Purity of Ethylene Prepared from Ethephon Decomposition and Ethanol Dehydration

Ethephon (2-chloroethylphosphonic acid) is a widely used chemical replacement for ethylene treatment in many physiological studies.1,2 The conversion of ethephon to ethylene takes place at higher pHs.3 Thus, ethylene can be easily prepared by hydrolyzing ethephon in buffered conditions. For industrial production, ethylene can be prepared by ethanol dehydration; however, the reaction takes place at harsh conditions and further purification is needed.4 The chromatograms for ethylene from a commercial source and prepared from ethephon decomposition are identical and no other hydrocarbon species were detectable (Fig. 1A). Gas collected from the ethanol dehydration gives multiple hydrocarbon species that can be detected by the FID (flame ionization detector). In addition to the ethylene and ethanol peaks we previously reported,5 another peak was prominent after the completion of the ethanol dehydration reaction (Fig. 1B). Results from GC-MS (Gas chromatography-mass spectrometry) analysis suggest that the hydrocarbon species is diethyl ether (Fig. 1C). The production of diethyl ether follows the equation: 2 CH₃CH₂OH → CH₃CH₂OCH₂CH₃ + H₂O (on 120°C).

Applications of Ethephon Decomposition in Ethylene Treatment

Ethylene inhibits the hypocotyl elongation in dark-grown seedlings and promotes the chlorophyll degradation in Arabidopsis and in many other plant species.6,7 The seedling triple-response assay and leaf senescence test provide an estimate for extent of ethylene response.6 Although ethylene can be easily prepared in a closed container by ethephon decomposition, it may not be convenient for some non-ethylene laboratories to handle the gas when various ethylene concentrations are needed for physiological treatments. The decomposition of ethephon to ethylene in a buffered condition is rapid, and the reaction nearly completes within 2 days.5 Conceivably, necessary amount of ethylene can be directly produced from ethephon decomposition within a closed container for the seedling triple-response assay and leaf senescence test (Fig. 2A). Direct ethylene treatment caused greater extent of leaf senescence than did the ethephon decomposition, possibly due to delayed ethylene release in the ethephon decomposition reaction (Fig. 2B). Although ethylene treatment resulted in stronger seedling growth inhibition, the difference was relatively minor and only occurred at low ethylene concentration range (Fig. 2C). At higher ethylene concentration (1 µL L⁻¹), there was no difference in the growth inhibition between the two treatments. The minor difference is conceivably caused by delayed ethylene release in the decomposition reaction. The leaf senescence test is a qualitative study and can be performed by directly decomposing necessary amount of ethephon in a closed container. For the seedling triple-response assay, direct ethephon decomposition can replace ethylene treatment when the necessary ethylene concentration is above 0.4 µL L⁻¹.

ERF1 Expression is Induced by High Concentration of ACC

Previously we showed that 10 µM ACC (1-aminocyclopropane-1-carboxylic acid) treatment, a commonly used ACC concentration in many experiments,7,8 was unable to induce ERF1 expression and leaf senescence by foliar spray. However, leaf senescence can be induced by ACC foliar spray when the concentration is extremely high (1 mM) in closed conditions.5 There are experiments where ethylene treatment is not feasible and the use of its replacement will be needed. For example, it is technically challenging to administer ethylene to tissues subjected to experiments involving microscopy. Figure 3A shows that ERF1 expression was highly induced by 1 mM ACC in open and closed conditions. Notably, ERF1 induction dropped quickly 8 hr after treatment in plants treated in open space. In contrast, ERF1 expression gradually increased in plants treated in closed conditions. At the completion of a 12-hr ACC treatment, the final ethylene concentration was 9.5 µL L⁻¹ (Fig. 3B), indicating that about 19% of the applied ACC was already converted to ethylene within 12 hr in closed conditions.

This result is in agreement with previous study that ACC undergoes early consumption and that ACC treatment is not ideal for experiments that are sensitive to response window or duration.9 In open conditions, ERF1 induction dropped quickly, implying quicker ACC conversion to ethylene, possibly due to more available oxygen for ACC oxidation. These results provide an explanation for why 1 mM ACC treatment failed to cause leaf senescence in open conditions, albeit ERF1 induction was initially high. For experiments where ethylene treatment is not feasible, such as observing movement or accumulation of GFP-fusion proteins in living cells, ACC of higher concentrations may be used as a replacement.