Comparison of Results Obtained with Acridine Orange and Quinacrine

The effect of temperature on the rate of proton transport and ATP hydrolysis by plasma membrane (PM) and tonoplast (TN) ATPases from barley (Hordeum vulgare L. cv CM 72) roots were compared. Rates of proton transport were estimated using the fluorescent amine dyes quinacrine and acridine orange. The ratio between rate of transport and ATP hydrolysis was found to depend on the dye, the temperature, and the type of membrane. The PM ATPase had an estimated Arrhenius energy of activation (Ea) of approximately 18 kilocalories per mole for ATP hydrolysis, and the Ea for proton transport was best estimated with acridine orange, which gave an Ea of 19 kilocalories per mole. The TN ATPase had an Ea for ATP hydrolysis of approximately 10 kilocalories per mole and the Ea for proton transport was best estimated with quinacrine, which gave an Ea of 10 kilocalories per mole. Acridine orange did not give an accurate estimate of Ea for the TN ATPase, nor did quinacrine for the PM ATPase. Reasons for the differences are discussed. Because it was suggested (AJ Pope, RA Leigh [1988] Plant Physiol 86: 1315-1322) that acridine orange interacts with anions to dissipate the pH gradient in TN vesicles, the complex effects of N03on the TN ATPase were also examined using acridine orange and quinacrine and membranes from oats and barley. Fluorescent amine dyes can be used to evaluate the effects of ions, substrates, inhibitors, and temperature on transport but caution is required in using rates of quench to make quantitative estimates of proton fluxes. Various membranes including the plasma membrane (PM2) and tonoplast (TN) have been hypothesized to be primary sites ofchilling injury because chilling temperatures may have direct effects on the fluidity, viscosity, or phase-state ofmembrane lipids (13, 23, 26 and references therein). Ion leakages caused by the effect of temperature on membrane lipids or proteins have been cited as potential causes of chilling injury or freezing damage (1 3, 28) and the process of cold acclimation may include modification of lipid and protein compo' This work was partially funded by U.S. Department ofAgriculture Competitive grant No. 85-CRCR-1-1647. 2Abbreviations: PM, plasma membrane; ApH, difference in pH across the vesicle membrane; Ea, Arrhenius energy of activation; TN, tonoplast; DCCD, dicyclohexylcarbodiimide. nents of the PM or TN (13, 21, 28). One method to test hypotheses about the direct effect of temperature on the TN and PM is to examine the effects of temperature on isolated vesicles (11). Analysis of the effect of temperature on isolated PM and TN vesicles from plant tissues also can provide information on the energy requirements of ion transport, the mechanisms of action of membrane transport systems (16), and the responses of plants to stressful high temperatures. Since barley has been used as an example of a plant that does not undergo chilling injury (26), it was of some interest to examine the effects of temperature on transport by barley membranes. In order to measure the effect of variables such as temperature and ions on the TN and PM ATPase, it is important to find methods to measure transport that are not affected significantly by the conditions being tested. If temperatureand ion-insensitive methods are not available, then the effects oftemperature and ions on the techniques that are used must be defined. Fluorescent dyes such as acridine orange and quinacrine are commonly used in procedures for estimating rates of proton transport by membrane vesicles. However, Pope and Leigh (25) discussed various artifacts that occurred when acridine orange was used to measure pH gradients formed in TN vesicles obtained from red beet storage tissue, and Grzesiek and Dencher (15) described the artifacts that can occur when 9-aminoacridine is used to measure pH gradients. In this paper, the effects of temperature on ATP hydrolysis and on proton transport by the TN and PM ATPases of barley roots are described. During the course of the experiments it became clear that estimates of the effect of temperature on transport rates obtained with acridine orange or quinacrine could be quite different, and that the same dye might not be the best choice for the PM as for the TN. Some limitations on the use of quinacrine and acridine orange are described with respect to the two different membranes. MATERIALS AND METHODS