Structure of malformin B, a phytotoxic metabolite produced by Aspergillus niger.

Malformin B, produced by Aspergillus niger, was separated into six compounds by HPLC. Their structures were determined by amino acid analyses, and by mass spectral and two-dimensional NMR experiments to be cyclic pentapeptides structurally related to malformin A1. Both the NMR and MS/MS experiments suggest cyclo-D-cysteinyl-D-cysteinyl-L-amino acid-D-amino acid-L-amino acid as the essential structure of malformins.

Structure of Malformin A, a Phytotoxic Metabolite Produced by Aspergillus niger.

Malformins-produced by Aspergillus niger were separated by HPLC and subjected to structural determination. Amino acid analyses and mass spectra suggested that all of them structurally resembled cyclic pentapeptide malformin A1. Two-dimensional NMR experiments and MS/MS experiments led us to deduce cyclo-D-cysteinyl-D-cysteinyl-L-amino acid-D-amino acid-L-amino acid as being the essential structure of malformins.

Allomalformin.

In an attempt to find explanation for the initial erroneous sequence assignment for malformin, a sequence-isomer of the natural product, 3-isoleucine-5-valine malformin or briefly "allomalformin" that on partial acid hydrolysis could have given rise to misleading fragments, was synthesized and compared with both natural and synthetic preparations of malformin. Allomalformin is identical to the parent microbial peptide (malformin A, or briefly malformin) with respect to biological activity and conformation (ORD and CD spectra) and is indistinguishable from it by high pressure liquid chromatography. Yet, the two isomers have slightly different Rf values on thin-layer chromatograms and by this method no allomalformin could be detected in samples of the natural product. On the other hand both high pressure liquid chromatography and thin-layer chromatography demonstrated the presence of the lower homolog, 5-valine malformin, in the samples examined. On partial acid hydrolysis this natural analog should liberate Val-Cys, while Cys-Val forms from malformin itself. Similarly, the corresponding desthio cyclopentapeptides should give rise to Val-Ala and Ala-Val respectively; the former being more resistant to further hydrolysis persists in the partial hydrolysates. The presence of Val-Cys in partial hydrolysates of malformin and of Val-Ala in the partial hydrolysates of desthiomalformin, both originating from the accompanying lower homolog rather than from malformin itself, is likely to have led to the postulation of the erroneous Cys-Val-Cys partial sequence.

Light Requirement for AgNO(3) Inhibition of Ethrel-Induced Leaf Abscission from Cuttings of Vigna radiata.

To obtain information regarding the antiethylene properties and binding site of Ag(+), studies were initiated to define conditions under which Ag(+) does or does not inhibit ethylene action. AgNO(3), applied as a leaf spray, inhibited 2-chloroethylphosphonic acid (Ethrel)-induced leaf abscission from green cuttings of Vigna radiata in white light but lost considerable activity in the dark. In the absence of Ethrel, AgNO(3) stimulated abscission in the dark. When cuttings were dark-aged for 24 hours prior to treatment with AgNO(3) and aged for an additional 24 hours in the dark after treatment, good inhibition of subsequent Ethrel-induced abscission was restored by returning the cuttings to light. However, when dark aging was preceded by far-red irradiation, considerably less inhibition of Ethrel-induced abscission was restored in the light. AgNO(3) was completely inactive on cuttings aged in the dark and treated with Ethrel in the dark. Light is required for the antiethylene activity of AgNO(3) with regard to leaf abscission of Vigna.

Induction of resistance to dark abscission by malformin in white light.

When cuttings or seedlings of Phaseolus aureus were treated proximally with malformin for 2 days in continuous white light, resistance to subsequent leaf abscission in the dark resulted. The amount of resistance diminished as the concentration of malformin decreased from 10 to 0.1 micromolar. Resistance to dark abscission persisted for 7 days in continuous light. Little resistance was obtained when cuttings were taken from seedlings grown under low irradiance and short photoperiods, but resistance gradually increased as the photoperiod increased. Resistance to dark abscission induced by malformin in light differs from inhibition of abscission by indoleacetic acid because when malformin is applied in the dark it stimulates abscission after distal or proximal application. Malformin induces resistance only in conjunction with light treatment.

Phytochrome involvement in the induction of resistance to dark abscission by malformin.

The active portion of the visible spectrum which is required for malformin to produce leaves which are resistant to dark abscission from cuttings of Phaseolus aureus is red light. Abscission resistance was partially to almost completely lost by far irradiation prior to dark incubation. Although Ethrel, an ethylene releasing compound, stimulated dark abscission of resistant and control leaves, resistance was not lost because control leaves always abscised at a greater rate. The participation of phytochrome in the induction of abscission resistance by malformin is indicated.

Phytochrome involvement in stimulation and alleviation of inhibition of plant growth by malformin.

Stimulation of stem elongation on green cuttings of Phaseolus aureus by malformin occurred only in red light and was specifically reversible by subsequent treatment with far red radiation. Inhibition of stem elongation of etiolated cuttings by malformin in the dark was alleviated by red light and was repeatedly reversible with far red irradiation. A direct or indirect effect of malformin on phytochrome action was suggested.

White light requirements for stimulation of plant growth by malformin.

Over a 3-day period, the minimum white fluorescent light intensity required for malformin-induced growth stimulation of etiolated and green cuttings of Phaseolus aureus was approximately 2.6 x 10(3) and 0.4 x 10(3) ergs/cm(2) . sec, respectively. High light intensities were unable to inhibit the ability of malformin to stimulate growth. Over 3 days, the minimum photoperiod for malformin-induced growth stimulation using etiolated and green cuttings and a light intensity of 13.5 x 10(3) ergs/cm(2) . sec was 4 hours and 1 hour, respectively. Malformin must be present in the area of growth stimulation during the time of light treatment. Those changes induced by light and required for malformin-induced growth stimulation were estimated to undergo almost complete decay within 1 hour in the dark. By manipulating the experimental technique, it was possible to stimulate the growth of green cuttings with malformin with a 10-min light treatment (13.5 x 10(3) ergs/cm(2) . sec). Although low light intensities and short photoperiods did not allow growth stimulation by malformin using etiolated cuttings, they prevented or alleviated growth inhibition induced by malformin in the dark.

Isolation and Identification of the Precursor of Ethane in Phaseolus vulgaris L.

Ethane production by homogenates of Phaseolus vulgaris L. cv. Harvester was studied. The precursor of ethane was identified as linolenic acid. The liberation of ethane was optimum at pH 4.2 and was highest from homogenates of leaves and apical buds. When roots were homogenized in linolenic acid solution, ethane and ethylene production were stimulated. In corn root homogenates, ethylene biosynthesis was stimulated nearly 8-fold by linolenic acid. The enzyme responsible for ethane production from oat root homogenates was soluble and had a high molecular weight.

A Mung Bean Assay for Malformin-induced Growth Stimulation.

A bioassay employing green or etiolated cuttings of Phaseolus aureus Roxb. was developed for determining malformin-induced growth stimulation in light. Growth enhancement of green cuttings was more rapid and relatively greater than that of etiolated cuttings. Cuttings from green seedlings responded less as seedlings aged; those from etiolated seedlings responded more. Malformin also stimulated the growth of green or etiolated seedlings in light. Most growth enhancement induced by malformin occurred in the upper 1 cm of the stem. Using green cuttings, malformin stimulated stem elongation relatively more when cotyledons, leaves, or especially apical buds were removed. Although malformin failed to stimulate elongation of 2-cm stem sections "floated" on solutions in Petri dishes, it stimulated elongation of sections when they were upright. High concentrations of indoleacetic acid inhibited growth enhancement by malformin. When gibberellin and malformin were combined, growth enhancement was nearly additive.

Malformin in Aspergillus niger-infected onion bulbs (Allium cepa).

Malformin was identified, by its biological activity and chromatography, in acetone extracts of the outer scales of onion bulbs infected with Aspergillus niger. Malformin was not detected in tissue underlying the infected areas or in the central portions of the bulbs, nor was malformein liberated from extracts or extracted tissues after reduction with zinc in acetic acid. This is the first report of naturally occurring malformin.

Potentiation and inhibition of the effects of 2-chloroethylphosphonic Acid by malformin.

Malformin completely inhibited Ethrel-induced swelling and fresh weight increase on the basal stem portion of Phaseolus vulgaris L. cuttings, but markedly potentiated Ethrel- or ethylene-induced abscission. With regard to abscission, malformin reacted synergistically with ethylene and dark aging, and in a manner which appeared to differ from that of ethylene and dark aging. The numerous effects of malformin on plant growth and development cannot be explained in simple terms of enhanced ethylene production.

Production of ethylene by fungi.

Ethylene was detected by gas chromatography, and verified by chemical means, as a metabolic product of 22 species of fungi. Because 58 of 228 species of fungi produced a gaseous compound with retention time identical to that of authentic ethylene, we believe that this compound is a common metabolic product of fungi.

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 CO(2). 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 CO(2), ethylene production is not stimulated by malformin. A role of ethylene in root curvatures induced by malformin is neither proposed nor disproved.