ACC synthase(ACS), which is a key enzyme in the pathway of ethylene biosynthesis, is regulated transcriptionally. However, recent studies have raised the possibility that ACS is regulated not only transcilptionally but also post--translationally. We found that enzyme activities of some ACS isozymes in cell were regulated by phosphorylation. LE-ACS2, which is tomato wound-inducible isozyme, was phosphorylated on serine-460 residue by protein kinase in the presence of Ca^<2+>. We prepared the antibody that recognized the phosphorylation state of LE-ACS2 using the synthetic phosphorylated peptide as antigen. Western blot analyses using this antitbody revealed that LE-ACS2 was immediately phosphorylated flier translation. Next, analyses by in vitro phosphorylation system demonstrated that not only LE-ACS2 but also LE-ACS1A, 3, 6, VR-ACS6 were phosphorylated, whereas LE-ACS4 was not phosphorylated. That is, ACS isozymes that were induced by external stimuli, such as wounding, touch, and exogenous auxin, were phosphorylated, where. as ACS isozymes that were induced by intemal stimuli, such as ripening: developmentally (genetically) program though the plant life, were not phosphorylated, suggesting the physiological meaning.

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Apical dominance is a phenomenon in which the terminal bud inhibits the outgrowth of axillary buds. The most widely accepted explanation of the mechanism of apical dominance is that auxin inhibits the growth of axillary buds, whereas cytokinin promotes the growth of axillary buds. It is thought that auxin acts to control the concentration of cytokinin derived from the roots. However, little is known about the molecular mechanism of dormancy in axillary buds. Auxin that is transported basipetally in stem from terminal buds, is not applied to axillary buds is required to inhibit outgrowth of the axillary buds. Based on this observation, we analyzed the genes that expressed in stem, especially node, before and after decapitation. We constructed the subtracted cDNA libraries from the second nod mRNAs before decapitation as tester and ones 3 hr atier decapitation as driver in pea (Pisum sativum L. CV. Alaska) seedlings, and vice versa. As the results from sequencing of more than 500 clones, we found that in node before decapitation auxin inducible genes including AUX/IAA families were expressed. On the other hand, in node after decapitation, adenylate isopentenyltransferase (IPT), which is thought to be a key enzyme of cytokinin biosynthesis, was expressed, implying that cytokinin was biosynthesized at node, not in roots after decapitation, and strongly suggesting that expression of some IPT was repressed by auxin. This result supported that cytokinin is naturally initiator of out growth of the axillary buds. We are investigating the expression patterns of these genes.

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In cultured green tobacco cells, cell growth and chloroplast development were maintained by phytohormone cytokinin. The cytokinin inducible gene (cig) were isolated by fluorescent differential display from the cultured green tobacco cells in early phase of the cytokinin stimulation. The expression of cig2 were stimulated by natural and synthetic cytokinin specifically compared to other phytohormone. Full length cDNA of cig2 suggested that it is a putative GDP/GTP exchange factor eIF2B which is a key regulator of translation initiation. But cig2 were could not complement the phenotype of yeast eIF2B disruptant, Antisense cig2 transgenic tobacco plants were altered timing of flowering. An Arabidopsis cig2 homolog (Atcig2) which have highly similarity to tobacco cig2 gene were also analysed. Transient expression of cig2::GFP and Atcig2::GFP were nulear-localized in tobacco leaf. Here we discussed possible function of cig2.

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3-hydroxy-3-methylglutaryl CoA reductase (HMGR) is a key enzyme in the mevalonate pathway. it is thought that HMGR regulates the biosynthesis of phytohormones and the farnesylation of some protein. It has been revealed that the inhibition of HMGR inhibits cell division by using tobacco BY-2 cells. When an inhibitor of HMGR is applied to whole plant, however, its effect has not been well understood. Here, we examined how the treatment of an inhibitor of HMGR, lovastatin, influences the growth of Arabidopsis thaliana plant. Seeds of Arabidopsis thaliana were cultured in MS liquid medium containing lovastatin 0 to lμM under the light or the dark. As a result, lovastatin inhibited elongation of roots and hypocotyls, enlargement of cotyledons, and the development of the inflorescence under the light, and also inhibited elongation of roots and hypocotyls under the dark. These results indicate that lovastatin inhibits not only cell division but also cell elongation. A treatment of geranyl pyrophosphate, gibberellin or brassinolide recovered the elongation of hypocotyls under the light but not under the dark. Therefore, there may be different mechanisms to elongate cells between under the light and the dark.

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Newly rapid determination methods for natural cytokinins in the plant was developed by LC-APCI/MS/MS. We separated and determinated 9 natural cytokinins and cytokinin derivatives. Mobile phase constituted 0.01M Ammonium hydrogen carbonate and 13%(v/v) acetonitrile. An analysis was done about the solvent which 80% extracted by the mothaol solvent from plants. And we used d_5-Zeatin for the internal standard, and a good straight line was shown about trans-cis-zeatin (t-Z, c-Z), trans- and cis-zeatin riboside (t-ZR, c-ZR), isopentenyladenin(iPA), isopentenyladenin riboside monophosphate(iPAMP), t-zeatin riboside monophosphate(ZRMP), t-zeatin-O-glucoside(ZoG) and t-zeatin-O-glucoside riboside(ZoGR), in the absolute quantity in the range of 10 pg from 1000 pg. These limit of detection (S/N=3) was about 3 pg (t-Z).

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For a higher sensitivity, the parameters of liquid chromatography-mass spectrometry-selected ion monitoring (LC-MS-SIM) usirtg atmospheric pressure chemical ionization (APCI) were examined. The area and height of a peak as sensitivity, the full width half maximum (FWHM) and the actual scan-time which was required for the scanning were measured. The suitable parameters for higher sensitivity on measurement limit level were as follows: 1) The scan-tlme might correspond to the value of FWHM. 2) The countling-number was over about 7,000. 3) The scan-range might correspond to a decimal fraction of the molecular weight of the sample substance. The results showed that the value obtained from repeated sampling-point-number was a total amount, that the value obtained from repeated counting-number was a mean.

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Phytosulfotdne (PSK) is the peptidyl plant growth factor and found in the culture medium from many dicot- and monocot- plants. But the function of PSK in whole plant is little known. In order to clarify the function of PSK in Arabidopsis thaliana ecotype Columbia plant, we investigated the effect of exogenously applied PSK to whole plant. Arabidopsis was cultured on Murashige and Skoog's (MS) medium containing PSK. On the mecdium containing PSK, the number of root tips increased significantly. The length of main root was longer than that of control at early stage (7 days after sowing), but not differentr at later stage (13 and 18 days after sowing). These results indicate that PSK enhances root growth and/or root branching. Expression analysis of PSK prectmsor genes was performed by RT-PCR. AtPSK3 was found to express in most organs examined, especially in flower stalk, flower, and silique. Expression of AtPSK2 was weak and limited in flower stalk. From these results, AtPSK3 may be a main PSK precursor gene in Arabidopsis. FXp. ression profile of AtPSK3 indicates that PSK has a role for stimulation of cell division in whole plant as same as in cultured cells, and might have another function in flower stalk, rosette leaf and mature seed. The present work indicates that PSK has a function not only in cultured cells but also in whole plant.

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Treatment of plant cells with an endogenous mitogenic peptide, phytosulfoldne (PSK), results in cell prolifera- tion in the presence of plant hormones. In the present study, we purified the carrot PSK receptor proteins using PSK-Sepharose column, and isolated 120- and 150 kD candidate proteins at 40% recovery. Molecular cloning of the corresponding full length cDNA based on the internal peptide sequence information revealed that these proteins have typical features of the receptor kinases.

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Phytosulfokine (PSK), a sulfated mitogenic peptide found in plants, strongly promotes proliferation of plant cells at very low concentrations. In this work, we describe cDNA cloning of Daucus carota PSK precursor proteins. PCR amplification of carrot cDNA library using a degenerate primer based on PSK amino acid se-quence and a vector T3 primer produced 370-400 bp DNA fragments. Subsequent screening of the cDNA library resulted in the isolation of two cDNA clones (DcPSK1 and DcPSK2) containing coding sequence for PSK and sulfation signals.

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Root culture of Menispermum dauricum exudes a germination stimulant for root parasitic weeds. Active ingredient was extracted from the culture filtrate and purified by sephadex LH-20 column chromatography and preparative HPLC. On the basis of physicochemical parameters, the germination stimulant was identified as (+)-strigol. Ten liter culture filrate yielded ca. 38 μg strigol.

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Fluridone added to the incubation media during conditioning induced seed germination of Slriga asiayica in the absence of a germination stimulant, strigol in the fiuridone-treated seeds, most of the radicles did not elongate and looked like haustria. Furthermore, fiuridione treatment increased sensitivity of the seeds to strigoi. Norfiurazon, another inhibitor of carotenoid biosynthesis, also increased the sensitivity but no germination occurred in the absence of strigoi. By contrast, uniconazole strongly inhibited these processes.

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A rice seedling (3-5day old) from a seed with husk has a long seminal root and four or five short crown roots, on the other hand a seedling from a seed without husk has longer crown roots and a shorter seminal root. When rice seeds without husks were grown on agar gel suspended with powdered husks or supplemented with water extracts of rice husks, the elongation of seminal roots were observed. The promotion rates were dependent on the amounts of husks added or extracts supplemented. These facts suggest that husks have plant growth substance(s). Isolation of plant growth substance(s) was attempted to afford an active substance (0.5mg)

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The main material used for mushroom production is sawdust, but the shortage of sawdust is now a serious problem caused by the rapid increase in demand. We cultivated Tamogitake and Hiratake mushroom (Pleurotus cornucopiae and PJeurotus ostreatus, respectively) on a glass-beads medium, instead of a saw-dust medium, with supplements of rice bran as nutrient supplement. The fruiting occurred on the glass-beads media, but the yield of mushrooms was lower than that of saw-dust media. When cellulose was added to a glass-beads medium with rice bran, the fruit-body formation was significantly improved. The fruit-body yield of mushroom increased with the increase of cellulose added to the medium. Vermiculite also significantly improved the fruit-body formation, when added to a glass-beads medium with rice bran. Vermiculite is not nutrient for the mushrooms. However, cellulose and vermiculite have high water-holding capacity force. Therefore, these results suggest that the high-water holding characteristic of cellulose powder are very suitable for mushroom cultures.

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Arbuscular mycorrhiza is the most widespread form of symbiotic associations between soilborne fungi in the order of Glomales and plant roots. This symbiosis confers benefits to tile host plant's growth and development through the acquisition of phosphate from the soil by the fungi. It was shown that the infection and intraradical growth of AM fungi are markedly inhibited in plant roots grown under high phosphate conditions possibly through phosphate-mediated physiological alterations of the roots. In order to better understand the role of secondary metabolites in the development and regulation of AM symbiosis, we investigated plant secondary metabolites that show a significant change in their levels during arbuscular mycorrhiza formation. So far, we had showed that a C-glycosylflavone, identified as isovitexin 2"-O-β-glucoside, significantly increased in melon roots grown under phosphate-limited condition, whereas no accumulation was observed in mycorrhizal roots and high phosphate-supplemented roots. Time course experiments showed that the compound started to accumulate from 30 days postinoculation and reached to 3.3 μg/g fresh weight at 40 days postinoculation in control roots. Its accumulation closely correlated with growth retardation of the plants observed from 30 days postinoculation. The flavonoid significantly stimulated mycorrhizal colonization in melon roots under low and high phosphate conditions when applied to the soil. These findings suggested that the phosphate deficiency-induced flavonoid is involved in the mechanism for phosphate-mediated regulation of AM fungal colonization in melon roots.

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Lemna paucicostata 441 that had been subjected to drought, heat or osmotic stress, released a substance into the surrounding water (Takimoto et alo 1994). This substance incubated with (-)- norepinephrine (NE) has strong flower-inducing activity in Lemna paucicostata 151. Yokoyama et al. (2000) clarified chemical structure of the substance and named FIF from the meaning of flower-inducing factor. In the present paper, we examined the effects of low temperature, salt UV or smell stress and the treatment of supersonic waves on the prodaction and release from Lemna paucicostata 151 of flower-inducing factor. The plants subjected to these stresses were floated on distilled water (WS) and WS was incubated with NE. WS incubation with NE induced flowering of Lemma paucicostata 151. Therefore, Lemna plants that had been subjected to low temperature, salt or smell stress and the treatment of supersonic waves also releases a flower-inducing factor capable of activating NE into the surrounding water. The flower-inducing factor was analyzed by HPLC on a CAPCELL PAK C18 column. Only one major peak was found and the retention time coincided with that of FIE suggesting that the flower-inducing factor is FIF.

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In Brassica self-incompatibility, recognition between pollen and the stigma is controlled by the Slocus which contains three highly polymorphic genes: S-receptor kinase gene (SRK), S-locus protein 11 gene (SP11) and S-locus glycoprotein gene (SLG). Here we show that a single form of SP11 of S8-haplotype (S8-SP11) specifically binds the stigma membrane of S8-haplotype to induce autophosphorylation of SRK8, and that SRK8 and SLG8 together form a high-affinity receptor complex for S8-SPll on the stigma membrane.

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In the course of our screening search for the regulators of plant reproductive development among fungal metabolites, using bioassay method with Arabidopsis thaliana, suitable for analysing floral structure and function, we found the presence of two inhibitors of pollen development in cultures of the fungus Aspergillus terreus. Bioassay-guided fractionorion led to isolation of the active compounds and succeeded in the structure determination of asptemc acid (1) and 6-hydroxymellein (2). 1 and 2 inhibited the pollen development at a concentration of l0 mg/l. The microscopic examination of pollen development suggested that the inhibition by the treatment with 1 might be caused at meiosis.

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In the course of screening for new bioactive compounds from plant-associated actinomycetes, we found that new antibiotics cedarmycins A (1) and B (2) (Fig.1) were produced in the fermentation broth of an actinomycete strain TP-A0456. The producing strain was isolated from the stem of Crypyomeria japonica collected in Toyama and identified as Streptomyces sp. based on the taxonomic study. The new compounds were purified by HP-20 resin, silica gel and preparative HPLC, consecutively. The structures of cedarmycins A and B were determined by spectroscopic methods such as 2D-NMR and FAB-MS as shown in Fig.1. These compounds showed antibiotic activity against Candida glabrata IFO 0622 with the MIC of 0.4 μg/ml, comparable to that of amphotericin B.

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Cultured broths of actinomycetes isolated from plants and rhizospheres were examined for plant growth-regulating activities in four bioassays. Approximate six hundred strains of streptomycetes and rare actinomycetes isolated from plants and rhizospheres were used in this experiment. Among these strains, the inhibitory effects of strain T-13, S-42 and S-59 were found in lettuce germination assay with the more potency than ABA. In rice germination seedling growth assay, strains T-5, T-6, T-13 and S-17 showed the inhibition in rice shoot elongation, and the root elongation was strongly inhibited by strain T-5 and S-17. Strain T-34 stimulated the callus growth of soybean hypocothle sections and retarded the chlorophyll degradation in rice leaf segments. These results indicate that the plant-associated actinomycetes may play the physiological function in whole plants through production of plant growth-regulating substances.

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Streptomyces galbus R-5, of the intense antagonistic activity against Pestalotiopsis sydowiana and Phytophthora cinnamomi, major pathogens of rhododendron, was used as a biocontrol agent. An appropriate amount of the mycelial suspension of R-5 was spread on the surface of multiplication medium in glass flasks where tissue-cultured rhododendron seedlings were growing The seedlings treated and untreated with R-5 were incubated a further 10 days in an illuminated incubator. The following inoculation tests were performed to examine whether resistance was induced in R-5 treated seedlings. (i) A mycelial disk of P.sydowiana was placed on the upper 4th leaves of treated and untreated seedlings, followed by incubation for 14 days. (ii) R-5 treated and untrealed seedlings were transplanted to soil in plastic boxes that was amended with an appropriate number of mycelial disks of Phy. cinnamomi. In 92% of R-5 pretreated seedlings only inoculated leaves showed a brownish symptom, but mycelia never spread to other leaves from the inoculated leaves. In 36% of untreated seedlings, leaves lower and upper than the inoculated leaves brownish and in addition 54% of seedlings entirely died. In the transplant experiment, 34% ofuntreated seedlings were killed by Phy. cinnamomi, while only 15% of treated seedlings became dead within a few weeks after transplant. These results show that pretreatment of tissue-cultured rhododendron seedlings with R-5 induced an intense resistance to both pathogens and that such an resistance is effective in tissue-culture flasks and even after transplanting in soil.

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Life cycle of plants is under the control of gravity. Whether or not the complete life cycle can be achieved under microgravity condition in space has not yet been elucidated, because many experiments done under microgravity condition in Cosmos and Space Shuttle have been carried out by comparing plant development between microgravity conditions in space and 1 g Condition on earth. The International Space Station (JSS) under construction is useful for elongation of the problcm, because Japanese Experiment Module (JEM) in ISS has plant growth faccilities to assess the effect of gravity on the life cycle of Arabidopsis plants in JEM of ISS. At present, we are aseptically eulturing Arabidopsis plants in the plant growth boxes (about 5 cubic cm), equipped with an illumination plate installed red and blue LED as light source. In the box illuminated from the side of the box, the hypocotyl grew toward lhe light source and elongated a little. In the box illuminated from the upper, the length of hypocotyls, the form and color of rosette leaves, and the growth rate of plants showed remarkable changes depending on light intcnsities.

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Nitrogen-trace elements mixed fertilizer containing 5-aminolevulinic acid (5-ALA), "PENTAKEEP" greately increaced dry weight of radish (Raphanus sativus vat. radicula). Data analysis by fitting to a response surface suggests that nutritional conditions may affect the growth promotive efficacy of 5-ALA on the fields.

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We already reported that the seasonal changes in the spectral composition of solar radiation might be one of the important factors in controling the flo-wering of long-day plants and short-day plants. So, we manufactured two type of photo-selective sheets. One type[N.S.Blue-sheet] works to become big the blue/red ratio and another type[N.S.Red-sheet] works to become small the blue/red ratio. N.S.Blue-sheet promoted the flowering in short day plants and N.S.Red-sheet promoted the flowering in in long-day plants. In this report, we verified that these photo--selective sheets had the different effects on vegetative growth in each season. We suggest that the effects of a photo-selective sheet on vegetative growth and flowering depend on season for using.

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The final goal of the present study is to remediate the heavy metal polluted soil by plants. For this purpose, seedlings ofArabidopsis thaliana were treated with 500μM Cd, and transcript populations that changed their levels were screened by a fluorescent differential display method. Among 170 cDNAs initially identified, 3l were further characterized for their identity, expression profile and response to other stresses. 10, 12 and 15 genes were related to signal transduction, protein denaturing stress, and responses to active oxygen species, respectively. They were glutathione S- transferase, monooxygenase, adenylylsulfate reductase, 2 HSPs, 4 transcription factors, 3 protein kinases and a putative calmodulin. Transcripts of some signal transduction factors were found to decrease in the presence of staurosporine, indicating that protein phosphorylation is involved in Cd stress signal transduction pathways. On introduction into yeast cells, 2 genes, encoding ATMEKKI and a putative farnesylated protein that has two metal binding motifs, endowed marked toleration of Cd toxicity. These results suggest that oxidative stress and protein denaturation are important components of Cd toxicity, and that, to cope with such stresses, plants activate a set of genes involved in metal detoxification, protein refolding and wound healing. The results also suggested temporarily and spatially well regulated protein phosphorylation and activation of transcription factors, accompanied by their transcription.

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In recent years, many reports have described expression of antibodies or antibody fragments in plants for various applications. We constructed a single-chain Fv (scFv) against chlorpropham, a preemergent herbicide, from the monoclonal antibody (mAb) producing hybridoma cell, K2158. The scFv showed the binding activity by ELISA. The binding affinity of the scFv was lower than that of the original mAb, but still high enough, and the scFv showed the similar binding specificity to the mAb. We introduced the scFv gene into tobacco. The expression vectors were designed to target the scFv into ER, cytoplasm or apoplastic space. The vectors for the expression of the scFv as a glycosyl-phosphatidyiinositol (GPI) anchored protein were also designed. So far, some transgenic plants have regenerated. We will analyze these plants to confirm the integration of the transferred gene and determine the expression level.

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M_1 seeds of tobacco which had been igadiated with N-ions, were used in the resistance test against BZB-OH; a herbicide inhibiting 4-hydroxyphenylpyruvate dioxygenase. Two strains of M_3 progeny showed strong resistance against BZB-OH and sulcotrione. In contrast, they were not resistant to norfiurazon, an inhibitor of phytoene desaturase.

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indanofan herbicide having oxirane moiety controls gramineous weeds in paddy rice, but its phytotoxic mode of action is not known yet. We investigated inhibition of very-long-chain fatty acid (VLCFA) formation by indanoran and its relative compounds, and found that they strongly inhibited VLCFA biosynthesis, exhibiting approx. I_50-values of 10^<-6> M〜10^<-7'gt;M. However, the compound 6 including CH_2-group except the oxirane moiety did not inhibit VLCFA biosynthesis even at 10^<-5>M, suggesting the oxirane group is essential to inhibkion of VLCFA elongation. Both indanofan and tridiphane, reference compound, inhibited more strongly C20:0 elongation (exhibiting approx. I_<50> = 10^<-6>M〜10^<-7>M), than C18:0 elongation (approx. I_<50> = 10^<-5>M〜10^<-6>M), possibly indicating that these compounds inhibited elongase II more strongly than elongase I.

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A variety of 2-aryl-l,3,4-oxadiazole-5-thiols were prepared and evaluated for their inhibitory activity on cinnamic acid 4-hydoxylase(CA4H) from Populus kitatcamiensis, which has been isolated and expressed in yeast. 2-Phenyl-1,3,4-oxadiazole-5-thiol(1) showed of 95.0% and 51.9% inhibitory activity at 10 and IμM, respectively. Studies on the structure -activity relationship indicated that benzene ring at 2-position of 1,3,4-oxadiazole was significant for activity. The introduction of chlorine atom at 3-position on the benzene ring increased the activity compared with that of 1, indicating 85.3% inhibitory activity at 1μM (I_<50> = 0.21μM). While the introduction of some substituents at 4-position on the benzene ring decreased the inhibitory activity.

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Cyclodextrins(CDs) are in a group of structually related oligosaccharides. The important characteristics of the cyclodextrin molecules are their cylindrical shape, somewhat hydrophobic central cavity and hydrophfiic outer surface. So, CDs is capable of forming inclusion complexes with many organic and inorganic compounds. The inclusion compounds enhance solubility, chemical stabihty and bioavailabiiity. In this study, one of the aims is to determine the usefulness of β-CD as an accelerator for plant germination and growth. The other aims are to determine the bactericidal activity of several CD-iodine complexes(CDIs), and to determine the deodrant activity of CDIs to some odor ingredients compared with β-CD. β-CD enhanced germination ratio and plant growth of Hatsuka-daikon(akind of radish). Bactericidal activity of CDIs was that, CDIs at concentration from 5ppm to 40ppm inhibited the proliferation of various bacteria. It is also shown that CDIs markedly decreased various odor ingredients better than β-CD.

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