Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance

Abstract

Salinity is considered one of the major limiting factors for plant growth and agricultural productivity. We are using salt cress (Thellungiella halophila) to identify biochemical mechanisms that enable plants to grow in saline conditions. Under salt stress, the major site of Na+ accumulation occurred in old leaves, followed by young leaves and taproots, with the least accumulation occurring in lateral roots. Salt treatment increased both the H+ transport and hydrolytic activity of salt cress tonoplast (TP) and plasma membrane (PM) H(+)-ATPases from leaves and roots. TP Na(+)/H+ exchange was greatly stimulated by growth of the plants in NaCl, both in leaves and roots. Expression of the PM H(+)-ATPase isoform AHA3, the Na+ transporter HKT1, and the Na(+)/H+ exchanger SOS1 were examined in PMs isolated from control and salt-treated salt cress roots and leaves. An increased expression of SOS1, but no changes in levels of AHA3 and HKT1, was observed. NHX1 was only detected in PM fractions of roots, and a salt-induced increase in protein expression was observed. Analysis of the levels of expression of vacuolar H(+)-translocating ATPase subunits showed no major changes in protein expression of subunits VHA-A or VHA-B with salt treatment; however, VHA-E showed an increased expression in leaf tissue, but not in roots, when the plants were treated with NaCl. Salt cress plants were able to distribute and store Na+ by a very strict control of ion movement across both the TP and PM.

Figure 1.

Effect of NaCl on growth of salt cress plants. Four-week-old plants were treated with NaCl (0–400 mm) for 1 (A) or 2 (B) weeks.

Figure 2.

Changes in cell sap Na⁺ concentrations in salt cress following 2 weeks of treatment with NaCl. Measurements of Na⁺ in cell sap extracted from leaves or roots as described in “Materials and Methods.” Values are means ± se of three independent experiments.

Figure 3.

Salt cress whole plant structure. A, Eight-week-old salt cress plant grown in hydroponics for 5 weeks. B, Close-up of the taproot.

Figure 4.

Purity of membrane fractions separated by Suc density gradients from leaf tissue of salt cress plants. Western-blot analysis was used to confirm the purity of the TP and PM fractions by immunodetection using antibodies directed against a PM P-ATPase (AHA3; 100 kD), and the subunit E of the TP V-ATPase (VHA-E; 31 kD). Immunodetection was carried out as described in “Materials and Methods.” Individual blots (eight lanes/blot) were digitally photographed (Kodak DC-120; Eastman-Kodak) and then images were aligned and joined using the imaging software Photo Impact SE 3.01 (Ulead Systems) in order to enable visualization of all representative fractions. Top, Suc concentrations in collected fractions. Middle, Immunological detection in the respective fractions of AHA3 and VHA-E. Bottom, Mean intensity of the protein bands detected, V-ATPase (▪) and P-ATPase (▴) in microsomal fractions. Blots are representative of at least four independent experiments.

Figure 5.

Expression of P-ATPase, V-ATPase, and V-PPase in salt cress under salt treatment. Plants were incubated for 2 weeks in the absence (C) or presence of 200 mm (2) or 400 mm (4) NaCl before isolation of TP and PM from leaves (A) or roots (B) on discontinuous Suc density gradients at the 0% to 22% (w/v) and the 34% to 38% (w/v) Suc interface, respectively. Western-blot analysis was carried out as described in “Materials and Methods” using antibodies directed against P-ATPase (AHA3), V-ATPase subunits (VHA-A, -B, and -E), and V-PPase (AVP1). Blots are representative of five independent experiments.

Figure 6.

Expression of HKT1, SOS1, and NHX1 in salt cress under salt treatment. Plants were incubated for 2 weeks in the absence (C) or presence of 200 (2) or 400 mm NaCl (4) before isolation of TP and PM fractions from leaves (A) and roots (B) on a discontinuous Suc density gradient at the 0% to 22% and the 34% to 38% Suc interface, respectively. Western-blot analysis was carried out as described in “Materials and Methods” using antibodies directed against the Na⁺ transporter HKT1, the PM Na⁺/H⁺ exchanger SOS1, and the TP Na⁺/H⁺ exchanger NHX1. Blots are representative of five independent experiments.