Method for Overcoming the Antiethylene Effects of Ag +

Abstract

A technique is descrbdW for eliminating the antiethylene effects of the Ag+ ion in the intact pea plant (Pisum saivum). The technique is based on the ability of the ethylene mimc, acetylene, to negate the antlethylene effect of Ag', presumably through salt formation, and subsequently to induce the ethylene response. The Ag+ ion effectively blocks ethylene action. In the initial work with cucumber and tomato (4) a single foliar application of AgNO3 greatly reduced ethylene sensitivity. Responses to exoge-nous ethylene counteracted by Ag+ included growth retardation, epinasty, senescence, and abscission. Ag+ also counteracted en-dogenous ethylene by shifting sex expression in gynoecious cucumber plants. In similar studies (2, 3), Ag+ pretreatment prevented ethylene-induced root growth inhibition and the classical "triple" response in etiolated peas. It also blocked abscission and/or senescence in cotton, bean, and orchids. Shortly after these reports, Halevy and Kofranek (7) reported that Ag+ delayed senescence of cut carnations. Additional work by Veen and van de Geijn (10) has recently suggested an improved method for extending flower longevity using the silverthiosulfate anionic complex rather than AgNO3. Liu (9) reported using Ag+ to counteract the effect of ethylene effectively in senescence spot development of bananas while Beutelmann and Kende (1) used Ag+ to inhibit ethylene induced rolling up of rib segments cut from the corolla of Ipomoea tricolor. More recently, Bradford and Dilley (5) applied AgNO3 to tomatoes to demonstrate convincingly that ethylene is responsible for waterlogging symptoms in these plants. These studies demonstrate the ability of Ag+ to prevent ethylene action. For certain applications it is often difficult to decide whether the Ag+ response really represents a specific antiethylene effect. For example, when ethylene promotes a response (e.g. abscission, ripening, seed germination, growth stimulation), the failure to observe the ethylene response following Ag+ treatment could be interpreted as either a specific blocking of ethylene action or a general inhibition of metabolism. To distinguish between these two possibilities a technique was developed that would eliminate the Ag+ effect and allow the ethylene response to develop, thereby demonstrating the viability of the tissue. The technique is based on the ability of acetylene to negate Ag+ action and to induce the ethylene response. Although it is presumed that acetylene negates Ag+ action through salt formation, this mechanism has not yet been demonstrated in vivo. MATERIALS AND METHODS Pea seeds (Pisum sativum cv. Alaska) were soaked in distilled H20 for 4 hr, planted in 15-cm pots containing Jiffy-Mix (Jiffy Products Inc., Chicago) combined with sand (3:1, v/v), and watered with distilled H20. Seeds were germinated and grown for 3 days in a dark room at a constant 22 C and 55% RH. After binding to 20 uniform seedlings 10 pots were transferred to each of three similar chambers purged with either ethylene-free air (air scrubbed with Purafil, H. E. Burroughs & Assoc., Inc. Chamblee, Ga.), ethylene (0.22 ,ld/l), or acetylene (240 ul/1). Following a 2-day exposure, five pots from each chamber were sprayed with water plus 0.01% Tween 20 and five pots were sprayed with 20 mg/l of AgNO3 plus surfactant. Immediately following treatment the seedlings were returned to their respective chambers. All transfers and treatments were made using a green safelight. The following day eight pots (four controls and four AgNO3-treated pots) were removed from each chamber and two control pots and two AgNO3-treated pots were placed in two large vacuum containers. One-half of the air was removed from one of the containers (about 380 mm Hg) and atmospheric pressure immediately restored by bleeding back in pure acetylene. After 15 min this vacuum treatment was repeated but atmospheric pressure was restored this time with room air. This procedure of bleeding air back into the chamber was repeated a third time resulting in three consecutive 15-min exposures to first 50%o acetylene, and then 25% and 12.5% acetylene. The two pots of control and AgNO3-treated seedlings in the other vacuum container were subjected to the same reduced pressures but were not exposed to acetylene. Immediately following these vacuum treatments most of the seedlings were returned for an additional 2 days to their respective chambers purged with either air, ethylene (0.22 ,ul/l), or acetylene (240 ,ul/l). However, one of the two pots of control and AgNO3-treated seedlings taken from the ethylene chamber for air or acetylene vacuum treatment was transferred back to the 240,u/i acetylene chamber for an equal period of 2 days. RESULTS AND DISCUSSION Acetylene rapidly reacts with Ag+ to form the very insoluble silver acetylide salt. Therefore, exposing Ag+-treated plants to acetylene gas is a potential method for eliminating the antiethylene effects of Ag+. To test this idea etiolated pea seedlings were first treated with 20 mg/l of AgNO3 and placed in 0.2 ,ul/l of ethylene for 2 days. Under these conditions the seedlings failed to respond to ethylene as previously reported (2). In an attempt to overcome this antiethylene effect of Ag+ the seedlings were removed from ethylene, treated with 50%o acetylene for 30 min, and then returned to ethylene. Although the AgNO3-treated seedlings clearly showed a greater sensitivity to ethylene when placed back in 0.2 ,ul/l of the gas, they did not respond fuily indicating incomplete removal of Ag+. Repeated vacuum treatment during acetylene exposure helped by facilitating the diffusion of acetylene into the tissue but even this treatment still did not completely eliminate the effect of Ag+. 616 www.plant.org on April 23, 2015-Published by www.plantphysiol.org Downloaded from