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 CO(2), and low O(2) 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 (14)C(2)H(4) 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 (14)C(2)H(4) into tissue metabolites, the oxidation of (14)C(2)H(4) to (14)CO(2) was unaffected in the same tissue. CO(2) (7%) inhibited ethylene-induced growth retardation but its effectiveness diminished at a greater rate than that of Ag(+) with increasing ethylene concentration. High CO(2) had just the opposite effect of Ag(+) since it inhibited (14)C(2)H(4) oxidation to (14)CO(2) without affecting tissue incorporation. In contrast to Ag(+), CO(2) did not inhibit ethylene action and metabolism to exactly the same extent, and the inhibition of metabolism did not rapidly decline with increasing (14)C(2)H(4) concentration. However, high CO(2) did alter the ratio of (14)C(2)H(4) tissue incorporation to (14)CO(2) production in a manner consistent with changes in ethylene effectiveness. Lowering the O(2) concentration to 5% reduced ethylene-induced growth retardation from 70 to 58% at 0.22 microliters per liter and inhibited (14)C(2)H(4) (0.25 microliters per liter) tissue incorporation and oxidation to (14)CO(2) by 26 and 45%, respectively. However, in contrast to Ag(+) and high CO(2) which slightly promoted growth in ethylene-free air, low O(2) reduced pea seedling growth under these conditions thereby severely limiting its usefulness as a specific antiethylene treatment. Collectively these data suggest that the metabolism of ethylene may be related to its action

Rapid Metabolism of Propylene by Pea Seedlings

Propylene uptake by intact pea seedlings (Pisum sativum L. cv. Alaska) was easily detected using standard gas chromatographic techniques suggesting rapid metabolism. Comparative studies with highly purified (14)C(3)H(6) and (14)C(2)H(4) under aseptic conditions verified that propylene was rapidly metabolized and indicated that some aspects of its metabolism were similar to that of ethylene since (14)C(3)H(6), like (14)C(2)H(4) (Beyer, Nature 1975, 255: 144-147), was oxidized to (14)CO(2) and incorporated into water-soluble tissue metabolites. However, (14)C(2)H(6) was metabolized at a substantially faster rate and unlike (14)C(2)H(4) the rate of (14)C(3)H(6) tissue incorporation exceeded its rate of oxidation to (14)CO(2). In addition the neutral (14)C-metabolites derived from (14)C(3)H(6) were chromatographically distinct from those formed from (14)C(2)H(4)

(14)C(2)H(4): Its Purification for Biological Studies

A gas chromatographic technique is described for obtaining ultra high purity (14)C(2)H(4) for use in biological studies. (14)C(2)H(4) purchased from commercial sources contained readily detectable impurities including radioactive acetylene. Following purification on two different columns, no impurities were detected by high sensitivity gas chromatographic analysis. However, shortly thereafter impurities were detected as a result of radiation decomposition. Trapping and immediately regenerating ultra high purity (14)C(2)H(4) from dilute, filtered Hg (CIO(4))(2) solutions did not cause the formation of impurities, whereas additional impurities were formed when unpurified (14)C(2)H(4) was used. Impurities were also formed when ultra high purity (14)C(2)H(4) was stored in such solutions prior to its regeneration or when it was trapped and immediately regenerated from more concentrated Hg(CIO(4))(2) solutions

A Potent Inhibitor of Ethylene Action in Plants

Ag(I), applied foliarly as AgNO(3), effectively blocked the ability of exogenously applied ethylene to elicit the classical “triple” response in intact etiolated peas (Pisum sativumcv. Alaska); stimulate leaf, flower, and fruit abscission in cotton (Gossypium hirsutumcv. Stoneville 213); and induce senescence of orchids (Hybrid white Cattleya, Louise Georgeianna). This property of Ag(I) surpasses that of the well known ethylene antagonist, CO(2), and its persistence, specificity, and lack of phytotoxicity at effective concentrations should prove useful in defining further the role of ethylene in plant growth

Auxin Transport: A New Synthetic Inhibitor

The new synthetic plant growth regulator DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo [5,1-a] isoindol-8-one) was examined for its effects on auxin transport. At a concentration of 0.5 mm in the receiver agar cylinders DPX1840 significantly inhibited the basipetal transport of naphthaleneacetic acid-1-(14)C in stem sections of Vigna sinensis Endl., Pisum sativum L., Phaseolus vulgaris L., Glycine max L., Helianthus annuus L., Gossypium hirsutum L., and Zea mays L. without significantly reducing total auxin uptake or recovery. The time sequence of the effect varied with the plant species. A similar inhibition of the basipetal movement of indoleacetic acid-1-(14)C was observed in intact seedlings of Phaseolus vulgaris L. In contrast to basipetal auxin transport DPX1840 had no significant effect on the acropetal movement of indoleacetic acid-1-(14)C in stem sections of Gossypium hirsutum L. Qualitatively the effect of DPX1840 on basipetal auxin transport was similar to that of other known auxin transport inhibitors. Quantitative differences, however, suggested the following order of activity: Naptalam>morphactin[unk]DPX1840>2,3,5-triiodobenzoic acid. DPX1840 also inhibited the lateral displacement of auxin. In horizontally placed stem sections of Helianthus annuus L. pretreated with DPX1840, the ratio of radioactivity from indoleacetic acid-1-(14)C in the upper versus the lower halves of the sections following basipetal indoleacetic acid-1-(14)C transport was approximately 50:50, whereas in the corresponding controls it was approximately 40:60. The data indicate that many of the characteristic effects of DPX1840 on plants, especially those which are known to involve auxin (e.g., epinasty, abscission, apical dominance, tropism), are due, at least in part, to its effects on auxin transport

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 (C(2)D(4)) and ordinary ethylene (C(2)H(4)) 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. Analyses by gas chromatography of gas samples obtained from chambers containing intact etiolated pea plants exposed to 2 microliters of C(2)D(4) per liter of air for up to 5 days resulted in no detectable exchange between the deuterium atoms of C(2)D(4) and the hydrogen atoms of the tissue. Similarly, infrared spectra of gas samples obtained from chambers containing plants exposed to either cis or trans-C(2)D(2)H(2) indicated that no conversion had occurred to the corresponding trans or cis isomer. These results suggest that the mechanism of ethylene action does not involve an intermediate ethylene complex resulting in hydrogen exchange or cis-trans isomerization during a possible catalytic activation of the receptor site(s)

Abscission: The Initial Effect of Ethylene Is in the Leaf Blade

The leaf blade of cotton (Gossypium hirsutum L. cv. Stoneville 213) was investigated as the initial site of ethylene action in abscission. Ethylene applied at 14 μl/l to intact 3-week-old plants caused abscission of the third true leaf within 3 days. However, keeping only the leaf blade of this leaf in air during ethylene treatment of the rest of the plant completely prevented its abscission for up to 7 days. This inhibition of abscission was apparently the result of continued auxin production in the blade since (a) the application of an auxin transport inhibitor to the petiole of the air-treated leaf blade restored ethylene sensitivity to the leaf in terms of abscission; (b) repeated applications of naphthaleneacetic acid to the leaf blade of the third true leaf, when the entire plant was exposed to ethylene, had the same preventive effect on abscission of this leaf as keeping its leaf blade in air; and (c) the inhibitory effect of ethylene on auxin transport in the petiole, which is reduced by auxin treatment, was also reduced by placing the leaf blade in air. The reverse treatment of exposing only the leaf blade of the third true leaf to 14 μl/l of ethylene, while the rest of the plant was kept in air, also did not cause abscission for up to 5 days. Auxin transport in the petioles of these leaves, however, was inhibited over 80% within 2 days and this effect presumably accounted for their increased sensitivity to ethylene during the subsequent exposures of the whole leaf to the gas. These results suggest that an initial and essential function of applied ethylene in abscission is to reduce the amount of auxin transported out of the leaf blade. This reduction together with the inhibitory effect of ethylene on auxin transport in the petiole reduces the auxin level at the abscission zone to a point where the cells in this region become responsive to the more direct action of the gas (e.g., enzyme induction and secretion). This sequence of events accounts for the lack of abscission unless ethylene is applied to both the leaf blade and the abscission zone

Abscission: Support for a Role of Ethylene Modification of Auxin Transport

Three types of whole plant experiments are presented to substantiate the concept that an important function of ethylene in abscission is to reduce the transport of auxin from the leaf to the abscission zone. (a) The inhibitory effect of ethylene on auxin transport, like ethylene-stimulated abscission, persists only as long as the gas is continuously present. Cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants placed in 14 μl/l of ethylene for 24 or 48 hours showed an increase in leaf abscission and a reduced capacity to transport auxin; but when returned to air, auxin transport gradually increased and abscission ceased. (b) Ethylene-induced abscission and auxin transport inhibition show similar sensitivities to temperature. A 24-hour exposure of cotton plants to 14 μl/l of ethylene at 8 C resulted in no abscission and no significant inhibition of auxin transport. Increasing the temperature during ethylene treatment resulted in a progressively greater reduction in auxin transport with abscission occurring at [unk]27 C where auxin transport was inhibited over 70%. (c) Auxin pretreatment reduced both ethylene-induced abscission and auxin transport inhibition. No abscission occurred, and auxin transport was inhibited only 18% in cotton plants which were pretreated with 250 mg/l of naphthalene acetic acid and then placed in 14 μl/l of ethylene for 24 hours. In contrast, over 30% abscission occurred, and auxin transport was inhibited 58% in the corresponding control plants. Collectively, the results presented here and elsewhere indicate that ethylene regulates the sensitivity of the cells in the abscission zone to the more direct actions of the gas (e.g., enzyme induction, secretion) by reducing auxin transport

(14)C(2)H(4): Its Incorporation and Metabolism by Pea Seedlings under Aseptic Conditions

The effects of various treatments on the recently reported system in pea (Pisum sativum cv. Alaska), which results in (a) the incorporation of (14)C(2)H(4) into the tissue and (b) the conversion of (14)C(2)H(4) to (14)CO(2), was investigated using 2-day-old etiolated seedlings which exhibit a maximum response. Heat treatment (80 C, 1 min) completely inhibited both a and b, whereas homogenization completely inhibited b but only partially inhibited a. Detaching the cotyledons from the root-shoot axis immediately before exposing the detached cotyledons together with the root-shoot axis to (14)C(2)H(4) markedly reduced both a and b. Increasing the (14)C(2)H(4) concentration from 0.14 to over 100 μl/l progressively increased the rate of a and b with tissue incorporation being greater than (14)C(2)H(4) to (14)CO(2) conversion only below 0.3 μl/l (14)C(2)H(4). Reduction of the O(2) concentration reduced both a and b, with over 99% inhibition occurring under anaerobic conditions. The addition of CO(2) (5%) severely inhibited (14)C(2)H(4) to (14)CO(2) conversion without significantly affecting tissue incorporation. Exposure of etiolated seedlings to fluorescent light during (14)C(2)H(4) treatment was without effect. Similarly, indoleacetic acid, gibberellic acid, benzyladenine, abscisic acid, and dibutyryl cyclic adenosine monophosphate had no significant effect on either a or b. The possibilities that the incorporation of (14)C(2)H(4) into pea tissues and its conversion to (14)CO(2) is linked to ethylene action, or that it represents a means of reducing the endogenous ethylene level, are discussed. Several problems encountered with the use of polyethylene vials, rubber serum stoppers, Clorox, and microbial contamination are also described

[(14)C]Ethylene Metabolism during Leaf Abscission in Cotton

Changes in (14)C(2)H(4) metabolism in the abscission zone were monitored during cotton (cv. Deltapine 16) leaf abscission. Rates of (14)C(2)H(4) oxidation to (14)CO(2) and tissue incorporation in abscission zone segments cut from the second true leaf of nonabscising leaves of intact plants were similar (about 200 disintegrations per minute per 0.1 gram dry weight per 5.5 hours) and relatively constant over a 5-day period. Deblading to induce abscission caused a dramatic rise in (14)C(2)H(4) oxidation, but tissue incorporation was not markedly affected. This rise occurred well before abscission, reaching a peak of 1,375 disintegrations per minute per 0.1 gram dry weight per 5.5 hours 2 days after deblading when abscission was 40%. The rate then gradually declined, but on day 5 when abscission reached completion, it was still nearly three times higher than in segments from nonabscising leaves. Application of 0.1 millimolar abscisic acid in lanolin to the debladed petiole ends increased the per cent abscission slightly and initially stimulated (14)C(2)H(4) oxidation. In contrast, naphthaleneacetic acid applied in a similar manner delayed and markedly inhibited both abscission and (14)C(2)H(4) oxidation. Petiole segments cut 1 centimeter from the abscission zone of intact second true leaves also incorporated and oxidized (14)C(2)H(4) to (14)CO(2) but at rates two and six times higher, respectively, than that of comparable adjacent abscission zone segments. However, in marked contrast to the abscission zone segments, no changes in oxidation were observed when the leaves were debladed to induce abscission. These results demonstrate that: (a) prior to abscission, the ethylene oxidation, but not the tissue incorporation pathway, rapidly increases in the abscission zone; (b) this increase does not occur in adjacent petiole tissue; and (c) changes in the rate of oxidation and per cent abscission brought about by hormone treatments parallel one another. The possible significance of these changes in ethylene metabolism is discussed with respect to the hypothesis that ethylene action and metabolism are directly related