Stimulation and inhibition of respiration by malformin: Relationship to enhanced ethane production

When apical bud sections of Phaseolus vulgaris were vacuum-infiltratedwith malformin, their rate of respiration increased significantlywithout a lag and without altering the respiratory quotient.When tissues were soaked in solutions, the effect of malforminwas concentration dependent. At high malformin concentration(200 µM), a marked inhibition of respiration for 8 to10 hr was followed by a rapid increase of oxygen uptake whichreached a level nearly 3-fold greater than that of controls.At lower concentration (20 µM) malformin had no effecton respiration for 8 to 10 hr, but then stimulated respirationmarkedly. Ca²⁺ completely blocked the early inhibition of respirationby malformin (200 µM), but had little or no effect onthe subsequent stimulation of respiration. Oxygen deficiencymay be a contributing factor to the enhancement of ethane productionby malformin. ¹Present address: Mobil Chemical Company, P.O. Box 240, Edison,New Jersey 08817, U.S.A. (Received August 28, 1979; )     

Effect of malformin on adventitious root formation and metabolism of indoleacetic acid-2-14C by Phaseolus vulgaris

Malformin inhibited rooting on cuttings of Phaseolus vulgaris.IAA antagonized malformin-induced inhibition of rooting, butmalformin inhibited IAA-induced swelling on the base of thecuttings. It was suggested that IAA-induced swelling was mediatedby ethylene. Malformin did not inhibit transport of root-promotingsubstances from upper portions of the cuttings or polar transportof IAA-2-¹⁴C, nor did it alter the melting point of DNA or thebinding of DNA to histone. Although malformin appeared to alterthe metabolism of IAA-2-¹⁴C, the effect may have been the resultof a marked and selective stimulation of efflux of IAA-2-¹⁴Cmetabolites by malformin. Efflux of IAA or its metabolites maycontribute toward inhibition of rooting by malformin. ¹ Journal Paper No. 4688 of the Purdue Agricultural ExperimentStation. Supported in part by grant GB-7158 from the NationalScience Foundation. ² Present address: Botanisches Institut der Technischen UniversitätBraunschweig, 3300 Braunschweig, Humboldtstraße 1. (Received March 9, 1972; )     

Synergistic effect of kinetin on IAA-induced ethylene production

Kinetin has a significant synergistic effect on IAA-inducedehtylene production in hypocotyl sections of eitolated mungbean(Phaseolus mungo L.) seedlings when it is administered immediatelyafter cutting. If the addition of kinetin was delayed, it becameless effective. Kinetin-pretreated sections followed by incubationeither in IAA or in IAA plus kinetin produced ehtylene essentiallyat an identical rate to the one treated with IAA plus kinetinat zero time. However, the buffer-preincubated sections followedby incubation in IAA or in IAA plus kinetin shows a much reducedrate of ehtylene production as compared with the one treatedwith IAA or with IAA plus kinetin at zero time, respectively.These results suggest that kinetin is required for the maximalstimulation of ethylene production only during the early incubationperiod while IAA is required continuously throughout the incubation;the removal of IAA from the IAA-treated tissues causes an immediatecessation of ethylene production. ¹Present address: Mann Laboratory, Department of Vegetable Crops,University of California, Davis, California 95616, U.S.A. (Received May 10, 1973; )     

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)     

Malformin negates the Inhibition of Ethrel-induced Leaf Abscission by AgNO3

In light, malformin completely abolished the ability of Ag⁺to inhibit Ethrel-induced leaf abscission from cuttings of Vignaradiata, even though Ag⁺ was applied 24 hr before malformin.Malformin itself did not induce abscission in the light. However,Ag⁺ was active on cuttings which had been pre-treated with malforminfor 2 days in the light. No evidence was obtained to suggestreaction between malformin and Ag⁺. In the dark, Ag⁺ had noeffect on stimulation of leaf abscission by malformin. (Received March 7, 1981; Accepted May 12, 1981)     

Isolation and characterization of the cell wall receptor of 14C-malformin in Phaseolus vulgaris L.

¹⁴C-malformin attaches to at least two cell wall receptors inPhaseolus vulgaris. One receptor was extracted with Tris buffer(pH 8.5) and the other with 0.1 N NaOH. In both cases, priortreatment of the walls with wall degrading enzymes (macerase,cellulysin) was required. The two receptors differed with regardto ultrafiltration and gel filtration chromatography. The Tris-extractedreceptor is a protein, probably a glycoprotein, which containshydroxyproline and sulfhydryl groups. Although cuttings nottreated with malformin had Tris-extractable receptor, formationof the receptor appeared to be enhanced by malformin. ¹ Present address: American Cyanamid, P. O. Box 400, Princeton,New Jersey 08540, U. S. A. (Received August 2, 1976; )     

Effect of malformin on the major constituents of Phaseolus vulgaris

Malformin inhibits wet and dry weight, nitrogen accumulation,and cell wall, RNA, DNA and protein synthesis in Phaseolus vulgaris.The relative proportion of dry matter and nitrogen in malformedtissues is increased in the ethanol soluble fraction and decreasedin the residue remaining after hydrolysis with 0.5 N HCl. Inhibitionof cell wall and protein synthesis was generally greater thaninhibition of nitrogen accumulation and RNA and DNA synthesis.The effects of malformin on the composition of P. vulgaris aresimilar to alterations in composition reported for ethylene,and opposite to those reported for gibberellic acid. ¹This research was supported by grant GB-7158 from the NationalScience Foundation and grant E-146-F from the American CancerSociety. ²Journal Paper No. 3509 of the Purdue Agricultural ExperimentStation. (Received October 23, 1968; )     

Factors Influencing Induction of Resistance to Dark Abscission by Malformin

Factors influencing induction of resistance to dark abscissionby malformin on cuttings of Vigna radiata during treatment inlight were examined. When light duration (13.5 W m^–2)increased from 0 to 48 h, the effect of malformin on subsequentdark abscission changed from stimulation only (0 to 4 h), stimulationfollowed by inhibition (8 to 12 h), to inhibition only (24 to48 h). Maximum abscission resistance occurred after 48 h whenirradiance was 6.6 W m^–2. Kinetin treatment in light reducedsubsequent dark abscission by controls but did not reduce abscissionon malformintreated cuttings. Hadacidin had no effect on inductionof abscission resistance by malformin. IAA, hydroxyproline,CaCl₂, sucrose, and NH₄NO₃ were inactive. ABA and ethephon completelyblocked induction of abscission resistance by malformin. Inhibitionof abscission induced by kinetin was also blocked by ABA. Becauseboth puromycin and malformin inhibited dark abscission followingtreatment in light, malformin may induce abscission resistanceby inhibiting protein synthesis or promoting formation of othersubstances which inhibit protein synthesis. Leaf blade removalfrom the distal end of the petioles abolished malformin-inducedabscission resistance. It is suggested that in light malformininduces formation of abscission-inhibiting compounds in leaveswhich are responsible for development of abscission resistance. (Received May 17, 1983; Accepted November 8, 1983)     

Effect of malformin on root growth

Malformin induces curvatures, stimulates root hair and lateralroot formation, promotes radial expansion, inhibits elongation,wet and dry weight, cell division and cell wall synthesis inroots of Zea mays, but has no effect on protein synthesis. Thegrowth curves (elongation, wet and dry weight) of Z. mays rootstreated with malformin are cubic. Processes which are involvedin inhibition of elongation are considered the primary causeof root curvatures by malformin. ¹This research was supported by grant GB-7158 from the NationalScience Foundation and grant E-146-F from the American CancerSociety. Journal Paper No. 3536 of the Purdue Agricultural ExperimentStation. ²Present address: Volcani Institute of Agricultural Research,P.O.B. 6, Bet-Dagan, Israel. (Received December 24, 1969; )     

Action of malformin A1 on gravitropic curvature in primary roots of maize (Zea mays L.)

Malformins, a small family of cyclic pentapeptides, are active plant growth regulators isolated from the fungusAspergillus niger. We purified malformin A₁ from the crude malformin A mixture, and studied its action in the gravitropic response of maize roots. Intact primary roots that had been pretreated vertically with malformin A₁ were placed in a humidified box in the horizontal position. Positive curvature (downward) was inhibited in the pretreated roots compared with the control. In addition, we measured the lateral transport of IAA in primary roots. Roots pretreated with malformin A, did not show asymmetric distribution of IAA between the upper and lower sides of the elongation zone. Malformin A, also stimulated ethylene production in maize root segments. Our results had suggested that malformin A₁ might inhibit the lateral transport of IAA across the roots from the upper to the lower side because of an increased level of ethylene. Therefore, we placed more IAA on the upper side at the initial phase of gravistimulation. These results were consistent with malformin A₁-pretreated roots showing inhibited positive gravitropic curvature.     

Time sequence of the effect of ethylene on transport, uptake and decarboxylation of auxin

The effect of ethylene on the uptake, decarboxylation and basipetaltransport of IAA-1-¹⁴C, IAA-2-¹⁴C and NAA-1-¹⁴C in cotton stemsections (Gossypium hirsutum L., var. Stoneville 213) was studied.A reduction in the capacity of cotton stem sections to transportauxin basipetally appears in sections excised from plants exposedto ethylene for only 3 hr and increases with fumigation time. In addition to reducing transport, increasing ethylene pretreatmentperiods from 3 to 15 hr also progressively reduced the uptakeof ¹⁴C and increased the release of ¹⁴C as ¹⁴CO₂ from IAA-1-¹⁴C.The effect of ethylene on the decarboxylation of IAA-1-¹⁴C wassignificant when expressed as either the cpm of ¹⁴C releasedper hr per mg dry weight or the cpm released per hr per mm²in contact with the IAA donor. Comparative experiments usingIAA-1-¹⁴C and IAA-2-¹⁴C demonstrated that the effect of ethyleneon the decarboxylation of IAA was primarily a cut surface effectwhich apparently contributes to the reduction of IAA uptakeby ethylene. Although ethylene significantly reduced the transport of NAA-1-¹⁴C,uptake was significantly increased rather than decreased aswith IAA-1-¹⁴C while decarboxylation was unaffected. Ethylene pretreatment caused no significant changes in the dryweight or the cross-sectional area of the absorbing surfaceof the transport tissue. ¹A contribution of the Texas Agricultural Experiment Station.Supported in part by Grant GB-5640, National Science Foundationand grants from the Cotton Producers Institute and the NationalCotton Council of America. ²Present address: Central Research Department, E. I. Du PontDe Nemours and Company, Wilmington, Delaware 19898, U. S. A. (Received May 29, 1969; )     

Regulation of ethylene production by phosphate in Penicillium digitatum

Inorganic phosphate regulated ethylene production in shake culturesof Penicillium digitatum. Decreasing the phosphate level ofthe medium from 100 to 0.01 mM markedly increased, about 100-fold,the rate of ethylene production, in 96 hr, which was confinedentirely to the fungal mycelium. Exogenous addition of between0.01 to 100 mM phosphate, to high ethylene producing, low-phosphatecultures strongly inhibited their ethylene production and increasedthe ATP content of the mycelium. Phosphate also inhibited ethyleneproduction in apple slices. Addition of calcium ions to theincubation medium stimulated the production of ethylene in appleslices, subhook epicotyl segments of pea and shake culturesof P. digitatum. We suggest that this stimulatory effect wascaused by the reduction of inhibitory levels of phosphate, whichcomplexed with calcium. Thus, phosphate in conjunction withcalcium may play an important role in regulating ethylene productionnot only in P. digitatum but also in higher plants. ¹ On leave from the Agricultural Research Organization, TheVolcani Center, Israel. ² On leave from the M.S. University of Baroda, India. (Received September 7, 1977; )     

Stimulation of Auxin Induced Ethylene Production in Mung Bean Hypocotyl Segments by 5,5' -Dithiobis-2-nitrobenzoic Acid

The non-permeant protein inhibitor 5,5'-dithiobis-2-nitrobenzoicacid (DTNB) was tested for its effects on auxin induced ethyleneproduction. There was a stimulation in the rate of auxin inducedethylene production at all concentrations of DTNB tested (1,2, 5, and 10 mM). The 5 mM DTNB treatment promoted the maximumstimulation of ethylene production with no further enhancementat the 10 mM concentration. After 12 hr ethylene productionplateaued with 0.1 mM indoleacetic acid (IAA) alone and in combinationwith 1 and 2 mM DTNB. Although the DTNB treatments plateauedit was at a higher level than IAA alone. Both the 5 and 10 mMtreatments of DTNB plus IAA did not show this leveling responseeven after 22 hr at which time these treatments were between90 and 100% higher than the control. There was no stimulationof ethylene production by DTNB in the absence of IAA. Segmentstreated with 10^–4 M rß-naphthaleneacetic acid(NAA) produced significantly higher levels of ethylene thanIAA at the same concentration. Stimulation of ethylene productionby DTNB was greatest at lower concentrations of IAA and NAA.The uptake of ¹⁴C-NAA by mung bean segments was 6-fold greaterin the presence of DTNB than in its absence. Ca^SS was requiredin the incubating media for DTNB to be effective. In the presenceof Ca^SS there was a highly significant increase in ethyleneproduction while in its absence there was no significant effect.The stimulation of IAA induced ethylene production appearedto have a pH optima of 4.6, at higher pH values this responsewas not shown. ¹ Approved for publication May 28, 1981 as paper number 6243in the journal series of the Pennyslvania Agricultural ExperimentStation. (Received June 10, 1981; Accepted January 5, 1982)     

Effect of isopropyl-N-(3-chlorophenyl) carbamate on the production of ethylene by mungbean hypocotyl sections

Isopropyl-N-(3-chlorophenyl) carbamate (CIPC) stimulates ethyleneproduction in hypocotyl sections of etiolated mungbean seedlings.The amount of ethylene produced is dependent on the concentrationsof CIPC applied. However, CIPC markedly inhibits IAA-inducedethylene production at relatively low concentration. It is possiblethat both CIPC and IAA are competing for the same cellular siteto induce ethylene production. However, the effectiveness ofCIPC in inducing ethylene production is much less compared toIAA treatment. The inhibition of ethylene production inducedby IAA at high concentration of CIPC may be solely due to thecomplete occupancy of the cellular site by CIPC and the effectof IAA is, therefore, eliminated. ¹ Present address: Mann Laboratory, University of California,Davis, California, U.S.A. (Received September 19, 1972; )     

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 THE GROWTH OF FRUIT BODY OF FUNGI: III. OCCURRENCE, FORMATION AND DESTRUCTION OF INDOLE ACETIC ACID IN THE FRUIT BODY OF AGARICUS BISPORUS (LANGE) SING

Considerable amounts of auxin, mostly IAA, in acid and boundforms, occur in gills, pilei and stipes of the mushroom, Agaricusbisporus. Stipe elongation is not stimulated by applicationof IAA. The juice squeezed from the fruit body has an activity to convertL-tryptophan into IAA. This activity is not lost by heating.The substance(s) responsible for it passes through cellophane,and is insoluble in some organic solvents, such as petroleumether and benzene. The amount of IAA produced depends exactlyon the amount of the extraction residue used for the reaction.Since the activity decreases as thiosulfate is added increasingly,the active principle seems to be some strong oxidizing substance(s). ¹ This paper was read on October 25th, 1958, at the 23rd annualmeeting of the Botanical Society of Japan, held at Fukuoka,Japan. ² This investigation has been aided by a grant from the ROCKEFELLERFoundaion. ³ Present Address: J. W. GIBBS Research Laboratory, Departmentof Botany, Yale University. New Haven, Conn., U.S.A. (Received June 6, 1961; )     

Does ethylene induce elongation of the rice coleoptile through auxin?

Ethylene stimulated the elongation of intact rice (Oryza sativaL.) coleoptiles in which endogenous growth had been stoppedcompletely by decapitation and red light. p-Chlorophenoxyisobutyricacid slightly inhibited endogenous growth, but not the ethyleneinduced growth. Thus, ethylene could stimulate the elongationof coleoptiles in which the auxin level was considered to bevery low. ¹ Present address: Institute for Agricultural Research, TohokuUniversity, Katahira, Sendai 980, Japan. (Received February 16, 1979; )     

Auxin and Ethylene-stimulated Adventitious Rooting in Relation to Tissue 9

We have previously shown that both endogenous auxin and ethylenepromote adventitious root formation in the hypocotyls of derootedsunflower (Helianthus annuus) seedlings. Experiments here showedthat promotive effects on rooting of the ethylene precursor,1-aminocyclopropane-l-carboxylic acid (ACC) and the ethylene-releasingcompound, ethephon (2-chloro-ethylphosphonic acid), dependedon the existence of cotyledons and apical bud (major sourcesof auxin) or the presence of exogenously applied indole-3-aceticacid (IAA). Ethephon, ACC, aminoethoxyvinylglycine (an inhibitorof ethylene biosynthesis), and silver thiosulphate (STS, aninhibitor of ethylene action), applied for a length of timethat significantly influenced adventitious rooting, showed noinhibitory effect on the basipetal transport of [³H]IAA. Theseregulators also had no effect on the metabolism of [³H]IAA andendogenous IAA levels measured by gas chromatography-mass spectrometry.ACC enhanced the rooting response of hypocotyls to exogenousIAA and decreased the inhibition of rooting by IAA transportinhibitor, N-1-naphthylphthalamic acid (NPA). STS reduced therooting response of hypocotyls to exogenous IAA and increasedthe inhibition of rooting by NPA. Exogenous auxins promotedethylene production in the rooting zone of the hypocotyls. Decapitationof the cuttings or application of NPA to the hypocotyl belowthe cotyledons did not alter ethylene production in the rootingzone, but greatly reduced the number of root primordia. We concludethat auxin is a primary controller of adventitious root formationin sunflower hypocotyls, while the effect of ethylene is mediatedby auxin. Key words: Auxin, ethylene, adventitious rooting, sunflower     

Cooperation of Ethylene and Auxin in the Growth Regulation of Rice Coleoptile Segments

Elongation of coleoptile segments, having or not having a tip,excised from rice (Oryza sativa L. cv. Sasanishiki) seedlingswas promoted by exogenous ethylene above 0.3 µl l^–1as well as by IAA above 0.1 µM. Ethylene production ofdecapitated segments was stimulated by IAA above 1.0µM,and this was strongly inhibited by 1.0 µM AVG. AVG inhibitedthe IAA-stimulated elongation of the decapitated segment witha 4 h lag period, and this was completely recovered by ethyleneapplied at the concentration of 0.03 µl l^–1, whichhad no effect on elongation without exogenous IAA. The effectsof IAA and ethylene on elongation were additive. These factsshow that ethylene produced in response to IAA promotes ricecoleoptile elongation in concert with IAA, probably by prolongingthe possible duration of the IAA-stimulated elongation, butthat they act independently of each other. Moreover, AVG stronglyinhibited the endogenous growth of coleoptile segments withtips and this effect was nullified by the exogenous applicationof 0.03 µl l^–1 ethylene. These data imply that theelongation of intact rice coleoptiles may be regulated cooperativelyby endogenous ethylene and auxin in the same manner as foundin the IAA-stimulated elongation of the decapitated coleoptilesegments. Key words: oryza sativa, Ethylene, Auxin, Coleoptile growth     

Mediation of a plant response to malformin by ethylene

Malformin and ethylene stimulate abscission of the primary leaves of Phaseolus aureus Roxb. in the dark, and abscission stimulation by both compounds is inhibited by indeleacetic acid and CO₂. Ethylene production by malformin-treated buds is stimulated within 4 hours. and up to 8 days, after treatment. Malformin-induced growth disturbances in P. vulgaris L. and abscission in P. aureus are considered mediated by ethylene. Although root curvatures of Zea mays L. are induced by both malformin and ethylene, and malformin is inhibited by CO₂, ethylene production is not stimulated by malformin. A role of ethylene in root curvatures induced by malformin is neither proposed nor disproved.