AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta

Abstract

Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na(+) homeostasis in planta and by this function modulates K(+) nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na(+) content in the shoot and lower content of the ion in the root, reducing the Na(+) imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K(+) to Na(+) ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K(+) to Na(+) ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na(+) homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K(+)-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K(+) deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na(+) homeostasis in planta and through this function regulates K(+) nutrient status.

Figure 1.

hkt1-1 is a genetic suppressor of sos mutations. In vitro grown seedlings (4 d on germination medium) were transferred to a basal medium (MS salts without CaCl₂, 30 g L⁻¹ Suc, and 12 g L⁻¹ Bacto Agar, DIFCO; Becton-Dickinson, Le Pont de Claix, France) supplemented with various amounts of NaCl, and either 3 mm (A, B, C, and D) or without CaCl₂ (A′, B′, C′, and D′). Root length increase was determined 5 d after transfer; values are the mean ± se (n ≥ 8).

Figure 2.

hkt1-1 suppresses NaCl hypersensitivity of soil-grown sos plants. Three-week-old plants in soil were irrigated by placing the pots in a container filled with deionized water without or with 50 mm NaCl until the soil was saturated by capillarity. Plants were rewatered weekly, and pictures were taken 3 weeks later.

Figure 3.

hkt1-4 suppresses NaCl sensitivity of sos3-1 plants but causes NaCl sensitivity of wild-type (gl1) plants. Stratified seeds in soil were transferred to a growth chamber for germination and plant growth. Plants were irrigated by placing the pots in a container of EXCEL solution until the soil was saturated. EXCEL includes 167 mg L⁻¹ K⁺, 67 mg L⁻¹ Ca²⁺, and no detectable Na⁺, according to the manufacturer's specifications. One-month-old plants were transplanted into Turface. After 1 week of acclimation, plants were watered every 3 or 4 d with EXCEL solution (0 mm NaCl) or supplemented with NaCl (50 mm NaCl for four treatments followed by 100 mm NaCl for 7 treatments). Photographs were taken 5 weeks after beginning of treatment.

Figure 4.

hkt1-4 restores Na⁺ content of sos3-1 plants to a level similar to that of wild-type plants (gl1). Plants were grown as described in Figure 3. Plants were harvested 5 weeks after the start of NaCl treatment. Shoots and roots (n ≥ 7 for each genotype per treatment) were separated, and materials were rinsed with deionized water and pooled into independent groups (minimum of approximately 50 mg after drying) for ion content analyses. A, Na⁺ content in the shoot; values are the mean ± se (n = 3). The fresh weight of the plants not treated with NaCl is gl1, 2.95 ± 0.2 g; sos3-1, 2.37 ± 0.25 g; sos3-1 hkt1-4, 2.32 ± 0.21 g; and hkt1-4, 2.63 ± 0.24 g, and of plants treated with NaCl (100 mm final concentration) is gl1, 2.34 ± 0.16 g; sos3-1, 0.44 ± 0.03 g; sos3-1 hkt1-4, 1.23 ± 0.09 g; and hkt1-4, 1.27 ± 0.04 g. B, Na⁺ content in the root; values are the mean ± se (n ≥ 2), except for sos3-1 treated with 100 mm NaCl. These data are representative of those obtained in replicate experiments.

Figure 5.

AtHKT1:AtHKT1 transgene expression increases NaCl hypersensitivity of sos3-1 seedlings. Four-day-old in vitro-grown seedlings were transferred to a basal medium (MS salts, 30 g L⁻¹ Suc, and 12 g L⁻¹ Bacto Agar) without or supplemented with NaCl; sos3-1, vector control (sos3-1-pCAM, two independent lines) and AtHKT1:AtHKT1 transformed lines (sos3-1-HKT1:HKT1, five independent lines). Root growth was determined 6 d after transfer; values are the mean ± se (n ≥ 11).

Figure 6.

AtHKT1:AtHKT1 transgene expression confers NaCl sensitivity to wild-type seedlings. Four-day-old seedlings were transferred to a basal medium (see Fig. 5 legend) without or supplemented with NaCl; wild-type (gl1), vector control (gl1-pCAM, three independent lines), and AtHKT1:AtHKT1 transformed lines (gl1-HKT1:HKT1, six independent lines). Root growth was determined 6 d after transfer; values are the mean ± se (n ≥ 10).

Figure 7.

AtHKT1:AtHKT1 transgene expression mediates Na⁺-specific salt sensitivity of wild-type and sos3-1 seedlings. Four-day-old seedlings were transferred to a basal medium (see Fig. 5 legend; white bars) or basal medium supplemented with 75 (hatched bars) or 125 (black bars) mm NaCl (A), 75 (hatched bars) or 125 (black bars) mm KCl (B), and 10 (hatched bars) or 15 (black bars) mm LiCl (C). Root growth of wild-type (gl1), wild-type vector control (gl1-pCAM, line no. 3-1 in Fig. 6), wild-type expressing AtHKT1 transgene (gl1-HKT1:HKT1, line no. 10-10 in Fig. 6), sos3-1, sos3-1 vector control (sos3-1-pCAM, line no. 13-5 in Fig. 5), and sos3-1 expressing AtHKT1 transgene (sos3-1-HKT1:HKT1, line no. 13-4 in Fig. 5) seedlings is illustrated. Root growth was determined 6 d after transfer; values are the mean ± se (n ≥ 10).

Figure 8.

AtHKT1 loss- or gain-of-function affects K⁺/Na⁺ homeostasis in planta. Na⁺ and K⁺ content in the shoot (A) and root (B) of wild-type (gl1), hkt1-4, or gl1 expressing the AtHKT1 transgene (gl1-HKT1:HKT1 line nos. 10-10 and 17-3 in Fig. 6) plants growing in Turface. Plants were grown as in Figure 3. Shoot content values are the mean ± se (n = 3), root content values are the mean ± se (n ≥ 2), and K⁺/Na⁺ was calculated from K⁺ and Na⁺ content.

Figure 9.

hkt1-1 suppresses the K⁺ deficiency of sos seedlings. Four-day-old seedlings were transferred to a basal medium containing 1/20× MS macronutrients [without KNO₃, and KH₂PO₄ replaced with (NH₄)₂HPO₄], MS micronutrients (KI replaced with NaI), 30 g L⁻¹ Suc, and 12 g L⁻¹ Bacto Agar, without or supplemented with various amounts of KCl. The basal medium contains 0.15 mm CaCl₂. Illustrated is hk1-1 suppression of sos1-1 (A), sos2-2 (B), and sos3-1 (C). Root growth was determined 9 d after transfer; values are the mean ± se (n ≥ 8).

Figure 10.

AtHKT1:AtHKT1 transgene expression increases the K⁺ deficiency of sos3-1 seedlings independent of Four-day-old seedlings were transferred to a basal medium (1/20× MS macronutrients [without KNO₃, without CaCl₂, KH₂PO₄ replaced with (NH₄)₂HPO₄, with 20 μm KCl], MS micronutrients [KI replaced with NaI], 30 g L⁻¹ Suc, and 12 g L⁻¹ Bacto Agar) and 0.15 (A) or 3 mm CaCl₂ (B); genotypes are as in Figure 5. Root growth was determined 6 d after transfer; values are the mean ± se (n ≥ 10).

Figure 11.

AtHKT1:AtHKT1 transgene expression-mediated K⁺ deficiency of wild-type (gl1) seedlings requires Four-day-old seedlings were transferred to a basal medium (1/20× MS macronutrients [without KNO₃, with 3 mm CaCl₂, KH₂PO₄ replaced with (NH₄)₂HPO₄], MS micronutrients [KI replaced with NaI], 30 g L⁻¹ Suc, and 12 g L⁻¹ Bacto Agar) without or supplemented with various amounts of KCl and without NaCl supplement (A) or with 20 mm NaCl (B); plants of wild-type (gl1), vector control (gl1-pCAM, line no. 11-3 in Fig. 6), AtHKT1 transgene expressing line (gl1-HKT1:HKT1, line no. 17-3 in Fig. 6), and sos3-1. Root growth was determined 6 d after transfer; values are the mean ± se (n ≥ 19).