zigzag (zig/sgr4) mutant exhibited abnormal gravitropism in both infloresence stems and hypocotyls. Additionally, the stems elongated in a zigzag fashion. The ZIG gene encodes a SNARE, AtVTI11 that is homologous to yeast Vti1p involved in vesicle transport to vacuoles. To understand how vesicle traffic to vacuoles are involved in morphogenesis and gravitropism, we have isolated some suppressor mutants of zig-1. A dominant mutation zig suppressor 1(zip1) could almost completely suppress the defects of zig-1 mutant in both morphogenesis and gravitropism. Based on the map positon, we find a point mutation in AtVTI12 of zip1 zig-1. AtVTI12 is homologous to ZIG/AtVTI11, but both are functionally different. To understand how zip1 suppress the defect of zig-1 at a molecular level, biochemical and cytological analyses are now in progress.

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Rice α-globulin is a 2S albumin-type seed storage protein that accumulates in the protein storage vacuole (PB-II) in starchy endosperm. We constructed GFP fusions with different regions of α-globulin to identify the vacuolar-targeting signal. The localizations of the fusion proteins and prolamin protein bodies (PB-I) stained with Rhodamine in developing endosperm were simultaneously analyzed by confocal microscopy. GFP fused with the N-terminal region (M1-S43) was mostly present in dilated ER till 12 DPA, in which single or multiple PB-I were frequently visualized. Subsequently, the GFP fusion protein was efficiently transported from the dilated ER to PB-II by 18 DPA. GFP in protein bodies was sequestered to the periphery and matrix that surrounds crystalloids containing glutelins. Since the signal peptide (M1-A22) is cotranslationaly cleaved, we conclude that an efficient vacuolar-targeting signal for α-globulin exists in the N-terminal region from Q23 to S43.

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Two major families of seed storage proteins, 2S-albumin and prolamin, have common regions termed A, B, and C, which respectively contain highly conserved CxxL, CCxxL, and PxxC motifs. In rice endosperm, the ABC-containing α-globulin and prolamins accumulate in the protein storage vacuole (PB-II) and ER-derived protein body (PB-I), respectively. Molecular mechanisms responsible for targeting storage proteins into PB-I or PB-II are still unclear. We found that partial α-globulin polypeptide (G21-Q111), containing the A and B regions, targeted GFP into PB-I. We hypothesized the protein-protein interaction between the B region and prolamins caused the accumulation of the fusion protein in PB-I. To clarify the role of CCxxL motif in the B region for PB-I targeting, we substituted each amino acid residue in the CCxxL motif. We report the analyses of their localizations and discuss the role of the CCxxL motif in protein accumulation in PB-I.

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Several reports have been shown that plants have the endocytotic pathway, although the details are still obscure. To clarify the mechanisms of endocytosis, we investigated endocytosis of BY-2 cells by visualizing endosomes with a fluorescent dye, FM4-64. When BY-2 cells were transferred to sucrose-starved or non-starved condition, FM4-64 was first bound to plasma membranes, then entered to small vesicles and finally transported to vacuoles. When BY-2 cells were treated with an inhibitor of papain family proteases, E64d, under sucrose-starved condition, FM4-64 was not transported to vacuoles. Instead, many vesicles with FM4-64 were accumulated in the cytosol. When BY-2 cells were treated with E64d under non-starved condition, no vesicles with FM4-64 were accumulated. These results suggest that papain family proteases were involved in endocytosis when BY-2 cells were starved.

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BY-2 cells transformed with a cytochrome B5 - red fluorescent protein fusion construct (CytB5-RFP). This protein distributed in the log-phase cells as punctate pattern. Upon reaching to the stationary phase, some of the RFP fluorescence was detectable in the lumen of the vacuole. To address what is the trigger of such the relocation of CytB5-RFP at the stationary-grown cells, we incubated the CytB5-RFP expressing cells at log phase in media with different composition. The result was that sucrose-free medium caused the relocation of RFP fluorescence into the vacuole. When the cysteine protease inhibitor was present in the starvation medium, the relocation of CytB5-RFP was blocked. These findings suggest that BY-2 cells have a mechanism to sense the amount of the nutrient in the medium and also suggest that the cells upon starvation activate a mechanism to transfer cytosolic organelles to the vacuole for degradation.

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Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. We have found that C-terminus of Apg8 is modified with lipid by ubiquitination-like reaction after cleavage of C-terminus by APG4 protease and this Apg8 lipidation system is essential for autophagy. Existence of APG8 and APG4 orthologues in Arabidopsis implicates that, in plant autophagy which is not well study, these molecules would function in a similar way. All of nine AtAPG8 and two AtAPG4 were expressed in almost all organs of Arabidopsis and induced by nitrogen-starvation. It has been known that yeast Apg8 is delivered to the vacuole by autophagy. So I made transgenic plants expressed GFP-AtAPG8 fusion protein and observed various tissues by fluorescence microscopy. GFP-AtAPG8 localized on some dot structures of cytoplasm and lumens of vacuole. Observation of GFP-AtAPG8 under nitrogen-starvation condition is in progress. We will also report behavior of AtAPG8 in double mutant of AtAPG4.

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Various types of mutants of precursor to the small subunit of RUBISCO (prSS) were designed and overexpressed in E. coli. The wild-type of Pisum sativum prSS has three Cystein residues, while these mutants have single Cystein residue at the various positions. These single-Cystein mutants (prSSC1) were modified with either the photoreactive crosslinker or the biotinylation reagent at the Cystein residue. The modifications didn't affect both the binding and the import activities of prSSC1. The protein close to one of the prSSC1 during binding step was crosslinked with prSSC1 via photoreactive crosslinker. Thus the identification of this crosslinked product is underway. On the other hand, one of the biotinylated prSSC1, incubated with avidin prior to the import assay, formed the protein translocation intermediate, in which prSSC1 was processed, but remained at the membrane. The characterization of the entire complex of this protein translocation intermediate is also underway.

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Most chloroplast proteins are encoded by the nuclear genome and synthesized in the cytosol as a preprotein which contains an amino-terminal transit peptide. Protein translocation across the outer and inner envelope membranes of chloroplast is mediated by the Toc and Tic complexes, respectively.
We reported previously that pea chloroplast Toc complexes consisting of at least Toc34, Toc75 and Toc159 were migrated at the sizes ranged from 700 to 800kDa on Blue Native PAGE. To elucidate molecular architecture of Toc complexes further, we determined the amount of each Toc component in the isolated chloroplasts and estimated their stoichiometry. Restricted proteolysis of the intact chloroplasts caused partial dissociation of some Toc components and revealed core components of the Toc complex. Also, we have obtained several Arabidopsis mutants of Toc genes. We are currently analyzing whether the mutant Toc complexes show any different molecular architecture as compared to that of the wild-type.

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At least 4 distinct protein targeting pathways to thylakoid membranes are known: 1) the Sec-dependent, 2) the signal recognition particle (SRP)-dependent, 3) the ΔpH-dependent, and 4) the spontaneous pathway. The SRP pathway, which is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) involves cpSRP43, cpSRP54 and a SRP receptor cpFtsY. The objective of this work is to gain the insight into the roles of cpFtsY on the biogenesis of chloroplast. We have succeeded in isolating csr (chloroplast SRP receptor) 1-1 and 1-3 mutants which possess a Mu transposon insertion into the cpFtsY gene. Null cpFtsY mutant csr1-1, showed a pale green phenotype, whereas csr1-3 was a low expression line which had pale yellow green leaves. Both mutants were seedling lethal. csr1-1 chloroplasts exhibited severe pleiotropic defects in the architecture of the thylakoid membrane.

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Importin β mediates the nuclear transport of proteins containing the nuclear localization signal (NLS). A trimeric complex, which consists of importin α /β and NLS-containing protein, is formed in the cytoplasm and migrates into the nucleoplasm through NPC in an importin β-dependent manner. To elucidate the mechanism of nuclear transport in plant cells, we isolated two cDNA encoding importin β from rice plant. Since the presence of two importin β genes was shown for the first time among a variety of eukaryotes, we are interested in the biological meaning of two importin βs. To address it, we carried three type of experiments: (1) Observation of the subcellular localization of them by using GFP fusion protein (2) GST pulldown assay to examine the interaction between rice importin αs and them (3) Northern hybridization analysis of their gene expression. The results indicate that two rice importin βs differentiate in their biological functions.

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In mammals, yeast and plant, the peroxisome biogenesis factors (peroxin) have been identified, but their functions remain unclear. Many peroxisomal proteins have peroxisomal targeting signal (PTS). The PTS are recognized by specific receptors. The PTS-containing proteins are delivered to peroxisomal membrane and translocated across the membrane via a translocation apparatus to the peroxisomal matrix.
So far, we attempted to identify peroxisomal targeting signal of pumpkin catalase (Cat1) by observing the cellular localization of the GFP-Cat1 fusion protein in transgenic tobacco cells and analyzed binding of Pex5p to Cat1 by yeast two-hybrid system. These results suggested that Cat1 is transported to peroxisomes by the PTS1 system. In this study, we focused on Pex10p and Pex12p. We isolated the cDNAs for tobacco peroxins and generated transgenic tobacco BY-2 cells lacking these peroxins. Furthermore, we analyzed the cellular localization of catalase in these transgenic tobacco cells.

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Chlamydomonas reinhardtii secretes arylsulfatase (ARS) to the periplasmic space in response to sulfate starvation. We have shown that the ARS transport is mediated by a mechanism similar to that observed in mammalian cells.
In this report, we studied the ARS secretion site by immunofluorescence using anti-ARS antibodies. Cells cultured in the sulfate-free medium had bright signals in a periplasmic region adjacent to the basal body, while control cells or oryzalin treated cells did not. This result suggests that ARS is transported by the microtubule-dependent polarized transport mechanism. To study effects of protein factors, we cloned a SAR1 homolog gene which encodes a GTP-binding protein required for the vesicle formation from the ER, and also constructed a dominant-negative version of the SAR1 gene.
We are doing the ultrastructural analysis of effects of reagents and the mutant SAR1 on the morphology of transported vesicles and organelle (Golgi and ER).

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Bacterial periplasmic proteins use the Sec machinery or the TAT pathway for translocation across the plasma membrane. Precursors transported by the Sec machinery are unfolded before translocation, while precursors transported by the TAT pathway have pre-bound cofactors or have been oligomerized. We have characterized periplasmic substrate-binding proteins (NrtA and CmpA) of ABC transporters in Synechococcus sp. PCC7942 that have 47% identity to each other. These proteins have characteristic signal peptide for the TAT pathway. We have shown that the *TAT* signal peptide was essential for the function of the NrtA protein. In this report, we show that the CmpA protein forms tetramer. These findings suggest that the TAT pathway is required for the tetrameric structure of the substrate-binding proteins and the tetrameric structure is required for the function of these proteins. We are characterizing the relationship between the TAT pathway and the tetrameric structure of these proteins.

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Bundle sheath cell (BSC) chloroplasts are more or less evenly distributed at immature region of an elongating leaf blade in finger millet (Eleusine coracana), an NAD-ME-type C₄plant, and the chloroplasts migrate to the vascular bundle and establish a centripetal arrangement after the elongation has stopped. The centripetal arrangement of BSC chloroplasts can be re-established after disturbance by centrifugation. Although it was reported that actin filaments concern in the arrangement, the molecular mechanism remains to be investigated. At the first step, we tried to visualize the actin cytoskeleton in leaf tissue of finger millet. We could visualize actin filaments that located around plasma membrane or connected to chloroplasts in BSC with immunofluoresence staining method. We also visualized actin filaments that encircled BSC chloroplasts in immature leaf blade or mature leaf after centrifugation. These actin filaments may mediate migration and arrangement of chloroplasts in bundle sheath cell.

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Root hairs are tubular extensions from root epidermal cells and elongate by tip growth. It has been suggested that actin cytoskeleton plays an important role in the process of tip growth. However, little is understood about a direct role of actin cytoskeleton in root-hair development. We isolated a novel Arabidopsis missense mutation in the ACT2 gene encoding a member of actin expressed in vegetative tissues. In the heterozygote, root-hair growth was inhibited and aerial parts of the plant showed dwarf phenotype. In the homozygote, any root hairs were not formed and growth of the plant was disturbed severely to result in seedling lethal. Transgenic plant expressing a mutant-formed ACT2 protein showed the phenotypes of the mutant as described above, indicating that the mutant protein have dominant-negative effects to cause morphological defects. We will discuss possible roles of actin cytoskeleton in root-hair development and cell expansion.

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Cell elongation is one of important factors for plant morphogenesis. In Arabidopsis, inflorescence stems elongate rapidly and straightly. To elucidate molecular mechanism of this process, we have isolated fiz1 (frizzy shoot1) mutant that shows frizzy or curling inflorescence stems and pedicels in Arabidopsis. The elongation of etiolated hypocotyl and root growth is also inhibited in this mutant. Moreover the root hair also developed abnormally. fis1 mutant is semi dominant. We also isolated fiz2 mutant similar to fiz1 in hypocotyl and root phenotypes. We found the missense mutation of ACT8 and ACT2 in fiz1 and fiz2, respectively. It is shown that actin family is strongly involved in cell elongation of shoot and root.

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γ-tubulin plays a role of microtubule nucleation in animal and fungal cells. We have shown that γ-tubulin predominantly localizes at the end of cortical microtubules in plants, but it is not proven that γ-tubulin is essential for microtubule nucleation and resulting cell morphogenesis. In the present study, we used virus-induced gene silencing system to examine roles of γ-tubulin in microtubule assembly, cell division and cell elongation.
Nicotiana benthamiana plants were infected with tomato mosaic virus vector with γ-tubulin partial mRNA. The infected plants continued to grow, probably because viruses cannot enter into the apical meristem. Leaves which expanded after spread of the viruses had many morphological defects including malformed epidermal cells and decreased number of palisade cells. Amount of γ-tubulin greatly decreased in the leaves. Control viruses without γ-tubulin mRNA sequence had no effect. Analysis of microtubule organization in such plants is in progress.

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The 3'-ends of the β-tubulin cDNA were amplified by 3'RACE and were grouped into five different isotypes, NTB1-5.
Individual isotype RNA levels during the progress of the cell cycle were quantitated by real time RT-PCR. The NTB1 mRNA kept the highest level throughout the cell cycle. NTB2 and 3 mRNAs were present at nearly equivalent levels, but the amplitude of variation of NTB2 was much higher than NTB3 mRNA. NTB4 and 5 isotypes were expressed at low levels. All of the five isotype RNAs decreased during or after mitosis.
The polyA attachment sites of NTB1-3 were found to be heterogeneous, giving different length of 3'-UTR for the same gene transcripts. The shortest mRNA of NTB2 and 3 existed around G2 to M phases.
β-tubulin isoforms in BY2 cells were separated into at least five spots on 2-D PAGE. Identification of each spot is now under investigation.

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The phragmoplast develops through repeating polymerization/depolymerization of phragmoplast microtubules, which are harmonized with the development of the cell plate. To know mechanisms that harmonize these phenomena, we examined the deposition of callose into the cell plate during the development of the phragmoplast in aniline-blue stained living BY-2 cells expressing YFP-tubulin. Until the leading edge of the expanding cell plate reached the parental cell wall, the central, microtubule-free portion of the cell plate was brightly stained with aniline blue. When the cell plate reached the wall, the marginal region of the cell plate where microtubules still remained became to be stained brighter, indicating that the callose deposition itself does not trigger the depolymerization of phragmoplast microtubules. These results together with the previous result that caffeine inhibits both the depolymerization of phragmoplast microtubules and the callose deposition suggest that both phenomena are triggered by a same signal.

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We have identified two types of myosins, 170-kDa and 175-kDa myosins, responsible for cytoplasmic streaming in BY-2 cells. On the basis of analysis of primary sequence of each myosin heavy chain gene, these myosins were classified into myosin XI. Localization studies using antibodies against 175-kDa myosin showed that this myosin is accumulated into phragmoplasts. The accumulation depended on actin filaments, because of the suppression by latrunculin B, while brefeldin A did not affect the accumulation, indicating that this phenomenon is not related to the transport of Golgi vesicles. We examined the effects of BDM to further explore the function of 175-kDa myosin in the phragmoplasts. Although the accumulation of this myosin into phragmoplasts was induced even in the presence of 30 mM BDM, the morphology of newly synthesized cell plates was appeared to be aberrant. We will discuss the role of 175-kDa myosin XI in the phragmoplasts.

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Butanedione monoxime (BDM) is well-known inhibitor of actomyosin interaction. However, it was reported that BDM up to 10 mM did not inhibit the cytoplasmic streaming of characean alga, well-known actomyosin driven system in plant (McCurdy, 1999). On the other hand, Tominaga et al. (2000) reported that the motile activity of myosin from lily pollen tube was inhibited when BDM at concentrations higher than 10 mM was applied. We examined if characean cytoplasmic streaming was inhibited by BDM at concentrations higher than 10 mM. It was found that cytoplasmic streaming of characean cells was inhibited by BDM at concentrations higher than 10 mM. This inhibition was reversible. Inhibition of characean actomyosin by BDM was confirmed by in vitro motility assay using crude characean myosin fraction. We concluded that characean actomyosin interaction is sensitive to BDM though its sensitivity is much lower than animal myosin.

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Cytoplasmic streaming in plant cells is the fastest known biological movement produced by actomyosin interaction. To reveal the molecular mechanism of plant myosin, we purified 175kDa myosin (myosin XI) from cultured tobacco BY-2 cells, its general morphology is similar to myosin V.
Forces and movement developed by single molecules of this myosin were measured by optical trap nanometry. Beads carrying just a single myosin molecule moved processively along actin filaments in 35nm steps. The maximal force measured was 0.5 pN, much smaller than that produced by skeletal muscle myosin IIs. The velocity produced by single 175kDa myosin was 7μm/s, correspond to the velocity of cytoplasmic streaming. This is the fastest known processive motors. Dwell-time analysis of single myosin molecules fitted the ATPase kinetics with ADP release being rate-limiting. These results indicate that this myosin XI is highly specialized for the production of fast processive movement with concomitant low force generation.

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The mammalian apoptotic protein Bax has been shown to induce cell death in plants as well as yeast and animal cells. This suggests parallels between PCD in plants and apoptosis in animals. To investigate the morphological changes involved in Bax-induced death in plants, we established the transgenic Arabidopsis possessing Bax and plastid- or mitochondria-targeted GFP. Upon DEX treatment, such plants exhibited morphological changes in mitochondria, the leakage of GFP proteins from chloroplasts, and etiolation of leaves. Interestingly, the transgenic plants treated with DEX under the dark condition showed similar events, but they did not present etiolation, which was the last step of plant cell death. Furthermore, non-photosynthetic Arabidopsis suspension cells expressing Bax were also induced to cell death. These results indicate that the developed chloroplast (or light) is not essential, and morphological changes of organelles occur in the early stage of Bax-induced cell death in plant.

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Although relatively little is known about the mechanistic details of cell death in plants, some aspects of the molecular machinery are conserved between plants and animals. We recently presented the evidence that the anti-apoptotic protein AtBI-1 (Arabidopsis Bax inhibitor-1) is biologically active in suppressing the lethal action of mammalian Bax in yeast and plant. Transgenic Arabidopsis expressing Bax exhibited marked cell death at the whole plant level. The plants possessing both Bax and AtBI-1 were able to maintain growth. Furthermore, tobacco suspension cells (BY-2) expressing AtBI-1 reduced hydrogen peroxide- or SA- induced cell death. To study the cellular localization of AtBI-1, a fusion protein with GFP was expressed in yeast and tobacco suspension cells. Microscopic analysis revealed that such protein was localized to the ER in each system. Involvement of ER-localized membrane protein in the response for oxidative stress will be discussed.

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Cultured cell line (GTH4) of F₁ hybrid between Nicotoana gossei Domin and N.tabacum L. grows normally at 37C, while dies at 26C. Quick loss of viability at 26C within 6 h was followed by slow cell death, resulting biphasic pattern, and eventually about 20% of cells were still alive at 72 h. Cell death was suppressed under anaerobic condition, suggesting involvement of superoxide intermediate (ROI) reduced from oxygen. The burst of O₂^- and H₂O₂ in the cell was detected as the peaks at 2 h and 0.5 h after transfer to 26C, respectively, while no increase at 37C. The burst of H₂O₂ was very low in the cell under anaerobic condition, but amount of O₂^- gradually increased in high level at later stage. Other findings and the results of pharmacological experiments including inhibitors of NADPH oxidase indicated that H₂O₂ was very probably involved in cell death process.

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Cell death of inviable F1 hybrid between Nicotiana gossei Domin and N.tabacum L. initiated from basal hypocotyl. Epidermal and cortex cells of hypocotyl were crushed, indicating loss of their protoplast. To more closely analyze collapse of cell during cell death, cultured cells (GTH4) from hypocotyl was used for further experiments. Loss of chloroplasts and mitochondria in the cells after transfer to 26C, condition which induce cell death, was observed by fluorescent microscopy, and number of cells showing low pH increased. GTH4 cell at 37C had high viability and complicate inner structure of which vacuole was divided into many vesicles by strands of cytoplasm. This structure was maintained just after transfer to 26C, but it turned into uniform density without any strands of cytoplasm. Finally, protoplasts were shrunk inside the cell. These findings indicated that execution of cell death is caused by quick collapse of vacuole under the lethal condition.

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During culture, Brassica napus leaf protoplasts become swollen and finally degrade. Both morphological and biochemical approaches revealed that the process through which the death occurred were subjected to apoptotic-like manner. This suggests that the recalcitrance of B. napus leaf protoplasts might be explained by apoptosis. The amounts of hydrogen peroxide and superoxide present in the medium during the isolation of leaf protoplasts from recalcitrant B. napus and regenerating Petunia hybrida were measured. This data showed that both active oxygen species (AOS) accumulated to greater levels in P. hybrida. Addition of radical scavengers to the isolation medium of B. napus protoplasts had no effect on the cell death. This suggests that neither of the two AOS generated during the isolation of leaf protoplasts might be involved in triggering apoptosis in B. napus leaf protoplasts. Intracellular ROS generated during the culture need to be further elucidated.

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Ozone, a major photochemical oxidant, induces salicylic acid (SA) synthesis prior to exhibit leaf damage. To identify the pathway for ozone-induced SA synthesis, the level of SA and mRNA for the enzymes shown to be participated in SA synthesis were investigated with ozone exposed tobacco leaves. The level of SA became a peak at 6h ozone-exposure. At the time, mRNA for phenylalanine ammonia-lyase (PAL) and chorismate mutase increased remarkably with concomitant increase in PAL activity. These phenomena were diminished in ozone-exposed transgenic tobacco that exhibited reduced ozone-induced ethylene production. The level of transcript for isochorismate synthase was decreased in both wild type and transgenic plants during 6h-ozone exposure. These results suggest that SA may be synthesized from phenylalanine in ozone-exposed tobacco and ozone-induced SA accumulation may be regulated by ethylene.

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Trichothecene mycotoxin family is synthesized by fungal plant pathogen Fusarium species. Trichothecene-contaminated cereals do harm to the health of animals and human. In animal cells, trichothecenes such as T-2 toxin trigger a ribotoxic stress response that activates JNK and p38MAPK and induces apoptosis. Trichothecenes are thought to be virulence factors in wheat head scab, their effects in plants are barely understood. We found that T-2 toxin also inhibited the protein synthesis in Arabidopsis suspension cells. Furthermore, T-2 toxin induced lesion formation in Arabidopsis leaves. T-2 toxin-induced lesions exhibited many dying cells, accumulation of callose deposition and autofluorescent materials, indicating that these lesions share many features with HR lesions. In addition, ROIs and SA/SAG were accumulated in T-2 toxin-injected leaves. In gel-kinase assays revealed that T-2 toxin activates the 47 and 44 kDa MAPK. Treatment of Arabidopsis with T-2 toxin induced expression of defense-related genes such as PR-1 and PDF1.2.

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A proteinaceous elicitor from a pathogenic fungus induced programmed cell death (PCD) accompanied by cytoplasmic condensation and shrinkage in suspension-cultured tobacco (BY-2) cells. Various plasma membrane ion fluxes such as Ca²⁺ influx, Cl^- efflux and H⁺ influx were induced within a few minutes after elicitation. Mitochondrial dysfunction was observed prior to PCD. Serial observations of vacuoles loaded with a fluorescent dye accumulated in the vacuolar lumen revealed that collapse of vacuoles were also induced prior to PCD. Possible functions of the vacuole in elicitor-induced PCD will be discussed.

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A proteinaceous elicitor, cryptogein induces hypersensitive response-like programmed cell death (PCD) in tobacco BY-2 cells. Effects of various protease inhibitors on this PCD were tested. Several kind of protease inhibitors significantly inhibited the PCD. Some of them were shown to inhibit early steps of the PCD signaling. Possible involvement of proteases in the elicitor-induced PCD will be discussed.

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Petal cell death may be based on the increase in the reactive oxygen species and the reduction in defense enzymes. Moreover, in aging process, breakdown of DNA and proteins is observed. In this research, we analyzed PCD parameters related to senescence such as petal oscillation ability, the anti-oxidization system, the amount of ethylene evolution, the DNA fragmentation, the hydrogen peroxide content. Although opening-and-closing movement of a petal was active in early stage, this ability declined from the middle stage of aging. DNA fragmentation was started at the same stage and DNase and protease activities also increased simultaneously. Following DNA fragmentation, hydrogen peroxide content increased and finally rapid evolution of ethylene was observed. In parallel to this, decrease of the ascorbate peroxidase activity started. So, the aging process in tulip petals is the same PCD process as in animal cells.

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