Effect of Sugars and Amino Acids on Membrane Potential in Two Clones of Sugarcane

Abstract

Sugarcane (Saccharnm officinarum L.) leaf parenchyma cells bathed in IX solution maintained an average membrane potential of-135 millivolts in the dark. No difference in membrane potential was found between clones 51 NG 97 and H50 7209. An electrogenic pump appears to contribute to membrane potential in these cells. Sugars (25 millimolar) added externally caused the folowing membrane potential depolarizations (in milivolts) in clone 51 NG 97: glucose, 18 + 4; galactose, 24 ± 7; 3-O-methylglucose, 10 ± 4; sucrose, 22 ± 3; fructose, 21 ± 7; raffinose, 9 ± 3; mannitol, 0, lactose, 0; melibiose, 0; and 1-O-methyl-a-galactose, 0. Glycine (25 millimolar) and serine (10 mHlmolar) caused depolarizations of 47 ± 7 and 23 ± 2 millivolts, respectively. Depolarization shows saturation kinetics with respect to glucose concentration, with a Km of 3 to 6 millimolar. The metabolic inhibitors KCN and salicyl hydroxamic acid together caused depolarization of the membrane potential and greatly inhibited depolari-zation by 25 millimolar glucose and 25 miHimolar raffinose. In a series of substitution experiments, glucose (25 millimolar) caused almost total inhibition of depolarization by raffinose, sucrose, and 3-0-methylglucose (all 25 millimolar), but only partial inhibition of depolarization to 25 millimolar glycine. Glycine (25 millimolar), also, only partially inhibited depolarization by 25 millmlar glucose. Total depolarization to 25 millimolar glycine and 25 miLlimolar glucose was comparable to the amount of depolarization of membrane potential caused by 1 millimolar KCN plus 1 millimolar salicyl hydroxamic acid. The results are consistent with a co-transport mechanism of membrane transport, with sugars and amino acids being transported by separate carrier systems. There is now overwhelming evidence for an electrogenic pump in bacteria, fungi, algae, bryophytes, and higher plants (10, 19). In all of these groups, the most important and universal electrogenic system appears to be a H+-efflux pump operating at the plasma membrane (19, 20, 23). The proton-efflux pump serves two important functions for the cell. The most obvious of these is the regulation of cytoplasmic pH since excess H+ ions are produced as byproducts ofcellular metabolism (20). The other main function of the pump is the coupling of metabolic energy to the active transport of solutes, which is predicted by Mitchell's chemiosmotic hypothesis (13). This hypothesis has led to the development of a cotransport theory for membrane transport in plant cells. Slayman (23) has proposed a model for proton dependent cotransport of an uncharged substrate. In this model, protons and substrate at the outer membrane surface combine sequentially with a membrane carrier, forming a positively charged complex which is ' The existence of H+-cotransport systems has been well documented for bacteria, fungi, and algae (19). Evidence has also accumulated for both sugar and amino acid cotransport in a number of higher plants. These plants include oats (7, 21), peas (5), maize (5), and Lemna (18). The data which support a cotrans-port mechanism are transient alkalinization of the bathing medium in the presence of sugars (5), depolarization of membrane potential during labeled transport of sugars (18) and amino acids (7, 21), increased transport in the presence of fusicoccin (5, 21) (which stimulates proton extrusion), and inhibition of transport by diethylstilbestrol (5), an inhibitor of the proton pump. Our objective was to examine the effects of sugars and amino acids on PD2 in sugarcane leaves in light of a possible cotransport mechanism for membrane transport of these substances. MATERIALS AND METHODS Plant Material. Sugarcane (Saccharum officinarum L.) clones 51 NG 97 and H50 7209 were provided by G. A. Strobel. These clones were chosen because they reportedly (24) differed in their ability to transport several sugars. Plants were maintained in the greenhouse in 25 cm plastic pots which contained a 2:1:1 mixture of loam, peat, and sand. Each plant was fertilized weekly with 2 to 3 g formulation 16-32-16 fertilizer (Start N Gro, Agway Inc., Syracuse, NY) in about 300 ml water. Tissue sections were cut from the third youngest leaf of a stalk using a new razor blade. Rectangular sections were cut from the area halfway between the basal end stem and the tip of the leaf and contained only the green tissue on either side of the midrib. Each section was 5 x 15 mm, with the vascular bundles parallel to the long axis. After cutting, tissue sections were aged by floating in 1X solution (see below) in the dark for at least 16 h but not more than 24 h. Perfusion Solutions. The bathing solution (6) was designated 1X and contained 1.0 mm KC1, 1.0 mm Ca(NO3)2, 0.25 mM MgSO4, 0.90 mm NaH2PO4, and 50 ylM Na2HPO4. The pH was 5.5 to 5.7. The following sugars, amino acids, and inhibitors were dissolved in IX solution: glucose, raffmose, galactose, melibiose, lactose, sucrose, fructose, mannitol, 3-O-methylglucose, 1-0-methyl-a-ga-lactose, glycine, alanine, serine, NEM, CCCP, and DNP. Other solutions were modified from IX to keep the final concentrations of H', K+, and Na+ the same as 1X. For 1 mm sodium azide solution, NaH2PO4 and Na2HPO4 were omitted, and Tris buffer was used to adjust the pH. When 1 mm KCN plus I mm SHAM were used, KCI was omitted. In all cases, the pH of the final 2 Abbreviations: PD, membrane potential; NEM, N-ethylmaleimide; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DNP, 2,4-dinitrophe-nol; SHAM, salicyl hydroxamic acid. 150 www.plant.org on March 8, 2015-Published by www.plantphysiol.org Downloaded from