Mechanism of Ethylene Action: Biological Activity of Deuterated Ethylene and Evidence against Isotopic Exchange and cis-trans-Isomerization

Deuterated ethylene was used to study the mechanism of ethylene action in etiolated pea seedlings (Pisum sativum L. cv. Alaska). No apparent differences were observed in the biological activity of tetradeuteroethylene (C₂D₄) and ordinary ethylene (C₂H₄) using the pea stem straight growth assay. The absence of an isotopic effect is discussed in relation to the possibility that ethylene binds to a metal or that carbon to hydrogen bonds of ethylene are broken during its mechanism of action.     

Effect of silver ion, carbon dioxide, and oxygen on ethylene action and metabolism

The relationship between ethylene action and metabolism was investigated in the etiolated pea seedling (Pisum sativum L. cv. Alaska) by inhibiting ethylene action with Ag⁺, high CO₂, and low O₂ and then determining if ethylene metabolism was inhibited in a similar manner. Ag⁺ (100 milligrams per liter) was clearly the most potent antiethylene treatment. Ag⁺ pretreatment inhibited the growth retarding action of 0.2 microliters per liter ethylene by 48% and it also inhibited the incorporation of 0.2 microliters per liter ¹⁴C₂H₄ into pea tips by the same amount. As the ethylene concentration was increased from 0.2 to 30 microliters per liter, the effectiveness of Ag⁺ in reducing ethylene action and metabolism declined in a similar fashion. Although Ag⁺ significantly inhibited the incorporation of ¹⁴C₂H₄ into tissue metabolites, the oxidation of ¹⁴C₂H₄ to ¹⁴CO₂ was unaffected in the same tissue.     

Molecular requirements for the biological activity of ethylene

The molecular requirements for ethylene action were investigated using the pea straight growth test. Biological activity requires an unsaturated bond adjacent to a terminal carbon atom, is inversely related to molecular size, and is decreased by substitutions which lower the electron density in the unsaturated position. Evidence is presented that ethylene binds to a metal containing receptor site. CO₂ is a competitive inhibitor of ethylene action, and prevents high concentrations of auxin (which stimulate ethylene formation) from retarding the elongation of etiolated pea stem sections. It is suggested that CO₂ delays fruit ripening by displacing the ripening hormone, ethylene, from its receptor site. Binding of ethylene to the receptor site is also impeded when the O₂ concentration is lowered, and this may explain why fruit ripening is delayed at low O₂ tensions.     

Enhancement of ethylene formation by selenoamino acids

Selenomethionine and selenoethionine enhanced ethylene production in senescing flower tissue of Ipomoea tricolor Cav. and in auxin-treated pea (Pisum sativum L.) stem sections. This enhancement was fully inhibited by the aminoethoxy analog of rhizobitoxine. Methionine did not have a comparable promotive effect, and ethionine partly inhibited ethylene production. When [¹⁴C]methionine was applied to flower or pea stem tissue followed by treatment with unlabeled selenomethionine or selenoethionine, the specific radioactivity of the ethylene evolved was considerably reduced. The dilution of the specific radioactivity of ethylene by selenomethionine, and in pea stem sections also by selenoethionine, was greater than the dilution by nonradioactive methionine at the same concentration. These results indicate that both selenoamino acids serve as precursors of ethylene and that they are converted to ethylene more efficiently than is methionine.     

Nonphysiological binding of ethylene by plants

Ethylene binding to seedling tissue of Vicia faba, Phaseolus vulgaris, Glycine max, and Triticum aestivum was demonstrated by determining transit time required for ethylene to move through a glass tube filled with seedling tissue. Transit time for ethylene was greater than that for methane indicating that these tissues had an affinity for ethylene. However, the following observations suggest that the binding was not physiological. Inhibitors of ethylene action such as Ag⁺ ions and CO₂ did not decrease binding. Mushrooms which have no known sites of ethylene action also demonstrated ethylene binding. The binding of acetylene, propylene, ethylene, propane, and ethane more closely followed their solubility in water than any known physiological activity.     

A role for ethylene in the metabolism of cyanide by higher plants

The action of ethylene on the capacity of plant tissues to metabolize cyanide to β-cyanoalanine was examined. Beta-cyanoalanine synthase (EC 4.4.1.9) catalyzes the reaction between cyanide and cysteine to form β-cyanoalanine and hydrogen sulfide. Levels of β-cyanoalanine synthase activity in tissues of 6 day old etiolated pea (Pisum sativum) seedlings were enhanced severalfold by 1 microliter per liter ethylene. The promotive effect of ethylene increased with increasing ethylene concentrations from 0.01 to 100 microliters per liter and with the period of exposure from 3 to 24 hours. Ethylene enhanced β-cyanoalanine synthase activity in all regions of the seedling (shoots and roots, internodal regions, cotyledons). The promotive effect was eliminated by norbornadiene, a competitive inhibitor of ethylene action. Levels of β-cyanoalanine synthase in seedlings of four other dicots (Phaseolus aureas, Glycine max, Lactuca sativa, Sinapis arvensis) and two monocots (Hordeum vulgares, Triticum aestivum) were also increased in response to ethylene. Our results suggest an important regulatory role for ethylene in the metabolism of cyanide by higher plants.     

Light level does not alter ethylene sensitivity in radish or pea

Ethylene accumulation occurs in many plant growth environments. In some instances, low photosynthetic photon flux (PPF) is also a stress factor. Ethylene helps regulate the shade-avoidance mechanism and synthesis rates can be altered by light. We thus hypothesized that ethylene sensitivity in whole plants may be altered in low light. Radish (Raphanus sativus) and pea (Pisum sativum) plants were selected as models due to their rapid growth, use in previous studies and difference in growth habit. We first characterized radish and pea sensitivity to ethylene. Radish vegetation was less sensitive to ethylene than pea vegetation. Pea reproductive yield was highly sensitive. Plants grown under low light levels are typically etiolated and less robust than plants grown under higher light. In a second series of studies we examined the interaction of ethylene (50 ppb pea, 200 ppb radish) with PPFs from 50 to 400 μmol m^?2 s^?1. There was no statistically significant interaction between ethylene sensitivity and PPF, indicating that high PPF does not mitigate the detrimental effects of chronic low-level ethylene exposure. This also suggests there is no crosstalk between the shade avoidance pathway and the primary ethylene signaling pathway.     

Inhibition by ethylene of polyamine biosynthetic enzymes enhanced lysine decarboxylase activity and cadaverine accumulation in pea seedlings

Exposing etiolated pea seedlings to ethylene which inhibited the activity of arginine decarboxylase and S-adenosylmethionine decarboxylase caused an increase in the level of cadaverine. The elevated level of cadaverine resulted from an increase in lysine decarboxylase activity in the tissue exposed to ethylene. The hormone did not affect the apparent K_m of the enzyme, but the apparent V_max was increased by 96%. While lysine decarboxylase activity in the ethylene-treated plants increased in both the meristematic and the elongation zone tissue, cadaverine accumulation was observed in the latter only. The enhancement by ethylene of the enzyme activity was reversed completely 24 hours after transferring the plants to an ethylene-free atmosphere. It is postulated that the increase in lysine decarboxylase activity, and the consequent accumulation of cadaverine in ethylene-treated plants, is of a compensatory nature as a response to the inhibition of arginine and S-adenosylmethionine decarboxylase activity provoked by ethylene.     

Effect of substrate-dependent microbial ethylene production on plant growth

Various compounds have been identified as precursors/substrates for the synthesis of ethylene (C₂H₄) in soil. This study was designed to compare the efficiency of four substrates, namely L-methionine (L-MET), 2-keto-4-methylthiobutyric acid (KMBA), 1-aminocyclopropane-1-carboxylic acid (ACC), and calcium carbide (CaC₂), for ethylene biosynthesis in a sandy clay loam soil by gas chromatography. The classic “triple” response in etiolated pea seedling was employed as a bioassay to demonstrate the effect of substrate-dependent microbial production of ethylene on plant growth. Results revealed that an amendment with L-MET, KMBA, ACC (up to 0.10 g/kg soil) and CaC₂ (0.20 g/kg soil) significantly stimulated ethylene biosynthesis in soil. Overall, ACC proved to be the most effective substrate for ethylene production (1434 nmol/kg soil), followed by KMBA, L-MET, and CaC₂ in descending order. Results further revealed that ethylene accumulation in soil from these substrates caused a classic “triple” response in etiolated pea seedlings with different degrees of efficacy. A more obvious classic “triple” response was observed at 0.15, 0.10, and 0.20 g/kg soil of L-MET, KMBA/ACC, and CaC₂, respectively. Similarly, direct exposure of etiolated pea seedlings to commercial ethylene gas also modified the growth pattern in the same way. A significant direct correlation (r = 0.86 to 0.97) between substrate-derived C₂H₄ and the classic triple response in etiolated pea seedlings was observed. This study demonstrated that the presence of substrate(s) in soil may lead to increased ethylene concentration in the air of the soil, which may affect plant growth in a desired direction. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 2, pp. 277–283. The text was submitted by the authors in English.     

The influence of ethylene and ethylene modulators on shoot organogenesis in tomato

The influence of ethylene and ethylene modulators on the in vitro organogenesis of tomato was studied using a highly regenerating accession of the wild tomato Solanum pennellii and an F1 plant resulting from a cross between Solanum pennellii and Solanum lycopersicum cv. Anl27, which is known to have a low regeneration frequency. Four ethylene-modulating compounds, each at four levels, were used, namely: cobalt chloride (CoCl₂), which inhibits the production of ethylene; AgNO₃ (SN), which inhibits ethylene action; and Ethephon and the precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which both promote ethylene synthesis. Leaf explants of each genotype were incubated on shoot induction medium supplemented with each of these compounds at 0, 10 or 15 days following bud induction. The results obtained in our assays indicate that ethylene has a significant influence on tomato organogenesis. Concentrations of ethylene lower than the optimum (according to genotype) at the beginning of the culture may decrease the percentage of explants with buds (B), produce a delay in their appearance, or indeed inhibit bud formation. This was observed in S. pennellii and the F1 explants cultured on media with SN (5.8–58.0 μM) as well as in the F1 explants cultured on medium with 21.0 μM CoCl₂. The percentage of explants with shoots (R) and the mean number of shoots per explant with shoots (PR) also diminished in media that contained SN. Shoots isolated from these explants were less developed compared to those isolated from control explants. On the other hand, ethylene supplementation may contribute to enhancing shoot development. The number of isolable shoots from S. pennellii explants doubled in media with ACC (9.8–98.0 μM). Shoots isolated from explants treated with ethylene releasing compounds showed a higher number of nodes when ACC and Ethephon were added at 10 days (in F1 explants) or at 15 days (in S. pennellii) after the beginning of culture. Thus, the importance of studying not only the concentration but also the timing of the application of regulators when developing regeneration protocols has been made manifest. An excess of ethylene supplementation may produce an inhibitory effect, as was observed when using Ethephon (17.2–69.0 μM). These results show the involvement of ethylene in tomato organogenesis and lead us to believe that ethylene supplementation may contribute to enhancing regeneration and shoot development in tomato.     

Abscission: the phytogerontological effects of ethylene

The role of ethylene in the aging of bean (Phaseolus vulgaris L. cv. Red Kidney) petiole abscission zone explants was examined. The data indicate that ethylene does accelerate aging in addition to inducing changes in break strength. Application of ethylene during the aging stage (stage 1) promoted abscission when followed by a second ethylene treatment during the cell separating stage (stage 2). The half-maximal effective concentration of ethylene to induce aging was around 0.3 microliter per liter; 10 microliters per liter was a saturating dose. CO₂ reversal of ethylene action during stage 1 was incomplete and gave ambiguous results. CO₂ (10%) reversed the effect of 10 microliters per liter ethylene but not 1 microliter per liter ethylene. The possibility that ethylene not only accelerated aging but was also a requirement for it was tested, and experimental evidence in favor of this idea was obtained. It was concluded that ethylene plays a dual role in the abscission of bean petiole explants: a phytogerontological effect and a cellulase-inducing effect.     

Effects of antagonists and inhibitors of ethylene biosynthesis on maize root elongation

During the first days of development, maize roots showed considerable variation in the production of ethylene and the rate of elongation. As endogenous ethylene increases, root elongation decreases. When these roots are treated with the precursor of ethylene aminocyclopropane- 1-carboxylic acid (ACC), or inhibitors of ethylene biosynthesis 2-aminoethoxyvinyl glycine (AVG) or cobalt ions, the root elongation is also inhibited. Because of root growth diminishes at high or reduced endogenous ethylene concentrations, it appears that this phytohormone must be maintained in a range of concentrations to support normal root growth. In spite of its known role as inhibitor of ethylene action, silver thiosulphate (STS) does not change significantly the root elongation rate. This suggests that the action of ethylene on root elongation should occur, at least partially, by interaction with other growth regulators.Key words: 2-aminoethoxyvinyl glycine, cobalt, ethylene, root elongation, silver thiosulphate, Zea mays     

The response of foliar gas exchange to exogenously applied ethylene

The responsiveness to ethylene of net photosynthesis and stomatal conductance to water vapor in intact plants was investigated in 13 herbaceous species representing seven plant families. Exposures were conducted in an open, whole-plant exposure system providing controlled levels of irradiance, air temperature, CO₂, relative humidity, and ethylene concentration. Net photosynthesis and stomatal conductance to water vapor in units of moles per square meter per second were measured on recently expanded leaves in control and ethylene-treated plants using a remotely operated single-leaf cuvette. The ethylene concentration was either 0 or 210 micromoles per cubic meter and was maintained for 4 hours. Species varied substantially in the response of their foliar gas exchange to ethylene. In 7 of the 13 species, net photosynthesis was inhibited statistically by 4 hours of ethylene exposure. As a function of the rate in control plants, the responses were most pronounced and statistically significant in Arachis hypogaea (−51.1%), Gossypium hirsutum (−31.7%), Glycine max (−24.8%), Cucurbita pepo (−20.4%), Phaseolus vulgaris (−18.4%), Setaria viridis (−17.5%), and Raphanus sativus (−4.4%). Whereas the responsiveness of net photosynthesis to ethylene among the 13 species showed no specific taxonomic associations, the responsiveness was positively correlated with the intrinsic rate of net photosynthesis. Stomatal conductance to water vapor after 4 hours of ethylene exposure declined statistically in 6 of the 13 species. As a function of control rates, the most marked and statistically significant responses of stomatal conductance were in Glycine max (−53.6%), Gossypium hirsutum (−51.2%), Arachis hypogaea (−42.7%), Phaseolus vulgaris (−38.6%), Raphanus sativus (−26.8%), and Solanum tuberosum (−23.4%). Although ethylene-induced changes in net photosynthesis and stomatal conductance were positively correlated, there were species-specific exceptions in which net photosynthesis declined after 4 hours of exposure without a concurrent change in stomatal conductance, stomatal conductance declined without a change in net photosynthesis, and the decline in stomatal conductance substantially exceeded the corresponding decline in net photosynthesis. Thus, the responsiveness to ethylene of net photosynthesis and stomatal conductance to water vapor were not consistently synchronous or equivalent among the 13 species. It is concluded that foliar gas exchange is responsive to exogenously applied ethylene in many plant species. The sensitivity of foliar gas exchange to ethylene may play a role in general plant response to environmental stress in which one of the physiological sites of action for endogenously produced stress ethylene in the leaf is the plant's photosynthetic capacity and/or stomatal conductance to water vapor.     

Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal regulation of plant growth

Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day. This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs. Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme. The inhibition was observed within 30 min of the hormone application, and maximal effect—90% inhibition—after 18 hours. Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 0.12 microliters per liter ethylene and maximal response at 1.2 microliters per liter. Ethylene provoked a 5-fold increase in the K_m^app of arginine decarboxylase for its substrate and reduced the V_max^app by 10-fold. However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere. Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings. On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed.     

On ethylene and stem elongation in green pea seedlings

Maximum elongation of excised internodal stem sections of light-grown pea (Pisum sativum L.) seedlings occurred at 10⁻⁵ molar indoleacetic acid (IAA), with submaximal responses occurring at 10⁻⁴ and 10⁻³ molar. Accompanying elongation at concentrations of IAA of 10⁻⁶ to 10⁻³ molar was production of ethylene, with the amount increasing up to 10⁻⁴ molar IAA and then becoming nearly constant. Elongation of light-grown sections was not inhibited by exogenous ethylene up to 10,000 ppm in the presence of 10⁻⁵ molar IAA. Marked (up to 50%) inhibition of elongation of internodal segments in situ was observed after treating whole light-grown seedlings with exogenous ethylene for 20 hours. It is concluded that ethylene is not responsible for the submaximal elongation responses of green pea stem sections at high auxin concentrations, but that IAA per se is accountable.     

Effects of gibberellic Acid, calcium, kinetic, and ethylene on growth and cell wall composition of pea epicotyls

The influence of gibberellic acid (GA), calcium, kinetin, and ethylene on growth and cell-wall composition of decapitated pea epicotyls (Pisum sativum L. var. Alaska) was investigated. Calcium, kinetin, and ethylene each caused an inhibition of GA-induced elongation of pea stems. Gibberellic acid did not reverse the induction of swelling by Ca²⁺, kinetin, or ethylene. Both Ca²⁺ and ethylene significantly inhibited the stimulatory effects of GA on the formation of residual wall material. Although GA promoted the development of walls relatively low in pectic substances and pectic uronic acid, Ca²⁺, kinetin, and ethylene favored the formation of walls rich in these constituents. Calcium, kinetin, and GA, alone or in combination, had no effect on the production of ethylene by pea epicotyls.     

Programme of senescence in petals and carpels of Pisum sativum L. flowers and its control by ethylene

The role of ethylene in the control of senescence of both petals and unpollinated carpels of pea was investigated. An increase in ethylene production accompanied senescence, and the inhibitors of ethylene action were effective in delaying senescence symptoms in different flower verticils. Pollination did not seem to trigger the senescence syndrome in the corolla as deduced from the observation that petals from pollinated and unpollinated flowers and from flowers whose carpels had been removed senesced at the same time. A cDNA clone encoding a putative ethylene-response sensor (psERS) was isolated from pea flowers, and the pattern of expression of its mRNA was studied during development and senescence of different flower tissues. The levels of psERS mRNA paralleled ethylene production (and also levels of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) mRNA) in both petals and styles. Silver thiosulfate treatments were efficient at preventing ACO and psERS mRNA induction in petals. However, the same inhibitor showed no ability to modify expression patterns in pea carpels around the anthesis stage, suggesting different controls for ethylene synthesis and sensitivity in different flower organs. Received: 18 June 1998 / Accepted: 22 December 1998     

Propylene-a competitor of ethylene action

Propylene competed with the ethylene-induced reduction in length growth of the epicotyl of the etiolated garden pea (Pisum sativum L. cv. Alaska). These results constitute further evidence that ethylene acts by attaching itself loosely to a site.     

Inhibitory action of auxin on root elongation not mediated by ethylene

The inhibitory effects of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) on elongation growth of pea (Pisum sativum L.) seedling roots were investigated in relation to the effects of these compounds on ethylene production by the root tips. When added to the growth solution both compounds caused a progressively increasing inhibition of growth within the concentration range of 0.01 to 1 micromolar. However, only ACC increased ethylene production in root tips excised from the treated seedlings after 24 hours. High auxin concentrations caused a transitory increase of ethylene production during a few hours in the beginning of the treatment period, but even in 1 micromolar IAA this increase was too low to have any appreciable effect on growth. ACC, but not IAA, caused growth curvatures, typical of ethylene treatment, in the root tips. IAA caused conspicuous swelling of the root tips while ACC did not. Cobalt and silver ions reversed the growth inhibitory effects induced by ACC but did not counteract the inhibition of elongation or swelling caused by IAA. The growth effects caused by the ACC treatments were obviously due to ethylene production. We found no evidence to indicate that the growth inhibition or swelling caused by IAA is mediated by ethylene. It is concluded that the inhibitory action of IAA on root growth is caused by this auxin per se.     

Exogenous Ethylene Inhibits Nodulation of Pisum sativum L. cv Sparkle

Exogenous ethylene inhibited nodulation on the primary and lateral roots of pea, Pisum sativum L. cv Sparkle. Ethylene was more inhibitory to nodule formation than to root growth; nodule number was reduced by half with only 0.07 μL/L ethylene applied continually to the roots for 3 weeks. The inhibition was overcome by treating roots with 1 μm Ag⁺, an inhibitor of ethylene action. Exogenous ethylene also inhibited nodulation on sweet clover (Melilotus alba) and on pea mutants that are hypernodulating or have ineffective nodules. Exogenous ethylene did not decrease the number of infections per centimeter of lateral pea root, but nearly all of the infections were blocked when the infection thread was in the basal epidermal cell or in the outer cortical cells.