Inhibition of IAA-induced ethylene production in etiolated mung bean hypocotyl segments by 2,3,5-triiodobenzoic acid and 2-(p-chlorophenoxy)-2-methyl propionic acid

The effect of two auxin antagonists, 2,3,5-triiodobenzoic acid (TIBA) and 2-( p -chlorophenoxy)-2-methyl propionic acid (CMPA) on IAA-induced ethylene production in etiolated mung bean hypocotyl ( Vigna radiata L. Rwilcz cv. Berken) segments was studied. Both TIBA and CMPA inhibited IAA-induced ethylene production and CO₂ production at concentrations from 0.001 m M to 0.1 m M and 0.01 m M to 1.0 m M , respectively. The optimum concentration for inhibition of ethylene production by TIBA was 0.05 m M and CMPA was 0.5 m M . At the optimum concentration of TIBA and CMPA, there was a significant decrease in IAA-induced ethylene production without a decrease in respiration rates below control levels. After 18 h, mung bean hypocotyl segments treated with 0.05 m M TIBA for 6 h or 0.5 m M CMPA for 8 h showed a maximum inhibition of IAA-induced ethylene production. Treatments longer than 8 h caused no further inhibition. The uptake of [¹⁴C]-naphthaleneacetic acid by mung bean segments was greatly reduced by the addition of either TIBA (0.05m M ) or CMPA (0.5 m M ) to the incubation media. The results of treatment sequences showed that TIBA needed to be applied prior to IAA in order to inhibit IAA-induced ethylene production, but CMPA caused the same inhibitory effect whether applied before or after IAA treatment. These findings provide evidence that TIBA inhibits auxin-induced ethylene production in etiolated mung bean hypocotyl segments by blocking auxin movement into the tissue whereas CMPA may work on both auxin transport and action.     

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 effect of brassinosteroid on auxin-induced ethylene production by etiolated mung bean segments

Brassinosteroid, an analogue of brassinolide, (BR) (2α, 3α, 22β, 23β-tetrahydroxy-24β-methyl-B-homo-7-oxa-5α-cholestan-6-one), was tested in conjunction with indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA), indole-3-propionic acid (IPA), indole-3-pyruvic acid (IPyA), indole-3-aldehyde (IAld), indole-3-carbinol (ICB) or tryptophan (TRP) for its effects on ethylene production by etiolated mung bean (Vigna radiata (L.) Rwilcz cv. Berken) hypocotyl segements. The enhancement of ethylene production due to BR was greatest in conjunction with 1 μM IBA, 2,4-D, IAA, or NAA (these increases were 2580, 2070, 890, and 300%, respectively). When increasing concentrations of IBA, 2,4-D, IAA, or NAA were used, there was a decrease in the percentage stimulation by BR. Both IPyA and IPA had different optimal concentrations than the other auxins tested. Their BR-enhanced maximum percentage stimulations (1430 and 1580%) were greatest with 5 μM IPya and 10 μM IPA, respectively. There was a marked reduction in the percentage stimulation by BR with either 100 μM IPyA or IPA. The inactive indoles (IAld, ICB, or TRP) did not synergize with BR at any of the concentrations tested. Four hours following treatment those segments in contact with 1 μM BR with or without the addition of 10 μM IAA began to show a stimulation in ethylene production above the control and this stimulation became greater over the following 20 h. It was necessary for BR to be in continual contact with the tissue to have a stimulatory effect on auxin-induced ethylene production. When segments excised from greater distances below the hypocotyl hook, were treated with either IAA alone or in combination with BR, there was a decrease in ethylene production with increasing distance. There was no effect of hypocotyl length on BR stimulation of auxin-induced ethylene production; however, there was a definite decrease in ethylene production when IAA was applied alone.     

Inhibition of ethylene production by osmotic shock. Further evidence for membrane control of ethylene production

Effects of osmotic shock applied to mung bean hypocotyl segmentswere examined to see if auxin-induced ethylene production isregulated by membrane activity. Uptake of -aminoisobutyric acidwas significantly inhibited by osmotic shock. While cold shockalone also inhibited the uptake, cold osmotic shock was mosteffective. Auxin-induced ethylene production by the osmoticallyshocked tissue was similarly inhibited. Shock treatment of auxin-pretreatedtissue also suppressed the subsequent ethylene production. Oxygenuptake was not affected by the shock treatment. Sorbitol andsucrose used as osmotic substance were as effective as mannitol.The results suggest that ethylene production is controlled bymembrane activity. (Received July 18, 1977; )     

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.     

Inhibitors of ethylene synthesis inhibit auxin-induced stomatal opening in epidermis detached from leaves of Vicia faba L

Using leaf epidermis from Vicia faba, we tested whether auxin-induced stomatal opening was initiated by auxin-induced ethylene synthesis. Epidermis was dark-incubated in buffered KNO3 containing 0.1 mM alpha-napthalene acetic acid or 1 mM indole-3-acetic acid. Maximum net opening was ca. 4 micron after 6 h. Opening was reversed by 20 microM ABA, 0.1 mM CaCl2. 1-Aminocyclopropane carboxylic acid (ACC) synthase catalyzes synthesis of ACC, the immediate precursor to ethylene. Auxin-induced stomatal opening was fully inhibited by 10 microM 1-aminoethoxyvinylglycine (AVG), an ACC synthase inhibitor. In solutions containing AVG, auxin-induced opening was restored in a concentration-dependent manner by exogenous ACC, but not in control solutions lacking an auxin. ACC-mediated reversal of AVG-inhibition of stomatal opening was inhibited by alpha-aminoisobutyric acid (AIB), an inhibitor of ACC oxidase, the last enzyme in the ethylene biosynthetic pathway, by 10 microM silver thiosulfate (STS), an inhibitor of ethylene action, and by 20 microM ABA, 0.1 mM CaCl2. CoCl2, an inhibitor of ethylene synthesis, also inhibited auxin-induced opening. Both STS and CoCl2 inhibited opening induced by light or by fusicoccin, but neither light- nor fusicoccin-induced opening was inhibited by AVG. These results support the hypothesis that auxin-induced stomatal opening is mediated through auxin-induced ethylene production by guard cells.     

Inhibition of ethylene production by cobaltous ion

The effect of Co²⁺ on ethylene production by mung bean (Phaseolus aureus Roxb.) and by apple tissues was studied. Co²⁺, depending on concentrations applied, effectively inhibited ethylene production by both tissues. It also strongly inhibited the ethylene production induced by IAA, kinetin, IAA plus kinetin, Ca²⁺, kinetin plus Ca²⁺, or Cu²⁺ treatments in mung bean hypocotyl segments. While Co²⁺ greatly inhibited ethylene production, it had little effect on the respiration of apple tissue, indicating that Co²⁺ does not exert its inhibitory effect as a general metabolic inhibitor. Ni²⁺, which belongs to the same group as Co²⁺ in the periodic table, also markedly curtailed both the basal and the induced ethylene production by apple and mung bean hypocotyl tissues.     

Cobalt and plant development: interactions with ethylene in hypocotyl growth

Co²⁺ promoted elongation of hypocotyl segments of light-grown cucumber (Cucumis sativus) seedlings. Time course and dose response data are presented and interactions with IAA, gibberellin, cyclohexanol, and cotyledons described. Segments without cotyledons responded to Co²⁺ only if grown in gas-tight vessels with IAA added. When bases of cotyledons were ringed with an inhibitor of auxin transport, Co²⁺ caused no growth promotion in the hypocotyl. Co²⁺ prevented lateral swelling of hypocotyls treated with supraoptimal IAA. Removal of ethylene from the atmosphere reduced the Co²⁺ response, but Co²⁺ did not counteract the inhibitory effect of increased ethylene levels. These results are consistent with the hypothesis that Co²⁺ promotes hypocotyl elongation by inhibiting ethylene production. The hypothesis was confirmed by a direct demonstration that Co²⁺, at growth-promoting concentrations, powerfully inhibited ethylene production in the cucumber hypocotyl.     

Regulation of Auxin-induced Ethylene Biosynthesis. Repression of Inductive Formation of 1-Aminocyclopropane-1-carboxylate Synthase by Ethylene

Changes in the 1-aminocyclopropane-1-carboxylate (ACC) synthaseactivity which regulates auxin-induced ethylene production werestudied in etiolated mung bean hypocotyl segments. Increasesboth in ethylene production and ACC synthase activity in tissuetreated with IAA and BA were severely inhibited by cycloheximide(CHI), 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide,actinomycin D and -amanitin. Aminoethoxyvinylglycine (AVG),a potent inhibitor of the ACC synthase reaction, increased theactivity of the enzyme in the tissue 3- to 4-fold. This stimulationalso was severely inhibited by the above inhibitors. Stimulationof the increase in the enzyme content by AVG was partially suppressedby an exogenous supply of ACC or ethylene. Suppression of theincrease in the enzyme took place with 0.3 µl/liter ethylene,and inhibition was increased to 10 µl/liter, which caused65% suppression. Air-flow incubation of the AVG-treated tissue,which greatly decreased the ethylene concentration surroundingthe tissue, further increased the amount of enzyme. Thus, oneeffect of AVG is to decrease the ethylene concentration insidethe tissue. The apparent half life of ACC synthase activity,measured by the administration of CHI, was estimated as about25 min. AVG lengthened the half life of the activity about 2-fold.Feedback repression by ethylene in the biosynthetic pathwayof auxin-induced ethylene is discussed in relation to the effectof AVG. (Received January 22, 1982; Accepted March 26, 1982)     

Mechanism of cytokinin action on auxin-induced ethylene production

The stimulative effect of cytokinin on ethylene production hasbeen examined in etiolated mungbean hypocotyl segments. Therate of auxin-induced production linearly increased with timein a certain range of exogenous IAA concentration. The rateof induced production doubled with a 10-fold increase in exogenousIAA concentration or addition of benzyladenine at 5 µM.Benzyladenine did not suppress inactivation of the induced ethyleneproducing system. Although the free IAA level within the tissueswas slightly increased by benzyladenine accompanied by a decreasein IAAsp formation, the increased free IAA level did not accountfor the doubling of the production rate. When the tissues werepreincubated with benzyladenine alone followed by postincubationwith auxin, the rate of induced production in pretreated tissueswas significantly higher than that in untreated or buffer-treatedtissues supplied with auxin and benzyladenine simultaneouslyin the post-incubation medium. Formation and disappearance ofthe cytokinin effect were temperature dependent. The rate ofendogenous production was constant over an experimental periodand benzyladenine simply enhanced the rate several-fold aftera lag period. Kinetics of the cytokinin stimulation was notthe inductive type. Based on these observations, a possiblemechanism of the stimulative effect of cytokinin was discussed. ¹This research was partly supported by grants from the Ministryof Education of Japan (C-856043 and C-956037) and the AsahiPress. (Received May 7, 1975; )     

Involvement of cellular membrane in regulation of ethylene production

Effects of various substances which would change membrane structureson auxin-induced ethylene production in etiolated mung beanhypocotyl segments were investigated. Auxin-induced ethyleneproduction was not affected by treatment of tissue segmentswith pronase, trypsin, Con A and amphotericin B. PhospholipaseD inhibited ethylene production and its action was reversible,suggesting that the inhibitory action may not be due to enzymaticaction. Lecithin, Tween 20, Triton X 100 and SDS also significantlyinhibited ethylene production. Inhibition by the former twosubstances was completely, and that of the latter two was partiallyreversed by removing them from the tissue segments. All thesubstances which inhibited ethylene production also suppressedIAAsp formation by the tissue. It was concluded that inhibitionresulted from structural changes of cell membranes caused byreversible interaction with the lipophilic substances and thatethylene production and IAAsp formation were under the controlof membrane function. ¹ Supported in part by grants from the Ministry of Educationand the Ministry of Agriculture and Forestry, Japan. (Received December 24, 1976; )     

Influence of cobalt on soybean hypocotyl growth and its ethylene evolution

Development of dark-grown “Clark” soybean (Glycine max [L.] Merr.) seedlings is abnormal at 25 C but normal at 20 and 30 C. At 25 C, hypocotyls swell and fail to elongate normally; lateral root formation and seedling ethylene evolution are enhanced.

Co²⁺ promoted hypocotyl elongation of etiolated “Clark” soybean seedlings by 28% when grown at 25 C. The same growth-promoting concentration reduced hypocotyl thickness and primary root elongation by 28 and 43%, respectively. Co²⁺ inhibited ethylene production both of intact seedlings and of apical 1-centimeter hypocotyl segments with attached epicotyls and cotyledons by 65 and 60%, respectively. These results suggest that Co²⁺ exerts its effects on the hypocotyl growth by inhibiting ethylene production, and also confirm our previous conclusion that abnormal ethylene production at 25 C is responsible for the inhibition of hypocotyl elongation and for its swelling.

     

Auxin-induced ethylene production by mungbean hypocotyl segments

Auxin induced an increase in the rate of ethylene productionby hypocotyl segments of etiolated mungbean seedlings aftera 1 hr lag period. The increase in the production rate was greatestwith segments immediately below the cotyledonary hook. Effectiveconcentrations of indoleacetic acid ranged from 1 µm to0.5 mM. Length of the lag period was the same regardless ofthe interval between segment excision and the addition of auxin.Auxin-induced ethylene production was inhibited by cycloheximidebut not by chloramphenicol. Auxin removal from, or cycloheximideaddition to, segments actively producing ethylene in responseto auxin caused a rapid decrease in the rate of ethylene production.These results suggest that the ethylene producing system inducedby auxin is rapidly turning over and that auxin acts at twosites to increase ethylene production ¹This work was supported in part by grant No. 0802 from Ministryof Education, Japan (Received November 30, 1970; )     

Interaction of ethylene and a cytokinin in promoting hypocotyl elongation in a dwarf strain of watermelon

Experiments were conducted to study the interaction of ethylene and the cytokinin N⁶-benzyladenine (BA) in promoting hypocotyl elongation in a dwarf strain of watermelon (Citrullus lanatus [Thunb] Matsu. and Nakai). Optimum promotion of hypocotyl elongation is elicited by an apical treatment with 0.2 microgram BA. At dosages above 0.3 microgram per apex, BA-enhancement of elongation is reduced concomitant with stimulation of ethylene production and lateral expansion of hypocotyls. Application of the ethylene biogenesis inhibitor, aminoethoxyvinylglycine, at dosages from 0.3 to 10 micrograms per apex inhibited BA-induced ethylene production. In seedlings treated with 0.2 microgram BA, 10 micrograms aminoethoxyvinylglycine per apex reduced ethylene production to about one-third of control levels and reduced BA stimulation of hypocotyl elongation by 74%. Exposure of watermelon seedlings to 60 ± 10 nanoliters per liter of ethylene in a flowing system nearly eliminated aminoethoxyvinylglycine inhibition of BA-promoted growth. The results suggest that physiological levels of internal ethylene are required for cytokinin promotion of hypocotyl elongation in watermelon.     

Lithium,aminoethoxyvinylglycine and cobalt reversal of the cotyledonary pricking-induced growth inhibition in the hypocotyl of Bidens pilosus in relation to ethylene and peroxidases

Pretreatment of young Bidens pilosus plants with lithium (Li), aminoethoxyvinylglycine (AVG) or cobalt (Co) prevents the cotyledonary pricking-induced growth inhibition of the hypocotyl. The effect is correlated with parallel prevention of the pricking-induced enhancement of peroxidase and ethylene production in the hypocotyl. Only Co prevents the increased capacity of hypocotyl segments or microsomes to convert aminocyclopropane carboxylic acid to ethylene. Li, AVG, and Co do not interfere with peroxidase and ethylene metabolism in pricked cotyledons. Although Li, AVG as well as Co are known to interfere with ethylene biosynthesis and action, they could well remove the wounding effect by different modes of action.     

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)     

Calcium ion dependency of ethylene production in segments of primary roots of Zea mays

We investigated the effect of Ca²⁺ on ethylene production in 2-cm long apical segments from primary roots of corn ( Zea mays L., B73 × Missouri 17) seedlings. The seedlings were raised under different conditions of Ca²⁺ availability. Low-Ca and high-Ca seedlings were raised by soaking the grains and watering the seedlings with distilled water or 10 m M CaCl₂, respectively. Segments from high-Ca roots produced more than twice as much ethylene as segments from low-Ca roots. Indoleacetic acid (IAA; 1 μ M ) enhanced ethylene production in segments from both low-Ca and high-Ca roots but auxin-induced promotion of ethylene production was consistently higher in segments from high-Ca roots. Addition of I-aminocyclopropane-I-carboxylic acid (ACC) to root segments from low-Ca seedlings doubled total ethylene production and the rate of production remained fairly constant during a 24 h period of monitoring. In segments from high-Ca seedlings ACC also increased total ethylene production but most of the ethylene was produced within the first 6 h. The data suggest that Ca²⁺ enhances the conversion of ACC to ethylene. The terminal 2 mm of the root tip were found to be especially important to ethylene biosynthesis by apical segments and, experiments using ⁴⁵Ca²⁺ as tracer indicated that the apical 2 mm of the root is the region of strongest Ca²⁺ accumulation. Other cations such as Mn²⁺, Mg²⁺, and K⁺ could largely substitute for Ca²⁺. The significance of these findings is discussed with respect to recent evidence for gravity-induced Ca²⁺ redistribution and its relationship to the establishment of asymmetric growth during gravitropic curvature.     

Auxin inhibition of flower induction of pharbitis is not mediated by ethylene

Treatment of whole Pharbitis nil seedlings or cotyledons with indole butyric acid (IBA) immediately before an inductive dark period greatly inhibited flowering. Treatment of the shoot tip alone with IBA had little or no effect. 1-Aminocyclopropane 1-carboxylic acid, which increased ethylene production by the seedlings much more than IBA, had no effect on the flowering response. Pretreatment of seedlings with the ethylene biosynthesis inhibitor aminooxyacetic acid or with the inhibitor of ethylene action silver thiosulfate did not reduce the inhibitory effect of IBA on flower induction. We concluded, therefore, that the auxin-induced inhibition of flowering of P. nil was not mediated by ethylene.     

Inhibition of Auxin-Induced Ethylene Production by Lycoricidinol

Lycoricidinol, a natural growth inhibitor isolated from bulbsof Lycoris radiata Herb. strongly suppressed auxin-induced ethyleneproduction from the hypocotyl segments of etiolated mung bean(Vigna radiata Wilczek) seedlings. The inhibitor did not significantlyinhibit ethylene formation from its immediate precursor, 1-aminocyclopropane-1-carboxylicacid (ACG), during short-term (up to 4 h) incubation. The ACCcontent in tissue treated with IAA was reduced by lycoricidinolin close parallel with the inhibition of ethylene production.Examination of radioactive metabolites in tissues labeled with3,4-¹⁴C-methionine indicated that reduction of the ACC contentwas not due to any possible promotive effect of lycoricidinolon conjugation of ACC with malonate. Lycoricidinol showed noinhibitory effect on the activity of ACC synthase if appliedin vitro, but it almost completely abolished the increase inthe enzyme activity when applied in vivo during incubation ofthe tissue with IAA. Lycoricidinol also strongly inhibited incorporationof ¹⁴C-leucine into protein in the tissue. The suppression ofthe enzyme induction and, in turn, that, of ethylene productionby lycoricidinol were interpreted as being due to the inhibitionof protein synthesis. (Received September 30, 1983; Accepted December 8, 1983)     

Role of oxygen in auxin-induced ethylene production

Ethylene production by IAA-treated mung bean hypocotyl segmentsunder various oxygen levels in the ambient atmosphere was examined.Rate of ethylene production was dependent upon oxygen levels,and gave a sigmoidal curve against oxygen levels. Tissue segmentspreincubated with IAA in low oxygen levels (1–10% O₂ inN₂) produced ethylene without a lag period at a rate higherthan that by control tissue segments preincubated in air, whenthey were exposed to a high oxygen level (air, 21% O₂). Theeffect of cycloheximide on tissue segments transferred froma low oxygen level to air was not much different from that onethylene production by control tissue segments previously incubatedin air. Incorporation of U-¹⁴C-leucine into the protein fractionby tissue segments placed in nitrogen was negligible, but thatin 2% oxygen was 10 to 14% of that in air. It was concluded that oxygen was an essential factor for boththe induction process of the ethylene producing system and thesynthesis of ethylene, and that although synthesis of ethyleneis dependent upon oxygen levels, formation of the ethylene producingsystem proceeded even under low oxygen levels. (Received January 13, 1977; )