Sodium influx and accumulation in Arabidopsis

Abstract

Arabidopsis is frequently used as a genetic model in plant salt tolerance studies, however, its physiological responses to salinity remain poorly characterized. This study presents a characterization of initial Na+ entry and the effects of Ca2+ on plant growth and net Na+ accumulation in saline conditions. Unidirectional Na+ influx was measured carefully using very short influx times in roots of 12-d-old seedlings. Influx showed three components with distinct sensitivities to Ca2+, diethylpyrocarbonate, and osmotic pretreatment. Pharmacological agents and known mutants were used to test the contribution of different transport pathways to Na+ uptake. Influx was stimulated by 4-aminobutyric acid and glutamic acid; was inhibited by flufenamate, quinine, and cGMP; and was insensitive to modulators of K+ and Ca2+ channels. Influx did not differ from wild type in akt1 and hkt1 insertional mutants. These data suggested that influx was mediated by several different types of nonselective cation channels. Na+ accumulation in plants grown in 50 mM NaCl was strongly reduced by increasing Ca2+ activity (from 0.05-3.0 mM), and plant survival was improved. However, plant biomass was not affected by shoot Na+ concentration, suggesting that in Arabidopsis Na+ toxicity is not dependent on shoot Na+ accumulation. These data suggest that Arabidopsis is a good model for investigation of Na+ transport, but may be of limited utility as a model for the study of Na+ toxicity.

Figure 1.

Increase in Na⁺ content (micromoles per gram) of excised roots of Arabidopsis after transfer into 200 mm NaCl plus 0.2 mm Ca²⁺ activity labeled with ²²Na⁺. a, Time course for up to 20 min; b, more detailed measures over the first 2 min, fitted with a line y = 3.31x + 0.16, r² = 0.96. Data represent means, and error bars the se (n = 4).

Figure 2.

Increasing unidirectional influx of Na⁺ (in micromoles per gram per minute) into excised roots of Arabidopsis with increasing concentrations of external Na⁺ and a constant Ca²⁺ activity of 0.2 mm, fitted with a line y = 0.023x + 0.15, r² = 0.97. Data represent means, and error bars the se (n = 4).

Figure 4.

The effect of 10-min or 3-h pretreatment in sorbitol or PEG (that are iso-osmotic with 50 mm NaCl) on influx of Na⁺ from 1 mm NaCl. All solutions contained 1 mm NaCl and 0.05 or 1.0 mm Ca²⁺ activity in addition to added osmotica. Influx was measured over 2 min into excised roots of Arabidopsis. Na⁺ influx was calculated as a percentage relative to control plants pretreated for 10 min in just 1 mm NaCl plus 0.05 or 1.0 mm Ca²⁺ activity. Data represent means, and error bars the se (n = 4). Numbers below the graph represent the percentage of total Na⁺ influx that was sensitive to a change in external Ca²⁺ activity from 0.05 to 1 mm, calculated according to the formula: [(Na⁺ influx [%] at 0.05 mm Ca²⁺) - (Na⁺ influx [%] at 1 mm Ca²⁺)]. Control fluxes were 74.9 ± 13.2 and 43.4 ± 5.5 nmol ^g-1 min^-1 at 0.05 and 1.0 Ca²⁺ activities, respectively.

Figure 3.

Inhibition of unidirectional influx of Na⁺ (in micromoles per gram per minute) into excised roots of Arabidopsis from 50 mm NaCl with increasing Ca²⁺ activity. Roots were either pretreated for 3 h in sorbitol before influx (white circles), or fluxes were performed after only 10 min of equilibration in non-radioactive influx solution (black circles). Data represent means, and error bars the se (n = 4 with sorbitol, n = 8 without sorbitol).

Figure 5.

The effect of a 3-h pretreatment in iso-osmotic sorbitol, polyethylene glycol (PEG), or 50 mm NaCl (Na pretr) or 12 d of growth in 50 mm NaCl (Na-grown), on unidirectional influx of Na⁺ into excised roots of Arabidopsis. Data represent means, and error bars the se (n = 4). Numbers below the graph represent the percentage of total influx that was sensitive to external Ca²⁺, calculated according to the formula: [(influx at 0.05 mm Ca²⁺ - influx at 1 mm Ca²⁺)/influx at 0.05 mm Ca²⁺] * 100.

Figure 6.

Effects of Ca²⁺ on growth and cation accumulation in 6-week-old Arabidopsis plants grown hydroponically in 50 mm NaCl after the 3rd week of planting. a, Shoot dry mass; b, shoot Na⁺ concentration; c, ratio of shoot K⁺ to Na⁺ concentration; d, ratio of shoot Na⁺ to Ca²⁺ concentration. Data represent means, and error bars the se (n = 24). Measurements were only made on surviving plants. To incorporate into the data the different mortalities with different treatments, shoot mass data (a) were multiplied by a “survival factor” of 0.58 for 0.05 mm Ca²⁺ activity treatment (14 of 24 plants survived the 50 mm NaCl treatment), 1 for 0.2 mm Ca²⁺ treatment (24 of 24 plants survived), and 0.875 for the 3.0 mm Ca²⁺ treatment (21 of 24 plants survived). Shoot mass includes the amount of Na⁺ in the tissue, but this accounted for 15.6%, 7.4%, and 3.0% of the total dry mass in the three Ca²⁺ activities used, having no significant effect on the overall trends. Ion concentrations in b to d were measured only in surviving plants and expressed on a dry weight basis for those plants, with no correction for survival.