Fusicoccin, an inhibitor of brassinosteroid-induced ethylene production

Fusicoccin was evaluated for its effects on brassinosteroid (BR), indole-3-acetic acid (IAA) and BR + IAA-induced ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC-synthase production by etiolated mung bean ( Vigna radiata L. Rwilez cv. Berken) hypocotyl segments. Fusicoccin inhibition of ethylene and ACC production induced by 2 μ M BR started at concentrations as low as 0.05 μ M . Maximum inhibition occurred at a 1 μ M concentration with no further inhibition at higher concentrations tested. Fusicoccin (1 μ M ) was effective in the inhibition of BR-induced ethylene, ACC and ACC-synthase production at low and high concentrations of BR.
Fusicoccin at concentrations as high as 2 μ M had no effect on ethylene and ACC production promoted by low concentrations of IAA (1 to 10 μ M ). When higher concentrations (100–1000 μ M ) of IAA were used, fusicoccin (1 μ M ) had an inhibitory effect on ethylene and ACC production. Interestingly, fusicoccin (1 μ M ) had little or no effect on ACC-synthase promoted by high concentrations of IAA (1000 μ M ).
When BR and IAA were used in combination, fusicoccin inhibited ethylene and ACC production at concentrations as low as 0.05 μ M with maximum inhibition occurring at 0.5 μ M . At a 1 μ M concentration, fusicoccin was effective in inhibiting the synergistic stimulation of ACC-synthase promoted by BR and IAA.     

Ca2+ acts synergistically with brassinosteroid and indole-3-acetic acid in stimulating ethylene production in etiolated mung bean hypocotyl segments

Brassinosteroid (BR) and indole-3-acetic acid (IAA) were used in combination with Ca²⁺ in order to determine if there was a synergistic effect in the stimulation of ethylene production in etiolated mung bean ( Vigna radiata L. Rwilez ev. Berken) hypocotyl segments. Ca²⁺ was found to act synergistically with BR. IAA or a combination of the two in promoting a stimulation in ethylene production. EDTA, which chelates Ca²⁺, greatly reduced the effectiveness of calcium salts in promoting ethylene production in the presene of either BR, IAA or a combination of the two. Neither K⁺, Mg²⁺ nor Mn²⁴ (chloride salts) acted synergistically with BR and IAA.     

Evidence that brassinosteroid stimulates auxin-induced ethylene synthesis in mung bean hypocotyls between S-adenosylmethionine and 1-aminocyclopropane-1-carboxylic acid

Brassinosteroid (BR) stimulation of auxin-induced ethylene production and the particular step at which BR acts to promote such synthesis were studied in mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. Increasing concentrations of methionine alone and in combination with 3 μ M BR and 10 μ M IAA had a minimal effect on ethylene production. With increasing concentrations of 1-aminocyclopro-pane-1-carboxylic acid (ACC), however, ethylene production increased. BR or IAA further enhanced ethylene production with maximum rates occurring when these compounds were added together with ACC. The addition of 10 μ M CoCl₂ in conjunction with BR and/or IAA resulted in 85–97% inhibition of ethylene production. When 20 μ M cycloheximide was used in conjunction with BR and/or IAA there was a complete inhibition of ethylene production. Total inhibition also resulted when 1.0 μ M aminoethoxy-vinylglycine (AVG) was used in combination with BR and/or IAA. AVG alone had no effect on ACC conversion to ethylene.     

Roles of auxin and gibberellic acid in growth and maturation of epicotyls of Vigna angularis: Cell wall changes

The effects of auxin and gibberellic acid on cell wall composition in various regions of epicotyls of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) were investigated with the following results. (1) Young segments excised from apical regions of the epicotyl elongated in response to added 10⁻⁴ M indole-3-acetic acid (IAA). When the segments were supplied with 50 m M sucrose, the IAA-induced segment growth was accompanied by enhanced overall synthesis of cell wall polysaccharides, such as xyloglucans, polyuronides and cellulose. This IAA effect on the cell wall synthesis is a consequence of extension growth induced by IAA. Gibberellic acid (GA) at 10⁻⁴ M synergistically enhanced the IAA-induced cell wall synthesis as well as IAA-induced extension growth, although GA by itself neither stimulated the cell wall synthesis nor extension growth. In the absence of sucrose, cell wall synthesis was not induced by IAA or GA. (2) In mature segments excised from basal regions of the epicotyl, no extension growth was induced by IAA or GA. GA enhanced the synthesis of xylans and cellulose when the segments were supplied with 50 m M sucrose. IAA had no effect on the cell wall synthesis. These findings indicate that synthesis of polyuronides, xyloglucans and cellulose, which occurs during extension growth of the apical region of the epicotyl, is regulated chiefly by auxin whereas synthesis of xylans and cellulose during cell maturation in the basal region of the epicotyl is regulated by GA.     

Biosynthesis of Auxin-Induced Ethylene. Effects of Indole-3-Acetic Acid, Benzyladenine and Abscisic Acid on Endogenous Levels of 1-Aminocyclopropane-l-Carboxylic Acid (ACC) and ACC Synthase

Auxin-induced ethylene biosynthesis and its regulatory stepsin etiolated mung bean hypocotyl segments were examined. Theendogenous content of 1-aminocyclopropane- 1-carboxylic acid(ACC), an immediate precursor of ethylene, increased correspondingto the rate of ethylene production. Benzyladenine (BA), whichis a synergistic stimulator of auxin-induced ethylene production,increased the ACC content parallel to the rate of ethylene productionin the presence of IAA, but failed to increase the ACC contentin the absence of IAA while ethylene production was significantlystimulated by BA. Abscisic acid (ABA) inhibited the formationof ACC. The ACC synthase activity in the tissue was increasedby IAA, and the increase was further promoted by the presenceof BA. Cycloheximide severely inhibited the development of auxin-inducedACC synthase. The enzymatic properties of mung bean ACC synthasewere similar to those of the tomato fruit enzyme. Aminoethoxyvinylglycine(AVG) and aminooxyacetic acid, which inhibit the ACC synthasereaction, stimulated the development of ACC synthase. The regulatorymechanisms of the growth regulators are discussed in relationto ACC formation. (Received December 3, 1980; Accepted January 22, 1981)     

High Temperature Sensitivity of Auxin-induced Ethylene Production in Mung Bean Hypocotyl Sections

High temperature sensitivities of IAA-induced and 1-aminocyclopropane-1-carboxylicacid (ACC)-dependent ethylene production in etiolated mung bean(Vigna radiata [L] Wilczek) hypocotyl sections were comparedat 30,40, 42.5°C. When ethylene production at 30°C wastaken as control, IAA-induced production at 40°C was firstenhanced and then suppressed after 3 h, whereas ACC-dependentproduction was enhanced two-fold throughout the 8 h experimentalperiod. However, when hypocotyl sections treated with 1 mM ACCat 30°C for several hours were transferred to 40°C,the ACC-dependent production rate fell below that at 30°C.An initial transient enhancement of IAA-induced ethylene productionat 40°C was supported by increased ACC synthase activityand thus by ACC content. At 42.5°C, both IAA-induced andACC-dependent production were almost completely suppressed.The results indicate that auxin-induced ethylene productionis affected by high temperatures in two different steps: a)at 40°C, the auxin action gradually deteriorates althoughconversion of ACC to ethylene is not affected at all, and at42.5°C, the conversion is nearly completely suppressed. (Received July 8, 1985; Accepted January 24, 1986)     

(+)-5-Hydroxy-dioxindole-3-acetic acid,a synergist from rice bran of auxin-induced ethylene production in plant tissue

(+)-5-Hydroxy-dioxindole-3-acetic acid (1) was isolated from rice bran as a substance synergistic with auxin in the auxin induced ethylene production by etiolated mungbean hypocotyl segments. 5-Hydroxy-oxindole-3-acetic acid (4) and IAA were also obtained. The importance of a hydroxyl group in the 5-position in the two compounds was suggested since synthesized (±)-dioxindole-3-acetic acid (6) was inactive.     

A brassinosteroid-cytokinin interaction on ethylene production by etiolated mung bean segments

Three cytokinins and their ribosides were tested in conjunction with brassinosteroid (BR) for their effects on ethylene production in etiolated mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. When varying concentrations of BR were tried in combination with a fixed amount of kinetin (10 μ M ), there was an increase in ethylene production with increasing concentrations of BR up to 3 μ M , and a decline beyond. A stimulation in ethylene production was observed with increasing concentrations of each of the cytokinins used. However, when applied in conjunction with 3 μ M BR, all cytokinin concentrations produced similar stimulatory patterns. When 3 μ M BR or 10 μ M 6-benzylaminopurine (BAP) was applied in conjunction with increasing concentrations of IAA, there was a stimulation in ethylene production with increasing concentrations of IAA. However, 3 μ M BR and 10 μ M BAP together with varying concentrations of IAA failed to alter the level of ethylene production.     

The effect of brassinosteroid and 2,4-D-L-amino acid conjugates on ethylene production by etiolated mung bean segments

Brassinosteroid (BR) an analogue of brassinolide was tested in combination with thirteen 2,4-dichlorophenoxyacetic acid-L-amino acid conjugates for its possible synergistic effects on ethylene production by etiolated mung bean (Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. When BR was used in combination with 2,4-D-L-amino acid conjugates the degree of enhanced ethylene production varied with the conjugate tested. In fact, the activity of the conjugate alone was directly related to its activity with BR.     

Relationship of steroidal structure to ethylene production by etiolated mung bean segments

Several brassinosteroid (BR) analogues, cholesterol and aldosterone were evaluated for their effectiveness alone and in combination with indole-3-acetic acid (IAA) in stimulating ethylene production by etiolated mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. Changing the conformation of the two hydroxyl groups on C-22 and C-23 positions from α to β did not greatly reduce the efficiency of these compounds to stimulate ethylene production alone or in combination with IAA. There was little difference in activity observed when the conformation of the methyl group in the C-24 position was changed from α to β. However, when hydroxyls were deleted from the side chain in the C-22 and C-23 positions, the compound was rendered inactive alone or in combination with IAA. The compound was also inactivated by removing the 7-oxa function on the B-ring and by substituting an ethyl group for the methyl group in the C-24 position. Both aldosterone and cholesterol were ineffective in promoting ethylene production. This study shows that very stringent structural features are required for a steroid to have BR-like activity and to act synergistically with auxin in the promotion of ethylene synthesis.     

Inactivity of Oxidation Products of Indole-3-acetic Acid on Ethylene Production in Mung Bean Hypocotyls

The suggestion that indole-3-acetic acid (IAA)-stimulated ethylene production is associated with oxidative degradation of IAA and is mediated by 3-methyleneoxindole (MOI) has been tested in mung bean (Phaseolus aureus Roxb.) hypocotyl segments. While IAA actively stimulated ethylene production, MOI and indole-3-aldehyde, the major products of IAA oxidation, were inactive. Tissues treated with a mixture of intermediates of IAA oxidation, obtained from a 1-hour incubation of IAA with peroxidase, failed to stimulate ethylene production. Furthermore, chlorogenic acid and p-coumaric acid, which are known to interfere with the enzymic oxidation of IAA to MOI, had no effect on IAA-stimulated ethylene production. Other oxidation products of IAA, including oxindole-3-acetic acid, indole-3-carboxylic acid, (2-sulfoindole)-3-acetic acid, and dioxindole-3-acetic acid, were all inactive. 1-Naphthaleneacetic acid was as active as IAA in stimulating ethylene production but was decarboxylated at a much lower rate than IAA, suggesting that oxidative decarboxylation of auxins is not linked to ethylene production. These results demonstrate that IAA-stimulated ethylene production in mung bean hypocotyl tissue is not mediated by MOI or other associated oxidative products of IAA.     

表油菜素内酯对绿豆上胚轴内源IAA及其氧化酶的影响

用0.5ppm表油菜素内酯处理绿豆幼苗,显著促进上胚轴伸长生长,若切除真叶则可抑制表油菜素内酯诱导的效应。三碘苯甲酸(TIBA)也可抑制表油菜素内酯促进的伸长生长。外源IAA能部分恢复TIBA的抑制效应。经处理的上胚轴内源IAA含量明显高于对照。暗示表油菜素内酯可能通过对内源IAA的调节来促进绿豆上胚轴的伸长生长。 表油菜素内酯处理的绿豆上胚轴组织中,与生长素降解密切相关的IAA氧化酶以及过氧化物酶活性均明显低于对照。     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

Isolation and expression of an elongation-dependent gene of mung bean (Vigna radiata) hypocotyl

A cDNA clone of an auxin up-regulated gene, ARG8 , was isolated from hypocotyl sections of etiolated mung bean [ Vigna radiata (L.) Wilczek] seedlings by differential screening. The deduced amino acid sequence suggested that ARG8 may encode a cell wall protein. The steady state mRNA level of ARG8 increased by treatment of hypocotyl sections not only with indole-3-acetic acid (IAA) but also with fusicoccin, and the auxin inducibility was inhibited by the addition of 0.3 M mannitol in the incubation medium. This indicated that it was not auxin but elongation that regulated the expression of ARG8 . The promoter activity of the 5'-flanking region of ARG8 was determined by assaying the transient expression of a luciferase fusion gene that was introduced into mung bean hypocotyl sections by the particle bombardment technique. The basal activity of the ARG8 upstream region was about a few tenths of that of a modified cauliflower mosaic virus 35S promoter, and it was increased a few fold by treatment with IAA. The auxin inducibility was completely suppressed by the addition of mannitol. A 5'-deletion analysis showed that a 53-bp region in the ARG8 promoter was important for the basal and elongation-dependent promoter activities.     

Relationships in actions of indoleacetic acid, benzyladenine and abscisic acid in ethylene production

The relationships between IAA and ABA, and between BA and ABAin their effects on ethylene production were examined with etiolatedmungbean hypocotyl segments. When ABA and IAA were simultaneouslyapplied to the tissues, ABA inhibited IAA-induced ethylene productionand the degree of inhibition was solely determined by the ABAconcentrations. Increasing concentrations of BA did not affectABA inhibition. Low concentrations of ABA slightly increasedendogenous ethylene production. When ABA and BA were appliedtogether in the presence of IAA, the degree of ABA inhibitionwas again determined by the ABA concentrations regardless ofthe BA concentrations. BA did not recover ABA inhibition andABA did not inhibit the stimulative effect of BA on both endogenousand IAA-induced ethylene production. Almost the same resultswere obtained with ABA and BA pretreatment of the tissues. Thisindicates that in the processes of IAA-induced ethylene production,IAA and ABA act in series, but that the actions at their respectivesites are independent. ¹ This research was partly supported by grants from the Ministryof Education (C-956037) and the Ministry of Agriculture (49–1330)of Japan, and by the Asahi Press. (Received June 14, 1975; )     

A Possible Regulation of Ethylene Production by the Epidermis in Mung Bean Hypocotyl Sections

IAA-induced and l-aminocyclopropane-l-carboxylic acid (ACC)-dependentethylene production in etiolated mung bean (Vigna radiata [L]Wilczek) hypocotyl sections does not occur in epidermal cells(Todaka and Imaseki 1985). Mung bean hypocotyls contain a proteinwhich inhibits auxin-induced ethylene biosynthesis in hypocotylsections (Sakai and Imaseki 1975a, b). This inhibitory proteinwas also found to inhibit ACC-dependent ethylene productionin hypocotyl sections, but not in hypocotyl sections from whichthe epidermis had been removed. Uptake of ACC by both unpeeledand peeled sections was not inhibited by the protein. Similarly,IAA-induced ethylene production was inhibited by the proteinin unpeeled hypocotyl sections, but not in peeled sections.The protein was not inactivated in peeled sections, as proteinsynthesis by peeled sections was inhibited to the same extentas in unpeeled sections. The protein inhibited incorporationof 3,4-[¹⁴C]-methionine into ACC and ethylene in unpeeled sections,but not in peeled sections, whereas oxidation of the labeledmethionine into CO₂ was inhibited by the protein to a similarextent in both types of hypocotyl sections. KCN, a potent inhibitorof ethylene production, inhibited both IAA-induced and ACC-dependentethylene production in both peeled and unpeeled hypocotyl sections.It is likely that the epidermis plays some role in controllingethylene production which occurs in stem cells other than epidermalcells. (Received July 16, 1985; Accepted October 21, 1985)     

Effects of the Inhibitory Protein of Ethylene Synthesis on ATP Levels in Mung Bean Hypocotyl Segments

The inhibitory protein of ethylene synthesis purified from mungbean seeds reduced ATP levels in mung bean hypocotyl segments.When the segments were incubated with 0.5mM IAA for 6 hr toinduce ethylene-producing activity, the presence of the inhibitoryprotein suppressed the ethylene production and ATP content inthe tissue about 82 and 60%, respectively. Similar suppressiveeffects were also observed for endogenous ethylene productionand ATP contents in tissue not treated with IAA. (Received June 20, 1981; Accepted October 24, 1981)     

Inhibition of auxin-induced ethylene production by salicylic acid in mung bean hypocotyls

Salicylic acid (SA), a common plant phenolic compound, influences diverse physiological and biochemical processes in plants. To gain insight into the mode of interaction between auxin, ethylene, and SA, the effect of SA on auxininduced ethylene production in mung bean hypocotyls was investigated. Auxin markedly induced ethylene production, while SA inhibited the auxin-induced ethylene synthesis in a dose-dependent manner. At 1 mM of SA, auxininduced ethylene production decreased more than 60% in hypocotyls. Results showed that the accumulation of ACC was not affected by SA during the entire period of auxin treatment, indicating that the inhibition of auxin-induced ethylene production by SA was not due to the decrease in ACC synthase activity, the rate-limiting step for ethylene biosynthesis. By contrast, SA effectively reduced not only the basal level of ACC oxidase activity but also the wound-and ethylene-induced ACC oxidase activity, the last step of ethylene production, in a dose-dependent manner. Northern and immuno blot analyses indicate that SA does not exert any inhibitory effect on the ACC oxidase gene expression, whereas it effectively inhibits both the in vivo and in vitro ACC oxidase enzyme activity, thereby abolishing auxin-induced ethylene production in mung bean hypocotyl tissue. It appears that SA inhibits ACC oxidase enzyme activity through the reversible interaction with Fe²⁺, an essential cofactor of this enzyme. These results are consistent with the notion that ethylene production is controlled by an intimate regulatory interaction between auxin and SA in mung bean hypocotyl tissue.     

The Changes of Ethylene Production and CaM Content in IAA-Treated Etiolated Mungbean Hypocotyl

When the segments of etiolated mungbean hypocotyl were treated with IAA it was observed that the contents of CaM in tissue which were determined by ELISA and ethylene production were increased with increasing the concentration of IAA. The time course of CaM content change was also similar to that of ethylene production. Some inhibitors, including CPZ, TFP, and CHI, inhibited both increased ethylene production and CaM content by IAA treatment. The activity of ACC synthase and EFE were inhibited by CPZ. Both of IAA-induced ethylene production and CaM content were affected by the level of Ca in segments as a result of pretreatment with EGTA, CaCI2 and H2O before the experiment. From these results it was suggested that the Ca and CaM play an important role in induction of ethylene production inmungbean hypocotyl by IAA.