Conservation of the salt overly sensitive pathway in rice

Abstract

The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots.

Figure 1.

Activation of rice SOS1 by the Arabidopsis SOS2/SOS3 kinase complex. A, AXT3K cells transformed with an empty vector (0) or with the indicated combination of SOS genes (1, OsSOS1; 2, AtSOS2; 3, AtSOS3) were grown overnight in selective synthetic dextrose medium. Five microliters of serial decimal dilutions were spotted onto plates of AP medium with 1 mm KCl and supplemented with 0 (data not shown), 100, or 200 mm NaCl. Plates were incubated at 28°C for 3 d. Plasmids used for expression of the SOS proteins were pDR195 for OsSOS1, pFL2T for AtSOS2, pFL3T for AtSOS3, and pFL32T for coexpression of AtSOS2 and AtSOS3. B, Intracellular Na⁺ content as determined by atomic emission spectrometry. Cells were grown in AP medium with 1 mm KCl and 30 mm NaCl and collected at OD₅₅₀ = 0.2 to 0.3. Values are the mean and se of three independent cultures of each combination of SOS genes. Units are nanomols of ions per milligram dry weight of cell samples. [See online article for color version of this figure.]

Figure 2.

Na⁺/H⁺ antiporter activity of rice SOS1. A, ATP-dependent pH gradient formation in membrane vesicles isolated from yeast cells expressing rice SOS1. An inside-acid ΔpH was formed after the addition of ATP to vesicles (arrow 1). Once fluorescence was stabilized, sodium salts were added to the cuvette (arrow 2) and fluorescence recovery, indicating proton exchange, was monitored for 2 min, after which ΔpH was disrupted by the addition of 25 mm (NH₄)₂SO₄ (arrow 3). Fluorescence is expressed as arbitrary units. B, Na⁺/H⁺ exchange as a function of Na₂SO₄ concentration and the presence of rice OsSOS1, with and without coexpression of the Arabidopsis SOS2/SOS3 kinase complex. Circles, AXT3K cells transformed with empty vector pDR195; squares, AXT3K cells expressing rice OsSOS1 alone; diamonds, AXT3K cells transformed to produce proteins OsSOS1, AtSOS2, and AtSOS3. Na⁺/H⁺ exchange activity is given as the proportion of dissipation of the preformed pH gradient per minute and milligrams of membrane protein. C, Specificity of sodium-induced proton exchange. Sodium was added to a final concentration of 75 mm as sulfate (gray bars) or gluconate (black bars) salt. Values are the mean and se of percent fluorescence dissipation of triplicate samples.

Figure 3.

Phosphorylation of rice SOS1 by the Arabidopsis kinase SOS2. A, Recombinant His-tagged OsSOS1, AtSOS2, and GST-fused AtSOS2T/DΔ308 were purified by affinity chromatography. Aliquots (1–4) of the first elution volumes were analyzed for protein purity by SDS-PAGE. Bands corresponding to OsSOS1:His-6x (128 kD), AtSOS2:His-6x (52 kD), and GST:AtSOS2T/DΔ308 (60 kD) are indicated. Standard M_rs are shown on the left. B, Purified proteins were combined as indicated in protein kinase reaction assays. Aliquots of phosphorylation reactions were resolved by SDS-PAGE and exposed to x-ray films. Arrowhead indicates the 128-kD band pertaining to phosphorylated OsSOS1.

Figure 4.

Complementation of Arabidopsis sos1-1 mutant by rice OsSOS1. A, Six-day-old seedlings grown on Murashige and Skoog agar medium were transferred to Murashige and Skoog medium supplemented with 50 mm NaCl and imaged after 14 d of salt treatment. Left, Transgenic sos1-1 mutant seedling expressing AtSOS1 from the cauliflower mosaic virus 35S promoter. Middle, five independent transgenic lines of sos1-1 mutants expressing the rice OsSOS1 gene under the control of the 35S promoter. Right, sos1-1 mutant seedling transformed with empty vector pBI321. B, Quantitation of seedling growth, expressed as fresh weight after 14 d in Murashige and Skoog medium with and without supplemental 65 mm NaCl. Data are the mean and se of fresh-weight values of three to six individual seedlings from each line. Dashed bars, sos1-1 mutant seedlings transformed with empty vector pBI321; gray bars, sos1-1 mutant seedlings transformed with the rice OsSOS1 gene under the control of the 35S promoter; black bars, sos1-1 mutant seedlings transformed with Arabidopsis SOS1. [See online article for color version of this figure.]

Figure 5.

Transcript abundance of OsSOS1 in response to salt stress. Total RNA purified from roots and shoots of rice plants subjected to salt stress with 100 mm NaCl in hydroponic culture medium for 0, 3, 15, and 48 h. Hybridization was performed with a probe prepared from OsSOS1 cDNA. RNA sample loading was normalized by hybridization with a probe derived from radish (Raphanus sativus) 18S rDNA.

Figure 6.

Functional interactions between Arabidopsis SOS proteins and rice counterparts. Strains YP890 carrying the integration of the PGK1_PRO:AtSOS1:CYC1_TER cassette (A and C), YP1021 with the analogous integration PMA1_PRO:OsSOS1:ADH1_TER with the SOS1 cDNA from rice (D), and AXT3K cells transformed with plasmid pSOS1-1 for the expression of AtSOS1 (B) were transformed with plasmids directing the expression of the regulatory proteins SOS2 and SOS3 from Arabidopsis or CIPK24 and CBL4 from rice, as indicated in each case. CIPK24Δ309 bears a C-terminal deletion rendering a constitutive, CBL-independent protein kinase. Yeast cells were grown overnight in selective synthetic dextrose medium. Five microliters of serial decimal dilutions were spotted onto plates of AP medium with 1 mm KCl and 200 mm NaCl. Plates were incubated at 28°C for 3 to 4 d.

Figure 7.

Complementation of Arabidopsis sos2 and sos3 mutants by rice CIPK24 and CBL4. Six-day-old seedlings of mutants sos3-1 (A) and sos2-2 (B) transformed with cDNAs of rice genes CBL4 (A) or CIPK24 (B), respectively, were transferred to Murashige and Skoog medium supplemented with 100 mm (CBL4 and sos3) or 75 mm NaCl (CIPK24 and sos2) and imaged after 14 d of growth. From left and right, two wild-type plants, five complemented lines, and two mutant plants transformed with empty vectors are depicted. The root length of seven individual plants from each of these lines was measured after 14 d in salinized media. C, sos3 mutant and rice CBL4. D, sos2 mutant and rice CIPK24. In both plots, dashed columns represent the root length attained by mutant lines; gray columns are complemented lines; and back columns are wild-type Col-0. [See online article for color version of this figure.]