Inactivation of 1-Aminocyclopropane-1-carboxylic Acid Synthase of Etiolated Mung Bean Hypocotyl Segments by its Substrate, S-Adenosyl-L-methionine

ACC synthase, isolated from mung bean hypocotyl segments treatedwith IAA and BA, was inactivated by its substrate, SAM, duringits catalytic action. The reaction products, ACC and MTA, hadno effect on ACC synthase activity. The half-life of the enzymewas 12 min with an initial concentration of 150µM SAM,but this was extended to 23.5 min when the SAM concentrationwas reduced to 40 µM, near to the endogenous concentrationof SAM in mung bean hypocotyl tissue. Addition of AVG, a competitiveinhibitor of ACC synthase, to the reaction mixture containing40 µM SAM, prevented ACC synthase inactivation and increasedthe half-life about 2-fold. We suggest that ACC synthase inactivationis caused by SAM acting as an enzyme-activated irreversibleinactivator (k_cat-type inactivator), besides being the substratefor the enzyme. This SAM-dependent inactivation of ACC synthasemay explain the rapid inactivation of the enzyme in intact mungbean hypocotyl segments previously found by Yoshii and Imaseki(1982). (Received October 15, 1985; Accepted December 6, 1985)     

Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)     

Auxin Transport Inhibitors and the Rooting of Hypocotyl Cuttings from Etiolated Mung-bean Vigna radiata (L.) Wilczek Seedlings

The auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA)and naphthylphthalamic acid (NPA) inhibited adventitious rootformation (ARF) induced by indol-3-butyric acid (IBA) on cuttingsfrom etiolated mung-bean seedlings floated on solutions of thegrowth regulators. The concentrations of TIBA and NPA requiredfor a 25 per cent reduction in ARF with 10 µM IBA wereestimated by linear interpolation to be 11.3 µm and 0.42µM respectively. NPA is a particularly potent inhibitorof IBA-induced ARF. The inhibitory effect of either compoundwas reversible by higher concentrations of IBA. NPA had no effectwhen applied after the auxin treatment. The inhibitory effects of TIBA or NPA could not be explainedby effects on the uptake or metabolism of [2-^14C]IAA. Consideringthis and other evidence, it is suggested that NPA and possiblyTIBA are acting as specific antagonists of auxin in the inductionof ARF. Vigna radiata (L.), mung-bean, root induction, hypocotyl cuttings, auxin inhibitors, indol-3-butyric acid, 2,3,5-triiodobenzoic acid, naphthylphthalamic acid, auxin uptake, auxin metabolism, adventitious roots     

Epidermal Cells Do Not Contribute to Auxin-Induced Ethylene Production in Mung Bean Stem Sections

Auxin-induced and 1-aminocyclopropane-1-carboxylic acid (ACC)-dependentethylene production in mung bean (Vigna radiata [L] Wilczek)hypocotyl sections, from which epidermis had been removed, wasinvestigated. Ethylene production in hypocotyl sections withoutepidermis was induced by treatment with IAA, and also occurredfrom exogenously supplied ACC in the presence of 0.2 M mannitol.Isolated epidermal strips alone failed to produce substantialamounts of ethylene in response to IAA or from exogenous ACC.3,4-[¹⁴C]-Methionone was incorporated into both ACC and ethylenein peeled sections treated with IAA, but not in the isolatedepidermal strips. Radioactive ACC, however, was detected inthe epidermal strips separated from the unpeeled sections previouslyfed with 3,4-[¹⁴C]-methionine in the presence of IAA. We concludethat the Site of auxin-induced ethylene production is not inthe epidermis, but in other hypocotyl cells, and that epidermalcells lack the activity which converts ACC to ethylene. (Received January 28, 1985; Accepted May 4, 1985)     

In vitro Hormonal Regulation of Laticifer Differentiation in Calotropis procera

Cultures of Calotropis procera were maintained on MS mediumsupplemented with 4·6 µM FAP and 3 µM NAA.Laticifer initials were observed in 2-week-old cultures whichwere converted into matured, branched, non-articulated laticifersin 4 weeks. It was observed that laticifer differentiation increasedwith the age of cultures, from 2·78% (in the second passage)to 15·11% (in the 12th passage). It has been establishedthat laticifer differentiation in vitro is a cytokinin-dependentprocess and among the cytokinins, FAP was more effective thanBA and 2-iP. But the type of auxin and its concentration alsoplay an important role in modifying the effect of cytokinin.Among the different auxins used IAA was more effective for laticiferdifferentiation than IBA and NAA, while 2,4D was inhibitory.Maximum laticifer differentiation (17·01% was observedon MS medium supplemented with 4·6 µM FAP and 1µM IAA.Copyright 1995, 1999 Academic Press Calotropis procera, callus culture, laticifer, differentiation, hormonal regulation     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

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.     

Interaction of a Brassinosteroid with IAA and GA3 in the Elongation of Cucumber Hypocotyl Sections

A synthetic brassinosteroid, 22,23(S,S)-homobrassinolide (hBR),was examined for its interaction with IAA and GA₃ in the elongationof hypocotyl sections of light-grown cucumber (Cucumis salivusL. cv. Aonagajibai) seedlings. hBR alone was less active thanIAA. Its optimal concentration was around 10 µM and thelowest effective concentration between 10 and 100 µM,which is more than 100 times higher than that of brassinolide.hBR was more active in sections from younger seedlings. Itsgrowth-promoting effect was negated or greatly reduced by inhibitorsof auxin-induced elongation such as p-chlorophenoxyisobutyricacid and kinetin. hBR acted synergistically with IAA and 2,4-Dbut not with GA₃ showing only an additive effect. Sequentialtreatment of sections with hBR and then with IAA also resultedin synergistic enhancement of auxininduced elongation, but whenthe order of treatment was reversed, hBR was inactive. The synergisticeffect was obtained with 1 h pretreatment with hBR and couldbe reduced by subsequent washing with water. There was no sequentialinteraction between hBR and GA₃. The synergistic pretreatmenteffects of hBR and GA₃ were simply additive to each other. Amembrane-bound ATPase inhibitor, dicyclohexylcarbodiimide, inhibitedthe hBR-induced elongation, but did not affect GA₃-induced elongation.The findings led to the conclusion that brassinosteroids enhanceauxin action and possess growth-promoting activity which isindependent of that of gibberellin. (Received November 9, 1984; Accepted February 18, 1985)     

The effect of 4-chlororesorcinol on the endogenous levels of IAA,ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3–4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.Abbreviations ABA - abscisic acid - 4-CR - 4-chlororesorcinol - IAA - indol-3-yl-acetic acid - IBA - indol-3-yl-butyric acid     

Abscisic Acid and Apical Dominance in Phaseolus coccineus L. The Role of Tissue Age

Abscisic acid (ABA) applied to the decapitated second internodeof runner bean plants enhanced outgrowth of lateral buds onlywhen internode stumps were no longer elongating. Applied toelongating internodes of slightly younger plants, ABA causesinhibition of bud outgrowth. Together with 10^–4 M indol-3-ylacetic acid (IAA), ABA stimulated internode elongation and interactedadditively in the inhibition of bud outgrowth. A mixture of10^–5 M ABA and 10^–6 M gibberellic acid (GA₃ ) causedsimilar effects on internode growth as IAA + ABA, but was mutuallyantagonistic in effect on growth of the lateral buds. Abscisic acid, apical dominance, gibberellic acid, indol-3yl acetic acid, Phaseolus coccineus, bean     

O-Benzylhydroxylamine: An Inhibitor of Phenylpropanoid Metabolism in Plants

O-Benzylhydroxylamine (OBHA) is a potent inhibitor of phenylalanineammonialyase (PAL, EC 4.3.1.5 [EC] ) and phenylpropanoid metabolismas evidenced by its effects on three plant species [soybean(Glycine max (L.) Merr.), buckwheat (Fagopyrum esculentum Moench.),and mung bean (Vigna radiata L.)]. When supplied to roots, OBHA(10^–5 M) did not significantly inhibit light- or dark-growthof soybean seedlings, but reduced (25%) soluble hydroxyphenoliccompound accumulation in light-grown axes. Higher concentrations(510^–5 M) of OBHA caused reductions (25%) in axis freshweight of light-grown seedlings (72 h), but did not lower axisweight of dark-grown seedlings. Anthocyanin accumulation inhypocotyls of intact mung bean seedlings was reduced by 25%after 3 days light growth after treatment with OBHA (10^–5M) via root feeding. Anthocyanin content of excised, etiolatedbuckwheat hypocotyls floated on solutions of OBHA (10^–5M) and incubated in the light for 24 h was reduced by 40%. L-Phenylalanineand t-cinnamic acid, intermediates of phenylpropanoid metabolism,were able to partially reverse this inhibition in buckwheat.Extractable PAL activity (specific activity basis) in soybeanaxes was substantially reduced (20% in dark, 40% in light) asearly as 24 h after root feeding with OBHA (10^–5 M). Reductionof PAL activity (specific activity or per axis basis) by OBHAcompared to control levels, continued throughout a time courseof 96 h. Kinetic studies on soybean PAL revealed a K_m of 1.1mM for L-phenylalanine and an apparent K_i of 3.5 µM forOBHA. (Received May 31, 1985; Accepted August 6, 1985)     

Effects of Some Growth Regulators and Benzoic Acid Derivatives on Flower Initiation and Root Elongation of Pharbitis nil, Strain Kidachi

Seedlings of Pharbitis nil, strain Kidachi, were grown undercontinuous light at 20°C in vessels containing 5,000-mlnutrient solution, 24 plants per vessel. NAA (0.005–0.5µM), GA₃ (0.1–0.5 µM), kinetin (0.5–5µM), benzyladenine (0.05–5 µM) or abscisicacid (4 µM) added to the nutrient solution induced long-dayflowering, and the flowering was always accompanied by suppressionof root elongation. 3,4-Dichlorobenzoic acid (0.05–10µM) and some other benzoic acid derivatives which arehighly effective for the induction of flowering in Lemna paucicostataalso showed similar effects. Neither NAA, kinetin nor 3,4-dichlorobenzoicacid applied via the apical part of the hypocotyl could causeflowering or suppression of root elongation. Thus, the flower-inducingeffect of the above substances was presumed to be secondaryto the suppression of root elongation. Ethrel (1–50 µM)added to the nutrient solution suppressed root elongation, butdid not induce flowering probably because it has flower-inhibitingactivity. ¹ This paper is dedicated to the memory of Dr. Joji Ashida,the first president of the Japanese Society of Plant Physiologists. (Received December 15, 1982; Accepted February 25, 1983)     

Novel inhibitors of ethylene production in higher plants

Of a number of O-substituted hydroxylamine derivatives, N-benzyloxycarbonyl-L-a-aminooxy-propionicacid and -aminooxyacetic acid inhibited ethylene productionby etiolated mung bean hypocotyls by 50% at 3 and 6 µmconcentrations, respectively. Their potency is thus similarto that of aminoethoxyvinylglycine (50% inhibition at 2 µM),the most potent inhibitor of ethylene production hitherto known.Methionine partially alleviated inhibition of ethylene productionby a-aminooxy-acetic acid. The results are in agreement withthe postulated involvement of pyridoxal phosphate in ethylenebiosynthesis. (Received August 31, 1979; )     

Dose-dependent effects of malformin A1 on IAA-induced ethylene production in mung bean (Vigna radiate L.) hypocotyl segments

Purified malformin A1 (cyclo-D-Cys-D-Cys-L-Val-D-Leu-L-lle), a cyclicpentapeptide toxin fromAspergillus niger, was applied to the hypocotyl segments of mung bean (Vigna radiata L.) seedlings to investigate its role in regulating ethylene biosynthesis. Production of ethylene was induced by treating the plants with 0.1 mM indole-3-acetic acid (1AA). When 0.1 μM malformin A1 was then applied, ethylene production increased and the activities of two key enzymes for its biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase (ACS) and ACC-oxidase (ACO), were also stimulated. However, at levels of 1 or 10 μM malformin A1, both ethylene production and enzymatic activities were significantly reduced. In the case of ACO,in vitro activity was regulated by malformin A1, independent of ACS activity or the influence of IAA. Furthermore, the conjugate form of ACC, N-malonyl ACC, was significantly promoted by treatment with 0.1 μM malformin A1. These data suggest that malformin A1 can modulate ethylene production through diverse paths and that its effect depends on the concentration of the treatment administered.     

Studies on soluble RNA binding indoleacetic acid in etiolated mung bean hypocotyl sections

Nucleic acid was extracted by the SLS-phenol method from Phaseolusaureus hypocotyl treated with IAA-2-¹⁴C. Radioactivity in thenucleic acid fraction was found at the positions of sRNA andrRNA on an MAK column. IAA-¹⁴C was released from the radioactivecompound(s) in the sRNA fraction, by alkaline hydrolysis, butnot by ethanol extraction, or by dialysis to 2 M NaCl, 8 M urea,and 0.1 M EDTA. When the radioactive compound at the positionof sRNA on an MAK column was further re-chromatographed on aDEAE-cellulose column and on a BD-cellulose column, it was alwayslocalized only in a settled part of the fraction of each column.From this fraction IAA-¹⁴C was released by alkaline hydrolysis.Also, IAA-¹⁴C was released from the radioactive compound insRNA fraction, by RNase digestion, but not by pronase treatment.Results of these experiments suggest the existence of some kindsof sRNA binding IAA. The genesis of this sRNA binding IAA-¹⁴Cwas observed within 30 min after the supply of IAA-¹⁴C, andthe sRNA became saturated with IAA-¹⁴C about 2 hr after thebeginning of incubation. The behavior of sRNA binding IAA, representedby sRNA binding IAA-¹⁴C, may have a role in IAA induced growthof mung bean hypocotyl sections. (Received July 6, 1971; )     

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.     

Brassinolide, a growth-promoting steroidal lactone

Brassinolide (BR), a naturally-occurring-steroidal lactone from rape ( Brassica napus L.) pollen, was compared with auxin for activity in a number of bioassay systems. Responses similar to IAA were elicited by BR in bioassays based upon bean hypocotyl hook opening, elongation of maize mesocotyl, pea epicotyl and azuki bean epicotyl sections, and fresh weight increase in Jerusalem artichoke (2,4-D used) and pea epicotyl sections. The azuki bean and dwarf pea epicotyl bioassays were much more responsive to BR than IAA (at 10 μ M ). Responses approximately two-fold greater in magnitude were elicited by IAA in the maize mesocotyl, bean hypocotyl hook and Jerusalem artichoke bioassays. Little or no response was elicited by BR (0.01 to μ M ) in the cress root or decapitated pea-lateral bud bioassays. A powerful synergism between BR and IAA was observed in the azuki bean, pea epicotyl and bean hypocotyl hook bioassays. Although, as previously reported, other steroidal substances are active in some of the bioassay systems tested, none compared with BR in magnitude and diversity of elicited responses.     

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)