Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency

Abstract

White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.

Figure 1

Hypothetical mechanism for the release of organic acids by proteoid root cells of white lupin.

Figure 2

Proteoid roots produced by white lupin with (A) and without (B) phosphate. Plants were grown in a solution culture for 3 weeks. Plasma membrane was isolated from different types of roots: proteoid roots of P-sufficient plants, marked as proteoid (+P); lateral roots of P-sufficient plants, marked as lateral (+P); active proteoid roots (the youngest, fully developed proteoid root) of P-deficient plants, marked as proteoid (−P); lateral roots of P-deficient plants, marked as lateral (−P).

Figure 3

H⁺ release by white lupin roots during a 3-week cultivation period. Plants were grown in nutrient solution, pH of which was kept constant at pH 6 by means of a pH stat system (Schott, Mainz, Germany). The amount of NaOH (or H₂SO₄) used for maintaining pH 6 was recorded daily and used for the calculation of H⁺ release. Values represent means ± se of four independent experiments.

Figure 4

Identification of active proteoid roots (A) and inhibitory effect of vanadate on rhizosphere acidification (B). Plants were grown in nutrient solution without phosphate for 3 weeks. After washing with deionized water, roots were carefully spread on the surface of agar sheet (0.75% [w/v] agar, 0.006% [w/v] bromocresol purple, 1 mm CaSO₄, and 2.5 mm K₂SO₄, pH 6) and gently pressed into the agar sheet and incubated for 5 h under light in a growth chamber. For study of the inhibitory effect of vanadate (B), 1 mm vanadate was included on the right side of the agar sheet.

Figure 5

Comparison of ATPase activity of plasma membranes derived from different types of white lupin roots. Plants were grown in nutrient solution at pH 6 for 3 weeks. Plasma membrane was isolated from proteoid roots of P-sufficient plants [proteoid (+P)], lateral roots of P-sufficient plants [lateral (+P)], active proteoid roots (the youngest, fully developed proteoid root) of P-deficient plants [proteoid (−P)], and lateral roots of P-deficient plants [lateral (−P)]. Plasma membrane ATPase activity was analyzed in the presence of 1 mm molybdate, 1 mm azide, and 50 mm nitrate at 30°C. Values represent means ± se of four independent experiments.

Figure 6

Comparison of the kinetic characteristics of plasma membrane ATPase from different types of white lupin roots (see legend of Fig. 5). A, Dependence of ATPase activity on ATP concentration. Plants were grown in nutrient solution at pH 6 for 3 weeks. Plasma membrane ATPase activity was analyzed in the presence of 1 mm molybdate, 1 mm azide, and 50 mm nitrate at 30°C. The concentration of ATP was kept constant in the range of 50 to 4,000 μm. Values represent means ± se of four independent experiments. B, Eadie-Hofstee plot of the data presented in A (r > 0.98).

Figure 7

Separation of plasma membrane proteins by SDS-PAGE (above) and immunodetection of plasma membrane H⁺ ATPase by western-blotting technique (below). M, Standard markers for molecular mass (Sigma, St. Louis); 1, plasma membrane from lateral roots of P-sufficient plants; 2, plasma membrane from active proteoid roots of P-deficient plants; 3, plasma membrane from lateral roots of P-deficient plants; 4, plasma membrane from proteoid roots of P-sufficient plants. Arrows indicate plasma membrane H⁺ ATPase. For separation of plasma membrane proteins, membrane vesicles (4-μg membrane proteins) were loaded onto polyacrylamide gel. After separation, the obtained gel was stained with Coomassie Brilliant Blue (above). For western-blot analysis (below), after separation on the gel the membrane proteins including molecular mass markers were transferred to polyvinylidene difluoride (PVDF) membrane filter (0.2 μm). For staining of the obtained blot, the lane of molecular mass markers was separated from other lanes of membrane proteins. The former was stained with Coomassie Brilliant Blue. The remaining blot with the lanes of plasma membrane proteins was incubated with a polyclonal antibody raised against the central portion of AHA2 (amino acids 340–650) and visualized with a secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG, Sigma). After separate staining, the blot of standard marker for molecular mass and the blot of plasma membrane proteins were combined.

Figure 8

Comparison of active H⁺ transport driven by plasma membrane H⁺ ATPase (A) and passive H⁺ transport by leakage (B) across the plasma membranes. Membrane vesicles were isolated from different types of roots of white lupin (see legend of Fig. 5). For the comparison of active H⁺ transport (A), the pH gradient formation across vesicle membranes was monitored by ΔA₄₉₂ of AO. At assay pH 6.5, intravesicular acidification was initiated by addition of 5 mm Mg-ATP. The established pH gradient was completely collapsed by 5 μm gramicidin (Gram.). For the comparison of passive H⁺ transport (B), the intravesicular acidification was initiated by addition of 5 mm Mg-ATP to create a pH gradient across plasma membrane vesicles. For a reliable comparison, ATPase activity was stopped by addition of 500 μm vanadate after quenching had reached 0.0300 A units for all four membranes. The resting pH gradient was collapsed by gramicidin (Gram., 5 μm).