Preparation of ethylene gas and comparison of ethylene responses induced by ethylene,ACC, and ethephon

Ethylene is a gaseous plant hormone used in many physiological studies examining its role in plant growth and development. However, ethylene gas may not be conveniently available to many laboratories for occasional use, and therefore several chemicals can be used as replacements. Here we report that the kinetics of the ethylene response induced by ethylene and two widely-used ethylene replacements are different. ACC failed to efficiently replace prolonged ethylene treatments, while the decomposition products of ethephon may cause non-specific responses and the efficiency of ethephon conversion to ethylene was relatively low. A cost-effective method to prepare ethylene gas was developed. Analyzed by gas chromatography, the chemically produced ethylene exhibited an identical chromatogram to that from the commercial source. Our synthetic ethylene gave the same dose-response curve in Arabidopsis as gaseous ethylene. Our study shows that the use of the ethylene gas is essential to experiments that are sensitive to treatment duration and dosage. When ACC and ethephon are used as replacements, caution should be taken in the experimental design. For laboratories that do not have an ethylene tank, ethylene gas can be easily prepared by a chemical approach without further purification.     

Inhibitory effect of methyl jasmonate on development of phytopathogen <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissl. and its reversal by ethephon and ACC

Methyl jasmonate (MeJA) was found to reduce spore germination, hyphal and mycelial growth in Alternaria alternata (Fr.) Keissl. The addition of ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene precursor, together with MeJA to the culture medium resulted in a promotion of all developmental stages of the fungus; these compounds partially or completely reversed the inhibition due to MeJA depending on the concentrations applied. MeJA alone had no effect on ethylene production by mycelium, but after 6 days of incubation in the presence of ACC, emanation of this gas increased significantly. Ethylene is involved in reversing the inhibition of A. alternata due to MeJA.     

Germination strategy of Striga hermonthica involves regulation of ethylene biosynthesis

Ethylene involvement in germination of Striga hermonthica (Del.) Benth., an important root parasitic weed on poaceous crops, was investigated at the physiological and molecular levels. Seeds, conditioned at 30°C for 14 days, were treated with ethylene, ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC). Ethylene consistently induced low germination. Ethephon and ACC effectively stimulated germination at concentrations of 0.01 and 1 m M , respectively. In contrast to ethylene, both ethephon and ACC acted in a concentration-dependent manner. Germination induced by the synthetic strigolactone GR24 was inhibited by aminoethoxyvinylglycine (AVG) and 1-methylcyclopropene. ACC reversed the inhibition caused by AVG. When seeds were treated with GR24 in sealed vials, ethylene concentration in headspace gas increased prior to the onset of germination. Total RNA extracted from germinating seeds 12 h after GR24 treatment was used for PCR-based amplification of cDNA fragments encoding the ACC synthase- and oxidase-active site domains. Two distinct cDNA fragments encoding ACC synthase ( SHACS1 and SHACS2 ) and one encoding ACC oxidase ( SHACO1 ) were cloned and sequenced. Southern analysis suggested that each of the cloned genes was present as a single copy in the genome of S. hermonthica . Northern analyses showed that SHACS1 exhibited a temporal change in expression peaking at 10 h after GR24 treatment, which coincided with a steady increase in ethylene concentration. SHACS2 was expressed at a low level with a similar trend. SHACO1 exhibited a temporal change in expression peaking at 15 days during conditioning, when seed response to GR24 was maximal. In summary, expression of ACC synthase and ACC oxidase genes was found to be responsive to a germination stimulant and to conditioning, respectively. The implications of these findings with respect to germination of S. hermonthica under field conditions are discussed.     

Regulation of Auxin-induced Ethylene Production in Mung Bean Hypocotyls: Role of 1-Aminocyclopropane-1-Carboxylic Acid

Ethylene production in mung bean hypocotyls was greatly increased by treatment with 1-aminocyclopropane-1-carboxylic acid (ACC), which was utilized as the ethylene precursor. Unlike auxin-stimulated ethylene production, ACC-dependent ethylene production was not inhibited by aminoethoxyvinylglycine, which is known to inhibit the conversion of S-adenosylmethionine to ACC. While the conversion of methionine to ethylene requires induction by auxin, the conversion of methionine to S-adenosylmethionine and the conversion of ACC to ethylene do not. It is proposed that the conversion of S-adenosylmethionine to ACC is the rate-limiting step in the biosynthesis of ethylene, and that auxin stimulates ethylene production by inducing the synthesis of the enzyme involved in this reaction.     

Light Stimulation of Ethylene Release from Leaves of Gomphrena globosa L

The effect of light and CO₂ on both the endogenous and 1-aminocyclopropane-1-carboxylic acid (ACC)-dependent ethylene evolution from metabolically active detached leaves and leaf discs of Gomphrena globosa L. is reported. Treatment with varying concentrations of ACC did not appear to inhibit photosynthesis, respiration, or stomatal behavior. In all treatments, more ethylene was released into a closed flask from ACC-treated tissue, but the pattern of ethylene release with respect to light/dark/CO₂ treatments was the same.

Leaf tissue in the light with a source of CO₂ sufficient to maintain photosynthesis always generates 3 to 4 times more ethylene than tissue in the dark. Conversely, the lowest rate of ethylene release occurs when leaf tissue is illuminated and photosynthetic activity depletes the CO₂ to the compensation point. Ethylene release in the dark is also stimulated by CO₂ either added to the flask as bicarbonate or generated by dark respiration. Ethylene release increases dramatically and in parallel with photosynthesis at increasing light intensities in this C₄ plant. Ethylene release appears dependent on CO₂ both in the light and in the dark. Therefore, it is suggested that the important factor regulating the evolution of ethylene gas from leaves of Gomphrena may be CO₂ metabolism rather than light per se.

     

Gravitropism in Higher Plant Shoots : IV. Further Studies on Participation of Ethylene

Ethylene at 1.0 and 10.0 cubic centimeters per cubic meter decreased the rate of gravitropic bending in stems of cocklebur (Xanthium strumarium L.) and tomato (Lycopersicon esculentum Mill), but 0.1 cubic centimeter per cubic meter ethylene had little effect. Treating cocklebur plants with 1.0 millimolar aminoethoxyvinylglycine (AVG) (ethylene synthesis inhibitor) delayed stem bending compared with controls, but adding 0.1 cubic centimeter per cubic meter ethylene in the surrounding atmosphere (or applying 0.1% ethephon solution) partially restored the rate of bending of AVG-treated plants. Ethylene increases in bending stems, and AVG inhibits this. Virtually all newly synthesized ethylene appeared in bottom halves of horizontal stems, where ethylene concentrations were as much as 100 times those in upright stems or in top halves of horizontal stems. This was especially true when horizontal stems were physically restrained from bending. Ethylene might promote cell elongation in bottom tissues of a horizontal stem or indicate other factors there (e.g. a large amount of `functioning' auxin). Or top and bottom tissues may become differentially sensitive to ethylene. Auxin applied to one side of a vertical stem caused extreme bending away from that side; gibberellic acid, kinetin, and abscisic acid were without effect. Acidic ethephon solutions applied to one side of young seedlings of cocklebur, tomato, sunflower (Helianthus annuus L.), and soybean (Glycine max [L.] Merr.) caused bending away from that side, but neutral ethephon solutions did not cause bending. Buffered or unbuffered acid (HCl) caused similar bending. Neutral ethephon solutions produced typical ethylene symptoms (i.e. epinasty, inhibition of stem elongation). HCl or acidic ethephon applied to the top of horizontal stems caused downward bending, but these substances applied to the bottom of such stems inhibited growth and upward bending—an unexpected result.     

Effects of ethylene on the tuberization of potato (Solanum tuberosum) cuttings

Ethylene, applied as ethephon, inhibited the elongation of etiolated, axillary potato shoots cultured in vitro and it stimulated radial growth along the whole length of these shoots. The same phenomena were observed when ACC, the precursor of ethylene, was added to the medium, whereas silver ions reversed these effects. However, tuber formation in vitro was suppressed by ethephon. This indicates a dual role of ethylene in the induction of tuber formation in potatoes: it had a positive effect by blocking the elongation of stolons and it suppressed tuber initiation.     

Interaction of karrikinolide and ethylene in controlling germination of dormant <Emphasis Type="Italic">Avena fatua</Emphasis> L. caryopses

Caryopses of Avena fatua L. are dormant after harvest and germinate poorly at 20 °C. Dormancy was released by after-ripening the dry caryopses in the dark at 25 °C for 3 months. Karrikinolide (butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, KAR₁), in contrast to exogenous ethylene and the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid (ACC), completely overcame dormancy. The effect of KAR₁ was not affected by aminoethoxyvinylglycine (AVG), α-aminoisobutyric acid (AIB) and CoCl₂, inhibitors of ACC synthase and oxidase, respectively. 2,5-Norbornadiene (NBD), a reversible inhibitor of ethylene binding to its receptor, counteracted the stimulatory effect of KAR₁. Ethylene, ethephon and ACC counteracted and AVG reinforced inhibition caused by norbornadiene. Inhibition due to norbornadiene, applied during the first 3 days of imbibition in the presence of KAR₁, disappeared after transfer to air or ethylene. The obtained results confirm that KAR₁ breaks dormancy and indicate that ethylene alone plays no role in releasing dormancy of Avena fatua caryopses. KAR₁ probably did not relieve dormancy via the stimulation of ethylene biosynthesis. Some level of endogenous ethylene is probably required for ethylene action, which might be required for releasing dormancy by KAR₁ or for subsequent germination of caryopses after removing dormancy.     

Influence of ACC and Ethephon on cell growth in etiolated lupin hypocotyls. dependence on cell growth state

The possible implication of ethylene on the growth regulation of etiolated lupin hypocotyls was investigated. Excised hypocotyl sections from actively growing seedlings produced ethylene at a rate of 3 nmol h^-1 g^-1 min^-1. The rate of ethylene production was increased about 7 times when sections were treated with 10 mM 1-aminocyclopropane-1-carboxylic acid (ACC). Measurement of endogenous ACC showed that 95 % of total ACC (64.2 nmol g^-1 min^-1) corresponded to conjugated ACC. Treatments to intact seedlings with the ethylene precursor ACC, and the ethylene generating compound, 2-chloroethyl phosphonic acid (ethephon) during the cell elongation phase of the hypocotyl (from 7 to 21 dage), modified the cell growth of the organ. ACC (1 or 5 mM) or low concentrations of ethephon (0.66 mM) produced a transient decrease in the growth rate without modifying the final length of the hypocotyls. Higher concentrations of ethephon reduced the final length; the younger the seedlings were, the greater the reduction. Simultaneously to inhibition of cell elongation, ethephon produced stimulation of the radial expansion of cells in pith and cortex. The growth inhibition period, which lasted for 2 days after the treatments, was followed by another period in which the growth rate of treated plants surpassed that of the control. In both cases differences were observed along the hypocotyls due to the different growth status of the cells. It is suggested that the sensitivity to ethylene and the metabolism of ethylene depend on the growth status of the cells.     

Ethylene in seed dormancy and germination

The role of ethylene in the release of primary and secondary dormancy and the germination of non-dormant seeds under normal and stressed conditions is considered. In many species, exogenous ethylene, or ethephon – an ethylene-releasing compound - stimulates seed germination that may be inhibited because of embryo or coat dormancy, adverse environmental conditions or inhibitors (e.g. abscisic acid, jasmonate). Ethylene can either act alone, or synergistically or additively with other factors. The immediate precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC), may also improve seed germination, but usually less effectively. Dormant or non-dormant inhibited seeds have a lower ethylene production ability, and ACC and ACC oxidase activity than non-dormant, uninhibited seeds. Aminoethoxyvinyl-glycine (AVG) partially or markedly inhibits ethylene biosynthesis in dormant or non-dormant seeds, but does not affect seed germination. Ethylene binding is required in seeds of many species for dormancy release or germination under optimal or adverse conditions. There are examples where induction of seed germination by some stimulators requires ethylene action. However, the mechanism of ethylene action is almost unknown.
The evidence presented here shows that ethylene performs a relatively vital role in dormancy release and seed germination of most plant species studied.     

Role of ethylene in stimulating stylar abscission in pistil explants of lemons

Abscission in styles of excised Citrus limon (cv. Lisbon) pistils was stimulated by addition of 70 μ M 2-chloroethylphosphonic acid (ethephon) or 0.1 m M 1-aminocyclopropane-1-carboxylic acid (ACC) to the defined medium of cultures. To study the relationship between ethylene and abscission, we used gas chromatography to analyze ethylene in cultures containing a test medium plus or minus abscission-active chemicals. In the presence of ethephon or ACC, ethylene levels in sealed tubes increased rapidly, suggesting that these compounds stimulated abscission because they were converted to ethylene. In the presence of test medium or the inhibitor of abscission 2 μ M picloram, the low ethylene levels found in sealed tubes did not differ strikingly in the two treatments. Ethylene production rates measured prior to abscission with test medium or in the presence of picloram were not markedly different either, although picloram completely inhibited abscission. Stylar abscission was delayed but not prevented by 50 μ M aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis, and by hypobaric conditions (280 mm Hg) which removed ethylene from cultures. We concluded that ethylene is an important factor regulating stylar abscission in vitro and suggest that the inhibitory effect of picloram involves a process other than detectable ethylene production. Our results do not exclude the possibility that picloram affects enodgenous ethylene biosynthesis and/or metabolism and/or tissue retention.     

Whole Plant and Leaf Steady State Gas Exchange during Ethylene Exposure in Xanthium strumarium L

The effects of ethylene evolved from ethephon on leaf and whole plant photosynthesis in Xanthium strumarium L. were examined. Ethylene-induced epinasty reduced light interception by the leaves of ethephon treated plants by up to 60%. Gas exchange values of individual, attached leaves under identical assay conditions were not inhibited even after 36 hours of ethylene exposure, although treated leaves required a longer induction period to achieve steady state photosynthesis. The speed of translocation of recently fixed ¹¹C-assimilate movement was not seriously impaired following ethephon treatment; however, a greater proportion of the assimilate was partitioned downward toward the roots. Within 24 hours of ethephon treatment, the whole plant net carbon exchange rate expressed on a per plant basis or a leaf area basis had dropped by 35%. The apparent inhibition of net carbon exchange rate was reversed by physically repositioning the leaves with respect to the light source. Ethylene exposure also inhibited expansion of young leaves which was partially reversed when the leaves were repositioned. The data indicated that ethylene indirectly affected net C gain and plant growth through modification of light interception and altered sink demand without directly inhibiting leaf photosynthesis.     

Ethylene Promotes the Capability To Malonylate 1-Aminocyclopropane-1-carboxylic Acid and d-Amino Acids in Preclimacteric Tomato Fruits

When whole unripe green tomato fruits (Lycopersicon esculentum Mill, cv T₃) were treated with ethylene (10 microliters per liter) for 18 hours, the fruit's ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to N-malonyl-ACC (MACC) increased markedly and such an effect was also observed in fruits of mutant nor, which cannot ripen normally. The promotion of the capability to malonylate ACC by ethylene increased with the increasing ethylene concentration from 0.1 to 100 microliters per liter and with increasing duration of ethylene treatment up to 8 hours; a longer duration of ethylene treatment did not further increase the malonylation capability. When ethylene was withdrawn, the promotion disappeared within 72 hours. Norbornadiene, a competitive inhibitor of ethylene action, effectively eliminated the promotive effect of ethylene. Ethylene treatment also promoted the fruits' capability to conjugate d-amino acids and α-amino-isobutyric acid. Since the increase in the tissue's capability to malonylate ACC was accompanied by an increase in the extractable activity of ACC and d-amino acid malonyltransferase, ethylene is thought to promote the development of ACC/d-amino acid malonyltransferase in unripe tomato fruits.     

Endogenous Levels and Transport of 1-Aminocyclopropane-1-Carboxylic Acid in Stamens of Ipomoea nil (Convolvulaceae)

Filament and corolla growth in flowers of Ipomoea nil are inhibited by ethylene production. Anthers inhibited filament growth in vitro during younger stages of development even in the presence of the growth promoter gibberellic acid (GA₃). To test whether the anthers could be sources of 1-aminocyclopropane-1-carboxylic acid (ACC) endogenous levels of ACC and ethylene production were monitored using gas chromatography. To also test whether the filaments could be transport vectors for ACC the movement of [¹⁴C]ACC was assessed by scintillation counting from donor agarose blocks, through filament sections, and into receiver agarose blocks. While ACC levels fluctuated in anthers 87 to 21 h before anthesis, anthers contained increased levels of ACC from 15 to 6 hours before anthesis. Ethylene production also fluctuated but peak levels were shifted about 6 hours closer to anthesis than ACC levels within the anthers. Both ACC and ethylene levels in filaments showed fluctuations similar to those in the anthers. [¹⁴C]ACC movement became increasingly basipetal during development. Older stages showed greater polar [¹⁴C]ACC efflux rates, while all stages showed constant polar influx rates. Low levels of endogenous ACC were transported basipetally from the anther through the filament into agarose blocks at all stages of development. Corresponding levels of endogenous ethylene production remained constant between the various stages during ACC transport. We have evidence that stamens of I. nil have a role as source tissues and transport vectors for ACC, to stimulate corolla growth, such as corolla unfolding and senescence.     

Autoinhibition of Ethylene Production in Citrus Peel Discs : SUPPRESSION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHESIS

Wound ethylene formation induced in flavede tissue of citrus fruit (Citrus paradisi MacFad. cv. Ruby Red) by slicing was almost completely inhibited by exogenous ethylene. The inhibition lasted for at least 6 hours after removal of exogenous ethylene and was then gradually relieved. The extent of inhibition was dependent upon the concentration of ethylene (1 to 10 microliters/liter) and the duration of treatment. The increase in wound ethylene production in control discs was paralleled by an increase in 1-aminocyclopropane-1-carboxylic acid (AAC) content, whereas in ethylene-treated discs there was little increase in ACC content. Application of ACC completely restored ethylene production in ethylene-pretreated discs, indicating that the conversion of ACC to ethylene is not impaired by the presence of ethylene. Thus, autoinhibition of ethylene synthesis was exerted by reducing the availability of ACC. Ethylene treatment resulted in a decrease in extractable ACC synthase activity, but this decrease was too small to account for the marked inhibition of ACC formation. The data indicate that autoinhibition of ethylene production in citrus flavede discs results from suppression of ACC formation through repression of the synthesis of ACC synthase and inhibition of its activity.     

Ethylene synthesis and growth in embryogenic tissue of Norway spruce: Effects of oxygen, 1-aminocyclopropane-1-carboxylic acid, benzyladenine and 2,4-dichlorophenoxyacetic acid

Ethylene production from an embryogenic culture of Norway spruce ( Picea abies L.) was generally low. ca 2.5 nl g⁻¹ h⁻¹, whereas 1-aminocyclopropane-1 -carboxylic acid (ACC) concentration was high, fluctuating between 50 and 500 nmol g⁻¹ during the 11-day incubation period. Hypoxia (2.5 and 5 kPa O₂) rapidly inhibited ethylene production without subsequent accumulation of ACC. Exogenous ACC (1, 10 and 100 μ M ) did not increase ethylene production, but the highest concentrations inhibited tissue growth. Ethylene (7 μl I⁻¹) did not inhibit growth either when supplied as ethephon in the medium or in a continuous flow system. Benzyladenine (BA) had little effect on ethylene production, although it was necessary for sustaining the ACC level. Omission of 2.4-dichloro-phenoxyacetic acid (2.4-D) from the medium caused ethylene production to increase from about 2.5 to 7 nl g⁻¹ h⁻¹ within the 11-day incubation period. Although 2.4-D did not specifically alter the endogenous level of ACC, the lowest ACC level, 33 nmol g⁻¹, was observed in tissue treated with 2.4-D (22.5 μ M ) and no BA for 11 days. Data from this treatment were used to estimate the kinetic constants for ACC oxidase, the apparent K_m was 50 μ M and V_max 2.7 nl g⁻¹ h⁻¹. Growth of the tissue was strongly inhibited by 2.4-D in the absence of BA, but weakly in the presence of BA (4.4 μ M ). The results suggest that ethylene or ACC may be involved in the induction of embryogenic tissue and in the early stages of embryo maturation.     

Effects of exogenous ethylene on ethylene production in citrus leaf tissue

Exogenous ethylene stimulated ethylene production in intact citrus (Citrus sinensis L. Osbeck cv. “Washington Navel”) leaves and leaf discs following a 24-hour exposure. Studies with leaf discs showed that ethylene production decreased when ethylene was removed by aeration. The extent of stimulation was dependent upon the concentration of exogenous ethylene (1-10 microliters per liter). Silver ion blocked the autocatalytic effect of ethylene at concentrations of 0.5 millimolar and lower, but increased ethylene production at higher concentrations. The stimulating effect of ethylene resulted from the enhancement of both 1-aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. Whereas autocatalysis was evident following 24 hours incubation, autoinhibition of wound- and mannitol-induced ethylene production was observed during the first 24-hour incubation. Ethylene treatment during this period resulted in a marked decrease in ACC levels and ethylene production rates. Furthermore, in leaf discs treated for 24 hours with ethylene, ethylene production rates increased greatly during the first 2 hours after removal of exogenous ethylene by aeration. This increase was eliminated if the discs were transferred to propylene instead of air, indicating that the autocatalytic effect of ethylene is counteracted by its autoinhibitory effect. It is suggested that autocatalysis involves increased synthesis of ACC synthase and the enzyme responsible for the conversion of ACC to ethylene, whereas autoinhibition involves suppression of the activity of these two enzymes.     

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.