Ethylene inhibitors enhance in vitro root formation from apple shoot cultures

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and three ethylene inhibitors, AgNO₃, aminoethoxyvinyglycine (AVG) and CoCl₂, on root formation were tested in vitro using shoot cultures of the apple (Malus×domestica Borkh.) cultivar Royal Gala. ACC inhibited root formation by delaying root emergence and increasing callus formation at the bases of shoots. In contrast, ethylene inhibitors promoted root formation. Both AgNO₃ and AVG at the appropriate concentrations increased the percentage of shoots producing roots and reduced callus formation at the base of these shoots. AgNO₃ stimulated root emergence and enhanced root growth, while AVG increased the number of roots per shoot. CoCl₂ slightly increased root number and rooting efficiency. These promotive effects may result from a reduction in ethylene concentration or inhibition of ethylene action. The results found in this study may be used to improve the rooting efficiency of other apple cultivars and rootstocks, and possibly of other plant species. Received: 2 March 1997 / Revision received: 1 July 1997 / Accepted: 18 July 1997     

Ethylene and in vitro rooting of rose shoots

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene biosynthesis inhibitor, (CoCl₂), and inhibitor of ethylene binding to receptors, 1-methylcyclopropene (1-MCP), on ethylene production and rooting in shoot culture of Rosa hybrida L. cv. Alba meidiland were studied. Additionally, effect of ethylene removal by KMnO₄ and HgClO₄ on rooting was tested. ACC increased ethylene production and delayed root formation, decreased the number of roots per shoot and inhibited root growth. In contrast, inhibition of ethylene production by CoCl₂ accelerated root emergence, and increased the number of roots per shoot. Likewise, removing ethylene from the ambient atmosphere improved root emergence and, increased root number of per shoot and markedly inhibited root growth. Blocking the ethylene receptors by 1-MCP increased ethylene level in the ambient atmosphere and increased both emergence and root formation. Both ethylene biosynthesis and action are involved in the control of rooting. Ethylene concentration in glass jars was too high for root emergence and formation, but was appropriate for root growth. CoCl₂ or 1-MPC can be recommended for regulation of rooting in rose shoot culture, since both emergence and number of roots were improved but root growth was not inhibited.     

1-Aminocyclopropane-1-Carboxylic Acid Enhanced Direct Somatic Embryogenesis from Oncidium Leaf Cultures

Influence of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and two ethylene inhibitors, silver nitrate (AgNO₃) and cobalt chloride (CoCl₂), on direct somatic embryogenesis were tested in vitro using leaf cultures of Oncidium cv. Gower Ramsey. Leaf cells of tips, adaxial sides and cut ends could directly form somatic embryos on a hormone-free 1/2-strength MS medium. The frequency of embryo-producing explants was 55, 52.5 and 30 %, respectively. The embryo numbers per embryo-producing explant was 20.3. ACC at lower concentrations (5 and 10 μM) significantly retarded direct embryo formation from cut ends. However, higher concentrations of ACC (20 and 50 μM) significantly promoted embryogenic response of leaf tips and adaxial sides. All concentrations of AgNO₃ and CoCl₂ significantly retarded direct embryo formation. The best response was found on 20 μM of ACC, and the frequency of embryo-producing explants were 90, 85 and 35 % on leaf tips, adaxial sides and cut ends, respectively. The embryo numbers per embryo-producing explant was 32.2. This revised version was published online in July 2006 with corrections to the Cover Date.     

Headspace ethylene accumulation on Stizolobium hassjoo hairy root culture producing L-3,4-dihydroxyphenylalanine

The addition of 1-aminocyclopropane-1-carboxylic acid (ethylene precursor), or 2-chloroethylphosphonic acid (ethephon, an ethylene-releasing compound) decreased root dry weight and l-DOPA (l-3,4-dihydroxyphenylalanine) accumulation in hairy root cultures of Stizolobium hassjoo. The inhibition caused by ethephon-mediated ethylene release was alleviated by 0.5 mg CoCl₂ l^–1 as an inhibitor of ethylene biosynthesis. The action of ethylene was inhibited by 1.5 mg AgNO₃ l^–1. Ethylene thus lowers hairy root formation and l-DOPA production; CoCl₂ decreases ethylene formation leading to a considerably improved root dry weight and l-DOPA production.     

Ethylene as an endogenous inhibitor of root regeneration in tomato leaf discs cultured in vitro

We examined ethylene effects on root regeneration in tomato leaf discs cultured in vitro. Applied ethylene or Ethephon did not stimulate rooting in the leaf discs. In the presence of indoleacetic acid. 5 × 10^-6M, these substances significantly inhibited root formation. Ethylene production (nl C₂H₄· (24 h)^-1. flask^-1) was positively correlated with increased IAA concentrations at various times during the culture period and, as a consequence, with the rooting response after 168 h. However, separate testing of equimolar concentrations of seven different auxins and auxin-like compounds showed no positive correlation between the rate of ethylene production and subsequent rooting response. Aeration of gas-tight flasks containing leaf discs and absorption of ethylene evolved from the discs by mercuric perchlorate in gas-tight flasks or pre-treatment of leaf discs with AgNO₃ significantly enhanced IAA induced root regeneration. Thus, these studies indicate that ethylene is not a rooting hormone per se. Furthermore, ethylene (whether applied externally or synthesized by the tissue) does not appear to account for the ability of auxin to stimulate rooting.     

Role of Ethylene on de Novo Shoot Regeneration from Cotyledonary Explants of Brassica campestris ssp. pekinensis (Lour) Olsson in Vitro

The promotive effect of AgNO₃ and aminoethoxyvinylglycine (AVG) on in vitro shoot regeneration from cotyledons of Brassica campestris ssp. pekinensis in relation to endogenous 1-amino-cyclopropane-1-carboxylic acid (ACC) synthase, ACC, and ethylene production was investigated. AgNO₃ enhanced ACC synthase activity and ACC accumulation, which reached a maximum after 3 to 7 days of culture. ACC accumulation was concomitant with increased emanation of ethylene which peaked after 14 days. In contrast, AVG was inhibitory to endogenous ACC synthase activity and reduced ACC and ethylene production. The promotive effect of AVG on shoot regeneration was reversed by 2-chloroethylphosphonic acid at 50 micromolar or higher concentrations, whereas explants grown on AgNO₃ medium were less affected by 2-chloroethylphosphonic acid. The distinctive effect of AgNO₃ and AVG on endogenous ACC synthase, ACC, and ethylene production and its possible mechanisms are discussed.     

Abscisic acid and Ethrel abolish the inhibition of adventitious root formation of paclobutrazol-treated bean primary leaf cuttings

Paclobutrazol (PB), a triazole growth retardant and an inhibitor of gibberellin biosynthesis, reduced at 17 μM concentration the adventitious root formation of bean primary leaf cuttings. Treatments with 5 μM ABA or 4 μM Ethrel, an ethylenereleasing compound, restored the rooting of PB-treated cuttings. Ethylene production and the content of the precursor 1-aminocyclopropane-l-carboxylic acid (ACC) were enhanced in root-forming tissues of PB-treated petioles 48 h after ABA application. The effect of ABA could be abolished by 10 μM CoCl₂, an inhibitor of ACC oxidase. Thus, ABA might stimulate rooting through its effect on ethylene release. 2 mM silver thiosulphate, an inhibitor of ethylene action, decreased the rooting of PB-treated cuttings similarly to Co²⁺, but failed to negate the ABA effect. These data indicate that the effect of PB on rhizogenesis is not associated directly with the inhibition of the biosynthesis of gibberellins Acknowledgements: We are grateful to Gabriella Biró. This work was supported by the Hungarian National Science Research Foundation (OTKA), Project No. 462.     

Role of auxins, polyamines and ethylene in root formation and growth in sweet orange

The primary objective of this work was to investigate the role of polyamines (PAs) on root formation and growth in two sweet orange (Citrus sinensis L. Osb.) cultivars Pineapple and Pêra. Adventitious shoots (30-d-old) derived from epicotyl explants were transferred to root induction medium containing Murashige and Skoog salts at different strengths and supplemented with different concentrations and combinations of auxins. Root formation and development decreased in both sweet orange cultivars concomitant with the reduction of medium strength. The α-naphtaleneacetic acid was important during the root differentiation phase, but its combination with indole-3-butyric acid was essential for root elongation. The addition of PAs significantly improved root formation and/or growth, depending on their concentration, whereas the presence of inhibitor of PAs biosynthesis α-difluoromethylornithine (DFMO) inhibited these processes. The rooting impairment caused by DFMO was partially reversed by the supplementation of putrescine. Aminoethoxyvinylglycine AVG and AgNO₃ also inhibited in vitro rooting in both sweet orange cultivars, indicating that ethylene was likewise important for rhizogenesis in sweet orange.     

Ethylene as an effector of wound-induced resistance to cellulase in oat leaves

Peeling the abaxial epidermis from oat leaves (Avena sativa var. Victory) induces the formation of wound ethylene and the development of resistance to cellulolytic digestion of mesophyll cell walls. Ethylene release begins between 1 and 2 hours after peeling in the light or dark. Aminoethoxyvinylglycine (AVG, 0.1 millimolar), CoCl₂ (1.0 millimolar), propyl gallate (PG, 1.0 millimolar) or aminooxyacetic acid (AOA, 1.0 millimolar) inhibits, whereas AgNO₃ stimulates wound ethylene formation. Incubation on inhibitors of ethylene biosynthesis (AVG, CoCl₂, PG, AOA) or action (AgNO₃, hypobaric pressure or the trapping of ethylene with HgClO₄) also prevents the development of wound-induced resistance to enzymic cell wall digestion. 1-Aminocyclopropane-1-carboxylic acid (ACC, 1.0 millimolar) reverses AVG (0.1 millimolar) inhibition of the development of resistance. Exogenous ethylene partially induces the development of resistance in unwounded oat leaves.

These results suggest that peeling of oat leaves induces ethylene biosynthesis, which in turn effects changes in the mesophyll cells resulting in the development of resistance to cellulolytic digestion.

     

Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors,silver and cobalt

The effects of CoCl₂, AgNO₃ and ethylene released by exogenous 2-chloroethylphosphonic acid (Ethephon), were studied on shoot regeneration from cotyledons of Helianthus annuus cv. E8206R, a poorly regenerative cultivar. Inhibition of ethylene biosynthesis by CoCl₂, at concentrations of 20 K, provoked a substantial enhancement of shoot regeneration (30 %): the control was poorly regenerative. However, CoCl₂ had no effect when Ethephon was supplied. Inhibition of ethylene action by AgNO₃, at concentrations of 10–25 M, caused a significant increase in plant regeneration: 25 % instead of 1.2 % in the control. Furthermore, addition of Ethephon to AgNO₃-treated tissues failed to reduce the stimulation of shoot regeneration caused by AgNO₃. On the basis of these findings, it is suggested that ethylene inhibits the regeneration process from cotyledons of sunflower.Abbreviations NAA 1-naphthalene acetic acid - BAP 6-benzylamino-purine - GA₃ gibberellic acid - Ethephon 2-chloroethylphosphonic acid - MS Murashige and Skoog medium - AVG aminoethoxyvinylglycine     

The influence of ethylene on proliferation and growth of rose shoot cultures

Ethylene accumulation in four different rose in vitro culture containers was evaluated. Multiplication rate was the highest, and axes most elongated, in the two containers where ethylene accumulation was limited. Pulse treatments of ethylene at various concentrations enhanced proliferation depending on concentration (5 ppm generally was the most favourable) and time of application, while reducing elongation of the shoots. An ethylene trap in the flask atmospheres of the cultures reduced rose shoot proliferation rate but increased elongation of the axes. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and cobalt chloride (CoCl₂), increased multiplication rate by providing a higher number of axes of a suitable size for subculture. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) had a beneficial effect on multiplication rate, although reducing longitudinal growth of the axes.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - BA benzyladenine - GA₃ gibberellic acid - IBA indolyl-3-butyric acid     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

Auxin-Ethylene Interaction in Adventitious Rooting and Related Changes in Anodic Peroxidase Isozymes in Sunflower Hypocotyls

Culturing the hypocotyl explants from 7-day-old; light-grown seedlings of sunflower (Helianthus annuus L. ) on auxin-supplemented MS medium leads to a marked stimulation in callus induction and root initiation. NAA proved more effective than IAA for both responses. Experiments employing ethylene precursors (methionine and ACC) and action Inhibitor (AgNO₃) revealed a significant role of endogenous ethylene levels in auxin-induced rooting. The auxin-ethylene interaction in root morphogenesis is accompanied with specific changes in anodic peroxidase isozymes.     

Promotion ofin vitro shoot formation from excised roots of silktree (Albizzia julibrissin) by an oxime ether derivative and other ethylene inhibitors

Summary This report describes the regeneration response of excised seedling roots of silktree (Albizzia julibrissin) to added ethylene precursors/generators (1-amino-cyclopropane-1-carboxylic acid [ACC], 2-chloroethylphosphonic acid [CEPA]), biosynthesis inhibitors (aminoethoxyvinylglycine [AVG], an oxime ether derivative [OED={[(ispropylidene)-amino]oxy}-acetic acid-2-(methoxy)-2-oxoethyl ester], CoCl² [Co⁺⁺]), and an ethylene action inhibitor (AgNO₃ [Ag⁺]). When placed on B5 medium, about 50% of the control explants formed shoot buds within 15 days. Addition of ACC or CEPA (1–10 µM) to the culture medium decreased both the percentage of cultures forming shoots and the number of shoots formed per culture. In contrast, AVG and OED (1–10 µM) increased shoot formation to almost 100% and increased the number of shoots formed per culture. Likewise, both Co⁺⁺ and Ag⁺ (1–10 µM) increased shoot regeneration, but the number of shoots produced after 30 days was less than with AVG or OED. The inhibitors of ethylene biosynthesis were partially effective in counteracting the inhibitory effect of ACC on shoot formation. These results suggest that modulation of ethylene biosynthesis and/or action can strongly influence the formation of adventitious shoots from excised roots of silktree.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CEPA 2-chloroethylphosphonic acid - OED oxime ether derivative     

Endogenous ethylene requirement for adventitious root induction and growth in tomato cotyledons and lavandin microcuttings in vitro

The role of ethylene in the formation of adventitious roots in vitro was studied in tomato (Lycopersicon esculentum Mill. cv. UC 105) cotyledons and lavandin (Lavandula officinalis Chaix × Lavandula latifolia microshoots. Both systems were able to form roots on hormone-free medium evolving low amounts of ethylene. The addition of 20–50 M indole-3-acetic acid (IAA) inhibited root formation in tomato cotyledons while increasing ethylene production. Naphthaleneacetic acid (NAA, 3 M) stimulated root number in lavandin explants and induced a transient rise in ethylene evolution. Enhanced ethylene levels via the endogenous precursors 1-aminocyclopropane-1-carboxylic acid (ACC, 25–50 M) drastically impaired root regeneration and growth in tomato. In lavandin, 10 M ACC stimulated ethylene production and significantly inhibited the rooting percentage and root growth. Conversely, ACC enhanced the root number in the presence of NAA only. Severe inhibition of rooting was also caused by ethylene reduction via biosynthetic inhibitors, aminoethoxyvinylglycine (AVG, 5–10 M) in tomato, and salicylic acid (SA, 100 M) in lavandin. A strict requirement of endogenous ethylene for adventitious root induction and growth is thus suggested.Abbreviations LS Linsmaier and Skoog medium - BA N⁶-benzyladenine - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - AVG Aminoethoxyvinylglycine - SA Salicylic acid - ACC 1-aminocyclopropane-1-carboxylic acid     

Evaluation of a closed system,diffusive and humidity-induced convective throughflow ventilation on the growth and physiology of cauliflower in vitro

The effects of ethylene inhibitors (silver nitrate – AgNO₃ and silver thiosulphate – Ag₂S₂O₃ as inhibitors of ethylene activity, cobalt chloride – CoCl₂ as inhibitor of ethylene biosynthesis) and ethylene stimulator (aminocyclopropane-1-carboxylic acid – ACC) were studied on the growth of cauliflower (Brassica oleracea L.) seedlings cultured in closed vessels (60 cm³). The addition of ethylene inhibitors have significant stimulatory effects on the growth and development of seedlings and the effects were greatest with 10 μM AgNO₃, the fresh weight of leaves was 2.6×, and the leaf area 2.8× those of the control (no additives). The effects of various methods of ventilation (humidity-induced convective through-flow ventilation, diffusive ventilation and sealed condition) on the growth and physiology of in vitrocauliflower seedlings were also investigated. The seedlings were cultured either in the presence or absence of AgNO₃ (inhibitors of ethylene activity) and ACC (a precursor). Ethylene and CO₂ levels in the head-space of the culture vessels were monitored. The humidity-induced through-flow ventilation system has shown to be effective for improving growth, leaf chlorophyll content and the rate of net photosynthesis and preventing symptoms of hyperhydricity, such as leaf epinasty, and franginess, reduction of leaf area etc. In contrast, the results also indicated that the sealing of culture vessels could have serious inhibitory effects on growth and development, induce hyperhydricity and reduce leaf chlorophyll content. In the light period, CO₂ depletion occurred in the head-space of the sealed vessels (ca. 40 μl l^-1), the CO₂ concentration increased with increasing efficiency of the ventilation. No ethylene accumulation was noticed in the head-space of the culture vessels when humidity-induced throughflow ventilation was applied; however, high ethylene accumulation occurred in sealed vessels. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sensitivity of chickpea and faba bean to root‐zone hypoxia,elevated ethylene,and carbon dioxide

During soil waterlogging, plants experience O₂ deficits, elevated ethylene, and high CO₂ in the root‐zone. The effects on chickpea (Cicer arietinum L.) and faba bean (Vicia faba L.) of ethylene (2 μL L^?1), CO₂ (2–20% v/v) or deoxygenated stagnant solution were evaluated. Ethylene and high CO₂ reduced root growth of both species, but O₂ deficiency had the most damaging effect and especially so for chickpea. Chickpea suffered root tip death when in deoxygenated stagnant solution. High CO₂ inhibited root respiration and reduced growth, whereas sugars accumulated in root tips, of both species. Gas‐filled porosity of the basal portion of the primary root of faba bean (23%, v/v) was greater than for chickpea (10%), and internal O₂ movement was more prominent in faba bean when in an O₂‐free medium. Ethylene treatment increased the porosity of roots. The damaging effects of low O₂, such as death of root tips, resulted in poor recovery of root growth upon reaeration. In conclusion, ethylene and high CO₂ partially inhibited root extension in both species, but low O₂ in deoxygenated stagnant solution had the most damaging effect, even causing death of root tips in chickpea, which was more sensitive to the low O₂ condition than faba bean.     

Ethylene and auxin-ethylene interaction in adventitious root formation in mung bean (Vigna radiata) cuttings

The role of ethylene in adventitious root formation and its involvement in auxin-induced rooting were investigated in cuttings ofVigna radiata (L.). Treatment with 30 M indole-3-acetic acid (IAA) for 24 h slightly inhibited rooting, whereas the same concentration of indole-3-butyric acid (IBA) significantly stimulated it. Ethylene derived from 1-aminocyclopropane-1-carboxylic acid (ACC) increased the number of adventitious roots but inhibited their emergence and elongation. Endogenous levels of ethylene, ACC, and malonyl-ACC (MACC) were initially higher in cuttings treated with IAA. This trend was quickly reversed, and cuttings, particularly hypocotyls, treated with IBA produced higher levels of ethylene and had more ACC and MACC during most of the rooting process. Aminoethoxyvinylglycine significantly inhibited rooting, but its inhibitory effect could not be reversed by ACC. The data suggest that the stimulating effect of IBA on rooting is closely associated with its induction of ACC and ethylene biosynthesis.     

The influence of oxygen deficiency on ethylene synthesis, 1-aminocyclopropane-1-carboxylic acid levels and aerenchyma formation in roots of Zea mays

The relationship between ethylene production, 1-aminocyclopropane-l-carboxylic acid (ACC) concentration and aerenchyma formation (ethylene-promoted cavitation of the cortex) was studied using nodal roots of maize (Zea mays L. cv. LG11) subjected to various O₂ treatments. Ethylene evolution was 7–8 fold faster in roots grown at 3 kPa O₂ than in those from aerated solution (21 kPa O₂), and transferring roots from aerated solution to 3 kPa O₂ enhanced ethylene synthesis within less than 2 h. Ethylene production and ACC accumulation were closely correlated in different zones of hypoxic roots, regardless of whether O₂ was furnished to the roots through aerenchyma or external solution. Both ethylene production and ACC concentrations (fresh weight basis) were more than 10-fold greater in the distal 0–10 mm than in the fully expanded zone of roots at 3 kPa O₂. Aerenchyma formation occurred in the apical 20 mm of these roots. Roots transferred from air to anoxia accumulated less than 0. 1 nmol ACC (mg protein)^-1 for the first 1.75 h; no ethylene was produced in this time. The subsequent rise in ACC levels shows that ACC can reach high concentrations even in the absence of O₂, presumably due to a de-repression of ACC synthase. The hypothesis was therefore tested that anoxia in the apical region of the root caused enhanced synthesis of ACC, which was transported to more mature regions (10–20 mm behind the apex), where ethylene could be produced and aerenchyma formation stimulated. Surprisingly, exposure of intact root tips to anoxia inhibited aerenchyma formation in the mature root axis. High osmotic pressures around the growing region or excision of apices had the same effect, demonstrating that a growing apex is required for high rates of aerenchyma formation in the adjacent tissue.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine