Inhibition of Ethylene Synthesis in Tomato Plants

Enhanced ethylene production and leaf epinasty are characteristic responses of tomato (Lycopersicon escukntum Mill.) to waterlogging. It has been proposed (Bradford, Yang 1980 Plant Physiol 65: 322-326) that this results from the synthesis of the immediate precursor of ethylene, 1aminocyclopropane-I-carboxylic acid (ACC), in the waterlogged roots, its export in the transpiration stream to the shoot, and its rapid conversion to ethylene. Inhibitors of the ethylene biosynthetic pathway are available for further testing of this ACC transport hypothesis: aminooxyacetic acid (AOA) or aminoethoxyvinylglycine (AVG) block the synthesis of ACC, whereas CO2 prevents its conversion to ethylene. AOA and AVG, supplied in the nutrient solution, were found to inhibit the synthesis and export of ACC from anaerobic roots, whereas Co2" had no effect, as predicted from their respective sites of action. Transport of the inhibitors to the shoot was demonstrated by their ability to block wound ethylene synthesis in excised petioles. Al three inhibitors reduced petiolar ethylene production and epinasty in anaerobically stressed tomato plants. With AOA and AVG, this was due to the prevention of ACC import from the roots as well as inhibition of ACC synthesis in the petioles. With Co21, conversion of both rootand petiole-synthesized ACC to ethylene was blocked. Collectively, these data support the hypothesis that the export of ACC from low 02 roots to the shoot is an important factor in the ethylene physiology of waterlogged tomato plants. Ethylene is an important endogenous regulator of plant responses to waterlogging (7, 14). Under the low 02 conditions in the soil during flooding, tomato roots synthesize ACC,3 the immediate precursor of ethylene, and transport it in the xylem to the shoot, where it is rapidly converted to ethylene (8, 9). This increase in the ethylene content of the shoot tissues results in petiole epinasty, aerenchyma formation, and other anatomical and morphological responses associated with waterlogging (6, 11, 14). The sites of inhibition in the biosynthetic pathway of ethylene are now known for several inhibitors. Application of these inhibitors allows further tests of the role of root-synthesized ACC in shoot responses to waterlogging. AOA and AVG effectively prevent the conversion of SAM to ACC, both in vivo (1, 22, 23) and with isolated ACC synthase (4, 21), but do not interfere with the ' Supported in part by Cooperative Western Regional Research Project W-54 and National Science Foundation grant PCM-8114933. 2Supported by a University of California Regents Fellowship. 3Abbreviations: ACC, l-aminocyclopropane-l-carboxylic acid; AOA, aminooxyacetic acid; AVG, aminoethoxyvinylglycine [L-2-amino-4-(2aminoethoxy)-trans-3-butenoic acid]; NAA, naphthyl-l-acetic acid; SAM; S-adenosylmethionine. conversion of ACC to ethlene. Co2" on the other hand, has no effect on ACC synthesis but inhibits the formation of ethylene from ACC (23). These compounds, therefore, permit inhibition of ethylene biosynthesis at specific steps in the pathway (see 18, 19, for reviews). In waterlogged plants, the locations of stress-induced ACC synthesis and its conversion to ethylene are separated spatially, with the formation of ACC from SAM occurring in the roots and conversion of ACC to ethylene occurring in the shoot. Thus, the effects of application of AOA, AVG, or Co2" to anaerobic roots should reflect their differing sites of action. In this study, these inhibitors were used to further characterize the role of root-synthesized ACC in the responses of tomato plants to root anaerobic stress. MATERUILS AND METHODS Plant Material. Tomato plants (Lycopersicon esculentum Mill.) of two cultivars, VFN8 and UC82, were germinated in vermiculite for 12 to 14 d. The seedlings were then transplanted into 1.75-1 plastic boxes containing full-strength nutrient solution [K+, 4 mM; N03, 12 mM; Ca2+, 5 mm; Mg2+, S02-, PO3-, 2 mM; Fe-chelate (sodium ferric ethylenediamine di-(o-hydroxyphenylacetate)), 0.04,UM micronutrients as for full-strength Hoagland solution]. Two seedlings were planted per box. The solutions were continuously aerated at a rate of 250 ml min-'. The pH was initially adjusted to 6.0, and the solutions were replenished with distilled H20 as needed. Two weeks after transplanting (five to seven leaf stage), the nutrient solutions were replaced and stress treatments were begun on the next day. The environmental conditions in the growth chamber were: light, 500 ,uE m-2 s-1 PAR; temperature, 27°C; photoperiod, 16 h; RH, 40%o. Inhibitor and Stress Treatments. Solutions of AOA, AVG, or CoCl2 were added to the nutrient solutions to give the final concentrations indicated. Due to the large quantities of the inhibitors required, most experiments used AOA rather than AVG. AOA was purchased from Sigma, and AVG was a gift from J. P. Scannel (Hoffman-LaRoche). After 4 h of aerobic uptake, root anaerobiosis was imposed by bubbling N2 through the solutions at 250 ml min-'. Measurements with an oxygen electrode indicated that deoxygenation was complete after 1 h. Twenty-four hours after the start of the anaerobic treatment, both plants from a box were sampled for ethylene and epinasty measurements and xylem sap was collected from one plant, as described below. Three or four replicate boxes were used for each treatment. Epinasty, Ethylene, and ACC Measurements. Epinasty was measured with a transparent protractor as the adaxial angle between the third oldest petiole and the stem. The increase in petiole angle over 24 h is plotted in the figures. Ethylene production from 3-cm proximal petiole sections was measured by placing the sections in 4.1 ml test tubes fitted with serum caps and determining the ethylene content of the air in the tube by GC after 0.5 h (9). Xylem sap was collected and analyzed for ACC as described 1503 D ow naded rom http/academ ic.p.com /plphys/art0/5/1503/6078828 by gest on 20 Feruary 2022 Plant Physiol. Vol. 70, 1982 previously (9), except that a suction of 0.5 rather than 0.9 bar was applied during sap collection. (HCI addition to the collected sap to stabilize the ACC was found to be unnecessary and, therefore, was omitted in these experiments.) The data are expressed as a flux rate (nmol h-1) by multiplying the ACC concentration in the sap by the rate of sap collection. Root ACC content was determined as before (9), except that the extract was directly used for ACC assay without passing through an ion exchange column. The data were analyzed by analysis of variance procedures. Error bars indicating ±i SE are shown in the figures as an indication of experimental variance. All experiments except that shown in Table I were performed at least twice with similar results.