The role of vacuolar malate-transport capacity in crassulacean acid metabolism and nitrate nutrition. Higher malate-transport capacity in ice plant after crassulacean acid metabolism-induction and in tobacco under nitrate nutrition

Abstract

Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H(+)-transport by H(+)-pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, Deltamu(H+), across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, J(mal)(rel), of apparent ATP-dependent malate-stimulated H(+)-transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. J(mal)(rel) was high in the obligate crassulacean acid metabolism (CAM) species Kalanchoë daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO(3)(-) nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher J(mal)(rel). It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO(3)(-) nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins.

Figure 1

Plant vacuolar malate transporters as described in the literature (c, Buser-Suter et al., 1982; , Martinoia et al., 1985; , Nishida and Tominaga, 1987; , Marigo et al., 1988; , Lahjouji and Canut, 1999; , Marigo and Bouyssou, 1989; , Martinoia and Vogt, 1989; , White and Smith, 1989; , Bouyssou et al., 1990; , Martinoia et al., 1991; , Rentsch and Martinoia, 1991; , Dietz et al., 1992; , Iwasaki et al., 1992; , Pantoja et al., 1992; , Blom-Zandstra et al., 1993; , Ratajczak et al., 1994a; , Cerana et al., 1995; , Lahjouji et al., 1996; , Cheffings et al., 1997; , Terrier et al., 1998; and i, Lahjouji and Canut, 1999).

Figure 2

Effects of chloride and malate on proton transport activity (A), Bafilomycin A₁-sensitive ATP-hydrolysis (B), and relative H⁺-transport/ATP-hydrolysis coupling ratios (C), i.e. (A):(B), of tonoplast vesicles of K. daigremontiana in the presence of Cl⁻ and malate at two different concentrations (20 and 50 mm, respectively) applied in different sequences, i.e. were the second anion was added after equilibrium was reached in the Palmgren test with the first anion, but the first anion always remained present in the assay when the second anion was given. White columns, first anion; black columns, first and second anion present. Errors are sd, n = 4 (two measurements of two independent membrane preparations).

Figure 3

Malate levels in leaves of tobacco grown under various nitrogen regimes as indicated. Samples were taken at the start of the light period. Data are mean values ± sd of three independent measurements. Columns marked by different letters (a, b, and c) are statistically significantly different at the P = 0.05 value.

Figure 4

Relative H⁺ transport rates (A) in the presence of 50 mm Cl⁻ alone (first anion; white bars) and 50 mm malate added subsequently (second anion; black bars) in the Palmgren assay. B, Concomitantly measured Bafilomycin A₁-sensitive ATP-hydrolysis. C, Calculated relative H⁺ transport/ATP hydrolysis ratios for the C₃/CAM intermediate ice plant in the C₃ and CAM state. Data are mean values ± sd: C₃ state nine measurements of four independent preparations, CAM state 10 measurements of two independent preparations.

Figure 5

Relative H⁺-transport rates (A), ATP hydrolysis (B), and coupling ratios (C) as in Figure 4, but for tobacco grown under various nitrogen regimes as indicated. Data are mean values ± sd: 2 mm NO₃⁻ 14 measurements of two independent preparations, 10 mm NO₃⁻ 16 measurements of three independent preparations, 20 mm NO₃⁻ 20 measurements of two independent preparations, 3 mm NH₄⁺ 11 measurements of three independent preparations, and 6 mm NH₄⁺ 18 measurements of three independent preparations.

Figure 6

Relative PP_i-dependent H⁺ transport rates in the presence of either 50 mm chloride (white bars) or 50 mm malate (black bars) alone in the assay for the obligate CAM plant K. daigremontiana (K.d.) and the tobacco grown under various nitrogen regimes as indicated. Data are mean values ± sd of one to three measurements of membrane vesicle preparations from K. daigremontiana (three independent preparations), 2 mm NO₃⁻ (three independent preparations), 10 mm NO₃⁻ (three independent preparations), 20 mm NO₃⁻ (three independent preparations), 3 mm NH₄⁺ (two independent preparations), and 6 mm NH₄⁺ (three independent preparations).

Figure 7

Relative rates (J_mal^rel) of ATP-dependent malate transport of tonoplast vesicles of K. daigremontiana calculated according to Equation 1 when malate was added in the presence of chloride as indicated (same experiments as the respective combinations of chloride and malate in Fig. 2).

Figure 8

Relative rates of ATP-dependent malate transport of tonoplast vesicles of ice plant in the C₃ and CAM state, and tobacco under different regimes of nitrogen nutrition calculated according to Equation 1 when 50 mm malate was added in the presence of 50 mm chloride (same experiment as in Figs. 4 and 5).

Figure 9

Patterns of polypeptides in the 30- to 45-kD range of vacuolar vesicles obtained from ice plant in the C₃ and CAM state after silver staining of the SDS-PAGE electropherogram and western blots immunostained with the antiserum against the hydroxyapatite eluate of K. daigremontiana (anti-HA) and the affinity-purified antiserum against the 32-kD polypeptide of the hydroxyapatite eluate (anti-32 kD). Diamonds and dots indicate a 32-kD polypeptide that was not present in the C₃ sample and a 33-kD polypeptide that increased in staining intensity after CAM induction, respectively. Numbers on the left-hand margin indicate molecular masses of standard proteins.

Figure 10

Example of simultaneous measurements of proton transport and ATP-hydrolysis in the Palmgren assay (A). Control experiment inhibiting the V-ATPase by Bafilomycin A₁ at the start (B). Proton transport, ●; left ordinate, acridine-orange absorption quenching (A₄₉₅). ATP-hydrolysis: ○, right ordinate absorption at 340 nm, A₃₄₀. Tonoplast vesicles of K. daigremontiana (50 μg protein) with 50 mm KCl present at the start. Additions of MgSO₄, 50 mm K₂malate, and Bafilomycin A₁ as indicated by arrows on top of the graph.