Plasma membrane H+-ATPase-dependent citrate exudation from cluster roots of phosphate-deficient white lupin

White lupin ( Lupinus albus L.) is able to grow on soils with sparingly available phosphate (P) by producing specialized structures called cluster roots. To mobilize sparingly soluble P forms in soils, cluster roots release substantial amounts of carboxylates and concomitantly acidify the rhizosphere. The relationship between acidification and carboxylate exudation is still largely unknown. In the present work, we studied the linkage between organic acids (malate and citrate) and proton exudations in cluster roots of P-deficient white lupin. After the illumination started, citrate exudation increased transiently and reached a maximum after 5 h. This effect was accompanied by a strong acidification of the external medium and alkalinization of the cytosol, as evidenced by in vivo nuclear magnetic resonance (NMR) analysis. Fusicoccin, an activator of the plasma membrane (PM) H⁺-ATPase, stimulated citrate exudation, whereas vanadate, an inhibitor of the H⁺-ATPase, reduced citrate exudation. The burst of citrate exudation was associated with an increase in expression of the LHA1 PM H⁺-ATPase gene, an increased amount of H⁺-ATPase protein, a shift in pH optimum of the enzyme and post-translational modification of an H⁺-ATPase protein involving binding of activating 14-3-3 protein. Taken together, our results indicate a close link in cluster roots of P-deficient white lupin between the burst of citrate exudation and PM H⁺-ATPase-catalysed proton efflux.     

Effect of Zinc Deficiency on Proton Fluxes in Plasma Membrane-Enriched Vesicles Isolated from Bean Roots

Plasma membrane-enriched vesicles were isolated by density gradientcentrifugation from roots of zinc-sufficient and zinc-deficientbean (Phaseolus vulgaris L. cv. Prélude) plants. Thetwo populations of vesicles had similar activities of specificmembrane marker enzymes and ATP hydrolysis and were competentfor proton transport. However, vesicles from zinc-deficientroots showed lower rates of ATP-dependent intravesicular acidificationand increases in passive permeability to protons as well asin the rate, of dissipation of a non-metabolic transmembranepH gradient. The decrease of the rate of proton accumulationin isolated vesicles closely paralleled the increase in potassiumleakage from intact roots and the appearance of visual zincdeficiency symptoms in the shoots. Re-supply of zinc to deficientplants for 24 h promoted shoot growth, reduced potassium leakagefrom roots and led to partial recovery of the proton accumulationcapacity and to a decrease in passive permeability to protonsin isolated vesicles. The results obtained with isolated vesiclesconfirm the previously observed ‘in vivo’ effectsof zinc deficiency and are consistent with the idea that analteration of plasma membrane lipids leads to an increase inpermeability and an impairment in trans-plasma membrane protongradient. Key words: Plasma membrane, H⁺ fluxes, ATPase, zinc deficiency