Inhibition of Ethylene Production in Penicillium digitatum

Production of ethylene by static cultures of Penicillium digitatum, which utilize glutamate and α-ketoglutarate as ethylene precursors, was inhibited by methionine, methionine sulfoxide, methionine sulfone, and methionine sulfoximine. Rhizobitoxine did not affect ethylene production but its ethoxy and methoxy analogues were effective inhibitors of ethylene production; its saturated methoxy analogue and kainic acid stimulated ethylene production. Tracer studies showed that the inhibitors blocked the conversion of [³H]glutamate into [³H]ethylene.

In shake cultures of this fungus, which utilize methionine as the ethylene precursor, rhizobitoxine and its unsaturated analogues all inhibited, while the saturated methoxy analogue stimulated ethylene production. In both types of cultures inhibition was irreversible and was diminished by increasing concentrations of the ethylene precursor. The inhibitory activity or lack of it by rhizobitoxine and its analogues appears to be a function of their structural resemblance to glutamate and methionine as well as of their size and stereoconfiguration. These data suggest similarities between the ethylene-forming system in the fungus and in higher plants despite differences in precursors under some cultural conditions.

     

Application of Hormetic UV-C for Delayed Ripening and Reduction of Rhizopus Soft Rot in Tomatoes: the Effect of Tomatine on Storage Rot Development

The application of ultraviolet-light C (UV-C, 254 nm) hormesis on tomato fruits to stimulate beneficial responses is a new method of inducing host resistance to Rhizopus soft rot, with maximum protection at 72 h following artificial inoculation with Rhizopus stolonifer and delay fruit ripening. In the tomato-Rhizopus soft rot pathosystem, UV-C induced resistance of tomatoes to soft rot was reversed by fluorescent light, and not by storage under dark condition. The present study was aimed at finding a possible fungal toxic mechanism that was involved in the control of Rhizopus soft rot. The development of carotenoids, loss of chlorophyll and ethylene production were significantly retarded during storage after treatment with hormetic doses of UV-C. The delayed ripening of UV-C treated fruits was attributed in part to the high level of putrescine and spermine polyamines. In a time-control study, it was found that the resistance of UV-C-treated tomatoes artificially inoculated with R. stolonifer correlated with a high concentration of tomatine which accumulated up to 72 h after UV-C treatment. However, there was a higher tomatine content in UV-C-treated fruits compared to untreated fruits where it was transitory at 96 h after treatment in the time-course study.     

Rhythmicity in ethylene production in cotton seedlings

Cotyledons of cotton (Gossypium hirsutum L.) seedlings grown under a photoperiod of 12 hour darkness and 12 hour light showed daily oscillations in ethylene evolution. The rate of ethylene evolution began to increase toward the end of the dark period and reached a maximum rate during the first third of the light period, then it declined and remained low until shortly before the end of the dark period. The oscillations in ethylene evolution occurred in young, mature, and old cotyledons (7 to 21 day old). These oscillations in ethylene evolution seemed to be endogenously controlled since they continued even when the photoperiod was inverted. Moreover, in continuous light the oscillations in ethylene evolution persisted, but with shorter intervals between the maximal points of ethylene evolution. In continuous darkness the oscillations in ethylene evolution disappeared. The conversion of [3,4-¹⁴C]methionine into [¹⁴C] ethylene followed the oscillations in ethylene evolution in the regular as well as the inverted photoperiod. On the other hand, the conversion of applied 1-aminocyclopropane-1-carboxylic acid into ethylene did not follow the oscillations in ethylene evolution, but was affected directly by the light conditions. Always, light decreased and darkness increased the conversion of applied 1-aminocyclopropane-1-carboxylic acid into ethylene. It is concluded that in the biosynthetic pathway of ethylene the conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene is directly affected by light while an earlier step is controlled by an endogenous rhythm.     

Methionine-induced Ethylene Production by Penicillium digitatum

Shake cultures, in contrast to static cultures of Penicillium digitatum grown in liquid medium, were induced by methionine to produce ethylene. The induction was concentration-dependent, and 7 mM was optimum for the methionine effect. In the presence of methionine, glucose (7 mM) enhanced ethylene production but did not itself induce ethylene production. The induction process lasted several hours, required the presence of viable mycelium, exhibited a lag period for ethylene production, and was effectively inhibited by cycloheximide and actinomycin D. Thus, the methionine-induced ethylene production appeared to involve induction of an enzyme system(s). Methionine not only induced ethylene production but was also utilized as a substrate since labeled ethylene was produced from [¹⁴C]methionine.     

Effects of lipophilic and water-soluble membrane probes on ethylene synthesis in apple and Penicillium digitatum

Several chemicals were used to probe the in situ ethylene formingenzyme systems in apple tissue and Penicillium digilatum. 2,4-Dinitrofluorobenzene,a membrane permeant probe, inhibited ethylene production effectivelyin apples but far less effectively in P. digitatum. In contrast,salicylaldehyde, another membrane permeant probe, effectivelyinhibited the P. digitatum system but, except at 0.1 mM concentration,little influenced the apple system. l,5-Difluoro-2,4-dinitrobenzene(DFDNB), a membrane permeant probe which cross-links proteinswith proteins and with phospholipids, strongly inhibited ethylenebiosynthesis in both apple and P. digitatum, whereas dimethylsuberimidate, the protein cross-linking reagent, inhibited slightlythe apple system but not P. digitatum system. Picrylsulfonate(TNBS), a non-permeant membrane probe, up to 0.1 mM, did notinhibit any of the two systems studied. However, in the presenceof exogenous methionine in the apple system and glutamate inP. digitatum, TNBS at 0.1 and 1 mM caused inhibition of ethylenesynthesis. These probes did not affect respiration of appleslices under similar incubating conditions, excepting for DFDNBwhich on longer incubation did inhibit respiration, but theeffect on ethylene synthesis was 15 times greater. Divalentcation ionophores, A23187 [GenBank] and X537 A, had no effect on ethylenesynthesis in both the systems. The water soluble iron chelatingagent, o-phenanthroline, was a more potent inhibitor of theapple system but minimally affected P. digitatum. In contrast,the lipophilic chelator, bathophenanthroline, was a more potentinhibitor of the P. digitatum system. Assay of the fatty acidcomposition of polar lipids from crude membrane fractions showedconsiderably greater linoleic to linolenic ratio in P. digitatumthan in apple. We suggest that the ethylene formations in appleand P. digitatum are sensitive to a modification of membranestructure and that specific chelator-sensitive metals (perhapsiron and copper) are involved in ethylene synthesis in boththese systems. ¹ On leave from the M.S. University of Baroda (India); presentaddress: Department of Plant Genetics, The Weizmann Instituteof Science, Rehovot, Israel. ²Present address: Agricultural Research Organization, The VolcaniCenter, Bet-Dagan, Israel. (Received February 23, 1979; )     

Enhancement by ethylene of cellulysin-induced ethylene production by tobacco leaf discs

Cellulysin-induced ethylene production in tobacco (Nicotiana tabacum L.) leaf discs was enhanced several-fold by prior exposure of the leaf tissue to ethylene. This enhancement in the response of the tissue to Cellulysin increased rapidly during 4 and 8 hours of pretreatment with ethylene and resulted from greater conversion of methionine to ethylene. On treatment with Cellulysin, the content of 1-aminocyclopropane-1-carboxylic acid (ACC) in leaf discs not pretreated with ethylene markedly increased while that of the ethylene-pretreated tissue was only slightly higher than in the tissue incubated in the absence of Cellulysin. Ethylene-treated tissue, however, converted ACC to ethylene at a faster rate than air controls. These data indicate that ethylene stimulates Cellulysin-induced ethylene production by stimulating the conversion of ACC to ethylene. Data are also presented on a possible relation of this phenomenon to ethylene produced by the tobacco leaf upon interaction with its pathogen, Alternaria alternata.     

Rhythmicity in cotton seedlings

Ethylene production by detached cotyledons of cotton (Gossypium hirsutum L.) seedlings grown under cycles of 12 h darkness and 12 h light has been shown to be rhythmic, with a minimum and maximum 4 and 16 h, respectively after the start of the cycle (Rikin, Chalutz and Anderson, 1984, Plant Physiol. 75, 493–495). Treatment with silver ions stimulated the rhythmic ethylene production in both regular and inverted cycles (i.e. dark period changed to light period, and vice versa). The rate of the conversion of [3,4-¹⁴C]methionine into ethylene also followed the stimulation of rhythmic ethylene evolution by silver ions in both regular and inverted cycles, while treatment with aminoethoxyvinylglycine (AVG) decreased this stimulation. Conversion of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene was not affected by silver ions, but was dependent upon the immediate light conditions, regardless of the time in the light-dark cycle, light decreasing and darkness increasing this process. It is concluded that silver ions stimulate the normal rhythmic ethylene production, and this stimulation is regulated at a step prior to the conversion of ACC into ethylene. The rhythmicity in other processes (cotyledon movement, phenylalanine ammonia-lyase activity, resistance to the herbicide 3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide [bentazon]) was not affected by a decrease in the rhythmic changes in ethylene production by AVG or interference in ethylene action by silver ions. Thus, these rhythmic changes were not regulated by the rhythmic changes in ethylene production.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethyoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Ethylene-induced Phenylalanine Ammonia-Lyase Activity in Carrot Roots

Ethylene enhanced the activity of phenylalanine ammonialyase in carrot (Daucus carota L., var. “Nauty”) root tissue. Slight increase in enzyme activity was exhibited by root discs incubated in ethylene-free air. It was probably due to the ethylene formed within the sliced tissue. Addition of ethylene to the air stream increased phenylalanine ammonia-lyase activity and the total protein content of the discs until maximum activity was reached after 36 to 48 hours of incubation. The continuous presence of ethylene was required to maintain high level of activity. Ethylene, at a concentration of 10 microliter per liter induced higher activity than at lower or higher concentrations. CO₂ partially inhibited the ethylene-induced activity. Cycloheximide or actinomycin D effectively inhibited the ethylene-induced activity in discs that had not previously been exposed to ethylene. The results appear to support the hypothesis that the mode of action of ethylene may involve both de novo synthesis of the enzyme protein and protection or regulation of activity of the induced enzyme.