Fig 1.
Structural analysis of SpSOS1 and SpAHA1 proteins.
(A) Phylogenetic tree of plasma membrane Na+/H+ antiporters from plants. SpSOS1 (JX674067), AtSOS1 (NM_126259), OsSOS1 (AY785147), PtSOS1 (XM_002315801), TaSOS1 (FN356232) and ThSOS1 (EF207775) indicate Na+/H+ antiporters from Sesuvium portulacastrum, Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Triticum aestivum and Thellungiella halophila, respectively. (B) Phylogenetic tree of plasma membrane H+-ATPases from plants. SpAHA1 (JX628604) indicates Sesuvium portulacastrum H+-ATPase. AtAHA1 (NM_127453), AtAHA2 (NM_001203941), AtAHA3 (NM_001203630), AtAHA4 (NM_114664), AtAHA5 (NM_128013), AtAHA6 (NM_126721), AtAHA7 (NM_115897), AtAHA8 (NM_114131), AtAHA9 (NM_001198521), AtAHA10 (NM_101587), and AtAHA11 (NM_125662) indicate Arabidopsis thaliana H+-ATPases. The phylogenetic trees were constructed using the MEGA5.0 software based on multiple alignments of full-length protein sequences. Evolutionary distances were computed using the neighbor joining method. Bootstrap values are indicated at the nodes of the tree and are expressed as percentages.
Fig 2.
SpSOS1 and SpAHA1 proteins are localized at the plasma membrane.
SpSOS1-GFP, SpAHA1-GFP and AtSOS1-GFP fusion proteins were produced as described in the Methods and Materials section. Tobacco leaves were transformed with the vector pCAMBIA1300 containing GFP alone (GFP), pCAMBIA1300-SpSOS1-GFP (SpSOS1-GFP), pCAMBIA1300-SpAHA1-GFP (SpAHA1-GFP) or pCAMBIA1300-AtSOS1-GFP (AtSOS1-GFP). GFP signals from epidermal cells (left panel; scale bars = 50 μm) and mesophyll protoplasts (right panel; scale bars = 10 μm) of tobacco leaves transiently expressing either GFP alone, SpSOS1-GFP, SpAHA1-GFP or AtSOS1-GFP were recorded using confocal microscopy.
Fig 3.
Changes in SpSOS1 and SpAHA1 mRNAs in S. portulacastrum exposed to salinity.
Seedlings were treated with 400, 600 or 800 mM NaCl. The expression of SpSOS1 (A, B) and SpAHA1 (C, D) was analyzed in roots (A, C) and leaves (B, D) at different time intervals (0, 3, 6, 9, 12, 24 and 48 h) under salt treatment using real-time PCR. The expression value at the initial time (0 h) was set to 1, and the expression levels at the other time points under salinity treatment were calculated as the fold of the value at 0 h. Values are expressed as the means±SE (n = 3).
Fig 4.
Effect of salt stress on the growth of yeast cells.
The wild-type strain (W303), mutant cells (AXT3K) and recombinant strains (SpSOS1, SpAHA1 and SpSOS1+SpAHA1) were grown to saturation, and then 10-μL serial decimal dilutions were spotted onto AP plates supplemented with 0, 50, 75, or 150 mM NaCl. SpSOS1: the AXT3K strain transformed with plasmid p416-SpSOS1; SpAHA1: the AXT3K strain transformed with plasmid p414-SpAHA1; SpSOS1+SpAHA1: mutant strain AXT3K co-transformed with plasmids p416-SpSOS1 and p414-AHA1; AXT3K: untransformed mutant strain AXT3K; W303: wild-type yeast strain W303.
Fig 5.
The Na+ content in unstressed yeast cells and yeast cells treated with 30 mM NaCl was measured. Values are expressed as the means±SE (n = 3). AXT3K: the non-transformed yeast strain; SpAHA1: the AXT3K strain transformed with plasmid p414-SpAHA1; SpSOS1: the AXT3K strain transformed with plasmid p416-SpSOS1; SpSOS1+SpAHA1: the mutant strain AXT3K co-transformed with plasmids p416-SpSOS1 and p414-AHA1.
Fig 6.
H+-ATPase and Na+/H+ antiport activities at plasma membrane vesicles.
Plasma membranes were isolated from mutant cells (AXT3K) and related transgenic strains (SpSOS1, SpAHA1 and SpSOS1+SpAHA1) using the aqueous two-phase system. Fluorescent quenching of quinacrine was used to monitor the acidification of plasma membrane vesicles from these yeast cells. Formation of a pH gradient, indicating that H+ was pumped into the vesicles, was initiated with ATP. Once fluorescence was stabilized, 50 mM Na2SO4 was added to the cuvette, and fluorescence recovery, indicating Na+/H+ exchange, was monitored. The reaction was terminated by adding 25 mM (NH4)2SO4, which dissipated the pH gradient. The change in fluorescence is expressed as arbitrary units (A). H+-ATPase activity was analyzed by measuring ΔpH across the membrane; it is expressed as the change in fluorescence quenching per minute per milligrams of membrane protein (ΔF/min/mg protein) (B). Na+/H+ exchange activity is given as the proportion of dissipation of the preformed pH gradient per minute per milligram of membrane protein (ΔF%/ min/mg protein) (C). Values are expressed as the means±SE of three replicates.