Differences in proton pumping and Na/H exchange at the leaf cell tonoplast between a halophyte and a glycophyte

Abstract

The tonoplast Na(+)/H(+) antiporter and tonoplast H(+) pumps are essential components of salt tolerance in plants. The objective of this study was to investigate the transport activity of the tonoplast Na(+)/H(+) antiporter and the tonoplast V-H(+)-ATPase and V-H(+)-PPase in a highly tolerant salt-accumulating halophyte, Salicornia dolichostachya, and to compare these transport activities with activities in the related glycophyte Spinacia oleracea. Vacuolar membrane vesicles were isolated by density gradient centrifugation, and the proton transport and hydrolytic activity of both H(+) pumps were studied. Furthermore, the Na(+)/H(+)-exchange capacity of the vesicles was investigated by 9-amino-6-chloro-2-methoxyacridine fluorescence. Salt treatment induced V-H(+)-ATPase and V-H(+)-PPase activity in vesicles derived from S. oleracea, whereas V-H(+)-ATPase and V-H(+)-PPase activity in S. dolichostachya was not affected by salt treatment. Na(+)/H(+)-exchange capacity followed the same pattern, i.e. induced in response to salt treatment (0 and 200 mM NaCl) in S. oleracea and not influenced by salt treatment (10 and 200 mM NaCl) in S. dolichostachya. Our results suggest that S. dolichostachya already generates a high tonoplast H(+) gradient at low external salinities, which is likely to contribute to the high cellular salt accumulation of this species at low external salinities. At high external salinities, S. dolichostachya showed improved growth compared with S. oleracea, but V-H(+)-ATPase, V-H(+)-PPase and Na(+)/H(+)-exchange activities were comparable between the species, which might imply that S. dolichostachya more efficiently retains Na(+) in the vacuole.

Keywords: Halophyte; NHX1; Salicornia; V-H+-ATPase; V-H+-PPase.; membrane transport; salt tolerance; vacuole.

Figure 1.

Ash-free dry mass of Spinacia oleracea and Salicornia dolichostachya after 8 days of growth. Plants were exposed for 8 days to 0 and 200 mM NaCl for S. oleracea, and 10 and 200 mM NaCl for S. dolichostachya. Values shown are means ± SEM with six replicate plants per treatment. Note that the ordinates of the two species have different scales. The treatment effect was statistically significant for both species (Student's t-test, *P < 0.05).

Figure 2.

Na⁺ and K⁺ concentrations in the shoot of Spinacia oleracea and Salicornia dolichostachya after 8 days of growth. Plants were exposed for 8 days to 0 and 200 mM NaCl for S. oleracea, and 10 and 200 mM NaCl for S. dolichostachya. Values shown are means ± SEM with six replicate plants per treatment. The treatment effect was statistically significant for shoot Na⁺ and shoot K⁺ concentrations for both species (Student's t-test, *P < 0.05).

Figure 3.

Shoot pH of Spinacia oleracea and Salicornia dolichostachya after 8 days of growth. Plants were exposed for 8 days to 0 and 200 mM NaCl for S. oleracea, and 10 and 200 mM NaCl for S. dolichostachya. Values shown are means ± SEM with six replicate plants per treatment. Different letters indicate a statistically significant treatment effect on leaf pH (one-way ANOVA, P < 0.05). Interaction effect: two-way ANOVA, species×treatment P < 0.05.

Figure 4.

Characteristics of the H⁺-ATPase- and H⁺-PPase-dependent ΔpH gradient. H⁺ translocation was determined by measuring the level of fluorescence quenching of ACMA. Formation of the ΔpH gradient was initiated by adding 1 mM ATP or 0.15 mM PP_i. When ATP-dependent fluorescence quenching reached a stable value, 50 mM KNO₃ or 0.1 mM Na₃VO₄ was added. Five micrograms of protein was used per assay. Recordings of vesicles derived from shoots of 200 mM grown Spinacia oleracea are shown.

Figure 5.

(A) Na⁺/H⁺-exchange capacity of membrane vesicles derived from shoots of the halophyte Salicornia dolichostachya grown at 200 mM NaCl. The Na⁺/H⁺-exchange capacity of the vesicles was measured as the level of recovery of ACMA-fluorescence quenching of a pre-established ΔpH gradient created by V-H⁺-ATPase after 1 mM ATP addition. In total, 2.5 μg of protein was used per assay. (B) Na⁺/H⁺-exchange capacity of membrane vesicles derived from shoots of the halophyte Salicornia dolichostachya and the glycophyte Spinacia oleracea. The Na⁺/H⁺-exchange capacity of the vesicles was measured as the level of recovery of ACMA-fluorescence quenching by addition of 250 mM NaCl of a pre-established ΔpH gradient created by V-H⁺-ATPase. Plants were grown under control conditions (10 mM NaCl for S. dolichostachya and 0 mM NaCl for S. oleracea) or saline conditions (200 mM NaCl for both species) for 8 days. Data points are means ± SEM of three independent membrane isolations per treatment. Different letters indicate a statistically significant treatment effect on Na⁺/H⁺-exchange capacity (one-way ANOVA, P < 0.05). Interaction effect: two-way ANOVA, species×treatment P < 0.05.

Figure 6.

H⁺-translocation activity of the tonoplast V-H⁺-ATPase of vesicles derived from shoots of the halophyte Salicornia dolichostachya (circles) and the glycophyte Spinacia oleracea (squares). The H⁺-translocation activity of V-H⁺-ATPase was determined by measuring the level of fluorescence quenching of ACMA. Plants were grown under control conditions (10 mM NaCl for S. dolichostachya and 0 mM NaCl for S. oleracea, open symbols) or saline conditions (200 mM NaCl for both species, closed symbols) for 8 days. Data points are means ± SEM of three independent membrane isolations per treatment. Kinetic analysis was performed by fitting a Michaelis–Menten curve through the data points. Kinetic parameters with standard errors were calculated with nls model fitting in R.

Figure 7.

Hydrolytic activity of the tonoplast V-H⁺-ATPase of vesicles derived from shoots of the halophyte Salicornia dolichostachya and the glycophyte Spinacia oleracea. The hydrolytic activity of V-H⁺-ATPase was determined by measuring the amount of inorganic phosphate (P_i) released. Plants were grown under control conditions (10 mM NaCl for S. dolichostachya and 0 mM NaCl for S. oleracea) or saline conditions (200 mM NaCl for both species) for 8 days. Data points are means ± SEM of four independent membrane isolations per treatment. Different letters indicate a statistically significant treatment effect on hydrolytic activity (one-way ANOVA, P < 0.05). Interaction effect: two-way ANOVA, species×treatment P < 0.05.

Figure 8.

H⁺-translocation activity of the tonoplast V-H⁺-PPase of vesicles derived from shoots of the halophyte Salicornia dolichostachya (circles) and the glycophyte Spinacia oleracea (squares). The H⁺-translocation activity of V-H⁺-PPase was determined by measuring the level of fluorescence quenching of ACMA. Plants were grown under control conditions (10 mM NaCl for S. dolichostachya and 0 mM NaCl for S. oleracea, open symbols) or saline conditions (200 mM NaCl for both species, closed symbols) for 8 days. Data points are means ± SEM of three independent membrane isolations per treatment. Kinetic analysis was performed by fitting a Michaelis–Menten curve through the data points. Kinetic parameters with standard errors were calculated with nls model fitting in R.