Kozo Ishizuka

Structure-activity relationships of benzoxazolinones with respect to auxin-induced growth and auxin-binding protein

Abstract The structure-activity relationships of the naturally occurring auxin-inhibiting substance, 6-methoxy-2-benzoxazolinone, isolated from maize shoots, and its artificial analogues with respect to auxin activity and membrane-bound auxin-binding protein, were studied. 6-Isobutoxy-2-benzoxazolinone strongly inhibited auxin (1-naphthylacetic acid, NAA)-induced growth of etiolated maize coleoptile segments. 6-Isopropoxy-2-benzoxazolinone, 6-ethoxy-2-benzoxazolinone and 6-methoxy-2-benzoxazolinone showed significant inhibitory activity. However, 2-benzoxazolin-one and 6-benzyloxy-2-benzoxazolinone did not inhibit auxin-induced growth. On the basis of these data, it seemed reasonable to assume that the auxin-inhibitory activity was enhanced by increasing the side chain length at position C-6 on the 2-benzoxazolinone, although the attachment of a ring at C-6 destroyed activity. Competition by benzoxazolinones with an alkoxy group at C-6 with 3 H-NAA at auxin-binding protein(s) isolated from endoplasmic reticulum (ER) membrane of maize shoots showed a positive correlation with their physiological effects. However, since the inhibitory activity of the benzoxazolinones for auxin-receptor(s) binding was small compared with their physiological activity, the benzoxazolinones may contribute to inhibition of auxin-induced growth through interference with other auxin-receptors.

The occurrence in plants of the growth inhibitors, the raphanusanins

Abstract The growth inhibitors, cis - and trans -raphanusanins are widespread in the genus Raphanus . However, no raphanusanins were detected in the other plant species tested. Effects of raphanusanins on the action of plant hormones, indole-3-acetic acid (IAA), gibberellic acid (GA 3 ) and benzyladenine (BA) were also studied using various bioassays. Raphanusanins significantly inhibited IAA activity in the Avena curvature test but had no efrect in the Avena section test. BA activity was strongly inhibited by raphanusanins in both the radish cotyledon and the Amaranthus betacyanin bioassays. In contrast, GA activity was not influenced in the range of concentrations of raphanusanins tested either in the leaf-sheath bioassay of dwarf rice or the rice endosperm bioassay. The present results suggest that raphanusanins cause growth inhibition through suppressing the action of auxin and/or cytokinin.

Plant regeneration through somatic embryogenesis in barnyardgrass, Echinochloa oryzicola vasing

Abstract Callus was obtained from leaf sheath segments in barnyardgrass (Echinochloa oryzicola Vasing) cultured on Murashige and Skoogs (MS) inorganic medium containing 2, 5 or 8 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3% (w/v) sucrose. Callus was subcultured for over 18 months on the medium containing vitamins and 2,4-D, and was then transferred to media containing cytokinin or auxin at various concentrations. Shoot formation and root formation, through somatic embryogenesis from the callus, were stimulated by addition of cytokinin and auxin, respectively. Regenerated plants were diploid and tetraploid. Diploid plants were green and grew to maturity in soil. Tetraploid plants were albino and did not grow into mature plants.

Lepidimoide promotes light-induced chlorophyll accumulation in cotyledons of sunflower seedlings

Abstract. The effect of disaccharide lepidimoide on light-induced chlorophyll accumulation was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings and detached cucumber (Cucumis sativus L.) cotyledons. From studies on the structure-activity relationships of lepidimoide, its analogs, and sucrose with respect to light-induced chlorophyll accumulation in the cotyledons of sunflower seedlings, both lepidimoide and the free carboxylic acid of lepidimoide (lepidimoic acid) showed the highest promoting activity, whereas the hydrogenated lepidimoide, which lacks a double bond in the C4, 5 position in uronic acid, showed lower activity than lepidimoide; however, sucrose exhibited very weak activity. These results suggest that lepidimoide acts as a new type of plant growth regulator, not simply as a carbon source providing energy. Lepidimoide promoted not only light-induced chlorophyll accumulation in sunflower cotyledons but also light-induced 5-aminolevulinic acid content, which is considered to be a rate-limiting step in chlorophyll biosynthesis. Lepidimoide with cytokinin stimulated the accumulation of chlorophyll and 5-aminolevulinic acid additively. In detached cucumber cotyledons, lepidimoide also promoted light-induced chlorophyll accumulation. These results indicate that lepidimoide, in cooperation with cytokinin, causes light-induced chlorophyll accumulation in the cotyledons of several dicot plant species by affecting the level of 5-aminolevulinic acid.

Trophodynamic structure of a swampy bog at the climax stage of limnological succession. II: Bacterioplankton dynamics

Matsumi-ike Bog is a swampy bog that has attained the climax stage of limnological succession. The dynamics of bacterioplankton were studied in relation to the influence of environment factors. The water temperature was shown primarily affect bacterial dynamics as a limiting factor throughout the year. The second most profound factor on the dynamics was the concentration of dissolved organic carbon (DOC) when dead phytoplankton were the major DOC source in the bog, whereas phosphate concentration was most important in the periods when the dead emergent plants were responsible for the DOC supply.

Light involvement in oxyfluorfen-induced protoporphyrin IX accumulation in several species of intact plants

Abstract Effect of light on diphenyl ether herbicide oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-trifluoromethylbenzene]-induced protoporphyrin IX accumulation in intact plants was investigated using five plant species which have different sensitivity to the herbicide. During 24 hr of dark incubation after 2 hr of shoot treatment with 1 μ M oxyfluorfen, protoporphyrin IX accumulation occurred in buckwheat and barnyard grass. When the plants were transferred to light after the dark incubation, a significant increase in protoporphyrin IX content was observed in rice and radish. In contrast, protoporphyrin IX was continuously decreased in buckwheat and barnyard grass in the light. Buckwheat was most susceptible to oxyfluorfen and its phytotoxic effect was detected after 2 hr of light exposure. In the plant, it was suggested that dark-accumulated protoporphyrin IX was involved in the rapid appearance of phytotoxicity in the light. By the determination of the accumulation rate in the light, it was shown that oxyfluorfen-induced protoporphyrin IX accumulation was increased by light in all plant species. This indicates that light acts as an enhancer of the accumulation in intact plants. However, profiles of protoporphyrin IX were considerably different among the tested species. Protoporphyrin IX contents in intact plants and the degree of phytotoxicity by oxyfluorfen were not always correlated.

Structure-activity relationships of raphanusanins and their analogues

Abstract The structure-activity relationship of raphanusanins A and B {(3 R *,6 R *)- and (3 R *,6 S *)-3-[methoxy(methylthio)methyl]-2-pyrrolidinethi

73 Cross-Tolerance of Oxyfluorfen-Tolerant Soybean Cell Line to Other Protoporphyrinogen Oxidase-Inhibiting Herbicides

Characterization of cross-tolerance of selected oxyfluorfen-tolerant and non-selected (normal) soybean cell lines to protoporphyrinogen oxidase (Protox) inhibiting herbicides (oxyfluorfen, 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl) benzene; bifenox, methyl 5-(2, 4-dichloro-phenoxy)-2-nitrobenzoate; nitrofen, 2, 4-dichloro-1-(4-nitrophenoxy) benzene; and oxadiazon, 3-[2, 4-dichloro-5-(1-methylethoxy) phenyl]-5-(1, 1-dimethylethyl)-1, 3, 4-oxadiazol-2-(3H)-one) or acetolactate synthase (ALS) inhibiting herbicide (bensulfuron-methyl, methyl α-[[3-(4, 6-dimethoxypyrimidin-2-yl) ureido] sulfonyl]-o-toluate, BSM) was determined. The sensitivities of both cell types to oxyfluorfen were compared by determination of the growth rates and target enzyme inhibition using various Protox inhibiting herbicides. On the I50 values of growth, the tolerant cells showed about 100-, 200-, 5, 000-, and >30, 000-fold more tolerance than the normal cells to oxyfluorfen, oxadiazon, bifenox, and nitrofen, respectively. The cells were found to have cross-tolerance to all Protox inhibiting herbicides tested, however, a lack of cross-tolerance (<1.0) to BSM was observed. Determination of the inhibition on Protox activity showed that the sensitivity of the enzyme preparations between the two cell types differed about 15-fold to oxyfluorfen, 30-fold to oxadiazon, 45-fold to bifenox, and 100-fold to nitrofen. There was a positive correlation between the tolerance ratio determined by growth rate and that at the enzyme level.

Bensulfuron Methyl Effects on Amino Acid and Protein Levels in Cultured Rice Cells.

Effects of bensulfuron methyl (BSM) on amino acid and protein content were compared in normal and 10-5M BSM tolerant rice cells. Culture in the presence of BSM led to a dramatic decrease in the level of branched-chain amino acids to 25-49% of untreated in normal rice cells over 48hr, while they increased in BSM-tolerant cells. The other amino acids in exposed normal cells increased substantially, but those amino acids in tolerant cells changed relatively little. The inhibition of protein biosynthesis by BSM appeared to cause a decreased level of protein content in normal cells, while almost no effect in tolerant cells. The feedback inhibition by one or more of the end-products, valine, leucine and isoleucine, of acetolactate synthase (ALS) from normal and tolerant cells was also investigated. The activity of extractable enzyme from both cell types was feedback inhibited by the three amino acids Val+Leu, Val, Leu, Ile: 39, 41, 45, 58% of control at 5mM, but the enzyme of tolerant cells had less feedback sensitivity: 49, 55, 57, 80%, respectively. Based on these findings, a high level of branched-chain amino acid biosynthesis by the BSM-insensitively modified ALS and less sensitivity of the enzyme to branched-chain amino acids may provide protection against BSM in the tolerant cells.

Absorption and Metabolism of Bensulfuron Methyl in Resistant Carrot Cells.

Carrot (Daucus carota L. cv. US-Harumakigosun) cell suspension cultures adapted to grow in 10-8M of bensulfuron methyl (BSM) were cultured in LS-medium by subculturing every 7 days. The absorption and the metabolic change of 14C-BSM in resistant and normal cells were investigated. 14C-BSM at 10-6 or 10-7M were added to the cells 4 days after subculture at their linear growth phase. 14C-BSM was rapidly absorbed during the initial 30-60min. The absorption rates of BSM by normal and resistant cells were not significantly different, though they were maximally 22 and 18% of the initially added amount at 1hr, respectively. For metabolism study, 14C-BSM and its metabolites were extracted by 80% acetone. Aqueous fraction after evaporation was partitioned by dichloromethane at pH 7 and then 3. 14C compounds of each fraction were analyzed with TLC. 14C-BSM was partially metabolized to O-demethyl BSM, sulfonamide, homosaccharin, water soluble and residual compounds in both cell groups to nearly the same extent. These results suggested that the absorption and metabolism of BSM did not much differ between the normal and resistant carrot suspension cultured cells.