Plant hormone ethylene and detection
The hydrocarbon ethylene C2H4 is a plant hormone that plays an important role in the regulation of many environmentally- and developmentally induced processes such as stress resistance, germination, ripening, senescence and abscission (Abeles et al., 1992). All tissue types and probably all cells of higher plants produce and liberate ethylene (Osborne, 1989). Tremendous progress is achieved during the last two decades in the biochemical and molecular characterization of the biosynthetic pathway for ethylene in higher plants (Yang and Hoffman, 1984; Kende, 1993).
Abeles FB, Morgan PW, Saltveit ME Jr. (1992) Ethylene in Plant Biology,Academic Press, London
Osborne D (1989) Abscission. Crit. Rev. Plant Sci. 8, 103-129.
Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Ann. Rev. Plant Physiol. 35, 155-189
Kende H (1993) Rthylene biosynthesis. Ann. Rev. Plant Physiol. Plant Mol. Biol. 44 283-307
Endogenous ethylene concentrations inside plant tissues depend on the activities of certain enzymes, the rate of outward diffusion and the rate of metabolization (Osborne, 1989). The rate limiting reactions of ethylene biosynthesis involve the conversion of S-adenosylmethionine into 1-aminocyclopropane-1-carboxylic acid (ACC) catalyzed by ACC synthase and the conversion of ACC into ethylene mediated by the enzyme ACC oxidase (Fluhr and Mattoo, 1996). Furthermore, ACC can be conjugated into 1-malonyl- aminocyclopropane-1-carboxylic acid (MACC) and 1-gamma-L-glutamylaminocyclopropane-1-carboxylic acid (Martin et al., 1995).
The action of ethylene is not only controlled by endogenous ethylene concentrations in tissues, but also by the tissue sensitivity. It is widely assumed that molecules involved in ethylene perception and in the transduction of the signal probably controls how much ethylene is required to evoke a physiological response.
Osborne D (1989) Abscission. Crit. Rev. Plant Sci. 8, 103-129
Fluhr R and Mattoo AK (1996) Ethylene: Biosynthesis and perception. Crit Rev. Plant Sci. 12 479-523
Martin MN, Cohen JD, and Safter RA (1995) A new aminocyclopropane-1-carboxylic acid conjugating activity in tomato fruit. Plant Physiology 109 917-926
The first chemical quantification of ethylene was performed more than 70 years ago on ripening apples (Gane, 1934). In the years hereafter, various techniques such as bio-assays, gravimetric analyses, manometric techniques and physico-chemical colorimetric assays were applied to quantify ethylene concentrations (see review Abeles, 1973). A major breakthrough in ethylene analysis was achieved in the late fifties when the gas chromatographic methodology was applied for the first time on ethylene. For a more detailed discussion of ethylene quantification by means of gas chromatography, the used columns and detectors and the potential sensitivity we refer to the methodological review of Bassi and Spencer (1985).
Despite the high sensitivity ((5-10 nl l-1) this gas chromatographical technique still requires accumulation of ethylene to obtain measurable quantities. For this purpose, it is common practice to incubate pieces of tissue for a few hours in small incubation vials. However, this procedure can disturb the rate of ethylene production due to wounding of tissue, disruption of transport processes, gravitropical disorientation and changes in gas composition around the tissue. The introduction of artifacts in measuring ethylene production rates with isolated plant tissues is elegantly demonstrated for the effect of plant water deficit on ethylene production (Morgan et al.,1990).
Gane R (1934) Production of ethylene by some ripening fruit. Nature 134,1008
Abeles FB (1973) Ethylene in Plant Bioology, Academic Press New York
Bassi PK, Spencer MS (1985) Comparative evaluation of photo-ionization and flame ionization detectors for ethylene analysis. Plant Cell Envir. 8, 161-165
Morgan PW, He C, de Greef JA, and de Proft MP (1990) Does water deficit stress promote ethylene synthesis by intact plants? Plant Physiology 94 1616-1624
Dynamic sampling system
To follow dynamic processes, it is necessary to measure ethylene directly and nearly continuously in the out-flowing air of continuous flow systems. This can be achieved if a flow-through system in line with a large sampling chamber is combined with the extremely sensitive laser photoacoustic detection technique. The detection limit of this system is three orders of magnitude better than gas chromatography, i.e. 6 pl l -1. Since the last decade laser photoacoustic spectroscopy is applied to determine ethylene production rates and concentrations in studies that focus on processes such as germination (Petruzzelli et al., 1994), flower senescence (Woltering et al., 1988), aerenchyma formation in roots (Brailsford et al., 1993), formation of adventitious roots (Visser et al., 1996), submergence-induced shoot elongation (Voesenek et al., 1990; Voesenek et al., 1993) and fruit ripening (de Vries et al., 1995; de Vries et al., 1996).
A gas flow system to flush the emitted ethylene from sampling cell to detection cell consisting of a bottle compressed air, a catalyst to remove all traces of hydrocarbons, a pressure reduction valve, tubing and connectors, mechanical flow controllers, cuvettes with biological sample, a KOH-scrubber between sampling cell and a detector to remove interfering CO2, a CaCl2-scrubber to remove water, a cold-trap (-150 C) to remove (when produced) ethanol and a mass flow controller.
The various parts of the laser-driven photoacoustic set-up are computer controlled, enabling a fully automated sampling of ethylene production rates of biological tissue. The sampling run starts by tuning the laser toward the ethylene absorbing laser line. Hereafter, the laser is tuned to a non-ethylene absorbing wavelength. The signals on both laser lines include ethylene absorption and background signal, the latter is generated from interfering gases. The interfering gases can cause problems to quantify the amount of ethylene. Therefore, they should be removed from the gas flow. The overall measuring sequence takes about one minute. The next step can be another run on the same cuvette or alternatively, a computer-controlled switch to another cuvette which is empty or containing control tissue. The latter signals should be subtracted from the experiment to extract the pure effect and cancel slow, external induced, variations (e.g. over hours or days).