Proton Fluxes and the Activity of a Stelar Proton Pump in Onion Roots

Abstract

The xylem vessels of excised adventitious roots of onion, Allium cepa, were perfused with unbuffered nutrient solution adjusted initially to either pH 9·3 or 3·9; the pH of the solution after passage through the xylem, at rates not less than 2 xylem volume changes min⁻¹, was close to pH 6·5 in both instances. The flux of H⁺ across the xylem/symplast boundary into mildly alkaline, phosphate-buffered solutions perfusing the vessels could be increased greatly with increasing buffer strength, up to a maximum value between 0·5–1·0 pmol H⁺ mm⁻² s⁻¹. The apparent neutralization of acidic malic acid buffers had a slightly lower maximum capacity, equivalent to −0·3 to −0·5 pmol H⁺ mm⁻² s⁻¹. The addition of 5·0 pmol m⁻³ fusicoccin (FC) to the xylem perfusion solution stimulated the entry of H⁺ into the xylem; in unbuffered perfusion solutions the pH fell to pH 3·6 after a lag of 25–35 min. FC additions to phosphate-buffered solutions also stimulated the H⁺ flux to an extent similar to that in unbuffered solution, viz. 0·2–0·4 pmol mm⁻² s⁻¹. The release of K⁺ (³⁶Rb-labelled) into xylem sap transiently increased as the [K⁺] in weakly buffered perfusion solutions was raised stepwise; a very marked increase being seen when the concentration was raised to 100 mol m⁻³ from 40 mol m⁻³. The addition of 5·0 mmol m⁻³ FC to the perfusing solution containing 100 mol m⁻³ K⁺ rapidly decreased the K⁺ flux to the xylem as the H⁺ flux increased. Fusicoccin also inhibited the flux of K⁺ into unbuffered perfusion solutions but the effect appeared reversible. Addition of 10 mmol m⁻³ abscisic acid (ABA) to the perfusion solution quickly produced transient increases in both K⁺ and H⁺ fluxes into the xylem. In this and other experiments using weakly phosphate-buffered perfusing solutions, H⁺ fluxes were comparable in size to those of K⁺ The results are consistent with the idea that the stele of onion roots contains a proton trarislocating ATPase whose activity responds to the pH of the xylem sap. It is evident that the activity of the proton secreting and proton neutralizing mechanisms in the xylem parenchyma control the movement of other ions across the xylem/symplast boundary.