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

The xylem vessels of excised adventitious roots of onion, Alliumcepa, were perfused with unbuffered nutrient solution adjustedinitially to either pH 9·3 or 3·9; the pH of thesolution after passage through the xylem, at rates not lessthan 2 xylem volume changes min^–1, was close to pH 6·5in both instances. The flux of H⁺ across the xylem/symplastboundary into mildly alkaline, phosphate-buffered solutionsperfusing the vessels could be increased greatly with increasingbuffer strength, up to a maximum value between 0·5–1·0pmol H⁺ mm^–2 s^–1. The apparent neutralization ofacidic 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^–3fusicoccin (FC) to the xylem perfusion solution stimulated theentry of H⁺ into the xylem; in unbuffered perfusion solutionsthe pH fell to pH 3·6 after a lag of 25–35 min.FC additions to phosphate-buffered solutions also stimulatedthe 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 transientlyincreased as the [K⁺] in weakly buffered perfusion solutionswas raised stepwise; a very marked increase being seen whenthe concentration was raised to 100 mol m^–3 from 40 molm^–3. The addition of 5·0 mmol m^–3 FC to theperfusing solution containing 100 mol m^–3 K⁺ rapidly decreasedthe K⁺ flux to the xylem as the H⁺ flux increased. Fusicoccinalso inhibited the flux of K⁺ into unbuffered perfusion solutionsbut the effect appeared reversible. Addition of 10 mmol m^–3abscisic acid (ABA) to the perfusion solution quickly producedtransient increases in both K⁺ and H⁺ fluxes into the xylem.In this and other experiments using weakly phosphate-bufferedperfusing solutions, H⁺ fluxes were comparable in size to thoseof K⁺ The results are consistent with the idea that the stele of onionroots contains a proton trarislocating ATPase whose activityresponds to the pH of the xylem sap. It is evident that theactivity of the proton secreting and proton neutralizing mechanismsin the xylem parenchyma control the movement of other ions acrossthe xylem/symplast boundary. Key words: Xylem perfusion, fusicoccin, abscisic acid, pH gradient